GIFT OF 
 
SEMICENTENNIAL PUBLICATIONS 
 
 OF THE 
 
 UNIVERSITY OF CALIFORNIA 
 
 1868-1918 
 
THE PHYSICAL CHEMISTET 
 
 OF 
 
 THE PROTEINS 
 
" From the point of view of the physicist, a theory of matter is a 
 policy rather than a creed; its object is to connect or co-ordinate 
 apparently diverse phenomena, and above all to suggest, stimu- 
 late and direct experiment. It ought to furnish a compass which, 
 if followed, will lead the observer further and further into pre- 
 viously unexplored regions. Whether these regions will be barren 
 or fertile experience alone will decide; but, at any rate, one who 
 is guided in this way will travel onward in a definite direction, and 
 will not wander aimlessly to and fro." J. J. Thomson, "The 
 Corpuscular Theory of Matter," 
 
THE PHYSICAL CHEMISTRY 
 
 OF 
 
 THE PROTEINS 
 
 BY 
 
 T. BRAILSFORD ROBERTSON, PH.D., D.Sc. 
 
 f 
 
 Professor of Biochemistry and Pharmacology in 
 the University of California, 
 
 LONGMANS, GREEN AND CO. 
 
 FOURTH AVENUE & SOxn STREET, NEW YORK 
 
 39 PATERNOSTER ROW, LONDON 
 
 BOMBAY, CALCUTTA, AND MADRAS 
 
 1918 
 
TU 
 
 COPYRIGHT, 1918, 
 
 BY 
 LONGMANS, GREEN & CO. 
 
DEDICATED TO MY MOTHER 
 
 IN AFFECTIONATE ACKNOWLEDGMENT 
 
 OF ENCOURAGEMENT AND 
 
 INSPIRATION 
 
 420161 
 
PREFACE 
 
 THE Proteins, with which, as its title indicates, this work has 
 most particularly to deal, have, ever since the publication of the 
 classic researches of Graham, been generally recognized as typical 
 examples of that class of substances which Graham designated 
 "Colloids." This work, therefore, although primarily concerned 
 with the physical chemistry of a limited section of the class, may 
 also, in some measure, be regarded as contributing to an analysis 
 of the properties and behavior of colloids in general, in so far as 
 these permit of illustration by the properties and behavior of 
 the various members of the protein group. 
 
 The leading problems in every field of chemical investigation are, 
 to a large extent, determined by the salient properties of the sub- 
 stances which form the subject of inquiry, and for this reason a 
 thorough acquaintance with what may be termed the " descriptive 
 chemistry" of any group of substances is prerequisite to a suc- 
 cessful attempt to interpret their behavior. If we were to en- 
 deavor to interpret the behavior of the proteins solely in the light 
 of preconceptions derived from the study of the chemistry of the 
 metals, for example, or the simpler organic compounds, we would 
 find that the behavior of the proteins displays merely a bewilder- 
 ing variety of inconsistencies. Their "amphoteric" character, 
 their multiple basicity and acidity, their instability in aqueous solu- 
 tion and, above all, the enormous mass and catenary configuration 
 of their molecules, confer upon them properties which are com- 
 paratively unfamiliar in other fields of chemistry or else exaggerate 
 properties which are normally displayed by the simpler chemical 
 bodies only to a comparatively negligible degree. 
 
 The proteins in this respect are not exceptional. Thus the mode 
 of investigation and the interpretation of the behavior of the 
 various lipoids must primarily be conditioned by their very general 
 insolubility in water and instability in the presence of oxygen. 
 The behavior of the complex polysaccharides is conditioned by 
 their relative stability and by the extraordinary tendency to op- 
 
 vii 
 
viii PREFACE 
 
 tical isomerism which is displayed by the simpler carbohydrate 
 radicals out of which they are built up, while, on the other hand, 
 the behavior of rubber and its congeners is primarily conditioned 
 by the enormous internal molecular friction which leads them to 
 display, to an exaggerated extent, phenomena analogous to hys- 
 teresis which, although not wholly negligible in other colloids, for 
 example in the protein group, nevertheless seldom present them- 
 selves as salient characteristics of their behavior. 
 
 The colloids are therefore an exceedingly heterogeneous group, 
 the only common distinguishing characteristic being the relatively 
 enormous mass and volume of the molecules of the most "typical" 
 representatives of the class, and of course as many gradations of 
 behavior exist as there are gradations in the mass and volume of 
 molecules. Nothing is to be gained, therefore, by endeavoring to 
 force the various members of the colloid group into artificial con- 
 formity with definitions which are designed to separate them, as 
 if they were a homogeneous group, from other classes of chemical 
 substances. To describe a particular property or mode of be- 
 havior as a " colloidal phenomenon" neither defines nor inter- 
 prets it and furthermore fails even to describe it, since there are 
 no phenomena which are distinctively " colloidal" and displayed 
 by every member of the colloid group, saving only those phenomena 
 which depend primarily upon the simple factor of the mass or 
 volume of the molecules, and which are therefore predictable 
 from and implied in the properties or behavior of the smaller 
 molecules of the non-colloidal substances. 
 
 Similarly, the use of the term "adsorption" to describe the 
 union between colloids and other substances implies a uniformity 
 where no uniformity exists and is, moreover, devoid of utility or 
 meaning unless we attach to the definition some distinct idea of 
 the nature of the underlying forces which condition the union, 
 whether these forces be regarded as consisting of chemical (i.e. y 
 atomic) attractions or of capillary (i.e., molecular) attractions. 
 But in forming such concrete ideas we are simply returning to 
 conceptions which are already familiar to us in the "crystalloid" 
 field of chemistry and which call for no definitions which we do not 
 already possess as the result' of our general acquaintance with the 
 physical and chemical phenomena which are displayed by simpler 
 and hitherto more familiar substances. 
 
 The investigations of recent years, not only upon the behavior 
 
PREFACE ix 
 
 of the proteins, but upon that of the colloids in general, have re- 
 sulted in the development of two rather sharply differentiated 
 schools of opinion. The one school endeavors, so far as technical 
 difficulties permit, to directly apply, with modifications suggested 
 by the properties and structure of the particular colloid under 
 investigation, the known laws of what may be termed "molec- 
 ular" physical chemistry to protein and other colloidal systems, 
 while the other school hesitates to do so. The essential question 
 at issue between these two schools, in so far as the proteins are 
 concerned, is, I think, simply this: Are we justified in assuming 
 that the rule of Avogadro is applicable to protein solutions or are 
 we not? In other words, are protein solutions molecularly dis- 
 persed systems or are they, rather, suspensions or emulsions? 
 The latter of the two schools to which I have referred avers from 
 a priori considerations, implicitly or explicitly, that Avogadro's 
 rule may not be applied to protein solutions, or, at least, that its 
 validity for these solutions should be established before we venture 
 to apply it. The former school prefers to assume that Avogadro's 
 rule does apply to these systems until actual inapplicability 
 demonstrates that it does not. Now the " proof " of the applica- 
 bility of Avogadro's rule to systems which are admittedly molec- 
 ularly dispersed has never consisted in anything but the appli- 
 cability, to these systems, of laws and deductions founded upon 
 Avogadro's rule. The procedure of the former school would 
 appear, therefore, to be sound and well justified by scientific 
 precedent. Following this procedure we will be enabled to cor- 
 relate and interpret the phenomena which are exhibited by such 
 protein systems as may chance to be molecularly dispersed, while, 
 on the other hand, we shall be enabled to accurately delimit the 
 conditions under which molecularly dispersed protein systems 
 exist and those under which they do not. 
 
 In this work I have endeavored to interpret the physico-chemi- 
 cal behavior of the proteins in the light of the laws of Boyle and of 
 Gay-Lussac, as they have been applied to solutions by van't Hoff, 
 and of the Guldberg and Waage mass-law which, as Larmor has 
 shown, is a direct consequence of Avogadro's rule and Boyle's 
 law.* I have also assumed the validity, in protein systems, of 
 the first and second laws of heat, albeit the applicability of the 
 
 * Larmor, J., Phil. Trans. Roy. Soc. London, 190 A (1887), p. 276. T. Brails- 
 ford Robertson, Journ. Physical Chem., 10 (1906), p. 521. 
 
X PREFACE 
 
 second law of heat to protein systems has, in some quarters,* been 
 questioned. In considering the electrochemical behavior of the 
 proteins I have assumed the applicability of Arrhenius' hypothesis 
 of electrolytic dissociation, of Kolrausch's law of the independent 
 motion of ions, of the Nernst theory of concentration-cells, and 
 further, although this has of recent years been very strongly 
 questioned,! the applicability of the Guldberg and Waage mass- 
 law to reactions between ions. I believe that the utility of these 
 hypotheses justifies us in applying them until still more useful 
 hypotheses shall have been elaborated to amplify or replace them. 
 
 A previous edition of this work appeared, in German, six years 
 ago.J Since that time our knowledge of the physico-chemical 
 behavior of the proteins has very considerably expanded and in- 
 creased in exactitude. Among the particularly important in- 
 vestigations which have appeared during this period may be men- 
 tioned the invention by Van Slyke of an accurate and simple 
 method of determining free amino-groups (Cf. Chapter I), the 
 work of Pauli upon the combining-capacity of deaminized pro- 
 teins (Cf. Chapters I, VIII and IX), the work of Schmidt and of 
 af Ugglas upon compound proteins (Cf. Chapter VII), the work 
 of Procter upon the swelling of protein jellies (Cf. Chapter XII), 
 the work of Reichert and Brown upon the crystallography of 
 haemoglobin (Cf. Chapter XII) and that of Dabrovsky upon the 
 molecular volumes of proteins dissolved in water and in solutions 
 of coagulating salts (Cf. Chapter XIII). The present edition 
 has been almost entirely rewritten and the literature has been 
 brought down to the middle of 1917. 
 
 In conclusion I wish to express my very great indebtedness to my 
 wife, for her assistance in preparing the manuscript for the press, 
 to Dr. Hardolph Wasteneys for his assistance in proof-reading, 
 and to Dr. C. L. A. Schmidt for his assistance in proof-reading 
 and in the verification of many references and formulas. 
 
 T. BRAILSFORD ROBERTSON. 
 
 BERKELEY, CALIFORNIA, 
 Nov. 1, 1917. 
 
 * von Schroeder, P., Zeit. f. physik. Chem., 45 (1903), p. 75. Dietz, W., 
 Zeit. f. physiol. Chem., 52 (1907), p. 279. Cf., however, Chapter XVII. 
 
 t Cf. for example, W. Sutherland, Phil. Mag. Series 6, 14 (1907), p. 1. 
 
 t " Die physikalische Chemie der Proteine," Dresden, 1912. Theodor Stein- 
 kopff. 
 
CONTENTS 
 
 PAQB 
 
 PREFACE vii 
 
 PART I. CHEMICAL STATICS IN PROTEIN SYSTEMS 
 
 CHAPTER I. THE CHEMICAL CONSTITUTION OP THE PROTEINS 3 
 
 1. The Chemical Homogeneity of the Protein Group 3 
 
 2. The Products of the Decomposition of the Proteins 4 
 
 3. The Connection between the Amino-acid Content and the Prop- 
 
 erties of the Proteins 8 
 
 4. The Synthesis of Proteins 9 
 
 5. The Occurrence of Peptids among the Products of Protein Hy- 
 
 drolysis 15 
 
 6. The Analysis and Characterization of Proteins by the Determi- 
 
 nation of the Chemical Groups Characteristic of the Different 
 
 Amino-acids 16 
 
 7. Types of Union in the Protein Molecule 17 
 
 8. Some Consequences of the Polypeptid Structure of the Protein 
 
 Molecule 20 
 
 9. "Racemized" Proteins 30 
 
 CHAPTER II. THE PREPARATION OF PURE PROTEINS 34 
 
 1. The Proteins as Chemical Individuals 34 
 
 2. Casein 37 
 
 3. "Insoluble" Serum Globulin 40 
 
 4. Fibrin 42 
 
 5. Haemoglobin . 43 
 
 6. Crystallizable Egg Albumin 44 
 
 7. Ovovitellin 44 
 
 8. The Vegetable Proteins 46 
 
 9. The Alcohol Soluble Vegetable Proteins (Gliadin, Zein, etc.) 48 
 
 10. Ovomucoid 49 
 
 11. Gelatin and Deaminized Gelatin 51 
 
 12. Globin 51 
 
 13. The Protamins 53 
 
 CHAPTER III. THE QUANTITATIVE ESTIMATION OF THE PROTEINS.. . 56 
 
 1. The General Principles Underlying the Quantitative Estimation 
 
 of Proteins 56 
 
 2. The Nephelometric Method of Estimating Proteins 58 
 
 3. The Refractometric Method of Estimating Proteins 60 
 
 xi 
 
xii CONTENTS 
 
 PAGE 
 
 CHAPTER IV. THE COMPOUNDS OF THE PROTEINS 67 
 
 1. The Amphoteric Character of the Proteins 67 
 
 2. The Direct Method of Demonstrating the Existence of Protein 
 
 Compounds by Precipitation or Coagulation 68 
 
 3. The Direct Method of Demonstrating the Existence of Protein 
 
 Compounds by the Solution of otherwise Insoluble Substances. 71 
 
 4. The Indirect Method of Precipitation 71 
 
 5. The Method of Electrical Conductivity 74 
 
 6. The Cryoscopic Method 74 
 
 7. The Potentiometric Method 76 
 
 8. The Method of Catalysis 79 
 
 9. The Indicator Method 81 
 
 10. The Method of "Masking" the Physiological Effects of Ions by 
 
 the Addition of Proteins to their Solutions 82 
 
 CHAPTER V. THE COMPOUNDS OF THE PROTEINS Continued 85 
 
 1. Stoichiometrical Relations in Protein Compounds 85 
 
 2. The Compounds of the Protamines with Inorganic Acids and 
 
 Bases 86 
 
 3. The Compounds of Casein with Bases, Acids, and Salts 88 
 
 4. The Compounds of Serum Globulin with Inorganic Acids and 
 
 Bases 98 
 
 5. The Compounds of Fibrin with Inorganic Acids and Bases 101 
 
 6. The Compounds of the Vegetable Proteins with Inorganic Acids 
 
 and Bases 101 
 
 7. The Compounds of Ovomucoid with Acids and Bases 103 
 
 8. Summary of Some of the Results Cited in this Chapter 103 
 
 CHAPTER VI. THE COMPOUNDS OF THE PROTEINS Continued 107 
 
 1. General Remarks on the Precipitation of the Proteins by Inorganic 
 
 Salts 107 
 
 2. Earlier Investigations on the Significance of the State of Hydra- 
 
 tion of the Proteins in Relation to their Coagulation by Salts. 108 
 
 3. The Influence of the Electrical Condition of the Proteins upon 
 
 their Precipitation and Coagulation by Electrolytes 112 
 
 4. Later Investigations on the Significance of the State of Hydration 
 
 of the Proteins in Relation to their Coagulation by Electro- 
 lytes 119 
 
 5. Application of the Phase-rule to Protein-salt-water Systems 123 
 
 6. The Chemical Mechanics of the Precipitation and Coagulation of 
 
 Proteins by Salts 125 
 
 CHAPTER VII. THE COMPOUNDS OF THE PROTEINS Continued 137 
 
 1. Compounds with the Heavy Metals 137 
 
 2. Compounds with the Phosphoric Acids 142 
 
 3. Compounds of the Proteins with Carbonic Acid 144 
 
CONTENTS xiii 
 
 PAGE 
 
 4. Compounds of the Proteins with the Alkaloidal Reagents, Dyes, 
 
 Alkaloids, etc 145 
 
 5. The Compounds of Proteins with Soaps and Lipoids 148 
 
 6. The Compounds of Proteins with Proteins and their Possible 
 
 Significance in Life-phenomena 148 
 
 7. Compounds of the Proteins with Toxins, Antibodies, Ferments, etc. 156 
 
 8. Methyl and Benzoyl Derivatives of the Proteins 156 
 
 9. The Halogen- and Nitro-substitution Compounds of the Proteins. 157 
 
 10. The Compounds of Proteins with Sulphur 159 
 
 11. The Compounds of Proteins with Oxygen 159 
 
 PART II. THE ELECTROCHEMISTRY OF THE PROTEINS 
 CHAPTER VIII. THE FORMATION AND DISSOCIATION OF PROTEIN SALTS 167 
 
 1. Compounds of the Proteins with Inorganic Bases and Acids; the 
 
 Non-dissociable Character of the Inorganic Radical 167 
 
 2. The Electrolysis of Protein Salts 176 
 
 3. The Relative Masses of Protein Anions and Cations 184 
 
 4. The Migration-velocity of Protein Ions 187 
 
 5. Objections to the above Theory of Protein lonization 189 
 
 6 Biological Applications; the "Selective" Action of Living Tissues. 192 
 
 CHAPTER IX. THE COMBINING-CAPACITY OF THE PROTEINS 195 
 
 1. The Electrochemical Determination of the Combining-capacity 
 
 of the Proteins 195 
 
 2. The Combining-capacity of Casein for Bases and of Ovomucoid 
 
 for HC1 at Absolute Neutrality 204 
 
 3. The Non-dependence of the Composition of the Compounds of 
 
 Protein with Acids and Bases upon the Dilution of their Solu- 
 tions 206 
 
 4. The "Isoelectric" Condition of Proteins at Certain H+ and OH' 
 
 Concentrations 209 
 
 5. Biological Applications; the Neutrality of the Tissues and of the 
 
 Tissue-fluids 211 
 
 CHAPTER X. THE ELECTRICAL CONDUCTIVITY OF SOLUTIONS OF PRO- 
 TEIN SALTS 220 
 
 1. The Influence of Dilution upon the Conductivity of Solutions of 
 
 Protein Salts 220 
 
 2. The Depression of the Freezing-point of Water which is Caused by 
 
 Dissolved Protein Salts, and the Stoichiometry of Protein Salts. 236 
 
 3. The Dependence of the Electrical Conductivity of Solutions of 
 
 Protein Salts upon the Proportion of Inorganic Acid or Base 
 which the Salts Contain 242 
 
 4. The Solubility and Minimal Combining-capacities of Casein and 
 
 of Serum Globulin in Solutions of Bases. . 248 
 
Xiv CONTENTS 
 
 PAGE 
 CHAPTER XI. THE ELECTROCHEMISTRY OF COAGULATION 252 
 
 1. The Coagulation of the Casemates by Alcohol 252 
 
 2. The Appli.cability.pf the Ostwald Dilution-Law to Solutions of the 
 
 Casemates in Alcohol-Water Mixtures 254 
 
 3. The Influence of the Concentration of Alcohol in the Solvent 
 
 upon the Conductivities of Solutions of Potassium Caseinate. . 257 
 
 4. The Interpretation of the Law x v = ^ 262 
 
 5. The Viscosities of Solutions of Potassium Caseinate in Alcohol- 
 
 Water Mixtures 266 
 
 6. The Molecular Condition of Potassium Caseinate in 75% Alcohol 268 
 
 7. The Chemical Mechanics of the Coagulation of Proteins by Al- 
 
 cohol. . 270 
 
 PART III. THE PHYSICAL PROPERTIES OF PROTEIN SYSTEMS 
 
 CHAPTER XII. THE PHENOMENA WHICH ACCOMPANY CHANGES IN 
 
 THE STATE OF AGGREGATION OF PROTEINS 275 
 
 1. The Passage of Dry Protein into Solution 275 
 
 2. The Swelling of Protein Jellies 292 
 
 3. The Gelatinization and Coagulation of Proteins 299 
 
 4. The Coagulation of Proteins by Heat, Light and Hydrostatic 
 
 Pressure 304 
 
 5. The Crystallization of Proteins 311 
 
 CHAPTER XIII. CERTAIN PHYSICAL PROPERTIES OF PROTEIN SOLU- 
 TIONS, ETC 320 
 
 1. The Viscosity of Protein Solutions 320 
 
 2. The Cohesiveness of Protein Solutions 328 
 
 3. The Elasticity of Protein Solutions 329 
 
 4. The Diffusion of Proteins in Solution 330 
 
 5. The Freezing- and Boiling-points of Protein Solutions 331 
 
 6. The Osmotic Pressure of Proteins in Solution 336 
 
 7. The Nature of Protein Solutions 340 
 
 8. The Opalescence of Protein Solutions; the Tyndall Effect 342 
 
 9. The Surface Tension of Protein Solutions . . . / 345 
 
 10. The Formation of Surface-films by Dissolved Proteins 345 
 
 11. The Specific Gravities of Protein Solutions 348 
 
 12. The Magnetic Properties of the Proteins 349 
 
 13. The "Gold Number" of Proteins 349 
 
 CHAPTER XIV. OPTICAL PROPERTIES OF PROTEIN SOLUTIONS 355 
 
 1. The Specific Rotatory Power of the Proteins 355 
 
 2. The Absorption of Light by Protein Solutions 358 
 
 3. The Refractive Indices of Protein Solutions 359 
 
CONTENTS XV 
 
 PART IV. CHEMICAL DYNAMICS IN PROTEIN SYSTEMS 
 
 PAGE 
 
 CHAPTER XV. THE HYDROLYSIS OF THE POLYPEPTIDS 371 
 
 1. The Hydrolysis of Polypeptids by Proteolytic Enzymes 371 
 
 2. The Kinetics of the Hydrolysis of Polypeptids by Proteolytic 
 
 Enzymes 376 
 
 3. The Order in which Amino-acids are Split off from Polypeptids by 
 
 Proteolytic Ferments 385 - 
 
 CHAPTER XVI. THE HYDROLYSIS OF THE PROTEINS 389 
 
 1. The Proteolytic Enzymes as Catalysors 4.389 
 
 2. The Evidence for the Existence of Intermediate Compounds 
 
 between the Proteolytic Ferments and their Substrates 398 
 
 3. The Kinetics of Protein Hydrolysis by Enzymes 400 
 
 4. The Influence of Acid and Alkali upon the Rate of Protein Hy- 
 
 drolysis by Enzymes 410 
 
 5. The Influence of Added Substances upon the Rate of Protein 
 
 Hydrolysis by Enzymes 415 
 
 6. The Action of the Coagulating Ferments, Rennet and Thrombin. 416 
 
 7. The Influence of Temperature upon the Velocity of Protein Hy- 
 
 drolysis 419 - 
 
 CHAPTER XVII. THE ENZYMATIC SYNTHESIS OF PROTEINS 425 ^~ 
 
 1. The Reversion of the Hydrolysis of Proteins by Pepsin and Tryp- 
 
 sin 425 
 
 2. The Probable Nature of the Coaguloses and Plasteins 437 
 
 3. The Chemical Mechanics of the Fermentative Synthesis of Pro- 
 
 teins 439 
 
 4. Reciprocal Catalysis 444 
 
 5. The Influence of Temperature upon the Enzymatic Synthesis 
 
 of Proteins 448 
 
 6. The Thermodynamics of the Enzymatic Hydrolysis and Synthesis 
 
 of Proteins 450 
 
 7. Biological Applications ./fTS^. 452 
 
 [ 
 APPENDIX. THE TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 
 
 IN PROTEIN SYSTEMS \ 456 
 
 INDEX OF AUTHORS > x 469 
 
 INDEX OF SUBJECTS 479 
 
PART I 
 CHEMICAL STATICS IN PROTEIN SYSTEMS 
 
CHAPTER I 
 THE CHEMICAL CONSTITUTION OF THE PROTEINS 
 
 1. The Chemical Homogeneity of the Protein Group. The 
 
 chemical similarity between the properties and behavior of 'the 
 various protein bodies, and the evidence thus afforded of their 
 close chemical relationship, early led protein chemists to ascribe 
 to the proteins a common and characteristic chemical structure, 
 and to endeavor to anticipate the nature of this structure. 
 
 The difficulty of securing a quantitative separation of protein 
 bodies is eloquent of their chemical similarity; one by one the 
 standard methods of separation brought forward by the physio- 
 logical chemist, such as fractional heat-coagulation, fractional 
 precipitation by salts, etc., have .been shown to possess but a 
 qualitative value; the separation is seldom or never complete, 
 and the critical temperatures, concentrations, etc., at which pre- 
 cipitations occur are seldom well-defined points, but appear, 
 rather, to represent portions of a continuous curve of solubility. 
 The close relationship between the various protein bodies is 
 furthermore evinced by the similarity of their physical properties, 
 and by the almost universal applicability, within the protein 
 group, of such typical color or other reactions as the proteins 
 display. The general physico-chemical characteristics of the vari- 
 ous proteins resemble one another, as we shall see in subsequent 
 chapters, quite as closely as their purely physical or purely chemi- 
 cal characteristics. One of the most striking of these physico- 
 chemical characteristics is their digestibility by the proteolytic 
 enzymes. All true proteins and a large number of the poly- 
 amino-acids are hydrolysable by trypsin, while the overwhelming 
 majority of the proteins are also hydrolysable by pepsin. The 
 chemical relationship between the various proteins would there- 
 fore appear to be even more intimate than that between the 
 various disaccharides, since the enzymes which accelerate the 
 hydrolysis of these are, as a rule, specific, the hydrolysis of each 
 
 3 
 
^CHEMICAL STATICS 
 
 sugar being accelerated to a high degree by one enzyme and by 
 that alone. 
 2. The Products of the Decomposition of the Proteins. The 
 
 proteins are notoriously liable to decomposition, and a complete 
 breaking down of the sparingly diffusible, difficultly crystallizable 
 proteins, into substances which readily diffuse and crystallize can 
 be brought about by a variety of agencies, such as the following: 
 
 I. Fusion with alkali. 
 
 II. Oxidation with permanganate, chromic acid, etc. 
 
 III. The action of halogens. 
 
 IV. Hydrolysis, through one or more of the following agencies: 
 
 a. Heating in acid solution, 
 
 6. Heating in alkaline solution, 
 
 c. Treatment with superheated steam. 
 
 d. Treatment with enzymes. 
 
 Of all these methods that of hydrolysis is the most satisfactory 
 and yields the most uniform and readily inter pretable results. 
 It appears that whatever be the method of hydrolysis employed, 
 the end-result, provided the hydrolysis has been complete, is 
 the same, namely, the production of a mixture of amino-acids. 
 
 Incomplete hydrolysis, however, results in the production of a 
 number of intermediate substances, variously designated, in the 
 order of decreasing complexity, proteoses (albumoses), peptones 
 and polypeptids. The hydrolysis of the proteins, therefore, 
 occurs in stages, just as, in the hydrolysis of starch, intermediary 
 stages (the dextrins and maltose) are passed through, before the 
 attainment of the last stage of hydrolysis and the complete con- 
 version of the starch into glucose. 
 
 It was early recognized that a predominating proportion of the 
 products of the complete hydrolysis of proteins consists of amino- 
 acids, and the most readily detected amino-acids, leucin and tyro- 
 sin, were discovered, respectively by Proust in 1818 (54), andjby 
 Liebig in 1846 (44). The chemical constitution of these sub- 
 stances, however, was a discovery of much later date, that of 
 leucin having been established in 1868 by Hiifner (31) and of 
 tyrosin in 1869 by Earth (3). 
 
 The older methods of isolating individual amino-acids from the 
 mixture which the complete hydrolysis of % a protein yields, de- 
 
PRODUCTS OF DECOMPOSITION 5 
 
 pended upon the fractional crystallization either of the free 
 ammo-acids or of their salts. They were not even approximately 
 quantitative and the isolation and identification of a given amino- 
 acid could only be effected with certainty when that acid was 
 present in relatively large amounts. Until 1890 only mono- 
 amino-acids were known, with certainty, to occur among the 
 products of protein hydrolysis. Then Drechsel (13) discovered 
 lysin and lysatinin, Hedin (29) isolated arginin and Kossel (34) 
 isolated histidin from among the dissociation products of proteins. 
 
 The attainment of our present relatively extensive knowledge 
 of the nature and yield of the products of the hydrolysis of pro- 
 teins is an achievement of the past twenty years, and we owe it 
 in the first place to the labors of Emil Fischer and of Kossel and 
 their pupils. 
 
 In 1900 Kossel and Kutscher (39) (40) succeeded in working 
 out a method for the quantitative separation and estimation of 
 the diamino-acids lysin, arginin and histidin which Kossel (35) 
 calls the hexone bases.* The method depends in principle upon 
 the precipitation of arginin and histidin in the form of their 
 silver salts, and of lysin first by phosphotungstic acid and then. 
 by picric acid. A partial but not quantitative separation of the 
 diamino-acids from the monoamino-acids in a protein digest, 
 can be procured by precipitation of the former with phospho- 
 tungstic acid. 
 
 In 1901 Emil Fischer (15) introduced a new method of sepa- 
 rating and estimating the monoamino-acids, which, with modi- 
 fications suggested by experience, is the one to which we owe the 
 greater part of our present knowledge of these products of protein 
 hydrolysis. The hydrolysis is carried out, as a rule, by boiling 
 with hydrochloric acid.f The amino-acids which result are then 
 converted into their esters by dissolving them in alcohol and 
 esterifying by saturation of the solution with dry hydrochloric 
 acid gas. The mixed esters thus obtained are separated by 
 
 * Owing to the fact that they each contain six atoms of carbon. An 
 especial interest attaches to these substances, since, unlike the monoamino 
 acids, they are predominantly basic in character, and their relation to the 
 hexoses suggests their possible importance in carbohydrate metabolism. 
 
 t N. Zelinsky (74) has suggested the employment of formic acid as a hy- 
 drolysing agent. According to this investigator the hydrolysis by formic acid 
 is much more rapid than that which is brought about by boiling the proteins 
 with hydrochloric acid. 
 
6 CHEMICAL STATICS 
 
 fractional distillation in vacuo* The different fractions which 
 are thus obtained each contain the esters of only a few amino- 
 acids. The esters in each fraction are now reconverted into the 
 free amino-acids, and the individual acids are separated, identi- 
 fied and estimated by appropriate methods which differ some- 
 what for the different acids. 
 
 The method, except so far as glutamic acid is concerned, is 
 by no means quantitative. The results which are obtained are 
 minimal yields. The extremely insoluble amino-acids, tyrosin, 
 cystin, and diaminotrioxydodecanic acid are separated from the 
 digest before esterification, and, so far as tyrosin is concerned, 
 our estimate is tolerably quantitative. 
 
 By a combination of these methods it has been shown that the 
 protein molecule is built up of a series of amino-acids, and the 
 following amino-acids have been isolated from amongst the 
 products of the hydrolysis of various proteins.f 
 
 A. Monoaminomonocarboxylic Acids 
 
 1. Glycin: C 2 H 5 NO 2 , or amino-acetic acid: 
 
 CH 2 .NH 2 .COOH. 
 
 2. Alanin: C 3 HyNO 2 , or a-aminopropionic acid: 
 
 CH 3 .CH(NH 2 ).COOH. 
 
 3. Valin: C 6 HnNO 2 , or a-aminoisovalerianic acid 
 
 8 X CH.CH(NH 2 )COOH. 
 
 4. Leucin: C 6 Hi 3 NO 2 , or a-aminoisocaproic acid: 
 
 CH.CH 2 .CH(NH 2 )COOH. 
 
 CH 3X 
 
 5. Isoleucin: C 6 Hi 3 NO 2 , or a-amino-/3-methyl-j8-ethylpropionic acid: 
 CH 3 , 
 
 X CH.CH(NH 2 ).COOH. 
 
 6. Phenylalanin : C9HnNO 2 , or /8-phenyl-a-aminopropionic acid: 
 
 C 6 H 5 .CH 2 .CH(NH 2 ).COOH. 
 
 7. Tyrosin: C 9 H U NO 3 , or 0-parahydroxyphenyl-a-aminopropionic acid: 
 
 HO.C 6 H 4 .CH 2 .CH(NH 2 )COOH. 
 
 * N. Zelinsky, A. Annenkov, and J. Kulikov (75) accomplish the separation 
 of the individual amino-acids by first neutralizing the excess of hydrochloric 
 acid with lead hydroxide and then subjecting the mixed hydrochlorides of the 
 ethyl esters to fractional distillation. 
 t Cited from Aders Plimmer (53). 
 
PRODUCTS OF DECOMPOSITION 
 
 8. Serin: C 3 H 7 NO 3 , or /S-hydroxy-a-aminopropionic acid: 
 
 CH 2 (OH).CH(NH 2 )COOH. 
 
 9. Cystin: C 6 Hi2N2O4S 2 or dicystein, or di-(/3-thio-a-aminopropionic 
 
 acid) : 
 HOOC.CH(NH 2 ).CH 2 .S - S.CH 2 .CH(NH 2 ).COOH 
 
 B. Monoaminodicarboxylic Acids 
 
 10. Aspartic acid, C 4 H 7 NO 4 , or aminosuccinic acid. 
 HOOC.CH 2 .CH(NH 2 ).COOH. 
 
 11. Glutamic acid, CsHgNO^ or a-aminoglutaric acid: 
 
 HOOC.CH 2 .CH 2 .CH(NH 2 )COOH. 
 
 C. Diaminomonocarboxylic Acids 
 
 12. Arginin, C 6 Hi 4 N 4 O 2 , or a-amino-5-guanidinvalerianic acid: 
 
 /NH 2 
 HN = C( 
 
 x NH.CH 2 .CH 2 .CH 2 .CH(NH 2 )COOH. 
 
 13. Lysin: CeHi 4 N 2 O 2 or a-e-diaminocaproic acid: 
 
 H 2 N.CH 2 .CH 2 .CH 2 .CH 2 .CH(NH 2 ).COOH. 
 
 D. Diamino-oxy-monocarboxylic Acids 
 
 14. Caseinic acid, or diaminotrioxydodecanic acid: 
 
 C 12 H 26 N 2 5 . 
 
 E. Heterocyclic Compounds 
 
 15. Histidin, C 6 H 9 N 3 O 2 , or /3-iminazolyl-a-aminopropionic acid: 
 
 CH 
 
 # \ 
 N NH 
 
 I I 
 CH = C.CH 2 .CH(NH 2 ).COOH 
 
 16. Prolin, C 5 H 9 NO 2 , or a-pyrrolidin carboxylic acid: 
 
 I I 
 
 CH 2 CH.COOH 
 VH 
 
 17. Oxyprolin, or oxypyrrolidin carboxylic acid: 
 
 C 6 H 9 N0 3 . 
 
 18. Tryptophane, CnHi 2 N 2 O 2 , or /3-indole-a-aminopropionic acid: 
 
 C.CH 2 .CH.NH 2 .COOH 
 
 / ^ 
 
 C 6 H 4 CH 
 
 VH 
 
8 CHEMICAL STATICS 
 
 3. The Connection between the Amino-Acid Content and the 
 Properties of the Proteins. The various proteins are found to 
 be built up of the several units enumerated above together, 
 possibly, with units which we have not yet succeeded in isolating. 
 Some of the proteins are built up out of all of these units in vary- 
 ing proportions, others out of a lesser number ; so that in some 
 cases amino-acids which are predominant among the products 
 of the hydrolysis of one protein are 'absent from among the prod- 
 ucts of the hydrolysis of another. Nevertheless the general 
 relationship of the various units and their occurrence in dif- 
 ferent proportions in the various proteins accounts not only 
 for the differences, but also for the similarity between 
 them. 
 
 In many instances a definite parallelism can be traced between 
 the chemical and physical behavior of the proteins and their 
 amino-acid content. Thus the albumins, which are soluble in 
 distilled water and are not coagulated by half-saturation of their 
 solutions with ammonium sulphate, contain no glycin, while the 
 globulins which are (when uncombined with bases or acids) 
 insoluble in distilled water, and are coagulated by half-saturation 
 of their solutions with ammonium sulphate, do contain this 
 amino-acid. The alcohol-soluble vegetable proteins contain only 
 a trace (probably attributable to associated impurities) of glycin 
 and some of them" contain no lysin, their content of diamino-acids 
 is very small, while their content of glutamic acid and of prolin 
 is very high. The phosphoproteins (casein, vitellin) are also 
 rather high in glutamic acid. Gelatin is characterized by its 
 high glycin content and keratin (the protein of hair and feathers) 
 by its high cystin content. The histones, which are predomi- 
 nantly basic substances, contain about 30 per cent of diamino- 
 acids, while the protamins, which are still more predominantly 
 basic, contain only small amounts of monoamino-acids, sal- 
 min containing over 80 per cent of arginin, while sturin con- 
 tains 67 per cent of its nitrogen as arginin, 10 per cent in 
 the form of histidin and from 6 to 7 per cent in the form of 
 lysin (41). 
 
 Kossel (35) has expressed the view that all proteins are built 
 up around a protamin nucleus and that this accounts for the 
 fact that the majority of the proteins yield, on hydrolysis, a 
 certain proportion of diamino-acids. 
 
SYNTHESIS OF PROTEINS 9 
 
 4. The Synthesis of Proteins. The marked predominance 
 of amino-acids in the products of protein hydrolysis, long ago 
 led protein chemists to surmise that the amino-acid structure, 
 or some derivative of that structure, must be represented in a 
 high degree in the protein molecule, and it was in following 
 this clue that Schiitzenberger (64) carried out one of the earliest 
 and most successful attempts to synthesize bodies of a protein 
 character. Recognizing that the decomposition of proteins into 
 amino-acids is essentially a phenomenon of hydrolysis, he re- 
 garded dehydration as an essential feature of any attempt at 
 protein synthesis, while the abundance of amino-acids among 
 the products of protein hydrolysis and the presence therein of 
 bodies related to urea, led him to believe that protein synthesis 
 must consist in the linkage of amino-acids with molecules of urea 
 and the elimination of water. Accordingly amino-acids were 
 mixed with urea and phosphorus pentoxide and heated to 125 C. 
 The product was a pasty solid, soluble in water and readily pre- 
 cipitated by alcohol. It was, furthermore, precipitated from aque- 
 ous solution by the usual protein precipitants and gave the biuret 
 and xanthoproteic reactions. 
 
 This experiment of Schiitzenberger's left us, however, very 
 much where we were, so far as real knowledge of the structure 
 of the protein molecule is concerned. The knowledge of the fact 
 that a mixture of amino-acids and urea yields, under certain 
 treatment, a body or bodies more or less closely resembling the 
 proteins, furnished us little or no information regarding the 
 structure of the protein molecule which we did not already 
 possess in the fact that the disintegration products of the pro- 
 teins are predominantly amino-acids. Prior to Schiitzenberger, 
 Grimaux (28) had shown that condensation-products of amino- 
 benzoic acid and of aspartic acid (probably comparable with the 
 octaspartic acid of Schiff (62)) resemble the proteins in many 
 of their properties; but these experiments also threw no light 
 upon the structure of the protein molecule beyond emphasizing 
 the already sufficiently evident probability that the amino-acid 
 grouping plays an important part in the building up of the protein 
 molecule. 
 
 The clue which led, through a series of remarkable researches 
 to our present comparatively extensive knowledge of the groupings 
 within the protein molecule, was obtained by Curtius (9) who, 
 
10 CHEMICAL STATICS 
 
 in 1883, observed that ethyl glycocollate, in watery solution, 
 tended to form glycocoll anhydride: 
 
 (In the absence of water) 
 NH 2 .CH 2 .COOH + C 2 H 5 OH = NH 2 .CH 2 .COOC 2 H5 + H 2 0. 
 
 (Glycocoll) (Ethyl glycocollate) 
 
 (In the presence of water) 
 NH 2 .CH 2 .COOC 2 H 5 + NH 2 .CH 2 COOC 2 H 5 = 
 
 (Ethyl glycocollate) (Ethyl glycocollate) 
 
 /CH 2 -NH. 
 
 O = C, X C = + 2C 2 H 5 OH. 
 
 N NH.CH 2 / 
 
 (Glycocoll anhydride) 
 
 Obviously, if the closed ring representing the glycocoll anhy- 
 dride molecule could be opened up without destroying the sta- 
 bility of the molecule, a new amino-acid would be formed, one 
 degree more complex than the original amino-acid (glycocoll). 
 This possibility occurred to Emil Fischer, and he found, in fact, 
 that if the glycocoll anhydride which is thus prepared be boiled 
 for a short time with concentrated hydrochloric acid, the fol- 
 lowing change occurs : 
 
 , CH 2 .NH x CH 2 .NH 2 .HC1 
 
 NH.CH 2 NH.CH 2 .COOH 
 
 (Glycocoll anhydride) (Glycyl-glycin chloride) 
 
 On now treating the glycyl-glycin chloride with silver oxide, 
 silver chloride is precipitated and free glycyl-glycin is obtained. 
 (23). If, however, the glycocoll anhydride be originally treated 
 with alcoholic instead of with watery HC1, the ethyl ester of 
 glycyl-glycin is obtained: 
 
 /CH2.NH, .CH 2 NH 2 
 
 NH.CH 2 NH.CH 2 .COOC 2 H 5 
 
 (Glycocoll anhydride) (Glycyl-glycin ester) 
 
 It would almost appear, therefore, as if we had only to repeat 
 this cycle of operations indefinitely in order to secure the most 
 complex poly-amino-acids; but this is not so easy as it might 
 appear at first sight; the instability of amino-acids consequent 
 upon the high reactivity of the NH 2 group, and the consequent 
 difficulty of obtaining simple anhydrides renders this procedure 
 
SYNTHESIS OF PROTEINS 11 
 
 impossible. Moreover the anhydride-ring is in many cases (e.g., 
 leucin anhydride) very difficult to break up when it has once 
 been formed. 
 
 In the search for methods of overcoming these difficulties, 
 Fischer found that the instability of the amino-acids could be 
 eliminated by the introduction of radicals into the NH 2 group, 
 and he and Fourneau synthesized phenyl-cyanate-glycyl-glycin 
 and carboxyethyl-glycyl-glycin ester (C 2 H 5 O.OC.NH.CH 2 .CO.- 
 NH.CH 2 .CO.OC 2 H 5 ) which are both chemically stable* bodies. 
 In subsequent investigations Fischer found that by gentle heat- 
 ing, combination between the esters of the carboxyethyl amino- 
 acids and other ammo-acid esters could be directly brought about 
 (25); in this way carboxyethyl-diglycyl-leucin ester was formed 
 (C 2 H 5 O.OC.NH.CH 2 CO.NH.CH 2 .CO.NH.CH.C4H 9 .CO.OC 2 H5). 
 
 The difficulty was here encountered, however, that the car- 
 boxyethyl group, once introduced, cannot be eliminated again. 
 
 The method which Fischer devised to overcome this difficulty 
 (25) was extremely ingenious. The introduction of a radical into 
 the NH 2 group appeared to be a necessity, forced upon us by 
 the impossibility of otherwise securing simple anhydrides of the 
 acids. It occurred to Fischer, however, that the radical thus 
 introduced into the NH 2 group might itself be made a carrier of 
 amino-acid groups into the molecule. This anticipation proved 
 to be correct. The radical which Fischer first utilized was the 
 chloracetyl group (C1CH 2 .CO ); when chloracetyl is allowed to 
 act upon glycyl-glycin ester (obtained by the methods described 
 above) chloracetyl-glycyl-glycin ester is obtained : 
 
 C1.CH 2 .COC1 + NH 2 .CH 2 .CO.NH.CH 2 .COOC 2 H 5 
 
 (Chloracetyl chloride) (Glycyl-glycin ester) 
 
 = C1CH 2 CO.NH.CH 2 .CO.NH.CH 2 .COOC 2 H 5 + HC1. 
 
 (Chloracetyl glycyl-glycin ester) 
 
 By saponification of this ester, free chloracetyl-glycyl-glycin is 
 obtained; on now treating this with a concentrated aqueous 
 solution of ammonia, the Cl atom in the chloracetyl group be- 
 comes replaced by an NH 2 group and diglycyl-glycin is obtained : 
 
 C1CH 2 .CO.NH.CH 2 .CO.NH.CH 2 .COOC 2 H 5 + 2NH 3 
 
 (Chloracetyl-glycyl-glycin ester) 
 
 = NH 2 .CH 2 .CO.NH.CH 2 CO.NH.CH2.COOC 2 H5 + NH 4 C1. 
 
 (Diglycyl-glycin ester) 
 
12 CHEMICAL STATICS 
 
 In other words, the chloracetyl group, introduced to protect 
 the NH 2 group of the amino-acid is, after it has performed its 
 protective function, itself transformed into an amino-acid group, 
 through the replacement of the halogen atom by NH 2 . Obvi- 
 ously, other halogen-containing acid groups may be used in place 
 of chloracetyl, and in this way a great variety of amino-acid groups 
 can be introduced into the NH 2 group. Thus Fischer employs: 
 
 Chloracetyl-chloride for the introduction of glycyl. 
 a-Bromopropionyl-chloride for the introduction of alanyl. 
 1-a-Bromopropionyl-chloride for the introduction of d-alanyl. 
 ex-Bromobutyryl-chloride for the introduction of a-aminobutyryl. 
 a-Bromisocapronyl-chloride for the introduction of leucyl. 
 a-Bromophenylacetyl-chloride for the introduction of phenylglycyl. 
 a-Bromohydrocinnamyl-chloride for the introduction of phenylalanyl. 
 a-Phenylbromopropionyl-chloride for the introduction of phenylalanyl. 
 a-5-Dibromovaleryl-chloride for the introduction of prolyl. 
 
 Fumaryl-chloride for the introduction of asparagyl. 
 
 By this method the chain of amino-acids is lengthened at the 
 amino-group end. Theoretically, it appeared possible tcValso. 
 lengthen the chain at the carboxyl end of the molecule, by acting 
 upon the esters of the amino-acids with the acid chlorides of 
 other amino-acids. Until 1904, however, the acid chlorides of 
 amino-acids were unknown and all attempts to prepare them had 
 failed, owing to the same reason which limits the use of the first 
 method of synthesizing poly-amino-acids, described above, namely 
 the reactivity of the NH 2 group. It will be recollected that 
 Fischer found that the NH 2 group could be protected by the 
 introduction of radicals, and, utilizing this fact, in 1904 Fischer 
 succeeded in devising a method of preparing the acid chlorides 
 of the amino-acids (17). The acid chlorides thus prepared react 
 with the esters of other amino- or poly-amino-acids to form poly- 
 ami no-acid chains of greater length. Thus: 
 
 C 4 H 9 .CHBr.CO.NH.CH 2 COCl + 2 NH 2 .CH 2 .COOC 2 H 5 
 
 (Bromisocapronylglycyl chloride) (Glycin ester) 
 
 = HC1NH 2 .CH 2 .COOC 2 H 5 + C 4 H 9 .CHBr.CO.NH.CH 2 CO.- 
 
 NH.CH 2 COOC 2 H 5 . 
 
 (Glycin ester hydrochloride) (Bromisocapronylglycyl-glycin ester) 
 
 Subsequent saponification of the bromisocapronyl-glycyl-glycin 
 ester and treatment with ammonia yields the poly-amino-acid (tri- 
 peptid) leucyl-glycyl-glycin: 
 
 C 4 H 9 .CH(NH 2 ).CO.NH.CH 2 .CO.NH.CH 2 .COOH. 
 
SYNTHESIS OF PROTEINS 13 
 
 If the bromisocapronylglycylchloride be made to act upon glycyl- 
 glycin ester, and the product be treated in the same way, the 
 tetrapeptid, leucyl-diglycyl glycin results: 
 
 C4H 9 .CH(NH2).CO.NH.CH2CO.NH.CH 2 CO.NH.CH 2 .COOH. 
 
 These methods of synthesis proved inadequate where hydroxy- 
 amino-acids are concerned, because the phosphorus penta- 
 chloride, used in the formation of the acid chloride, attacks the 
 OH group. It was, however, ascertained that the OH could be 
 protected by acting upon the hydroxy amino-acid with methyl 
 chlorcarbonate (20), which converts the OH into OC0 2 CH 3 , 
 which is not attacked by PCU and is readily removed by hy- 
 drolysis. In this way it has proved possible to introduce phenyl- 
 carboxylic acids, such as tyrosin, into poly-amino-acid chains. 
 
 By these methods, and modifications of these methods, Fischer 
 has succeeded in building up long chains of amino-acid groups, 
 these chains being collectively termed, by Fischer, peptids. 
 Chains consisting of two links, i.e., combinations of two amino- 
 acids, Fischer terms dipeptids; such, for example, are glycyl- 
 glycin, alanyl-alanin and leucyl-leucin; chains consisting of three 
 links he terms tripeptids, such being, for example, diglycyl- 
 glycin, and leucyl-glycyl-glycin; chains consisting of four links 
 are termed tetrapeptids, and so on, the higher members of the 
 series being collectively termed poly peptids. 
 
 The surpassing interest of these investigations lies in the fact 
 that Fischer considers many of his polypeptids to be, in all proba- 
 bility, identical with some of the natural peptones and sub- 
 peptones; while others probably merit inclusion among the, 
 proteins themselves. Thus the octadecapeptid 1-leucyl-triglycyl- 
 1-leucyl-triglycyl-l-leucyl-octaglycyl-glycin, and the tetradeca- 
 peptid 1-leucyl-triglycyl-l-leucyl-octaglycyl-glycin so closely re- 
 semble, in general properties, the ordinary proteins, that, as 
 Fischer puts it, they would have been classed as proteins had 
 they been first met with in nature (18) (19). Thus they give 
 the biuret reaction, form opalescent watery solutions, and the 
 tetradecapeptid is precipitated by ammonium sulphate, by tannic 
 acid and by phosphotungstic acid. As they do not contain 
 tyrosin, tryptophane or cystin they fail to give such protein 
 color reactions as depend upon the presence of these groups. 
 The molecular weight of the octadecapeptid is 1213, and the 
 
14 CHEMICAL STATICS 
 
 substitution of phenylalanin, tyrosin and cystin in the place of 
 glycin groups would increase this weight two or three times, 
 giving a value which is of the same order as that of the more 
 modern estimations of the (minimal) molecular weights of many 
 of the natural proteins.* 
 
 A whole series of the polypeptids give the typical peptone 
 biuret reaction, and such as contain tyrosin give also Millon's 
 reaction. The biuret reaction is, with the glycin compounds, 
 first encountered in the tetrapeptid, but it is given by other 
 tripeptids. It is more intense the greater the length of the poly- 
 peptid chain, and it is also intensified by the carboxyl group or 
 by conversion of the carboxyl group into an acid amide group. 
 The majority of the polypeptids are readily soluble in water, 
 and such as are with difficulty soluble in water are readily soluble 
 in dilute mineral acids and alkalies with which they combine; 
 they are less soluble in solutions of acetic acid. As a rule they 
 are insoluble in absolute alcohol, but in alcohol containing a 
 little watery ammonia they are generally soluble; on boiling off 
 the ammonia they are precipitated again. Under conditions 
 involving dehydration, e.g., heating or treatment of the esters 
 with alcoholic ammonia, the dipeptids are converted into diketo- 
 piperazines which are ring-compounds. Under similar conditions 
 the polypeptids are modified in an analogous manner, with the 
 formation of ring-compounds. 
 
 Upon hydrolysis the peptids break down into their constituent 
 amino-acids, the imino groups in the polypeptid molecule being 
 converted, by the taking up of water, into amino groups. A 
 
 * Emil Fischer strongly inclines to the opinion that the higher estimates, 
 such as 12,000 and 15,000 for the minimal weight of the protein molecule, 
 which are freely cited in the older literature upon proteins, are in error, since 
 the admixture of a small quantity of another protein might easily raise the 
 calculated value to this magnitude, and we have no proof of the chemical 
 individuality of the majority of our protein preparations, even when, as some- 
 times happens, they are crystallizable. An exception appears to be afforded 
 by haemoglobin, the minimal weight of which is placed by many observers, 
 employing adequate chemical and physico-chemical technique, at or in the 
 neighborhood of 16,000. But then haemoglobin is probably to be regarded as 
 a salt-like compound of, possibly, two or more molecules of a basic, histone- 
 like protein (globin) with a non-protein acid, namely hsematin. In a similar 
 way, we shall see (Chaps. V and IX) that casein, which is possessed of a 
 minimal molecular weight of 4000-4400, forms, under certain conditions, salts 
 of which the molecular weight is double or four times this. 
 
OCCURRENCE OF PEPTIDS 15 
 
 very large number of them are hydrolysed by the proteolytic 
 enzymes, pepsin, trypsin, etc., and in some cases, at all events, 
 it is certain that the hydrolysis takes place in stages, as it does 
 with the proteins and peptones (1). Further discussion of the 
 mode of hydrolysis of the polypeptids by enzymes will be found 
 in the chapters dealing with the chemical dynamics of protein 
 systems. 
 
 5. The Occurrence of Peptids among the Products of Protein 
 Hydrolysis. It is highly probable that many of the bodies 
 which are known to the biochemist as "proteoses" " peptones, " 
 etc., will turn out to be identical with some of the polypeptids 
 already synthesized; this identity has, however, not yet been 
 proven. But several of the simpler peptids^and one tetrapeptid 
 have already been isolated from protein digests. These are to 
 be regarded as products of incomplete hydrolysis, intermediate 
 between the higher complexes and the simple amino-acids which 
 result from their complete decomposition. 
 
 The presence of a dipeptid amongst the products of the hy- 
 drolysis of a protein of silk, silk-fibroin, was detected by Fischer 
 and Bergell in 1902 (16). Later, Fischer and Abderhalden (9) 
 showed that its anhydride, which they isolated, was the diketo- 
 piperazine of glycyl-alanin, and that it could not possibly have 
 arisen, by synthesis from glycin and alanin, during the process 
 of its isolation. At the same time they isolated another dipep- 
 tid from among the products of the incomplete hydrolysis of 
 silk-fibroin, namely glycyl-1-tyrosin, while from among the prod- 
 ucts of the incomplete hydrolysis of elastin glycyl-1-leucin was 
 obtained. Later, Fischer and Abderhalden (22), by partial 
 hydrolysis of the silk-fibroin, obtained a peptone-like substance, 
 precipitable by phosphotungstic acid, easily soluble in water, 
 insoluble in alcohol, precipitable from its aqueous solution by 
 saturation with ammonium sulphate or sodium chloride, which 
 proved to be a tetrapeptid, yielding, on hydrolysis, two molecules 
 of glycin, one of alanin and one of tyrosin. Its molecular weight, 
 determined by the cryoscopic method, was 350. The synthetic 
 pentapeptid, 1-leucyl-triglycyl-l-tyrosin, possesses very similar 
 properties, so that the peptones are not necessarily exceedingly 
 complex substances, nor is excessive complexity necessary in 
 order that substances of this type may be precipitable from their 
 aqueous solutions by saturation with ammonium sulphate. 
 
16 CHEMICAL STATICS 
 
 A number of other peptids have been isolated by various 
 observers (42) (48) from among the products of the incomplete 
 hydrolysis of proteins. 
 
 6. The Analysis and Characterization of Proteins by the Deter- 
 mination of the Chemical Groups Characteristic of the Different 
 Ammo-acids. The hydrolysis of the proteins is accompanied 
 by a very marked increase in the total number of free amino 
 groups. This is due to the fact, now incontestibly established, 
 that the various amino-acid radicals of the protein molecule 
 are attached to one another in an end to end or catenary linkage, 
 through the union of the amino group of one amino-acid with the 
 carboxyl group of the adjacent acid, in accordance with the 
 general equation: 
 
 HaNRiCOOH+H.HNRaCOOH = HaNRiCOHNR^COOH+HaO. 
 
 The reversion of this reaction, in hydrolysis, leads, of course, to 
 the transformation of an imino or potentially imino group into 
 a free amino group and the series of such transformations which 
 constitutes the process of the hydrolysis of a protein leads to the 
 appearance of a large number of free amino groups which were 
 not present as such in the unhydrolysed protein molecule. 
 
 Free amino groups in the aliphatic series have the well-known 
 property of reacting with nitrous acid with the liberation of 
 nitrogen, in accordance with the equation: 
 
 RNH 2 + HN0 2 = ROH + N 2 + H 2 O. 
 
 Very ingenious advantage has been taken of this fact by D. D. 
 Van Slyke (69) (70) (2) in the method which he has devised and 
 which is now very widely employed for the determination of the 
 distribution and partition of nitrogen within the protein molecule. 
 
 This method consists essentially in the following process. The 
 protein having been in the first place subjected to complete 
 hydrolysis, the ammonia in the mixture of products (derived 
 from "amid" nitrogen in the protein molecule) is first removed 
 by vacuum distillation and separately determined. The residual 
 mixture of products is then treated with phosphotungstic acid, 
 which results in the precipitation of the diamino-acids, namely 
 cystin, arginin, lysin and histidin. A determination of sulphur 
 yields a measure of the cystin-content. Arginin has the property 
 of yielding one-half of its nitrogen in the form of ammonia on 
 
TYPES OF UNION 17 
 
 boiling with alkali. The quantity of ammonia developed on 
 boiling the precipitate with alkali therefore affords a measure of 
 the content of arginin. The total nitrogen in the precipitate 
 is now determined and from it is subtracted the proportion of 
 nitrogen which is contributed by the cystin and arginin content. 
 The residual nitrogen is derived from lysin (= x) and histidin 
 (= y). On treatment with nitrous acid lysin yields a volume 
 of free nitrogen corresponding to the whole of its nitrogen 
 content (= x), while histidin yields a volume of free nitrogen 
 which corresponds to two-thirds of its nitrogen content (= f y). 
 The amino-nitrogen content of the precipitate is therefore deter- 
 mined by the nitrogen yield on treatment with nitrous acid and 
 after subtraction of the amino-nitrogen contents of arginin 
 (= one-fourth of its total nitrogen) and of cystin (= the whole 
 of its nitrogen content), the residual amino nitrogen evidently 
 represents the whole of the lysin nitrogen plus three-fourths 
 of the histidin nitrogen. But the determination of the total 
 nitrogen in the precipitate and the subtraction therefrom of the 
 cystin and arginin nitrogen has already given us a measure of 
 the total nitrogen yielded by the lysin and histidin. Subtract- 
 ing, therefore, the amino-nitrogen yield of these amino-acids 
 the difference evidently corresponds to one-third of the histidin 
 nitrogen, from which the contents of histidin and lysin may 
 readily be computed. 
 
 jr. In the filtrate, after the separation of the diamino-acid precipi- 
 tate, the total nitrogen and the amino-nitrogen are separately 
 determined. The difference yields a measure of the nitrogen 
 contained in pyrrollidine (prolin and oxyprolin) or indol (trypto- 
 phane) rings. 
 
 , During the hydrolysis by hydrochloric acid a small amount 
 of a very deeply colored precipitate separates out. The nitro- 
 gen content of this precipitate is the so-called " melanin " or 
 "humin" nitrogen. According to Gortner and Blish (27) this 
 is derived from a portion of the tryptophane and, in the presence 
 of a sufficiency of carbohydrate, the yield of melanin nitrogen 
 is a quantitative measure of the tryptophane content of the 
 protein. 
 
 7. Types of Union in the Protein Molecule. Following the 
 recognition of the fact that the proteins are complexes built up 
 by the union of amino-acids, the question of the mode of union 
 
18 CHEMICAL STATICS 
 
 between them became one of paramount importance. Hof- 
 meister (30) has pointed out that it is possible to conceive of 
 several ways in which the acids may be linked together such as: 
 
 A. Direct union of the carbon atoms, as: 
 
 I I 
 -C-C- 
 
 I I 
 
 under which condition the molecule would be an immense chain 
 of carbon atoms, and would not be readily hydrolysable, as the 
 proteins are, into its constituent amino-acids. 
 
 B. Ether-like unions, as : 
 
 I I 
 
 -C-O-C- 
 
 I I 
 
 Such unions, however, would only be possible when one of the 
 two amino-acids contained a hydroxyl group. Only tyrosin, 
 serin and oxyprolin, among the amino-acids which occur in the 
 proteins contain such groups, however, therefore this mode of 
 union cannot be of general occurrence in the proteins. 
 
 C. The carbon atoms may be united by a nitrogen atom, as: 
 
 I I I 
 -C-N-C- 
 
 I I 
 
 Several varieties of this mode of union are possible, such as: 
 -CH 2 -NH-CH 2 - -CH 2 -NH-CO- -CH 2 -NH-C(NH) 
 I II III 
 
 The syntheses accomplished by Emil Fischer, which I have 
 described above, the occurrence of peptids among the products 
 of the partial hydrolysis of proteins, and their digestibility by 
 proteolytic enzymes, demonstrate that type II is the most gen- 
 eral mode of union among the amino-acids which constitute 
 the protein molecule. The fact that the proteins give the biuret 
 reaction also supports this view. It has been shown by Schiff (61) 
 that only those substances which contain two CO NH groups, 
 or two CS NH , or two C (NH) NH groups, or, under certain 
 conditions, two CH NH groups yield the biuret test. That 
 such groups as: 
 
 I 
 -COHN-C-COHN- 
 
 I 
 
TYPES OF UNION 19 
 
 occur in the protein molecules, as they do in the molecules of the 
 polypeptids, is therefore highly probable and is furthermore 
 confirmed by the extreme paucity of free amino groups in the 
 native protein molecule. 
 
 The content of free amino groups in the native protein molecule 
 may be determined either by the yield of nitrogen on treatment 
 with nitrous acid (43) (69) (38) (71) or by the method of titra- 
 tion with formaldehyde, originally proposed by Sorensen (66) 
 (67) (46) (38) which latter method also enables us to indirectly 
 estimate the free carboxyl groups. An examination of the vari- 
 ous proteins by either of these methods reveals the fact that the 
 content of free amino groups in the unhydrolysed protein mole- 
 cule is very small indeed. Thus Van Slyke and Birchard have 
 obtained the following results (71) : 
 
 PERCENTAGE OF TOTAL NITROGEN PRESENT IN FREE AMINO GROUPS 
 
 Haemoglobin 6.0 
 
 Casein 5.5 
 
 Hsemocyanin 4.3 
 
 Gelatin 3.1 
 
 Edestin 1.8 
 
 Gliadin 1.1 
 
 Zein 0.0 
 
 Heteroalbumose 8.1 
 
 Protoalbumose 9.9 
 
 On the other hand, edestin, after complete hydrolysis by hydro- 
 chloric acid, yields a volume of free nitrogen, on treatment with 
 nitrous acid, corresponding to no less than 79 per cent of its 
 total nitrogen content (69). The investigations of Kossel and 
 Gavrilov and of Van Slyke and Birchard have in fact shown 
 that the free amino nitrogen in the unaltered protein molecule 
 exactly corresponds in quantity with one-half the lysin nitrogen. 
 Hence zein, which contains no lysin (49) yields no free nitrogen 
 on treatment with nitrous acid. The period required for complete 
 interaction of proteins with nitrous acid (30 minutes) is longer 
 than that required by the a-amino groups (3 to 4 minutes), but 
 corresponds to that found for the co-amino group of lysin. From 
 these facts Van Slyke and Birchard infer that one of the two 
 amino groups of lysin, the o> group, exists free in the protein 
 molecule, and that this group represents, within at most a frac- 
 tion of a per cent of the total protein nitrogen, the entire amount 
 
20 CHEMICAL STATICS 
 
 of the free amino nitrogen determinable in the native proteins 
 by the nitrous acid method. The a-amino groups which con- 
 stitute the remaining and greater part of the free amino nitrogen 
 found after complete hydrolysis are, in the intact protein mole- 
 cule, practically all condensed into peptid linkings. 
 
 In the primary albumoses or first split-products of protein 
 hydrolysis, the relations are different. The free amino groups 
 in hetero- and protoalbumose exceed one-half the content of 
 lysin nitrogen by 3.0 and 4.8 per cent of the total nitrogen re- 
 spectively, indicating that an appreciable proportion of the 
 a-amino groups are uncovered in even the first steps of hydrolysis. 
 
 8. Some Consequences of the Polypeptid Structure of the 
 Protein Molecule. The polypeptids are as essentially amino- 
 acids as the amino-acids out of which they are built up. Thus 
 glycyl-glycin is as typically an amino-acid as glycocoll itself, 
 since it possesses an NH 2 group as well as a COOH group 
 and for this reason is presumably capable of forming compounds, 
 not only with acids and bases, but also with neutral salts (52). 
 On undergoing electrolytic dissociation it may be supposed to 
 yield either hydrogen (H + ) ions, or hydroxyl (OH 7 ) ions, owing 
 to the occurrence of a reaction with water of the type: 
 
 NH 3 OH 
 
 / 
 
 R/ 
 COOH X COOH 
 
 just as ammonia, in aqueous solution, partially reacts with water 
 to form NH 4 OH. 
 
 It is usually conceded that these elements in the structure of 
 the proteins afford an explanation of the power which they pos- 
 sess of neutralizing both acids and bases, in other words the 
 "amphoteric" character of the proteins. To this opinion I have 
 also formerly inclined, but an accumulation of data irreconcil- 
 able with this view induced me some years ago (57) to abandon 
 it. Since that time evidence of a perfectly conclusive character 
 has been obtained and we may now regard it as an established 
 fact that some elements in the protein molecule other than ter- 
 minal NH2 or COOH groups are responsible for the acid- and 
 base-neutralizing power which is possessed in such a marked 
 degree by many proteins (vide Chapters IV and V). 
 
 In the first place, the investigations of Levites and D. D. Van 
 Slyke, referred to above, have shown that only a very small 
 
CONSEQUENCES OF THE POLYPEPTID STRUCTURE 21 
 
 proportion of the nitrogen in proteins is present within their 
 molecules in the form of NH 2 groups. Thus in the case of 
 edestin, as we have seen, only 1.8 per cent of the total nitrogen 
 is present in the form of free NH 2 groups. 
 
 Now edestin, as Osborne (47) has shown, is insoluble, when 
 in the free condition, in water. It forms an insoluble hydro- 
 chloride containing 14 X 10~ 5 equivalents of HC1 per gram 
 and, on further addition of acid, passes into solution. Its com- 
 bining-capacity for acids does not remain constant, however, for 
 at neutrality to tropseolin, which corresponds to a free acidity 
 of from 0.01 to 0.001 H+ (60) it neutralizes 127 X 10~ 5 equiva- 
 lents of acid per gram. Hence, if the acid is neutralized by 
 
 NH 2 groups of edestin, the number of these groups in each 
 molecule of edestin must be at least *- = 9.* From the former 
 determination it would appear that the molecular weight of 
 edestin is 7000, and this corresponds with the molecular weight 
 indicated by its tyrosin and glutamic acid content (40) (1 mol. 
 tyrosin + 3 mols. glutamic acid +) Nine NH 2 groups 
 in this molecule would correspond to over ten per cent of the 
 total nitrogen, or almost exactly to the whole NH 2 content 
 of the arginin, calculated as the free diamino-acid, which the 
 edestin molecule contains. Since only 1.8 per cent of the total 
 nitrogen of the edestin molecule is present therein in the form 
 of NH 2 groups no less than eighty per cent of the neutralizing 
 power of the edestin for acids must be accounted for in some 
 other fashion than by the assumption of a union of the acid with 
 free NH 2 groups. 
 
 From the investigations of Erb (14), although the exact in- 
 terpretation' of his results is in some respects open to question 
 (Chap. IV), it would appear that the combining-weight of egg 
 albumin for acids may be as low as 152, while its molecular weight 
 is, according to Hofmeister, 5400 or some multiple of this. Hence 
 upon the assumption that terminal NH 2 groups bind these 
 acids, there must be at least 35 of them in egg albumin (45), 
 which would correspond to no less than 69 per cent of the total 
 nitrogen in this protein. 
 
 * Osborne believes that an insoluble "monochlorhydrate" is also formed, 
 containing 7 X 10" 6 equivalents per gram which would raise the number of 
 
 NH 2 groups to 18, but would, at the same time, double the estimate of the 
 molecular weight. 
 
22 CHEMICAL STATICS 
 
 It has been pointed out by Kossel and Cameron (37) that the 
 acid-combining capacity of the protamin, salmin, is equal to the 
 combining capacity of the guanidin groups of the arginin radicals 
 which it contains, yet salmin yields no nitrogen on treatment 
 with nitrous acid. Sturin (41) contains 67 per cent of its nitro- 
 gen in the form of arginin, 10 per cent in the form of histidin 
 and 6 to 7 per cent in the form of lysin. It yields nitrogen on 
 treatment with nitrous acid corresponding to the co-amino group 
 of the lysin. Only about three out of every hundred nitrogen 
 atoms in sturin are therefore present in the form of free NH 2 
 groups. Yet one hundred nitrogen atoms in sturin will neu- 
 tralize no less than 24 equivalents of acid. All of these pro- 
 tamins, moreover, possess a certain, although relatively inferior 
 power of neutralizing bases. 
 
 The number of terminal COOH groups in any protein can- 
 not be much in excess of the terminal NH 2 groups, as the formol- 
 titration and behavior towards cupric hydroxide (32) (33) (66) 
 (67) show, and also because the protein would otherwise be 
 overwhelmingly acid in character (30) and the majority of the 
 proteins possess a distinct capacity for neutralizing acids, even 
 when they are themselves predominantly acid. Now free casein * 
 is insoluble in water, but when combined with acids or with 
 bases it is soluble. To carry one gram of casein into solution 
 11.4 X 10~ 5 equivalents of base just suffice, indicating a com- 
 bining-weight for casein of about 8800. The tyrosin, glutamic 
 acid and sulphur contents of casein indicate a minimal molecu- 
 lar weight of from 4000 to 4400. 
 
 In the presence of excess of base, however, casein attains a 
 maximal combining capacity (measured by the gas-chain) of 
 180 X 10~ 5 equivalents per gram, so that it behaves like a 16- 
 basic acid, and if COOH groups bind the base there must 
 be 16 of them in the molecule, corresponding to 25 per cent of 
 the total oxygen, or, almost exactly, to the percentage of the total 
 oxygen which is contained in the COOH groups of the glutamic 
 add in the casein molecule, calculated as the free acid. In order 
 to provide so many free carboxyls, the form of the casein mole- 
 cule would necessarily be that of a branched chain, or the radi- 
 ating spokes of a wheel, at the centre of which must exist unions 
 of the type: C C 
 
 * For references to the sources of these data Cf. Chap. IV. 
 
CONSEQUENCES OF THE POLYPEPTID STRUCTURE 23 
 
 and the regular decomposition of the proteins into their con- 
 stituent amino-acids, upon hydrolysis, would be unintelligible 
 (30). Moreover, in the synthetical polypeptids, which closely 
 resemble the natural peptones in their behavior, the linkage of 
 the amino-acids is not radial but catenary in character* and 
 the peptids which have been isolated from the mixed products 
 of partial protein hydrolysis are likewise catenary in structure. 
 
 In the second place, as Vernon has pointed out (72), although 
 the power of the sum of the decomposition-products of a protein 
 to neutralize bases is greater, yet it is only very slightly greater 
 than that of the unhydrolysed protein. Now in the process of 
 hydrolysis the COHN groups of the protein are split into 
 NH 2 and COOH groups; yet this results in no pronounced 
 gain of combining-capacity for bases. The obvious conclusion is 
 that the COHN group within the protein molecule must be 
 nearly as efficient in accomplishing the neutralization of bases 
 as the COOH group of the constituent amino-acids out of 
 which the protein is built up. 
 
 Direct proof, however, that terminal NH 2 groups are not 
 responsible for any appreciable proportion of the acid-combining 
 capacity of proteins has been furnished by the recent experiments 
 of Blasel and Matula (5) and Pauli and Hirschfeld (51). These 
 investigators prepared deaminized gelatin by acting upon gelatin 
 with nitrous acid, thus destroying all the free NH 2 groups in 
 the molecule. They then compared, with the aid of the gas- 
 chain, the acid-combining capacity of the deaminized gelatin 
 with that of normal gelatin. They found that the combining- 
 capacity of deaminized gelatin for acids is but slightly inferior 
 to that of ordinary gelatin, indicating, beyond any question, that 
 the combining-capacity of gelatin for acids is, in very large pro- 
 portion, attributable to elements of the molecule other than 
 free NH 2 groups. The inference is unavoidable that the ele- 
 ments of the molecule which actually participate in the union 
 with acids are, in very large proportion, the COHN groups 
 within the body of the protein molecule. 
 
 Very strong evidence that the same structural elements of the 
 protein molecule are responsible for the neutralization of bases 
 by proteins has also been recently afforded by the investigations 
 
 * Even when dicarboxylic acids enter into the compounds, Cf . Fischer and 
 Koenigs (24), and Fischer and Schmidlin (26). 
 
24 CHEMICAL STATICS 
 
 of Osborne and Leavenworth (50) who have shown that edestin 
 combines with, or holds in solution, 34.67 per cent of its weight 
 of copper in the form of cupric hydroxide. This, if we assume 
 that each copper atom unites with one nitrogen atom, involves 
 the union of cupric hydroxide with ten out of every sixteen atoms 
 of nitrogen in the edestin molecule. Now this is exactly the 
 proportion of nitrogen which edestin yields in the form of amino- 
 nitrogen after complete hydrolysis. In other words it is exactly 
 equal to the proportion of COHN groups which the unhydro- 
 lysed molecule contains. Precisely similar results were obtained 
 with gliadin. 
 
 To account for the high acid- and base-combining capacity of 
 the proteins we must therefore look, not to the terminal NH 2 
 or COOH groups, but to the COHN groups within the body 
 of the protein molecule. Now two varieties of this union can be 
 conceived, between which it has not proved possible as yet to 
 decide by any direct method of analysis. Thus glycyl-glycin 
 may conceivably be either: 
 
 Keto-form 
 H 2 N.CH 2 .CO - HN.CH 2 .COOH 
 
 or Enol-form 
 
 H 2 N.CH 2 .C(OH) = N.CH 2 .COOH 
 
 and our analytical data, and the modes of decomposition and 
 synthesis of the proteins and peptids do not enable us with any 
 degree of certainty to distinguish between them. Neither form 
 is therefore inconsistent with our present knowledge of the syn- 
 thesis and hydrolysis of proteins and polypeptids, but while 
 the keto-form of the COHN group would conceivably possess 
 the power of neutralizing acids it offers no probable point of union 
 for bases. The enol-form, on the contrary, would provide a 
 point of union for either acids or bases. 
 
 According to Werner's theory of valencies the nitrogen in 
 either of these unions contains two latent valencies, positive and 
 negative, which, while the nitrogen is trivalent, neutralize one 
 another, but which, when .the nitrogen becomes pentavalent are 
 capable, respectively, of neutralizing a negative and a positive 
 radical. The enol type of union carries with it the possibility 
 of the following types of reaction : 
 
CONSEQUENCES OF THE POLYPEPTID STRUCTURE 25 
 
 H 
 
 I 
 -COH=N- + Na+ + OH' = -CONa++ + ^N- (i) 
 
 I 
 
 OH 
 and 
 
 H 
 
 -COH = N- + H++C1' = -COH+++ ^N- (ii) 
 
 I 
 
 Cl 
 yielding, in each case, only protein ions. 
 
 I have already incidentally dwelt upon the fact, in connection 
 with Kossel's theory of a protamin nucleus of proteins and in 
 connection with the neutralizing powers of edestin for acids and 
 of casein for bases, that there is reason to suspect that diamino 
 and dicarboxylic radicals in the protein molecule play a pre- 
 dominant part in accomplishing the neutralization of acids and 
 bases, and electrochemical data, as we shall see, lend further 
 support to this view. Accordingly, the above formulae, which 
 represent the reaction when only a single COHN bond is 
 involved should probably, at least in many instances, be doubled, 
 and in the following possibilities would then exist: 
 In combination with bases, 
 
 H OH 
 \ / 
 
 .COH.N- /COK++ ^N- 
 
 R' + KOH + H 2 = R' + (iii) 
 
 X COH.N- N COH++ <*N- 
 
 / \ 
 H OH 
 
 H OH 
 \ / 
 
 /COH.N- /COK++ ^N- 
 
 R' +2KOH = R^ + (iv) 
 
 X COH.N- X COK++ ^N- 
 
 / \ 
 H OH 
 In combination with acids, 
 
 H Cl 
 
 \ / 
 -COH.N. -COH++ ^N v 
 
 )R + HC1 + H 2 0= + )R (v) 
 
 -COH.N X -COH++ ^N x 
 
 / \ 
 H OH 
 
26 CHEMICAL STATICS 
 
 H Cl 
 \ / 
 -COH.N. -COH++ ^N. 
 
 / R + 2 HC1 = -f ^ R (vi) 
 
 -COH.N X -COH++ ^N x 
 
 H Cl 
 
 It is obvious that in reactions (iii) and (v) the molecules of 
 water may or may not participate in the reaction; also that the 
 anionic groups in reactions (iii) and (iv) may or may not be 
 united to form a single quadrivalent cation (derived from a 
 dicarboxylic acid group). As we shall see (Chapter X.), no evi- 
 dence has been found (at least among the compounds of casein 
 or of serum globulin with bases) of the occurrence of reactions 
 of the type represented by equation (iii). Equation (iv) faith- 
 fully represents, so far as the electrochemical data are concerned, 
 the mode of combination of bases with these proteins, and the 
 anionic groups are probably united to form one quadrivalent 
 union. The union of serum globulin and ovomucoid with acids 
 follows equation (v) (with or without the molecule of water) 
 when the concentration of acid is low; but at higher acidities 
 ovomucoid, at least, combines with hydrochloric acid in the 
 manner indicated by equation (vi). 
 
 In the succeeding chapters of this work we shall see that this 
 hypothesis regarding the mode of union between the proteins and 
 acids and bases is supported by the following facts : 
 
 (i) The compounds which the proteins form with acids and 
 bases, when dissolved in water, are excellent conductors of elec- 
 tricity, and true electrolytes (65) (55) (4), yet they do not, for 
 example, yield chlorine ions, when the compound in question is a 
 hydrochloric acid compound (8) (59), nor does the potassium 
 hydrate compound yield potassium ions, or the calcium hydrate 
 compound calcium ions (56). The equivalent conductivities of 
 the compounds at infinite dilution are such as would indicate 
 the presence only of bulky organic ions, travelling under a given 
 fall of potential at the constant minimal equivalent velocity of 
 about 20 X 10~ 5 cm. per sec. per volt per cm. fall in potential 
 at 30 C. which is characteristic for such ions (6). 
 
 (ii) Edestin will displace NaOH from its combination with 
 hydrochloric acid (T. B. Osborne, Cf. Chapter V) and casein, 
 
CONSEQUENCES OF THE POLYPEPTID STRUCTURE 27 
 
 although insoluble in water, will displace carbonic acid from its 
 combination with calcium hydrate (W. A. Osborne, Cf. Chap. V). 
 Solutions of caseinates of the bases may be obtained which are 
 pronouncedly acid in reaction (Robertson, Cf. Chap. V). Since 
 in all these cases the molecular concentration of the protein 
 is very low, and the compounds which are formed are quite 
 highly electrolytically dissociated (Cf. above), were the forma- 
 tion of these compounds due to the replacement of OH' groups of 
 NH 3 OH groups by acid radicals or of H + groups in COOH 
 groups by bases, then the "strength," i.e., the degree of disso- 
 ciation of edestin as a base must be equal to or greater than 
 that of NaOH, while that of casein as an acid must be much 
 greater than that of H 2 C0 3 and comparable with the degree of 
 dissociation of NaOH at very high dilutions (since Na caseinate 
 may be prepared which is acid in reaction). Such conclusions, 
 applied to bodies which are amphoteric, are, of course, absurd. 
 Were the formation of potassium caseinate due to the formation 
 of a salt such as 
 
 RCOO' + K+ 
 
 an acid solution of this compound could no more exist than an 
 acid solution of potassium aluminate. As in similar cases which 
 occur in the domain of inorganic chemistry, we can interpret 
 these phenomena only by assuming that the basic radical in the 
 casein compounds, and the acid radical in the edestin compound 
 are bound up in a non-dissociable form. Since the casein com- 
 pounds, at least, when in solution in water, are notable conduc- 
 tors of electricity, they must dissociate at some other point in 
 the molecule than that of the union between the base and the 
 protein. 
 
 (iii) Each equivalent * of a monobasic acid or monacid base 
 neutralized by serum globulin or casein yields two equivalents of 
 the protein compound (Cf. Chap. X). This obviously corre- 
 sponds with the mode of dissociation depicted above, while, if 
 terminal NH 2 or COOH groups accomplished the union, 
 each equivalent of neutralized acid or base would produce only 
 one equivalent of salt. 
 
 (iv) On successive additions of 1, 2, 3, etc., equivalents of 
 
 * That is, gram-molecule divided by the valency of the combining radical, 
 in this instance unity. 
 
28 [CHEMICAL STATICS 
 
 monacid bases to a solution of an organic polybasic acid of the 
 type R(COOH) m , and the formation of salts by the replacement 
 of H atoms in the COOH groups, the osmotic pressure of the 
 solution would increase (provided the salts were highly dissociated) 
 in the same proportion, namely 2, 3, 4, etc. The experimental 
 fact, for casein (58), is that the osmotic pressure (= depression 
 of the freezing-point) increases in the proportion 2, 4, 6, etc., 
 i.e., each successive equivalent of neutralized base gives rise to 
 an equal number of ions. This obviously corresponds with what 
 would be expected were the union and its mode of dissociation 
 of the type outlined above. 
 
 In addition to these, a host of minor details in the behavior 
 of the protein salts, which would be very hard to explain upon 
 any other basis, admit, as we shall see, of a simple explanation 
 on the basis of the hypothesis outlined above. 
 
 The poly-amino-acid structure of the proteins, however, carries 
 with it other possibilities which are of importance in interpreting 
 their behavior. Some measure of the dissociations 
 
 /NH 2 
 
 R' +H+ 
 X COO' 
 
 and 
 
 ,NH 2 /NH 3 + 
 
 T5 ' I TT S~\ "R I OTT' 
 
 X COOH X COOH 
 
 must undoubtedly occur, although, in the majority of cases (a 
 notable exception being afforded by the protamins) these modes 
 of dissociation must play a very subordinate part. Then the 
 terminal NH 2 and COOH groups (and at least one of these 
 must exist at either end of a chain of amino-acids) may neutralize 
 themselves internally, thus : 
 
 NH 3 
 
 R-COO 
 
 forming what Winkelblech terms an " internal salt" (73). Such a 
 molecule, whatever the mode of union, would of course form 
 compounds with acids or bases with much more difficulty than 
 the protein which had not undergone such internal neutralization, 
 since, before dissociation could occur the ring-formation would 
 
CONSEQUENCES OF THE POLYPEPTID STRUCTURE 29 
 
 have to be opened up. The internal-salt formation may go a 
 step further, with the formation of anhydrides such as 
 
 NH 
 
 such anhydride formation being frequently observed in the poly- 
 peptids. Then two molecules of an amino-acid, and therefore, 
 of a poly-amino-acid such as protein, may unite with one another 
 in either of two ways, thus : 
 
 /NH 2 HOOC. /NH.OC.R.NH 2 
 
 R' + ^R = R' + H 2 
 
 X COOH R 2 ^ / N COOH 
 
 or 
 
 y NH 2 HOOC V /NH.OC, 
 
 R' + ^R = R' X R + 2H 2 
 
 X COOH HJX' X CO.HN X 
 
 the product being, in the first instance, a poly-amino-acid of a 
 higher order and greater molecular weight, and in the second 
 an " internal salt" or anhydride.* Another type of anhydride 
 which may be formed is that of a diketopiperazin, of the general 
 
 formula: 
 
 / CH(R).C(X 
 NH/ X NH 
 
 O.CH(R). 
 
 and these anhydrides may exist in two isomeric forms, the keto 
 and enol forms, the former being represented by the above for- 
 mula, and the latter by the general formula : 
 
 The enol form carrying with it the possibility (in the presence 
 of water) of the transient formation of salts with bases before the 
 ring breaks up.f 
 
 * Such as leucyl-glycin anhydride. 
 
 t Cf. R. H. Aders Plimmer (53), Part 2, p. 36. 
 
30 CHEMICAL STATICS 
 
 9. "Racemized" Proteins. It has recently been shown by 
 Kossel (36) and by Dakin (11) (12) that treatment of proteins 
 with rather concentrated alkali leads to a progressive diminu- 
 tion of the optical rotary power of the solutions. This Dakin 
 attributes to an internal racemization or, more correctly, enoli- 
 zation of the protein molecule analogous to that which occurs in 
 the hydantoins (10). He depicts the reaction as follows: 
 
 NH-CO- 
 I 
 R-CH-CO-NH-CHR-COOH 
 
 NH-CO- 
 I 
 <=R-C = C(OH)-NH-CHR-COOH 
 
 the alkali tending to shift the equilibrium towards the right. 
 The central carbon atom instead of being attached by its valencies 
 to four different groups is now attached to three groups, to one 
 of them by a double bond, and any optical activity which it may 
 have possessed owing to assymetry must have been lost. 
 
 The "racemized" proteins thus prepared are totally resistant 
 to hydrolysis by pepsin, trypsin or erepsin. Putrefactive bac- 
 teria do not attack "racemized" casein although they slowly 
 decompose the "racemized" caseose (proteose) which is simul- 
 taneously formed through partial hydrolysis of the casein and 
 subsequent "racemization" of the higher products of the hydroly- 
 sis. "Racemized" egg albumin (7) and casein (63) are non- 
 antigenic. The amino-acids resulting from the complete hy- 
 drolysis of "racemized" protein by acid are for the most part 
 optically inactive (12). 
 
 A number of objections have been urged by Kober, (32) against 
 Dakin's interpretation of the progressive loss of optical activity 
 of proteins in alkaline solutions. In the first place he points 
 out that the presence of an oscillating hydrogen atom within 
 the molecule might be anticipated to lead to the development of 
 bands in the absorption spectrum and no absorption-bands are 
 observable in protein solutions excepting those in the ultra- 
 violet spectrum which are attributable to the phenylalanin and 
 tyrosin radicals. This objection is deprived of force, however, 
 by the fact that in the hydantoins, in which analogous "enoli- 
 zation" does demonstrably occur, no absorption-band is developed. 
 
"RACEMIZED" PROTEINS 31 
 
 The objection, also raised by Kober, that racemized polypetids 
 are hydrolysable by trypsin is not valid because the reaction 
 depicted by Dakin is not, correctly speaking, racemization but 
 " enolization " and the structures of polypeptids formed from 
 racemic amino-acids and the so-called " racemized proteins" are 
 probably not in the least analogous. Finally, the fact, also 
 cited by Kober as an objection to Dakin's hypothesis, that not 
 all of the * amino-acids resulting from the complete hydrolysis 
 of enolized proteins are optically inactive, merely tends to show 
 that not all COHN groups are equally affected by alkali. 
 
 According to Underbill and Hendrix (68) crude "racemized" 
 proteins produce toxic symptoms when introduced into the cir- 
 culation of animals. Purified "racemized" proteins exert no 
 toxic action. A portion, but not all of the toxic substance can 
 be removed from "racemized" proteins by extraction with alcohol. 
 Evidently the main reaction is complicated by the occurrence 
 of side-reactions chief among which must be hydrolysis which is 
 rather rapid in solutions of the alkalinity employed. 
 
 In terms of the hypothesis of protein ionization outlined above 
 the "racemization" of proteins by alkali must consist in an ex- 
 change of hydrogen atoms in the enol group in accordance with 
 an equation such as: 
 
 NH-CO- 
 I 
 R-CH-C(OH) = N-CH-RCOOH 
 
 NH-CO- 
 I 
 R_C = C(OH)-NH-CH-R-COOH 
 
 with consequent transformation of the double bond connecting 
 carbon and nitrogen in the COHN group into a single bond. 
 
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32 CHEMICAL STATICS 
 
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 (31) Hufner, G., Zeit. f. Chem. (1868), p.[391. 
 
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 1580. 
 
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 siol. Chem. 25 (1898), p. 165. 
 
 (36) Kossel, A., Zeit. f. physiol. Chem. 78 (1912), p. 402. 
 
 (37) Kossel, A., and Cameron, A. T., Zeit. f. physiol. Chem. 76 (1912), p. 457. 
 
 (38) Kossel, A., and Gavrilov, N., Zeit. f. physiol. Chem. 81 (1912), p. 274. 
 
 (39) Kossel, A., and Kutscher, F., Zeit. f. physiol. Chem. 31 (1900), p. 165. 
 
 (40) Kossel, A., and Patten, A. J., Zeit. f. physiol. Chem. 38 (1903), p. 39. 
 
 (41) Kossel, A., and Weiss, F., Zeit. f. physiol. Chem. 78 (1912), p. 402. 
 
 (42) Levene, P. A., and Beatty, W. A., Ber. d. d. Chem. 39 (1906), p. 2060. 
 
 (43) Levites, S., Zeit. f. physiol. Chem. 43 (1904), p. 202. Biochem. Zeit. 20 
 
 (1909), p. 224. 
 
 (44) Liebig, J., Annalen der Chem. 57 (1846), p. 127. 
 
 (45) Mann, G., "Chemistry of the Proteins," London (1906), p. 147. 
 
LITERATURE CITED 33 
 
 (46) Obermeyer, F., and Willheim, R., Biochem. Zeit. 38 (1912), p. 331. 
 
 (47) Osborne, T. B., Journ. Amer. Chem. Soc. 24 (1902), p. 39. 
 
 (48) Osborne, T. B., and Clapp, S. H., Amer. Journ. Physiol. 18 (1907), p. 
 
 123. 
 
 (49) Osborne, T. B., and Leavenworth, C. S., Journ. Biol. Chem. 14 (1913), 
 
 p. 481. 
 
 (50) Osborne, T. B., and Leavenworth, C. S., Journ. Biol. Chem. 28 (1916), 
 
 p. 109. 
 
 (51) Pauli, W., and Hirschfeld, M., Biochem. Zeit. 62 (1914), p. 245. 
 
 (52) Pfeiffer, P., and von Modelski, J., Zeit. f. physiol. Chem. 81 (1912), p. 
 
 239; 85 (1913), p. 1. 
 
 (53) Plimmer, R. H. Aders, "The Chemical Constitution of the Proteins," 
 
 London, 1908. 
 
 (54) Proust, M., Ann. de Chim. et de Phys. 10 (1819), p. 29. 
 
 (55) Robertson, T. Brailsford, Journ. physical Chem. 15 (1911), p. 179. 
 
 (56) Robertson, T. Brailsford, Journ. physical Chem. 15 (1911), p. 521. 
 
 (57) Robertson, T. Brailsford, "Die physikalische Chemie der Proteine," 
 
 Dresden, 1912. 
 
 (58) Robertson, T. Brailsford, and Burnett, Theo. C., Journ. Biol. Chem. 6 
 
 (1909), p. 105. 
 
 (59) Rohonyi, H., Biochem. Zeit. 44 (1912), p. 165. 
 
 (60) Salm, E., Zeit. f. physik, Chem. 57 (1906), 471. 
 
 (61) Schiff, H., Ber. d. d. Chem. Ges. 29 (1896), p. 298. Annalen der Chem. 
 
 299 (1897), p. 239; 319 (1901), p. 300. 
 
 (62) Schiff, H., Ber. d. d. chem. Ges. 30 (1897), p. 2449. Annalen der Chem. 
 
 303 (1898), p. 183; 307 (1899), p. 231; 310 (1899), p. 301. 
 
 (63) Schmidt, C. L. A., Proc. Soc. Exper. Biol. and Med. 14 (1917), p. 104. 
 
 (64) Schutzenberger, P., C. R. Acad. Sci. 106 (1888), p. 1407; 112 (1891), 
 
 p. 198. 
 
 (65) Sjoquist, J., Skan. Arch. f. physiol. 15 (1895), p. 277. 
 
 (66) Sorensen, S. P. L., Biochem Zeit. 7 (1907), p. 45. 
 
 (67) Sorensen, S. P. L., and Jessen-Hansen, H., 7 (1907), p. 407. 
 
 (68) Underbill, F. P., and Hendrix, B. M., Journ. Biol. Chem. 22 (1915), p. 
 
 543. 
 
 (69) Van Slyke, D. D., Journ. Biol. Chem. 9 (1911), p. 185. 
 
 (70) Van Slyke, D. D., Journ. Biol. Chem. 10 (1911), p. 15; 12 (1912), p. 
 
 275; 22 (1915), p. 281; 23 (1915), p. 407. 
 
 (71) Van Slyke, D. D., and Birchard, F. J., Journ. Biol. Chem. 16 (1913), 
 
 p. 539. 
 
 (72) Vernon, H. M., Journ. of Physiol. 31 (1904), p. 346. 
 
 (73) Winkelblech, K., Zeit. f. physik. Chem. 36 (1910), p. 546. 
 
 (74) Zelinsky, N., Chem. Ztng. 36 (1914), p. 824. 
 
 (75) Zelinsky, N., Annenkov, A. and Kulikov, J., Zeit. f . physiol. Chem. 73 
 
 (1911), p. 459. 
 
CHAPTER II 
 THE PREPARATION OF PURE PROTEINS 
 
 1. The Proteins as Chemical Individuals. With the excep- 
 tion of casein and the protamins and those proteins such as 
 haemoglobin, fibrin, egg albumin and certain of the vegetable 
 proteins which may be prepared in crystalline condition, it can- 
 not be positively affirmed that any of the proteins which have 
 hitherto been isolated in a "pure" condition are, in reality, 
 chemical individuals and not mixtures, tolerably constant in 
 composition, of two or more proteins or of one protein with the 
 products of its partial hydrolysis or with other colloidal sub- 
 stances. Our methods of isolation and purification are admittedly 
 inadequate, and the most diverse opinions exist regarding the 
 appropriate criteria of the purity of any given protein. The 
 reason for this latter fact is probably to be sought in the imper- 
 fection of our knowledge of the properties, chemical, physical, 
 and physico-chemical, of the proteins as a class; lack of knowledge 
 implying, of course, the lack of a basis for comparison and of a 
 standard for calibration. 
 
 The proteins are, as a rule, non-crystallizable or only crystal- 
 lizable with difficulty and under such conditions as to involve 
 contamination with a variable and usually indeterminate pro- 
 portion of impurities, while repeated re-crystallization usually 
 leads to a more or less sensible alteration in the properties of the 
 protein, which may be attributable, in the majority of cases, to 
 a partial hydrolysis. The salts which the proteins form with the 
 inorganic bases and acids are, as a rule, either soluble or else, if 
 insoluble, of such a nature (for example, the protein salts of the 
 heavy metals) that the inorganic constituent cannot be removed 
 again from the molecule without altering its properties. 
 
 A limited number of the proteins are insoluble in distilled water 
 when uncombined with bases or acids, and these, of course, 
 afford exceptionally favorable opportunities for isolation and puri- 
 fication. Precipitation of these proteins in the free condition can 
 
 34 
 
PROTEINS AS CHEMICAL INDIVIDUALS 35 
 
 usually be brought about by inducing a certain reaction (i.e., 
 hydrogen or hydroxyl ion concentration) in their solution, through 
 the addition of acids or of bases, the hydrogen- or hydroxyl-ion 
 concentration being sufficient to set the protein free from its 
 combination with bases or acids respectively and insufficient to 
 lead to the formation of a new salt with the acid or base employed 
 for its precipitation. Thus, for example, casein is precipitated 
 from milk by the addition of acetic acid, "insoluble" serum globulin 
 from serum by dilution and the passage of C0 2 , histones from cell- 
 extracts by the addition of ammonia. 
 
 The isolation of proteins of this class is usually most conven- 
 iently carried out by employing for the precipitation an acid or 
 base which is insufficiently dissociated to transform the protein 
 into a base, if an acid is employed, or into an acid if a base is 
 employed for the precipitation, so that the protein which is at 
 first precipitated does not, on adding an excess of the acid or base, 
 form a salt and pass into solution again. For instance, if a 
 " strong" acid such as hydrochloric acid be added to milk, or to 
 any solution of a caseinate of an alkali or an alkaline earth, free 
 casein is at first precipitated. But if the addition of the hydro- 
 chloric acid be continued the casein passes into solution again 
 and it is now found that the casein is behaving as a base and that 
 a certain proportion of the acid is neutralized by it. If a "weak" 
 acid such as acetic be employed, however, the acidity of the mix- 
 ture is insufficient to transform the casein into a base, and a 
 considerable excess of acetic acid may be added to the mixture 
 without danger of loss of material owing to resolution of the protein. 
 
 The acid or base which is employed in setting free the insoluble 
 uncombined proteins of this class must, however, be sufficiently 
 strong to set free the protein from its salts; for example, C0 2 
 cannot be employed to precipitate casein because casein is a 
 stronger acid than C0 2 and displaces it from carbonates. But 
 C0 2 can be and is successfully employed to precipitate "insoluble" 
 serum- -globulin because this protein is a weaker acid than casein 
 and can be displaced from its salts by so feeble an acid as car- 
 bonic acid. Undoubtedly correlated with this is the fact that 
 serum globulin is more readily transformed into a base than 
 casein, so that acetic acid redissolves and is partially neutralized 
 by serum globulin, although it is insufficiently strong except in 
 very high concentrations to redissolve casein. 
 
36 CHEMICAL STATICS 
 
 It is clear, therefore, that proteins of this class must be pre- 
 cipitable only within a very narrow range of hydrogen- and 
 hydroxyl-ion concentrations and that the probability of simul- 
 taneous precipitation of more than one protein is very slight 
 unless the proteins chance to be acids or bases of very nearly 
 equal strength. In very few cases, however, do we possess, at 
 present, any very reliable guarantee that this does not occur, 
 and in a few instances, at all events, we are positive that it does 
 occur. For example, the various members of the paranuclein 
 series, which are derived from casein by its partial hydrolysis, 
 are all precipitable by acetic acid, and, some of them at least, 
 are not redissolved by the addition of moderate excess of acetic 
 acid. True, there are recognizable differences between the basic 
 functions of the various paranucleins and casein. The para- 
 nucleins are precipitated in solutions of lower acidity than casein, 
 and doubtless an acid could be found the solution of which, at 
 a concentration sufficiently acid to precipitate paranuclein, would 
 not be sufficiently acid to precipitate casein. But this acid has 
 not, as yet, been discovered and so a reliable method for sepa- 
 rating casein from paranuclein in a solution containing a mixture 
 of these proteins is yet to seek; while the separation of the dif- 
 ferent members of the paranuclein group from one another is a 
 task replete with difficulties and uncertainties.* 
 
 It is not intended to imply, by the above remarks, that the 
 isolation of free casein is as a rule attended with danger of con- 
 tamination with paranuclein, because, fortunately, the presence 
 of paranuclein in solutions of casein is not to be feared unless the 
 casein has undergone appreciable hydrolysis in an acid medium. 
 I am merely pointing out the imaginary difficulties which would 
 be encountered in endeavoring to separate casein from para- 
 nuclein, were they found in nature in the same fluid, as an illus- 
 tration of the very real difficulties which unquestionably attend 
 the separation and isolation of other proteins which are insoluble 
 in distilled water when uncombined with acids or bases. 
 
 All that can be said, therefore, when a protein isolated by means 
 of a certain procedure, is found repeatedly to yield the same 
 physical, physico-chemical and chemical constants, is that it is 
 a protein or mixture of proteins of constant composition provided 
 that particular method of isolation be employed and no other. 
 * Vide T. Brailsford Robertson (31), 
 
CASEIN 37 
 
 Only when a protein has been prepared in crystalline form or 
 by many different observers in many different ways and has 
 always been found to possess the same physical, physico-chemical 
 and chemical constants, can we pronounce it, with any degree 
 of safety, a definite chemical individual. So far this has only 
 been accomplished for a very limited number of proteins. 
 
 It cannot with propriety be assumed, therefore, that the fol- 
 lowing methods for the isolation of "pure" proteins yield definite 
 chemical individuals, save those for the isolation of the crystal- 
 line proteins and of casein and of the various members of the 
 protamin group. But the methods which follow are those which, 
 to the knowledge of the author, have been shown to yield, at 
 least, mixtures of appreciably uniform composition, appreciably free 
 from non-protein contamination. 
 
 Very many well-known methods for the isolation and purifi- 
 cation of various proteins are, of course, omitted, not because 
 they do not in all probability yield products conforming to the 
 above restrictions, but because either evidence of this fact is 
 lacking, or they have never been employed in research of a dis- 
 tinctly physico-chemical character.* 
 
 2. Casein. The following is the method of preparation 
 employed by Hammarsten (8) : 
 
 "Milk is diluted with four volumes of water and the mixture 
 treated with acetic acid to 0.75 to 1 per mille. Casein thus ob- 
 tained is purified by repeatedly dissolving in water with the aid 
 of the smallest quantity of alkali possible, by filtering and re- 
 precipitating with acetic acid and thoroughly washing with water. 
 Most of the milk-fat is retained by the filter on the first filtration, 
 and the casein contaminated with traces of fat is purified by 
 treating with alcohol and ether." 
 
 According to Bosworth and Van Slyke (3) casein prepared in 
 this manner is always contaminated by a small admixture of 
 dicalcium phosphate and contains about 0.14 per cent of phos- 
 phorus attributable to this source. The method of preparation 
 which these investigators employ to secure a product free from 
 this contamination is as follows: 
 
 Separator skim-milk is diluted with seven or eight times its 
 volume of distilled water, and dilute acetic acid (6 cc. of glacial 
 
 * For a number of methods for the separation of various animal proteins 
 vide Hammarsten (8). 
 
38 CHEMICAL STATICS 
 
 acetic acid diluted to 1 litre) is carefully added until the casein 
 separates completely, after which the clear solution is removed 
 by syphon as soon as the precipitate settles. Distilled water is 
 then added, the mixture stirred vigorously and the precipitate 
 allowed to settle, after which the wash water is syphoned off. 
 Water is again added, and the casein is dissolved by adding, for 
 each litre of milk used, 1 litre of dilute NH 4 OH (6 cc. of strong 
 reagent diluted to 1 litre). When the solution is complete the 
 whole is filtered through a thick layer of absorbent cotton. The 
 casein is then precipitated again with dilute acetic acid; the 
 precipitate is allowed to settle, and is then washed, redissolved 
 in dilute NH 4 OH and filtered, the process of precipitation, wash- 
 ing, dissolving, etc., being repeated not less than four times. 
 Finally an excess of strong NH 4 OH (10 cc.) is added and then 
 20 cc. of a saturated solution of ammonium oxalate. The mix- 
 ture is allowed to stand twelve hours or more. Calcium is pre- 
 cipitated as oxalate in very finely divided condition, too fine to 
 permit its satisfactory removal by ordinary methods of filtration. 
 Better aggregation of the precipitate can, however, be effected 
 by means of centrifugal force. The centrifuged mixture is then 
 filtered through double thickness of filter paper. The filtered 
 solution is next treated with dilute HC1 (10 cc. HC1, sp. gr. 1.20 
 diluted to 1 litre) until the casein is precipitated. The precipi- 
 tate is washed with distilled water until free from chloride and is 
 then placed on a hardened filter paper in a Buchner funnel, as 
 much water as possible being now removed by suction. The mass 
 is next transferred to a large mortar and thoroughly triturated 
 with 95 per cent alcohol. The alcohol is then removed by suc- 
 tion on a Buchner funnel and the casein is placed in a mortar 
 and triturated with absolute alcohol. Most of the alcohol is 
 removed and the casein treated twice with ether in a mortar 
 by trituration, the ether being removed each time by means of 
 suction on a Buchner funnel. The material is then placed in a 
 large evaporating dish and spread out in a layer as thin as possible, 
 allowed to stand twelve hours or more in a warm place, and 
 then finely ground in a mortar until the particles pass through a 
 40-mesh sieve and dried for two days over H 2 S04 in a desiccator 
 under diminished pressure. 
 
 Casein prepared in this manner contains only 0.71 per cent of 
 phosphorus; corresponding almost exactly to the theoretical per- 
 
CASEIN 39 
 
 centage calculated on the supposition that casein contains two 
 atoms each of phosphorus and sulphur. Previous estimates, 
 derived from analyses of casein prepared by Hammarsten's 
 method, indicated a phosphorus content of about 0.85 per 
 cent. 
 
 Casein "nach Hammarsten" is obtainable upon the market in 
 a high degree of purity. For the investigations described in this 
 work I have always employed the C. P. casein "nach Hammar- 
 sten" which is manufactured in Germany for Messrs. Eimer and 
 Amend of New York. This substance is free from appreciable 
 fat and would appear to be contaminated only by a small amount 
 of an acid, water-soluble substance. This I remove by further 
 purification in the following manner (30) (35) : 
 
 Half a pound of casein is triturated with about 12 litres of dis- 
 tilled water, the water being added in six successive portions. 
 On each addition of water the casein is well stirred up in a 
 porcelain mortar and allowed to settle, the supernatant water 
 is then poured off and fresh water is added. It is next washed 
 in a similar manner in 5 kilos of absolute alcohol, and then in 
 5 kilos of ether " distilled over sodium." The mortar, containing 
 the casein drained as free from superfluous ether as possible,* is 
 then placed in an incubator over sulphuric acid at 40-50 C., 
 the flame is turned out under the incubator, and it is allowed to 
 cool for about 24 hours. The casein is now found, if these op- 
 erations have been conducted carefully, to be in the form of a 
 dry, pure white powder, still containing, however, a considerable 
 quantity of ether. The presence of a color in the powder indi- 
 cates contamination, either with moisture or with some other 
 impurity. 
 
 The casein is now spread out, within the incubator, in a layer 
 not over 1 cm. deep, the flame under the incubator is lighted and 
 it is allowed to stand for 24 hours at 40-50 C. The casein is 
 then found to be free from appreciable water or ether. 
 
 The casein which is thus prepared gives every indication of being 
 a pure product and a definite chemical individual. It is insoluble 
 in distilled water, save in traces which adhere to the casein par- 
 
 * At this point it is necessary to avoid exposing the mortar to the moist air 
 of the room for a moment longer than is necessary, otherwise the evaporating 
 ether causes the condensation of sufficient moisture to spoil the product unless 
 it is again treated with alcohol and ether. 
 
40 CHEMICAL STATICS 
 
 tides,* and it neutralizes, to phenolphthalein and to litmus, exactly 
 the quantities of alkali determined by Soldner (41), Lacquer and 
 Sackur (15) and Van Slyke and Hart (45). 
 
 Casein which has been carefully prepared in the manner out- 
 lined above, floats upon the top of and is not readily wetted by 
 water or watery solutions of bases; if, however, it contains a 
 mere trace of moisture, it is readily wetted by all save the most 
 alkaline solutions. In order to successfully and completely dis- 
 solve perfectly anhydrous casein it is necessary to first add to it 
 a very little of the solution in which it is to be dissolved, rub it 
 up into a paste, and then add, while stirring, the remainder of 
 the solution. 
 
 If casein which is readily wetted by water be desired (34) it is 
 necessary to omit the desiccation over H 2 SO4. For this purpose 
 CaCl 2 should be employed as the desiccating agent, and desic- 
 cation should be continued for 12 hours at about 30 C. Casein 
 prepared in this way readily sinks in and is wetted by water and 
 aqueous solutions; it contains, however, not very significant traces 
 of moisture and a considerable amount of adherent ether. 
 
 3. * Insoluble" Serum Globulin. There appear to be at 
 least two globulins in serum, the one insoluble in distilled water, 
 which is precipitated on dialysis of the serum, or dilution and the 
 subsequent addition of a weak acid; the other soluble in water, 
 precipitable by saturation with magnesium sulphate after the 
 removal of the insoluble fraction (16) (5) (27). 
 
 According to Hardy these globulins differ markedly in their 
 phosphorus content, the insoluble globulin containing from 0.07 
 to 0.08 per cent phosphorus; the soluble globulin only a trace 
 (about 0.009 per cent) of phosphorus (9). 
 
 According to Hammarsten (7) the individuality of these pro- 
 teins, and the freedom of any given preparation of the one from 
 an admixture of the other is open to serious doubt. Moreover, 
 as Taylor has shown (44), the "insoluble" globulin is readily 
 converted, by hydrolysis, into the "soluble" form. The relation- 
 ship between the various serum globulins would therefore appear 
 to be somewhat analogous to that between the various members 
 of the paranuclein group. . 
 
 * So that when litmus paper is dipped into a suspension of casein particles 
 in distilled water the paper is reddened where it is touched by the undissolved 
 particles, while the fluid which bathes them remains clear and neutral (30). 
 
SERUM GLOBULIN 41 
 
 The following method of preparing serum globulin is based 
 upon the observation of Alexander Schmidt (38) and Kuhne (14) 
 that " insoluble" serum globulin can be precipitated from serum, 
 previously diluted with ten volumes of water, by the passage of 
 C0 2 . It appears to be difficult or impossible to bring about this 
 precipitation in the total absence of salts of stronger acids, e.g., 
 NaCl. I have repeatedly observed that if serum globulin, pre- 
 cipitated by C0 2 and carefully washed, be dissolved in a minimal 
 quantity of NaOH or KOH it cannot be reprecipitated by dilu- 
 tion and the passage of C02, although a distinctly acid and very 
 opaque solution results. 
 
 Three litres of ox-serum are diluted with ten volumes of dis- 
 tilled water, and C0 2 is bubbled through it at a good rate for 
 about half an hour. The globulin which is thus precipitated 
 is allowed to settle in tall glass cylinders,* the supernatant 
 fluid being syphoned off after settling. The precipitate is then 
 washed with about 60 litres of distilled water, in two portions. 
 The globulin is then dissolved in a minimal quantity of 
 N / 10 HClf and immediately reprecipitated by cautious neutral- 
 ization with AT/10 KOH. This precipitate, after settling and de- 
 cantation of the supernatant fluid, is washed in 60 litres of distilled 
 
 * For the settling and washing of protein precipitates I employ wide- 
 mouthed glass cylinders from 50 to 60 cm. high and about 10 cm. in diameter, 
 closed by ground glass stoppers. The syphon is provided with a side tube 
 through which it can be filled and can then be closed by a rubber tube provided 
 with a pinch-cock. When the protein is being washed with alcohol or with 
 ether, the syphon is filled with alcohol so as to avoid accidental contamination 
 of the contents of the cylinder with water. The syphon is supported on a 
 stand which is so arranged that the height of the opening of the syphon above 
 the precipitate can be adjusted by turning a screw. The greater part of the 
 liquid is first rapidly run off with the opening of the syphon a good way above 
 the precipitate. The syphon is then lowered and the remainder of the liquid 
 run off more slowly. 
 
 f This can be calculated from the fact that 1 litre of serum yields about 
 5 grams of globulin and that about 20 X 10~ 6 gram equivalents of the strong 
 monobasic acids just suffice to dissolve one gram. If acetic acid be preferred, 
 about 100 X 10~ 5 equivalents are required to dissolve 1 gram (9). 
 
 It has been shown by Hammarsten (7) that "insoluble" serum globulin 
 is not readily "denatured," i.e., altered in properties, by acids. "A solution 
 of serum globulin containing 0.2-0.3 per cent acetic acid remains unchanged 
 for days at a low temperature, and one can recover the serum globulin unal- 
 tered by neutralization." In the presence of stronger acids denaturation is a 
 matter of some hours. 
 
42 CHEMICAL STATICS 
 
 water in 6 successive washings, the precipitate, after each agi- 
 tation with distilled water, being allowed to settle for 24 hours 
 in the presence of toluol, after which the supernatant fluid is 
 drawn off and the globulin suspended in a fresh quantum of dis- 
 tilled water.* The thick suspension of globulin which is thus 
 obtained after the final washing is kept, in the presence of toluol, 
 in a stoppered bottle and used in this form, since globulin, if 
 washed with alcohol and ether and dried, is redissolved only with 
 difficulty. 
 
 The suspension is well shaken before withdrawing a measured 
 sample. The globulin content of the suspension is determined to 
 within 0.01 gram per 100 cc. by placing 25 cc. samples in small 
 and accurately weighed beakers, evaporating the fluid to dry- 
 ness on a water-bath, and then drying the residue over H 2 S04 at 
 70 until its weight becomes constant. 
 
 4. Fibrin. The following is the method of preparing fibrin 
 which is employed by Bosworth (2). 
 
 Fresh ox-blood is collected in a large bottle and, as soon as 
 possible, transferred to wide-mouthed precipitating jars and 
 allowed to coagulate. The clots are then removed, broken into 
 small pieces, and washed in running water to remove the serum 
 and blood corpuscles. The washed masses of fibrin are passed 
 through a mincing machine, placed in a large (8-litre) bottle, a 
 little toluol added, and the bottle filled with 0.2 per cent sodium 
 hydroxide solution. This solution causes the fibrin to swell and 
 after about 36 hours the whole content of the bottle resembles a 
 thin jelly. This jelly is broken up, one-half transferred to another 
 8-litre bottle, and after the two bottles are filled by the addition 
 of water they are allowed to stand for an additional 36 hours. 
 The jelly is by then almost completely dissolved and the contents 
 of both bottles are filtered, first through cheese-cloth, then linen 
 and finally paper. The clear filtrate is then diluted with an equal 
 volume of water and placed in tall wide-mouthed precipitating 
 jars. Dilute acetic acid (0.3 per cent) is then added cautiously. 
 At a certain point a flocculent precipitate of fibrin appears which 
 quickly settles to the bottom of the jars. 
 
 * This suspension is, of course, equivalent to a prolonged dialysis of the 
 protein, the precipitate acting as a dialyser of enormously extended surface, 
 permitting the passage of associated diffusible impurities into the distilled 
 water and retaining the protein. 
 
HAEMOGLOBIN 43 
 
 The supernatant liquid is syphoned off, the precipitate washed 
 with water, dissolved in dilute sodium hydroxide (0.05 per cent) 
 and again precipitated with acetic acid. This process is repeated 
 three times, the final precipitate being washed with alcohol and 
 ether and dried over sulphuric acid in an evacuated desiccator. 
 
 The behavior of fibrin strongly resembles that of casein in 
 all stages of its preparation except in its extreme sensitiveness 
 to a slight excess of acid or alkali, for unlike casein, it is readily 
 soluble in weak acetic acid. 
 
 5. Haemoglobin. The following is the method of prepara- 
 tion recommended by Preyer (26). 
 
 The blood is collected in a vessel and allowed to coagulate 
 and to stand for several hours (or, better, for a day) in a cool 
 place. Then the serum with the white corpuscles and the fat 
 which have collected above the clot is removed and the coag- 
 ulum is washed with distilled water and then cut into very small 
 pieces, and these pieces in turn are repeatedly washed with cold 
 distilled water. Then the clot is comminuted, best by freezing 
 and reducing the frozen mass to powder. This powder is placed 
 in a filter paper and washed with cold distilled water until the 
 filtrate no longer gives any very bulky precipitate with bichloride 
 of mercury. The coagulum is extracted by water heated to from 
 30 to 40 degrees and filtered, and the filtrate is collected in a 
 large cylindrical vessel standing in ice. A small measured portion of 
 the red solution thus obtained is gradually mixed during constant 
 agitation with small quantities of alcohol until a slight precipi- 
 tate forms. This determines how much alcohol may be added to 
 the whole solution without a precipitate appearing. A slightly 
 smaller proportion of alcohol is now added to the remaining 
 filtrate and the mixture is placed in a cooling, medium. After a 
 few hours the crystals separate in great abundance. The crys- 
 tals, owing to the large volume of water used, are very easily 
 filtered off in the cold. They are then washed with cold water 
 containing a little alcohol until the filtrate yields only an insig- 
 nificant cloudiness upon the addition of acetate of lead or mer- 
 curic chloride. The product yielded is a very large one. The 
 crystals may be purified by repeated washing by decantation 
 until the wash-water does not become cloudy with bichloride of 
 mercury, lead acetate or silver nitrate. They are then nearly 
 pure, and the ash is free from phosphoric acid and consists of 
 
44 CHEMICAL STATICS 
 
 pure iron oxide. If this is not the case, then they must be dis- 
 solved in warm water and recrystallized. At a temperature of 
 less than C. the crystals can be dried in the air without de- 
 composition. 
 
 Preyer states that, of all kinds of blood, that of the horse is 
 best adapted for the production of very large quantities of pure 
 haemoglobin. 
 
 For a variety of rapid methods of preparing small quantities 
 of crystals from the blood of different species the reader is re- 
 ferred to the monograph on the Crystallography of Haemoglobins 
 by Reichert and Brown (28). 
 
 6. Crystallizable Egg- Albumin. The method of Hopkins and 
 Pinkus ( 1 0) is universally employed for the preparation of this protein . 
 
 Two hundred cc. of egg-white obtained from newly-laid fowls' 
 eggs are mixed with an equal bulk of saturated ammonium sul- 
 phate solution, the latter being very gradually added and the mix- 
 ture stirred briskly with an egg-beater between each addition. 
 It is then allowed to stand over night. The mixture is then filtered 
 and to the clear filtrate more saturated ammonium sulphate solu- 
 tion is added until a permanent precipitate is obtained. Distilled 
 water is then added, a few drops at a time, until the solution is 
 just clear again. Ten per cent acetic acid solution is then added 
 drop by drop until a slight but definite precipitate has appeared. 
 The bottle is then immediately stoppered and allowed to stand. 
 After 24 hours an abundance of uniformly crystalline precipitate 
 has settled out consisting of rosettes of needles. If a somewhat 
 greater proportion of acetic acid is employed the rosettes are 
 mixed with sheaths and fan-shaped aggregates of crystals. Re- 
 crystallization of this first product is carried out by filtering off 
 the crystals from the mother-liquor, redissolving in a moderate 
 amount of water, acidifying with a few drops of dilute acetic 
 acid and adding saturated ammonium sulphate solution until a 
 faint turbidity is produced. In 24 hours a large proportion of 
 the protein will have recrystallized. This process may be re- 
 peated 5 or 6 times in the course of a week. The crystals which 
 are finally obtained may be freed from inorganic contamination 
 by prolonged dialysis against running distilled water. 
 
 7. Ovovitellin. The following method of preparation is that 
 employed by Osborne and Campbell (23), modified by Plimmer 
 (25) and further modified by the author (33). 
 
OVOVITELLIN 45 
 
 Twenty-five yolks of eggs are carefully washed, without break- 
 ing the enveloping membrane, in a stream of water, so as to re- 
 move all traces of the whites. To the yolk is then added an equal 
 volume of 10 per cent sodium chloride solution and the solution 
 which is thus obtained is extracted from ten to twelve times 
 with twice its volume of ether in separatory funnels, extracting 
 several times after the ethereal layers fail to yield a precipitate, 
 due to the presence of lecithin, upon the addition of acetone. 
 The complete extraction occupies a period of from two to three 
 weeks. The watery layer which is finally obtained is then poured 
 into twenty volumes of distilled water, and the precipitate of 
 ovovitellin which is thus obtained is allowed to settle in tall 
 glass cylinders. The supernatant fluid is then syphoned off and 
 the precipitate redissolved in 10 per cent sodium chloride and 
 reprecipitated in the same manner. This process is repeated. 
 Finally the vitellin is dissolved in very dilute sodium hydrate 
 and the solution filtered, the filtrate being allowed to drop directly 
 into dilute acetic acid, thus reprecipitating the vitellin. This 
 precipitate is suspended in distilled water and allowed to settle 
 in tall glass cylinders. The supernatant water is then drawn off 
 and the washing with water repeated several times. The pre- 
 cipitate is then washed in 6 litres of 99.8 per cent alcohol. After 
 allowing the precipitate to settle, the supernatant alcohol is 
 syphoned off and the washing in alcohol repeated twice. The 
 vitellin is then washed twice in ether "distilled over sodium," 
 employing 6 litres each time. The thick suspension of vitellin 
 in ether finally obtained is quickly poured into a hardened filter 
 and allowed to filter and dry over sulphuric acid at 40 degrees 
 for 48 hours. The vitellin is thus obtained as a white, somewhat 
 coarse powder. 
 
 About a gram of this ovovitellin was placed in about 30 cc. 
 of alcohol and boiled for some five minutes. About 5 cc. of the 
 alcohol, after filtration, was then tested directly for lecithin by 
 the addition of several volumes of acetone. The remainder of 
 the alcohol was evaporated down to dryness and the residue 
 (barely visible) taken up in about 10 cc. of ether. This ether 
 was then tested by the addition of several volumes of acetone. 
 Both tests proved entirely negative, not the slightest opalescence 
 being produced by the acetone; thus the ovovitellin did not con- 
 tain any lecithin. Hence the older statements to the effect that 
 
46 CHEMICAL STATICS 
 
 vitellin is always associated and probably combined with lecithin 
 are attributable to inadequate extraction of the yolks with ether 
 and the consequent incomplete removal of the lecithin. 
 
 It is extremely difficult to prepare homogeneous solutions of 
 ovo vitellin. A five per cent solution in alkali (JV/10 KOH) is a 
 thick jelly, opalescent owing to the presence of a multitude of 
 air bubbles entangled in it while stirring. In endeavoring to 
 prepare more dilute solutions it is found very difficult to avoid 
 the formation of small lumps of jelly within the solutions, and 
 these are exceedingly difficult to break up, and dissolve with ex- 
 treme slowness. Although vitellin is soluble in dilute solutions 
 of the strong acids, yet when the powder is directly mixed with 
 an acid solution it will not dissolve or does so with extreme 
 slowness; it is soluble in acid solutions only when freshly pre- 
 cipitated and still wet.* 
 
 It is possible to obtain clear, homogeneous 1 per cent solutions 
 of ovovitellin (not more concentrated) by gradually dropping 
 the vitellin from above into the solvent while undergoing violent 
 stirring, and maintaining the stirring for about an hour. 
 
 8. The Vegetable Proteins. The following methods of 
 preparing the globulin of flax-seed will serve as an illustration of 
 the general nature of the technique which is employed in pre- 
 paring the various vegetable proteins. For further information 
 concerning this subject the reader is referred to the monograph 
 by Osborne (22) in which he summarizes the result of his impor- 
 tant and extensive researches upon the preparation and properties 
 of the vegetable proteins. According to Osborne (20) flax-seed 
 globulin may be extracted from flax-seed meal in either of three 
 ways, namely by extraction with distilled water, or with sodium 
 chloride solution or with dilute KOH. The following are the 
 methods of preparation employed by him. 
 
 (a) Extraction with distilled water. 
 
 The ground flax-seed is freed from oil by extracting with ben- 
 zene and from the greater part of the outer coating of the seed 
 by sifting through a fine sieve. One hundred grams of this 
 meal are extracted for about 24 hours with distilled water at 
 temperatures between 20 and 40 C. The filtered extract has 
 a yellow color, is acid in reaction and very slightly turbid. 
 
 * Exactly the same phenomenon is encountered with casein. Cf. Van 
 Slyke and Van Slyke (46), T. Brailsford Robertson (32). 
 
VEGETABLE PROTEINS 47 
 
 This extract is then saturated with pure ammonium sulphate. 
 The copious precipitate is collected on a filter and, after the com- 
 pletion of filtration, dissolved in about 800 cc. of water. The 
 solution is then filtered and placed in a dialyser. After twenty- 
 four hours a considerable precipitate has formed, which is seen 
 under the microscope to consist entirely of perfectly formed 
 octahedral crystals. After dialysing for about five days the solu- 
 tion is found to be free from chlorides, and the precipitate is then 
 filtered off and washed with water, followed by dilute alcohol 
 gradually increased in strength to absolute alcohol; it is finally 
 washed with ether and dried over sulphuric acid. 
 
 The preparation, after drying, still retains its crystalline char- 
 acter. The meal yields about 10 per cent of its weight in globulin. 
 
 (6) Extraction with sodium chloride solution. 
 
 One hundred grams of the flax-seed meal are extracted three 
 successive times with 20 per cent sodium chloride solution, and 
 the clear, filtered, bright yellow extracts are united and dissolved 
 in 10 per cent sodium chloride, and this solution, after filtering 
 off an insoluble residue, is submitted to dialysis. After five days 
 the solution, free from chlorides, is filtered and the precipitate 
 washed with water, alcohol and ether and dried. The prepara- 
 tion, after drying, still retains its crystalline character. One 
 hundred grams of meal yield about 11.6 grams of protein. This 
 protein is identical with the globulin obtained by extraction with 
 water. 
 
 (c) Extraction with dilute KOH solution. 
 
 Flax-seed globulin, prepared by either of the above methods, is 
 soluble in dilute KOH and can be precipitated from this solution 
 unchanged in composition or properties, by cautious neutraliza- 
 tion with hydrochloric acid. 
 
 Flax-seed meal is extracted with 100 cc. of 0.01 KOH per 
 gram. After filtration it is saturated with ammonium sulphate 
 and the precipitate filtered off and dissolved as completely as 
 possible in 10 per cent sodium chloride. This solution is dialysed 
 until the chlorides are removed and the subsequent treatment is 
 the same as that employed in the two previous preparations. 
 
 This preparation is imperfectly crystalline, but identical in 
 composition with the other preparations. One hundred grams 
 of meal yield about 17.5 grams of protein. 
 
 The plant globulins are, like serum globulin, precipitable from 
 
48 CHEMICAL STATICS 
 
 their solutions in sodium chloride by dilution and the passage of 
 C0 2 . It is stated by Osborne, however, that the separation which 
 is secured in this manner is rarely so complete as that which is 
 accomplished by dialysis (22). The plant globulins are, accord- 
 ing to Osborne, very readily "denatured" by strong acids, even 
 when diluted. 
 
 For the preparation of some of the seed-proteins it is sufficient 
 to extract the crushed seeds with hot sodium chloride solution; 
 on cooling, the protein crystallizes out spontaneously (6) (19) 
 (29). 
 
 Osborne inclines to the view that the majority of the vegetable 
 globulins, instead of being predominantly acid substances, as they 
 are generally believed to be, are, in reality, predominantly basic. 
 This question will be commented upon in a later chapter (Chap. V). 
 
 9. The Alcohol Soluble Vegetable Proteins (Gliadin, Zein, 
 etc.). The following method of preparing gliadin, based upon 
 that employed by Osborne and Harris (24), has been communi- 
 cated to me by Dr. J. E. Greaves: 
 
 Gluten is prepared by kneading dough, made from wheat flour, 
 in a stream of cold water until all of the starch has been washed 
 out; it is then partially dried and a moisture determination is 
 made. The moist gluten thus obtained is finely chopped and 
 mixed with twenty times its weight of alcohol of such a strength 
 that with the water in the gluten it forms an alcoholic solution 
 containing 70 per cent of alcohol by volume. The mixture of gluten 
 and 70 per cent alcohol is then allowed to stand, with frequent 
 shaking, for 48 hours. After allowing the mixture to stand for 
 10 hours the supernatant alcoholic solution of gliadin is syphoned 
 off and filtered through very finely shredded and well-washed and 
 dried asbestos until a perfectly clear filtrate is obtained. The fil- 
 trate is then evaporated, under a pressure of about \ atmosphere 
 until frothing prevents further concentration. It is then cooled and 
 very slowly poured, with constant stirring, into about one hundred 
 times its volume of ice-cold distilled water, containing 10 grams 
 of sodium chloride per litre. The gummy mass, which usually 
 collects on the stirring-rod, is dissolved by the addition of the 
 least possible amount of- absolute alcohol, and this solution is 
 evaporated, under reduced pressure, to a thick syrup; the syrup 
 is then cooled and poured, in a very fine stream, with constant 
 stirring, into hot absolute alcohol. The precipitate is dissolved 
 
OVOMUCOID 49 
 
 in 70 per cent alcohol and this solution is evaporated under re- 
 duced pressure, with the occasional addition of absolute alcohol, 
 until a thick syrup is again obtained. The gliadin is precipitated 
 from this syrup by the method just described, washed three times 
 with ether (distilled over sodium), partially dried over sulphuric 
 acid, ground up as finely as possible, and then completely dried 
 over sulphuric acid, in partial vacuum, at room temperature. 
 The conditions of the precipitation, of course, involve contami- 
 nation with NaCl. According to Osborne (21) there is only one 
 alcohol-soluble protein in flour; on standing in alcoholic solution, 
 however, a protein insoluble in alcohol or alcohol-water mixtures 
 is precipitated within a few hours. Kosutany (13) believes that 
 this substance is derived from gliadin by the splitting off of water 
 and is identical with glutenin, the alcohol-insoluble and water- 
 insoluble protein constituent of gluten. According to Osborne 
 (21), however, glutenin differs qualitatively from gliadin in that, 
 upon hydrolysis with hydrochloric acid, glutenin yields glycocoll 
 and lysin, while gliadin yields neither of these amino-acids. 
 
 10. Ovomucoid. The following method of preparing ovo- 
 mucoid is a modification of that originally employed by Morner 
 (17) (33) (36). 
 
 The whites of eggs are beaten up to a froth and allowed to 
 stand in shallow vessels over night. The subnatant fluid is then 
 poured off, the froth being rejected. This fluid is diluted to five 
 times its volume with distilled water, and to every litre of the 
 diluted fluid is added 130 cc. of approximately N/IQ acetic acid 
 (made up by diluting 10 cc. of glacial acetic acid to 1750 cc.). 
 This mixture is heated slowly to boiling point, being rapidly 
 and uniformly stirred meanwhile, and after being allowed to boil 
 for about 3 to 5 minutes, is put aside in rather shallow vessels 
 for about 12 hours. At the end of this time most of the coagulum 
 has floated to the top and the clear pale yellow subnatant fluid is 
 filtered through hardened filter paper. Filtration is very rapid, 
 and the filtered fluid, when boiled, either with or without further 
 addition of acetic acid, remains perfectly clear. The fluid which 
 is thus obtained is now slowly evaporated to one-fifth of its volume, 
 the temperature of the fluid never being allowed to rise above 
 55 C. After allowing this fluid to cool, the protein is precipitated 
 from it by the addition of ten volumes of 99.8 per cent -alcohol 
 and is allowed to settle in tall glass cylinders. The supernatant 
 
50 CHEMICAL STATICS 
 
 fluid is then syphoned off and the precipitate is washed in the 
 same volume of alcohol as that employed in the precipitation. 
 This washing is repeated, again employing the same volume of 
 alcohol, and the precipitate is allowed to steep in this alcohol for 
 about 24 hours, in order to remove all adherent or combined 
 acetic acid. The alcohol is then syphoned off and the precipitate 
 is washed in the same volume of ether (distilled over sodium). 
 This washing is repeated. The ether is then syphoned off and 
 the thick suspension of protein in ether thus obtained is rapidly 
 poured into a hardened filter, the filter and the contained sus- 
 pension of protein in ether being at once transferred to an incu- 
 bator and the filtration continued over sulphuric acid at 40 degrees 
 (to avoid condensation of atmospheric moisture on the filter). 
 
 After the completion of filtration, the ether which has filtered 
 off is removed from the incubator, and the precipitate is allowed 
 to dry for 24 hours. The protein is then obtained in the form 
 of chalky cakes which are very readily broken up into impal- 
 pable powder. This powder is passed through a fine sieve and 
 conserved in a glass stoppered bottle. 
 
 It is found inadvisable to work with fewer than six dozen eggs 
 at one time as, otherwise, the amount of precipitate which is 
 finally obtained is so small that the danger of excessive caking 
 and partial decomposition, in drying, due to the deposition of 
 traces of moisture upon the filter, is very great. 
 
 Twenty-four dozen eggs of average size yield about 40 grams of 
 ovomucoid. 
 
 About a gram of ovomucoid, prepared in this manner, was 
 dissolved in about 100 cc. of N/2 hydrochloric acid, and this 
 solution was boiled until 30 cc. of fluid had distilled over. This 
 distillate was then tested for acetic acid. It contained a trace 
 of an acid of the fatty series, sufficient to yield a slight coloration 
 with ferric chloride, but insufficient to yield a precipitate of ferric 
 hydrate on boiling, or to yield the ethyl acetate test.* 
 
 Ovomucoid is a gluco-protein, and yields, on boiling with 
 strong acid, about 30 per cent of glucosamin (47) (40) (18) (42) 
 which, according to Steudel (42), is contained in the molecule not 
 in the form of glucosamin but in the form of an antecedent which 
 yields glucosamin on treatment with strong acids. 
 
 * The solutions of ovomucoid prepared in this manner have themselves a 
 distinct odor of ethyl acetate. 
 
GELATIN 51 
 
 11. Gelatin and Deaminized Gelatin. The only practicable 
 method of purifying gelatin which has been devised consists in 
 subjecting the best qualities of commercial gelatin to very pro- 
 longed dialysis against running distilled water (4). Deaminized 
 gelatin is prepared by Blasel and Matula (1) by the following 
 method : 
 
 About 200 grams of the best commercial gelatin is dissolved 
 in one litre of warm water. To this solution is added 200 grams 
 of sodium nitrite dissolved in one litre of water. After cooling, 
 140 grams of glacial acetic acid is carefully added. The mix- 
 ture is allowed to stand for twelve hours and then heated on 
 a water-bath for two hours. The deaminized gelatin is then 
 precipitated by the addition of solid ammonium sulphate and 
 purified by prolonged dialysis (2 weeks) against running distilled 
 water. 
 
 12. Globin. Globin may best be prepared by the following 
 modification of the method devised by Schulz (39) (37). 
 
 A thick suspension of ox-corpuscles is obtained by centrif- 
 ugalizing freshly defibrinated ox-blood, the volume of the sus- 
 pension being about one-third that of the blood from which it 
 is derived. 
 
 After pipetting off the supernatant serum the suspension is 
 diluted to the original volume of the blood from which it was 
 derived by the addition of N/Q NaCl solution and the centrif- 
 ugalization is repeated, the supernatant fluid being removed as 
 before. This is repeated six times in order to free the corpuscles 
 from adherent serum. After the last centrifugalization the cor- 
 puscle suspension is not again diluted with NaCl. 
 
 The thick suspension of corpuscles which is thus obtained is 
 diluted to ten times its volume by the addition of distilled water. 
 The corpuscles are thus "laked" and the contained haemoglobin 
 is discharged into the water, forming a clear solution which is 
 allowed to stand in tall glass vessels for twenty-four hours in 
 order to permit the leucocytes to settle. The upper portion of 
 the fluid is then decanted and employed, the lower portion of 
 the fluid being rejected. 
 
 Two and one-half litre portions of this fluid are placed in six- 
 litre bottles, to the contents of each bottle are added 56 cc. of 
 concentrated HC1 (sp. gr. 1.18) and the mixture is thoroughly 
 shaken and allowed to stand at room temperature for one hour. 
 
52 CHEMICAL STATICS 
 
 The addition of the acid causes a flocculent precipitate to appear 
 and the mixture turns dark brown. Two and one-half litres of 
 ether are then added to each bottle and the contents shaken 
 thoroughly until they attain an oily consistency. Rubber stop- 
 pers with two perforations are then fitted into the necks of the 
 bottles. Through one perforation is inserted a long glass tube 
 reaching to the bottom of the bottle, to act subsequently as an 
 air inlet, and through the other is inserted a short tube just reach- 
 ing to the bottom of the stopper and provided with a rubber 
 tube and pinch-cock. The stoppers are then tied down and the 
 bottles quickly inverted and allowed- to stand at room tempera- 
 ture for twenty-four hours. By this time the contents of the 
 bottles, if the temperature of the room is not too warm, should 
 have separated into two layers, an upper, jelly-like, very deeply 
 colored ether layer containing the greater part of the hsematin 
 hydrochloride, and a lower, aqueous layer containing the globin 
 to some extent contaminated by hsematin hydrochloride. The 
 latter is then drawn off through the shorter of the two tubes 
 inserted through the stopper.* 
 
 To this fluid is added four volumes of a mixture of equal parts 
 by volume of alcohol and ether. A light-colored precipitate is 
 obtained, leaving the fluid very deeply colored. This precipi- 
 tate is collected upon a hardened filter, washed once with a volume 
 of alcohol equal to that of the watery fluid from which it was 
 precipitated, drained and then scraped off the paper and dissolved 
 in a volume of JV/10 HC1 equal to that of the fluid from which 
 the globin was precipitated. This solution is now diluted by 
 the addition of three times its volume of distilled water, and 
 20 per cent ammonia solution is carefully added until a precipitate 
 just appears. One cubic centimetre of strong ammonia per litre 
 is then added and the dense, flocculent precipitate is collected 
 on a filter. The precipitate is then washed in large volumes of 
 absolute alcohol, alcohol-ether mixture and ether. After drain- 
 ing in a dry atmosphere (preferably within an incubator over 
 H 2 SO 4 ) the precipitate is allowed to dry for 24 hours in an incu- 
 bator over H 2 S04, then pulverized and sifted and returned to the 
 incubator to dry over H 2 SO 4 for one week. 
 
 * The separation of the fluid into two layers is sometimes very slow. In 
 such cases separation may be hastened by centrifugalization. 
 
PROTAMINS 53 
 
 13. The Protamins. Kossel, to whose investigations we 
 owe our comparatively extensive knowledge of these, "the sim- 
 plest proteins," prepares them in the following manner (12). 
 
 The minced ripe testicles of herrings (yielding clupein), salmon 
 (yielding salmin) or sturgeons (yielding sturin) are shaken up in 
 water, whereby a suspension of spermatozoa is obtained. This 
 fluid is coagulated with acetic acid and the precipitate washed 
 with alcohol and ether and dried. About 100 grams of the dry 
 mass is then shaken up in 500 cc. of a 1 per cent solution of 
 H 2 S04 for one-half hour and filtered; this extraction is repeated 
 six times and the extracts are mixed. The filtered extract is 
 precipitated with three times its mass of alcohol, the fluid is 
 syphoned off and the precipitate is dissolved in hot water and 
 again precipitated with alcohol. 
 
 This precipitate of protamin sulphate is dissolved in about one 
 and one-half litres of hot water and the solution is allowed to 
 cool, when a small part of the sulphate separates out as a yellow 
 or brownish colored oil. This least soluble portion of the pro- 
 tamin sulphate is rejected. The supernatant fluid is collected 
 and evaporated to a small volume when the greater part of the 
 protamin sulphate separates out as an oil. The mixture is placed 
 in a separatory funnel and the oil drawn off. 
 
 This preparation may be freed from the last traces of associated 
 nucleic acid in the following manner: It is dissolved in hot 
 water and then precipitated with sodium picrate. This precipi- 
 tate is well washed with water and then freed from picric acid 
 by shaking it up with ether in the presence of an excess of H 2 S04. 
 The protamin sulphate is precipitated out of the sulphuric acid 
 solution by the addition of alcohol. This precipitation should be 
 repeated. The protamin sulphate should now come down as a 
 loose white precipitate. If the precipitate -has a glutinous ap- 
 pearance, the solution in water and precipitation by alcohol 
 should be repeated. Finally, the precipitate is dried at 110 to 
 120 C. 
 
 According to Kossel, clupein and salmin are identical, the 
 formula of the sulphate being a multiple of CsoHsTNiyOe, 2 H 2 SC>4. 
 The most probable formula for sturin sulphate is, according to 
 the same author, 4 CseHegNigO: + 11 H 2 S0 4 . 
 
 These substances are strong bases; when one precipitates the 
 H 2 S0 4 out of a solution of clupein sulphate by the addition of 
 
54 CHEMICAL STATICS 
 
 the equivalent mass of baryta, the resultant solution of free 
 clupein has a strongly alkaline reaction (12) (43). 
 
 Salmin carbonate yields an alkaline solution owing to hydro- 
 lytic dissociation (43). Taylor states that the salmin yielded by 
 the Pacific salmon is identical with that which is yielded by the 
 European salmon. 
 
 The protamins readily form " basic salts" in which the pro- 
 portion of H 2 SO 4 to protein is less than in the fully neutralized 
 compound; they also tend to combine with water to form vari- 
 ous hydrates. Thus salmin sulphate which is insufficiently dried 
 has the formula C 3 oH 5 7Ni 7 06, 2 H 2 SO 4 + H 2 O. 
 
 LITERATURE CITED 
 
 (1) Blasel, L., and Matula, J., Biochem. Zeit. 58 (1914), p. 417. 
 
 (2) Bosworth, A. W., Journ. Biol. Chem. 20 (1915), p. 91. 
 
 (3) Bosworth, A. W., and Van Slyke, L. L., Journ. Biol. Chem. 14 (1913), 
 
 p. 203; 19 (1914), p. 67. 
 
 (4) Chiari, R., Biochem. Zeit. 33 (1911), p. 167. 
 
 (5) Freund, E., and Joachim, J., Zeit. fiir Physiol. Chem. 36 (1902), p. 407. 
 
 (6) Gruebler, G., Journ. Prakt. Chem. 23 (1881), p. 97. 
 
 (7) Hammarsten, O., Ergeb. der physiol. 1 Abt. 1 (1902), p. 330. 
 
 (8) Hammarsten, O., "A Text-book of Physiological Chemistry," trans. 
 
 by J. A. Mandel, 6th English Edn. New York, 1911. 
 
 (9) Hardy, W. B., Journ. of Physiol. 33 (1905), p. 330. 
 
 (10) Hopkins, F. Gowland, and Pinkus, S. N., Journ. of Physiol. 23 (1898), 
 
 p. 130. 
 
 (11) Hoppe-Seyler, F., Med.-chem. Untersuch. (1866), p. 216. 
 
 (12) Kossel, A., Zeit. f. physiol. Chem. 25 (1898), p. 165. 
 
 (13) Kosutany, T., Journ. Landw. 51 (1903), p. 130. 
 
 (14) Kuhne, W., "Lehrbuch der physiol. Chem.," Leipzig (1868), pp. 168 
 
 and 174. 
 
 (15) Lacqueur, E., and Sackur, 0., Beitr. z. chem. Physiol. und Pathol. 3 
 
 (1902), p. 196. 
 
 (16) Marcus, E., Zeit. f. physiol. Chem. 28 (1899), p. 559. 
 
 (17) Morner, C. T., Zeit. f. physiol. Chem. 18 (1894), p. 535. 
 
 (18) Muller, F., Zeit. f. Biol. 42 (1901), p. 468. 
 
 ;(19) Osborne, T. B., Amer. Chem. Journ. 13 (1891), p. 408; 14 (1892), pp. 
 212, 662. 
 
 (20) Osborne, T. B., Amer. Chem. Journ. 14 (1892), p. 629. 
 
 (21) Osborne, T. B., "The Proteins of the Wheat Kernel," Carnegie Inst. 
 
 Publ., Washington (19CJY). 
 
 (22) Osborne, T. B., "The Vegetable Proteins," London (1910). 
 
 (23) Osborne, T. B., and .Campbell, G. F., Journ. Amer. Chem. Soc. 22 
 
 (1900), p. 413. 
 
LITERATURE CITED 55 
 
 (24) Osborne, T. B., and Harris, I. F., Amer. Journ. of Physiol. 17 (1906), 
 
 p. 233. 
 
 (25) Plimmer, H. A., Journ. Chem. Soc. London (1908), p. 1500. 
 
 (26) Preyer, W. T., "Die Blutkrystalle," Jena, 1871. 
 
 (27) Quinan, C., Univ. of California Publ. Pathol. 1 (1903), p. 1. 
 
 (28) Reichert, E. T., and Brown, A. P., Carnegie Inst. Publ. Nr. 116, Wash- 
 
 ington (1909). 
 
 (29) Ritthausen, H., Journ. Prakt. Chem. 23 (1881), p. 481. 
 
 (30) Robertson, T. Brailsford, Journ. Biol. Chem. 2 (1907), p. 317. 
 
 (31) Robertson, T. Brailsford, Journ. Biol. Chem. 2 (1907), p. 337; 5 (1909), 
 
 p. 493; 8 (1910), p. 287. 
 
 (32) Robertson, T. Brailsford, Journ. Physical Chem. 13 (1909), p. 469. 
 
 (33) Robertson, T. Brailsford, Journ. Biol. Chem. 7 (1910), p. 359. 
 
 (34) Robertson, T. Brailsford, Journ. Physical. Chem. 14 (1910), p. 377. 
 
 (35) Robertson, T. Brailsford, Journ. Physical Chem. 14 (1910), p. 528. 
 
 (36) Robertson, T. Brailsford, Journ. Physical Chem. 14 (1910), p. 709. 
 
 (37) Robertson, T. Brailsford, Journ. Biol. Chem. 13 (1913), p. 455. 
 
 (38) Schmidt, A., Arch. f. (Anat. und) Physiol. (1862), p. 428. 
 
 (39) Schulz, F. N., Zeit. f. physiol. Chem. 24 (1898), p. 449. 
 
 (40) Seeman, T., Arch. f. Verdanungskrankheiten. Bd. 4 (1898), cited after 
 
 Muller (18). 
 
 (41) Soldner, F., Landw. Versuchsst. 35 (1881), p. 351. 
 
 (42) Steudel, H., Zeit. f. physiol. Chem. 34 (1901), p. 353. 
 
 (43) Taylor, A. E., Univ. of California Publ. Pathol. 1 (1904), p. 7. 
 
 (44) Taylor, A. E., Journ. Biol. Chem. 1 (1906), p. 345. 
 
 (45) Van Slyke, L. L., and Hart, E. B., Amer. Chem. Journ. 33 (1905), p. 461. 
 
 (46) Van Slyke, L. L., and Van Slyke, D. D., Amer. Chem. Journ. 38 (1907), 
 
 p. 393. 
 
 (47) Zanetti, C. U., Ann. di. Chim. E. Farm. (1897), Nr. 12, cited after 
 
 Steudel (42). 
 
CHAPTER III 
 THE QUANTITATIVE ESTIMATION OF THE PROTEINS 
 
 1. The General Principles Underlying the Quantitative 
 Estimation of Proteins. The extraordinary bulk of the protein 
 molecule in comparison with the molecules of the simpler sub- 
 stances which were the first to claim investigation by analytical 
 chemists, places unusual difficulties in the way of the estimation 
 of proteins by the traditional methods of analytical chemistry. 
 Although the proteins act as acids and bases, yet their amphoteric 
 character seriously interferes with direct titration by acidimetric 
 methods, for both the acidic and basic functions of the proteins 
 are weakened and modified by their simultaneous presence in the 
 same molecule, and it is but rarely, in cases such as those afforded 
 by casein and the various members of the protamin group, that 
 the predominance of the one function is sufficiently great to 
 permit of its utilization in the determination of the protein by 
 direct titration (1) (18) (32). Even in these cases we are addi- 
 tionally hampered by the great magnitude of the combining- 
 weight of the protein, leading to a proportionate enhancement 
 of the normal margin of acidimetric inexactitude, and also by the 
 fact that many of the indicators which are customarily employed 
 in alkalimetry or acidimetry form compounds with proteins, either 
 ceasing to function as indicators, or displaying novel or abnormal 
 color-changes or even directly entering into and disturbing the 
 very equilibrium, between protein on the one hand and an acid 
 or base on the other hand, which we desire to measure. 
 
 Since the nitrogen-content of the proteins is high and nitrogen 
 is one of the elements which we are able to determine with the 
 greatest precision, nearly all the efforts of analytical chemists 
 in this field have until recently been aimed at reducing the esti- 
 mation of protein to an estimation of nitrogen. The problem 
 thus resolved itself into one of separating the protein in question 
 from other proteins and from nitrogenous contaminations. In 
 cases in which this preliminary isolation is not difficult of attain- 
 
 56 
 
QUANTITATIVE ESTIMATION 57 
 
 ment, as in the case of the casein in milk, no more accurate and 
 generally reliable method of procedure has yet been devised. 
 Unfortunately in many, if not in the majority of, instances it is 
 the fulfilment of this preliminary condition of complete separa- 
 tion from nitrogenous contaminations which presents the most 
 serious difficulties. For instance the most valuable reagent for 
 accomplishing the separation of various members of the globulin 
 group from one another and from other proteins is ammonium 
 sulphate, but the coagulum which is produced by this reagent, 
 necessarily employed in high concentration, is heavily contami- 
 nated by the ammonium salt and must be freed therefrom by 
 very prolonged dialysis before it can be utilized for a determination 
 of nitrogen. Prolonged dialysis, on the other hand, is not only 
 time-consuming but also involves the possibility of serious errors 
 arising from autohydrolysis or even hydrolysis due to enzymes 
 or to bacterial contamination, due to the protracted period during 
 which the labile protein must be exposed to the action of water. 
 Any alternative method of freeing the protein from contamina- 
 tion by ammonium salts, such as resolution followed by recoagu- 
 lation with some other reagent, is attended with many difficulties 
 and uncertainties attributable in large proportion to the modi- 
 fications of the actions of coagulating agents which are brought 
 about by the saline contamination which we desire to remove. 
 
 The high combining-weight of the proteins attributable to the 
 mass of their molecules, is, as we have seen, the chief obstacle 
 to the application of chemical methods in their quantitative 
 estimation. In the application of physical methods for this pur- 
 pose, however, the bulk of the protein molecule, far from being 
 a disadvantage, is frequently a positive advantage and may indeed 
 enable us to employ physical methods of estimation not ordi- 
 narily applicable to the smaller molecules of the majority of 
 inorganic or the simpler classes of organic substances, for the 
 great majority of the physical properties of a substance are 
 determined by the mass or volume of its molecules and those 
 properties which are magnified by increasing mass of the molecule 
 are of course displayed exceptionally prominently by the proteins. 
 
 It appears extremely probable therefore that in the future 
 we will come to rely more and more upon physical methods for 
 the estimation of the characteristically bulky molecules of col- 
 loidal substances. Many attempts have been made to estimate 
 
58 CHEMICAL STATICS 
 
 the protein-content of fluids by their optical rotation. This 
 particular physical property of the proteins was, however, a 
 most unfortunate choice, since it is one of the few physical prop- 
 erties which is independent of the absolute mass or volume of 
 the molecules displaying it, and the specific rotatory power of 
 proteins is extremely low in comparison with that of a large 
 proportion of optically active substances of much smaller molec- 
 ular dimensions. 
 
 In recent years two methods of estimating protein, based upon 
 the measurement of physical qualities of their solutions or sus- 
 pensions, have been suggested and applied to specific problems 
 in very considerable detail. These are the nephelometric method, 
 based upon measurements of opacity, and the refractometric 
 method. Both methods are capable of attaining a high degree 
 of accuracy and may be rendered to a great extent independent 
 of contamination by non-protein substances. It therefore ap- 
 pears probable that they may eventually prove applicable 
 to the estimation of a variety of proteins under conditions which 
 would preclude, or render exceedingly difficult, reduction of the 
 estimation to a determination of nitrogen. 
 
 2. The Nephelometric Method of Estimating Proteins. 
 This method, which we owe to Kober and his collaborators (6) 
 (7) (8) (9) (10), depends upon the measurement of the relative 
 opacities of suspensions of coagulated proteins, the measurement 
 of relative opacity being carried out in a modified form of color- 
 imeter. A full description of this instrument will be found in a 
 recent article by Kober and Graves (9). 
 
 The success of the nephelometric method depends upon the 
 discovery of specific coagulating agents which will bring the 
 protein into stable suspension in masses of approximately uni- 
 form, or at least reproducible, magnitude, of sufficiently large 
 diameter to scatter incident light-rays and insufficiently large to 
 result in instability of the suspension. The discovery of the most 
 suitable coagulating agent for each protein and under each set 
 of modifying circumstances constitutes, indeed, the chief diffi- 
 culty of the method, but, once the correct coagulant is found 
 and the circumstances attending its employment have been 
 standardized, the method is susceptible of a very high degree 
 of accuracy, far surpassing indeed that attainable through the 
 estimation of nitrogen. 
 
NEPHELOMETRIC METHOD 59 
 
 The coagulating reagent employed by Kober is sulpho- 
 salicylic acid. The following are the details of the method 
 as applied by Kober to the estimation of the various proteins in 
 milk (8). 
 
 Five cubic centimetres of milk are carefully measured into a 
 250-cc. graduated flask and after adding 200 cc. of distilled water 
 and 10 cc. of decinormal sodium hydroxide solution, water is 
 added to the mark and the solution is thoroughly shaken. Ten 
 cubic centimetres of this mixture is then placed, together with 
 2 cc. of ether (which has previously been washed with a 10 per 
 cent aqueous solution of sodium hydroxide) in a centrifuge tube 
 which is then tightly stoppered with a cork and vigorously shaken. 
 After allowing the mixture to stand until the layers have sepa- 
 rated, or after centrifuging for one to two minutes, the cork is 
 removed and 5 cc. of the aqueous layer is withdrawn. This is 
 done by closing the top of the pipette with the finger and insert- 
 ing it quickly into the centrifuge tube. If this is done correctly 
 the ether solution will not contaminate the sample. This 5 cc. 
 of the aqueous layer is then diluted in a volumetric flask to ex- 
 actly 50 cc. 
 
 The milk treated in this way has an almost inappreciable tur- 
 bidity, not more than 1.6 per cent of the turbidity which is sub- 
 sequently produced by the coagulating agent. Such residual 
 turbidity as it possesses is almost exactly equal to that of the 
 standard solution of casein with which it is subsequently com- 
 pared, and is therefore without influence upon the accuracy of 
 the comparison with the standard. 
 
 To 10 cc. of this solution is now added 10 cc. of a 3 per cent 
 solution of sulphosalicylic acid and the suspension of coagulated 
 milk protein which is thus obtained is matched in the nephel- 
 ometer with the following standard: One volume (5 cc.) of a 
 0.01 per cent casein solution to which is added two volumes 
 (10 cc.) of 3 per cent sulphosalicylic acid. 
 
 The volume of solution employed is not a correct aliquot of 
 the original sample of milk, owing to the fact that in the extrac- 
 tion with ether some ether is dissolved by the aqueous layer and 
 some water by the ether layer. A determination of the volume 
 increase of a solution of sodium caseinate shaken up with ether 
 shows that 10 cc. of the clear aqueous layer of defatted diluted 
 milk is equal to 9.1 cc. of the diluted milk before extraction with 
 
60 CHEMICAL STATICS 
 
 ether. Correction must be made for this change of volume in 
 the estimate of protein-content. 
 
 In order to obtain the casein-content of the milk, the casein 
 from a fresh portion of milk is precipitated in accordance with the 
 "official" method by the addition of acetic acid, and the opacity 
 of the suspension obtained in an aliquot part of the filtrate, by 
 adding four volumes of 3 per cent sulphosalicylic acid solution, 
 is determined nephelometrically. This determination yields an 
 estimate of the " globulin and albumin" fraction which, sub- 
 tracted from the " total proteins" estimated above, yields the 
 amount of casein precipitable by the " official " method. 
 
 A modification of this method of protein estimation has been 
 utilized by Pfeiffer, Kober and Field (10) in the estimation of 
 the globulins and total proteins in cerebrospinal fluid. 
 
 It will be seen that the chief difficulty attaching to the nephelo- 
 metric method arises from the necessity of achieving constant 
 conditions of coagulation, a difficulty which is especially serious 
 when the coagulating process has to be superadded to the pro- 
 cedures incident to the separation from one another of the vari- 
 ous proteins in a mixture in which it is desired to determine 
 each protein separately. This difficulty does not attach to the 
 refractometric method described below, in which, once the sepa- 
 ration of the various protein fractions has been achieved, no 
 further procedures other than dilution, etc., are necessitated 
 before a determination can be made. On the other hand, the 
 refractometric method does not permit the extreme accuracy 
 which may be attained with the nephelometric method under 
 favorable conditions. 
 
 3. The Refractometric Method of Estimating Proteins. 
 This method, originally employed by Reiss (11) (12) (13) (14) 
 (15) (16) for the estimation of the total proteins in blood-serum, 
 has been modified by the author so as to render it applicable to 
 the separate determination of the non-proteins, globulins, albu- 
 mins and total proteins in a little over 1.5 cc. of blood-serum (24). 
 The following are the details of the method : 
 
 (a) Estimation of the non-proteins. 
 
 Glass tubes 25 cm. long, having an inside diameter of 5 mm. and 
 walls about 1 mm. thick, are sealed at one end. It is well to blow 
 gently into the tube while the sealed end is still soft, thus making 
 the contour of the bottom of the tube hemispherical and dimin- 
 
REFRACTOMETRIC METHOD 61 
 
 ishing the tendency to crack on cooling. Into one of these tubes, 
 which, has been carefully cleaned and dried, is now introduced 
 exactly 1 cc. of serum with the aid of an accurately calibrated 
 pipette with a capillary tip. Such pipettes may be prepared by 
 taking lengths of narrow-bore glass tubing drawn out to a capil- 
 lary at one end, introducing 1 cc. of mercury, and marking with 
 a diamond the extremities of the mercury column. The mercury 
 is then delivered into another similar tube, which is similarly 
 marked, and the operation repeated until the desired number of 
 pipettes is obtained. Prepared in this way the pipettes all deliver 
 (between the marks) the same volume of fluid, and if this is the 
 case the exact volume employed is immaterial provided it is in 
 the neighborhood of 1 cc. 
 
 In delivering the serum, wetting the upper part of the tube and 
 the formation of air-bubbles should be carefully avoided. 
 
 The serum having been introduced, with another pipette cali- 
 brated against the first, deliver 1 cc. of N/25 acetic acid solution, 
 which may be made up with sufficient accuracy by diluting 4 cc. 
 of glacial acetic acid to 1750 cc. 
 
 A short length of thick platinum, silver, or nickel wire is now 
 introduced into the tube and the upper end is sealed off in a flame, 
 taking care not to heat the contents. After cooling, the tube is 
 shaken and the short length of wire contained in it brings about 
 a thorough admixture of the contents. The tube is now placed 
 in a beaker of cold water of such a depth as to immerse the top 
 of the contained column of liquid several millimeters below the 
 surface. It is well to rest the bottom of the tube upon a wad of 
 glass wool or a piece of wire gauze to avoid the cracking of the 
 tube by bumping during the subsequent boiling. The water is 
 now slowly heated to boiling and allowed to boil energetically 
 for exactly two minutes. The tube is then removed from the 
 boiling water and allowed to cool to room temperature. 
 
 When cool the tube is broken open a little above the surface 
 of its contents and the coagulum is broken up. This is best 
 accomplished with the aid of a platinum wire about 0.6-0.7 mm. 
 in diameter and provided with several slight bends. This is 
 inserted into the tube and the upper end twirled between the 
 thumb and forefinger. The fluid and the coagulum are now 
 separated by centrifugalization, the fluid is withdrawn by the 
 aid of a dry, clean pipette, and the refractive indices of the fluid 
 
62 CHEMICAL STATICS 
 
 and of N/5Q acetic acid solution (prepared by diluting the N/25 
 acetic acid used above to exactly one-half with distilled water) 
 are determined simultaneously. By determining the refractive 
 indices of the fluid and of JY/50 acetic acid simultaneously, the 
 necessity for regulation of the temperature at which the readings 
 are made is obviated, for although the absolute values of the 
 refractive indices are affected by temperature, the differences 
 between them are independent of the temperature at which they 
 are determined (19). In carrying out a large number of esti- 
 mations, however, it is necessary to eliminate the possible error 
 due to progressive changes in the temperature of the dark-room by 
 redetermining the refractive index of the solvent (in this instance 
 JV/50 acetic acid) at frequent intervals. 
 
 The refractive index of a 1 per cent solution of NaCl is 0.00160 
 greater than that of distilled water. The refractivities of 1 per 
 cent solutions of other inorganic salts and of glucose and urea are 
 of very similar magnitude. To within a sufficient degree of 
 accuracy, therefore, the percentage of non-protein substances 
 in the serum may be estimated by dividing their refractivity 
 by the factor 0.00160. The result must be multiplied by 2, 
 because the serum has been diluted to one-half with N/25 acetic 
 acid. 
 
 For the determination of the refractive index, I employ a 
 Pulfrich refractometer, which reads the angle of total reflection 
 to within one minute. A sodium flame is used as the source of 
 light. The refractive index may be estimated from the angle 
 of total reflection with the aid of the table which is supplied with 
 the instrument. 
 
 When it is desired to make a number of successive determina- 
 tions, the cup of the refractometer should be carefully dried with 
 absorbent cotton and lens paper before a new sample of fluid is 
 introduced. 
 
 (b) Estimation of the albumins. 
 
 Glass tubes 9-10 cm. long are prepared, having an inside diam- 
 eter of 5 mm. and one end closed. Tubes which have been em- 
 ployed in the estimation of the non-protein constituents, after 
 having been cleaned and. dried, may be cut down'to the proper 
 length and utilized for this purpose. Small corks or short pieces 
 of glass tubing sealed at one end, the sealed end being pressed 
 against the open end of the longer tubes and held in position by 
 
REFRACTOMETRIC METHOD 63 
 
 short pieces of rubber tubing, are employed as stoppers. Into 
 one of these tubes is introduced, with the aid of a pipette similar 
 to that described above, 0.5 cc. of a saturated solution of ammo- 
 nium sulphate (prepared by dissolving an excess of ammonium 
 sulphate in hot water, allowing the excess to crystallize out on 
 cooling and then removing the fluid from the subnatant crystals 
 and keeping in a well-stoppered container). With another pi- 
 pette which has been calibrated against the first, introduce 0.5 cc. 
 of serum, drop in a piece of platinum, silver or nickel wire, affix 
 the stopper and shake thoroughly with as little delay as possible. 
 It is necessary to introduce the ammonium sulphate first, as other- 
 wise, being of greater specific gravity than the serum, it sinks 
 through the serum, portions of which are thus exposed for some 
 time to ammonium sulphate of higher concentration than one- 
 half saturated. This leads to a precipitation of albumins which 
 do not readily redissolve, and the results obtained are erroneous 
 and irregular. If the ammonium sulphate is introduced first 
 the serum floats on the top of it and energetic shaking brings 
 about almost immediate admixture of the serum and the reagent. 
 It is well, while shaking, to hold the thumb against the bottom 
 of the tube, thus diminishing the danger of cracking the tube 
 by the impacts of the heavy piece of wire. 
 
 The tube, with the stopper still affixed, is now centrifuged. 
 The precipitate soon settles and sufficient supernatant fluid may 
 ,be drawn off to fill the tip of a pipette and the space between 
 two marks known to hold about 0.25 cc. (prepared as above). 
 This quantity of the supernatant fluid is delivered into another 
 clean, dry tube of the type employed in the precipitation, and 
 0.25 cc. of distilled water is added with the aid of a pipette cali- 
 brated against the first. A piece of wire is dropped in, a clean 
 stopper affixed, and the contents are shaken. We now deter- 
 mine the refractive index of this fluid and that of one-quarter 
 saturated ammonium sulphate solution prepared (and kept as a 
 stock solution) by mixing equal volumes of saturated ammonium 
 sulphate solution and distilled water and adding to this mixture 
 an equal volume of distilled water. The difference between these 
 refractive indices represents one-fourth of the combined refrac- 
 tivities of the albumins and of the non-protein constituents. 
 Multiplying by 4, therefore, and subtracting the refractivity 
 of the non-protein constituents, we have the refractivity of the 
 
64 CHEMICAL STATICS 
 
 albumins. Dividing this by 0.00177, we obtain the percentage 
 of albumin in the serum. It is necessary to dilute the ammonium 
 sulphate solution of albumins because the refractivity of proteins 
 in half -saturated ammonium sulphate solutions is abnormal (21). 
 
 (c) Estimation of the globulins. 
 
 Determine the refractive index of the serum and that of dis- 
 tilled water. Subtracting from the difference the known refrac- 
 tivity of the non-proteins and the known refractivity of the 
 albumins, we obtain the refractivity of the globulins. Dividing 
 this by 0.00229 we obtain the percentage of globulin in the serum. 
 Adding together the percentages of albumins and globulins we 
 obtain the percentage of total proteins. 
 
 The following is an illustrative determination, employing ox- 
 serum obtained by whipping and centrifuging freshly-drawn 
 blood : 
 
 Refractive index of the fluid obtained after acidifying with 
 
 acetic acid and boiling (3 determinations) 1 . 33024 
 
 Refractive index of AT/50 acetic acid 1 . 32902 
 
 Difference X 2 0.00244 
 
 Estimated concentration of non-proteins = fff = 1.5 per cent. 
 
 Refractive index of the fluid obtained after precipitation of the globulins 
 and subsequent dilution: 
 
 1st determination 1 . 35189 
 
 2nd determination 1 . 35172 
 
 3rd determination 1 . 35181 
 
 4th determination 1 . 35189 
 
 Refractive index of | saturated (NH 4 ) 2 SO 4 1 . 34932 
 
 Differences X 4: 
 
 1st determination 0.01028 
 
 2nd determination 0.00960 
 
 3rd determination 0.00996 
 
 4th determination 0.01028 
 
 Refractivity of the albumins: 
 
 1st determination 0.01028 - 0.00244 = 0.00784 
 2nd determination 0.00960 - 0.00244 = 0.00716 
 3rd determination 0.00096 - 0.00244 = 0.00752 
 4th determination 0.01028 - 0.00244 = 0.00784 
 Average 0.00759 
 
 Concentration of albumins: 
 
 1st determination = ^ = 4.4 per cent 
 2nd determination = rf = 4.1 per cent 
 3rd determination = ff =4.3 per cent 
 4th determination = ty$ = 4.4 per cent 
 Average 4 . 3 per cent 
 
REFRACTOMETRIC METHOD 65 
 
 Refractive index of the serum 1 . 34686 
 
 Refractive index of distilled water 1 . 32887 
 
 Difference 0.01799 
 
 Refractivity of the proteins = 0.01799 - 0.00244 = 0.01555 
 Refractivity of the globulins = 0.01555 - 0.00759 = 0.00796 
 Concentration of globulins = Iff = 3.5 per cent 
 Concentration of total proteins = 3.5 + 4.3 = 7.8 per cent 
 
 Since the percentage of total proteins is dependent upon the 
 dilution of the blood and is therefore highly variable, while the 
 relative proportions of globulin and albumin are much more 
 constant, it is convenient to express these in percentages of the 
 total proteins. Thus in the above result, taking the total pro- 
 teins as 100, we find that the globulins formed 45 per cent and 
 the albumins 55 per cent of the total proteins. 
 
 This method has been applied to the study of the proteins in 
 human blood-serum under normal conditions and a variety of 
 disease-conditions by A. H. Rowe (25) (26) (27) (31). It has 
 also been applied to the study of the changes in the ratio of 
 globulin to albumin in blood-serum during immunization and 
 infection by Righetti (17), Hurwitz and Meyer (4) and Schmidt 
 (28). The globulins, albumins and non-proteins in the blood- 
 sera of a variety of mammals and birds have also been estimated 
 refractometrically and characteristic differences found in the 
 relative proportions of these constituents in widely-separated 
 classes of animals (2) (3) (5) (23) (30) (33) (34). 
 
 The refractometric method has also been applied to the esti- 
 mation of casein in milk (20) and to the estimation of the diges- 
 tive activity of proteolytic ferments (22) (29). 
 
 LITERATURE CITED 
 
 (1) Arny, H. V., and Pratt, T. M., Amer. Journ. of Pharmacy 78 (1906), 
 
 p. 121. 
 
 (2) Briggs, R. S., Journ. Biol. Chem. 20 (1914), p. 7. 
 
 (3) Buck, L. W., Journ. of Pharmacology and Exper. Therap. 5 (1913), 
 
 p. 553. 
 
 (4) Hurwitz, S. H., and Meyer, K. F., Journ. Exper. Med. 24 (1916), p. 
 
 515. 
 
 (5) Jewett, R. M., Journ. Biol. Chem. 25 (1916), p. 21. 
 
 (6) Kober, P. A., Journ. Biol. Chem. 13 (1912), p. 485. 
 
 (7) Kober, P. A., Journ. Amer. Chem. Soc. 35 (1913), p. 290. 
 
 (8) Kober, P. A., Journ. Amer. Chem. Soc. 35 (1913), p. 1585. 
 
66 CHEMICAL STATICS 
 
 (9) Kober, P. A., and Graves, S. S., Journ. Indust. and Engineering Chem. 
 7 (1915), p. 843. 
 
 (10) Pfeiffer, J. A. F., Kober, P. A., and Field, C. W., Proc. Soc. Exper. Biol. 
 
 and Med. 12 (1915), p. 153. 
 
 (11) Reiss, E., Beitr. z. Chem. Physiol. und Pathol. 4 (1904), p. 150. 
 
 (12) Reiss, E., Arch. f. exper. Path, und Pharm. 51 (1904), p. 18. 
 
 (13) Reiss, E., Miinchener Med. Wochenschr. 55 (1908), p. 1853. 
 
 (14) Reiss, E., Jahrb. f. Kinderheilkunde 70 (1909), p. 174. 
 
 (15) Reiss, E., Ergeb. d. inn. Med. und Kinderh.10 (1913), p. 531. 
 
 (16) Reiss, E., Deutsch. Arch. f. Klin. Med. 117 (1915), p. 175. 
 
 (17) Righetti, H., University of California Publ. Pathol. 2 (1916), p. 205. 
 
 (18) Robertson, T. Brailsford, Journ. Biol. Chem. 2 (1907), p. 371. 
 
 (19) Robertson, T. Brailsford, Journ. Physical Chem. 13 (1909), p. 469. 
 
 (20) Robertson, T. Brailsford, Journ. Indust. and Engineering Chem. 1 
 
 (1909), p. 723. 
 
 (21) Robertson, T. Brailsford, Journ. Biol. Chem. 11 (1912), p. 179. 
 
 (22) Robertson, T. Brailsford, Journ. Biol. Chem. 12 (1912), p. 23. 
 
 (23) Robertson, T. Brailsford, Journ. Biol. Chem. 13 (1912), p. 325. 
 
 (24) Robertson, T. Brailsford, Journ. Biol. Chem. 22 (1915), p. 233. 
 
 (25) Rowe, A. H., Journ. of Laboratory and Clin. Med. 1 (1916), p. 439. 
 
 (26) Rowe, A. H., Arch. Int. Med. 18 (1916), p. 455. 
 
 (27) Rowe, A. H., Arch. Int. Med. 19 (1917), p. 354. 
 
 (28) Schmidt, E. S., and C. L. A., Journ. of Immunology 2 (1917), p. 343. 
 
 (29) Schorer, G., "Ueber refraktometrische Pepsinbestimmungen " aus der 
 
 med. Klinik der Universitat Bern (1908). 
 
 (30) Thompson, W. B., Journ. Biol. Chem. 20 (1914), p. 1. 
 
 (31) Tranter, C. L., and Rowe, A. H., Journ. Amer. Med. Assn. 65 (1915), 
 
 p. 1433. 
 
 (32) Van Slyke, L. L., and Bosworth, A. W., Journ. Indust. and Engineering 
 
 Chem. 1 (1909), p. 768. 
 
 (33) Wells, C. E,, Journ. Biol. Chem., 15 (1913), p. 37. 
 
 (34) Woolsey, J. H., Journ. Biol. Chem. 14 (1913), p. 433. 
 
CHAPTER IV 
 THE COMPOUNDS OF THE PROTEINS 
 
 1. The Amphoteric Character of the Proteins. The fact that 
 one and the same protein can combine either with a base or with 
 an acid appears to have been first clearly stated by Platner (33) 
 in 1866. The term "amphoteric" which is now used to desig- 
 nate substances possessing this power is due to Bredig (4). 
 
 The source of this dual combining-capacity of the proteins 
 has been discussed in Chap. I. In 1886, Strecker (51) pointed 
 out that amino-acids can, like other substituted ammonias, bind 
 acids by their NH 2 groups, or, as we now know, through the trans- 
 formation of trivalent into pentavalent nitrogen; while they can 
 also bind bases in virtue of their possession of a COOH group. 
 It is probable that in most cases a proportion of the amino- 
 acid, when dissolved in water, combines with water to form a 
 compound analogous to ammonium hydrate, which is in chemi- 
 cal equilibrium with the unhydrated form also present in the 
 solution. Thus amino-acetic acid, 
 
 NH 2 
 
 COOH 
 
 when dissolved in water, is believed to partially undergo the 
 reaction 
 
 /NH 2 /NH 3 .OH 
 
 CH>' + H.OH = CH</ 
 
 X COOH X COOH 
 
 the trivalent nitrogen becoming pentavalent, and the molecule 
 splitting off both hydrogen and hydroxyl ions in definite pro- 
 portions. As I have stated, however, the transformation is not 
 complete, nor is it by any means an easy matter, as a rule, to 
 determine its extent (25) (39). 
 
 Nevertheless, as I have pointed out in Chap. I and as will be 
 more clearly revealed in the chapters dealing with the electro- 
 chemistry of the proteins, it is very improbable that terminal 
 NH 3 OH or COOH groups in the proteins molecule are involved 
 
 67 
 
68 CHEMICAL STATICS 
 
 in the union of the majority of proteins with inorganic acids and 
 bases. The active agents in accomplishing these unions are, 
 more probably, COH.N groups within the protein molecule. 
 
 Whatever may be the method of union between inorganic sub- 
 stances and the proteins, however, there is now no longer any 
 doubt that it is of very general occurrence. The methods which 
 have been employed by various investigators to demonstrate 
 the existence of compounds of the proteins with inorganic sub- 
 stances are very diverse in nature, but they may be conveniently 
 classed as direct and indirect. The direct methods consist in the 
 precipitation of the (supposedly) unaltered compound by appro- 
 priate reagents (in the case of compounds such as those with the 
 heavy metals, which are insoluble in water, addition of a reagent 
 is of course unnecessary), followed by the elementary analysis 
 of the precipitate, or in the analysis of soluble compounds of 
 proteins with substances which, when uncombined, are insoluble 
 in water. The indirect methods may be variously designated: 
 the indirect method of precipitation, the method of electrical 
 conductivity, the cryoscopic method, the potentiometric method, 
 the method of catalysis, the indicator method and the method 
 of " masking" the physiological effects of inorganic substances 
 by the addition of proteins to their solutions. The results ob- 
 tained by these methods will be considered separately. 
 
 2. The Direct Method of Demonstrating the Existence of 
 Protein Compounds by Precipitation or Coagulation. This, 
 which is the method most frequently employed, consists, usually, 
 in precipitating or coagulating the protein salt by the addition 
 of suitable reagents, the reagent which is most commonly em- 
 ployed being alcohol. 
 
 The very important question immediately suggests itself, 
 whether or not the reagent which is employed for this purpose 
 precipitates the protein compound unaltered, as it exists in the 
 solution, or whether the compound is altered in the process of 
 precipitation. On the whole it may be said that too little atten- 
 tion has been given to this question by investigators who have 
 employed this method and that in consequence we possess very 
 little accurate information on the subject. 
 
 It appears, however, that alcohol may be relied upon, at all 
 events within certain limits, to precipitate the salts of some 
 proteins with bases in an unaltered condition, that is, containing 
 
DIRECT METHOD OF PRECIPITATION 69 
 
 the same relative proportion of base to protein as the pre-existing 
 compound in aqueous solution. Thus Van Slyke and Hart (52) 
 employed alcohol to precipitate calcium caseinate, and they 
 found that the precipitate from solutions neutral to litmus con- 
 tained exactly the quantity of calcium (uncombined with hydro- 
 chloric acid) which corresponds with the quantity of calcium 
 hydrate which casein will neutralize to litmus while in aqueous 
 solution. Similarly the precipitate from solutions neutral to 
 phenolphthalein contained the calculated quantity of calcium. 
 As we shall see in considering the electrochemical phenomena 
 which accompany the coagulation of the caseinates by alcohol, 
 coagulation of these salts through the addition of alcohol to their 
 aqueous solutions is preceded by a profound decrease in their 
 degree of dissociation (43); hence the caseinate is precipitated 
 in combination with the proportion of base which was bound 
 up by it in its aqueous solution, because, immediately preceding 
 precipitation, the combined base is bound up in an undissociated 
 molecule. 
 
 It has also been shown that serum-globulin is precipitated 
 from serum by alcohol in the form of a salt with a base, and not 
 in the form of the free protein, since the mixed proteins which 
 may thus be precipitated are completely soluble in distilled water, 
 and this solution yields a precipitate on passing CO 2 through it 
 (42). 
 
 It would not be safe to assume, however, without special in- 
 vestigation, that alcohol precipitates all protein salts without 
 alteration of the relative proportion of base or acid to protein 
 which subsists in these compounds as they exist in aqueous 
 solution. So far this can only be positively affirmed for the 
 "neutral" and "basic" casemates, that is, the caseinates the 
 solutions of which are respectively neutral to litmus and to 
 phenolphthalein. 
 
 The influence of other coagulating agents upon the composition 
 of the protein salts which they precipitate is even more uncertain 
 than that of alcohol, and in one case at all events, namely that 
 of the precipitation of sodium caseinate from solutions containing 
 a considerable excess of sodium hydrate by means of ammonium 
 sulphate, we are in possession of definite evidence that the act 
 of coagulation is accompanied by a change in the composition 
 of the protein salts. 
 
70 
 
 CHEMICAL STATICS 
 
 Spiro and Pemsel (49) employed ammonium sulphate to precipi- 
 tate protein salts; their procedure was as follows: A given weight 
 of protein was dissolved in a measured quantity of standardized 
 NaOH solution, and the protein-base compound was then precipi- 
 tated by the addition of a suitable excess of a saturated solution 
 of ammonium sulphate. The quantity of alkali bound by the 
 protein and precipitated with it was then determined by titration 
 of the filtrate. The following were their results, employing casein: 
 
 Solution 
 
 Mg of NaOH pre- 
 cipitated with 1 
 gram of casein 
 
 Equivalent grm. mol. 
 of NaOH precipitated 
 with 1 gram casein 
 
 1.14 grams casein in 10 cc. N/5 NaOH. . 
 1.41 grams casein in 15 cc. N/5 NaOH. . 
 1.24 grams casein in 20 cc. N/5 NaOH. . 
 1.31 grams casein in 30 cc. N/5 NaOH. . 
 
 26.1 
 29.2 
 33.1 
 34.3 
 
 65 X 10- 6 
 73 X 10~ 5 
 82 X 10~ 5 
 36 X 10~ 5 
 
 Now I have shown (40) employing the potentiometric method 
 (vide infra), which is a static measurement, not involving any 
 change in the dynamical condition or composition of the body 
 of the solution, that in all of the above solutions investigated by 
 Spiro and Pemsel the amount of alkali which was actually bound 
 by one gram of casein while in solution must have been at 
 least 160 X 10~ 5 gram equivalents. Hence it is clear that in 
 the act of coagulation by ammonium sulphate these salts of casein 
 are very materially altered in composition. 
 
 The method of coagulation by heat has been comparatively 
 little employed for the investigation of protein compounds with 
 acids. It cannot be employed for the investigation of compounds 
 of proteins with bases because although proteins in alkaline 
 solution are denatured by heat the denatured protein is only 
 flocculated in an acid medium (6). It has been shown by Chick 
 and Martin that in acid media the protein coagulum binds a 
 certain proportion of the acid, but whether or not the protein 
 salt, as it exists in the solution prior to coagulation, is flocculated 
 without alteration, cannot at present be definitely stated. 
 
 At the present stage of our knowledge therefore, the direct 
 method of demonstrating the existence of protein compounds 
 cannot be trusted to yield accurate quantitative data, and re- 
 sults and conclusions based upon this method are to be accepted 
 with the greatest caution. 
 
INDIRECT METHOD OF PRECIPITATION 71 
 
 3. The Direct Method of Demonstrating the Existence of 
 Protein Compounds by the Solution of Otherwise Insoluble 
 Substances. This method has been employed by W. A. 
 Osborne (30) who has shown that casein, when triturated with 
 calcium carbonate, carries a definite proportion of the calcium 
 into solution in the form of the soluble calcium caseinate with 
 the evolution of COg. Strychnin is similarly carried into solu- 
 tion by casein. 
 
 Osborne and Leavenworth (31) have shown that edestin will 
 hold in solution a quantity of cupric hydroxide corresponding 
 to no less than 34.67 per cent of its weight of copper, which, 
 assuming that each atom of copper is united to one nitrogen 
 atom, implies that no less than ten out of every sixteen nitrogen 
 atoms contained in the edestin molecule participate in forming 
 the compound with cupric hydroxide. This is exactly the pro- 
 portion of nitrogen atoms which is present in edestin in the 
 form of COHN groups. Precisely similar results were ob- 
 tained with gliadin. 
 
 4. The Indirect Method of Precipitation. Cohnheim and 
 Krieger (9) have elaborated an ingenious method of determining 
 the acid-binding capacity of the proteins which is based upon 
 the fact that phosphotungstic acid forms insoluble salts with 
 proteins. If the protein is combined with an acid, and calcium 
 phosphotungstate is employed, double decomposition takes place 
 as follows: 
 
 Protein hydrochloride -f- calcium phosphotungstate 
 
 (soluble) (soluble) 
 
 = protein phosphotungstate -+- CaC^ 
 
 (insoluble) 
 
 hence the quantity of calcium chloride present in the filtrate, 
 after filtering off the precipitate and carefully washing it (34) 
 is taken as a measure of the quantity of hydrochloric acid which 
 was bound by the protein just before precipitation. The pro- 
 cedure is as follows: Weighed amounts of protein are dissolved 
 in measured volumes of acid of known concentration. The 
 protein is then precipitated by the addition of an excess of the 
 phosphotungstate and the filtrate, plus the washings of the pre- 
 cipitate, is titrated against standard alkali. An excess of the 
 phosphotungstate is stated to be necessary because the phospho- 
 tungstate of protein is believed to undergo extensive hydrolytic 
 
72 
 
 CHEMICAL STATICS 
 
 dissociation so that it is stable, and precipitation is complete, 
 only in the presence of an excess of the reagent. 
 
 It is obvious that this method is open to all of the objections 
 attaching to the direct method of precipitation considered above. 
 In our present stage of knowledge we are very much in the dark 
 concerning the actual chemical mechanism of the precipitation 
 of proteins by reagents such as phosphotungstic acid and we are 
 by no means certain, indeed it is highly improbable, that only 
 one compound of a given protein with phosphotungstic acid 
 exists or that its composition is independent of the excess of 
 phosphotungstic acid employed. Hence it is not certain that in 
 the substitution of phosphotungstic acid for hydrochloric acid, 
 under the conditions outlined above, one equivalent of phospho- 
 tungstic acid replaces one or even a constant number of equiva- 
 lents of hydrochloric acid. Results obtained by this method are 
 at present, therefore, only of qualitative value. 
 
 The experiments of Erb (11), who employed this method, 
 must nevertheless be quoted here in order to point out a mis- 
 conception which his interpretation of the results involves. One 
 cubic centimetre of a 5 per cent watery solution of vitellin was 
 mixed with 1, 2, 3, etc., cubic centimetres of normal HC1 and the 
 volume of each solution was made up to 10 cc. To these were 
 added a constant excess of calcium phosphotungstate, the pre- 
 cipitate was filtered off and washed, and the filtrate ti'trated. 
 The following is the way in which Erb expresses his results : 
 
 Cc. of normal 
 HC1 added 
 
 Cc. AT/10 NaOH re- 
 quired to neutral- 
 ize the filtrate 
 
 HCl bound 
 in cc. AT/10 
 HCl 
 
 Theoretical 
 excess of N/10 
 HCl 
 
 Hydrolytic 
 dissociation 
 per cent 
 
 1 gram 
 protein binds 
 mg. of HCl 
 
 0.1 
 
 0.1 
 
 0.9 
 
 -1.9 
 
 69.0 
 
 63 
 
 0.2 
 
 0.8 
 
 1.2 
 
 -0.9 
 
 59.6 
 
 87 
 
 0.3 
 
 1.5 
 
 1.5 
 
 0.1 
 
 48.3 
 
 108 
 
 0.5 
 
 3.0 
 
 2.0 
 
 2.1 
 
 31.0 
 
 146 
 
 0.7 
 
 4.5 
 
 2.5 
 
 4.1 
 
 13.7 
 
 183 
 
 0.9 
 
 6.2 
 
 2.8 
 
 6.1 
 
 3.4 
 
 204 
 
 1.0 
 
 7.2 
 
 2.8 
 
 7.1 
 
 3.4 
 
 204 
 
 1.5 
 
 12.1 
 
 2.9 
 
 12.1 
 
 0.0 
 
 212 
 
 It will be seen that the -quantity of HCl which is bound by 
 one gram of vitellin increases with increasing concentration of 
 HCl in its solution. We shall see that this phenomenon is a 
 general one where proteins are concerned. It appears that the 
 
INDIRECT METHOD OF PRECIPITATION 73 
 
 binding-capacity of the vitellin reaches a maximum in the higher 
 concentrations of acid. Assuming that this is the true binding- 
 capacity of vitellin but that the compound which is formed under- 
 goes hydrolytic dissociation according to the balanced equation: 
 
 Protein hydrochloride + water + Protein + HC1. 
 
 Erb calculates, in the fourth and fifth columns of the above 
 table, respectively, the " theoretical excess" of HC1 (unbound) 
 and the percentage hydrolytic dissociation of the protein salt in 
 each mixture. This assumption is, however, unquestionably 
 incorrect. The acid- or base-combining capacity of the proteins 
 is a function of the concentration of acid or base in their solution 
 and not independent of it, as it is in the case of a strong mono- 
 basic acid which is incapable of undergoing polymerization. This 
 is very readily shown in the case of casein, which is insoluble 
 when uncombined and which would therefore be precipitated if 
 its salts underwent appreciable hydrolytic dissociation. If the 
 alkali-binding capacity of casein were independent of the con- 
 centration of alkali in its solution, therefore, the addition of any 
 quantity of alkali beyond that necessary to carry the casein into 
 solution should be without effect upon the number of equivalents 
 of alkali neutralized by one gram of casein. As one increases 
 the amount of alkali in its solution, however, the alkali-binding 
 capacity of casein, as estimated by the potentiometric method, 
 increases from 11.4 X 10~ 5 equivalents per gram to 180 X 10~ 5 
 equivalents per gram, or 16 times the amount of alkali neces- 
 sary to carry it into solution (40). The alkali-binding capacity 
 of casein is therefore not a constant in the presence of varying 
 amounts of alkali. The same considerations apply, of course, 
 to the varying combining-capacity of serum globulin in the 
 presence of a varying excess of acid or alkali (39). 
 
 In the case of proteins which are soluble in water when un- 
 combined with acids or with bases the demonstration of the fact 
 that their combining-capacities vary with the quantity of acid 
 or alkali in their solutions is not such a simple matter, but it is 
 not difficult to infer, from data to which reference will be made 
 in succeeding chapters, that such variations in combining-capac- 
 ity are an essential feature of the behavior of these proteins also. 
 
 It is obvious that almost any sufficiently neutral salt of an 
 acid which forms insoluble compounds with the proteins might 
 
74 
 
 CHEMICAL STATICS 
 
 be employed in investigations such as those cited above, in the 
 place of calcium phosphotungstate. von Rhorer has employed 
 potassium mercury iodide (K 2 HgI 4 ) and calcium picrate. 
 
 5. The Method of Electrical Conductivity. This method is 
 based upon the fact that the salts of the proteins are, in solution, 
 less highly ionized than the majority of the strong inorganic 
 acids or bases in equivalent concentration. Hence on adding 
 proteins to a solution of one of these latter, the total number 
 of ions per liter of the solution is diminished. Moreover, the 
 protein ions which are formed have a low velocity of migration 
 (16) (37) (38) (40) (41) as might be anticipated, having regard 
 to the magnitude of their mass and volume. Hence, on adding 
 protein to a solution of a strong acid or of a strong base, the 
 conductivity of the solution is diminished and this diminution 
 affords a measure of, although it is not directly proportional 
 to, the quantity of acid or base bound by the protein. This 
 method was first employed in a systematic manner by Sjoqvist 
 (47) . The following figures are illustrative of his results : 
 
 
 Egg albumin dissolved in 0.025 N HCl. 
 
 Grams albumin 
 in 100 cc 
 Mol. Cond. X10 7 . 
 
 
 
 340.5 
 
 0.53 
 271 
 
 1.06 
 207 
 
 1.59 
 151 
 
 2.13 
 108 
 
 2.55 
 
 86 
 
 3.19 
 71.5 
 
 4.25 
 67 
 
 5.23 
 
 67 
 
 6.38 
 68 
 
 The molecular conductivities were calculated for a 0.025 m. 
 HCl solution. It will be observed that after a certain quantity 
 of albumin has been dissolved in the acid (4.25 per cent) the 
 molecular conductivity of the solution approaches constancy, 
 indicating that all of the acid is bound by the protein; the infer- 
 ence being that the conductivity thereafter measured is that of 
 the protein hydrochloride alone, the addition of further albumin 
 contributing but little to the conductivity of the solution, since 
 free, unneutralized protein is but sparingly ionized. 
 
 Details of more recent results obtained by this method and 
 their interpretation will be found in the chapters dealing with 
 the electrochemistry of the proteins. Since this measurement 
 is a static one, the objections which apply to the two methods 
 previously described do not apply to it. 
 
 6. The Cryoscopic Method. This method depends upon the 
 diminution in the total ionic + molecular concentration in solu- 
 
CRYOSCOPIC METHOD 75 
 
 tions of strong acids or bases, upon the addition to them of pro- 
 teins. As in the previous method, the measurement is a static 
 one, with this difference, however, that the measurement is 
 necessarily conducted at or in the neighborhood of C. and 
 that the dynamical equilibrium of the system under investigation 
 is shifted to the equilibrium pertaining at this temperature. 
 Regarding the magnitude of this shift for any given temperature 
 range we have but meagre data, but in the light of results ob- 
 tained with the caseinates and with the salts of ovomucoid (Chap. 
 IX) it is probably in these instances and for the temperature- 
 range 30-0 C., not greater than the experimental error of the 
 method. 
 
 The cryoscopic method was first employed, for this purpose, 
 by Bugarzsky and Liebermann (5), who found that upon adding 
 6.4 grams of egg albumin :to 100 cc. of 0.05 N HC1 or NaOH 
 the difference between the freezing-point of the solution and that 
 of water is reduced nearly 50 per cent, indicating a diminution 
 by nearly 50 per cent of the total number of ions plus molecules 
 per liter of the solution. Upon adding similar quantities of 
 protein to solutions of sodium chloride, little or no alteration in 
 the freezing-point of the solutions could be detected. From 
 these results it is usually inferred that the proteins do not bind 
 neutral salts. This, however, is not an altogether justifiable 
 inference. Hardy (loc. cit.) has detected a slight, but what he 
 considers unmistakable, depression in the electrical conductivities 
 of salt solutions upon saturation with serum globulin, and he 
 assumes that a compound with the neutral salt is actually formed, 
 but that it is only stable in the presence of excess of the salt, so 
 that at any given salt-concentration only a small proportion of 
 the salt is held in combination. Mellanby (28) has arrived at 
 similar conclusions. This possibility becomes more plausible 
 when we view it in the light of the Guldberg-Waage mass law. 
 Representing the reaction between protein and NaCl thus: 
 
 Protein + NaCl <= Protein NaCl Compound, 
 
 we may suppose that the station of equilibrium lies far to the 
 right. An excess of NaCl would displace this equilibrium towards 
 the left and a greater proportion of the protein would exist in 
 the solution in the form of the protein-salt compound, i.e., the 
 compound would be more "stable." Turning now, to the above- 
 
76 
 
 CHEMICAL STATICS 
 
 quoted results of Bugarzsky and Liebermann, it is evident that, 
 save in the presence of a considerable excess of neutral salts, 
 little or no alteration in the freezing-point of the solution would 
 result from the addition to it of protein, since the depression of 
 conductivity observed by Hardy only amounted to 2 per cent 
 of that of a N/ 10 solution, and the cryoscopic method is not 
 sufficiently sensitive to reveal, with certainty, such slight varia- 
 tions in the freezing-point of solutions. For this purpose the 
 method of electrical conductivity is much to be preferred. An 
 alternative interpretation of these results will be found in Chap. VI. 
 
 7. The Potentiometric Method. This method is also based 
 upon the fact that the addition of protein to a solution of a strong 
 base or acid diminishes the concentrations of the ions of the 
 inorganic constituent of the mixture. As in the previous methods, 
 the measurement is static. But, instead of involving the meas- 
 urement of the total ionic or ionic plus molecular concentration 
 of the mixture this method enables us to directly determine the 
 concentration of a given ion. The results are therefore much 
 more readily interpreted than those obtained by the two methods 
 previously described and, indeed, furnish us with a direct meas- 
 ure of the quantity of a (highly dissociated) base or acid which 
 is actually bound in the solution, by the protein. For a brief 
 description of the principles underlying potentiometric measure- 
 ments in concentration-chains the reader is referred to the appen- 
 dix; for fuller details to such works as those of Hamburger (15) 
 and Michaelis (29).* 
 
 The potentiometric method was first employed for this pur- 
 pose by Bugarzsky and Liebermann (5). These investigators 
 employed two different concentration-chains. The one, the 
 mercury-chain, was built up as follows: 
 
 Hg 
 
 HgCl (solid), HC1 
 
 NaCl 
 
 NaBr, HgBr (solid) 
 
 Hg 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 In the first instance the potential of the chain for a certain con- 
 centration of HCi in 2 was measured, then a weighed amount 
 
 * For the modifications in technique imposed by the presence of proteins 
 in the solutions under investigation vide Appendix. 
 
POTENTIOMETRIC METHOD 
 
 77 
 
 of protein was introduced into 2 and the potential again deter- 
 mined. The difference between the two readings, in accordance 
 with the Nernst formula, affords a measure of the number of 
 Cl ions bound by the protein. The other concentration-cell 
 was the ordinary gas-chain. 
 
 Pt saturated with H 2 
 
 Acid 
 
 Alkali 
 
 Pt saturated with H 2 
 
 1 
 
 2 
 
 3 
 
 4 
 
 As before, the potential of the chain was first measured for a 
 known concentration of acid in 2, then a weighed amount of 
 protein was added to the acid and the potential of the chain was 
 again determined, the difference between the two readings afford- 
 ing, in this case, a measure of the diminution in the number of 
 H ions due to the introduction of the protein. When it is desired 
 to determine the base-binding capacity of the protein, the latter 
 is added to the alkali in 3, instead of to the acid in 2. 
 
 The following are representative of the results obtained by 
 Bugarzsky and Liebermann. In the tables G signifies the number 
 of grams of protein in "100 cc. of solution, p the percentage of 
 acid or base bound by the protein, and r the ratio. 
 
 grams of acid or base bound 
 grams of protein 
 
 I. GAS-CHAIN. EGG ALBUMIN DISSOLVED IN 0.05 N HCI 
 
 G 
 
 p 
 
 r 
 
 G 
 
 P 
 
 r 
 
 
 
 0.4 
 0.8 
 1.6 
 
 
 9.0 
 18.9 
 33.3 
 
 0'042" 
 0.044 
 0.038 
 
 3.2 
 6.4 
 12.8 
 
 60.2 
 96.6 
 99.7 
 
 0.034 
 0.027 
 0.014 
 
 
 
 
 
 
 
 II. MERCURY-CHAIN. EGG ALBUMIN DISSOLVED IN 0.05 N HCI 
 
 G 
 
 p 
 
 r 
 
 G 
 
 p 
 
 r 
 
 
 
 
 
 
 1.6 
 
 38.0 
 
 0.044 
 
 0.4 
 
 10.7 
 
 0.048 
 
 3.2 
 
 64.0 
 
 0.037 
 
 0.8 
 
 20.2 
 
 0.046 
 
 6.4 
 
 76.0 
 
 0.022 
 
78 
 
 CHEMICAL STATICS 
 
 III. GAS-CHAIN. EGG ALBUMIN DISSOLVED IN 0.05 N NaOH 
 
 G 
 
 p 
 
 r 
 
 G 
 
 p 
 
 r 
 
 
 
 
 
 
 
 
 3.2 
 
 60.2 
 
 0.037 
 
 0.8 
 
 14.4 
 
 0.035 
 
 6.4 
 
 97.0 
 
 0.030 
 
 1.6 
 
 27.4 
 
 0.034 
 
 12.8 
 
 99.9 
 
 0.016 
 
 It will be seen that as the concentration of protein rises, i.e., 
 as the proportion of base or acid to protein diminishes, the base- 
 or acid-binding capacity of the protein diminishes. We have 
 already had occasion to note this fact in connection with the 
 results of Erb (11). 
 
 Employing a mercury chain containing the element (HgCl 
 (solid), NaCl) instead of the element (HgCl (solid), HC1) used 
 above, and adding to the NaCl solution in this element varying 
 quantities of albumin, the following results were obtained: 
 
 IV. MERCURY-CHAIN. EGG ALBUMIN DISSOLVED IN 0.05 N NaCl 
 
 G 
 
 E (electromotive force 
 of chain) 
 
 G 
 
 E (electromotive force 
 of chain) 
 
 
 
 0.4 
 0.8 
 
 0.1287 
 0.1280 
 0.1279 
 
 1.6 
 3.2 
 6.4 
 
 0.1278 
 0.1281 
 0.1275 
 
 It will be seen that the electromotive force of the chain is but 
 little affected by the addition of egg albumin to the element 
 containing NaCl. 
 
 The method employed by Bugarzsky and Liebermann involved, 
 as we have seen, a direct comparison of the ionic concentrations 
 in the protein solution and in the solvent, respectively. This 
 comparison may be avoided and the existence of a variety of 
 protein compounds inferred from the changes in curvature of 
 the curve of ionic concentration consequent upon the admixture 
 with the protein solution of varying proportions of the substance 
 with which compounds are formed. This method, which is 
 essentially the same as that now widely employed in various 
 branches of analytical chemistry (3) (46) (18) (19), was first 
 applied to protein solutions by D'Agostino and Quagliariello 
 (10) and has recently been extensively employed by C. L. A. 
 
METHOD OF CATALYSIS 79 
 
 Schmidt (45) who, with its aid, has been able to sharply dis- 
 tinguish between the precipitation of a protein through the 
 formation of a true, insoluble compound, and flocculation of the 
 protein through alteration of its physical condition or degree 
 of hydration; the so-called " salting-out" effect. 
 
 The details of recent investigations by the potentiometric 
 method and their interpretation will be found in the chapters 
 dealing with the electrochemistry of the proteins. 
 
 8. The Method of Catalysis. In this method, as in the 
 above, direct measurements of the number of hydrogen or 
 hydroxyl ions bound by the protein are secured. The measure- 
 ment is not, however, altogether a static one, since a foreign 
 substance is added to the solution of the protein, and the rate 
 of change of this substance is the quantity actually measured. 
 When the change in this substance leads to the setting free of 
 an acid (as, for example, in the catalysis of methyl acetate) the 
 method is open to serious objection. 
 
 As is well known, many reactions are accelerated by hydroxyl 
 and hydrogen ions, and the velocity-constants of these reactions 
 (such as the inversion of sugar by acids, the saponification of 
 methyl acetate by alkalies, etc.) are directly proportional to the 
 concentration of the hydroxyl or hydrogen ions in the reacting 
 system. If, therefore, we act upon such a mixture with a given 
 concentration of an acid, or of a base, as the case may be, and 
 observe the velocity of the transformation, and, if we then act 
 upon a similar mixture with the same quantity of acid, to 
 which, however, a weighed amount of protein has been added, 
 and again determine the velocity of the transformation, the 
 difference between the two velocities affords a measure of the 
 numbers of H ions (or of OH ions, as the case may be) which 
 have been bound through the introduction of the protein. This 
 method was employed by Cohnheim (8) who states that it was 
 first used for this purpose by Hoffman, at the suggestion of 
 Wilh. Ostwald. 
 
 As a reaction which is accelerated by H + ions Cohnheun em- 
 ployed the inversion of cane sugar. This reaction, in the presence 
 of free acid, obeys the monomolecular formula 
 
80 CHEMICAL STATICS 
 
 where A is the initial concentration of the cane-sugar, X is the 
 amount inverted after a time t, and k is a constant (at constant 
 temperature) which varies directly as the hydrion concentration 
 of the solution. The period during which the reaction proceeded 
 was constant (4 hours) ; hence, if X be the amount of sugar trans- 
 formed after the addition to its solution of a measured volume 
 of HCl-solution, and X' that after the addition to the same con- 
 centration of cane-sugar of the same volume of the HCl-solution, 
 to which, however, protein has been added then: 
 
 C log A -log (A -30 
 C' logA-log(A-JT)' 
 
 where C is the concentration of hydrions in the pure acid, and 
 C" that in the acid to which protein has been added. Knowing 
 the value of C that of C" can be immediately calculated. From 
 his results Cohnheim concluded that 0.25 gram of protalbu- 
 mose, dissolved in 5 cc. of solution containing 0.025 gram of 
 HC1, combines with 4.16 per cent of its own weight of the acid, 
 similar determinations were made with other albumoses. 
 
 Hardy (16) has employed the catalysis of the inversion of cane 
 sugar by H + ions in the measurement of the acid-binding capacity 
 of serum-globulin, and that of the saponification of methyl acetate 
 by OH' ions in the measurement of the base-combining capacity 
 of this protein. 
 
 In this connection (although, owing to the complexity of the 
 experimental conditions, they have, as yet, but a qualitative 
 significance) the results obtained by a number of observers (12) 
 (7) (22) (23) (2) (1) (53) (35) on the protection conferred by 
 proteins against the destruction of enzymes by acid or alkali may 
 be mentioned. As is well known, the proteolytic enzymes, for 
 example, are very rapidly destroyed by an excess of free acid 
 or base, the action of the acid or base being, probably, that of 
 catalysing the destruction (hydrolysis?) of the enzyme which 
 occurs even in pure water. If, however, protein be added to 
 acid or alkaline solutions containing these enzymes, the rate of 
 destruction of the enzyme is greatly diminished, and this has 
 been attributed by Falk (12), Langley and Edkins (22), Langley 
 and Eves (23) and Vernon (53) to a binding of the free acid or 
 alkali by the added protein. It must not be forgotten, however, 
 that the proteolytic enzymes are, in the presence of protein, 
 
INDICATOR METHOD 81 
 
 probably combined in some proportion with the protein, which 
 may thus serve in a double manner to protect them from de- 
 struction. This fact, however, only adds to the complexity of 
 the conditions, without militating against the correctness of the 
 view urged by the investigators quoted above that the protein 
 protects the enzyme by binding some of the excess of acid or base. 
 
 9. The Indicator Method. This is simply the ordinary 
 method of acidimetry or alkalimetry applied to solutions contain- 
 ing protein, and has been very extensively used; unfortunately, 
 in the past, without a very clear understanding of the exact sig- 
 nificance of the data obtained. In order to appreciate this fact 
 it is necessary to recollect that the method, as ordinarily applied, 
 is not a static one. Not only is a foreign substance, namely 
 the indicator, added to the system under investigation, but while 
 acid or alkali is being added to the system to secure neutrality 
 to the chosen indicator, the combining capacity of the protein 
 is continually changing in response to the altering reaction of its 
 solution. The final result obtained in this manner with a given 
 indicator tells us nothing save the condition of equilibrium in 
 the solution at the precise H + or OH' concentration at which that 
 indicator changes color; it cannot yield us information concern- 
 ing the acid or alkali binding capacity of the protein at any other 
 H+ or OH' concentration. In the further application of this 
 method, the methods of acidimetry and alkalimetry devised by 
 Friedenthal and Salm (14) (13) (44) and elaborated by Sorensen 
 (48) are indubitably destined to prove of the greatest utility, and 
 arouse the hope that the indicator method may be of more service 
 to the protein chemist in the future than it has been in the past. 
 
 The use of indicators in protein solutions is, however, accom- 
 panied by two notable drawbacks. In the first place, owing to 
 the amphoteric character of the proteins and also to their mul- 
 tiple combining capacities, the changes in the hydrogen- or 
 hydroxyl-ion content of protein solutions, upon the addition of 
 acid or of alkali, over a considerable range, are relatively slight, 
 and, for this reason, sharp end-reactions are rarely obtained with 
 indicators in protein solutions, unless their color changes occur at 
 H+ or OH' concentrations lying without or upon the boundaries of 
 this range. Then, again, many of the substances commonly used 
 as indicators in acidimetry and alkalimetry combine chemically 
 with the proteins, and the compounds thus formed are not in- 
 
82 CHEMICAL STATICS 
 
 frequently either insoluble or of a different color from the free 
 indicator or its combination with inorganic acids or bases (27) 
 (17) (36). 
 
 10. The Method of "Masking" the Physiological Effects of 
 Ions by the Addition of Proteins to their Solutions. This 
 method although, as yet, only of qualitative importance, is 
 nevertheless of surpassing interest to the physiologist, since the 
 fluids which bathe the tissues contain notable quantities of 
 protein, which may be supposed to modify, in a greater or less 
 degree, the physiological action of the inorganic substances 
 which they contain. This method appears to have first been 
 employed by Loeb (24), and it has since been utilized by Stiles 
 and Beers (50) who, among other observations, have shown that 
 the onset of rigor, which rapidly occurs when frogs' muscles are 
 immersed in solutions of barium salts, is greatly delayed when 
 protein is added to the solutions. These experiments are, how- 
 ever, not conclusive since they are open to the criticism that the 
 added protein may alter the permeability of the tissue for inor- 
 ganic ions, for example, by clogging up the pores of the external 
 limiting membranes. This criticism, although a serious one from 
 the chemical standpoint, does not, however, detract from the 
 interest to physiologists of such experiments, since, whatever the 
 mechanism may be which leads to modification by proteins of 
 the physiological effects of inorganic substances in solution, the 
 probable importance of such effects in life-phenomena is the 
 same. 
 
 La Franca (21) has published the results of a series of experi- 
 ments which, in the light of the important investigations of 
 Madsen and Nyman (26) hold out the hope that this method 
 may ultimately be employed in a quantitative manner. This 
 observer, employing the method of Paul and Kronig (20) (32), 
 determined the toxicity, for bacteria, of solutions of copper 
 sulphate, mercurous nitrate and silver nitrate to which varying 
 amounts of protein had been added; at the same time he meas- 
 ured the concentration of heavy-metal ions in the solution by 
 the potentiometric method. His results show a satisfactory 
 parallelism between the diminution in the toxicities of these 
 solutions caused by the addition of the protein, and the number 
 of heavy-metal ions bound by the protein, as revealed by the 
 potentiometric measurements. 
 
LITERATURE CITED 
 
 LITERATURE CITED 
 
 (1) Bayliss, W. M., and Starling, E. H., Journ. of Physiol. 30 (1903), p. 61. 
 
 (2) Biernacke, E., Zeit. f. Biol. 28 (1891), p. 49. 
 
 (3) Bottger, W., Zeit. f. Physik. Chem. 24 (1897), p. 253. 
 
 (4) Bredig, G., Zeit. f. Elektrochem. 6 (1899), p. 33. 
 
 (5) Bugarszky, S., and Liebermann, L., Arch. f. d. ges. Physiol. 72 (1898), 
 
 p. 51. 
 
 (6) Chick, H., and Martin, C. J., Journ. Physiol. 45 (19i2), pp. 61 and 261. 
 
 (7) Chittenden, R. H., and Ely, J. S., Amer. Chem. Journ. 4 (1882), p. 107. 
 
 (8) Cohnheim, O., Zeit. f. Biol. 33 (1896), p. 489. 
 
 (9) Cohnheim, O., and Krieger, H., Zeit. f. Biol. 40 (1900), p. 95. 
 
 (10) D'Agostino, E., and Quagliariello, G., Nernst Festschrift. (1912), p. 27. 
 
 (11) Erb, W., Zeit. f. Biol. 41 (1910), p. 309. 
 
 (12) Falk, F., Virchow's Arch. 84 (1881), p. 119. 
 
 (13) Fels, B., Zeit. f. Elektrochem. 10 (1904), p. 208. 
 
 (14) Friedenthal, H., Zeit. f. Allgem. Physiol. 4 (1904), p. 44; Zeit. f. Elek- 
 
 trochem. 10 (1904), p. 113. 
 
 (15) Hamburger, H. J., "Osmotischer Druck und lonenlehre." Wiesbaden, 
 
 1907. 
 
 (16) Hardy, W. B., Journ. of Physiol. 33 (1905), p. 251. 
 
 (17) Heidenhain, M., Arch. f. d. ges. Physiol. 90 (1902), p. 115. 
 
 (18) Hildebrand, J. H., Journ. Amer. Chem. Soc. 35 (1913), p. 847. 
 
 (19) Hildebrand, J. H., and Bowers, W. G., Journ. Amer. Chem. Soc. 38 
 
 (1916), p. 785. 
 
 (20) Kronig, B., and Paul, Th., Zeit. f. Hyg. und Infekt. Krankheiten 25 
 
 (1897), p. 1. 
 
 (21) La Franca, S., Zeit. f. physiol. Chem. 48 (1906), p. 481. 
 
 (22) Langley, J. N., and Edkins, J. S., Journ. of Physiol. 7 (1886), p. 371. 
 
 (23) Langley, J. N., and Eves, F., Journ. of Physiol. 4 (1883), p. 18. 
 
 (24) Loeb, J., Amer. Journ. of Physiol. 3 (1900), p. 327. 
 
 (25) LundSn, H., Journ. Biol. Chem. 4 (1908), p. 267. 
 
 (26) Madsen, T., and Nyman, M., Zeit. f. Hyg. und Infekt. Krankheiten. 57 
 
 (1907), p. 388; Communication de 1'Inst. Serotherap. de PEtat 
 Danois 2 (1908). 
 
 (27) Mathews, A. P., Amer. Journ. Physiol. 1 (1898), p. 445. 
 
 (28) Mellanby, J., Journ. of Physiol. 33 (1905), p. 338. 
 
 (29) Michaelis, L., "Die Wasserstoffionen konzentration," Berlin (1914). 
 
 (30) Osborne, W. A., Journ. of Physiol. 27 (1901), p. 398. 
 
 (31) Osborne, T. B., and Leavenworth, C. S., Journ. Biol. Chem. 28 (1916), 
 
 p. 109. 
 
 (32) Paul, T., and Kronig, B. Zeit. f. physik. Chem. 21 (1896), p. 421. 
 
 (33) Platner, A. E., Zeit. f. Biol. 2 (1866), p. 417. 
 
 (34) Von Rhorer, L., Arch. f. d. ges. Physiol. 90 (1902), p. 368. 
 
 (35) Rosenthaler, L., Biochem. Zeit. 26 (1910), p. 9. 
 
 (36) Robertson, T. Brailsford, Journ. Biol. Chem. 4 (1904), p. 1. 
 
 (37) Robertson, T. Brailsford, Journ. Physical Chem. 11 (1907), pp. 437 
 
 and 542. 
 
84 CHEMICAL STATICS 
 
 (38) Robertson, T. Brailsford, Journ. Physical Chem. 12 (1908), p. 473. 
 
 (39) Robertson, T. Brailsford, Journ. Biol. Chem. 5 (1908), p. 155. 
 
 (40) Robertson, T. Brailsford, Journ. Physical Chem. 14 (1910), p. 528. 
 
 (41) Robertson, T. Brailsford, Journ. Physical Chem. 14 (1910), p. 601. 
 
 (42) Robertson, T. Brailsford, Journ. Biol. Chem. 7 (1910), p. 351. 
 
 (43) Robertson, T. Brailsford, Journ. Physical Chem. 15 (1911), p. 387. 
 
 (44) Salm, E., Zeit. f. Elektrochem. 10 (1904), p. 341; Zeit. f. physikal 
 
 Chem. 57 (1905), p. 471., cf. also Clark, W. M., and Lubs, H. A., 
 Journ. Bact. 2 (1917), p. 1. 
 
 (45) Schmidt, C. L. A., Journ. Biol. Chem. 25 (1916), p. 63. Univ. of 
 
 Calif. Publ. Pathol. 2 (1916), p. 157. 
 
 (46) Schmidt, C. L. A., and Finger, C. P., Journ. Physical Chem. 12 (1908), 
 
 p. 406. 
 
 (47) Sjoqvist, J., Skand. Arch. f. Physiol. 5 (1895), p. 277. 
 
 (48) Sorensen, S. P. L., Ergeb. d. Physiol. 12 (1912), p. 393. 
 
 (49) Spiro, K., and Pemsel, W., Zeit. f. physiol. Chem. 26 (1898), p. 233. 
 
 (50) Stiles, P. G., and Beers, W. H., Amer. Journ. Physiol. 14 (1905), p. 133. 
 
 (51) Strecker, A., Annalen der Chem. 148 (1868), p. 87. 
 
 (52) Van Slyke, L. L., and Hart, E. B., Amer. Chem. Journ. 33 (1905), p. 461 . 
 
 (53) Vernon, H. M., Journ. of Physiol. 31 (1904), p. 346. 
 
CHAPTER V 
 THE COMPOUNDS OF THE PROTEINS (Continued) 
 
 1. Stoichiometrical Relations in Protein Compounds. With 
 the aid of the various methods outlined in the previous chapter, the 
 existence of a number of compounds of the proteins with inorganic 
 acids, bases and salts has been conclusively demonstrated. Of 
 these compounds, however, comparatively few have been exten- 
 sively studied, and in equally few cases have the Stoichiometrical 
 relations which pertain between the constituents of the compounds 
 been elucidated. This is due in part, no doubt, to the complexity 
 and amphoteric character of the proteins themselves, and in part 
 to the difficulty of sharply characterizing and of isolating the 
 individual proteins in a pure condition, but in the main, I believe, 
 to the fact that physical chemistry has only recently been in a 
 position to supply us with the implements which the investigation 
 of these compounds demands. We have seen that the successful 
 investigation of these compounds requires, as a rule, the employ- 
 ment of static methods of measurement; methods, that is, which 
 do not involve a disturbance of the equilibria in the system while 
 these equilibria are being determined. Many investigators in this 
 field have, in the past, employed methods of determining the exist- 
 ence of protein compounds, which, to our modern perceptions, 
 clearly involved a variable and uncontrolled interference with the 
 very equilibrium which was the subject of investigation. Exam- 
 ination of more modern literature, however, cannot fail to impress 
 the reader with the conviction that in proportion to the adequacy 
 of the chemical or physico-chemical technique employed, Stoi- 
 chiometrical relations between the proteins and the substances with 
 which they combine are revealed or indicated. 
 
 The problem of determining the nature of the compounds 
 formed by the proteins with inorganic acids or bases is complicated 
 by the fact that, as a rule, a protein can combine with not only one 
 but several equivalents of a base or acid, so that on adding acid or 
 alkali to a solution of a protein we obtain a continuously varying 
 mixture of the various possible salts. Precisely analogous phenom- 
 
 85 
 
86 CHEMICAL STATICS 
 
 ena are, however, met with among the complex and double salts, 
 and the salts of polybasic acids or poiyacid bases in the domain of 
 inorganic chemistry, and they afford no legitimate grounds for the 
 belief that the protein compounds are not molecular in character 
 and consequently do not obey the law of constant combining pro- 
 portions. All of the accurate data which we possess lend decided 
 support to the view that although the proteins may enter into 
 combination in multiple proportions, these proportions are con- 
 stant and indicate that under definite conditions of reaction and 
 concentration the protein molecules possess a definite and measur- 
 able equivalent weight. 
 
 For the investigation of such compounds exactly the type of 
 technique must be employed which has proved to be successful in 
 dealing with the inorganic compounds of analogous complexity, 
 modified, however, by the limitations imposed upon us by the 
 instability of the proteins themselves. From the previous chapter 
 it will be clear that in the majority of instances the characteriza- 
 tion of the protein compounds must, for the present and pending 
 further elaboration of our technique, be based upon electrochemical 
 measurements. A discussion of the data derived from measure- 
 ments of this type must necessarily be deferred until later chapters 
 in which the electrochemical behavior of the protein salts will be 
 taken up in detail. For the present, therefore, except in the case 
 of the protamins, of which the salts with acids lend themselves to 
 sharp characterization by ordinary chemical methods, we shall 
 simply dwell upon the existence and general properties of certain 
 protein compounds without seeking to decide, in any specific 
 instance, how many equivalents of the protein or of the remaining 
 constituent are bound up in one molecule of the salt. 
 
 2. The Compounds of the Protamins with Inorganic Acids 
 and Bases. The protamins occur combined with nucleic 
 acid, in the nuclei of spermatozoa (27) (28) (13). According to 
 Burian (10), the nucleo-protein in the spermatozoa of the salmon 
 is formed by the combination of one molecule of nucleic acid with 
 one molecule of salmin. 
 
 The protamins (19) (20) are predominantly basic substances, 
 the acid function being very small in comparison. They are 
 soluble in water, yielding strongly alkaline solutions. The formula 
 of salmin sulphate is a multiple of: 
 
 C3oH 67 N 17 6 , 2 H 2 S0 4 + H 2 
 
PROTAMINS 87 
 
 while that of clupein sulphate is a multiple of 
 C 3 oH 57 N 17 06, 2 H 2 SO 4 ; 
 
 but if an insufficient quantity of acid is present to completely 
 neutralize the protamin, "basic" salts, containing fewer equiva- 
 lents of acid, are readily formed. 
 
 The sulphuric acid in these compounds is readily replaced by an 
 equivalent amount of hydrochloric (13), nitric, carbonic, or 
 chromic acids. The nitrates, chlorides and carbonates are readily 
 soluble, the sulphates sparingly soluble in cold, but more soluble 
 in hot, water, while the chromates are insoluble. The chloride of 
 salmin is soluble in methyl alcohol (13). 
 
 The formula of sturin sulphate is, according to Kossel, a multiple, 
 either of 
 
 2C 3 3H 61 N 17 7 
 
 or, more probably, of 
 
 4C 3 6H 69 N 19 7 
 
 Scombrin (derived from mackerel) sulphate possesses, according 
 to Kurajeff (21), the formula: 
 
 C 3 oH 6 oN 16 6 ,2H 2 S04. 
 
 It is soluble in water. The chromate is insoluble in water and 
 possesses the formula: 
 
 CsoHgsNieOs, 2 H 2 Cr0 4 , 
 
 differing, therefore, from the sulphate, not only in the acid com- 
 ponent, but also in the absence of one molecule of water for every 
 two molecules of combined acid. It is to be particularly noted 
 that the less soluble protein salt contains less water combined with the 
 protein. Remarkably analogous to this is the fact that whereas 
 salmin sulphate is freely diffusible through parchment paper (53), 
 clupein sulphate, which contains one molecule less of water for 
 every two molecules of combined acid, is almost indiffusible (49). 
 We shall have occasion to refer to analogous facts when considering 
 the mechanism of the coagulation of proteins (Chap. XII). 
 
 The protamins also form compounds with chloroplatinic acid (28) ; 
 these compounds are insoluble in neutral water, alcohol or ether, 
 but are soluble in water containing an excess of acid. According 
 to Goto (13), the formula of the chloroplatinate of salmin is: 
 
 C3oH 57 N 17 6 , 4 HC1, 2 PtCl 4 . 
 
88 CHEMICAL STATICS 
 
 Although the acid function of the protamins is very weak in 
 comparison with the basic function, salts with inorganic bases are 
 nevertheless formed. The salts of the heavy metals (except cupric 
 salts) produce from alkaline solutions of free protamin precipi- 
 tates of the corresponding salts of the protein. The compound 
 with divalent copper is soluble in water, and its solutions are 
 violet in color. The compounds with monovalent copper are very 
 sparingly soluble. On adding alkali (sodium hydrate) in excess 
 of one-half the equivalent of the protein (clupein) molecule, to the 
 precipitate of the silver salt, a yellow color is produced, indicating 
 that sodium has displaced some of the silver from the molecule and 
 that the silver protaminate is now mixed with silver oxide. 
 
 Other compounds which the protamins form will be considered 
 in the succeeding chapter. The physical properties of the pro- 
 tamins and their salts will be discussed in the chapters dealing with 
 the physical properties of protein systems. 
 
 3. The Compounds of Casein with Bases, Acids, and Salts. 
 Casein, in the dry state, forms a white powder, the general proper- 
 ties of which have been described in Chap. II. Uncombined and 
 purified casein is insoluble in distilled water or in very dilute salt 
 solutions (11) (34) (22) (36). A suspension of casein, thoroughly 
 shaken up in distilled water, reddens litmus paper wherever the 
 suspended particles touch it, but if this suspension be filtered the 
 filtrate contains no detectable casein and is neutral to litmus. 
 Casein is therefore probably soluble to a very slight extent in dis- 
 tilled water, but this slight amount is practically entirely con- 
 centrated at the surface of the water which is in immediate contact 
 with the solid particles, owing to the diminution in surface energy 
 which is thus brought about, so that the litmus is only reddened 
 where it touches these surfaces. 
 
 Uncombined casein is soluble in warm 5 per cent salt-solution 
 and in hot 50 per cent alcohol. When freshly prepared and 
 warmed it is very plastic and is capable of being drawn out into 
 long, fine threads (59). 
 
 Although it is so insoluble in water, uncombined casein, when 
 suspended in water, nevertheless acts as an acid in expelling carbon 
 dioxide from carbonates and bicarbonates, forming a salt with the 
 base (51), and in this way, according to Osborne (34), the ammo- 
 nium, potassium, sodium, lithium, magnesium, strontium and 
 calcium caseinates can be prepared. The salts of the alkaline 
 
CASEIN 89 
 
 earths can be precipitated without alteration of their composition 
 by the addition of alcohol to their solutions in water.* By direct 
 analysis of this precipitate Van Slyke and Hart (59) have shown 
 that the calcium salt which is formed in this way contains 2.4 per 
 cent of calcium oxide or from 80 X 10~ 5 to 90 X 10~ 5 equivalents of 
 calcium per gram (7). This salt, in solution, is neutral to phenol- 
 phthalein, and the alcohol precipitate from a lime-water solution of 
 casein which has been neutralized to phenolphthalein contains ex- 
 actly the same percentage of CaO (unneutralized by mineral acids) 
 as that from the solution of casein in a carbonate-solution. Similar 
 results had previously been obtained by Soldner (51) (52), de Jager 
 (18) and Laquer and Sackur (22), who, however, employed volu- 
 metric methods of measurement. Other 6gures for the percentage 
 of CaO in this compound which have been obtained by various 
 observers are: Courant (12), 2.91 and Timpe (54), 2.62. These 
 figures are not very divergent, and since the great majority of 
 observers agree in placing the percentage at 2.4, we may assume 
 that this result is not far from correct. The percentage of casein 
 in alkaline solutions can be fairly accurately determined by titra- 
 tion to neutrality to phenolphthalein, upon the assumption that in 
 solutions of this alkalinity (OH concentration) casein is combined 
 with 2.4 per cent of CaO or with an equivalent quantity of other 
 (strong) bases. 
 
 If, however, a lime-water solution of casein be neutralized to 
 litmus instead of to phenolphthalein, by the addition of mineral 
 acid or of casein, the alcoholic precipitate now contains only 1.5 
 per cent of CaO (Van Slyke and Hart (59)). The same estimate of 
 the combining-capacity of casein at absolute neutrality (i.e., in 
 solutions neutral to litmus) has been made by Soldner (51) (52) 
 and by myself (42) employing the potentiometric method. The 
 casein from goats' milk combines with bases in the same propor- 
 tions as the casein from cows' milk (9). The compound of casein 
 with 2.4 per cent of CaO, or an equivalent quantity of any other 
 strong base, is termed by Soldner and by Van Slyke and Hart the 
 ''basic" caseinate, by Courant the " tricaseinate " of the base. 
 That with 1.5 per cent of CaO, or an equivalent quantity of other 
 strong bases is termed by Soldner and by Van Slyke and Hart the 
 
 * Cf. Chap. II. The "basic" salt of potassium (i.e., neutral to phenol- 
 phthalein) is soluble in 98.6 per cent alcohol, but it may be precipitated from 
 this solution by the addition of ether (45). 
 
90 CHEMICAL STATICS 
 
 "neutral" caseinate; by Courant the " dicaseinate " of the base. 
 Both terminologies are objectionable since they imply or suggest 
 hypothetical stoichiometrical relations. Neutrality of its solutions 
 to an arbitrarily chosen indicator is no evidence of the unity of a 
 substance. As we shall see in considering the electrochemistry of 
 these substances, both that containing 2.4 per cent CaO and that 
 containing 1.5 per cent CaO are mixtures of two or more individual 
 salts of casein. We shall, however, for the sake of brevity in allu- 
 sion, employ the nomenclature of Soldner, as being the less objec- 
 tionable of the two. 
 
 At neutrality either to litmus or to phenolphthalein all observers 
 agree that casein binds the alkalies and alkaline earths in equiva- 
 lent-molecular proportions. Thus regular stoichiometrical rela- 
 tions between the protein and these metals are clearly indicated 
 although the above cited data are insufficient to define them. With 
 regard to ammonium caseinate, however, some confusion exists 
 since Bechamp (3) states that it contains 1.17 to 1.21 per cent of 
 ammonia (= 1.94 to 1.99 per cent of CaO), while Salkowski (47) 
 states that it contains only 0.35 per cent of ammonia. Salkowski 's 
 method of preparing this compound, however, was faulty since he 
 precipitated it from alcoholic solution by the addition of NaCl, a 
 procedure which involved the possibility of double decomposition 
 and the partial substitution of sodium for ammonia in the casein 
 compound.* 
 
 The quantitative results for casein, which we have so far cited, 
 may be stated in other words thus: At neutrality to litmus one 
 gram of casein binds 50 X 10~ 5 equivalent gram molecules of a 
 base, and at neutrality to phenolphthalein 80 X 10~ 5 equivalent 
 gram molecules. f 
 
 It was formerly stated by the author (36) that if a given con- 
 centration of alkali be " saturated" with casein, that is, if it be 
 shaken up with excess of casein until no more of the protein will 
 dissolve, and then filtered, the filtrate is always neutral to litmus 
 and contains the quantity of casein required to form the "neutral" 
 caseinate of the base. In later communications (41) (42), I have 
 shown, however, that this conclusion was erroneous, the source of 
 
 * Regarding the actual occurrence of such types of interaction between 
 casein salts and inorganic salts, Cf. W. A. Osborne (35) and Van Slyke and 
 Bosworth (57). 
 
 t That is, gram molecules divided by the valency of the combining ion. 
 
CASEIN 91 
 
 error lying in the fact that after the attainment of neutrality to 
 litmus additional casein dissolves in the solution with extreme 
 slowness. Many hours of rapid shaking or stirring are quite 
 insufficient, at room temperature, to bring about complete " satu- 
 ration " of the alkali with casein. Resource was had, therefore, to 
 the device of dissolving weighed amounts of casein in measured 
 volumes of alkali of known concentration, and then neutralizing 
 the excess of alkali by the addition of standardized acid, stirring 
 vigorously the while, until the casein just began to be precipitated. 
 The point at which precipitation began was readily determined, 
 with considerable accuracy, by the change (diminution) of the re- 
 fractive index of the solution. In this way it was ascertained that 
 at complete ''saturation" of an alkali (NaOH or LiOH) one gram 
 of casein binds 11.4 X 10~ 5 equivalent gram molecules of the base. 
 
 These determinations have been repeated by Van Slyke and 
 Bosworth (57), whose results very closely coincide with mine. 
 They find that at the point at which further diminution of the 
 proportion of base to casein results in the precipitation of free 
 casein one gram of casein is combined with between 11.0 X 10~ 5 
 and 11.5 X 10~ 5 equivalents of ammonium, sodium or potassium. 
 
 These "saturated" compounds of casein with bases react in 
 solution to indicators as follows (41): 
 
 Dimethylaminoazobenzol yellow 
 
 Congo red red 
 
 Sodium alizarinsulphonate. red 
 
 Cochineal rose 
 
 Paranitrophenol colorless 
 
 Rosolic acid yellow 
 
 indicating, according to the determinations of Salm (48) an acidity 
 (H+ concentration) of about 10~ 5 . According to Michaelis and 
 Rona (26) and Allemann (1), the optimum reaction for the complete 
 precipitation of casein is 2 X 10~ 5 H + . 
 
 It is not possible with equal simplicity of technique to determine 
 the combining capacity of casein for the alkaline earths at "satura- 
 tion " of these bases with casein, because the chloride of the alkaline 
 earth, which is necessarily formed in the solution at the same time, 
 precipitates the caseinate.* Thus four grams of casein were dis- 
 
 * The "neutral" and "basic" casemates of the alkaline earths are likewise 
 precipitated by the addition to their solutions of a somewhat greater propor- 
 tion of the corresponding alkaline earth. Cf. A. S. Loevenhart (24) and Van 
 Slyke and Hart (59). 
 
92 CHEMICAL STATICS 
 
 solved in 100 cc. of 0.048 N Ca(OH) 2 solution and then 40 cc. of 
 AT/10 HC1 were cautiously delivered into the solution by means of 
 a pipette of which the opening was held below the surface of the 
 fluid, the fluid being rapidly and continuously stirred meanwhile 
 through the agency of a small motor. The total volume was then 
 made up to 200 cc. and the mixture filtered through soft filter- 
 paper. By measurement of the refractive index of this filtrate it 
 was found that the 0.0008 equivalent of Ca(OH) 2 unneutralized 
 by HC1 had, under these conditions, only held in solution 0.2 
 gram of casein; although, as we have seen, in the absence of 
 CaCl2, 0.0008 equivalent of Ca(OH) 2 will readily dissolve one 
 gram of casein, rendering the solution neutral to phenolphthalein. 
 In a subsequent chapter (X) it will be shown that a definite 
 relationship subsists between the initial alkalinity of a solution 
 and the depression of its electrical conductivity which is brought 
 about by the addition to it of a definite proportion of casein. This 
 relationship is of such a character that by exterpolation from the 
 measurements made upon solutions of potassium caseinate con- 
 taining from 25 X 10~ 5 to 300 X 10~ 5 equivalents of base per 
 gram of casein, it would appear that the depression of con- 
 ductivity caused by casein is zero when the proportion of base is 
 sufficient and only just sufficient to hold the casein in solution (42), 
 namely, 11.4 X 10~ 5 equivalents per gram. Similar exterpola- 
 tion from measurements of the depression of the conductivity of 
 Ca(OH) 2 solutions consequent upon the addition of casein leads to 
 the conclusion that the depression of conductivity would vanish 
 at an initial alkalinity corresponding to a proportion of 11.9 X 10~ 5 
 equivalents of Ca(OH) 2 per gram of casein, a value so close to 
 that obtained for the combining-capacity of casein for the alkalies 
 at "saturation" with casein that I have previously inferred that 
 bases dissolve casein in equivalent-molecular proportions (46). 
 Van Slyke and Bosworth have, however, measured the combining- 
 capacity of casein for alkaline earth bases at " saturation" of the 
 base with casein by the more direct method of first dissolving the 
 casein in an excess of the base and then neutralizing the excess with 
 hydrochloric acid and dialysing the mixture until free from soluble 
 chlorides (57) . In this way- they have found that a compound of 
 casein with calcium hydrate which contains 11.25 X 10~ 5 equiva- 
 lents of base per gram exists but is not soluble in water, the 
 lowest proportion of base yielding a soluble compound being 
 
CASEIN 93 
 
 22.5 X 10~ 5 equivalents per gram, or exactly double the pro- 
 portion of alkaline earth contained in the insoluble compound. 
 The insoluble compound will, however, dissolve in solutions of 
 NaCl, NH 4 C1, KC1, etc., probably owing to exchange of bases, 
 with the formation of soluble caseinates of the alkalies (56) (57). 
 
 Magnesium hydrate forms a similar series of compounds with 
 casein, namely a compound insoluble in water but soluble in 5 per 
 cent NaCl solution and containing 11.25 X 10~ 5 equivalents of 
 base per gram, a soluble compound containing 22.5 X 10~ 5 
 equivalents of base per gram, a compound neutral to litmus 
 containing about 56 X 10~ 5 equivalents of magnesium per gram 
 and a compound neutral to phenolphthalein containing between 
 80 XlO~ 5 and 90 X 10~ 5 equivalents of magnesium per gram (62). 
 
 From the phosphorus content of casein (8), we would infer a 
 minimal combining- weight of 4430. The combining- weight corre- 
 sponding to the proportion of NaOH or KOH combined with casein 
 at " saturation " of the base with protein is 8888. This corresponds 
 so closely with twice the minimal combining-weight calculated on 
 the assumption that the casein molecule contains only one atom 
 of phosphorus that we may infer, with Bosworth and Van Slyke, 
 that the molecule of casein contains two atoms of phosphorus. 
 
 The maximum combining-capacity of casein for bases (KOH) 
 has been measured by the author (42), employing the potentio- 
 metric method. In the presence of a considerable excess of KOH, 
 the combining-capacity of casein attains the constant maximum of 
 180 X 10~ 5 equivalents per gram. In passing from its minimum 
 to its maximum, therefore, the combining-capacity of casein is 
 multiplied sixteen times. 
 
 The compounds of casein with adds have not been so extensively 
 studied as those with bases. Hammarsten (14) held that there is 
 no true combination between casein and acid, because by pro- 
 longed trituration with water he could remove all of the acid 
 contained in the casein. Our modern conception of balanced 
 reactions deprives this consideration of its weight (59) (36) . Since, 
 however, it has been frequently applied to other protein com- 
 pounds,* it may be worth while to dwell briefly upon the implica- 
 tions of such a deduction. 
 
 * For example by Hans Trunkel (55): When so much of the published 
 evidence on behalf of the "adsorption theory" is of this unfortunate and 
 inconclusive character, advocates of "adsorption" cannot complain if their 
 hypotheses are viewed with scepticism by physical chemists. 
 
94 CHEMICAL STATICS 
 
 When any two or more substances, for example, A and B react 
 to form other substances A' and B', equilibrium is reached, i.e., no 
 further change occurs in the composition of the system, when the 
 reaction 
 
 proceeds at exactly the same velocity from left to right as it 
 from right to left. Since the velocity with which a chemical 
 reaction proceeds varies directly as the active masses of the react- 
 ing substances (Guldberg and Waage's law) the velocities of the 
 two opposing reactions gradually approach each other as the 
 reaction proceeds, until they become equal, when the reaction 
 apparently ceases. According to the relative magnitude of the 
 proportionality-factors expressing the ratio of mass to velocity of 
 each of the reactions concerned, equilibrium may occur so far to 
 the left or right that, with our limited precision of measurement, 
 the reaction appears to be complete or not to occur at all as the 
 case may be, or equilibrium may occur when the active masses of 
 the reacting components on either side of the equation are nearly 
 equal; and, of course, every type of equilibrium intermediate be- 
 tween these extremes is found to occur. If, after the attainment 
 of equilibrium, one of the reacting components be wholly or par- 
 tially removed from the system the reaction will recommence more 
 or less rapidly, restoring a certain proportion of the abstracted 
 component, and attaining a fresh station of equilibrium deter- 
 mined by the new relative masses of the components. 
 
 If, now, after the attainment of equilibrium in such a reaction as : 
 
 Casein + HC1 <=* Casein hydrochloride 
 
 (Insoluble) (Soluble) (Insoluble or sparingly soluble) 
 
 even if only a small proportion of free HC1 and casein exist in the 
 system, and HC1 be removed by trituration with water, the re- 
 action will recommence, now proceeding from right to left, and 
 restore a proportion of the HC1 which has been washed away. If 
 trituration be prolonged and the velocity-constant (proportionality 
 between mass and velocity) for the decomposition of the casein 
 hydrochloride be tolerably high, it is easy to understand how the 
 compound might ultimately be completely decomposed and 
 deprived of its combined acid. Similarly, the acid components of 
 substances so universally admitted to be chemical compounds as 
 mercuric sulphate and cuprous chloride can be completely removed 
 
CASEIN 95 
 
 from them by prolonged trituration with water (23) (16). To 
 argue from this fact that these combinations are not chemical in 
 character would be analogous to concluding that, because salicylic 
 acid can be completely removed from its solution in water by 
 repeated extraction with ether, therefore the salicylic acid was not 
 truly dissolved in the water. 
 
 To return to the more specific problem of the occurrence of 
 combinations between inorganic acids and casein. Van Slyke and 
 Hart (59) and Van Slyke and Van Slyke (60) have described a 
 series of compounds of casein with inorganic acids which are 
 insoluble in distilled water. When solid casein is suspended in a 
 dilute solution of an inorganic acid, the protein abstracts some of 
 the acid from the solution, forming these insoluble compounds. 
 Van Slyke and Van Slyke measured the velocity with which the 
 acid is abstracted from the solution and the final equilibrium at- 
 tained by abstracting portions of the well-stirred suspension, from 
 time to time filtering, and measuring the electrical conductivity 
 of the filtrate. The decrease in conductivity, from the moment of 
 introduction of the casein, afforded a measure of the amount of 
 acid bound. They found that the acid is bound at first rapidly 
 and later more slowly (Cf . Chap. XII) and at equilibrium the ratio 
 
 acid bound by one gram casein 
 acid in one cc. of solution 
 
 is nearly constant between the limits of concentration of the acid 
 employed (AT/125 and Af/lOOO) for hydrochloric acid at constant 
 temperatures. When sulphuric acid is employed the ratio increases 
 with dilution of the acid, the quantity of acid bound by one gram 
 varying as the square root* instead of directly as the concentration 
 of the acid. The equilibrium is the same from whichever direction 
 it is approached, whether by suspending the protein compound in 
 more dilute acid than that in which it was formed, in which case 
 it yields acid to the solution, or by suspending uncombined casein 
 directly in the solution of acid. When one gram of casein is sus- 
 pended in 100 cc. of AT/500 acid at C., it takes up 17.4 X 10~ 5 
 equivalents of sulphuric, 11.9 X 10~ 5 equivalents of hydrochloric, 
 8.9 X 10~ 5 equivalents of lactic or 5.3 X 10~ 5 equivalents of acetic 
 
 * The exact exponent obtained by Van Slyke and Van Slyke is 1/1.95; this 
 experimental value is so nearly | that it affords no grounds for preferring the 
 experimental value to that which is indicated by stoichiometry. 
 
96 CHEMICAL STATICS 
 
 acid. Increase in temperature increases the rate at which equi- 
 librium is approached but decreases the final amount of acid 
 taken up. 
 
 It is evident that the concentration of acid was not sufficient in 
 any of these solutions to bring about complete neutralization of the 
 casein. When more concentrated solutions were employed, how- 
 ever, a proportion of the casein was dissolved, and the methods of 
 estimation utilized by Van Slyke and Van Slyke could not be 
 employed. From considerations which follow below (Cf. Chap. 
 XII), it appears possible that the compounds of casein with acids 
 which are formed in experiments such as these are in reality soluble, 
 but that while a sufficient proportion of casein remains uncom- 
 bined they are mechanically hindered from passing out of the 
 casein particles into the solvent (38) (40). Van Slyke and Van 
 Slyke, however, believe that the binding of casein by acids, under 
 these conditions, is an "adsorption" phenomenon (60) (61). 
 
 If one stirs excess of casein in 0.1 AT HC1 (or more dilute) rapidly 
 and continuously for over an hour, at room temperature very little 
 or none of the casein passes out into the solvent; the solution 
 remains perfectly clear on filtration and its refractive index is only 
 very slightly changed. If, however, the casein be previously 
 dissolved in dilute NaOH and then precipitated with HC1, the 
 addition of the slightest excess of HC1 will then carry it into solu- 
 tion. In other words, wet, freshly precipitated casein dissolves 
 rapidly in dilute acid, but the physical properties of dry, granular 
 casein, hinder the compounds which are formed from passing out 
 of the particles.* Dry serum globulin similarly dissolves with 
 extreme slowness in acids, although, when wet and freshly pre- 
 cipitated, it dissolves in dilute solutions of acids with great readi- 
 ness. 
 
 I have determined the acid-equivalent of casein at " saturation" 
 of the acid with protein in the following manner (41): Weighed 
 amounts of casein were dissolved in measured volumes of alkali of 
 known concentration; the excess of acid over that sufficient to 
 
 * The fact that the casein hydrochloride which is formed is in reality soluble, 
 although it does not pass out into the solution, explains the dependence of the 
 amount formed upon the dilation of the acid, which was observed by Van 
 Slyke and Van Slyke. Were the casein salt insoluble, as these authors point 
 out, the amount formed should be invariable, or else equivalent to the total 
 amount of acid in the system. 
 
CASEIN 97 
 
 neutralize the alkali, which it was necessary to add to the solution 
 in order to just redissolve the casein was then determined. The 
 point of complete re-solution of the casein was determined by 
 noting the point at which the refractive index of the filtered mix- 
 ture attained a constant (maximum) value. It was found that at 
 " saturation" in solutions containing 1.25 per cent of casein, 1 
 gram of casein = approximately 32 X 10~ 5 equivalent gram mole- 
 cules of HC1. The acid-equivalent in more dilute solutions ap- 
 pears to be somewhat higher, although I have not measured it 
 directly.* This is attributable to the fact that, as the observa- 
 tions of Van Slyke and Van Slyke show, the hydrochloride of 
 casein tends in some measure to decompose into casein and hydro- 
 chloric acid, and, since free casein is insoluble, an excess of acid 
 over that actually combined with the casein is necessary in order 
 to diminish this (hydrolytic) dissociation sufficiently to keep the 
 casein in solution. The same phenomenon, as we shall see, is 
 encountered in acid solutions of serum globulin. 
 
 Schlossmann (50) and Arny and Pratt (2) have described com- 
 pounds of casein with alums which, especially that with ferric 
 alum, are but sparingly soluble. Upon this fact Arny and Pratt 
 have based a method of estimating casein volumetrically. 
 
 Paracasein, the product of the action of rennet upon casein, 
 resembles casein very closely, save in the greater insolubility of 
 the calcium salt in the presence of an excess of calcium ions. 
 Calcium caseinate is precipitated by calcium chloride, a smaller 
 amount of calcium chloride being required to bring about complete 
 precipitation, the smaller the proportion of base in the caseinate 
 (i.e., the more acid the solution). This precipitation is still more 
 readily accomplished at higher temperatures (24) (59). Calcium 
 paracaseinate differs from calcium caseinate in that it is precipi- 
 tated by a smaller excess of calcium ions, and, by a given excess, 
 at a lower temperature (24). Paracasein differs, however, still 
 more markedly from casein in the proportion of base which it 
 binds at " saturation" of the base with protein, i.e., when the 
 proportion of inorganic base is sufficient and only just sufficient to 
 
 * The accuracy of this estimate of the combining-capacity of casein for 
 acids at "saturation" of the acid by protein cannot be relied upon, since the 
 solution necessarily contained a certain quantity of the chloride of the alkali 
 employed to dissolve the casein, and the compounds of casein with acids are 
 very readily precipitated by salts. 
 
98 
 
 CHEMICAL STATICS 
 
 hold the paracasein in solution. We have seen that the proportion 
 of alkali which just suffices to hold casein in solution in water is 
 about 11.25 X 10~ 5 equivalents per gram. Paracasein, however, 
 requires exactly double this proportion of alkali to hold it in 
 solution (57) indicating, as Van Slyke and Bosworth believe, that 
 the molecule of paracasein is one-half the weight of the molecule 
 of casein. From this it would follow that the molecule of para- 
 casein contains but one atom of phosphorus (58). 
 
 4. The Compounds of Serum Globulin with Inorganic Acids 
 and Bases. The compounds of the globulin which is precipi- 
 tated from diluted serum by the passage of C0 2 through it, and 
 which, in the free condition, is insoluble in distilled water, have 
 been extensively studied by W. B. Hardy (15). This investigator 
 has measured the quantities of various acids which are required 
 to dissolve one gram of globulin at various concentrations. The 
 mean values, measured in gram molecules, taking the quantity of 
 HC1 required to dissolve one gram of the globulin as unity, are 
 quoted below: 
 
 HC1 1.0 
 
 HNO 3 0.995 
 
 CHC1 2 COOH 1.0 
 
 CH 2 C1COOH....1.05 
 
 HCOOH 1.25 
 
 CHaCOOH 5.2 
 
 CH 3 CH 2 COOH...7.56 
 
 It is evident that the acid-equivalent of serum-globulin is the 
 same for all of the strong monobasic acids investigated, although 
 it is much higher for the weak acids. This equivalent is, for one 
 gram of the protein, about 18 X 10~ 5 gram-equivalents (mean 
 value). It is, however, somewhat higher the more dilute the 
 globulin, and this effect is more marked with the weak than with 
 the stronger acids. Thus : 
 
 H 2 SO 4 1.91 Citric... 
 
 Tartaric 1 . 994 H 3 PO 4 . . 
 
 Oxalic.. ..1.9 H 3 BO 3 .. 
 
 3 
 
 2.9 
 
 , . very great 
 excess 
 
 Concentration of protein, 
 per cent 
 
 Gram-equivalents of acid necessary to dissolve one 
 gram of protein 
 
 Acetic acid 
 
 Hydrochloric acid 
 
 0.28 
 1.46 
 4.18 
 
 183 X10- 5 
 56X10~ 5 
 40X10- 6 
 
 23X10-* 
 15X10- 5 
 13X10- 6 
 
SERUM GLOBULIN 
 
 99 
 
 We have already had occasion to discuss the interpretation of simi- 
 lar phenomena which are displayed by the compounds of casein 
 with acids. 
 
 The acid-equivalent of serum-globulin for dibasic (strong) acids 
 is twice that for monobasic acids, and for tribasic (strong) acids 
 it is three times that for monobasic acids. Hence serum-globulin 
 combines with acids, at "saturation" in molecular and not equiva- 
 lent molecular proportions. 
 
 Solutions of acids " saturated" with serum-globulin are faintly 
 acid to methyl orange. 
 
 Employing the potentiometric method (37) (39) I have deter- 
 mined the quantity of acid neutralized by globulin in solutions of 
 hydrochloric acid of various concentrations. The following are 
 among my results, the concentration of globulin in each solution 
 being 0.496 per cent. 
 
 Concentrations of HC1 in 
 which globulin was dissolved 
 
 Concentration of H ions in 
 solution containing globulin 
 
 Equivalents of HC1 neutral- 
 ized per gram of globulin 
 
 38.5 X 10- 4 
 57.8 X 10~ 4 
 77.0 X 10~ 4 
 120.3 X 10~ 4 
 
 12.4 X 10~ 4 
 23.8 X 10~ 4 
 36.8 X 10~ 4 
 72.7 X 10~ 4 
 
 53 X 10~ 5 
 69 X 10~ 5 
 81 X 10~ 5 
 96 X 10~ 5 
 
 We see that the acid-equivalent of serum-globulin rises with in- 
 creasing acidity of its solution. 
 
 The compounds of serum-globulin with bases have also been 
 investigated by Hardy. He finds that at " saturation " of solutions 
 of bases with serum-globulin the bases are bound by the globulin 
 in molecular, not equivalent molecular proportions. Solutions of 
 monacid bases " saturated" with globulin are neutral to litmus, 
 the alkali equivalent for monacid bases being approximately 
 10 X 10~ 5 gram-equivalents of the base per gram of globulin, that 
 for di-acid bases being approximately 20 X 10~ 5 in the same units. 
 
 At neutrality to phenolphthalein, however, that is, at an alka- 
 linity corresponding to an OH' concentration of 20 X 10~ 7 (Cf. 
 Salm (48)), the bases are bound in equivalent molecular pro- 
 portions, such that one gram of globulin binds 20 X 10~ 5 gram- 
 equivalents of the base. Thus the point of complete saturation of 
 Ba(OH) 2 solutions by globulin and that of neutrality to phenol- 
 phthalein coincide, while solutions of the monacid bases which are 
 
100 CHEMICAL STATICS 
 
 saturated with globulin contain only half the proportion of base 
 to protein which is contained in those which are neutral to phenol- 
 phthalein. Hardy has suggested, in explanation of this, that 
 globulin has at least two replaceable hydrogens and that its acid 
 salts of sodium or potassium are soluble, while its acid salt of 
 barium is relatively insoluble, the " neutral " salt being soluble 
 and neutral to phenolphthalein. Since complete solution of the 
 globulin is just attained at neutrality to litmus and the ratio 
 of the alkali-binding capacity of serum at neutrality to phenol- 
 phthalein to its capacity at neutrality to litmus is 2:1, this 
 hypothesis, although based only upon results obtained with arbi- 
 trarily chosen indicators, would appear to be justifiable. We 
 shall have occasion to further discuss this question from a some- 
 what different point of view in Chap. IX. 
 
 Employing the potentiometric method (37) I have determined 
 the quantity of alkali neutralized by globulin in solutions contain- 
 ing an excess of KOH. The following were the results obtained, 
 the concentration of globulin in each solution being 0.208 per cent. 
 
 Concentration of KOH in which 
 globulin was dissolved 
 
 Equivalents of KOH bound by 
 one gram of globulin 
 
 12.9 X 10~ 4 
 16.1 X 10~ 4 
 19.3 X lO- 4 
 25.8 X 10~ 4 
 
 47 X 10- 5 
 46 X 10~ 5 
 50 X 10~ 5 
 45 X 10-* 
 
 The alkali-equivalent of the globulin, therefore, remained appreci- 
 ably constant throughout the range of alkalinities mentioned. Its 
 mean value is 47 X 10~ 5 gram-equivalents of KOH per gram 
 of globulin, which may therefore be regarded as the alkali-equiva- 
 lent of globulin in the presence of excess of base. 
 
 The ratio of the alkali to the acid-equivalent of globulin at 
 "saturation" is, according to Hardy's determinations 10 : 18. He 
 considers that the true ratio is probably 1 : 2. 
 
 The compounds of globulin with salts have been studied by 
 Hardy (15) and by Mellanby (25). But as these compounds have 
 not yet been clearly defined, and their discussion involves a con- 
 sideration of the general nature of the mechanism of the solution 
 and precipitation of protein by salts, the description of the results 
 obtained by these observers will be deferred to the succeeding 
 chapter. 
 
VEGETABLE PROTEINS ' '> . '< : ^ 10 1 / 
 
 6. The Compounds of Fibrin with Inorganic Acids and Bases. 
 Employing methods similar to those utilized by Van Slyke and 
 Bosworth in the investigation of the compounds of casein with in- 
 organic bases, Bosworth has investigated the combining-capacity 
 of fibrin for inorganic bases and acids (6). He finds that at neu- 
 trality to phenolphthalein one gram of fibrin neutralizes 61.4 X 
 10~ 5 equivalents of NaOH or 61.6 X 10~ 5 equivalents of Ca(OH) 2 . 
 Fibrin, therefore, neutralizes bases, in the presence of excess of the 
 base, in equivalent-molecular proportions. At " saturation," i.e., 
 when the proportion of base is only just sufficient to hold the pro- 
 tein in solution, fibrin combines with 15.2 X 10~ 5 equivalents of 
 NaOH, and the precipitate produced on addition of HC1 to this 
 solution is free from sodium. In other words no insoluble salts of 
 sodium are formed. The case is quite otherwise with calcium 
 which forms with fibrin an insoluble salt (soluble, however, in 
 5 per cent NaCl solution), containing 30 X 10~ 5 equivalents of the 
 base, and a soluble salt containing 45 X 10~ 5 equivalents of the 
 base. The proportion of 1:2:3 encountered in these various 
 compounds is irresistible evidence of the existence of definite 
 stoichiometrical relations between fibrin and the bases with which 
 it combines. 
 
 The sulphur-content of fibrin would indicate a minimal com- 
 bining- weight of 6751. The proportion of base in the " saturated" 
 compound of fibrin with NaOH would indicate a combining-weight 
 of 6667. 
 
 Calcium and barium fibrinates yield a precipitate of fibrin on the 
 passage of CO2 through them (6) (17), sodium, potassium and 
 ammonium fibrinates do not. Fibrin does not decompose CaCO 3 , 
 however, as casein does on trituration with this salt in the presence 
 of water. 
 
 Fibrin forms a compound with hydrochloric acid, which is just 
 soluble in water, containing 15 X 10~ 5 equivalents of the acid. 
 
 6. The Compounds of the Vegetable Proteins with Inorganic 
 Acids and Bases. These have been investigated by T. B. 
 Osborne (29) (30) (31) (32), who has found evidence of combina- 
 tion between inorganic acids and the following vegetable proteins : 
 Edestin, legumin, excelsin, amandin, corylin, phaseolin, gliadin, 
 hordein and zein. He finds that when edestin, deposited from 
 salt-solutions, is suspended in water and made neutral to phenol- 
 phthalein, edestin itself remains undissolved, while the added 
 alkali carries into solution its equivalent of the acid or acids which 
 
102 CHEMICAL STATICS 
 
 had been united with the edestin. The character of 'the acid 
 united with the edestin varies with that of the salt employed for 
 solution of the edestin. When the salt is sodium chloride, the acid 
 is HC1, when it is ammonium sulphate, the acid is H 2 S0 4 . The 
 combination is apparently facilitated by a weakly acid reaction, 
 not necessarily induced by the same acid as that with which the 
 edestin combines. Osborne attributes this phenomenon to the 
 strength of edestin as a base; the compound which is formed, 
 however, must be practically undissociated so far as Cl' ions are 
 concerned, otherwise, having regard to the extremely low equiv- 
 alent-concentration of protein in the solutions in which it is 
 formed, we should attribute to edestin a basic function of a magni- 
 tude wholly inconsistent with the fact that it is an amphoteric 
 acid. Therefore, it does not follow that these proteins are stronger 
 bases than they are acids because they can displace hydrochloric 
 acid from its combination with sodium hydrate, as Osborne 
 assumes, for this phenomenon may be interpreted by merely 
 supposing that the protein can combine with either constituent 
 of the organic salt, but that the compound with HC1 produces 
 fewer Cl' ions than the Na + ions produced by the compound with 
 NaOH. A further discussion of this phenomenon will be found in 
 a later chapter. (Chap. VI, section 6.) 
 
 The fact that the occurrence of decomposition of neutral mineral 
 salts through the agency of animal proteins has not been generally 
 recognized, is probably attributable to the fact that it has not been 
 carefully looked for, and that the proteins which are most readily 
 obtained in the pure condition are not usually prepared under 
 conditions favoring the formation of protein compounds through 
 the decomposition of inorganic salts. 
 
 Two compounds of edestin with acid (hydrochloric) appear to 
 exist. The one, which Osborne terms the monohydrochloride, 
 contains an amount of acid-equivalent to 0.7 cc. N/IQ HC1 per 
 gram of the protein, and is insoluble in water; it is, however, 
 soluble in salt solution and is deposited therefrom in crystals on 
 dialysis. The other compound of edestin with hydrochloric acid 
 contains just sufficient acid to hold the protein in solution, and is 
 termed by Osborne the dihydrochloride. The quantity of HC1 
 required to just hold one gram of edestin in solution is 1.4 cc. 
 of AT/10 HC1. The acid-equivalent, for monobasic acids, at 
 " saturation " of the acid with edestin is therefore 14 X 10~ 5 
 gram-equivalents per gram. The quantity of alkali which will 
 
SUMMARY 103 
 
 just hold one gram of edestin in solution is equivalent to the 
 quantity of acid with which it combines to form Osborne' s "edes- 
 tin monohydrochloride," that is, 7 X 10~ 5 equivalents. Hence the 
 ratio of the alkali- to the acid-equivalent at " saturation " is 1 : 2. 
 
 From the observations of Osborne it would appear that acids 
 dissolve edestin, as they do serum-globulin, in molecular propor- 
 tions, not in equivalent molecular proportions. 
 
 Osborne has also determined the hydrochloric acid equivalents 
 at neutrality to tropceolin (10~ 2 to 10~ 3 H + according to Salm 
 (48)), of a variety of vegetable proteins. That of edestin was 
 found to be 127 X 10~ 5 gram-equivalents per gram, of excelsin 
 124 X 10- 5 , of legumin 129 X 10~ 5 , of amandin 103 X 10~ 5 , of 
 crystallized egg-albumin 90 X 10~ 5 in the same units. Recol- 
 lecting that these determinations are made in the presence of a 
 considerable excess of acid they may be considered as furnishing, 
 possibly, a measure of the constant maximum acid-equivalents of 
 these proteins in the presence of excess of acid. However this may 
 be, it is evident that the acid-equivalent of edestin increases very 
 considerably with the acidity of its solution. 
 
 The compounds which edestin and gliadin form with cupric 
 hydroxide (33) have already been discussed in Chap. I. 
 
 7. The Compounds of Ovomucoid with Acids and Bases. 
 These have so far chiefly been studied by electrochemical methods 
 (43). Uncombined ovomucoid is freely soluble in distilled water, 
 but on adding small quantities of acid or alkali to this solution 
 a proportion of the acid or base is neutralized by the protein. 
 Employing the potentiometric method I have shown that at neu- 
 trality to litmus (i.e., absolute neutrality) 1 gram of ovomucoid 
 neutralizes 7 X 10~ 5 equivalents of HC1. Its basic function, there- 
 fore, predominates over its acid function. 
 
 The maximum (constant) combining-capacity of ovomucoid for 
 KOH, in solutions containing excess of the base, is about 50 X 10~ 5 
 equivalents per gram. 
 
 The maximum combining-capacity of ovomucoid for HC1 was 
 not attained in any of the solutions investigated, but is probably 
 in excess of 100 X 10~ 5 equivalents per gram. 
 
 In the chapters dealing with the electrochemistry of the proteins 
 the reader will find a more detailed discussion of these compounds. 
 
 8. Summary of Some of the Results Cited in this Chapter. 
 Some of the more important determinations which have been cited 
 in this chapter are summarized in the following table: 
 
104 
 
 CHEMICAL STATICS 
 
 ill 
 
 xjl : 
 
 
 1 I - 
 
 ?Jg f Jg~ 
 -H i O cS O O 03 
 
 a^ 
 
 in 
 
 E'o 
 
 > - 
 
 ISs 
 
 
 la a 
 
 .9 SS 
 
 >0 rH 
 
 i 
 
 2S 
 X"o 
 
 f 1 
 
 S. -i 
 
 u. I 
 
 a 
 
 I 
 f 5 8 
 
 2! 
 
 xa xa 
 
 I I I I 
 
 16 
 
 1 1 
 
 - 
 
 xa 
 
 i-I 
 
 rl^^ 
 
 H 03 
 
 
 
 a -s 
 
 & in 11 ll i 
 
 
LITERATURE CITED 105 
 
 LITERATURE CITED 
 
 (1) Allemann, O., Biochem. Zeit. 45 (1912), p. 346. 
 
 (2) Arny, H. V., and Pratt, T. M., Amer. Journ. of Pharmacy, 78 (1906), 
 
 p. 121. 
 
 (3) Be'champ, A., Bull. Soc. Chem. (3) 11 (1894), p. 153. 
 
 (4) Bosworth, A. W., Journ. Biol. Chem. 15 (1913), p. 231. 
 
 (5) Bosworth, A. W., Journ. Biol. Chem. 19 (1914), p. 397. 
 
 (6) Bosworth, A. W., Journ. Biol. Chem. 20 (1915), p. 91. 
 
 (7) Bosworth, A. W., and Van Slyke, L. L., Journ. Biol. Chem. 14 (1913), 
 
 p. 207. 
 
 (8) Bosworth, A. W., and Van Slyke, L. L., Journ. Biol. Chem. 19 (1914), 
 
 p. 67. 
 
 (9) Bosworth, A. W., and Van Slyke, L. L., Journ. Biol. Chem. 24 (1916), 
 
 p. 173. 
 
 (10) Burian, R., Ergeb. d. physiol. 5 (1906), p. 768. 
 
 (11) Cohnheim, O., "Chemie der Eweisskorper " (1900), p. 173. 
 
 (12) Courant, G., Arch. f. d. Ges. physiol. 50 (1891), p. 109. 
 
 (13) Goto, M., Zeit. f. physiol. Chem. 37 (1902), p. 94. 
 
 (14) Hammarsten, O. Nov. Act. Reg. Soc. Upsala (1877), cited after Maly's 
 
 Jahresber. f. Tierchem. (1877), p. 158. 
 
 (15) Hardy, W. B., Journ. of Physiol. 33 (1905), p. 251. 
 
 (16) Haywood, J. K., Journ. of Physical Chem. 1 (1897), p. 411. 
 
 (17) Hekma, E., Koninklijke Akad. Van Wetenschappen Te Amsterdam 
 
 16 (1913), p. 172. 
 
 (18) De Jager, L., Nederl. Tijdschr. Voor Geneeskunde. 2 (1897), p. 253, 
 
 cited after Maly's Jahresber. f. Tierchem. 27 (1897), p. 276. 
 
 (19) Kossel, A., Zeit. f. physiol. Chem. 25 (1898), p. 165. 
 
 (20) Kossel, A., and Weiss, F., Zeit. f. physiol. Chem. 78 (1912), p. 402. 
 
 (21) Kurajeff, D., Zeit. f. physiol. Chem. 26 (1899), p. 524. 
 
 (22) Laqueur, E., and Sackur, O., Beitr. z. chem. Physiol. und Pathol. 3 
 
 (1902), p. 193. 
 
 (23) Lescoeur, H., Ann. de chim. et de phys. 7 Ser. 2 (1884), p. 78. 
 
 (24) Loevenhart, A. S., Zeit. f. physiol. Chem. 41 (1904), p. 177. 
 
 (25) Mellanby, J., Journ. of Physiol. 33 (1905), p. 338. 
 
 (26) Michaelis, L., and Rona, P., Biochem. Zeit. 28 (1910), p. 197. 
 
 (27) Miescher, F., Verhand. Naturforsch. Ges. in Basle 6 (1874), p. 138. 
 
 (28) Miescher, F., Arch. f. Exper. Path, und Pharm. 37 (1896), p. 100. 
 
 (29) Osborne, T. B., Journ. Amer. Chem. Soc. 21 (1899), p. 486. 
 
 (30) Osborne, T. B., Zeit. f. physiol. Chem. 33 (1901), p. 240. 
 
 (31) Osborne, T. B., "The Vegetable Proteins," London (1909). 
 
 (32) Osborne, T. B., Ergeb. d. physiol. 10 (1910), p. 47. 
 
 (33) Osborne, T. B., and Leavenworth, C. S., Journ. Biol. Chem. 28 (1916), 
 
 p. 109. 
 
 (34) Osborne, W. A., Journ. of Physiol. 27 (1901), p. 398. 
 
 (35) Osborne, W. A., Proc. physiol. Soc.; Journ. of Physiol. 33 (1905), p. 
 
 10; 34 (1906), p. 84. 
 
 (36) Robertson, T. Brailsford, Journ. Biol. Chem. 2 (1907), p. 317. 
 
106 CHEMICAL STATICS 
 
 (37) Robertson, T. Brailsford, Journ. Physical Chem. 11 (1907), p. 437. 
 
 (38) Robertson, T. Brailsford, Journ. Biol. Chem. 4 (1908), p. 35. 
 
 (39) Robertson, T. Brailsford, Journ. Biol. Chem. 5 (1908), p. 155. 
 
 (40) Robertson, T. Brailsford, Zeit. f. Kolloidchem. 3 (1908), Heft. 2. 
 
 (41) Robertson, T. Brailsford, Journ. Physical Chem. 13 (1909), p. 469. 
 
 (42) Robertson, T. Brailsford, Journ. Physical Chem. 14 (1910), p. 528. 
 
 (43) Robertson, T. Brailsford, Journ. Physical Chem. 14 (1910), p. 709. 
 
 (44) Robertson, T. Brailsford, Journ. Physical Chem. 15 (1911), p. 179. 
 
 (45) Robertson, T. Brailsford, Journ. Physical Chem. 15 (1911), p. 387. 
 
 (46) Robertson, T. Brailsford, "Die physikalische Chemie der Proteine," 
 
 Dresden (1912), p. 71. 
 
 (47) Salkowski, E., Zeit. f. Biol. 37 (1899), p. 404. 
 
 (48) Salm, E., Zeit. f. physikal. Chem. 57 (1906), p. 471. 
 
 (49) Samuely, F., Oppenheimer's Handbuch der Biochem. (1909), Bd. 1, 
 
 p. 35. 
 
 (50) Schlossmann, A., Zeit. f. physiol. Chem. 22 (1896), p. 197. 
 
 (51) Soldner, F., Landwirthschaft. Versuchst. 35 (1888), p. 351. 
 
 (52) Soldner, F., Zeit. f. angew. Chem. (1895), p. 370. 
 
 (53) Taylor, A. E., Univ. of California Publ. Pathol. 1 (1904), p. 7. 
 
 (54) Timpe, H., Arch. f. Hyg. 18 (1893), p. 1. 
 
 (55) Trunkel, H., Biochem. Zeit. 26 (1910), p. 493. 
 
 (56) Van Slyke, L. L., and Bosworth, A. W., New York Agric. Expt. Stn. 
 
 Technic. Bull. Nr. 26 (1912). 
 
 (57) Van Slyke, L. L., and Bosworth, A. W., Journ. Biol. Chem. 14 (1913), 
 
 p. 211. 
 
 (58) Van Slyke, L. L., and Bosworth, A. W., Journ. Biol. Chem. 14 (1913), 
 
 p. 227. 
 
 (59) Van Slyke, L. L., and Hart, E. B., Amer. Chem. Journ. 33 (1905), p. 461. 
 
 (60) Van Slyke, L. L., and Van Slyke, D. D., Amer. Chem. Journ. 38 (1907), 
 
 p. 383. 
 
 (61) Van Slyke, L. L., and Van Slyke, D. D., Journ. Biol. Chem. 4 (1908), 
 
 p. 259. 
 
 (62) Van Slyke, L. L., and Winter, O. B., Journ. Biol. Chem. 17 (1914), p. 
 
 287. 
 
CHAPTER VI 
 THE COMPOUNDS OF THE PROTEINS (Continued) 
 
 1. General Remarks on the Precipitation of Proteins by 
 Inorganic Salts. It was pointed out by Hardy (24) in his 
 exhaustive communication on globulin that the precipitation 
 of proteins and, indeed, of colloids in general may be of two 
 kinds. The first is clearly accompanied by decomposition of 
 the precipitating agent; it will not occur, as Pauli has demon- 
 strated (56) unless the protein is in some proportion ionized, 
 and relatively small quantities of the precipitating agent are 
 required to bring about the precipitation. The second kind of 
 precipitation, however, whether accompanied by decomposition 
 of the precipitating agent or not, occurs even when the protein 
 is non-ionic and requires relatively large amounts of the pre- 
 cipitating agent. Precipitation of the first kind is, generally 
 speaking, only brought about by electrolytes, while precipitation 
 of the second kind, although as a rule, more readily brought 
 about by electrolytes than by non-electrolytes, may nevertheless 
 be brought about by certain non-electrolytes, for example, by 
 alcohol. 
 
 For this latter type of precipitation we shall henceforth, when- 
 ever possible, reserve the term coagulation.* Both precipitation 
 and coagulation of a protein may be brought about by one and 
 the same inorganic salt. In such a case the gradual addition of 
 salt to the originally salt-free solution which contains ionic pro- 
 tein, i.e., protein which drifts to one electrode or to the other in 
 an electric field, first brings about precipitation and then reso- 
 lution of the protein. In this new solution the protein is, accord- 
 
 * Much confusion exists in the literature on this subject on account of the 
 fact that the distinction between the precipitation of a protein through chemical 
 interaction with the added salt, and its coagulation through the change in the 
 nature of the solvent resulting from the further addition of salt has not invari- 
 ably been recognized. 
 
 107 
 
108 CHEMICAL STATICS 
 
 ing to Hardy, non-ionic and it can be coagulated by still further 
 addition of the salt.* 
 
 The first kind of precipitation appears to be undoubtedly 
 chemical in character and in mechanism. The mechanism of 
 coagulation is, however, far from clear, and for the attainment 
 of an adequate understanding of this phenomenon we shall 
 doubtless have to wait until the physico-chemical theory of solu- 
 tions in general has reached a more mature stage of development 
 than it has at present. At least three possibilities exist. 
 
 (i) The coagulation of proteins by salts is a purely physical 
 phenomenon due either to an alteration in the electrical condition 
 of the protein (Bredig, Billitzer, Freundlich) or to a physical 
 alteration in the nature of the solvent. 
 
 (ii) The coagulation of proteins by salts is partly a physical 
 and partly a chemical phenomenon depending upon the forma- 
 tion of various compounds between the protein and the salt, 
 and upon their varying solubilities in salt solutions (Spiro, 
 Galeotti). 
 
 (iii) The coagulation of proteins by salts is indirectly a chemi- 
 cal phenomenon, attributable to a disturbance in the chemical 
 equilibrium between the protein and its solvent (Hofmeister, 
 Pauli). 
 
 In reviewing the various factors which have been ascertained 
 to be of importance in determining the precipitation and coagu- 
 lation of proteins by electrolytes we shall incidentally discuss the 
 applicability of these several hypotheses. 
 
 2. Earlier Investigations on the Significance of the State of 
 Hydration of the Proteins in Relation to their Coagulation by 
 Salts. The fact that proteins can be thrown out of solution by 
 the addition thereto of inorganic salts appears to have first been 
 pointed out by Claude Bernard (2), who employed, among others, 
 magnesium sulphate, sodium sulphate and ammonium carbonate. 
 As early as 1854, Virchow (76) suggested that the inorganic 
 
 * An interesting example of the dependence of the ionization of a protein 
 upon concentration (i.e., available mass of water) of the medium in which it 
 is dissolved is afforded by serum-globulin dissolved in solutions of sodium 
 citrate. In solutions containing low concentrations of sodium citrate the 
 protein is ionized and drifts in an electric field, while the same protein when 
 dissolved in more concentrated sodium citrate solution is found to be no longer 
 ionized (-9). 
 
EARLIER INVESTIGATIONS 
 
 109 
 
 salts render proteins insoluble by extracting water from them. 
 This suggestion was again put forward in 1888 by F. Hofmeister 
 (30), who, together with his pupils, advanced in the immediately 
 succeeding years, a number of experimental data in support of 
 his thesis (30) (31) (38) (62) (43). 
 
 Kiihne (39) and Kauder (38), having shown that not only the 
 concentration of the added salt but also that of the protein is 
 of importance in determining the amount of salt required for its 
 coagulation, care was taken in these and in the majority of suc- 
 ceeding investigations, when comparing the coagulating power of 
 different salts, to maintain the concentration of the protein 
 constant, the procedure being to add to a given volume of protein- 
 containing fluid (e.g., blood serum) varying amounts of different 
 salt solutions, and then dilute the mixture to a standard volume. 
 
 Lewith showed that the relative efficacy of the different salts 
 which he employed in coagulating the proteins of blood serum 
 was the same for serum globulins as for serum-albumins, sul- 
 phates and acetates being more powerful coagulants than nitrates 
 or chlorides. His results have been tabulated as follows by 
 Gustav Mann (46), a (+) indicating that the salts do, and a ( ) 
 that they do not precipitate serum-albumin. 
 
 
 Potassium 
 
 Sodium 
 
 Ammonium 
 
 Magnesium 
 
 Calcium 
 
 Barium 
 
 Acetate 
 
 + 
 
 + 
 
 
 
 
 
 Chloride. . .. 
 
 + 
 
 + 
 
 
 
 
 
 + 
 
 , 
 
 Nitrate 
 
 
 + 
 
 
 
 
 
 i 
 
 ^^ 
 
 Phosphate 
 
 
 + 
 
 
 
 
 
 Sulphate 
 
 _ 
 
 + 
 
 -f 
 
 + 
 
 
 
 Sulphocyanate 
 
 
 
 
 
 
 
 Iodide 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Bromide 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Chromate 
 
 
 
 _ 
 
 
 
 
 Bicarbonate 
 
 
 _ 
 
 
 
 
 
 
 
 
 
 
 
 
 Hofmeister, in 1888, extended and confirmed these observations, 
 employing not only serum-proteins but also egg-albumin, gelatin 
 and other colloids, namely colloidal ferric hydrate and sodium 
 oleate. He found that whatever the colloid employed the rela- 
 tive coagulating power of the various salts was the same. Excep- 
 tions to this rule were noted, however, when salts of di- or tri- 
 basic acids were employed, the order of their efficacy in coagu- 
 lating egg-globulin and gelatin being the same, but differing 
 
110 
 
 CHEMICAL STATICS 
 
 when ferric hydrate and sodium oleate were employed. Nasse 
 (49), in discussing the hypothesis that the coagulation of proteins 
 is due to the withdrawal of water from the colloid, had raised the 
 objection that the ratio of the concentrations at which the mag- 
 nesium and ammonium sulphates bring about coagulation of 
 different colloids is not always exactly the same. For different 
 colloids Nasse found the ratio 
 
 cone, of (NH 4 )2S04 just sufficient to coagulate 
 cone, of MgSO 4 just sufficient to coagulate 
 
 possessed the following values: 
 
 Gelatin 
 
 Egg-albumin 
 
 Serum-albumin 
 
 Hemi-albumose 
 
 Peptone 
 
 0.84 
 
 1.03 
 
 0.94 
 
 0.85 
 
 1.00 
 
 Hofmeister, however, pointed out that the absolute concen- 
 trations which are required to bring about coagulation in these 
 cases are very different, and an exact quantitative relation of 
 this kind could not be expected to hold good, since the condition 
 of the salts in solution, or, as we should now express it, their 
 relative degree of electrolytic dissociation, differs at different 
 absolute concentrations. 
 
 Following up the idea that the coagulation of colloids by salts 
 is attributable to the possession by the salts, in the concentra- 
 tions employed, of a greater power of binding water than that 
 possessed by colloid, Hofmeister (1890-91) took up the study of 
 the swelling or absorption of water by colloids in various solutions, 
 the degree of swelling in different solutions being regarded as a 
 measure of the relative binding capacities of the colloid and of 
 the dissolved salts. He found that in solutions of sulphates, tar- 
 trates, acetates, alcohol, cane-sugar or grape-sugar gelatin-plates 
 take up less water than they do when immersed in distilled 
 water, while in solutions of potassium, sodium or ammonium 
 chlorides, sodium chlorate, sodium nitrate and sodium bromide 
 they take up more water than they do when immersed in distilled 
 water. It will be recollected that the sulphates, tartrates and 
 acetates are the most energetic coagulants of gelatin. Regarding 
 their high coagulating power as being attributable to their power 
 of abstracting water from the protein, the interpretation of these 
 results -of Hofmeister's becomes clear. 
 
EARLIER INVESTIGATIONS 
 
 111 
 
 In 1898 Pauli (51) (58) published the results of a number of 
 investigations upon the influence of various salts on the gelatin- 
 izing- and melting-temperatures of gelatin solutions. He found 
 that effects of different salts upon the gelatinizing and melting 
 of strong gelatin solutions ran parallel with their power of coagu- 
 lating gelatin and of inhibiting the swelling of gelatin plates. 
 Thus chlorides, bromides, and iodides of potassium, sodium, am- 
 monium and magnesium lower the temperatures of gelatinization 
 and of melting, in the following order, the most effective being 
 placed first.* 
 
 Sodium sulphate, magnesium sulphate, sodium citrate, ammo- 
 nium sulphate, magnesium sulphate, sodium tartrate, sodium 
 acetate. The following is the order in which these salts bring 
 about the coagulation of gelatin, the most effective- being, placed 
 first: 
 
 Sodium sulphate, potassium sulphate, sodium citrate, mag- 
 nesium chloride, sodium tartrate, magnesium sulphate, ammonium 
 sulphate, sodium acetate^ potassium chloride, sodium chloride. 
 
 There is evidently a close, although not an absolute parallel- 
 ism between the two series. Urea and alcohol lowered, but 
 glycerin markedly raised the temperatures of gelatinization and 
 of melting. 
 
 In a later communication (52) Pauli showed that the order in 
 which various salts affect the coagulation-temperature of egg- 
 globulin is similar to that in which they occur in the above series. 
 The following table, cited after Pauli, gives the order in which 
 the various salts bring about the transformation of the proteins 
 from the dissolved into the solid condition: 
 
 Coagulation of 
 egg-globulin 
 (Hofmeister) 
 
 Coagulation of 
 gelatin 
 (Pauli) 
 
 Heat coagulation 
 of egg-globulin 
 (Pauli) 
 
 Gelatinization of 
 gelatin 
 (Pauli) 
 
 Swelling of gela- 
 tin 
 (Hofmeister) 
 
 Sulphates 
 Acetates 
 Citrates 
 Tartrates 
 Chlorides 
 Chlorates 
 
 Sulphates 
 Citrates 
 Tartrates 
 Acetates 
 Chlorides 
 
 Chlorides 
 Acetates 
 Sulphates 
 Chromates 
 Chlorates 
 Nitrates 
 
 Sulphates 
 Citrates 
 Tartrates 
 Acetates 
 Chlorides 
 Chlorates 
 
 Sulphates 
 Citrates 
 Tartrates 
 Acetates 
 Chorides 
 Chlorates 
 
 Nitrates 
 
 
 Bromides 
 
 Nitrates 
 
 Nitrates 
 
 
 
 Iodides 
 
 Bromides 
 
 Bromides 
 
 
 
 
 Iodides 
 
 
 
 
 
 
 
 All of these substances were employed in equimolecular solutions. 
 
112 'CHEMICAL STATICS 
 
 The nature of the cation was, however, found to be not wholly 
 without influence, since the temperature at which egg-globulin 
 coagulates is lowest in the presence of ammonium chloride, and 
 highest in the presence of magnesium chloride, the series being 
 as follows: NH 4 , K, Na, Li, Ba, Mg. The curves expressing the 
 relationship between the concentrations of the salts employed 
 and the temperature of coagulation are, however, not parallel, 
 and in some cases cut one another, so that at higher concentra- 
 tions (4 N) the series runs as follows : Na, NH 4 , Li, Ba, Mg. 
 
 Pauli also investigated the effect of mixtures of two salts, and 
 found, as had already been indicated by the results of Schafer 
 (71), that the coagulating action of salts upon proteins is additive, 
 that is, each salt exerts its separate effect and the precipitating 
 (or dissolving) power of the mixture is the algebraic sum of the sepa- 
 rate effects exerted by its components except when the two salts 
 have a common ion and so diminish each other's degree of disso- 
 ciation. This result has since been more fully confirmed by 
 Pauli (53) and by Mellanby (47). 
 
 The globulins, as a class, are insoluble in distilled water, but 
 are soluble in dilute saline solutions. Further addition of salt 
 results in coagulation of the globulin. Non-electrolytes can bring 
 about the coagulation but not the solution of the globulin. From 
 these facts Pauli concluded that the solution of globulin is due 
 to the formation of compounds of the globulin with salts, of the 
 
 tyP e: Na - globulin - Cl. 
 
 the further addition of salts to this solution resulting in the 
 precipitation of this compound. 
 
 3. The Influence of the Electrical Condition of the Proteins 
 upon their Precipitation and Coagulation by Electrolytes. The 
 action of very small quantities of salts in bringing about the 
 precipitation of colloids from their solution was first studied by 
 Graham (20). He ascertained, for example, that a solution of 
 colloidal aluminium hydrate prepared by dialysing the chloride 
 is so sensitive to the presence of salts that the mere addition of 
 a few drops of undistilled water suffices to precipitate it. This 
 type of action of salts upon colloids was further investigated by 
 Schultz (73), Prost (66), and Linder and Picton (44). 
 
 Defining the precipitating-power of a salt as the reciprocal 
 of the concentration in gram-molecules per litre necessary to 
 coagulate a given solution of the hydrosol of sulphide of arsenic, 
 Schultz found that the relative precipitating-powers of the uni- 
 
INFLUENCE OF ELECTRICAL CONDITION 
 
 113 
 
 valent, divalent and trivalent metals are in the ratios 1: 30: 1650. 
 Prost, employing the hydrosol of cadmium sulphide, obtained a 
 similar relation, while Linder and Picton found that the precipi- 
 tating powers of different salts of a given metal are proportional 
 to their equivalent conductivities and that the relative precipi- 
 tating powers of the sulphates of univalent, divalent, and trivalent 
 metals can be expressed by the ratios 1 : 35 : 1023. 
 
 In all of these cases the colloid employed was electronegative, 
 that is, on electrolysis it migrated to the anode. The experi- 
 ments which we have cited show that in such cases the ion of 
 the added electrolyte which is effective in bringing about precipi- 
 tation is the cation. In 1899, however, Hardy (21) showed that 
 egg-albumin, modified by heating its solution so as to partially 
 coagulate the protein, may be induced to travel either to the 
 anode or to the cathode by simply changing the reaction of its 
 solutions. In acid solutions the protein behaves like a cation, 
 migrating to the cathode; in alkaline solutions it behaves like 
 an anion, migrating to the anode. In alkaline solutions, the 
 cations of added salts proved to be the active agents in precipi- 
 tating egg-albumin, while in acid solutions the precipitating 
 power of the cation proved to be altogether subordinate to that 
 of the anion (22). The following illustrate his results: 
 
 PROTEIN IN PRESENCE OF TRACE OF ALKALI, ELECTRO- 
 NEGATIVE 
 Temperature 16 degrees. Coagulating salt 1 gram-mol. in 80,000 cc. 
 
 Coagulated at once 
 
 On slightly warming 
 
 Did not coagulate 
 
 A1 2 (S0 4 ) 3 
 Cd(N0 3 ) 2 
 CuSO 4 - 
 CuCl 2 
 
 MgS0 4 
 BaCl 2 
 CaCl 2 
 
 Na2S0 4 
 K 2 S0 4 
 NaCl 
 
 
 
 
 PROTEIN IN PRESENCE OF TRACE OF ACID, ELECTRO- 
 POSITIVE 
 
 Coagulated instantly 
 
 No effect 
 
 A1 2 (S0 4 ) 3 
 CaSO 4 
 K 2 S0 4 
 Na 2 SO 4 
 MgSO 4 
 
 CuCl 2 
 
 Cd(N0 3 ) 2 
 BaCl 2 
 NaCl 
 
 
 
114 CHEMICAL STATICS 
 
 Similar results were obtained with other colloids. Electroneg- 
 ative colloids are precipitated, if at all, by cations; electropositive 
 colloids, by anions. 
 
 Whetham (78) (26) explained these phenomena in the following 
 way: He assumes that at the surface of the colloid particles there 
 exists a double electrical layer. When the particles move towards 
 the anode they are negatively charged, when they move to the 
 cathode they are positively charged. The surface energy of the 
 colloidal phase is reduced by the presence of the electrical double 
 layer and therefore its tendency to contract. The existence of 
 the double layer, therefore, conduces to the stability of a system 
 in which the surface of the colloid is greatly extended, i.e., to the 
 stability of the " colloidal solution." The cations of the added elec- 
 trolyte, in the case of electronegative colloids, or the anions in the 
 case of electropositive colloids, neutralize this charge and therefore 
 increase the energy of the surface of the colloidal phase and its 
 tendency to contract. Hence the finely suspended colloidal par- 
 ticles unite to form large aggregates having a less extended surface, 
 and these aggregates finally become so large as to assume the 
 properties of matter in mass, and hence are carried out of solu- 
 tion by the action of gravity. In this way the dependence of 
 the precipitating-power of an electrolyte upon its dissociation, 
 found by Linder and Picton and by Hardy, and also the reversal 
 in the relative precipitating-powers of the ions of the added 
 electrolyte upon reversion of the sign of the electrical charge 
 carried by the colloid, found an explanation. In interpreting 
 the valency rule discovered by Schultz, Whetham develops his 
 theory as follows: 
 
 "In a solution where ions are moving freely, the probability 
 that an ion is at any instant within reach of a fixed point is, 
 putting certainty equal to unity, approximately represented by 
 a fraction proportional to the ratio between the volume occupied 
 by the spheres of influence of the ions and the whole volume of 
 the solution, and may be written as AC, where A is a constant 
 and C represents the concentration of the solution. The chance 
 that two such ions should be present together is the product of 
 their separate chances, -that is (AC) 2 . Similarly the chance for 
 the conjunction of three ions is (AC) 3 , and for the conjunction 
 of n ions is (AC) n ." 
 
 "In order that three solutions, containing trivalent, divalent, 
 
INFLUENCE OF ELECTRICAL CONDITION 115 
 
 and univalent ions respectively should have equal coagulative 
 powers, the frequency with which the necessary conjunctions 
 should occur must be the same in each solution. We should 
 then have, the constant being assumed equal in each case, 
 
 A 2 "C 3 2n = A 3 "C 2 3n = A 6n d &n = a constant = B. 
 
 Therefore 
 
 B~. B^ B^ 
 
 T> '"A' X' 
 
 Cij Cz, Ca, representing the concentrations of monads, diads., 
 and triads in their respective solutions. Thus we get for the 
 ratios of the concentrations of equi-coagulative solutions : 
 
 J_ _L J 
 B Gn : B* n : B 2 
 
 _ 
 Let us put B 6n = -', the ratios can then be written: 
 
 The reciprocals of the numbers expressing the relative concen- 
 trations of equi-coagulative solutions give values proportional to 
 the coagulative-powers of solutions of equal concentration; so 
 that, calling the coagulative-powers of equivalent solutions con- 
 taining monovalent, divalent, and trivalent ions respectively, pi, 
 P2, PS, we get: 
 
 Pi'.pz'.pa = l:x:x 2 , 
 
 Let us now take some numerical examples. 
 Putting x = 32 we get the series: 
 
 1:32:1024. 
 
 Which agrees very well with Linder and Picton's results for 
 colloidal solutions of antimony sulphide: 
 
 1:35:1023; 
 and putting x 40, we get 
 
 1:40:1600, 
 
 numbers comparable to Schultze's values for sulphide of arsenic" 
 (79). 
 
116 CHEMICAL STATICS 
 
 It is curious that during the discussions which immediately 
 followed these discoveries the fact should have been so generally 
 overlooked that the above hypothesis of Whetham's is simply 
 a statement, in kinetic terms, of the Guldberg and Waage mass- 
 law. According to this law, the velocity with which any given 
 reaction proceeds varies directly as the active masses of each 
 of the reacting molecules. In the case under consideration, 
 presuming that a given number (e.g., one) of molecules of protein 
 react with one molecule of a salt of a monovalent metal to form 
 a compound, then twice as many molecules of the protein may be 
 supposed to react with a molecule of a salt of a divalent metal, 
 and three times as many with a salt of a trivalent metal. Assum- 
 ing that the active mass of the colloid (the molecular concentra- 
 tion multiplied by the degree of dissociation) is the same in each 
 of these cases (which is also assumed in Whetham's theory) and 
 equal to A, calling the initial velocities of the respective reactions 
 Vi t v z , and v s and the concentrations of the mono-, di- and trivalent 
 ions Ci, Czj and Cs, we have: 
 
 Vi is proportional to Aci, 
 Vz is proportional to A 2 Cz, 
 3 is proportional to A 3 c 3 , 
 
 whence it follows that if Vi = v 2 = VB and the velocity-constants of 
 the three reactions are equal (which is also assumed in Whetham's 
 hypothesis) :* 
 
 and , and , i.e., the dilutions of the mono-, di- and trivalent 
 
 Ci ' C 2 C 3 ' 
 
 ions at which combination proceeds with equal velocity are re- 
 lated to one another in the same way. Now in the experiments 
 
 described above, - is denned as p, the precipitating-power of the 
 
 salt, hence: 
 
 Pi'.pz'.ps: : I: A: A 2 , 
 
 which is exactly the relation deduced by Whetham. The rela- 
 tions found by Schultze, Prost, Linder and Picton and others, 
 are, therefore, just as explicable upon the assumption that the 
 
 * In the terminology of Whetham's hypothesis, the term "velocity con- 
 stant" would read "proportion of effective collisions." 
 
INFLUENCE OF ELECTRICAL CONDITION 117 
 
 colloid reacts chemically with the precipitating ion as upon 
 the assumption that the precipitating ion acts merely through 
 altering the electrical condition of the colloidal particles. The 
 former view attributes to the colloids no especial qualities which 
 differentiate them from other chemical systems, the latter view 
 necessarily involves assumptions regarding the nature of col- 
 loidal solutions which have hitherto proved incapable of veri- 
 fication. 
 
 It is not surprising that velocities rather than equilibria deter- 
 mine the "precipitating-powers" of reagents for colloids, when 
 we recollect, firstly, the enormous part played by the velocity of 
 change in the final physical condition of a colloid, f and secondly 
 the method by which these "precipitating-powers" are measured. 
 Linder and Picton measured the precipitating-power of a salt by 
 titration, running the solution of the salt into the solution of 
 arsenic sulphide until coagulation just began to be perceived. 
 They expressly state that unless the time occupied in the titra- 
 tion be kept approximately the same, serious deviations from the 
 above rule occur, "as a quantity of coagulant insufficient to pro- 
 duce coagulation immediately will do so in the course of time." 
 Under these conditions, what is actually measured is the con- 
 centration of the precipitating agent which is requisite to bring 
 about a given degree of change (visible precipitation) within a 
 given brief period, that is, a velocity and not an equilibrium. 
 Data regarding the degree of precipitation after varying periods 
 and the equilibria which are attained in such reactions as these 
 are lacking, but Linder and Picton have shown that the cation 
 which brings about the precipitation of colloidal arsenic is bound 
 by the colloid and carried down with it. It cannot be washed out 
 with water, but it can be replaced by another metal. Whitney 
 and Ober (80) obtained similar results and, moreover, showed that 
 different metals are carried down by this colloid in equivalent- 
 molecular proportions. 
 
 That these results are susceptible of purely chemical inter- 
 pretation was pointed out by Hardy (22) in 1900 and this view 
 was further developed by him in his communications on globulin 
 referred to above (24). He showed that in a series of salts of 
 
 * According to W. B. Hardy (23), the more slowly the division into two 
 phases occurs the smaller and less curved is the surface of separation. Cf. 
 also Freundlich (15). 
 
118 CHEMICAL STATICS 
 
 the same valency the power of precipitating an electronegative 
 colloid varies directly as the equivalent conductivity of the salt, 
 that is, as the active mass of the dissociated metal ions, Since 
 electronegative colloids appear to behave, electrically, like the 
 anions of an acid, and electropositive colloids like the cations 
 of a base, their respective affinities for the basic and acid radicals 
 of salts are readily explicable. This fact appears, however, to 
 have been first clearly pointed out by Loeb (45). 
 
 An interpretation of the phenomena attending the precipita- 
 tion of ionic (" electrically active") colloids by electrolytes prac- 
 tically identical with that of Whetham was afterwards brought 
 forward by Bredig (7). 
 
 Modifications of Whetham's theory have been advanced by 
 Billitzer (3) (4) and by Freundlich (15) (16). Billitzer objected 
 to the assumption which was made by Whetham (although as 
 we have seen, it was not at all essential to his hypothesis) that 
 an electrical double layer exists at the surface of colloid particles 
 in aqueous solution and showed that this assumption is inadequate 
 to explain the migration of the particles in an electrical field. 
 The charge on the colloid particles must therefore be supplied 
 by the particles themselves, by giving up oppositely charged 
 ions to the solution, i.e., the colloids must be electrolytes. So far 
 his view is identical with that which is developed above. But 
 he adds to this the assumption that the charge which is carried 
 by the colloid particles is not a full atomic charge. The precipi- 
 tating action of oppositely charged ions he attributes to the 
 electrostatic attraction of the colloid particles by the oppositely 
 charged crystalloid ion. Since the colloid particle is assumed not 
 to possess a full atomic charge, a number of particles must, he 
 considers, be attracted by one crystalloid ion, thus forming a 
 molecular group large enough to be appreciably acted on by 
 gravity. How the colloid particles come to acquire a fraction 
 of an atomic charge Billitzer does not explain, nor does our ex- 
 perience of the behavior of electrolytes afford any legitimate 
 precedent for such an assumption. 
 
 Freundlich believes that the precipitating ion must penetrate 
 (be "adsorbed" by) the surface of the colloid particle and that 
 the surfaces of colloids are permeable to ions possessed of an 
 opposite but not to ions possessed of the same charge as their 
 own. The precipitating powers of various salts are stated to 
 
LATER INVESTIGATIONS 119 
 
 run parallel with the degree to which they are "adsorbed" by 
 finely pulverized charcoal. 
 
 Biltz (5) also considers that the precipitation of colloids by 
 electrolytes and by other colloids is due to the formation of 
 "adsorption compounds." 
 
 4. Later Investigations on the Significance of the State of 
 Hydration of the Proteins in Relation to their Coagulation by 
 Electrolytes. Continuing his investigations, cited above, on the 
 coagulating action of pairs of salts on egg-white, Pauli (53) found 
 that a number of salts which will not coagulate egg-white by 
 themselves will increase the coagulating power of other salts 
 when mixed with them, while others markedly diminish the 
 coagulating power of salts which, in their absence, readily coagu- 
 late egg-albumin. Moreover certain salts, although very soluble, 
 do not in any concentration cause coagulation of egg-white. 
 The possibility suggested itself that the coagulating action of 
 salts might depend upon two antagonistic factors, respectively 
 attributable to the cations and the anions of the salts. In con- 
 firmation of this view it was found that the NEU ion, when com- 
 bined with S0 4 will coagulate egg-albumin, although when com- 
 bined with acetanion it will not. Acetanion coagulates when 
 combined with sodium, but not when combined with ammonium. 
 Pursuing this line of reasoning and Investigation Pauli concluded 
 that if the coagulating powers of a series of cations be indicated 
 by /, /', /", . . . and the opposite (solvent) powers of a series 
 of anions by h, h', h", . . . , then in a mixture of electrolytes the 
 following possibilities exist : 
 
 2 (/,/', A . . .)iz(M',ft", 0, 
 
 the mixture being such as to coagulate, leave unaffected or in- 
 hibit the coagulation of the albumin. Pauli found that in egg- 
 white (in which the protein is electronegative) the cations of 
 added electrolytes are the active agents in inducing coagulation, 
 while the anions inhibit coagulation. In the following table of 
 Pauli's the cations are arranged in ascending order of precipi- 
 tating-power from left to right, while the anions are arranged 
 vertically, the weakest inhibitor coming first and the strongest 
 last. A (+) indicates that the salt which results from the union 
 of the cation and anion causes coagulation of egg-albumin; while 
 a ( ) indicates that it does not. 
 
120 
 
 CHEMICAL STATICS 
 
 
 
 
 Cations 
 
 
 
 
 Mg 
 
 NH 4 
 
 K 
 
 Na 
 
 Li 
 
 Fluoride 
 
 
 4- 
 
 4. 
 
 4. 
 
 
 Sulphate . 
 
 4- 
 
 4- 
 
 4- 
 
 4. 
 
 4- 
 
 Phosphate 
 
 
 4- 
 
 4- 
 
 4- 
 
 
 Citrate .... 
 
 
 4- 
 
 4- 
 
 4- 
 
 
 Tartrate 
 
 
 4- 
 
 4- 
 
 4- 
 
 
 Acetate 
 
 _ 
 
 
 4- 
 
 4- 
 
 
 Chloride 
 
 _ 
 
 _ 
 
 4- 
 
 4- 
 
 4- 
 
 Nitrate 
 
 _ 
 
 _ 
 
 
 4- 
 
 4- 
 
 Chlorate 
 
 
 
 
 4- 
 
 
 Bromide 
 
 _ 
 
 
 
 
 + 
 
 Iodide 
 
 
 
 
 
 
 Thiocyanate . 
 
 _ 
 
 _ 
 
 _ 
 
 _ 
 
 
 
 
 
 
 
 
 The valency of the coagulating ion would appear to be quanti- 
 tatively of less importance in the coagulation of proteins by 
 salts than it is in the precipitation of ionic protein by small 
 quantities of salts, since magnesium resembles the alkalies closely 
 in its coagulative power, while lithium approaches the alkaline 
 earths. 
 
 The order of effectiveness of the different salts in bringing 
 about the coagulation of electropositive protein is, however, exactly 
 the reverse of their order of effectiveness in bringing about the 
 coagulation of electronegative protein, such as the albumin in egg- 
 white. This was first shown by Posternak (64), who employed 
 the reserve-material of the seeds of Picea excelsa, dissolved in 
 very dilute hydrochloric acid. Posternak's observation has been 
 confirmed by Pauli (53) and by Hoeber (29). Acidifying a 
 solution of egg-white reverses the functions of the coagulating 
 ions, those which coagulated electronegative protein most strongly 
 now inhibit its coagulation most strongly. Those which inhibited 
 its coagulation now induce coagulation. The series is reversed 
 in every respect; the anions now induce coagulation and the 
 cations inhibit it. The anions precipitate in the order: 
 
 CNS > I > Br > NO 3 > Cl > Acetate, 
 while the cations inhibit precipitation in the order: 
 Li > Na > K > NH 4 > Mg. 
 
 When electronegative protein (egg-white) is acted upon by 
 salts of the alkaline earths "irreversible" precipitates or coagula 
 
LATER INVESTIGATIONS 121 
 
 are formed, insoluble, that is, on dilution of the system with 
 water. The alkaline earths, moreover, afford a strong contrast 
 to the alkalies, in that the precipitating-power of the cation is 
 increased by the anions, in the series which is characteristic for 
 electropositive protein (54). In other words, when egg-white is 
 acted upon by salts of the alkaline earths the protein behaves, so 
 far as the action of anions upon it is concerned, as though it were 
 in acid solution. Pauli believes that the alkaline earth reacts 
 with OH groups of the protein to form the comparatively slightly 
 dissociated hydrates of the alkaline earths, the acid which is set 
 free inducing the acid reaction of the medium. In other words, 
 Pauli's view is that protein aids the action of water in bringing 
 about hydrolytic dissociation. Other colloids would appear to 
 act similarly, since Whitney and Ober (80) found that on the 
 addition of neutral halogen salts of the alkaline earths to col- 
 loidal arsenic sulphide the reaction of the solution becomes acid. 
 The influence of added salts of the alkalies and magnesium 
 upon the precipitation of proteins by heavy metals varies with 
 the concentration of salt employed (55). At low concentrations 
 (0.005 m.) the salts inhibit precipitation in the order: 
 
 S0 4 < Cl< C 2 H 3 2 < N0 3 < Br < I< SON, 
 
 while in high concentrations (4 m.) they encourage precipitation 
 in the order: 
 
 SO 4 > Cl > C 2 H 3 2 > NO 3 > Br > I > SON. 
 
 This is simply a particular instance of the rule, to which the 
 attention of the reader was drawn in the earlier part of this 
 chapter, that the salts may act as precipitants and as coagulants 
 at low and at high concentrations respectively, acting as solvents 
 at intermediate concentrations. The heavy metal salts afford no 
 exception to this rule. At low concentrations they precipitate, at 
 higher concentrations they dissolve, and at still higher concentra- 
 tions they coagulate the proteins of egg-white (55). Copper sul- 
 phate acting upon egg-albumin would appear to afford an excep- 
 tion since it does not coagulate even in saturated solution. That 
 the exception is apparent and not real is, however, shown by the 
 observation of Galeotti (17) that in sufficiently supersaturated 
 solution copper albuminate is coagulated by copper sulphate, 
 addition of merely saturated copper sulphate solution redissolves 
 
122 CHEMICAL STATICS 
 
 this coagulum, addition of water then results in the precipitation 
 of copper albuminate. 
 
 The concentration-range throughout which the salt acts as 
 solvent may be evanescent, as it is in the case of silver nitrate 
 acting upon egg-albumin (Pauli). 
 
 It was already shown in 1833 by Rose (68) that haemoglobin 
 is coagulated by concentrated mercuric chloride, but may be 
 redissoived by dilution, to be precipitated on further dilution. 
 
 The very important observation has been made by Pauli (56) 
 that absolutely electrolyte free egg-albumin is not ionic (i.e., 
 does not drift in an electric field) and that under these conditions 
 it is not precipitable by heavy metals. It is, however, coagulated 
 by highly concentrated salts.* 
 
 According to Pauli and Handovsky (57) (60) the number of 
 ionized protein particles in a solution of ionic protein is dimin- 
 ished by the addition of salts; at the same time the viscosity of 
 the solution diminishes and the coagulability of the protein by 
 the usual coagulating agents is increased. When salts are added 
 to a solution of electropositive protein (i.e., protein combined 
 with acid) an increase in the acidity of the solution results (24). 
 They believe that acid-protein reacts with salts as follows: 
 
 H H 
 
 NH 2 ( NH 2 ( 
 
 / X C1 / X C1 
 
 R( + NaN0 3 = R\ + HN0 3 , 
 
 X COOH XX)ONa 
 
 while alkali-protein reacts as follows: 
 
 / H / K 
 
 NH 2 ( NH 2 ' 
 
 / OH / X C1 
 
 R( \ + KC1 = R\ + H 2 0. 
 
 ' X COONa N COONa 
 
 On the basis of the more probable view of the electrolytic 
 dissociation of proteins outlined in Chap. I, this hypothesis 
 would be represented by the following schematic formulae: 
 
 * Egg-albumin is nearly equally basic and acid. It is probable that the 
 proteins which are either predominantly acid or basic may still, in some 
 measure, be electrolytically dissociated when uncombined with bases or acids. 
 
APPLICATIONS OF THE PHASE-RULE 123 
 
 Cl Cl 
 
 ^ I ' 
 
 -COH++ + ^N- + NaN0 3 = -CONa = N- + HN0 8 
 
 I I 
 
 H H 
 
 OH Cl 
 
 -CONa++ + ^N- + KC1 = -CONa = N-'+ H 2 O. 
 I I 
 
 H K 
 
 The symmetry of the action of the salts upon the acid and 
 alkali protein compounds would appear, however, to indicate a 
 greater similarity in the structure of the compounds formed 
 than that which is suggested by these formulae. 
 
 5. Applications of the Phase-rule to Protein-salt-water Sys- 
 tems. In addition to the above investigations attempts have 
 been made to interpret the behavior of the proteins in the pres- 
 ence of salts, in the light of the phase-rule, starting from the 
 view developed by Spiro (74) (10) that the coagulation of pro- 
 teins by salts might be regarded as a separation of the system 
 into two phases, a solid phase rich in protein, or a protein-salt 
 compound, and poor in salt and water, and a liquid phase poor 
 in protein, rich in salt and water. 
 
 The phase-rule, as developed by Willard Gibbs (19), van't 
 Hoff and Roozeboom may be enunciated as follows: A system of 
 r coexistent phases containing n independently variable compo- 
 nents is capable of n + 2 r variations in the temperature, 
 total pressure of the system or concentration of the components 
 of its phases. A " phase" is a portion of the system separated 
 from the rest by a definite surface, the " independently variable 
 components" are the least number of different substances with 
 which it is possible to represent, in a chemical equation, the 
 composition of each phase in the equilibrium (14). Only equi- 
 libria are contemplated in the derivation of the phase-rule, con- 
 sequently it is not applicable to systems which by reason of 
 hysteresis or limited reaction- velocity * are in a condition of 
 incomplete equilibrium. A discussion of the theoretical basis of 
 
 * Unless the reaction-velocity in question is so small as to be negligible in 
 comparison with the velocity with which the equilibrium under examination 
 is attained; for example, the hydrolysis of protein by water in the system 
 protein-salt-water. 
 
124 CHEMICAL STATICS 
 
 the phase-rule would be out of place here, since an exposition 
 of the principles underlying its application can be found in any 
 general work on physical chemistry. 
 
 Galeotti (17) (18) has applied the phase-rule to the systems 
 egg-albumin, CuSO 4 , H 2 0, serum-albumin CuS0 4 , H 2 O; serum- 
 albumin, AgN0 3 , H 2 0; egg-albumin, AgN0 3 , H 2 0; while Hardy 
 (25) has applied it to the systems serum-globulin, salts, H 2 0. 
 Both observers employ the triangular co-ordinates recommended 
 by Roozeboom, in which the three sides of an equilateral triangle, 
 each equal to unity, represent the components of the system and 
 the distances of a point P from these axes, measured in a direc- 
 tion parallel with the sides of the triangle, express the compo- 
 sition of a ternary mixture corresponding to the point. Since 
 each of the systems investigated by these observers is a system 
 consisting of two immiscible bodies, protein and salt, which are 
 both partially miscible in a third, water, they are comparable 
 with the system water, sodium chloride and succinic nitrile, 
 which has been studied by Schreinemakers (72). The form of 
 the isotherm obtained by Hardy and by Galeotti is exactly 
 that obtained by Schreinemakers in the above-named system. 
 
 The temperature and pressure in these investigations being 
 arbitrarily fixed and the components being three in number and 
 in equilibrium with water-vapor at the pressure and temperature 
 employed, the number of degrees of freedom is 3 r, where r 
 is the number of phases. From this it follows that for a given 
 temperature and pressure there can be, in general, not more than 
 three phases in equilibrium with water-vapor coexisting in the 
 system at one time. 
 
 Now Galeotti, as has been mentioned above, finds that solid 
 CuSC>4 + 5 H 2 0, solid protein and water can coexist in the sys- 
 tem at the same time, the water being saturated with CuS0 4 + 
 5 H 2 O andralso containing some dissolved protein, as is shown by 
 the fact that dilution of the fluid phase causes further precipitation 
 of protein. From this it follows that the protein, while in solution, does 
 not constitute a separate phase and is not divided off from the solvent 
 by a surface enwrapping molecules which are not in physical con- 
 tact with the fluid, for otherwise there would be four coexistent 
 phases; this simple consideration appears to have been over- 
 looked by the majority of writers on colloids and by many of 
 the advocates of "adsorption." 
 
PRECIPITATION AND COAGULATION 125 
 
 The general thermodynamic conditions of equilibrium in sys- 
 tems in which the protein or other colloid forms a region divided 
 from the remainder of the system by a boundary at which abrupt 
 change of properties takes place (as in a suspended coagulum 
 or a jelly) have been elaborated by Tolman (75). 
 
 In his first communication (17) Galeotti arrived at the con- 
 clusion that proteins do not form unions of definite composition 
 with copper, since the copper can be partially and progressively 
 removed from the protein by washing. I have dwelt in a pre- 
 vious chapter (Chap. V) upon the fallibility of this reasoning. 
 In his second communication (18), however, Galeotti obtained 
 data which indicate that at the moment of precipitation of egg- 
 albumin by AgN0 3 , a compound is formed of perfectly definite 
 molecular weight and solubility in distilled water. These data 
 will be more fully discussed in the succeeding chapter. 
 
 Keeping the quantities AgN0 3 and water constant and alter- 
 ing the concentration of protein (egg-albumin) Galeotti finds 
 that as protein is successively added a rapid diminution in the 
 number of silver ions occurs, but with the appearance of a pre- 
 cipitate the diminution proceeds more slowly and finally tends to a 
 constant maximum. 
 
 6. The Chemical Mechanics of the Precipitation and Coagu- 
 lation of Proteins by Salts. We have seen that in order that 
 precipitation of a protein by salts may occur the protein must 
 be ionized, but for coagulation this condition is not requisite. 
 In determining the rate of precipitation the valency of the pre- 
 cipitating ion is of prime importance, in determining the rate 
 of coagulation it is of comparatively subordinate importance. 
 For precipitation very low concentrations of the precipitating 
 salt suffice, for coagulation high concentrations of the salt are 
 required. This latter fact, and the fact that the presence of 
 coagulating salts aids coagulation by alcohol and by heat suggests, 
 as it did to Hofmeister, that coagulation is dependent upon 
 dehydration of the protein. 
 
 Starting from the observation of Jones and Ota (35) that 
 certain salts, when dissolved in water, produce an abnormal 
 depression of the freezing-point, Jones and his pupils have built 
 up a very large body of evidence for the existence of hydrates 
 (or "solvates") of substances in solution (32) (37). These 
 investigators find that both ions and undissociated molecules can 
 
126 CHEMICAL STATICS 
 
 form "solvates," and that these hydrates or "solvates" are 
 readily decomposed at temperatures which approach the boiling 
 point of the solvent and by the presence of other agents in the 
 solution which compete for the solvent.* An interesting attempt 
 has been made, upon the basis of this hypothesis, to explain the 
 color changes which many salts undergo in the presence of vary- 
 ing amounts of water or of dehydrating agents (37) (33) (36) 
 (42) (34). As is well known, anhydrous cobalt chloride is blue, 
 but on taking up water it becomes violet or red. Ostwald (50) 
 believed that the undissociated cobalt chloride is blue, while the 
 cobalt ion is red. Since, however, the color of a concentrated 
 solution of cobalt chloride can be changed from purplish red to 
 blue by the addition of small amounts of calcium, or still smaller 
 amounts of aluminium chlorides, or by the addition of a few 
 drops of alcohol (1) Lewis concludes that this change in color 
 is due to dehydration of the cobalt chloride molecule in solution, 
 by the abstraction of water from it by the added substance, 
 similar conclusions had previously been reached by other observ- 
 ers (70) (63) (81) (40) (27) (28). Similarly the progressive 
 change in color of cupric chloride solutions, from blue to greenish 
 brown, on concentration or dehydration is attributed to the loss 
 of water on the part of cupric chloride-water complexes. Lewis 
 finds that if various bromides be added to concentrated solutions 
 of cupric bromide the copper salt is dehydrated (turned brown) 
 by the salts of monovalent metals in the order Li > (Na, NH 4 ) > K. 
 For the chlorides the order was Li > Na > NH 4 > K. Di- 
 valent metals dehydrate more strongly, the order being Mg > 
 
 * A very striking experiment illustrating the formation of "solvates" is 
 that cited by Pickering (6). If a mixture of propyl alcohol and water be 
 placed in a semipermeable vessel and surrounded with water, it is found that 
 water enters the cell, but that no propyl alcohol escapes. If, however, the 
 same semipermeable vessel, containing the same mixture of alcohol and water 
 be immersed in propyl alcohol, propyl alcohol enters the cell and water does 
 not leave it. In other words the vessel is permeable to either propyl alcohol 
 or water when these are pure, but it is impermeable to mixtures of the two, 
 the inference being that large molecular complexes are formed on mixing these 
 reagents, which cannot pass through the pores of the vessel. From these and 
 similar experiments Poynting (65) concludes that osmotic pressure is an ex- 
 pression of the diminution in tKe active mass of the solvent due to the formation 
 of compounds with the dissolved substance. For a fuller discussion of these 
 and similar hypotheses the reader is referred to current works on physical 
 chemistry. 
 
PRECIPITATION AND COAGULATION 127 
 
 Ca > Sr > Ba while trivalent metals (Al) act still more ener- 
 getically. In opposition to this view Donnan (11) (12) (41) (48) 
 (13) advances the hypothesis that the blue color of concen- 
 trated solutions of cobalt chloride is due to the formation of 
 complex ions of the type CoCl 2 .Cl 2 ". Lewis points out, however, 
 that this view is inconsistent with the fact that it is possible to 
 change the color of the solution from blue to red by mere dilu- 
 tion, without altering the active mass of any component of the 
 system except water. Other objections against Donnan's view 
 have been urged by Hartley and H. C. Jones. 
 
 The peculiar interest to the biological chemist of the possi- 
 bility thus indicated, that substances dissolved in water form 
 loose combinations with the solvent, lies in the especial signifi- 
 cance of water in relation to the protein and polypeptid struc- 
 ture. As indicated in the first chapter, dehydration of a protein 
 may result in the following reactions: 
 
 /NH 3 OH HOOC. /NH 3 .OOC.R,NH 3 OH 
 
 R; + / R = R v + H 2 
 
 N COOH HOH 3 N X N COOH 
 
 , NH 3 OOC.R.NH 3 OH , NH.OC.R.NH 3 OH 
 R' = R( + H 2 
 
 N COOH X COOH 
 
 , NH.OC.R.NH 3 OH , NH.OC.R.NH 2 
 R' = R' + H 2 
 
 X COOH X COOH 
 
 , NH.OC.R.NH 2 / NH.OC.R.NH 
 R' = R' I +H 2 
 
 X COOH X CO- 
 
 and hydration, of course, may result in the reversion of this 
 series of changes. 
 
 That proteins may be thrown out of solution in two very dif- 
 ferent conditions of hydration is evident from the researches 
 cited in the previous sections of this chapter; it is even more 
 clearly shown by the following experiments (67): 
 
 Anhydrous casein dissolves readily in cold anhydrous * formic 
 acid; still more readily in hot formic acid If, to a two per cent 
 solution of casein in formic acid, we add a fairly concentrated 
 
 * Anhydrous, that is, save for traces of moisture derived from the atmos- 
 phere. 
 
128 CHEMICAL STATICS 
 
 solution of cupric chloride, the mixture is at first green, indicating 
 the presence of lower hydrates of cupric chloride, but on adding 
 more of the solution it becomes blue, and simultaneously with 
 the appearance of a pure blue color, but not before, precipitation 
 of cupric caseinate occurs. If, to 5 cc. of a 2 per cent solution in 
 formic acid, we add 1J, 2, or 2\ cc. of a saturated solution of 
 cupric chloride, no precipitation of the caseinate occurs, but on 
 diluting this mixture with water a precipitate results, and the 
 appearance of this precipitate coincides with the attainment of a 
 clear blue color on the part of the mixture. 
 
 About six cubic centimetres of water are required to produce 
 a permanent precipitate. This precipitate redissolves on heat- 
 ing and the mixture simultaneously becomes green; on cooling the 
 blue color reappears and with it the precipitate. If formic 
 acid be added to the mixture the precipitate redissolves as soon 
 as the mixture becomes green. If the precipitate be very slight 
 it will redissolve on adding alcohol. It cannot be urged that the 
 formation of cupric caseinate requires the presence of a sufficient 
 concentration of cupric ions, because green solutions of cupric 
 chloride contain an abundance of ions * and casein will react 
 with very small amounts of metal ions, for although it is itself 
 insoluble it will drive carbonic acid out of the sparingly soluble 
 calcium carbonate to form a freely soluble caseinate of calcium 
 (vide Chap. V). 
 
 If instead of adding water to a mixture of 5 cc. of 2 per cent 
 casein in formic acid and 2 cc. of saturated CuCU, we add alcohol, 
 no coagulation occurs until the mixture changes in color from 
 green to brown when a coagulum of cupric caseinate is produced 
 which redissolves on adding water. 
 
 Similar results are obtained when a 2 m. solution of cobalt 
 chloride is employed instead of a saturated solution of cupric 
 chloride. If to 5 cc. of a 2 per cent solution of casein in formic 
 acid we add 2 to 3 cc. CoCl 2 we obtain a blue-purple solution; 
 
 * Green solutions containing, probably, a mixture of the anhydrous brown 
 salt and the fully hydrated blue salt. Even on the basis of the hypothesis 
 urged by Donnan (11), (12), therefore, a considerable number of cupric ions 
 must exist in green solutions. It is important to notice that the precipitation, 
 as stated above, does not occur until the solution is pure blue in color; mix- 
 tures so slightly green that they appear wholly blue until viewed alongside 
 pure blue mixtures produce no precipitate. 
 
PRECIPITATION AND COAGULATION 129 
 
 on adding water to this mixture it changes in color from blue- 
 purple through red-purple to clear pink. Not until a pure pink 
 color is obtained does a precipitate result. If, instead of add- 
 ing water, we add a considerable volume of alcohol. (10 volumes) 
 the mixture rather abruptly changes to a clear pale blue and 
 then, but not before, we obtain a coagulwn of cobalt caseinate. 
 
 Electronegative casein is not precipitated by the salts of the 
 alkalies, though it is readily precipitated by salts of the alkaline 
 earths. Electropositive casein (i.e., casein dissolved in acids) is, 
 however, very readily precipitated by salts and these precipitates 
 are not soluble upon dilution. Thus if 2 cc. of N/1Q HC1 be 
 added to 5 cc. of a 1 per cent solution of casein in 0.008 N KOH, 
 a clear acid solution of casein results. The casein is precipitated 
 from this by the addition of four drops of a saturated solution 
 of sodium chloride, or by one drop of a saturated solution of 
 ammonium sulphate; this latter precipitate does not dissolve 
 on diluting the mixture to one-sixteenth. 
 
 Casein formate is no exception to the other salts of casein with 
 acids, but the precipitation will only occur in the presence of a 
 sufficiency of water. If to 5 cc. of a 2 per cent solution of casein 
 in formic acid we add a saturated solution of ammonium sulphate, 
 3 cc. of this solution just suffice to produce a coagulum; this 
 becomes more abundant on adding water, and redissolves on 
 adding formic acid. If, however, instead of adding 3, we add 
 2 cc. of the saturated ammonium sulphate solution, a clear solu- 
 tion is obtained. On adding water to this a precipitate results 
 which redissolves on heating and reappears on cooling. 
 
 Analogous results may be obtained with ovomucoid. 
 
 It is clear, therefore, that protein may be thrown out of solu- 
 tion by electrolytes in two grades of hydration, the one of high, 
 the other of very low hydration. The former process is what 
 we have termed precipitation, the latter we have defined as 
 coagulation. At grades of hydration intermediate between these 
 extremes the protein may be soluble. Dehydration, partial or 
 complete, leading to resolution or to coagulation, may be induced 
 by heat, by non-electrolytes possessing an affinity for water or 
 by electrolytes.* 
 
 The importance of a high degree of dehydration in the pro- 
 
 * See also Chick and Martin (10). 
 
130 CHEMICAL STATICS 
 
 duction of coagula irresistibly suggests that this phenomenon is 
 dependent upon the formation of anhydrides * analogous to the 
 diketopiperazines (vide Chap. I) and of the general formula: 
 
 /NH /NH.OC, 
 
 "R I nr "R T? 
 
 X CO X CO.HN X 
 
 Such bodies may exist either in the keto form, illustrated by 
 the above formulae, or in the enol form, such as: 
 
 ,N.(HO)C X 
 Rx C(OH).N x 
 
 If this be granted then the fact that alcohol throws down the 
 protein salts in an unaltered condition (vide Chap. IV) lends 
 strong support to the view which I have advanced (Chap. I) 
 regarding the mode of formation and structure of the protein 
 salts, since according to that view the metal ions in a protein- 
 metal compound would be bound up in COH.N groups pre- 
 viously to dehydration, which could only affect terminal NH 2 
 or NH 3 OH and COOH groups and would leave the union be- 
 tween the protein and the metal unaffected. 
 
 In this connection, and with reference to the "solvate 77 theory 
 of Jones, it is of interest to note that the relative efficacy of the 
 various salts in bringing about the coagulation of electronegative 
 protein is exactly their relative efficacy in bringing about dimi- 
 nution of the solubility of phenylthiocarbamid (69) (53). On the 
 other hand, evidence of the affinity of proteins for water is afforded 
 by the observation of Pauli and Samec (59) that the solubility 
 of highly soluble inorganic salts, such as ammonium chloride, 
 magnesium chloride or ammonium sulphocyanate in water is 
 diminished by the presence of gelatin or of blood-serum proteins. 
 The solubility of very slightly soluble substances (calcium sul- 
 phate, phosphate, or carbonate and uric acid) is, however, defi- 
 nitely increased. 
 
 As regards the precipitation of proteins by salts; it appears 
 probable that acid and alkali protein react with salts as follows: 
 
 * Cf. also Gustav Mann (46). 
 
PRECIPITATION AND COAGULATION 131 
 
 H 
 
 H 2 N.R.COH++ + ^N.KCOOH + 2 NaN0 8 
 I 
 Cl 
 
 Na 
 I 
 
 = H 2 N.R.CONa = N.R.COOH + 2 HNO 3 (i) 
 I 
 
 Cl 
 H 
 
 H 2 N.R.CONa++ + ^N.R.COOH + NaCl 
 
 I 
 OH 
 
 Na 
 I 
 
 = H 2 N.R.CONa = N.R.COOH + H 2 O (ii) 
 
 I 
 Cl 
 
 Since proteins, dissolved in salt solutions, are electrically neutral 
 (Hardy), it appears probable that this compound undergoes 
 internal neutralization thus (vide Chap. I) : 
 
 Na Na 
 
 I I 
 
 H 2 N.R.CONa = N.R.COOH = H 3 N.R.CONa = N.R.COO (iii) 
 
 I 
 
 Cl 
 
 Cl 
 
 It will be observed that this hypothesis is a slight modification 
 of that advanced by Pauli and Handovsky (57) in that, in the 
 first place, cognizance is taken of the fact that the proteins ionize, 
 not at terminal NH 2 or COOH groups but at internal enol 
 groups and, in the second place, the compounds which are formed 
 with acid- and alkali-protein respectively are symmetrical in 
 structure, so that the symmetry of the effects of salts in dissolv- 
 ing and coagulating these compounds, which has been observed 
 by Pauli, is readily accounted for. 
 
 In dilute solutions, that is, solutions in which the active mass 
 of water is great, these compounds undergo hydrolytic dissocia- 
 tion in the following way: 
 
132 CHEMICAL STATICS 
 
 Na 
 I 
 
 H 2 N.R.CONa=N.R.COOH + H 2 O 
 I 
 Cl 
 
 H 
 I 
 
 = H 2 N.R.CONa = N.R.COOH + NaOH. (iv) 
 I 
 Cl 
 
 This decomposition will, naturally, take place more readily 
 in dilute acid than in alkaline solution; more concentrated acid 
 would, of course, abstract the sodium from the compound and 
 convert the whole of it into the acid-protein compound. In 
 either case the neutral compound which results may be insoluble. 
 When the active mass of water is diminished, however, for ex- 
 ample by the addition of a dehydrating agent or of a salt with 
 an affinity for water, this hydrolytic decomposition is prevented, 
 and the complex salt: 
 
 Na 
 I 
 HsN.R.CONa = N.R.COO 
 
 I Cl I 
 
 may pass into solution. 
 
 Further dehydration leads to the loss of H and OH by 
 terminal NH 2 and COOH groups, as depicted above, and the 
 formation of complex insoluble anhydrides. 
 
 This hypothesis furnishes an explanation of the following facts: 
 
 (i) That the addition of small concentrations of neutral salts 
 to a solution of acid-protein increases the acidity of the solution, 
 while the addition of salts to alkali-protein solutions does not 
 increase their alkalinity (vide equations i and ii). 
 
 (ii) That acid-protein is precipitated by cations, alkali-protein 
 by anions (vide equation iii). 
 
 (iii) Since the union between the salt and the protein is chemi- 
 cal in character, Schultzes' valency-rule would apply to the rate 
 of precipitation (Cf. section 3). 
 
 (iv) The reaction (acidity or alkalinity) of the system being 
 maintained constant, the precipitation of the protein depends 
 only upon the active mass of water and not upon the active 
 
PRECIPITATION AND COAGULATION 133 
 
 mass of salt, provided this is sufficient to enter into combination 
 with the protein (vide equation iii), i.e., it is possible to bring 
 about precipitation by mere dilution, the relative masses of 
 protein and salt being unaltered. 
 
 (v) The precipitation of proteins by salts occurs more readily 
 in acid than in alkaline solutions (vide equation iii). 
 
 (vi) The observation of Bonamartini and Lombardi (6)* that 
 egg-albumin in neutral solution combines with both the basic 
 and the acid radicals of copper sulphate in equivalent propor- 
 tions to form an insoluble compound but that in alkaline solu- 
 tion it combines with excess of copper to form a soluble compound 
 (vide equation iv). In alkaline solution hydrolytic dissociation 
 of the complex salt is pushed back in accordance with the fol- 
 lowing equations. 
 
 CuS0 4 + 2KOH - K 2 SO 4 + Cu(OH) 2 
 H 2 N.R.CO.N.R.COOH H 2 N.R.CO.N.R.COOH 
 
 / ' \ 
 
 Cu(OH) 2 -f Cu( S0 4 ;l - Cu( SO )Cu +2H 2 O 
 
 \ i / 
 
 H 2 N.R.CO.N.R.COOH H 2 N.R.CO.N.R.COOH 
 
 (vii) The observation of Osborne (Cf. Chap. V) that edestin 
 crystallized from concentrated salt-solutions will decompose the 
 salt, binding the base, is probably attributable to the reaction: 
 
 H 
 
 H 2 N.R.CONa.N.R.COOH + NaCl 
 I 
 Cl Na 
 
 I 
 
 = H 2 N.R.CONaN.R.COOH + HC1 
 
 I 
 
 Cl 
 
 the complex salt being in this case insoluble. 
 
 (viii) The observation of Pauli that precipitation of a protein 
 by salts when it is non-ionic is impossible. For, when the pro- 
 tein is not ionized the nitrogen is bound up in undissociated 
 COH.N groups and is not attached to H + and OH' groups 
 replaceable by the ions of the salt. 
 
 * The egg-albumin employed by these observers is not ash free; it must, 
 therefore, according to Pauli have been ionic. 
 
134 CHEMICAL STATICS 
 
 This fact also explains the observation of Bugarszky and Lieber- 
 mann (8) that uncombined and, presumably, unionized proteins 
 do not combine with salts in neutral solutions. These observers 
 used egg-albumin, which, according to Pauli, is electrically neutral 
 when uncombined with acids or bases. 
 
 (ix) The observation of Hardy and Pauli that coagulation of a 
 protein by salts is possible whether it be ionic or not, since the 
 dehydration of terminal NH 2 and COOH groups does not 
 depend upon the dissociation of COH.N groups. 
 
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 (1) Babo, P., Jahresber. d. Chem. (1857), p. 72. 
 
 (2) Bernard, C., "Lemons sur les phe"nomenes de la Vie" (1885) (1st. Edn. 
 
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 (3) Billitzer, J., Zeit. f. physik. Chem. 45 (1903), p. 307;~51 (1905), p. 129. 
 
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 (15) Freundlich, H., Zeit. f. physik. Chem. 44 (1903), p. 129. 
 
 (16) Freundlich, H., Zeit. f. Chem. und Ind. d. Koll. 1 (1907), p. 321. 
 
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 (19) Gibbs, J. Willard, "Collected Papers," London (1906), Vol. 1, p. 96. 
 
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 (22) Hardy, W. B., Proc. Roy. Soc. London, 66 (1900), p. 110. 
 
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 (24) Hardy, W. B., Journ. of Physiol. 33 (1905), p. 251. 
 
 (25) Hardy, W. B., Proc. Roy. Soc. London, 79B (1907), p. 413. 
 
 (26) Hardy, W. B., and Whetham, W. C. D., Journ. of Physiol. 23 (1899), 
 
 p. 288. 
 
 (27) Hartley, W. N., Trans. Roy. Soc. Dublin, II, 7 (1900), p. 253. 
 
LITERATURE CITED 135 
 
 (28) Hartley, W. N., Journ. Chem. Soc. London, 53 (1888), p. 641. 
 
 (29) Hober, R., Beitr. z. chem, Physiol. und Path. 2 (1904), p. 35. 
 
 (30) Hofmeister, F. f Arch. f. Exper. Path, und Pharm. 24 (1888), p. 247; 
 
 25 (1888), p. 1; 27 (1890), p. 395; 28 (1891), p. 210. 
 
 (31) Hofmeister, F., Zeit. f. physiol. Chem. 14 (1890), p. 165. 
 
 (32) Jones, H. C., Zeit. f. physik. Chem. 74 (1910), p. 325. 
 
 (33) Jones, H. C., and Anderson, J. A., Amer. Chem. Journ. 37 (1907), p. 126. 
 
 (34) Jones, H. C., and Anderson, J. A., Carnegie Inst. Publ. Nr. 110, Wash- 
 
 ington (1909). 
 
 (35) Jones, H. C., and Ota K., Amer. Chem. Journ. 22 (1899), p. 5. 
 
 (36) Jones,' H. C., and Strong, W. W., Carnegie Inst. Publ. Nr. 130, Wash- 
 
 ington (1910). 
 
 (37) Jones, H. C., and Uhler, H. S., Amer. Chem. Journ. 34 (1905), p. 291. 
 
 (38) Kauder, G., Arch. f. exper. Path, und Pharm. 20 (1886), p. 411. 
 
 (39) Kuhne, W., and Chittenden, R. H., Zeit. f . Biol. 20 (1884), p. 11; 22 
 
 (1886), p. 423. 
 
 (40) Le Chatelier, H. L., Bull. Soc. Chim. (3) 6 (1891), p. 84. 
 
 (41) Lewin, M., Journ. Chem. Soc. London, 55 (1906), p. 513. 
 
 (42) Lewis, G. N., Zeit. f. physik. Chem. 52 (1905), p. 224; 56 (1906), p. 
 
 223. 
 
 (43) Lewith, I., Arch. f. Exper. Path, und Pharm. 24 (1888), p. 1. 
 
 (44) Linder, S. E., and Picton, H., Journ. Chem. Soc. London, 61 (1892), 
 
 p. 137. 
 
 (45) Loeb, J., Univ. of California Publ. Physiol. 1 (1904), p. 149. 
 
 (46) Mann, G., "Physiological Histology," Oxford (1902), p. 31. 
 
 (47) Mellanby, J., Journ. of Physiol. 33 (1905), p. 338. 
 
 (48) Moore, B. S., Journ. Chem. Soc. London, 55 (1906), p. 641. 
 
 (49) Nasse, O., Arch. f. d. Ges. Physiol. 41 (1887), p. 504. 
 
 (50) Ostwald, Wilh., "Grundlinien d. Anorgan. Chemie," Leipzig, 1900, 
 
 p. 620. 
 
 (51) Pauli, W., Arch. f. d. Ges. Physiol. 71 (1898), p. 333. 
 
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 (53) Pauli, W., Beitr. z. chem. Physiol. und Path. 3 (1903), p. 225. 
 
 (54) Pauli, W., Beitr. z. chem. Physiol. und Path. 5 (1904), p. 27. 
 
 (55) Pauli, W., Beitr. z. chem. Physiol. und Path. 6 (1905), p. 233. 
 
 (56) Pauli, W., Beitr. z. chem. Physiol. und Path. 7 (1906), p. 531. 
 
 (57) Pauli, W., and Handovsky, H., Biochem. Zeit. 18 (1909), p. 340; 24 
 
 (1910), p. 239. 
 
 (58) Pauli, W., and Rona, P., Beitr. z. chem. Physiol. und Path. 2 (1902), 
 
 p. 1. 
 
 (59) Pauli, W., and Samec, M., Biochem. Zeit. 17 (1909), p. 235. 
 
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 (61) Pickering, S. U., Ber. d. d. chem. Ges. 24 (1891), p. 3639. 
 
 (62) Pohl, J., Arch. f. exper. Path, und Pharm. 20 (1886), p. 426. 
 
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 (3), 6 (1891), p. 264. 
 
 (64) Posternak, S., Ann. de PInst. Pasteur 15 (1901), pp. 85, 169, 451 and 
 
 570. 
 
136 CHEMICAL STATICS 
 
 (65) Poynting, J. H., Phil. Mag. 42 (1896), p. 289. 
 
 (66) Prost, E., Bull. Acad. Roy. Sci. de Beige. (3), 14 (1887), p. 312. 
 
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 (69) Rothmund, V., Zeit. f. physik. Chem. 33 (1900), p. 401. 
 
 (70) Russell, W. S., Proc. Roy. Soc. London, 32 (1881), p. 258. 
 
 (71) Schafer, E. A., Journ. of Physiol. 3 (1880), p. 181. 
 
 (72) Schreinemakers, F. A. H., Zeit. f. physik. Chem. 23 (1897), p. 417. 
 
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 p. 27. 
 
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 (77) Walden, P., Trans. Faraday Soc. 6 (1910), p. 71. 
 
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 and 398. 
 
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 p. 630. 
 
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CHAPTER VII 
 THE COMPOUNDS OF THE PROTEINS (Continued) 
 
 1. Compounds with the Heavy Metals. From what has 
 been said in the previous chapter concerning the mechanism of the 
 precipitation and coagulation of proteins by salts, it will be clear 
 that the percentage of a heavy metal which is bound by protein 
 must depend very intimately upon the mode of preparation of the 
 compound. For instance, in the presence of silver nitrate under 
 conditions involving moderate hydration (e.g., a fairly high con- 
 centration of the silver nitrate or the presence of some other 
 dehydrating agent), or in alkaline solution, the following reaction 
 may occur: jj 
 
 I 
 H 2 N.R.CONa.N.R.COOH + 2 AgNO 3 
 
 I Ag 
 
 OH I 
 
 = H 2 N.R.COAg.N.R.COOH + NaNO 3 
 I 
 N0 3 
 
 in an acid medium, or under conditions involving less hydration, 
 this compound could not exist and the reaction 
 
 Ag 
 
 I 
 H 2 N.R.COAg.N.R.GOOH + H 2 O 
 
 I H 
 
 N0 3 I 
 
 ^H 2 N.R.COAg.N.R.COOH+ AgOH 
 I 
 N0 3 
 
 must occur. In a still more acid medium the silver would be 
 altogether abstracted from this compound, while in a fairly 
 strongly alkaline medium the compound 
 
 H 
 
 I 
 H 2 N.R.COAg.N.R.COOH 
 
 I 
 
 OH 
 137 
 
138 CHEMICAL STATICS 
 
 would doubtless be formed. In the majority of investigations 
 upon this subject no care has been taken to maintain a neutral or 
 otherwise constant reaction in the medium from which the protein- 
 heavy metal compound is thrown out, each observer has prepared 
 these compounds under different conditions, and no clear dis- 
 tinction has been made between the precipitation of the protein by 
 the heavy metal salt and its coagulation. It is not matter either 
 for surprise or bewilderment, therefore, that different observers 
 have obtained very conflicting results in determining the heavy 
 metal content of these compounds. In the light of our present 
 knowledge the majority of these results are perceived to be mean- 
 ingless, since they were obtained with compounds, or mixtures of 
 different compounds, prepared under inadequately controlled 
 conditions.* 
 
 From the observations of Galeotti (30), we may infer that at 
 the moment when precipitation begins, the compound of egg-albumin 
 with silver (or silver nitrate) is of constant molecular weight and 
 solubility in water. This investigator has applied to the system 
 protein, egg-albumin, water, the law of Jahn, that the influence 
 which an electrolyte exerts upon the solubility of another substance 
 (its dehydrating or hydrating action) is a function of three vari- 
 ables, namely, the concentration of the undissociated molecule of 
 the electrolyte, that of its cation, and that of its anion. Gal- 
 eotti measured the percentage of protein precipitated and also the 
 concentration of silver ions, determining the latter directly with 
 the aid of a concentration-chain (potentiometer). Calling C the 
 molecular concentration of a saturated solution of the protein- 
 silver-nitrate, and C' its solubility in the presence of silver nitrate, 
 applying Jahn's law we have 
 
 lognat. = [CotK + C k fou + 
 
 in which R is the gas-constant; <foi (at constant temperature and 
 pressure) is the constant ratio between the concentration of the 
 non-dissociated molecule of AgNOs and its effect upon the solu- 
 bility of the protein-compound; $n and <fci are similar factors for 
 the cation and the anion of the silver nitrate; and C and Ck are 
 the concentrations, respectively, of the undissociated and the 
 
 * For a review of the older literature upon the heavy metal compounds of 
 proteins, Cf. O. Cohnheim (25). 
 
HEAVY METALS 
 
 139 
 
 dissociated AgNO 3 , the concentration of the cation being assumed 
 to be equal to that of the anion. 
 
 Calling C g the total concentration of silver nitrate, then C = 
 C g Ck, and we have 
 
 = [CW> i + C k fo 
 
 where C is the molecular concentration of the protein-silver-nitrate 
 in saturated watery solution (a quantity which naturally, cannot 
 be measured directly) and C' is the molecular concentration of the 
 protein-silver-nitrate in the AgN0 3 solution. 
 
 Assuming that the molecular weight of the compound, in saturated 
 solution, is always the same, whether excess of silver nitrate be present 
 or not, .we can take C' as the percentage concentration of the com- 
 
 Ql 
 
 pound, since the ratio ^- will then not involve the molecular weight 
 
 of the compound. 
 Putting 
 
 1 2 m <f>Qi 2m 
 
 ~logC" a; IT'logC"^' "R" 
 
 we have, from the above equation, 
 alogC' + 0(V 
 
 logC 
 
 1=0, 
 
 in which a, 0, and 7 are constants, and C 1 ', C g , and C k are all directly 
 measurable. The following results of Galeotti's demonstrate the 
 validity of this equation, the most probable values of the constants 
 (a = -1.507; = +37.27; 7 = -72.06) having been determined 
 from all the observations by least squares. 
 
 X log C' 
 
 fiXC, 
 
 ,xc k 
 
 Sum 
 
 -0.207 
 -0.136 
 +0.140 
 +0.501 
 +0.741 
 
 +0.933 
 +1.465 
 +1.759 
 +2.250 
 +3.063 
 
 -1.741 
 -2.341 
 -2.872 
 -3.741 
 -4.813 
 
 -1.015 
 -1.012 
 -0.973 
 -0.990 
 -1.009 
 
 Averace 
 
 
 
 -1.000 
 
 
 
 
 
 From this result we^may draw the following conclusions: 
 (a) The substance precipitated is a compound and not the free 
 protein, since the value of a in the above equation indicates a 
 
140 CHEMICAL STATICS 
 
 limited solubility in water and free egg-albumin is miscible with 
 water in all proportions.* 
 
 (b) This compound is of the same molecular weight in all of the 
 
 C' 
 
 solutions investigated, otherwise the ratio -~- would include a 
 
 
 
 factor expressing the relation between the molecular weight of the 
 compound and the excess of AgN0 3 in its saturated solution. Also 
 the constancy of C (i.e., the constancy of a) shows that it is always 
 the same substance that is present "in solution at the moment 
 preceding precipitation. 
 
 (c) This compound is probably not ionized, since Jahn's law 
 applies only to the influence of electrolytes upon the solubility of 
 non-electrolytes . 
 
 (d) The quantity of NO 3 ' bound by the protein just before 
 precipitation must be the same as the quantity of Ag + which is 
 bound by it at the same moment, otherwise the concentration of 
 silver ions in its saturated solution would not be the same as that 
 of the N(Y ions. 
 
 All of these conclusions obviously answer to the formation of 
 the electrically neutral compound: 
 
 H 
 I 
 H 3 N.R.COAg.N.R.COO 
 
 N0 3 
 and its precipitation. 
 
 The last of the above conclusions also agrees with the finding 
 of Bonamartini and Lombardi (22), that in neutral solutions egg- 
 albumin binds the entire CuS04 molecule to form an insoluble 
 compound, although in alkaline solutions it binds an excess of 
 copper, forming a soluble compound. 
 
 Rohmann and Hirschstein (105) have described compounds 
 of casein with silver, or silver nitrate, formed by acting upon 
 solutions of casein, neutral to phenolphthalein, with excess of 
 AgNOs. The resultant precipitate when prepared from solutions 
 of sodium caseinate contained 85 X 10~ 5 equivalents of silver per 
 gram, while that prepared. from ammonium caseinate contained 
 77 X 10- 6 equivalents of silver. It will be recollected (Cf. Chap. 
 V) that at neutrality to phenolphthalein ca*sein neutralizes 80 X 
 
 * For it not only dissolves in water, but swells in water. Cf. Chap. XII. 
 
HEAVY METALS 141 
 
 10~ 5 equivalents of base. This corresponds, therefore, with the 
 direct interaction of one molecule of silver nitrate with one molecule 
 of the caseinate, according to the equation: 
 
 H 
 I 
 
 H 2 N.R.CONa.N.R.COOH + AgNO 3 
 I 
 
 OH H 
 
 I 
 
 = H 2 N.R.COAg.N.R.COOH + NaNO 3 . 
 I 
 OH 
 
 This compound is sparingly soluble in water (0.47 per cent) 
 and its solution requires the addition of a small amount of NaOH 
 before it becomes neutral to phenolphthalein.* If to this solu- 
 tion, neutralized to phenolphthalein, more AgN0 3 be added, a 
 fresh precipitate is obtained containing a higher percentage of 
 silver than the first, and the additional silver content is exactly 
 equivalent to the quantity of alkali which was added to the first 
 to render it neutral to phenolphthalein. The active mass of 
 caseinate in this reaction is directly proportional to the number of 
 COH.N groups which have been opened up by combination 
 with a base, i.e., which are electrolytically dissociated. Conversely, 
 undissociated COH.N groups cannot react with neutral silver 
 nitrate. 
 
 The compounds which proteins form with zinc sulphate have 
 been studied by Lippich (72) who considers that the reaction 
 between zinc sulphate and protein results in an equilibrium which 
 is determined by a variety of factors amongst which he enumerates 
 ionization, hydrolytic dissociation, complex salt formation, etc. 
 He finds that serum proteins combine with zinc in alkaline solu- 
 tion to form insoluble salts which dissolve in excess (J saturated) 
 of ammonium sulphate. This soluble salt he believes to be of the 
 type ZnS0 4 -protein, the insoluble salt formed at a higher degree 
 of hydration (absence of a dehydrating agent such as ammonium 
 sulphate) being of the type Zn-protein. 
 
 The compounds which copper salts form with amino-acids, 
 
 * Since AgOH is a very weak base, a caseinate of silver corresponding to the 
 caseinate of a strong base which is neutral to phenolphthalein, would neces- 
 sarily be acid to phenolphthalein. 
 
142 CHEMICAL STATICS 
 
 peptids, peptones and proteins have been extensively investi- 
 gated by Kober (57) (58) (59) (60) and, more recently, by Osborne 
 and Leaven worth (88). Kober has shown that the copper salts 
 of amino-acids in alkaline solutions yield their copper quanti- 
 tatively in the form of a precipitate of cupric hydrate on heating 
 the solution or on addition of an excess of alkali. Under similar 
 conditions the peptones and peptids yield little or no precipitate. 
 Osborne and Leaven worth have shown that the maximum amount 
 of cupric hydrate which edestin or gliadin will hold in solution 
 exactly corresponds with the number of COHN groups in 
 the molecule of these proteins, assuming that one atom of copper 
 combines with each nitrogen atom. This fact would appear 
 to be at variance with the view expressed by Kober and Sugiura 
 (59) that the pink "biuret" color which alkaline solutions of the 
 peptone compounds of copper yield on heating is due to a union 
 of copper with four nitrogen atoms, for if this were the case the 
 development of the pink color would lead to the precipitation of 
 an excess of cupric hydrate previously held in solution by the 
 COHN groups of the peptone. 
 
 Neumann (84) has studied the compounds which ovomucoid 
 forms with various metal salts (ZnCl 2 , CuCl 2 , MgCl 2 , A1C1 3 , FeCl 3 ) 
 and finds that in alkaline solutions this protein forms insoluble 
 compounds with the heavy metal, the weight of the metal com- 
 bined being proportionate to the molecular weight of its hydrox- 
 ide. In other words, ovomucoid and the various hydroxides of 
 the heavy metals combine in stoichiometrical proportions. 
 
 Rona and Michaelis (106) have shown that ferric hydrate will 
 displace Ca(OH) 2 from its combination with casein, forming 
 insoluble ferric caseinate. Benedicenti and Rebello-Alves (15) 
 (16) have shown that the magnetic properties of iron in solution 
 or even of finely pulverized iron suspended in water are "masked" 
 by the presence of proteins. They believe that in the latter 
 case this phenomenon is attributable to a direct fixation of the 
 metallic ions by the protein. 
 
 Henze (42) has described an interesting chromogenic protein 
 in the blood corpuscles of ascidians in which vanadium would 
 appear to play the part of the iron in the haemoglobin of mam- 
 malian corpuscles. 
 
 2. The Compounds with the Phosphoric Acids. It was 
 pointed out by Graham in 1861 that metaphosphoric acid unites 
 
PHOSPHORIC ACIDS 143 
 
 with gelatin to form an insoluble compound (34), 100 parts of 
 gelatin uniting, according to this observer, with 3.6 parts of 
 metaphosphoric acid. Graham suggested that the difference be- 
 tween the behavior of metaphosphoric and other phosphoric 
 acids in this respect might be connected with the possibly col- 
 loidal character of the latter.* This suggestion has more recently 
 been reiterated by Mylius (82). There appears, however, no 
 adequate reason for adopting such a conclusion. Metaphosphoric 
 acid differs from orthophosphoric acid in other respects, and these 
 differences are not attributable to the "colloidal" character of 
 its solutions. As Graham pointed out, the low equivalent of 
 metaphosphoric acid which combines with gelatin suggests that 
 the metaphosphoric acid enters into the compound in a crystal- 
 loid and not a colloidal (polymerized) form. Colloidal silicic acid 
 combines with gelatin in its colloidal form and the compound con- 
 tains nearly equal weights of gelatin and of silicic acid (Graham, 
 loc. cit., p. 206). 
 
 The compounds of metaphosphoric acid with the proteins 
 were at one time regarded with peculiar interest on account 
 of their supposed identity with the nucleo-proteins (69) (74). 
 Pohl (96) and Kossel (61), however, showed that these compounds 
 differ fundamentally from the true nucleins in that, on hydrolysis, 
 they yield no purin bases. Thanks to the work of Miescher, 
 Kossel and their pupils, it has now been shown that the nucleins 
 are not protein salts of phosphoric or of metaphosphoric acid 
 but of a complex substituted phosphoric acid, nucleic acid (6). 
 
 The compounds of the protein with metaphosphoric acid 
 appear, as a rule, to be unstable save in the presence of excess 
 of the acid (Graham, loc. cit., Malfatti, loc. cit.) ; if this excess be 
 removed by washing then the compound undergoes decompo- 
 sition, liberating metaphosphoric acid. Progressive removal of 
 the free acid by washing, therefore, results finally in complete 
 decomposition of the product. Conversely, in the presence of 
 a variable excess of metaphosphoric acid the quantity which is 
 bound by a protein is greater the greater the excess (28). 
 
 Bechhold (14) has described compounds of egg-albumin with 
 
 * "It will be an interesting inquiry whether metaphosphoric acid is a 
 colloid, and enters into the compound described in that character, or is a 
 crystalloid, as the small proportion and low equivalent of the acid would 
 suggest," loc. cit. page 221. 
 
144 CHEMICAL STATICS 
 
 orthophosphoric acid prepared by treating crystallized egg- 
 albumin, dissolved in NaOH, with POC1 3 . 
 
 3. Compounds of the Proteins with Carbonic Acid. Siegfried 
 (113) has described a special form of combination between inor- 
 ganic salts and amino- or polyamino-acids which is probably 
 destined to assume considerable importance in the eyes of physi- 
 ologists. He observed that if CO 2 be passed through solutions 
 of various amino-acids in barium hydrate, provided that the 
 total concentration of Ba(OH) 2 is not greater than twice that 
 of the amino-acid no precipitate of barium carbonate is obtained. 
 Similar results are obtained with Ca(OH) 2 , and the peptones 
 and the proteins of the blood behave in a manner analogous to 
 the simple amino-acids. On standing, BaC0 3 or CaC0 3 , as the 
 case may be, is slowly liberated, and this process is accelerated 
 by heating. Direct analysis of the products obtained when 
 CaC0 3 acts upon glycocoll and other amino-acids showed that the 
 compounds can be represented by the general formula 
 
 H 
 
 R 
 
 I I 
 
 COO -- Ba 
 
 being the barium salts of carbamino-acids. 
 For the monoamino-acids the ratio 
 
 molecules C0 2 bound 
 atoms of N 
 
 is 1, indicating that the NH 2 group reacts quantitatively with 
 the carbonate. For diamino-acids, such as lysin, the ratio is 
 also 1, showing that both NH 2 groups react quantitatively. 
 For arginin, which contains 4 atoms of nitrogen, the ratio is J, 
 indicating that only one of the NH 2 groups and neither of the 
 imino-groups react. For the different dipeptids the ratio varies 
 between 
 
 1 , 1 
 L63 and L79* 
 
 If the N.HOC groups. did not react at all the ratio would be 
 i, if they reacted quantitatively it would be 1. For tripeptids 
 
 the ratio is -==, whereas it would be if the N.HOC groups 
 
ALKALOIDAL REAGENTS, ETC. 145 
 
 did not react at all. For the tetrapeptid triglycyl glycin the 
 
 ratio is 
 
 When C0 2 is passed into a solution of a protein in Ca(OH) 2 
 the conductivity of the mixture diminishes. From this Siegfried 
 draws the conclusion that the proteins react with carbonates 
 to form carbamino acids. From what has been said in the pre- 
 vious chapter concerning the mode of combination of proteins 
 with salts, however, the reader will gather that the validity of 
 this conclusion does not admit of being established in so simple 
 a manner. 
 
 4. Compounds of the Proteins with the Alkaloidal Reagents, 
 Dyes, Alkaloids, etc. In order that a protein may react with 
 an acid which is insufficiently dissociated to break up its 
 N.HOC groups, some stronger acid must be present, com- 
 bined with the protein, and which can then be displaced by the 
 weaker acid through the agency of one of its more strongly ionized 
 salts. Thus: 
 
 H 
 
 -N.HOC- -h HA = -N"+ HOC- 
 
 H H 
 
 I ++ I ++ 
 
 -N" + HOC- + BA' = -N" + HOC- + BA 
 I I 
 
 A A' 
 
 HA being a strong acid, HA' a very weak acid, and BA' a salt 
 of that acid. Similar remarks apply, of course, to very weak 
 bases. Hence it is observed that proteins will combine with 
 very weak acids or with the acid radical of salts more readily 
 in acid than in neutral or alkaline solution, and with very weak 
 bases or the basic radical of salts in an alkaline rather than a 
 neutral or acid medium. Similarly a protein may not be able 
 to decompose a salt of an acid, binding the acid (or base as the 
 case may be), but, in the presence of a free acid it may be able 
 to do so, partly because the first acid is partially set free from 
 its salt by the second, but also because the ionization of the 
 protein is increased. 
 
146 CHEMICAL STATICS 
 
 Hence, on adding free picric, molybdic, tungstic, phospho- 
 tungstic, tannic, stearic or chromic acids to a neutral solution of 
 protein a precipitate or coagulum is, as a rule, formed immedi- 
 ately. But if these acids be added in the form of their (neutral) 
 salts then no precipitate results until the reaction of the solution 
 is rendered acid, when the protein at once combines with the 
 acid radical of the salt. Similarly, neutral lead acetate will 
 not, as a rule, precipitate protein (egg-albumin) from neutral 
 solutions, but it will precipitate it readily from faintly alkaline 
 solutions (77) (solutions, that is,, in which the protein is in the 
 form of an ionized compound with a base). 
 
 Upon these facts probably depend certain phenomena which 
 are encountered in the tanning of leather. As is well known, 
 chromium unites firmly with the hide-substance only in the 
 form of its cation Cr +++ , and not when it is united with oxygen 
 to form an anion as in chromic acid. Now it is observed that 
 firm union only occurs when the chromium salt is "basic" (such 
 as Cr 2 Cl 3 (OH) 3 ), i.e., when the salt is neutral in reaction or even, 
 at the moment of union, faintly alkaline (97). Further addition 
 of alkali results in a displacement of the chromium by the added 
 base. On the other hand, tannic acid unites with the hide sub- 
 stance in a faintly acid medium. 
 
 Similar principles apply to the union of dyes with proteins (77) 
 (39). Proteins which are about equally acid and basic in charac- 
 ter, such as gelatin and egg-albumin, combine with basic dyes only 
 in faintly alkaline solutions. Predominantly acid proteins, such 
 as casein or the nucleins, will combine with basic dyes even in 
 neutral or faintly acid solutions (80) since (ionized) salts of these 
 proteins with bases can exist even in solutions acid to litmus 
 (Cf . Chap. V) ; on the other hand, they combine with acid dyes 
 with great difficulty. Lilienfeld has shown that so long as nucleic 
 acid is not saturated with albumins to form neutral nuclein an 
 insoluble compound with methyl green is formed when this dye 
 is added to a solution of the nuclein, but after saturation with 
 protein the nucleins show a greater affinity for acid dyes (71). 
 
 An interesting compound of azolitmin and mucoid has been 
 obtained by Rosenbloom and Gies (109). I have found that in 
 faintly alkaline solution trypsin, or some constituent of Gruebler ; s 
 trypsin (nach Spateholz), forms an insoluble compound with 
 safranin (99) (44). 
 
ALKALOIDAL REAGENTS, ETC. 147 
 
 Evidence of the existence of protein dye compounds even 
 when these are freely soluble is afforded by the influence of added 
 protein upon the distribution of the dye between water and some 
 other appropriate solvent (100). Thus if erythrosin in acidulated 
 water is shaken up with ethyl acetate it passes completely into 
 the ethyl acetate, forming a yellow solution and leaving the 
 water colorless; if, however, neutral sodium caseinate be added 
 to the water, the watery layer remains pink upon shaking it up 
 with erythrosin. Similarly, when gentian violet in alkaline 
 watery solution is shaken up with ethyl acetate a large proportion 
 of the gentian violet passes into the ethyl acetate, forming a 
 red solution but leaving the water blue; if, however, casein or 
 gelatin be added to the water very little of the gentian violet 
 passes into the ethyl acetate and that which does so only stains 
 it faintly violet. Similarly neutral red can be shown to combine 
 with casein, and bismarck brown with casein and gelatin. I 
 have observed that casein forms insoluble compounds with acid 
 fuchsin and orange G, gelatin with crystal violet and acid fuchsin, 
 and protamin with orange G and carminic acid. The compound 
 of protamin with carminic acid is very interesting because it is 
 of a very different color from the free acid or its compounds 
 with inorganic bases; as is well known, free carminic acid, in 
 solution, is golden in color, while its salts with inorganic bases 
 have the familiar carmine tint; the compound with protamin 
 is, however, deep violet. I have observed in several cases, also, 
 that the colors of solutions of dyes to which the proteins have 
 been added are not identical with the colors of pure watery 
 solutions. 
 
 Many authors have doubted the chemical nature of the process 
 of staining of tissues, preferring to regard it as a physical phe- 
 nomenon of "solid solution" or "adsorption." Having regard, 
 however, to the unquestionable chemical combinations between 
 dyes and proteins which occur, as we have seen, in vitro, and 
 recollecting that, as Mathews has shown, even proteins coagu- 
 lated by fixatives show similar phenomena, there can remain 
 very little doubt that similar combinations must frequently occur 
 between the dyes used by cytologists and the protein constituents 
 of the tissues.* 
 
 * For a discussion of this interesting question Cf. Gustav Mann (75), also 
 Pelet-Jolivet, L., "Die Theorie des Farbeprozesses," Dresden (1910). 
 
148 CHEMICAL STATICS 
 
 That casein will combine with various alkaloids to form water- 
 soluble compounds has been shown by W. A. Osborne (89). 
 From the marked effects of caffeine upon the viscosity and con- 
 ductivity of gelatin in hydrochloric acid solutions Pauli and 
 Falek (93) infer that caffeine enters into combination with gelatin 
 hydrochloride. The compounds which the various dyes form 
 with amino-acids have been studied by Suida (116). 
 
 5. The Compounds of Proteins with Soap and Lipoids. It 
 has been pointed out by Rona and Michaelis (106) that the 
 power of soaps, such as sodium oleate, to reduce the surface 
 tension of water, is much diminished by the presence of proteins, 
 and they infer that the soaps form compounds with protein. 
 No such effect was observed when esters, such as amyl acetate, 
 tributyrin, propyl acetate or ethyl butyrate were employed 
 instead of soaps. These results have been confirmed by Berczeller 
 (17). It has long been known that it is an extremely difficult 
 matter to free proteins from contamination with lecithin, and 
 the existence of lecithin-protein compounds has frequently been 
 inferred from this fact (48) (87) (95). More decisive evidence 
 of the existence of such compounds has recently been brought 
 forward by Feinschmidt (27) and Allemann (5) who have shown 
 that the optimal H+ concentration for the precipitation of vari- 
 ous proteins is very considerably modified by the presence of 
 lecithin. 
 
 Fatty acid compounds of the peptones have been prepared by 
 Izar and di Zuattro (54) by the action of chlorides of the fatty 
 acids upon peptones at low temperatures. The compounds thus 
 prepared contain from 18 to 20 per cent of fatty acid. 
 
 6. The Compounds of Proteins with Proteins and their Pos- 
 sible Significance in Life Phenomena. It has been shown by 
 Kossel (62) that the protamins, which, it will be recollected, 
 are strongly basic proteins, when added to weakly alkaline solu- 
 tions of other proteins give rise to precipitates. Kossel believes 
 that the compounds which are thus formed between the pro- 
 tamins and less basic proteins are analogous to the naturally 
 occurring histones (63) (66). Hunter (53) has further investi- 
 gated these compounds -and finds that while crystallized egg- 
 albumin, casein, hemi-elastin, gelatin, edestin, heteroalbumose, 
 protalbumose, "alkali-albuminate" and histone sulphate yield a 
 precipitate in alkaline solution upon the addition of clupein. 
 
COMPOUND PROTEINS 
 
 149 
 
 Elastin, peptone, deuteroalbumose, histopeptone and several 
 peptids fail to yield a precipitate. Upon digestion of these 
 precipitates with pepsin the protamin is set free * and the remain- 
 der of the compound is converted into deuteroalbumose and 
 peptones which are not precipitable by picric acid. The pro- 
 tamin which was held in the compound before digestion can now 
 be determined quantitatively by precipitation with picric acid. 
 The following are among the results which were obtained by 
 Hunter : 
 
 Protein combined with clupein 
 
 In 100 parts of nitrogen 
 in the compound the pro- 
 tamin moiety contains 
 
 Weight of protein which 
 combines with one part 
 by weight of clupein 
 
 Alkalialbuminate 
 
 34.36 
 
 
 Ovalbumin 
 Gelatin 
 
 29.31 
 22.79 
 
 4.1 
 
 4.8 
 
 Hemi-elastin 
 
 22 71 
 
 5 2 
 
 Casein 
 
 39 17 
 
 2 5 
 
 Edestin 
 
 13 95 
 
 8 5 
 
 
 
 
 An attempt made by Gay and Robertson (32) to prepare 
 protamin (salmin) caseinate by the method of Hunter failed to 
 yield a product containing the high proportion of protamin which 
 he describes. The nitrogen content of the compound protein 
 indicating a protamin content of less than ten per cent. The 
 origin of this discrepancy has been ascertained by af Ugglas 
 (117) who has shown that the preparations described by Hunter 
 contained a considerable excess of the protamin precipitated in 
 a condition of admixture with the compound. The compound 
 of protamin (clupein) with casein prepared by af Ugglas con- 
 tained 94 per cent of casein and 6 per cent of the protamin 
 while the compound with haemoglobin contained 5 per cent of 
 protamin. Schmidt, who has prepared protamin (salmin) edesti- 
 nate (110) (116), finds that it contains about ten per cent of the 
 protamin. 
 
 When globin and casein are mixed in faintly acid solution 
 (110) a precipitate of globin caseinate is produced which is soluble 
 in excess of acid or in dilute alkalies (103). That this precipi- 
 tation is accompanied by true compound formation has been 
 demonstrated by the method of electrometric titration (Schmidt 
 
 * The protamins, although readily digestible by trypsin, are not digestible 
 by pepsin. 
 
150 CHEMICAL STATICS 
 
 (110) (111)). The precipitate produced by admixture of an 
 excess of globin with sodium caseinate in solution contains about 
 34.5 per cent of casein. A compound of globin with deutero- 
 albumose has been prepared by Schmidt (111). 
 
 Thymus histone combines with haemoglobin, according to 
 af Ugglas (117), in the proportion of one part of thymus histone 
 to two of haemoglobin, and with casein to form a compound 
 containing about 30 per cent of the histone. 
 
 A particularly interesting compound protein is the haemo- 
 globin caseinate which has been prepared by af Ugglas. To a 
 solution of casein in alkali an excess of hydrochloric acid is added 
 until the precipitate of free casein which is at first formed is 
 redissolved. The casein hydrochloride is precipitated from this 
 solution by the addition of sodium chloride and the precipitate 
 redissolved and reprecipitated until the washings from the pre- 
 cipitate are perfectly neutral. A solution of this substance 
 added to an excess of a solution of haemoglobin, produces a pre- 
 cipitate containing 33 per cent of casein and 66 per cent of 
 haemoglobin. The commonly accepted molecular weight of haemo- 
 globin, which has now been confirmed by a variety of measure- 
 ments (Cf. section 11), is about 16,700. We have seen that the 
 minimal molecular weight of casein is about 8800 (Chap. V). 
 It would, therefore, appear that casein and haemoglobin combine 
 with one another in molecular proportions. 
 
 From important observations of Hardy's it appears extremely 
 probable that many of the protein constituents which may be 
 isolated from the various tissues and tissue fluids do not pre- 
 exist there but are bound up in complex compounds of proteins 
 with proteins. I quote from the appendix to Hardy's article 
 on globulin (35). 
 
 "The facts of the case are these. The proteins of serum are 
 electrically inactive. Neither the whole nor any fraction moves 
 in a field. It is not possible to detect a trace of 'ionic' protein. 
 Dialysis or dilution disturbs the equilibrium and 'ionic' globulin 
 appears, and can be swept out of the general mass of proteins as 
 an opalescent cloud before dialysis has been pushed to the point 
 where precipitation occurs. The development of even minute 
 quantities of 'ionic' globulin can be detected in this way. The 
 direction of the movement is towards the anode, therefore the 
 globulin which appears is the anion of alkali-globulin." 
 
COMPOUND PROTEINS 151 
 
 "Serum which has been dialyzed against water with very low 
 carbonic acid content until it ceases to give any precipitate, 
 but which can still give with acid a large yield of globulin, is 
 in a most interesting condition. The whole remaining protein 
 is now ionic. It moves towards the anode quite uniformly, 
 therefore it behaves as a whole as the protein ion of an alkali 
 protein compound." 
 
 "Now what does this mean? The presence of other proteins 
 does not interfere with the movement of ionic protein. This 
 point cannot be too much insisted on.* It lies at the root of 
 the evidence. Therefore one starts with protein which behaves 
 uniformly and is electrically inactive, one ends with protein 
 which behaves uniformly and is electrically active, and in the 
 final stage there is no evidence of more than one kind of protein 
 ion. But this residue of uniform character still contains a globulin 
 fraction. It can be split by saturation with a neutral salt or 
 by acidulation into fractions differing in properties according to 
 the mode of separation." 
 
 "When in the cell used for these experiments, which is de- 
 scribed on p. 289,f the upper layer of fluid is a solution of ^^th 
 normal acetic acid and the lower layer is serum and a current 
 is passed for 24 hours, the serum protein becomes slowly charged 
 in such a way that it is repelled from both poles. Therefore 
 in the anodic limb it becomes charged positively, in the cathodic 
 limb negatively. The result of the double repulsion is that the 
 protein is condensed into a hard plate of rubber-like consistency 
 just midway between the electrodes." 
 
 "The phenomena would be explained, on the theory which has 
 been outlined, in this way. In the anodic limb an excess of 
 hydroxyl ions are liberated owing to the electric convection, and 
 these, reacting with the serum protein, convert it into cationic 
 protein. In the cathodic limb the converse reaction with hydrions 
 occurs and is possible owing to the amphoteric nature of proteins. 
 The entire mass of serum protein is thus thrown into the ionic 
 state and in this condition moves with uniform motion, the one 
 half as cationic protein, the other half as anionic protein. The 
 electric current, the most subtle of analyses, detects only one 
 substance, and this substance, owing to its amphoteric nature 
 can exist in either the cationic or the anionic state." 
 
 * Cf. also Chap. XIII. (Author.) f Cf. Hardy's paper. (Author.) 
 
152 CHEMICAL STATICS 
 
 "The concentrated hard rubber-like mass of protein obtained 
 in this way cannot be discriminated from serum protein. It 
 is easily soluble in distilled water and in dilute salt solutions. 
 From the solution in water a globulin fraction can be precipitated 
 by acid, or by saturation with magnesium sulphate. From the 
 solution in dilute salt solution a globulin fraction can be pre- 
 cipitated by saturation with salt (NaCl, or MgSO^. The ni- 
 trate, after removal of the globulin by saturation, contains a 
 protein which is precipitated by acetic acid." 
 
 "The position is that in serum one has protein which can be 
 thrown into the ionic state and which then moves in a field as a 
 single substance. From it an electrically active fraction, namely 
 globulin, can be split off, and the protein thereby becomes elec- 
 trically heterogeneous." 
 
 "Now the globulin fraction has an abiding characteristic. In 
 all its solutions its molecular state is so gross as to cause the 
 molecules to be arrested by a porous pot. They will not pass 
 such a filter even under pressure. In this it is sharply distinct 
 from the parent serum protein, which is readily filterable. If 
 globulin be present as such in serum it is not only non-ionic, but 
 the agent which dissolves it must be something more than alkali 
 and salt since either alone or together they will not produce so 
 high a grade of solution (78)." 
 
 "The difference in the molecular grade of globulin when once 
 separated, and the electrical homogeneity of serum protein and 
 of the fraction (still capable of further subdivision by salting 
 out) which remains after the alkaline globulin fraction which 
 most readily appears has been removed suggests that serum 
 protein is a complex unit. If such a unit exists it is not satu- 
 rated with globulin. Fresh ox-serum has an extraordinary power 
 of dissolving globulin, it will take up almost its own volume of 
 the thick cake at the bottom of a centrifuge tube; and in ox- 
 serum so saturated there is not a trace of alkali globulin nor of 
 any ionic protein." 
 
 The phenomena observed by Hardy appear to admit of in- 
 terpretation on the view (102) that the protein-complex in serum 
 is formed by the union of a number of alkali protein compounds, 
 the union taking place in a manner strictly analogous to the 
 combination of neutral salts with protein (Cf. Chap. VI), alkali 
 protein molecules behaving like inorganic salt molecules thus: 
 
COMPOUND PROTEINS 153 
 
 H H 
 
 I I 
 
 2HOOC.Ri.N" + 2-H-NaOC.Ri.NH2 + HOOC.R.N" + 
 I I 
 
 OH OH 
 
 RLCOOH 
 
 I 
 
 H N = NaOC.R!.NH 2 
 
 ++NaOC.R.NH 2 = HOOC.R.N ( ) NaOC.R.NH 2 + 2 H 2 O 
 
 I \/ 
 
 OH N = NaOCRi.NH 2 
 I 
 Rx.COOH 
 
 Hydrolytic dissociation of this complex, just as in the case of the 
 salt-alkali-protein compounds described in the previous chapter, 
 would result in its decomposition, partial or complete, and hence, 
 if the complex were non-ionic (and, as we have seen, the homol- 
 ogous compounds with inorganic salts are non-ionic) mere dilu- 
 tion of its solution or the removal of dehydrating agents (salts) 
 might result, as in Hardy's experiments, in the splitting off of 
 fraction after fraction of ionic protein. A very faintly alkaline 
 reaction would probably favor its stability, a mere trace of acid 
 might be expected to disrupt the complex (Cf. equation iv, Chap. 
 VI).* It will be found, I believe, that the presence of such 
 protein-complexes as these, in the tissues and tissue-fluids, affords 
 
 * The fact that this complex will pass through the pores of a porcelain 
 filter, while the simpler, ionic protein will not, is due to its non-ionic character. 
 The thesis will be developed in a later chapter (Chap. XI), upon a very 
 extensive experimental basis, that the colloidal, non-filterable, viscous character 
 of solutions of ionic protein is attributable not to the size of the protein particles 
 but to the size of the associated complex of water molecules, which is much larger 
 in solutions of ionic than in solutions of non-ionic protein. The non-filterable 
 character of ionic globulin is to be interpreted in the same way as the non- 
 permeability of a porous pot for mixtures of propyl alcohol and water, although 
 it is permeable to both water and propyl alcohol when they are pure (S. U. 
 Pickering (94)). Just as, in Pickering's experiment, propyl alcohol, when dis- 
 solved in water, will not pass out through the walls of a porous pot into the 
 surrounding water, not because the wall of the pot is impermeable to propyl 
 alcohol, for pure propyl alcohol will pass through the pot, but because it is 
 impermeable to the molecule of propyl alcohol plus its associated complex of 
 water molecules, so ionic globulin will not pass through porcelain because the 
 porcelain is impermeable to the globulin ion plus its associated complex of 
 water molecules. 
 
154 CHEMICAL STATICS 
 
 a simple and readily intelligible explanation of an otherwise 
 exceedingly puzzling fact. I refer to the individuality of the 
 tissues and tissue-fluids. Despite the fact that the individual 
 proteins which are found in the tissue-fluids of tolerably nearly 
 related animals (e.g., the mammalia) appear, on analysis, to be 
 identical with one another,* yet the tissue-fluids of one mammal, 
 when injected into the circulation of another, are treated as 
 foreign intrusions and give rise to "antibodies"; while the indi- 
 vidual protein constituents of these fluids are also treated as 
 foreign matter when introduced into the circulation, giving rise 
 to " precipitations " and to phenomena such as " anaphylaxis " 
 (120). 
 
 On the basis of the view developed above, however, not only 
 the constitution of the individual components of the protein 
 complex is of importance in determining its characteristics, but 
 also the relative proportion of these components.! Two protein- 
 complexes of this type might well be built up out of identical 
 units and yet differ fundamentally owing to differences in the 
 combining proportions and, consequently, in the mode of linkage 
 of these units. Any one constituent of the complex would, of 
 course, be a totally different substance from the complex itself 
 and its introduction into the tissues or tissue-fluids would result 
 in a more or less extensive disruption of the equilibrium of which 
 the complex is an expression and which lends it, in each tissue, 
 tissue fluid and species, its own distinctive character. 
 
 In pursuance of this idea Gay and Robertson (32) (33) and 
 C. L. A. Schmidt (110) (111) have investigated the antigenic 
 properties of several compound proteins. If compound proteins 
 differ in their biological specificity from their constituents then 
 
 * Thus the casein of human milk is chemically identical with the casein of 
 cows' milk and with that of goats' milk (2) (1). The fibrin of ox-blood ap- 
 pears to be identical with that of horse-blood (4). The serum albumins and 
 globulins of goose-blood are identical with those of horse-blood (3). Whether 
 chemically identical proteins derived from different animal species are also 
 antigenically identical or not has not yet been satisfactorily established. 
 
 t Just as the different proteins and polypeptids are built up out of very 
 similar amino-acids and yet differ widely from one another in their character- 
 istics because the proportions in which the amino-acids enter into the mole- 
 cules are different. These protein-complexes may be regarded as bearing the 
 same relationship to the simple proteins which they yield on decomposition as 
 the polypeptids do to the amino-acids. 
 
COMPOUND PROTEINS 155 
 
 a compound protein should represent a new antigen giving rise 
 to antibodies for itself as distinguished from the antibodies for 
 its constituents. Unfortunately a formidable technical difficulty 
 stands in the way of clearly recognizing the presence of anti- 
 bodies which are specific for the compound protein. This is the 
 difficulty which is constituted by the fact that any protein which 
 is capable of being split by hydrolysis into moieties which are 
 still proteins (in the sense that they are antigenic) gives rise on 
 injection into animals to antibodies^ not only for itself, but also 
 for these split-products (31). Analogously, a compound protein 
 gives rise to antibodies for its constituent parts and it is only 
 possible to distinguish between these, which would appear in 
 the blood of immunized animals after injection of the separate 
 constituents, and any antibodies which may be formed for the 
 compound as a whole in those doubtless exceptional instances 
 in which the antibody for the compound reacts with a constituent 
 which is not normally antigenic. 
 
 We have therefore investigated from this point of view certain 
 compound proteins in which one constituent is non-antigenic, 
 such as protamin caseinate, of which the protamin constituent 
 is non-antigenic and toxic while the casein constituent is anti- 
 genic and non-toxic, and globin caseinate of which the globin 
 constituent is toxic and non-antigenic.* 
 
 Protamin caseinate displays no antigenic characteristics which 
 enable it to be distinguished from casein. It is non-toxic, but 
 whether this lack of toxicity is attributable to the masking of 
 the toxic properties of protamin by its combination with casein, 
 or to the smallness of the proportion of protamin contained in 
 the compound has not yet been definitely established. It gives 
 rise to antibodies for casein by virtue of its casein-content, just as 
 casein gives rise to antibodies for its split-product paranuclein, but 
 it does not give rise to antibodies for protamin. Similarly, pro- 
 tamin edestinate (111) is non-toxic and gives rise to antibodies 
 for edestin but does not give rise to any antibody which will 
 react with its protamin constituent. Globin caseinate, however, 
 
 * It is asserted by C. H. Browning and G. H. Wilson (23) that globin is 
 antigenic. The negative results of Gay and Robertson which have been 
 repeated and confirmed by Schmidt show that the antigenic character of the 
 globin prepared by Browning and Wilson must have been due to contamination 
 by protein impurities. 
 
156 CHEMICAL STATICS 
 
 differs very markedly in its antigenic behavior from either of 
 its constituents. In the first place it is non-toxic, and the failure 
 to exhibit toxicity can hardly be attributable to dilution of the 
 globin constituent by admixture with casein since, as we have 
 seen, globin caseinate contains 65.5 per cent of globin (103). 
 Still more striking is the fact that it yields antibodies which 
 react (i.e., display alexin-fixation) not only with the casein con- 
 stituent of the compound, but also with the globin constituent. 
 It would appear evident, therefore,' that injection of globin 
 caseinate into animals gives rise to an antibody which does not 
 appear in response to separate injections of its constituents. 
 Schmidt (111) has investigated the antigenic behavior of a com- 
 pound of globin (toxic and non-antigenic) with deutero-albumose 
 (non-toxic and non-antigenic) . The compound retains the toxicity 
 of globin and is non-antigenic. 
 
 7. Compounds of the Proteins with Toxins, Antibodies, 
 Ferments, etc. Very extensive evidence has been advanced by 
 numerous authors * in support of the view that the antitoxins 
 are true proteins. If this be true, and the probabilities are 
 immensely in favor of its truth, then the entire series of toxin- 
 antitoxin reactions are reactions in which proteins play a leading 
 part. To consider these here would be manifestly out of place; 
 for an analysis of the physico-chemical laws which govern these 
 reactions the reader is referred to Arrhenius (8). 
 
 The question of the occurrence of combinations between the 
 proteolytic ferments and proteins and their significance in the 
 mechanism of protein hydrolysis will fall under consideration 
 in a later chapter (Chap. XVI) but the reader's attention is here 
 drawn to the work of Hedin (37) (38) upon this subject. 
 
 8. Methyl and Benzoyl Derivatives of the Proteins. Ac- 
 cording to Rogozinski (104), methylation of clupein causes pro- 
 found alteration in the composition of the molecule, reducing 
 especially the proportion of arginin yielded on subsequent hy- 
 drolysis. Skraup and Krause (114) have shown that not only 
 is arginin reduced but also tyrosin, lysin and histidin, while the 
 glutamic acid and leucin yields are unaffected. 
 
 Blum and Umbach (19). have prepared benzoyl derivatives of 
 native and iodized proteins. 
 
 " Cf. for literature Carl Oppenheimer (86). 
 
HALOGEN AND NITRO COMPOUNDS 157 
 
 9. The Halogen and Nitro Substitution-Compounds of the 
 Proteins. Besides the combinations of the proteins with 
 inorganic substances which are formed through the ionization 
 of N.HOC groups, other types of combination between 
 proteins and inorganic substances are unquestionably possible. 
 Among these the best known are the substitution-compounds of 
 the proteins with the halogens and with N(>2. 
 
 According to Blum (18) (20) and to Hofmeister (43) (112) (41) 
 (85), the halogen compounds are formed by the replacement 
 of hydrogen atoms in the aromatic groups of the proteins by 
 halogen atoms. The best known examples, both artificially pre- 
 pared and naturally occurring, are the iodoalbumins. They can 
 be prepared (Cf. papers just cited) by allowing a mixture of 
 potassium iodate and potassium iodide to act upon proteins at 
 a moderately high temperature (40 degrees). Hopkins and 
 Brook (46), however, prepared iodoproteins by allowing powdered 
 iodine to act directly on the protein, in solution. When the 
 former method of preparation is employed, part of the iodine 
 enters into combination with the protein and part is liberated 
 as HI. Hence excess of a carbonate, of an alkali, or of an alka- 
 line earth must be added to the mixture to neutralize the hydro- 
 gen iodide set free, and prevent it from hydrolysing the protein, 
 through the catalytic action of hydrogen ions. The iodo-proteins 
 are, in the dry condition, similar in appearance to ordinary pro- 
 teins; they are insoluble in water or alcohol, and they appear 
 to be more predominantly acid than the non-iodized proteins 
 from which they are derived, since they are readily soluble in 
 dilute solutions of alkalies or of alkaline carbonates, but are 
 soluble in acid only in the presence of a considerable excess. 
 
 The percentage of iodine contained in the iodo-proteins varies, 
 as might be expected, with the nature of the protein, it also 
 appears to vary somewhat with the mode of preparation or 
 subsequent treatment, since two types of compounds are formed, 
 the one containing a very high percentage of iodine, partly in 
 firm and partly in loose combination (the per-iodo-casein of 
 Liebrecht (70)), and the other a lower percentage of iodine en- 
 tirely in firm combination. 
 
 Improved methods of preparing iodized proteins have recently 
 been described by Oswald (92). 
 
 The bromine and chlorine substitution-products of the proteins 
 
158 CHEMICAL STATICS 
 
 have been specially studied by Hopkins (45) (46) (47) and Blum 
 and Vaubel (20). The following were the percentages of the 
 different halogens found by Hopkins and by Blum in firm com- 
 bination with egg-albumin.* 
 
 Hopkins 
 
 Blum 
 
 6.2 per cent iodine. 
 2.84 per cent bromine. 
 1.93 per cent chlorine. 
 
 6-7 per cent iodine. 
 4-5 per cent bromine. 
 2 per cent chlorine. 
 1.2 per cent fluorine. 
 
 As in the case of the iodo-proteins, two series of bromine com- 
 pounds exist: the one in which the bromine is firmly bound and 
 the percentage of bromine is low, the other in which a part of 
 the bromine is loosely bound and the percentage is high. Hop- 
 kins and Brook found in this latter type of brominated egg- 
 albumin 14.89 per cent of bromine. These compounds, as also 
 the chlorinated egg-albumin, are soluble in hot absolute alcohol, 
 the halogen-protein being precipitated from this solution by the 
 addition of ether (Hopkins and Brook). 
 
 The best-known naturally occurring halogen-protein is the 
 thyreoglobulin of the thyroid gland. It contains only 1.75 per 
 cent of iodine (91). The physiological action of this protein 
 does not appear to be primarily dependent upon its iodine con- 
 tent (108) and it is doubtful whether this is the physiologically 
 active iodine-containing principle of the thyroid (56) . An iodized 
 keratin, gorgonin, is found in corals (26) (40) (79) (81). It 
 contains a high (about 8) percentage of iodine. Sponges also 
 contain iodo-proteins (36) (52) (115). 
 
 Nitro substitution-products of the proteins were first prepared 
 by Loew (73). VonFtirth (29) prepared them by acting upon 
 the protein with nitrous acid, at the same time adding urea to 
 prevent the formation of nitric acid. He obtained a product con- 
 taining 1.78 per cent of N0 2 . The nitro derivatives of the pro- 
 tamins have been very extensively studied by Kossel (65) (64) 
 (67) (68) (119), who believes that the nitration of protein leads 
 to the entry of N0 2 into the guanidine radical of the arginin. 
 The nitrated proteins therefore yield nitroarginin on hydrolysis. 
 On treatment with alkali the nitro-guanidine radical breaks up 
 yielding carbon dioxide, nitrous oxide and ammonia. 
 
 * Cited after Gustav Mann (76). 
 
SULPHUR 159 
 
 10. The Compounds of Proteins with Sulphur. Uhl (118) 
 has prepared sulphur compounds of the proteins by utilizing 
 the reaction: 
 
 < H 
 
 I X R + CS 2 = I R 
 
 H C = S 
 
 I 
 SH 
 
 which occurs in alkaline solution. These compounds combine 
 with heavy metals; the salts thus formed contain a high pro- 
 portion of the heavy metal and are soluble in water. 
 
 11. The Compounds of Proteins with Oxygen. The best 
 known compound of this type is that of oxygen with the coloring 
 matter of the blood, haemoglobin. This has been especially 
 studied by Hiifner (50) who employed the spectrophotometric 
 method of measurement, and found that at high pressures (by 
 interpolation) the amount of oxygen bound up by one gramme of 
 haemoglobin is 1.338 cc., reduced to standard temperature and 
 pressure. Carbon monoxide combines with haemoglobin in 
 equivalent proportions. Hiifner considered that the reaction 
 between haemoglobin and oxygen is a balanced reaction, namely, 
 Hb -j- 2 ?=* Hb0 2 . From this, if the concentration of reduced 
 haemoglobin be Cr and of oxyhaemoglobin C and that of oxygen 
 
 where P is the partial pressure of oxygen and a t the absorption- 
 coefficient of the gas at temperature t it would follow that if K 
 is the velocity constant 
 
 Co = Ka t 
 CrXP 760' 
 
 the quantities on the right of this equation being constant at 
 constant temperatures, the relation between the oxygen tension 
 and the percentage of haemoglobin converted into oxyhaemoglobin 
 should be expressed by a rectangular hyperbola. Bohr (21), 
 however, found that the curve of dissociation of oxyhaemoglobin 
 is not a rectangular hyperbola, and he explains his results by 
 supposing that the haemoglobin, in the presence of oxygen, first 
 splits off a haematin-containing moiety and that this then sub- 
 
160 CHEMICAL STATICS 
 
 sequently unites with the oxygen. Barcroft and Roberts (9) (10) 
 (11) (12) (13) have, however, shown that the irregularities ob- 
 tained by Bohr were due to the presence of electrolytes in the 
 haemoglobin solutions; in dialysed solutions the curve of dis- 
 sociation is exactly that demanded by Hiifner's theory. The 
 velocity of dissociation of oxyhsemoglobin obeys the equation 
 indicated by the mass law. The variations of the equilibrium- 
 constant (K) with change of absolute temperature follow the 
 van't Hoff equation 
 
 K dt 2T 2 
 
 where q is constant and equal to 28,000 calories. This is there- 
 fore the heat of combination of one gram-molecule of haemoglobin 
 with oxygen. Since the amount of heat which is actually given 
 out when one gram of haemoglobin unites with oxygen is 1.85 
 calories, the molecular weight of haemoglobin, in dialysed solu- 
 tion is, according to these results, 15,000. The minimum weight 
 indicated by Hiifner's results, cited above, is 16,669. A similar 
 figure is indicated by the iron-content of the haemoglobin mole- 
 cule (55) (121) (49) (50) and by the osmotic pressure measure- 
 ments of Hufner and Gausser (51). Weymouth Reid (98), 
 however, found a value, as indicated by osmotic pressure meas- 
 urements, three times as great. Barcroft and Hill (11) suggest 
 that haemoglobin may possibly exist as a polymer of itself, under 
 certain conditions, and that the irregularities observed in the 
 curve of dissociation of oxyhaemoglobin in the presence of elec- 
 trolytes may be due to the breaking down of such aggregates. 
 The temperature-coefficient of the dissociation of oxyhaemoglobin 
 is large, about 4 per 10 degrees rise in temperature. 
 
 These masterly investigations of Barcroft effectually demon- 
 strate two things; in the first place that the union between 
 haemoglobin and oxygen is not an "adsorption combination" 
 as Wo. Ostwald has suggested (90) (101) (.13) and, in the second 
 place that, since the derivation of the van't Hoff law (reaction 
 isochore) involves the assumption that the gas laws apply strictly 
 to the system under consideration,* haemoglobin, although a 
 colloid, does not form a 'separate phase within its solution,! but 
 
 * Cf. W. Nernst (83). 
 
 t It may be remarked that under such conditions haemoglobin cannot 
 present any surface with which to "adsorb." 
 
OXYGEN 161 
 
 is molecularly distributed throughout the solution in accordance 
 with the law of Avogadro. Again we see, therefore, as we saw 
 in considering the copper albuminates (Chap. VI, section 5), that 
 the distinction which has been built up by certain authors be- 
 tween the crystalloids which form homogeneous, and the colloids 
 which are supposed to form heterogeneous, solutions, is entirely 
 artificial and not in accordance with the facts. 
 
 The results obtained by Barcroft and his co-workers have 
 received decisive confirmation at the hands of Butterfield (24) 
 who, using the spectrophotometric method, has shown with a 
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 dilution and therefore in accordance with the mass law the pro- 
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PART II 
 THE ELECTROCHEMISTRY OF THE PROTEINS 
 
CHAPTER VIII 
 THE FORMATION AND DISSOCIATION OF PROTEIN SALTS 
 
 1. Compounds of the Proteins with Inorganic Bases and 
 Acids ; the Non-Dissociable Character of the Inorganic Radical. 
 -We have already had occasion to incidentally dwell upon the 
 fact that the inorganic radical of the protein base and protein 
 acid compounds is not electrically dissociated as such. The most 
 direct proof which we possess of this fact is that which was ob- 
 tained by Bugarszky and Liebermann (4). These observers 
 employed the potentiometric method, using two different con- 
 centration chains. The first was the ordinary gas-chain: 
 
 Pt saturated with Hz 
 
 Acid (HC1) 
 
 Base (NaOH) 
 
 Pt saturated with H 2 
 
 1 
 
 2 
 
 3 
 
 4 
 
 the potential being measured, first with pure acid in 2 and then 
 with acid plus protein; the difference between the two readings * 
 yielding by computation from the Nernst formula, the number 
 of hydrogen ions bound by the protein. The second concentration- 
 chain was built up as follows: 
 
 Hg 
 
 HgCl (solid), HC1 
 
 NaCl 
 
 NaBr, HgBr (solid) 
 
 Hg 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 and enabled them to estimate, in a similar way, the number of Cl f 
 ions bound by the protein. Now the number of CV ions bound by 
 a given mass of protein dissolved in dilute HCl was found to be 
 exactly equal to the number of H + ions which it binds. The fol- 
 lowing data are compiled from those obtained by Bugarszky and 
 Liebermann : 
 
 * Less a correction expressing the potential between 2 and 3. 
 
 167 
 
168 
 
 ELECTROCHEMISTRY 
 
 EGG-ALBUMIN IN 0.05 N HC1. 
 
 Grams protein in 100 
 cc. 
 
 Per cent of H + bound 
 by the protein 
 
 Per cent of Cl' bound 
 by the protein 
 
 
 
 
 
 
 
 0.4 
 
 9.0 
 
 10.7 
 
 0.8 
 
 18.9 
 
 20.2 
 
 1.6 
 
 33.3 
 
 38.0 
 
 3.2 
 
 60.2 
 
 64.0 
 
 6.4 
 
 96.6 
 
 76.0 
 
 The slight irregularities are, save in the last observation, no 
 greater than might have arisen out of the uncertain magnitude 
 of the correction for the potential between the elements 2 and 3 
 in the gas-chain. 
 
 These striking results have been confirmed by Rohonyi (31) 
 employing Merck's crystallized ovalbumin and Witte's peptone; 
 the following were the results obtained, the substances being 
 dissolved in JV/20 HC1 solution : 
 
 Substance 
 
 Per cent 
 
 Per cent of chlorine 
 ions bound 
 
 Per cent of hydrogen 
 ions bound 
 
 Ovalbumin 
 Albumose 
 Alanin 
 
 1.9 
 1.8 
 1 5 
 
 21.9 
 37.2 
 37 9 
 
 22.6 
 35.9 
 66 2 
 
 
 
 
 
 A decided difference between the modes of combination of pro- 
 teins and an amino-acid with hydrochloric acid is thus very 
 clearly revealed. 
 
 Manabe and Matula (17) and Blasel and Matula (2) have 
 shown that at low H+ ion concentrations a greater proportion 
 of H + is bound by serum albumin or gelatin than of Cl'. Ringer 
 (33) has confirmed this observation for albumoses, but he also 
 finds that in higher concentrations of hydrochloric acid (1 per 
 cent of albumose in 0.1 N HC1) the H + and Cl' are bound equally. 
 The same tendency is shown in the observations of Bugarszky 
 and Liebermann which are quoted above, for in the solution 
 containing the lowest proportion of hydrochloric acid to protein 
 the Cl' bound by the protein was about 20 per cent less than the 
 proportion of H + which was bound. 
 
 It has been shown by Blasel and Matula (2) that deaminized 
 
NON-DISSOCIABLE INORGANIC RADICAL 169 
 
 gelatin, prepared by the action of nitrous acid upon gelatin, 
 notwithstanding the absence of end NH 2 groups, still retains the 
 power of binding Cl' ions in solutions of hydrochloric acid. 
 
 Oryng and Pauli (20) have shown that serum albumin, gelatin 
 and deaminized gelatin, dissolved in solutions of potassium chlo- 
 ride, bind a definite proportion of Cl' ions, and this proportion 
 is greatly increased by the addition of acids (such as sulphuric 
 acid) to the solution. 
 
 Confirmatory evidence is not lacking. Loevenhart (15) and 
 others * have found that rennet will not coagulate calcium casein- 
 ate unless a small amount of a dissociable salt of calcium is present. 
 The calcium bound by the casein itself is not available for this 
 purpose, but if a small quantity of acid be added then a pro- 
 portion of the calcium is freed from its combination with casein 
 and, if it forms a dissociable salt with the added acid, it is able 
 to bring about coagulation. 
 
 W. A. Osborne (21) has shown that if calcium caseinate be 
 placed inside a dialysing tube which is then immersed in a very 
 dilute solution of mercuric chloride the mercury diffuses into the 
 tube and is there held in an undissociated form, since the con- 
 centration of mercury within the tube is found, after some time, 
 to considerably exceed that of the mercury in the outside fluid. 
 
 Similarly, it has been shown by Moore, Roaf and Webster 
 (18) that if casein dissolved in dilute sodium hydroxide be placed 
 inside an osmometer of which the membrane is permeable to crys- 
 talloids but not to colloids and the concentration of NaOH be 
 rendered initially equal on both sides of the membrane, then 
 NaOH will move into the osmometer against the osmotic pressure 
 gradient and actually lead to a pronounced increase in the pres- 
 sure within the osmometer. It is evident, therefore, that not 
 only OH' but also Na + ions must have been bound by the protein, 
 since otherwise no movement of Na + across the membrane could 
 have occurred. 
 
 * The statement of van Dam (6). that the extent of coagulation depends 
 upon the quantity of Ca bound by the casein is not irreconcilable with Loeven- 
 hart 's results. As is well known, the calcium bound in casein as calcium 
 caseinate does not suffice to bring about coagulation. A dissociable salt of 
 calcium must also be present. That this salt may combine with the calcium 
 paracaseinate to form a double salt analogous to those described in the 
 previous chapters, is not at all unlikely. 
 
170 ELECTROCHEMISTRY 
 
 Rohmann and Hirschstein (30) have shown that solutions of 
 silver caseinate contain no silver ions, since they fail to yield a 
 precipitate on adding sodium chloride. 
 
 The most obvious conclusions to be drawn from these results 
 are (a) that the protein-base or protein-acid compounds are not 
 subject to dissociation at all; or else (6) that they dissociate the 
 positive and negative ions of the inorganic constituent in equiva- 
 lent proportions, i.e., undergo hydrolytic dissociation. 
 
 That these assumptions are incorrect, however, is shown by 
 a large number of experiments which demonstrate that the pro- 
 tein compounds with inorganic bases and acids are true electro- 
 lytes, independently of any hydrolytic dissociation which they 
 may undergo in solution. For example, Sjoqvist (35) has shown 
 that if egg-albumin be dissolved in dilute hydrochloric acid, as 
 the concentration of albumin is increased, keeping that of the 
 HCl-solution constant, the molecular conductivity (calculated for 
 0.025 AT HC1) diminishes until it reaches a constant minimum 
 value, which is attained when about four grams are dissolved 
 in 100 cc. of 0.025 N HC1. Now the above quoted results of 
 Bugarszky and Liebermann show that in this solution at least 97 
 per cent of the hydrochloric acid is bound by the egg-albumin. 
 The observed " molecular" conductivity (67 X 10~ 3 ) is at least 7 
 times greater than could be accounted for by the maximum 
 possible residuum of unneutralized hydrochloric acid and must 
 therefore be due to the protein-acid compound. 
 
 Solutions of the caseinates of the alkalies and alkaline earths 
 can be obtained which are neutral or even acid to litmus (Cf. 
 Chap. V), these solutions therefore contain no free base; never- 
 theless they are excellent conductors of electricity (34) (23) (27) 
 (28) since a 2 per cent solution of potassium caseinate which is 
 neutral to litmus possesses a conductivity of 92.7 X 10~ 3 recip- 
 rocal ohms per equivalent of base neutralized at 30 C. That 
 this conductivity is not attributable to associated impurities, 
 inorganic or otherwise, is shown by the following facts: 
 
 (i) It bears a definite relation to the amount of base neutralized 
 by the protein (27). 
 
 (ii) The conduction of electricity is accompanied by migration 
 of the casein to the anode, and the amount of casein transported 
 to the anode is directly proportional to the quantity of electricity 
 which is transported through the solution (29). 
 
NON-DISSOCIABLE INORGANIC RADICAL 171 
 
 Similarly, solutions of the serum-globulinates of the alkalies 
 and alkaline earths may be obtained which are neutral to litmus 
 and which nevertheless conduct electricity, the passage of a 
 direct current through these solutions being accompanied by 
 transport of the protein to the anode (10). 
 
 Only one conclusion is left open to us, therefore, namely, that 
 the salts which proteins form with inorganic acids and bases do 
 not dissociate at the point of union of the inorganic radical with 
 the protein, but elsewhere, within the protein molecule itself, 
 yielding, not an inorganic and a protein ion, but two or more 
 protein ions, in one or more of which the inorganic radical is 
 bound up in a non-dissociable form.* 
 
 On examining the details of the behavior of the protein salts, 
 as electrolytes, we are speedily compelled, by inference, to reach 
 precisely the same conclusion. 
 
 In the first place, the conductivity of solutions of certain 
 protein salts, for example potassium caseinate, is not at all affected 
 by the presence, in the solution, of an excess of the ions of the 
 inorganic radical. This fact is very clearly shown by the fol- 
 lowing experiments (27). 
 
 Two and a half grams of pure casein were dissolved in solu- 
 tions containing varying amounts of KOH of which, in each 
 instance, so much was neutralized by 0.1 N HC1 as to leave the 
 equivalent of 25 cc. of 0.1 N KOH unneutralized by the acid. 
 These solutions were then each diluted to 250 cc., so that the final 
 solutions consisted of 1 per cent casein dissolved in 0.01 N KOH 
 plus varying amounts of KC1. The conductivities ( = x) of these 
 solutions (at 30 degrees) were then determined and also the 
 conductivities (= Xi) of solutions similarly made up without the 
 introduction of casein. The conductivity of the original solu- 
 tion, before the introduction of casein, is the sum of two quanti- 
 ties, i.e., the conductivity of 0.01 KOH + the conductivity of 
 the KC1; that of the solution of the caseinate is the sum of three 
 quantities, i.e., the conductivity of the unneutralized KOH and 
 the conductivity of the KC1 + the conductivity of the caseinate. 
 Subtracting the latter from the former, therefore, we obtain 
 Xi x, which is the conductivity of the 0.01 KOH minus the 
 conductivity of the KOH unneutralized by 1 per cent casein 
 and the conductivity of the casemate itself; in other words, the 
 
 * Cf. also Chap. IX, 3. 
 
172 
 
 ELECTROCHEMISTRY 
 
 conductivity of a solution of the neutralized KOH minus the 
 conductivity of the caseinate. If we subtract this quantity 
 (xi x) from the conductivity of the neutralized KOH, therefore, 
 we obtain the conductivity of the caseinate itself. Now in these 
 solutions, as determined by the gas-chain, the concentration of 
 neutralized KOH was 0.00993, and the conductivity at 30 degrees 
 of a solution of free KOH of this concentration would be 280 X 10~ 5 
 reciprocal ohms. Subtracting Xi x from 280 X 10~ 5 , therefore, 
 we obtain the conductivity, in each of the solutions investigated, 
 of the caseinate itself. In the following table some experimental 
 results are given. In the first column is given the concentration 
 of KC1 which was present in the solution of caseinate; in the 
 second the conductivity (= Xi) of the solution containing no 
 casein; in the third the conductivity (= x) of the solution con- 
 taining casein; in the fourth the conductivity (= 280 X 10~ 5 
 (xi x)) of the caseinate itself; in the fifth the deviation of this 
 conductivity from its average value in the different solutions; 
 in the sixth the experimental error of the conductivity meas- 
 urement itself, exclusive of any possible deviations arising out 
 of errors in weighing, measurements of volume, or decomposition 
 of the casein. 
 
 Concen- 
 tration of 
 KC1 
 
 Xi (recip. ohms) 
 
 x (recip. ohms) 
 
 Conductivity 
 of caseinate it- 
 self 
 
 = Deviation 
 from average 
 
 = Experimen- 
 tal error 
 
 0.00 AT 
 
 o.oi AT 
 
 0.02 AT 
 0.03 AT 
 
 277.4X10- 5 
 417.0X10- 5 
 548.0X10- 5 
 698.0X10- 5 
 
 81.2X10- 5 
 222.6X10- 5 
 360.5X10- 5 
 494.5X10~ 5 
 
 83.8X10- 5 
 85.6X10- 5 
 91.8X10- 5 
 76.1X10- 5 
 
 -0.5X10- 5 
 + 1.3X10- 5 
 +7.5X10- 5 
 -8.2X10- 5 
 
 0. 7X10~ 5 
 1.8X10- 5 
 3.5X10-5 
 5.9X10- 6 
 
 It is evident that the estimates of the conductivity of the 
 caseinate itself which were thus obtained are appreciably con- 
 stant, the deviation from the average being irregular and less or 
 only very slightly greater than the purely metrical error of the 
 observation. In other words, whether the concentration of KC1 
 in its solution is 0.00 or 0.03 the share taken by the potassium 
 caseinate itself in conducting the current is the same within the 
 experimental error, that is, at the very highest estimate, within 
 10 per cent. 
 
 Now suppose that potassium caseinate, containing this pro- 
 portion of potassium, yields, on dissociation, only one potassium 
 
NON-DISSOCIABLE INORGANIC RADICAL 173 
 
 ion and one casein ion, then applying the ordinary laws of dis- 
 sociation, since in the solution containing no KC1 the total con- 
 centration of K ions must have been very nearly 0.01 and calling 
 A the concentration of protein ions in this solution, C the dissoci- 
 ation-constant of the caseinate and X the concentration of the 
 undissociated casemate we have, very nearly: 
 
 0.01 A = KX 
 
 while the solution containing 0.04 N potassium (0.03 N KC1) we 
 have, very nearly, 
 
 whence A = 26 X , or the caseinate must, in the first solution, have 
 been f f ths dissociated. Now it should be particularly observed 
 that this is most decidedly a minimal estimate of the degree of 
 dissociation of this salt, if the assumption upon which we have 
 proceeded is correct. For free casein is insoluble and 1 gram of 
 casein is just carried into solution by 11.4 X 10~ 5 equivalents 
 of base, whereas these solutions contained as we have seen nearly 
 nine times this amount of neutralized KOH. If we suppose that, 
 in reality, nine COOH groups in the casein molecule have been 
 neutralized by KOH in these solutions, so that the caseinate 
 yields nine K + ions, then the above solution (i.e., that which 
 contained no KC1) must have been no less than fMffffths 
 dissociated. Hence, at the very lowest estimate, if we assume 
 that it dissociates potassium ions, a solution of potassium casein- 
 ate containing 99 X 10~ 5 equivalents of potassium per gram of 
 caseinate must be f ths dissociated. 
 
 Upon dilution, therefore, its conductivity could only increase 
 by sVth, or 4 per cent and, moreover, no solution of potassium 
 caseinate could be possessed of a greater equivalent conductivity 
 than that of the above solution plus 4 per cent, i.e., about 90 X 10~ 3 
 at 30 C. 
 
 But, as we shall see in succeeding chapters, the equivalent con- 
 ductivity of a 1 per cent solution of caseinate in 0.01 N KOH 
 increases very much more than 4 per cent on dilution, nor can 
 this increase, as we shall see, be attributed to hydrolytic dissocia- 
 tion. Moreover solutions of potassium caseinate can readily be 
 obtained which are add to litmus and which possess an equivalent 
 
1 74 ELECTROCHEMISTRY 
 
 conductivity (calculated upon the basis of the potassium which 
 they contain) of over 120 X 10~ 3 or 30 per cent more than that 
 of the solution considered above. 
 
 Hence we have no resource but to conclude that our initial 
 assumption was erroneous and that potassium caseinate dis- 
 solved in water does not yield potassium ions.* 
 
 Again, we may reach the same conclusion by quite a different 
 process of reasoning and from very different experimental data. 
 The conductivity of solutions of the caseinates and globulinates 
 of the alkalies and alkaline earths and of the salts which ovo- 
 mucoid forms with acids does not decrease in direct proportion 
 with the dilution, but more slowly, indicating a progressively 
 increasing dissociation of the caseinate on dilution. From the 
 curve expressing the relation between the equivalent-conduc- 
 tivity (calculated on the basis of the inorganic radical) and the 
 dilution we can, by exterpolation, estimate the maximum equiva- 
 lent conductivity, i.e., the equivalent conductivity at infinite 
 dilution of the salt; this we can do rather accurately, since at 
 readily attainable dilutions the equivalent conductivity already 
 increases very slowly with dilution and obviously tends to ap- 
 proach a constant maximum. Now, as is well known, this maxi- 
 mum bears a constant proportion to the sum of the equivalent 
 conductivities of the ions into which the salt dissociates. 
 
 If the inorganic radical is dissociated as such, the equivalent 
 conductivity of these salts cannot be less than that of the inor- 
 ganic radical itself, but must exceed it by a quantity equal to 
 the equivalent conductivity of the protein ion or ions. In the 
 following table are compared the observed equivalent conduc- 
 tivities (at infinite dilution) of a number of protein salts at 30 
 degrees, f and those of the inorganic radicals which they contain, 
 calculated from the data given by Kohlrausch and Holborn (12)4 
 
 * It may be mentioned in passing, that in the solutions investigated 
 potassium caseinate evidently does not form a double salt with potassium 
 chloride. Were such a double salt formed, and we have seen that in many 
 cases salts of this type may be formed in protein solutions, then, of course, the 
 KC1 would not be without effect upon the conductivity of the caseinate. 
 
 t Cf. data cited in Chap. X. 
 
 t The migration-velocities of the ions at 18 degrees (p. 200) increased by 
 2 per cent per degree to reduce to the temperature employed and multiplied 
 by the proportionality between ionic velocity and reciprocal ohms per cc., i.e., 
 96.44. 
 
NON-DISSOCIABLE INORGANIC RADICAL 
 
 175 
 
 Salt 
 
 Temp., 
 Degrees 
 C. 
 
 Equivalent con- 
 ductivity at in- 
 finite dilution, 
 equivalent cone, 
 taken as that of the 
 inorganic radical. 
 Reciprocal ohms 
 per cc. per equiva- 
 lent per litre 
 
 Equivalent con- 
 ductivity at in- 
 finite dilution of 
 the inorganic radi- 
 cal. Reciprocal 
 ohms per cc. per 
 equivalent per 
 litre 
 
 Sodium caseinate (80X10" 5 equivs. 
 per gram) 
 
 25 
 
 63 5X10~ 3 
 
 50 6X10~ 3 
 
 Ammonium caseinate (80X10~ 5 
 equivs. per gram) 
 
 25 
 
 79 4X10~ 3 
 
 73 2X10- 3 
 
 Potassium caseinate (80X10~ 5 
 equivs. per gram) . . 
 
 30 
 
 80.6X10~ 3 
 
 81.0X10-3 
 
 Calcium caseinate (80X10~ 5 equivs. 
 per gram) 
 
 30 
 
 35.9X10~ 3 
 
 65.7X10-3 
 
 Strontium caseinate (80X10~ 5 
 equivs. per gram) 
 
 30 
 
 30.7X10- 3 
 
 67.0X10-3 . 
 
 Barium caseinate (80X10" 5 equivs. 
 per gram) 
 
 30 
 
 42 1X10-3 
 
 71 1X10-3 
 
 Potassium serum-globulinate 
 (20X10~ 5 equivs. per gram) 
 
 30 
 
 51 OX1Q-3 
 
 81 0X10-3 
 
 Calcium serum-globulinate 
 (20XlO~ 5 equivs. per gram). 
 
 30 
 
 23.5X10- 3 
 
 65.7X10- 3 
 
 Strontium serum-globulinate 
 (20X10" 5 equivs. per gram) 
 Barium serum-globulinate 
 (20X10~ 5 equivs. per gram) 
 Ovomucoid chloride (45X10~ 5 
 equivs. per gram) 
 
 30 
 30 
 30 
 
 27.5X10- 3 
 23.4X10-3 
 196 0X10-3 
 
 67.X 1C- 3 
 71.1X10- 3 
 81 7X10- 3 
 
 
 
 
 
 It is evident that in many cases the equivalent conductivity 
 of a protein salt is actually less, in the salts of the alkaline earths 
 very considerably less than that of its inorganic radical alone. 
 On the supposition that the protein salt splits off the inorganic 
 radical as an ion, not only the inorganic radical but also the 
 protein must be participating in the conduction of electricity 
 through its solution, and its equivalent conductivity, when com- 
 pletely dissociated, must be greater than that of the inorganic 
 radical by the amount contributed by the protein ion. Hence 
 the assumption that the salts of the proteins split off the inorganic 
 radical as an ion must be incorrect. 
 
 We have seen, in considering the constitution of the protein 
 molecule (Chap. I), that the protein molecule does not contain 
 a sufficient number of terminal NH 2 and COOH groups to 
 account for its high combining capacity for acids and bases, and 
 the suggestion was put forward that the true point of union with 
 acids and bases is the N.HOC group, and that union with 
 
176 ELECTROCHEMISTRY 
 
 bases and acids and the dissociation of the resultant salts take 
 place according to the following schemes: 
 
 H 
 
 I ++ 
 
 -N.HOC- -|- NaOH = -N" + NaOC- 
 
 I 
 OH 
 
 H 
 
 I ++ 
 -N.HOC- + HC1 = -N" + HOC- 
 
 I 
 Cl. 
 
 or in accordance with modifications of these schemes arising out 
 of the participation of dicarboxylic- and diamino-radicals in the 
 reactions. 
 
 In the succeeding pages the endeavor will be made to interpret 
 the electrochemical behavior of the proteins and their salts with 
 the aid of this hypothesis. 
 
 2. The Electrolysis of Protein Salts. It was shown by 
 Hardy (9) in 1899 that if a trace of acid be added to a solution 
 of dialysed white of egg, modified by dilution and boiling, on 
 passing a direct current through the solution the whole of the 
 protein finally moves over to the cathode, where it is precipitated. 
 If a trace of alkali is added, however, instead of acid, the whole 
 of the protein finally migrates, under the influence of the current, 
 to the anode. He later showed that serum-globulin in solution 
 behaves similarly (10). Under such circumstances, therefore, the 
 protein behaves like the anion or cation of a salt; like the cation 
 when combined with an acid; like the anion when combined 
 with a base. 
 
 Here an apparent, but not a real difficulty confronts us in the 
 application of the hypothesis outlined above to the electrolysis 
 of protein solutions. On the basis of this hypothesis the protein 
 should migrate in both directions, the cation, when the protein 
 is combined with a base, and the anion, when the protein is com- 
 bined with an acid, carrying the inorganic constituent with it. 
 At first sight it might appear as if the protein should be deposited 
 at both electrodes, but not when we look more closely into the 
 matter. In both of the cases just cited the free, uncombined 
 
ELECTROLYSIS 177 
 
 protein is insoluble, while the combined protein is soluble. Con- 
 sider the electrolysis of a compound of such a protein with a base. 
 
 The anion 
 
 rl 
 
 I 
 
 R.N" 
 I 
 OH 
 
 will migrate to the anode, and after neutralizing any excess of 
 base which may be present (since the ion, obviously, must contain 
 N.HOC groups and therefore, like the entire molecule, is am- 
 photeric) must eventually, when the film in immediate contact with 
 the electrode has become saturated with protein, be precipitated 
 
 as the uncombined and therefore insoluble protein. The case is 
 
 ++ 
 very different with the cation R.CONa, for this, on arriving at 
 
 the cathode, will bring it an excess of base and the cathode region 
 must become alkaline and, therefore, free protein cannot be pre- 
 cipitated there. Similarly, in the electrolysis of a salt of such a 
 protein with an acid the uncombined protein must be precipi- 
 tated at the cathode, but not at the anode. If the protein is 
 of such a type that the free protein is soluble, for example ovo- 
 mucoid, I have observed that no deposition of protein occurs at 
 either electrode, but only the evolution of gas, presumably hydro- 
 gen at the cathode and oxygen at the anode. That under such 
 conditions the protein nevertheless migrates to both electrodes has 
 been shown by Stirling and Brito (36) and by Howell (11). 
 
 If a direct current of about one milliampere be passed through 
 a solution of potassium caseinate, which is neutral to litmus, 
 gas may be observed to be evolved at both electrodes, but a firm 
 white spongy precipitate is deposited upon the anode, the cellu- 
 lar texture of which is attributable to entangled bubbles of gas, 
 presumably oxygen (29). 
 
 A quantity of this precipitate was collected. The anode con- 
 sisted of a spiral of platinum wire some 9 cm. long. This wire, 
 when coated with the precipitate, was well washed in a stream 
 of distilled water, and the precipitate was then scraped off into 
 99.8 per cent alcohol. The precipitate which was thus collected 
 under alcohol was washed with alcohol and ether and dried at 
 30 degrees over sulphuric acid for 48 hours. 
 
 The precipitate proved to be uncombined (base free) casein. 
 
1 78 ELECTROCHEMISTRY 
 
 In fact so nearly was it devoid of mineral content as to practi- 
 cally realize that elusive ideal, the " ash-free protein," for one 
 gram of the precipitate yielded less than two milligrams of ash. 
 Yet this casein, in every way in which it was tested, proved to 
 be perfectly normal. The possibility was thus indicated of esti- 
 mating the electrochemical equivalent of casein. 
 
 Solutions of potassium caseinate were placed in a U-tube of 
 about 30 cc. capacity, 25 cc. of solution being employed in each 
 experiment. The anode consisted of a spiral of platinum wire 
 about \ mm. thick and 9 cm. long, the diameter of the spiral 
 being about \ cm. and its " pitch" about 45 degrees. The cathode 
 simply consisted of a platinum wire dipped in the fluid in the 
 other arm of the U-tube. The U-tube was provided at the 
 bottom with a 3-way stop-cock, which could either be turned so 
 as to provide fluid communication between the two arms of the 
 tube, or else so as to permit the contents of the anodal limb to 
 escape into a receptacle. In this way it was possible to investi- 
 gate separately, if desired, the contents of each arm of the U-tube. 
 It was found that after electrolysis the fluid in the anodal arm, 
 in which the deposition of casein occurred, was practically un- 
 altered in reaction, although its casein-content was much dimin- 
 ished. In the cathodal arm, not only did the casein-content 
 diminish, but the alkalinity of the fluid was markedly increased. 
 
 The current was led from the terminals of a 110-volt circuit 
 through a 16 candle-power lamp, a milliamperemeter, a silver 
 titration voltameter containing 30 cc. of JV/100 AgN0 3 , and 
 through the solution of caseinate. 
 
 The silver was then determined by titration with a AT/100 
 ammonium thiocyanate in the presence of a constant excess of 
 nitric acid, a constant quantity (5 cc. of saturated solution} of 
 ferric alum being employed as indicator. 
 
 The amount of casein which had been precipitated by the 
 current was estimated by determining the refractive indices of 
 the original and of the electrolysed solutions at the same tem- 
 perature; the differences between the refractive indices divided 
 by 0.00152 yielding the decrease in the percentage of casein con- 
 tained in the solution consequent upon electrolysis.* The quan- 
 tity of solution employed was always 25 cc. Hence the decrease 
 
 * Cf. Chap. XIV. Also T. Brailsford Robertson (24). 
 
ELECTROLYSIS 179 
 
 in the percentage-content of casein, divided by 4, was the amount 
 of casein precipitated by the current. 
 
 The experiments were all conducted at 30 C. 
 
 Varying amounts of casein were dissolved in 100 cc. each of 
 KOH solutions of varying concentrations so that the proportion 
 of base to casein was 50 X 10~ 5 equivalents per gram (neutral 
 to litmus),* or 80 X 10~ 5 equivalents per gram (neutral to phenol- 
 phthalein),f or 100 X 10~ 5 equivalents per gram. In estimating 
 the current employed, the electrochemical equivalent of silver, 
 in grams per coulomb, is taken as 0.001118 (8). 
 
 It was found that the solutions containing the higher pro- 
 portions of base to casein yielded an apparently lower electro- 
 chemical equivalent for the casein. This was speedily traced to 
 resolution of the casein from the electrode after precipitation. 
 The anode, after having been coated with casein by the action 
 of a current of one milliampere passing through a 3 per cent 
 solution of casein, neutral to litmus, was washed in water, alcohol 
 and ether, dried and weighed. It was then immersed in a solu- 
 tion of casein (3 per cent) neutral to litmus for 2 hours, then 
 withdrawn and washed, dried and weighed as before. It was 
 found to have lost 11 milligrams in weight. Similar experiments 
 were conducted in which the solutions in which the coated wire 
 was immersed were neutral to phenolphthalein and alkaline to 
 phenolphthalein. The following were the results obtained: 
 
 Current em- 
 ployed for pre- 
 cipitation 
 
 Solution 
 
 Loss after 
 two hours 
 
 1 milliamp. 
 1 milliamp. 
 2 milliamp. 
 1 milliamp. 
 
 3 per cent casein in 
 3 per cent casein in 
 3 per cent casein in 
 3 per cent casein in 
 
 50XKr 5 equivs. 
 80XlO~ 5 equivs. 
 80X10~ 5 equivs. 
 100XlO~ 5 equivs. 
 
 KOH grm. 
 KOH grm. 
 KOH grm. 
 KOH grm. 
 
 11 mg. 
 54 mg. 
 59 mg. 
 97 mg. 
 
 Hence doubling the rate of deposition makes very little, if 
 any, difference to the rate of resolution of the casein, but increas- 
 ing alkalinity of the solution in which the deposition occurs 
 increases the rate of resolution very markedly.! 
 
 * Cf. Chap. V. Also T. Brailsford Robertson (27). 
 
 t Cf. Chap. V. 
 
 $ If the anode be much less than 9 cm. in length there is a tendency, after 
 prolonged electrolysis, to what may be termed "flocculent deposition," or 
 precipitation of the casern within the body of the fluid in the anodal arm and 
 
180 ELECTROCHEMISTRY 
 
 Assuming the rate of resolution to be constant, since the con- 
 tents of the anodic arm are not appreciably altered in reaction 
 in the electrolysis, it is possible to calculate from the above data 
 for each of the solutions and periods employed the loss due to 
 resolution. In the column headed "Loss due to resolution" in 
 the accompanying tables these quantities are given. On adding 
 them to the amounts of casein lost from the solution from which 
 the "apparent" values of the electrochemical equivalent are 
 estimated one obtains the "corrected" values corresponding to 
 the total precipitation actually induced by the passage of the 
 current. 
 
 The possible error in the refractometer reading is 1' of the 
 angle of total reflection, this corresponds to an error of, 0.00010 in 
 the refractive index, that is, to an error of 0.07 in the estimated 
 decrease in the percentage of casein due to electrolysis of the 
 solution, and to an error of 0.0175 in the estimate of the amount 
 of casein deposited by the current. The possible error in each 
 estimate of the electrochemical equivalent, arising from this source, 
 is indicated in the tabulated results. 
 
 The average values for the electrochemical equivalent in the 
 different solutions are obviously, within the experimental error, 
 identical. Now in solutions containing 50 X 10~ 5 equivalents 
 of base per gram, the combining capacity of casein is 50 X 10~ 5 
 equivalents per gram, in solutions containing 80 X 10~ 5 equiva- 
 lents per gram, it is 80 X 10~ 5 equivalents per gram, and in solu- 
 tions containing 100 X 10~ 5 equivalents per gram it is between 
 
 99 and 100 X 10~ 5 equivalents per gram.* In the solutions 
 investigated, therefore, the combining weight of the casein varies 
 
 100 per cent, yet the electrochemical equivalent, measured in this 
 way, remains the same. 
 
 These facts are to be interpreted as follows : We have seen that 
 the casein anion, which is free from base, must migrate to the 
 anode. There it may be presumed to react with water, liber- 
 ating oxygen and free casein, which combines with the excess 
 
 not upon the wire. Under these conditions, resolution is apparently more 
 rapid and even if the flocculent deposit be filtered off from the anodal fluid 
 before the alkaline cathodal fluid is mixed with it, the values of the electro- 
 chemical equivalent are low. No such phenomenon was observed when the 
 anode was of sufficient length. 
 
 * Cf. Chap. IX. Also T. Brailsford Robertson (27). 
 
ELECTROLYSIS 
 
 181 
 
 a 
 
 X 
 
 
 
 s> 
 
 3 5. 
 
 O 
 
 o 
 
 fi-8 
 
 8888888 
 
 ddddddo 
 
 O CO (N CO -^ (N <M 
 
 ddddddo 
 -H -H -H -H -H -H -H 
 
 o 
 
 1C iO iO 1C C 1C O 
 
 poppopp 
 ddddddo 
 -H -H - -H -H -H -H 
 
 iO 10 *O O iC O OO 
 i I CO OO OO i I OO CO 
 
 0} T-( CO T-H CO T-l T-H 
 
 ddddddo 
 
 S S.S.S.S.S.S 
 S S p B S S 9 
 oojco o 
 
 02 02 CO 02 GQ 02 
 
 (N <N (M <N (M (N T-H 
 
 OOOOOOO 
 
 XXXXXXX 
 
 T-H O5 00 O I^ T-H 10 
 
 CO Tt< * CO CO <N C<l 
 
182 
 
 ELECTROCHEMISTRY 
 
 98 
 
 X 
 
 II 
 
 0.0" 
 
 C<1 lO 1C ^ CO CO to 
 
 ddddddd 
 -H -H -H -H -H -H -H 
 
 ^b 
 
 ooooooo 
 
 -H 
 
 gs^ g 
 
 iO *O TH CO CO O 
 T I 1-H T ( 1 I C^ C? 
 
 ddddddd 
 
 -H-U-H-H-H-H-H 
 o i 1 1^ co o oo o 
 
 O5 l> O O5 tO CO OO 
 
 ppppopp 
 ddddddd 
 
 ppppopp 
 ddddddd 
 -H -H -U -H -H -H -H 
 
 O CO O OO CO <N O 
 
 OO l>- T ( CD !> lO 1* 
 -* O5 00 * Oi O CO 
 
 ooooooo 
 
 <N (N T}< Tj< <N (N T-H 
 
 mmi 
 
 XXXXXXX 
 
 Sp^w^ 
 
 O CO 00 00 OO O O 
 
 CO CO CO CO CO CO CO 
 
 
 i 
 I? 
 
 ii 
 
 
 
 
 8-2 
 
 do 
 
 O 
 
 
 ll 
 
 -H4J 
 
 41 
 
 
 n 
 
 -O oj 
 
 11 
 (M 
 
 1 
 
 
 I 
 
 do 
 
 d 
 
 
 ill 
 
 MJ 
 
 
 ill 
 
 .2 
 
 |i 
 
 ss 
 
 
 
 
 11 
 
 do 
 
 
 *o 
 
 o.S 
 
 -H-H 
 
 
 a 
 
 'w'fl o 
 
 ^HO 
 
 
 c3 
 & 
 
 If 
 
 1 t 1 1 
 
 OO 
 
 do 
 
 
 1 
 
 II 
 
 
 
 
 
 g 
 
 
 
 M 
 
 g 
 
 b-b- 
 
 
 S 
 
 
 
 T 1 TH 
 OO 
 
 
 1 
 
 || 
 
 do 
 
 4H 
 
 
 a 
 
 y O 
 
 OO CO 
 00 O5 
 
 o^ ^ 
 
 
 "3 
 cr 
 
 
 
 do 
 
 
 
 o 
 
 
 
 X 
 
 1 
 
 Time of 
 passage 
 
 S ^ 
 
 
 9 
 
 .s 
 
 T 1 T I 
 
 XX 
 
 
 
 
 <N<M 
 
 T 
 
 t 
 
 
 1! 
 
 CO <N 
 
ELECTROLYSIS 183 
 
 of base until the proportion of base to casein in the film in im- 
 mediate contact with the anode falls to that which obtains at 
 "saturation" of the base with casein. Any additional casein 
 thus migrating into the film in contact with the anode must be 
 precipitated as uncombined casein. The cations, containing the 
 potassium, migrate to the cathode and there react with water, 
 liberating KOH, casein and hydrogen, the casein reacting with 
 the excess of KOH to again form potassium caseinate and to 
 again participate in carrying the current in each direction. 
 
 Hence the electrochemical equivalent which is actually meas- 
 ured in solutions of all reactions is that of casein at "saturation" 
 of the base with the protein. 
 
 Rejecting the data obtained in the solutions alkaline to phenol- 
 phthalein, on account of the possible error arising from hydro- 
 lytic decomposition of the protein due to the excess of alkali, 
 the average of all the determinations yields the value 0.0242 
 0.0019 for the electrochemical equivalent of casein. 
 
 Multiplying this by the Faraday constant, 96,530, we obtain 
 the weight of casein in grams which transports one atomic charge. 
 This is 2336 =t 183. 
 
 Now at "saturation" of a base by casein, the proportion of 
 base to casein is 11.4 X 10~ 5 equivalents per gram,* correspond- 
 ing, if at this reaction casein combines with only one molecule 
 of base, with the molecular weight of 8772. If we assume that 
 at "saturation" of the base with casein two, three, or four, etc., 
 molecules of base are bound up in one molecule of caseinate the 
 molecular weight of the casein would be two, three, or four, etc., 
 times 8772. Either of two assumptions may now be made: 
 
 (i) The potassium caseinate dissociates into potassium and 
 casein ions. If this be the case then the weight of the casein 
 anion must be that of the molecule of casein, i.e., a multiple of 
 8772, and the valency of the casein ions must be a multiple of 
 
 8772 
 2336 183 
 
 i.e., of 3.8 dz 0.3, or, in round numbers, 4. 
 
 (ii) The potassium caseinate dissociates into two protein ions 
 of approximately equal weight. If this be the case then the 
 weight of the casein anion must be half that of the molecule of 
 
 * Cf. Chap. V. Also T. Brailsford Robertson (24) (27). 
 
184 ELECTROCHEMISTRY 
 
 casein, i.e., a multiple of 4386, and the valency of the casein ions 
 
 must be a multiple of g, i- e -> f 1-9 0-15, or, in round 
 
 numbers, 2. 
 
 Since, as we have seen in the earlier part of this chapter, the 
 former of these two assumptions is inadmissible, we may con- 
 clude that the valency of the casein ions, in solutions of a base 
 11 saturated" with casein, is a multiple of 2. This obviously cor- 
 responds with the view that the caseinate dissociates into two 
 protein ions in accordance with the scheme: 
 
 H 
 
 I ++ 
 
 Ri.N = C.R2 + KOH = RjN" + KOCR* 
 I i 
 
 OH OH 
 
 3. The Relative Masses of Protein Anions and Cations. 
 
 From the above experimental results it appears that the masses 
 of the protein cation and anion must be nearly equal, for other- 
 wise the weight of one ion would not be one-half but some other 
 fraction of the entire molecule and the valency, deduced from 
 the above experimental data, would not be a whole number but 
 some fraction. Since the valency of an ion is necessarily a whole 
 number and not a fraction, the weights of the cations and anions 
 which the protein molecule yields must be nearly equal. The 
 above data do not enable us, however, to decide whether or not 
 the weights of the cations and anions are exactly equal, since the 
 precision attained in these experiments is not sufficient to reveal 
 with certainty a difference of less than 10 per cent between the 
 weights of the two ions. 
 
 It has been shown by Bredig (3) that the equivalent migration- 
 velocities * of very heavy ions under unit potential gradient at 
 constant temperature tend to approach a minimal constant value 
 of about 20 X 10~ 5 cm. per sec. at 18 C. The conception de- 
 veloped above, therefore, of the mode of dissociation of the salts 
 of a protein leads to the conclusion that the velocities of migra- 
 tions of both the cation and the anion of a protein salt must be 
 
 * That is, the migration-velocity under unit force. For a divalent ion 
 under unit potential gradient the force exerted is twice as great as that which 
 is exerted on a univalent ion; the absolute velocity is, therefore, twice as great 
 as that of a univalent ion, but the equivalent velocity is the same. 
 
RELATIVE MASSES OF PROTEIN IONS 
 
 185 
 
 equal. Equal numbers of protein ions must therefore migrate 
 to the anode and to the cathode respectively. Now since only 
 one of these ions is precipitated it would appear possible to de- 
 termine whether or not they are of equal weight by measuring 
 the change in concentration at the anodal and cathodal regions. 
 Employing Hittorf s method of representation, in the accompany- 
 ing diagram let the region to the right represent the cathodal and 
 that to the left the anodal region. Let the black spots repre- 
 sent anions and the white cations. The initial state of the solu- 
 tion is represented by the first double row of spots and its final 
 condition, after the decomposition of six molecules by the current, 
 by the second double row of spots. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 o 
 
 o 
 
 o 
 
 
 
 O 
 
 o 
 
 o 
 
 o 
 
 o 
 
 
 
 o 
 
 o 
 
 
 
 
 
 
 
 
 
 
 O 
 
 o 
 
 o 
 
 o 
 
 o 
 
 o 
 
 o 
 
 o 
 
 
 
 o 
 
 
 
 
 
 Since the velocities of the ions are presumed to be equal, the 
 number of undecomposed molecules of the original caseinate 
 must, after the passage of the current, be the same on each side. 
 The ions which have separated to the left (anions) have been 
 precipitated while those which have migrated to the right have 
 remained in solution. It is clear from the above diagram that 
 on the right six anions have been lost and six cations have been 
 gained. If they were equal in weight, therefore, it would appear 
 as if the concentration of casein in the right (cathodal) half should 
 remain unaltered by the passage of electricity. A moment's con- 
 sideration, however, will suffice to show that this is not correct. 
 In attaining this conclusion the assumption has been made that 
 the casein cations after reaching the cathode, no longer partici- 
 pate in the carrying of current. Unquestionably this is not the 
 case. As we shall see in the two succeeding chapters, the com- 
 bining capacity of casein increases markedly with increasing 
 
186 ELECTROCHEMISTRY 
 
 alkalinity of its solution, at first in direct proportion, each fresh 
 equivalent of combined alkali splitting another N.HOC union. 
 Therefore the potassium or other metal which is transported 
 along with the casein to the cathode, on being transformed there 
 into the hydrate, will split the ion with which it has travelled 
 into two, thus: 
 
 + 2 H 2 O 
 = H 2 N.Ri.COH.NR2.COOH + KOH + H 2 
 
 + KOH H 
 
 = H 2 N.R 1 .COK + + ^N.R 2 .COOH 
 I 
 OH 
 
 the resultant cation may be presumed to be retained in the cath- 
 ode region while the anion migrates to the anodal region. Each 
 cation which the cathodal region gains, therefore, yields up, 
 provided the masses of the cations and anions are in every case 
 equal, one-half of its mass to the anodal region again. Referring 
 to the diagram again, therefore, the true gain by the cathodal 
 region will be only half of that represented, equal in weight to 
 only three of the six ions which have been deposited on the anode, 
 while its loss will have been three molecules, together equal to 
 the total weight of casein deposited on the anode. Provided, 
 therefore, the anions and cations in a solution of potassium 
 caseinate are equal in weight, no matter what their absolute 
 weight, then it is clear that the loss of casein from the cathodal 
 region must be exactly half the loss from the anodal region. 
 
 I have endeavored to test this theoretical conclusion by de- 
 termining the loss of casein from the anodal and cathodal limbs 
 of the U-tube employed in the experiments described in the 
 preceding section of this chapter. The following are some ex- 
 perimental results. The measurements of the amounts of casein 
 were made by means of the refractometer, as described in the 
 preceding section. 
 
 (1) Solution of 3.75 per cent casein in 0.03 N KOH; neutral to phenol- 
 
 phthalein. 
 
 Electrolysis for 2 hours at 30 degrees. Current approx. 1 milliampere. 
 Gram of casein lost from the anodal arm . 062 . 007 
 
 Gram of casein lost from the cathodal arm 0.036 0.009 
 
 Ratio 1.9 0.7 
 
MIGRATION-VELOCITY 187 
 
 (2) Solution of 4 per cent casein in 0.02 N KOH; neutral to litmus. 
 Electrolysis for 2 hours at 30 degrees. Current approx. 1 milliampere. 
 
 Gram of casein lost from the anodal arm . 093 . 008 
 
 Gram of casein lost from the cathodal arm . 045 . 007 
 
 Ratio 2. 15 0.55 
 
 (3) Solution of 3.75 per cent casein in 0.03 N KOH; neutral to phenol- 
 
 phthalein. 
 
 Electrolysis for 2 hours at 30 degrees. Current approx. 1 milliampere. 
 Gram of casein lost from the anodal arm . 070 db . 008 
 
 Gram of casein lost from the cathodal arm . 045 . 009 
 
 Ratio 1 . 65 . 55 
 
 Within the experimental error, therefore, the ratio of the anodal 
 to the cathodal loss is 2, as demanded by theory. The experi- 
 mental error in determining the ratio is, however, large, as the 
 above figures reveal, and although these results may be taken as 
 confirmatory of the general correctness of the above outline of 
 the mechanism of electrolysis in these solutions, yet they do not 
 suffice to enable us to determine whether or not the protein anions 
 and cations are absolutely equal in mass. Further elaboration 
 and refinement in the technique of these measurements will 
 doubtless enable us in the future, however, to measure the rela- 
 tive masses of the protein anions and cations with considerable 
 precision. 
 
 In passing it may be pointed out that these results afford a 
 striking confirmation of the view which I have developed above 
 that the protein salts in solution in water do not yield protein 
 and inorganic ions but only protein ions. Referring to the 
 Hittorf diagram again it will be evident that if the cations 
 were potassium ions the loss of casein from the anodal region 
 should be at least four times that from the cathodal region, since 
 the equivalent velocity of potassium ions is at least four times 
 that of heavy organic ions. The experimental fact that the loss 
 from the anodal region is only about twice as great as that from 
 the cathodal region can only be interpreted by assuming that 
 protein material is transported into the cathodal region by the 
 current, in other words, that the current is transported in both direc- 
 tions by protein ions. 
 
 4. The Migration- Velocity of Protein Ions. W. B. Hardy 
 endeavored to measure the migration-velocities of serum-globu- 
 lin ions directly (10) by placing solutions of serum-globulin, 
 combined with acids or bases, at the bottom of a U-tube and 
 
188 ELECTROCHEMISTRY 
 
 placing above the solutions, in both arms of the U-tube, a solu- 
 tion of the acid or base employed to dissolve the protein. A 
 potential gradient was placed across the U-tube and it was ob- 
 served that in acid solutions both boundaries of the protein solu- 
 tion migrated towards the cathode, while in alkaline solutions 
 both boundaries of the protein solution migrated towards the 
 anode. The rate of migration varied between 7 to 10 X 10~ 5 cm. 
 per sec. for unit potential gradient when strong acids or bases 
 (HC1 and NaOH) were employed and about 20 X 10~ 5 cm. per 
 sec. when a weak acid (acetic acid) was employed. Following 
 the view which I have developed of the mode of dissociation of 
 protein salts, it would appear as if the boundaries should move 
 in opposite directions at approximately equal velocities. A 
 moment's consideration of the method of measurement employed 
 by Hardy shows, however, that under the conditions of his ex- 
 periment this would not occur. At the boundary of the protein 
 solution (nearly neutral) and the HC1 solution (for example) a 
 difference of potential of considerable magnitude would exist 
 owing to the much more rapid diffusion of H + into the protein 
 solution than of Cl'. This difference of potential would lower the 
 potential gradient at the cathodal boundary and raise it at the 
 anodal boundary. Bearing in mind the fact that the two protein 
 ions are possessed of the same equivalent migration-velocities 
 under unit potential fall, the effect of these inequalities in poten- 
 tial gradient must have been, at the anodal boundary, to urge 
 the protein anions toward the anode more rapidly than the cat- 
 ions were repelled from the boundary, and, at the cathodal bound- 
 ary, to repel the protein anions from the boundary into the protein 
 solution more rapidly than the cations crossed the boundary. 
 The net result of these processes would be, obviously, a migration 
 of the protein, as a whole, towards the anode. The contact- 
 differences of potential at the boundaries would be equal in 
 magnitude but opposite in sense and hence both boundaries 
 would migrate at the same velocity, but in the same direction. 
 The fact that -the highest velocities were obtained when weak 
 acids (acetic) were employed, in which the difference between 
 the equivalent velocities- of the ions (H + and acetanion) is great- 
 est, strongly supports this interpretation of Hardy's results. 
 Alkali-globulin, in contact with alkaline solutions would, of 
 course, move as a whole, but somewhat more slowly (since the 
 
THEORY OF PROTEIN IONIZATION 189 
 
 difference between the velocity of OH 7 and that of Na+ is not 
 so great as that between H+ and Cl') towards the cathode. This 
 'accords with the experimental observations of Hardy. The 
 velocities measured by Hardy, therefore, do not afford a meas- 
 ure of the migration-velocities of protein ions under a uniform 
 potential gradient. 
 
 5. Objections to the above Theory of Protein lonization. 
 Several objections to the hypothesis of protein ionization which 
 has been presented above have been urged by Pauli, Samec and 
 Strauss (22). These objections may be summarized as follows: 
 
 (i) The hypothesis involves the splitting in solution of a poly- 
 amino-acid into parts which are not amino-acids. Enzymatic 
 splitting of a protein, if it took place in the same way, would not 
 yield amino-acid end-products. This objection is really analo- 
 gous to that which was originally advanced against Arrhenius' 
 theory of the electrolytic dissociation of inorganic substances. 
 It will be remembered that at the time that that hypothesis was 
 first advanced, it was argued that the ionization of KC1 into 
 K + and Cl' would imply the presence of free potassium and chlo- 
 rine in the solution, whereas the characteristic properties of these 
 substances are not displayed by a solution of KC1. The answer 
 to this was simply that the properties of an ion are not to be 
 estimated in terms of those of a molecule and furthermore the 
 ability of ions in a solution to display properties independent of 
 those of the oppositely charged ion is limited by electrostatic 
 forces. We may well suppose that protein ions, like other ions 
 in solution, cannot react with other substances independently 
 of the corresponding ion of opposite charge. In considering the 
 reaction between proteins and water which leads to the decompo- 
 sition of the polyamino-acid chains and is accelerated by enzymes 
 we must consider the two ions of the protein molecule together, 
 and not each ion separately. Undoubtedly the hydrolytic de- 
 composition of a protein must involve the interaction of the 
 protein ions with accompanying transfer of a labile hydrogen 
 atom from the cation to the anion in accordance with the scheme : 
 
 H 
 
 I 
 
 -C = N- + HO.H= -C-OH + N- 
 
 I II I 
 
 OH OH 
 
1 90 ELECTROCHEMISTRY 
 
 The presence of a labile atom in a molecule leads, not unusually, 
 to the development of color in solution, due to the absorption 
 of light-vibrations by the vibrating atom. The development or 
 non-development of absorption-bands in the visible or photo- 
 graphic spectrum will, of course, depend upon the ratio of the 
 frequency of vibration of the labile atom to the frequency of the 
 light-waves which impinge upon it. That the presence of ab- 
 sorption-bands in the visible spectrum is not an inevitable con- 
 sequence of the presence within the molecule of a labile hydro- 
 gen atom is shown by the analogous instance of an equilibrium 
 between the keto- and enol-forms in the colorless hydantoins (5). 
 
 (ii) The above-mentioned authors still consider that electro- 
 phoresis experiments show the presence of only one protein ion. 
 Now this is unquestionably the reverse of the fact. While it is 
 true that a superficial consideration of Hardy's results, alluded to 
 in the preceding section of this chapter, might encourage such a 
 supposition, we have seen that more careful analysis of the condi- 
 tions of the experiment shows that his results admit of a very 
 different interpretation, while the experiment cited at the end of 
 section 3 showing that in solutions of casein in which the minimal 
 valency of the casein ion, on the supposition that potassium casein- 
 ate yields only one protein ion, must be 4, the loss of protein in 
 the cathodal arm during electrolysis is only one-half the loss in 
 the anodal arm, is totally inconsistent with the view that protein, 
 during electrolysis, migrates in only one direction. 
 
 Direct proof, however, of the simultaneous migration of protein 
 to both poles under the influence of an electric current is fortu- 
 nately available. Stirling and Brito (35) have shown that if a 
 direct current be allowed to traverse a solution of haemoglobin, 
 deposition of crystals of haemoglobin occurs at both electrodes. 
 An alternating current is without effect. Howell (11) has fur- 
 thermore shown that if a direct current be passed through a 
 solution of fibrinogen, the fibrinogen increases in concentration 
 at both poles although, as might be anticipated, the solutions of 
 fibrinogen obtained from the neighborhood of the two poles differ 
 in some particulars from one another in their behavior towards 
 thrombin. Since haemoglobin is a crystalline protein and fibrin- 
 ogen, as Howell has recently shown, is converted by thrombin 
 into a crystalline protein (fibrin), it cannot be urged that in these 
 cases we are dealing with the opposite migrations of two distinct 
 
THEORY OF PROTEIN IONIZATION 191 
 
 and oppositely charged colloids. The phenomena can only be 
 interpreted to mean that solutions of these proteins contain oppo- 
 sitely charged but otherwise similar moieties, i.e., oppositely 
 charged protein ions. 
 
 (iii) Pauli, Samec and Strauss also urge the objection that 
 under certain conditions (a low proportion of acid to protein) 
 H + and Cl' may not be bound equally in solutions of protein in 
 hydrochloric acid. This, however, does not really affect the 
 question of the mode of ionization of the protein molecule but 
 rather the question of the relative affinity of the nitrogen atom 
 for H + and Cl' ions. It is not inconceivable that some measure 
 of "Z witter ion" or doubly and oppositely charged ion formation 
 may occur in protein solutions (37). Yet this is not the normal, 
 but rather the exceptional form of ionization and it is especially 
 remarkable, and quite inconsistent with the view that proteins 
 normally dissociate into protein and non-protein ions, that the 
 inferiority of Cl' binding to H + binding is dependent solely upon 
 the proportion of HC1 to protein and not at all upon the dilution 
 of the system, although the equivalent conductivity of protein 
 solutions is very decidedly influenced by dilution (Cf. section 1). 
 
 Perhaps the most obvious objection to the hypothesis which 
 I have advanced is that a form of ionization involving the break- 
 ing of a double bond between carbon and nitrogen is very unusual 
 and in fact without precedent in other fields of chemistry. It 
 must be remembered, however, that as the experiments of Gom- 
 berg (7) have so beautifully and decisively shown, the precise 
 point within a molecule at which ionization may occur is de- 
 termined by the strains to which the molecule is subjected, and 
 that when the strain is unusually great the break involved in 
 ionization may occur at points which resist the tension due to 
 strains of normal magnitude. Thus ethane, H 3 C CH 3 , is a sub- 
 stance of remarkable stability, and not only ethane, but tetra- 
 phenyl ethane, (C 6 H5) 2 HC CH(C 6 H 5 )2, and pentaphenyl ethane 
 (CeH 5 ) 3 C CH(C 6 H 5 ) 2 , are stable substances. Yet all attempts to 
 prepare hexaphenylethane have failed for the reason that the 
 compound breaks in the middle, the bond between the carbons, 
 which we are accustomed to regard as one of the most stable 
 types of union, being ruptured by the strain imposed upon it by 
 the great weight of three phenyl groups at either end of the 
 molecule. The rupture of this bond is all the more remarkable 
 
1 92 ELECTROCHEMISTRY 
 
 because it involves the creation of a trivalent carbon atom and 
 the independent existence of an unsaturated radical. Free radi- 
 cals containing trivalent carbon have been prepared from com- 
 pounds belonging to a wide variety of types, and, more recently 
 the existence of divalent nitrogen has also been recognized. 
 With facts such as these in mind the customary stability of any 
 linkage in the simpler types of organic compounds no longer 
 suffices as a priori evidence of the impossibility of its rupture in 
 compounds of such a type as to expose it to strains of very un- 
 usual magnitude. Now the protein molecule weighing no less 
 than from 10,000 to 20,000 and consisting of a large number of 
 amino-acids linked together in long chains must evidently be 
 subject to exceptional strains. Just as a long thin bar of ma- 
 terial, no matter how great its strength, will, ultimately, if suffi- 
 ciently long and unsupported, break of its own weight, so in the 
 long chain of atoms composing a protein molecule, rupture of 
 linkages may occur which are sufficiently strong to resist all the 
 strains to which shorter or more symmetrical molecules are com- 
 monly subjected. It may of course be urged that this argument 
 proves too much, since, by parity of reasoning, the split of the 
 linkage between carbon and nitrogen should occur at all times and 
 not be dependent upon salt-formation, the additional strain im- 
 posed by the weight of a molecule of hydrochloric acid or sodium 
 hydroxide being negligible in comparison with the total strain to 
 which the molecule is subjected. It must be remembered, how- 
 ever, that the additional strains which a molecule of acid or base 
 introduces into the molecule are not merely those commensurate 
 with and attributable to its weight, but also strains of electrostatic 
 origin, since the salt which is formed unquestionably undergoes 
 ionization. It may very possibly be true that the first step in 
 salt formation consists in the neutralization of end NH 2 or 
 COOH groups, but that the ionization of the compound formed, 
 leading to the development of electrostatic tension at the very 
 places at which it must exert the greatest strain, namely the ex- 
 tremities of the molecule, results in the splitting of the otherwise 
 stable linkage C = N and the redistribution of the compo- 
 
 I 
 nents of the molecule and the strains to which it is subjected. 
 
 6. Biological Applications; the * 'Selective" Action of Living 
 Tissues. The non-dissociable character of the inorganic con- 
 
BIOLOGICAL APPLICATIONS 193 
 
 stituent of the protein salts affords, I believe, an explanation of 
 an important physiological phenomenon; I refer to the well- 
 known power which living tissues possess of " selecting" or storing 
 up certain inorganic constituents in a concentration greater than 
 that in which these substances are found in the surrounding 
 liquid medium (23) (25) (26). Thus the skeletal muscles and the 
 red blood corpuscles contain a marked excess of potassium over 
 sodium, while the plasma which bathes them contains much less 
 potassium than sodium. Again, although in fresh- water streams 
 the concentration of potassium salts is often very low, the plants 
 which live in them are capable of storing up a comparatively 
 large amount of potassium in their tissues. 
 
 If we place within a dialyser an excess of diffusible potassium 
 salts over diffusible sodium salts and dialyse against a solution 
 containing excess of diffusible sodium salts, the proportions of 
 sodium to potassium within and without the dialyser sooner or 
 later readjust themselves, approaching equality. Hence the above- 
 mentioned phenomenon, which is met with in living tissues, 
 admits, as Loeb has pointed out (14) of only one explanation, 
 the inorganic constituents of a tissue which are found therein in 
 excess of their concentration in the fluids which bathe it must 
 exist within the tissue in the form of non-dissociated non-diffu- 
 sible compounds. "If a tissue utilizes one kind of metal in this 
 way, for example K, while another metal, for example Na, is 
 chiefly used for the formation of dissociable compounds with 
 Na as the free ion, the consequence will be that the ashes of the 
 tissue contain K and Na in altogether different proportions from 
 those in which they are contained in the surrounding solution. 
 I think we may take it for granted that, at least, potassium forms 
 a non-dissociable constituent of the protoplasm of a number of 
 tissues of animals and plants" (Loeb. loc. cit.). 
 
 If we admit that, as many investigators now believe (13) (14) 
 (32) (26), the inorganic constituents of living tissues are partly 
 united with proteins, the fact that such unions dissociate only 
 into protein and not into protein and inorganic ions affords a 
 sufficient explanation of the above phenomenon. 
 
 Loeb (13) (14) and W. A. Osborne (21) have advanced an 
 analogous explanation of the " oligodynamic " (16) (19) action of 
 many highly toxic heavy metals. 
 
194 ELECTROCHEMISTRY 
 
 LITERATURE CITED 
 
 (1) Beutner, R., Biochem. Zeit. 47 (1912), p. 73. 
 
 (2) Blasel, L., and Matula, J., Biochem. Zeit. 58 (1914), p. 417. 
 
 (3) Bredig, G., Zeit. f. physik. Chem. 13 (1894), p. 191. "" ... _ 
 
 (4) Bugarszky, S., and Liebermann, L., Arch, f . d. Ges. Physiol. 72 (1898), 
 
 p. 51. 
 
 (5) Dakin, H. D., Journ. Biol. Chem. 13 (1912-13), p. 357; Amer. Chem. 
 
 Journ. 44 (1910), p. 48. 
 
 (6) van Dam, W. Zeit. f . physiol. Chem. 58 (1908), p. 295; 61 (1909), p. 147. 
 
 (7) Gomberg, M., Journ. of Indust. & Engineering Chem. 6 (1914), p. 33. 
 
 (8) Guthe, K. E., Bull. U. S. Bureau of Standards (1905), p. 362. 
 
 (9) Hardy, W. B., Journ. of Physiol. 24 (1899), p. 288. 
 
 (10) Hardy, W. B., Journ. of Physiol. 33 (1905), p. 286. 
 
 (11) Howell, W. H., Amer. Journ. of Physiol. 40 (1916), p. 526. 
 
 (12) Kohlrausch, F., and Holborn, L., "Das Lietvermogen der Elektro- 
 
 lyte," Leipzig, 1898. 
 
 (13) Loeb, Jacques, Amer. Journ. of Physiol. 3 (1900), p. 327. 
 
 (14) Loeb, Jacques, "The Dynamics of Living Matter," New York (1906). 
 
 (15) Loevenhart, A. S., Zeit. f. physiol. Chem. 41 (1904), p. 176. 
 
 (16) Loew, O., Landw. Jahrb. 20 (1891), p. 235. 
 
 (17) Manabe, K., and Matula, J., Biochem. Zeit. 52 (1913), p. 269. 
 
 (18) Moore, P., Roaf, H. E., and Webster, A., Biochem. Journ. 6 (1912), 
 
 p. 110. 
 
 (19) Nageli, O., Denkschr. der Schweiz. Naturforsch. Ges. 33 (1893), p. 1. 
 
 (20) Oryng, T., and Pauli, W., Biochem. Zeit. 70 (1915), p. 368. 
 
 (21) Osborne, W. A., Journ. of Physiol. 34 (1906), p. 84. 
 
 (22) Pauli, W., Samec, M., and Strauss, E., Biochem. Zeit. 59](1914), p. 470. 
 
 (23) Robertson, T. Brailsford, Journ. of physical Chem. 11 (1907), p. 542. 
 
 (24) Robertson, T. Brailsford, Journ. of physical Chem. 13 (1909), p. 469. 
 
 (25) Robertson, T. Brailsford, Univ. of Calif. Publ. Physiol. 3 (1909), p. 
 
 170. 
 
 (26) Robertson, T. Brailsford, Ergeb. d. Physiol. 10 (1910), p. 334. 
 
 (27) Robertson, T. Brailsford, Journ. of physical Chem. 14 (1910), p. 528. 
 
 (28) Robertson, T. Brailsford, Journ. of physical Chem. 14 (1910), p. 601. 
 
 (29) Robertson, T. Brailsford, Journ. of physical Chem. 15 (1911), p. 179. 
 
 (30) Rohmann, F., and Hirschstein, L., Beitr. z. chem. Physiol. und Path. 
 
 3 (1902), p. 288. 
 
 (31) Rohonyi, H., Biochem. Zeit. 44 (1912), p. 165. 
 
 (32) Richards, T. W., Journ. of physical Chem. 4 (1900), p. 207. 
 
 (33) Ringer, W. E., Zeit. f. physiol. Chem. 95 (1915), p. 195. 
 
 (34) Sackur, O., Zeit. f. physik. Chem. 41 (1902), p. 672. 
 
 (35) Sjoquist, J., Skand. Arch. f. physiol. 5 (1895), p. 277. 
 
 (36) Stirling W., and Brito, P. S., Journ. Anat. and Physiol. 16 (1882), p. 446. 
 
 (37) Winkelblech, K., Zeit. f. physik. Chem. 36 (1910), p, 546, 
 
CHAPTER IX 
 THE COMBINING CAPACITY OF THE PROTEINS 
 
 1. The Electrochemical Determination of the Combining 
 Capacity of the Proteins. The potentiometric method was 
 employed by Bugarszky and Liebermann (5) for the purpose of 
 demonstrating that proteins possess a true combining capacity 
 for acids and bases. I have utilized this method in determining 
 the relationship of the combining capacity of the proteins to the 
 alkalinity or acidity as well as the absolute concentration of their 
 solutions (54) (59) (60). In brief, the principles upon which the 
 method depends are as follows:* 
 
 When a metal or hydrogen is brought into contact with a liquid 
 a certain amount of the metal tends to pass over into the liquid 
 and it strives to do so with a certain measurable and character- 
 istic force, analogous to gas- or osmotic-pressure, which Nernst 
 (47) (48) has termed the solution pressure. The particles which 
 this pressure tends to bring into solution are all, in the case of 
 the metals and hydrogen, charged electropositively. The number 
 of these particles which actually pass into solution, provided no 
 current is allowed to traverse the liquid and the metal, and no 
 chemical work is performed, must be very small and, in proportion 
 to weighable qualities, quite evanescent; for as the positively 
 charged particles pass out into the liquid an electrostatic tension 
 is necessarily developed. The film of liquid which is in immediate 
 contact with the metal becomes positively charged; the metal, 
 having lost positive charges, acquires a corresponding negative 
 charge, and the force driving fresh metal or hydrogen ions into 
 the liquid becomes balanced by the electrostatic repulsion of the 
 positive ions, from the film of liquid which is in contact with the 
 metal into the surface of the metal again. Hence there is quickly 
 developed a certain constant difference of electric potential be- 
 
 * For a fuller discussion of these principles and of the forms of apparatus 
 used the reader is referred to standard works of general physical chemistry 
 (21) (41) (64) (67). 
 
 195 
 
1 96 ELECTROCHEMISTRY 
 
 tween the liquid (charged positively) and the metal (charged 
 negatively) which is characteristic for each metal and each liquid. 
 
 The force which tends to drive the metal ions back into the 
 metal again is the osmotic pressure of the dissolved ions. If, 
 through the introduction of a dissociable salt of the metal, the 
 concentration (osmotic pressure) of the metal ions in the liquid 
 is increased, this may equal or even exceed the solution pressure 
 of the metal itself. In the former case the potential difference 
 between the metal and the solution is abolished; in the latter it 
 is renewed again, but in the opposite sense, the metal being now 
 charged positively and the solution negatively. 
 
 If, now, we build up a galvanic element as follows: Metallic 
 silver in contact with dilute AgN0 3 , in conducting communi- 
 cation with strong AgN0 3 , the latter solution being again in con- 
 tact with metallic silver, it is evident that there must be a differ- 
 ence in potential between the two portions of metallic silver, a 
 potential which will be greater the greater the difference between 
 the concentrations of the two silver nitrate solutions. Provided 
 the potential between the two silver nitrate solutions themselves 
 (due to the unequal migration-velocities of silver and N0 3 ions) 
 can be neglected or made to vanish (and we will shortly explain 
 how this may be approximately achieved), it has been shown 
 by Nernst that the potential of this " concentration chain" can 
 be expressed by the formula: 
 
 _ RT C 2 
 
 It 77 ' lOgnat. s*i 
 
 r TI (_/ i 
 
 where R is the gas constant, T the absolute temperature, F the 
 Faraday constant, 77 the valency of the ion (in this case 1), Cz the 
 concentration of the stronger solution of silver nitrate, Ci that of 
 the weaker and TT the potential in volts. 
 
 In this way the absolute concentrations of two different solu- 
 tions of an ion can be accurately computed, provided only a 
 minute amount of current be allowed to pass^ during the potential 
 determination, without any significant alteration in the compo- 
 sition of the solutions. The measurement is, in other words, 
 static; and static methods of measurement must be employed in 
 determining the combining capacities of proteins because the 
 combining capacity of proteins is not possessed of one fixed value 
 but of fixed minimum and maximum values and, between these 
 
COMBINING CAPACITY 197 
 
 intermediate values which are determined by the alkalinity or 
 acidity (H + concentration) of their solutions. 
 
 In applying this method to the comparison of the acidity or 
 alkalinity (H + concentration) of solutions the terminal electrodes 
 must be hydrogen. A hydrogen electrode can be obtained by 
 employing a saturated solution of hydrogen in a metal, e.g., 
 platinum. In order to obtain a large surface, platinum gauze 
 coated with platinum black is usually employed and a stream 
 of pure hydrogen is passed over or through the gauze. The 
 potential between the two acid or alkaline solutions themselves 
 (as distinguished from that between the hydrogen electrodes and 
 these solutions) may be abolished or at any rate greatly diminished 
 by connecting them by means of a U-tube filled with agar satu- 
 rated with KC1 (3) (1). 
 
 I have utilized this method in determining the combining 
 capacities of casein and of ovomucoid for acids or bases in solu- 
 tions of varying acidity and alkalinity (59) (60). For further 
 details regarding the technique employed and the precautions to 
 be taken in carrying out such determinations in solutions which 
 contain proteins, the reader is referred to the appendix. 
 
 In the following tables, which enumerate the results of these 
 experiments, the symbols which are employed have the following 
 significance : 
 
 61 == The concentration of a solution of base in which protein 
 
 is dissolved, 
 ai = The concentration of a solution of acid in which protein 
 
 is dissolved. 
 TT = The potential of the chain : 
 
 acid or base 
 plus protein 
 
 acid or base 
 alone 
 
 in volts. At 30 degrees, with monacid bases or mono- 
 basic acids 
 
 = 0.0601 lo glo j or 0.0601 lo glo ~ 
 
 b = The hydroxyl concentration (unneutralized base) in the 
 
 solution of base containing protein. 
 a = The hydrogen concentration (unneutralized acid) in the 
 
 solution of acid containing protein. 
 m = bi b or a\ a = the concentration of base or acid 
 
 neutralized by the protein. 
 
198 
 
 ELECTROCHEMISTRY 
 
 To all the solutions employed a small amount of KC1 was added. 
 This is necessitated in the preparation of the casein solutions 
 by technical difficulties of which an account will be found in the 
 appendix, but it serves also to confer upon the solutions a higher 
 conductivity and thus to render more easy the detection of the 
 zero point upon the potentiometer bridge wire by means of a 
 galvanometer. So far as the solutions of casein in KOH are 
 concerned, the presence of KC1 in the concentrations employed 
 evidently did not appreciably affect the combining capacity of 
 the protein for the base, since, as the tables reveal, in solutions 
 of very different KCl-content (0.015 N to 0.030 N) the combining 
 capacity at absolute neutrality proved to be the same. In the 
 tables the total amount of potassium present in the solvent as 
 KOH or KC1, is designated by the symbol r. 
 
 All of these determinations were made at 30 C. 
 
 0.5 PER CENT CASEIN DISSOLVED IN KOH SOLUTIONS 
 
 (r = 0.020) 
 
 &1 
 
 it 
 
 b 
 
 m 
 
 0.02000 
 
 0.0160 
 
 1.083X10- 2 
 
 0.00917 
 
 0.01000 
 
 0.0462 
 
 1.701X10- 3 
 
 0.00830 
 
 0.00750 
 
 0.0708 
 
 4.987X10- 4 
 
 0.00700 
 
 0.00500 
 
 0.1181 
 
 5.429X10" 5 
 
 0.00495 
 
 0.00250 
 
 0.2600 
 
 1.181X10- 7 
 
 0.00250 
 
 0.00150 
 
 0.2710 
 
 4.648X10- 8 
 
 0.00150 
 
 1.0 PER CENT CASEIN IN KOH SOLUTIONS (FIRST SERIES) 
 
 bi 
 
 7T 
 
 b 
 
 m 
 
 r 
 
 Remarks 
 
 0.03028 
 
 0.0239 
 
 1.213X10- 2 
 
 0.01815 
 
 0.03028 
 
 
 0.02907 
 
 0.0273 
 
 1.021X10- 2 
 
 0.01886 
 
 0.03028 
 
 
 0.02422 
 
 0.0320 
 
 7.111X10- 3 
 
 0.01711 
 
 0.02422 
 
 
 0.01817 
 
 0.0538 
 
 2.314X10- 3 
 
 0.01586 
 
 0.01817 
 
 
 0.01212 
 
 0.0937 
 
 3.351X10- 4 
 
 0.01178 
 
 0.01817 
 
 
 0.01091 
 
 0.1042 
 
 2.015X10- 4 
 
 0.01071 
 
 0.01817 
 
 
 0.00970 
 
 0.1332 
 
 5.907X10- 5 
 
 0.00964 
 
 0.01817 
 
 
 0.00849 
 
 0.1736 
 
 1.096X10- 5 
 
 0.00848 
 
 0.01817 
 
 
 0.00728 
 
 0.2276 
 
 1.191X10- 6 
 
 0.00728 
 
 0.01817 
 
 
 0.00607 
 
 0.2542 
 
 3.583X10- 7 
 
 0.00607 
 
 0.01817 
 
 
 0.00486 
 
 0.2758 
 
 1.251X10- 7 
 
 0.00486 
 
 0.01817 ) 
 
 Av. value of 
 
 0.00486 
 
 0.2753 
 
 1.276X10- 7 
 
 0.00486 
 
 0.01817 f 
 
 7r = 0.2756 
 
 0.00365 
 
 0.3034 
 
 3.265X10- 8 
 
 0.00365 
 
 0.01817 
 
 
 0.00244 
 
 0.3130 
 
 1.514X10~ 8 
 
 0.00244 
 
 0.01817 
 
 
COMBINING CAPACITY 
 
 199 
 
 1.0 PER CENT CASEIN KOH SOLUTIONS (SECOND SERIES) 
 
 (r = 0.030) 
 
 6) 
 
 K 
 
 6 
 
 TO 
 
 0.03000 
 
 0231 
 
 1.237X10- 2 
 
 0.01763 
 
 0.02500 
 
 0313 
 
 7.523X10- 
 
 0.01748 
 
 0.02000 
 
 0471 
 
 3.292X10- 
 
 0.01671 
 
 0.01750 
 
 0603 
 
 1.736X10- 
 
 0.01576 
 
 01500 
 
 0.0741 
 
 8.782X10- 
 
 0.01412 
 
 0.01250 
 
 0954 
 
 3.233X1 - 
 
 0.01218 
 
 0.01000 
 
 0.1299 
 
 6.892X10- 
 
 0.00993 
 
 00750 
 
 0.2265 
 
 1.276X10- 
 
 0.00750 
 
 00500 
 
 0.2596 
 
 2.398X10- 
 
 0.00500 
 
 00250 
 
 2903 
 
 3.699X10- 
 
 0.00250 
 
 1.5 PER CENT CASEIN IN KOH SOLUTIONS (r = 0.030) 
 
 61 
 
 a 
 
 b 
 
 TO 
 
 03000 
 02000 
 01500 
 0.01000 
 0.00750 
 0.00500 
 
 0.0439 
 0.0881 
 0.1280 
 2523 
 0.2897 
 0.3128 
 
 5.578X10- 3 
 6.842X10- 4 
 1. 113X10-' 
 6.337X10- 7 
 1.136X10- 7 
 3. 119X10- 8 
 
 0.02442 
 0.01932 
 0.01489 
 0.01000 
 0.00750 
 0.00500 
 
 2.0 PER CENT CASEIN IN KOH SOLUTIONS 
 
 6, 
 
 TT 
 
 6 
 
 TO 
 
 r 
 
 0.05000 
 0.03000 
 0.02000 
 0.01500 
 0.01000 
 00500 
 
 0.0311 
 0.0778 
 0.1343 
 0.2425 
 0.3000 
 0.3152 
 
 1.522X10- 2 
 1.525X10- 3 
 1.167X10- 4 
 1. 383X10-' 
 1.020X10- 7 
 2.846X10- 8 
 
 0.03478 
 0.02847 
 0.01988 
 0.01500 
 0.01000 
 0.00500 
 
 0.050 
 0.030 
 0.040 
 0.040 
 0.040 
 0.040 
 
 3.0 PER CENT CASEIN IN KOH SOLUTIONS 
 
 & 
 
 ir 
 
 b 
 
 TO 
 
 r 
 
 
 o.osooo 
 
 0.0733 
 
 3.011X10- 3 
 
 0.04699 
 
 0.050) 
 
 Av.value of 
 
 0.05000 
 
 0.0739 
 
 2.943X10' 3 
 
 0.04706 
 
 o.osot 
 
 m= 0.04703 
 
 0.03000 
 
 0.1438 
 
 1.217X10' 4 
 
 0.02988 
 
 0.040 
 
 
 0.02500 
 
 0.2343 
 
 3.156X10- 6 
 
 0.02500 
 
 0.040 
 
 
 0.02000 
 
 0.2823 
 
 4.017X10- 7 
 
 0.02000 
 
 0.040 
 
 
 01500 
 
 0.3172 
 
 7.920X10' 8 
 
 0.01500 
 
 0.040 
 
 
 01000 
 
 0.3413 
 
 2.096X10- 8 
 
 0.01000 
 
 0.040 
 
 
200 
 
 ELECTROCHEMISTRY 
 
 The relation between m, the amount of alkali neutralized by the 
 casein, and 61, the alkalinity of the solution in which the casein 
 was dissolved, is shown graphically, for all of the concentrations 
 of casein employed, in the accompanying figure : 
 
 0.05 
 
 0.04 
 
 0.03 
 
 0.02 
 
 0.01 
 
 b, 
 
 0.01 
 
 0.02 
 
 0.03 
 
 0.04 
 
 0.05 
 
 The ordinates = m = the concentration of KOH neutralized 
 
 by 0.5%, 1%, 1.5%, 2.0% and 3% casein. 
 
 The abscissae = 6 : = the concentration of the KOH solution 
 
 in which the casein was dissolved. 
 
 It will be understood, of course, that the curves only represent 
 this relation for the alkalinities of the original solutions in excess 
 of that necessary to dissolve all of the casein. For an ordinary 
 acid, forming only one salt with the base, which did not undergo 
 hydrolytic dissociation, the curve would, of course, be a straight 
 line parallel with the horizontal axis. It will be seen that as the 
 proportion of base to casein declines, the combining capacity of 
 the casein tends to become directly proportional to the concen- 
 tration of the base, but that as the proportion of base to casein 
 (and the excess of unneutralized base) becomes large, the com- 
 bining capacity of the casein tends towards constancy, i.e., in 
 comparatively strongly alkaline solutions the behavior of casein 
 approximates more and more to that of an ordinary acid. 
 
 These results are to be interpreted as follows: When just 
 enough alkali (= 11.4 X 10~ 5 equivalents per gram, Cf. Chap. V) 
 
COMBINING CAPACITY 201 
 
 has reacted with casein to dissolve it, we may suppose that one 
 
 N.HOC group (or two such groups, if dicarboxylic radicals 
 are involved) in each molecule of casein has been opened up, 
 in accordance with the equation: jj 
 
 ^ ' ++ 
 -N.HOC- + KOH = - ^N + KOC- 
 
 I 
 OH 
 
 From the Guldberg and Waage mass law we know that if this 
 equation represents the true nature of the reaction which occurs, 
 then the mass of caseinate formed must be dependent (the tem- 
 perature and other conditions of the reaction being constant) 
 only upon the reacting masses of free casein and KOH and not 
 upon the total dilution of the system. At the completion of the 
 reaction these masses are both very small, since free casein is 
 inappreciably soluble in water and the solution of this salt of 
 casein is acid in reaction (Cf. Chap. V). Upon further addition 
 of alkali, however, another N.HOC group (or pair of groups) 
 is opened up and when another 11.4 X 10~ 5 equivalents of alkali 
 have been added per gram of casein we have now four ions of 
 caseinate instead of two. Whether these ions are derived from 
 one, or from two, molecules of caseinate these data do not enable 
 us to decide. As the tables show, the solution of this salt is also 
 acid to litmus. Upon still further addition of alkali yet another 
 
 N.HOC group (or pair of groups) in each casein molecule 
 is opened up, and when 22.8 X 10~ 5 more equivalents of KOH 
 (= 45.6 X 10~ 5 per gram in all) have been added all of the ions 
 of caseinate have again been split in half. The solution of this 
 salt is, as we shall see, almost exactly neutral to litmus. Again 
 doubling the alkali-content the ions will be split again, yielding 
 a salt containing 91.2 X 10~ 5 equivalents of KOH per gram, the 
 solution of which, as the above tables reveal, is alkaline to phenol- 
 phthalein (62) and which, therefore, presumably, requires the 
 presence of a slight excess of KOH to maintain its stability, i.e., 
 to push the equilibrium in the above reaction sufficiently over 
 to the right. We may suppose that this type of reaction is re- 
 peated until the last N.HOC group that is dissociable by 
 alkali has been opened up, when the combining capacity of the 
 casein reaches, as we have seen, a constant maximal value. This 
 maximum value of the combining capacity must necessarily, if 
 
202 
 
 ELECTROCHEMISTRY 
 
 the above picture of the chain of events is correct, be an even 
 multiple (1, 2, 4, 8, 16, etc.) of the quantity of alkali which is 
 just sufficient to form the first salt, i.e., to carry the casein into 
 solution. By interpolation from the above tables and by con- 
 tinuing the curves it is seen that for all of the percentages of casein 
 employed the constant maximal value of the combining capacity 
 for KOH is, as nearly as can be estimated, 180 X 10~ 5 equiva- 
 
 1 80 
 
 lents per gram. Now -^r-r = 15.8 or, within the error of esti- 
 mation, 16. The experimental results are therefore in good 
 agreement with the theory and we may conclude that casein 
 forms the following salts with KOH: 
 
 I containing 11.4 X 10~ 6 equivalents of KOH per gram of casein 
 II " 22.8 X 10- 5 
 
 III " 45.6X10- 6 " " " " 
 
 IV " 91.2X10- 5 " " " " 
 V " 182.4 X ID" 6 " " " " 
 
 In forming the last of these salts 16 N.HOC groups (or a 
 multiple of this number) have been opened up. 
 
 Similar experiments were carried out upon solutions of ovo- 
 mucoid in dilute acid and alkali; they are tabulated below: 
 
 1 PER CENT OVOMUCOID IN KOH SOLUTIONS 
 (KC1 in each solution = 0.01 N) 
 
 61 
 
 IT 
 
 b 
 
 m 
 
 0.0005 
 
 0.0755 
 
 2.77X10- 5 
 
 0.00047 
 
 0.0010 
 
 0.0475 
 
 1.62X10- 4 
 
 0.00084 
 
 0.0020 
 
 0.0500 
 
 2.95X10- 4 
 
 0.00171 
 
 0.0030 
 
 0.0367 
 
 7.34X10- 4 
 
 0.00227 
 
 0.0050 
 
 0.0356 
 
 1.28X10- 3 
 
 0.00372 
 
 0.0100 
 
 0.0185 
 
 4.92X10- 3 
 
 0.00508 
 
 1 PER CENT OVOMUCOID IN HCL SOLUTIONS 
 (KC1 in each solution = 0.01 N) 
 
 fll 
 
 tc 
 
 a 
 
 m 
 
 6.95XlO- 
 
 0.1087 
 
 1.08X10- 7 
 
 6.84X10- 6 
 
 0.0005 
 
 0.2261 
 
 8.65X10- 8 
 
 0.00050 
 
 0.0010 
 
 0.2107 
 
 3.12X10- 7 
 
 . 0.00100 
 
 0.0020 
 
 0.17Z6 
 
 2.69X10- 6 
 
 0.00198 
 
 0.0030 
 
 0.1492 
 
 9.88X10-* 
 
 0.00299 
 
 0.0050 
 
 0.1190 
 
 5.24X10- 5 
 
 0.00495 
 
 0.0100 
 
 0.0610 
 
 9.66X10- 4 
 
 0-00903 
 
COMBINING CAPACITY 203 
 
 The results are shown graphically in the accompanying figure: 
 
 0.01 
 
 0.005 
 
 b, . 
 
 0.01 
 
 0.005 
 
 0.01 
 
 0.005 
 
 61 is the concentration of KOH in 
 which 1 per cent ovomucoid is dis- 
 solved; m is the number of gram- 
 equivalents of KOH which is neutral- 
 ized per litre. 
 
 0.006 0.01 
 
 i is the concentration of HC1 in 
 which 1 per cent ovomucoid is dis- 
 solved; m is the number of gram- 
 equivalents of HC1 which is neutral- 
 ized per litre. 
 
 It is obvious that the relationship between the amount of 
 acid or base neutralized by the ovomucoid and the total amount 
 of base present is of very much the same general nature as the 
 corresponding relationship for solutions of casein in KOH 
 solutions. Ovomucoid, however, differs very strikingly from 
 casein in the fact that it is predominantly basic, while casein is 
 predominantly acid. The maximum combining capacity of ovo- 
 mucoid for KOH is obviously about 50 X 10~ 5 equivalents per 
 gram, but its maximum combining capacity for acids was not 
 attained in any of the solutions investigated and must be at least 
 100 X 10~ 5 equivalents per gram. Since free ovomucoid, unlike 
 free casein, is soluble in water the quantity of a base or acid which 
 enters into one N.HOC group cannot be estimated from 
 these data alone, but it is evident that it cannot be in excess of 
 50 X 10~ 5 per gram of protein. 
 
 The combining capacities of serum albumin, gelatin and de- 
 aminized gelatin for acids have been determined electrometri- 
 cally by Pauli and Hirschfeld (49). They find that, as in the 
 case of casein, the maximum combining capacities of these pro- 
 teins are independent of their dilution and, moreover, that in 
 isohydric solutions acetic acid, despite the fact that it yields a 
 much inferior proportion of hydrogen ions, is bound by proteins 
 in greater proportion than hydrochloric acid, a fact which in 
 
204 ELECTROCHEMISTRY 
 
 itself speaks very strongly in favor of the view that the protein 
 compounds which are formed do not yield, by electrolytic disso- 
 ciation, the corresponding acid anions. They, moreover, find 
 that the combining capacity of deaminized gelatin is hardly 
 inferior to that of normal gelatin. 
 
 The electrometric method of measuring the combining capaci- 
 ties of proteins for acids and bases has also been employed by 
 D'Agostino and Quagliariello (2), Rohonyi (61), Manabe and 
 Matula (38), Blasel and Matula (4), Ringer (52) and Schmidt 
 (63) (64). 
 
 2. The Combining Capacity of Casein for Bases and of Ovo- 
 mucoid for HC1 at Absolute Neutrality. A number of investi- 
 gators have shown (66) (34) (71), by direct titration to neutrality 
 to litmus, that the combining capacity of casein at absolute 
 neutrality is constant, that is, independent of the total dilu- 
 tion of the system. The potentiometric determinations, cited in 
 the above tables, enable us to confirm these observations. It 
 will be recollected that the OH' concentration in the solutions 
 containing casein was determined by measuring the potential 
 between two hydrogen electrodes, the one dipped in a solution 
 containing a given concentration of casein, the other in an ex- 
 actly similar solution to which no casein had been added. Plot- 
 ting a curve in which the reaction of the solutions containing 
 no casein form the abscissae and the potentials between the two 
 solutions the ordinates, the reactions ( = x) of the solution to which 
 the given concentration of casein had to be added in order to 
 procure an exactly neutral solution * is given by the intersection 
 of this curve with the curve defined by the formula: 
 
 y = 0.4107 - 0.0601 Iog 10 x. 
 
 The points of intersection of these curves may be found, with 
 sufficient approach to accuracy, in the following way: The values 
 of y in the above curve corresponding to x (alkalinity of the 
 solution containing no casein) = 0.0025; 0.005; 0.0075; 0.010; 
 0.015 and 0.020 are computed and these points are marked upon 
 accurately ruled "squared paper" and joined by straight lines. 
 Experimental values of TT- lying upon each side of its value at 
 neutrality, in each solution, are then also marked off upon the 
 
 * Taking the H+ concentration at absolute neutrality at 30 degrees as 
 1.47 X 10-*; cf. Kohlrauch and Heydweiller (33). 
 
CASEIN AND OVOMUCOID 
 
 205 
 
 paper and joined by straight lines. The abscissa of the point of 
 intersection of the two broken curves thus drawn yields the number 
 of gram equivalents of KOH which are bound by the given per- 
 centage of casein in the production of an absolutely neutral 
 solution.* Dividing the number of gram equivalents of KOH 
 neutralized by the casein by the percentage concentration of the 
 casein we obtain the number of gram equivalents of KOH 
 neutralized by 1 gram of casein at absolute neutrality. The 
 following are the results computed from the data tabulated 
 above: 
 
 Concentration of casein, per cent 
 
 Gram equiv. of KOH neutralized 
 by 1 gram of casein at absolute neu- 
 trality at 30 degrees 
 
 0.5 
 1.0 (first series) 
 1.0 (second series) 
 1.5 
 2.0 
 3.0 
 
 52X10- 6 
 50X10~ 5 
 43X10- 5 
 53X10- 5 
 54X10-* 
 56X10- 5 
 
 Average 51X1Q- 5 
 
 The values of the combining capacities of the casein at neutrality 
 to litmus computed in this manner are seen to be appreciably 
 constant. The average value is 51 X 10~ 5 equivalents per gram 
 of casein, which is in excellent agreement with that obtained by 
 fcitration, namely, 50 X 10~ 5 equivalent gram molecules per gram. 
 Solutions of ovomucoid require the addition of a small quantity 
 of add to obtain an absolutely neutral solution. f The amount 
 
 * It was at first thought necessary to pa~s the curve y = ax? + bx z + ex + 
 d, through four of the points of the experimental curve TT = f(bi) and to deter- 
 mine algebraically the point of intersection of this curve with the curve 
 y = 4107 0.060 logio %', this was, however, found to be an unnecessary 
 refinement, as the results obtained did not differ appreciably from those 
 obtained by the simpler method described above. 
 
 t In this connection attention may be called to the fact, which is revealed 
 in the above tables, that when 1 per cent ovomucoid is dissolved in 6.95 X 
 10" 6 N acid, although the ovomucoid neutralizes a little of this acid, yet this 
 solution is actually more acid than the solution which is obtained by dissolving 
 1 per cent ovomucoid in 0.0005 N HC1. A similar phenomenon was observed 
 by W. B. Hardy in solutions of serum globulin (22). It is possible that this 
 indicates a slight decomposition of the salt which was present in these solu- 
 tions by the protein, or, more probably that it is due to a small proportion of 
 the free protein ionizing according to the equation R.COOH = RCOO' + H+. 
 
206 ELECTROCHEMISTRY 
 
 of HC1 bound by ovomucoid at absolute neutrality, estimated 
 in the above manner, is 7 X 10~ 5 equivalents per gram. 
 
 3. The Non-Dependence of the Composition of the Compounds 
 of Protein with Acids and Bases upon the Dilution of their 
 Solutions. The view would appear to be very generally held 
 (35) (9) (51) that the salts which proteins form with acids and 
 bases are subject in a very high measure to hydrolytic dissoci- 
 ation. This view is nevertheless erroneous. When a base unites 
 with an organic acid to form a salt by the neutralization of a 
 COOH group the reaction may be expressed as follows: 
 
 R.COOH + KOH < RCOOK + H.OH. 
 
 If the acid is a very strong one then the reaction will proceed 
 completely from left to right and the salt will not be appreciably 
 decomposed by dilution. If, however, the acid is tolerably weak 
 (e.g., acetic acid) so that the water itself, acting as "an acid, is 
 able, if present in large amounts, to displace it partially from its 
 combination with the base, then the reaction will not proceed 
 completely from left to right but will pause, in accordance with 
 the mass law, in a condition of balance, which is shifted towards 
 the right by the addition of excess of base and towards the left 
 by dilution. On adding excess of base to a dilute solution of an 
 acid of this type, therefore, more of the base will be observed 
 to be neutralized, while the addition of water will result in partial 
 decomposition of the salt. Similar considerations hold good, of 
 course, for the salts of weak bases. When one examines the 
 evidence which has been brought forward by different observers 
 in support of the thesis that protein salts are subject to hydrolytic 
 dissociation one finds that it is all of the first kind, that is, con- 
 sists in the fact that upon the addition of more acid or base to the 
 protein solution, or of more protein, more of the acid or base is 
 bound. From this the somewhat illogical assumption has been 
 made that the protein salts must exhibit the other characteristic 
 property of salts of weak acids and bases, namely, decomposa- 
 bility by water. We have, however, seen that the fact that pro- 
 tein will bind more of an acid or a base in the presence of an 
 
 This mode of dissociation would, of course, be prevented by dissociation of the 
 molecule through its N.HOC groups, and, since this is brought about 
 by the addition of acid, the addition of small quantities of acid might well 
 decrease the acidity (H+ concentration) of the solution. 
 
NON-DEPENDENCE OF COMPOSITION ON DILUTION 207 
 
 excess of these reagents admits of a very different explanation, 
 and the data cited above also enable us to conclude that water 
 is not able to appreciably decompose the potassium salt of casein. 
 We have seen that whether the percentage of casein in solution 
 is 0.5 or 3.0 or intermediate between these, the quantity of potas- 
 sium hydrate which is bound by a given weight of casein at 
 absolute neutrality is the same. The dilution (relative mass of 
 water to salt) may vary 600 per cent and yet the salt remains 
 unaltered; it is not decomposed by the water in any preceptible 
 degree. This result has also been obtained for other bases, 
 including comparatively weak bases such as Ca(OH) 2 , by the 
 observers, cited in the previous section, who have employed the 
 method of direct titration and these observers have also shown 
 that at neutrality to phenolphthalein the composition of the 
 casein salt (= 80 X 10~ 5 equivalents of base per gram) is always 
 the same no matter what the dilution of the system. Moreover, 
 as the curves in the figure on p. 200 clearly reveal, the compo- 
 sition of the casein salt, when the casein is exerting its maximum 
 combining-capacity, is the same for very different concentrations 
 of this salt. That this should be so in alkaline solution is not 
 surprising, since the excess of alkali might be expected to drive 
 the reaction: 
 
 R.COOH + KOH <=> RCOOK + H.OH 
 
 over towards the right; but that it is so in absolutely neutral 
 solution, when the concentration of free KOH is evanescently 
 small, is an extremely striking fact. 
 
 But, not only is the composition of the caseinate of potassium 
 independent of its dilution in neutral solution, it is also inde- 
 pendent of its dilution in a solution which is pronouncedly acid 
 in reaction. It will be shown in the succeeding chapter that the 
 amount of a base which is bound by casein at "saturation" of the 
 base with casein, that is, when there is just sufficient base to hold 
 the casein in solution is also independent of the dilution of the 
 system. Now in this solution the acidity is about 10~ 5 H + 
 (cf. the table of reactions to indicators on p. 91). A glance at 
 the formula 
 
 R.COOH + KOH<=R.COOK + H.OH 
 
 suffices to show that in acid solutions, when the KOH concen- 
 tration is excessively small and the H+ concentration large the 
 
208 ELECTROCHEMISTRY 
 
 tendency to hydrolysis of a salt of the type R.COOK must be 
 exceptionally great, yet potassium caseinate does not reveal 
 this tendency, and the conclusion is forced upon us that potas- 
 sium caseinate is not formed in accordance with an equation of 
 the above type. 
 
 Reverting, now, to the hypothesis which I have advanced 
 regarding the mode of formation of protein salts, we shall see 
 that in the equation : 
 
 H 
 
 I +4- 
 
 -N.HOC- H- KOH <= N> - + KOC- 
 
 I 
 OH 
 
 no water is involved, and, consequently, the composition of the salt 
 must be dependent only upon the relative concentrations of protein 
 and base and not upon the total dilution. Similarly in the forma- 
 tion of salts with acids: 
 
 H 
 
 I ++ 
 
 -N.HOC- + HC1<= -N^+ HOC- 
 
 Cl 
 
 no water enters into the reaction. Hence both the dependence 
 of the composition of the salt upon the excess of acid or base 
 and its non-dependence upon dilution receive a simple interpre- 
 tation in the light of this hypothesis. 
 
 It will I think be clear from the foregoing discussion, that the 
 non-dependence of the composition of protein salts upon their 
 dilution is one of the most emphatic proofs we possess of the 
 fact that terminal COOH and NH 2 groups are not respon- 
 sible for their formation. 
 
 An experiment which demonstrates in a very striking manner the 
 fact that protein salts do not undergo an appreciable amount of 
 hydrolytic dissociation in solution in water, nor split off the inor- 
 ganic radical as an ion, is the following. It will be recollected that 
 casein, deprived of its combined base or acid, is insoluble, and that 
 if, to a solution of a caseinate of a base, exactly enough free acid 
 be added (e.g., HC1) to completely neutralize the combined base 
 the free casein is entirely precipitated. Now one gram of ovo- 
 mucoid (60) combines with 45 X 10~ 5 equivalents of HC1 to form 
 
" ISOELECTRIC " CONDITION 209 
 
 a compound such that less than 1 per cent of the acid remains 
 uncombined (as estimated by gas-chain measurements). One 
 gram of casein combines with 90 X 10~ 5 equivalents of KOH to 
 form a compound such that less than ^ per cent of the KOH 
 remains uncombined (59). If, now, these salts were appreciably 
 subject to hydrolytic dissociation, or even if they yielded Cl' and 
 H + ions respectively, then on mixing two volumes of a solution 
 of the ovomucoid salt with one volume of a solution of the casein 
 salt (each of the same percentage concentration) the K + pro- 
 vided by the caseinate would be exactly neutralized by the Cl' 
 provided by the ovomucoid salt and it might be anticipated 
 that free uncombined casein would be precipitated. Nothing 
 of the sort occurs, however. If to 25 cc. of a 2 per cent solution 
 of the casein salt are added 50 cc. of a 2 per cent solution of the 
 ovomucoid salt the mixture is no more opalescent than its con- 
 stituent parts and the conductivity of the mixture is the sum of the 
 separate conductivities of the two protein salts* If the mixture 
 be allowed to stand in the presence of toluol for a considerable 
 period at 36 degrees, however, after 24 to 45 hours a marked 
 increase in its opalescence is observed; after two or three days 
 traces of casein begin to be deposited, and after three to four 
 days all of the casein is found to have been precipitated. The 
 precipitation of the casein is accompanied by a marked increase 
 in the conductivity of the mixture, attributable to the setting 
 free of KC1. It is therefore evident that at the beginning the 
 mixture must contain only minute traces of K+ and Cl' ions and 
 that the protein salts only yield up these ions with extreme 
 slowness. 
 
 4. The "Isoelectric" Condition of Proteins at Certain H+ and 
 OH' Concentrations. If we assume that the proteins dissociate 
 H + and OH' ions according to the formula: 
 
 X NH 3 OH^ /NH 3 OH 
 
 N COOH ' N COO' 
 and 
 
 /NH 3 OH 
 
 R ^R + OH' 
 
 N COOH N COOH 
 
 * Conductivity of a 0.5 per cent solution of potassium casemate containing 
 90 X 10~ 5 equivalents of KOH per gram at 30 degrees equals 38.0 X 10~ 6 
 reciprocal ohms. Conductivity of a 1 per cent solution of ovomucoid chloride 
 
210 ELECTROCHEMISTRY 
 
 then the dissociation of H+ ions must be inhibited and that of 
 OH' ions encouraged by the presence of an excess of H + ions, 
 while that of OH' must be inhibited and of H + ions encouraged 
 by an excess of OH' ions, and a reaction must exist at which 
 H+ and OH' are split off in exactly equal quantities so that the 
 salt is "isoelectric," i.e., wanders equally in both directions in an 
 electric field. A simple calculation suffices (53) to show that if 
 the mode of dissociation of the protein is actually that repre- 
 sented by the above formulae, then the number of protein ions 
 in a solution of proteins must attain a minimum in solutions in 
 which it is "isoelectric." 
 
 A number of observations, to which more detailed reference 
 will be made in a later chapter, tend to establish the fact that the 
 viscosity of protein solutions is in the greater proportion attributa- 
 ble to its ions, and that, consequently, when the ionization of a 
 protein is at a minimum the viscosity of its solution will tend 
 to a minimum also. 
 
 Applying these considerations, and also employing direct obser- 
 vation of the movement of the protein in an electric field, Michaelis 
 and co-workers (40) (41) (42) (43) (44) (45) have endeavored 
 to ascertain the hydrogen ion concentration of the solution in 
 which egg-albumin is isoelectric. 
 
 In the light of the view which I have developed concerning 
 the mode of formation and dissociation of the protein salts a 
 somewhat different interpretation must be placed upon these 
 results. A glance at the figure on p. 185 will show that if the 
 protein is soluble in the free condition and uncombined with 
 bases or acids it must, if it is ionized at all, migrate equally in 
 both directions through the solution under the influence of an 
 electric current, reacting at the electrodes as follows : 
 
 (Anode) 2 HOOC.R.N^ = 2 HOOC.R.NH 2 + 2 
 I 
 OH 
 
 (Cathode) H 2 N.R.COH + H 2 O = H 2 N.R.COOH + H 2 
 
 containing 45 X 10~ 5 equivalents of HC1 per gram at 30 degrees equals 80.1 X 
 10~ 5 reciprocal ohms. Sum of these conductivities equals 118.1 X 10~ 6 recip- 
 rocal ohms. Conductivity of a mixture containing the two salts in the above 
 concentrations at 30 degrees equals 108.5 X 10~ 5 reciprocal ohms. 
 
BIOLOGICAL APPLICATIONS 211 
 
 and regenerating the free protein to again participate in carrying 
 the current both ways. Provided, therefore, the two ions are 
 of nearly equal weight (and we have seen that in the case of the 
 ions of potassium caseinate the ions are nearly equal in weight, 
 cf. previous chapter) no noticeable change in the distribution 
 of the protein in the electric field will be observed. If the ions 
 should be of unequal weight, then a trace of acid or alkali com- 
 bined with the protein might suffice to neutralize this inequality 
 and render the protein apparently "isoelectric." The "iso- 
 electric" protein of Michaelis is, therefore, in all probability, 
 merely free protein uncombined with acids or bases. This view 
 of Michaelis' result is supported by Pauli and Wagner (50) who 
 point out that the egg-albumin employed by Michaelis is, in 
 reality, an albuminate of a base, which therefore requires the 
 addition of some acid to set the protein free. The free protein 
 is sparingly ionized and hence the viscosity of its solution is low. 
 
 5. Biological Applications; the Neutrality of the Tissues and 
 of the Tissue Fluids. The power which the protein salts 
 possess of neutralizing additional equivalents of acid or base by 
 the opening up of fresh N.HOC bonds confers upon them, 
 since the reaction of free protein is usually nearly neutral, a 
 remarkable power of maintaining the neutrality of their solu- 
 tions, and it appears probable that this characteristic of the 
 protein salts plays an important part in maintaining the neu- 
 trality of the tissues and a not insignificant part in maintaining 
 the neutrality of the tissue fluids. 
 
 The older statements which are found in physiological and 
 medical literature concerning the reactions of the blood are totally 
 unreliable, since they were based upon the erroneous belief that 
 it is possible to ascertain the reaction of a fluid such as the blood 
 by titration. The determinations of Hoeber (31), Farkas (10) 
 (11) (12) (13), Fraenkel (18), Szily (69) (70), Hasselbalch (23) 
 (24) and de Corral (8) carried out by the potentiometric method, 
 have shown that the H + and OH' ion concentrations of normal 
 blood are very close to those at neutrality (neutral point = 
 8 X 10~ 8 H+ and OH'), the alkalinity first found by Hoeber having 
 been traced to the fact that the stream of hydrogen, used to 
 impregnate the platinum electrode of the gas-chain, washed the 
 CO 2 out of the blood. On eliminating this source of error the 
 blood is found to be almost absolutely neutral, the exact OH' 
 
212 ELECTROCHEMISTRY 
 
 concentration in normal blood at physiological C02 pressures 
 (0.028 to 0.054 atmosphere) being, according to recent deter- 
 minations, about 0.37 X 10~ 7 . 
 
 Friedenthal (19) and von Szily (69), employing a somewhat 
 different method of investigation, have reached the same con- 
 clusion as the above quoted observers. They utilized the indi- 
 cator-method of Friedenthal, Fels and Salm. Mixtures of acid 
 and basic phosphates of known H + and OH' content (determined 
 by Salm, using the potentiometric method) were tinged with a 
 number of different indicators and the samples of the body- 
 fluid under examination were tinged with equal quantities of 
 the same indicators the colors of the two series of fluids were 
 then compared. The phosphate-mixture which approximated 
 most closely in its indicator reactions to the normal blood-serum 
 had an acidity corresponding to 6.5 X 10~ 8 H + , absolute neutrality 
 being 8 X 10~ 8 H+. 
 
 Moreover the investigations of Friedenthal (19) (20) and of 
 Foa (15) (16) (17) have shown that not only is the blood almost 
 exactly neutral but that almost all of the tissue-fluids are also 
 approximately neutral. Thus, the pancreatic juice of a dog, one 
 of the most alkaline of the body-fluids, contained 5 X 10~ 9 H + , 
 corresponding to an alkalinity of 13 X 10~ 7 OH'. Hitherto, ac- 
 cording to Friedenthal (20), no animal fluid has been found con- 
 taining less than 10~ 10 N H+, that is, more than about 6 X 10~ 5 
 N OH'. The gastric juice of a dog was, however, found to be 
 1000 times more acid than the pancreatic juice is alkaline, that 
 is, it possessed the acidity of, approximately, a hundredth normal 
 solution of HC1. 
 
 With few exceptions, therefore, the tissue fluids are practically 
 neutral in reaction. It is a significant fact also (Friedenthal 
 (20), Loeb (37)) that the naturally occurring waters, in which 
 the lives of aquatic animals are spent, are also very nearly neutral 
 in reaction. "We may therefore draw the conclusion that life- 
 phenomena occur in a neutral liquid" (Loeb, loc. cit.), and, more- 
 over, save in certain special localities, such as the internal wall 
 of the stomach, the life-process occurs exclusively in a neutral 
 milieu; thus even an increase of 50 per cent in the minute H + 
 concentration of normal blood induces profound toxic symptoms, 
 and an increase to 1 X 10~ 7 N H + induces fatal acidosis. 
 
 In view of the fact that the products of metabolism include a 
 
BIOLOGICAL APPLICATIONS 213 
 
 large proportion of acid substances, the mechanism which pre- 
 serves the neutrality of the tissues and tissue-fluids despite the 
 relatively enormous daily and hourly fluctuations in acid-pro- 
 duction or consumption which necessarily occur within the body, 
 is, in all probability, one of prime physiological importance. 
 
 The acid which is produced most copiously in the body is 
 carbonic acid. The classic theory regarding the neutralization 
 of this acid is that of Fernet (14), Heidenhain and Mayer (25) 
 (26) and von Bunge (6) (7) who supposed that it is bound by the 
 disodium phosphate in the blood, according to the balanced 
 reaction : 
 
 C0 2 + H 2 + Na2HP0 4 ? NaHC0 3 + NaH 2 P0 4 , 
 
 the reaction proceeding towards the right in the tissues, where 
 the C0 2 tension is high, towards the left in the lungs, where the 
 C0 2 tension (active mass of CO 2 ) is low. Sertoli (65), however, 
 and Mroczkovsky (46) pointed out that the quantity of phos- 
 phoric acid in the blood of many animals, e.g., the ox and the 
 pig,* is too small to play the important r61e assigned to it by 
 these investigators. 
 
 We have seen that the alkali- and acid-equivalent of the pro- 
 teins varies very markedly with the hydroxyl- or hydrogen- 
 contents of their solutions. Thus one gram of serum-globulin 
 (Cf. Chap. V) in solutions neutral, or approximately neutral, to 
 litmus (absolute neutrality: H+ = OH' = 8 X 10~ 8 ) neutralizes 
 10 X 10~ 5 equivalent gram molecules of a base, while in solutions 
 neutral to phenolphthalein (OH 7 = 20 X 10~ 7 ) 1 gram neutralizes 
 20 X 10~ 5 equivalent gram molecules of a base. The significance 
 of these figures may, perhaps, be more fully realized when stated 
 thus: in one litre of NaOH solution containing 1 per cent of 
 globulin and neutral to litmus, addition of 100 X 10~ 5 gram- 
 equivalents of NaOH only raises the alkalinity (hydroxyl-content) 
 of the solution by about 0.2 X 10~ 5 , that is, by two-tenths of a 
 per cent of the change in the sodium content of the solution. 
 Casein, at neutrality to litmus, neutralizes 50 X 10~ 5 gram- 
 equivalents of base per gram, and at "saturation," i.e., at an 
 acidity of about 50 X 10~ 7 H+, it neutralizes only 11.4 X 10~ 5 
 gram-equivalents of base per gram. Hence to a litre of a 1 per 
 cent solution of sodium caseinate, neutral to litmus, we should 
 * Cf . von Bunge (7) . 
 
214 
 
 ELECTROCHEMISTRY 
 
 have to add 400 X 10~ 5 equivalents of HC1 to reach an acidity 
 of 0.5 X 10~ 5 equivalents just over one-tenth of a per cent of 
 the acid added. Were the casein present in a concentration of 
 8 per cent (the percentage concentration of the proteins in the 
 blood), 3200 X 10~ 5 gram equivalents of HC1, or 320 cc. of a 
 tenth normal solution, would have to be added to a litre of the 
 solution to attain an acidity of 0.5 X 10~ 5 equivalents. One 
 twentieth of a cubic centimeter of tenth normal HC1, added to a 
 litre of pure distilled water, would produce the same acidity. The 
 power of the proteins to maintain the neutrality of the fluids in 
 which they occur is therefore very remarkable, and Loeb (36), 
 myself (55) (56) (57) (58) and others have suggested that they 
 play an important part in regulating the neutrality of the tissues 
 and tissue-fluids, while Hoppe-Seyler (32), Sertoli (65), Zuntz (72) 
 (73) and myself (55) (57) have advanced the view that the liber- 
 ation of C0 2 from the blood in the lungs is accompanied by a 
 transport of sodium from the carbonic acid to the proteins of the 
 plasma. 
 
 Friedenthal (19) (20), Spiro (68), Loeb (36) and Henderson 
 (27) (28) (29) (30) have pointed out that the bicarbonates of the 
 blood and other media in which organisms live must also be of 
 importance in preserving their neutrality. Henderson points out 
 that the rate of change in the alkalinity or acidity of a solution 
 of an acid is a minimum when the dissociation constant of the 
 acid is equal to the hydrogen ion concentration (8 X 10~ 8 N) at 
 neutrality. He illustrates this by the following table, showing 
 the amount of tenth normal alkali required to secure a definite 
 but arbitrarily chosen change in alkalinity when added to equal 
 amounts of the undermentioned acids. 
 
 Acid 
 
 Dissociation-con- 
 stant X 10~ 7 
 
 Cubic centimeters 
 of alkali required 
 
 Phenol 
 
 0.0013 
 
 0.01 
 
 Boric acid. 
 
 017 
 
 08 
 
 Hydrogen sulphide 
 
 57 
 
 1 10 
 
 Mono-sodium phosphate 
 
 2 
 
 1.00 
 
 Carbonic acid 
 
 3 
 
 0.72 
 
 Picolinic acid 
 
 18.0 
 
 0.10 
 
 Acetic acid . 
 
 180.0 
 
 0.03 
 
 
 
 
 Henderson believes that the neutrality of the tissue fluids is 
 chiefly maintained by the bicarbonates which they contain 
 
BIOLOGICAL APPLICATIONS 215 
 
 and in a minor degree by the phosphates and by the proteins. 
 He illustrates his hypothesis by reference to a system tenth- 
 molecular in total carbonic acid and equally concentrated in 
 total phosphoric acid, combined or uncombined with sodium, 
 and he shows that in order to appreciably change the acidity of 
 this system a quantity of acid comparable with the total amount 
 of sodium which is present at neutrality must be added. He 
 estimates, from known quantitative data, the quantity of acid 
 which would be neutralized by the bicarbonates of the blood in 
 passing from the reaction of normal blood (= 0.37 X 10~ 7 H + at 
 38 degrees) to that of blood in advanced acid intoxication (about 
 1.00 X 10~ 7 H+ at 38 degrees). At the same time, from data 
 derived from experiments in which he employed indicators to 
 determine the change in the reaction of solutions of the serum- 
 proteins to which varying amounts of acid and alkali had been 
 added, he estimated the amount of acid which is neutralized by 
 the proteins of the blood, and their salts, as the reaction of their 
 solutions changes by the same amount, and he concludes that 
 it cannot be more than one-fifth of the amount of acid which 
 would be neutralized by the bicarbonates. 
 
 Experimental results which involve the use of indicators to 
 determine such slight changes in reaction as these afford an un- 
 satisfactory basis for so important a theoretical conclusion. Ac- 
 cordingly, employing the potentiometric method, I carried out a 
 series of determinations of the reactions of solutions of the pro- 
 teins of serum, of the concentration in which they occur in serum, 
 to which varying amounts of acid or alkali had been added (58). 
 The results of these experiments entirely confirm Henderson's 
 estimate of the neutralizing power of the proteins of blood-serum 
 between the reactions mentioned above. 
 
 The proteins of ox-blood serum were coagulated by alcohol, 
 carefully washed with alcohol and ether and dried. They were 
 then dissolved in 0.01 N KC1 containing varying concentrations 
 of KOH or HC1, each solution containing 8 per cent of the pro- 
 teins (= percentage concentration in blood). The following were 
 the results obtained.* 
 
 * The value of K, the constant ionic product for water at 34 degrees, was 
 taken as (1.47 X lO" 7 ) 2 = 2.16 X 10~ 14 (Cf. Kohlrausch and Heydweiller 
 (33) ) . The experimental error in the determination of C H + in the " unknown " 
 is not more than 6 per cent of CH+. 
 
216 
 
 ELECTROCHEMISTRY 
 
 Known element of 
 chain 
 
 "Unknown" element of 
 chain 
 
 Potential of 
 chain, volt 
 
 C H + in "un- 
 known" ele- 
 ment 
 
 [Log 10 C H + in 
 "unknown" 
 element 
 
 0.01 ATKOH 
 
 0.01ATKOH+8per 
 cent serum pro- 
 teins. 
 
 0.0920 
 
 7.35X10- 11 
 
 -10.13 
 
 0.01JVKOH 
 
 0.005 ATKOH+8 per 
 cent serum pro- 
 teins. 
 
 0.1186 
 
 2.03X10- 10 
 
 - 9.79 
 
 0.01 N KOH 
 
 8 per cent serum 
 proteins. 
 
 0.1710 
 
 1. 52X10- 9 
 
 - 8.82 
 
 0.01ATKOH 
 
 0.005 N HC1+8 per 
 cent serum pro- 
 teins. 
 
 0.2277 
 
 1. 33X10- 8 
 
 - 7.88 
 
 0.01 N HC1 
 
 0.01 AT HC1+8 per 
 cent serum pro- 
 teins. 
 
 0.2968 
 
 1.15X10- 7 
 
 - 6.94 
 
 0.01 ATHC1 
 
 0.02 N HC1+8 per 
 cent serum pro- 
 teins. 
 
 0.2198 
 
 2.20X10-* 
 
 - 5.66 
 
 If, taking the solution of 8 per cent serum-proteins in neutral 
 0.01 N KC1 as the zero-point on the axis of the abscissae, we 
 measure off to the right of this point the concentrations of acid 
 in which the proteins were dissolved, and to the left of this point 
 the concentrations of alkali in which they were dissolved and 
 plot the values of log CH + given in the above table, we obtain 
 a curve,* by interpolation from which it is readily found that in 
 passing from the reaction 0.37 X 10~ 7 H+ to the reaction 1.00 X 
 10~ 7 H + , 100 cc. of an 8 per cent solution of the serum protein 
 neutralizes 2.25 cc. of JV/10 HC1, that is 22.5 cc., or about one- 
 fifth of its own volume of N/WO HC1. According to Henderson, 
 in passing through the same range of H + concentrations the 
 bicarbonates in 100 cc. of blood will neutralize the equivalent 
 of 100 cc. of N/WQ HC1. Hence we must conclude that, between 
 the reactions mentioned, the proteins of the serum are only one- 
 fifth as efficient in maintaining its neutrality as the bicarbonates. 
 To the neutralizing power of the serum proteins must be added, 
 in circulating plasma, that of the fibrinogen. This is, however, 
 probably very slight, since only a very small percentage of fibrin- 
 ogen is contained in the blood. 
 
 * For the form of the curve see my original communication (58). 
 
BIOLOGICAL APPLICATIONS 217 
 
 It has been shown by Marshall (39) that the major part of the 
 neutralizing power of saliva is likewise attributable to its inor- 
 ganic constituents. 
 
 It would not be safe, however, to conclude from these results 
 that the part played by the proteins in maintaining the neutrality 
 of the tissues is not, possibly, equal in magnitude to, or even 
 greater than, that played by the bicarbonates and acid phosphates. 
 Thus, referring to the tables in section 2 it will be seen that 
 100 cc. of a 1.5 per cent solution of potassium caseinate, in pass- 
 ing from the reaction 0.34 X 10~ 7 H+ to the reaction 1.14 X 10~ 7 
 H+ neutralizes the equivalent of 25 cc. of N/ 10 HC1; a 3 per 
 cent solution (the concentration of casein in cow's milk) would 
 therefore neutralize, in passing from the former to the latter of 
 the above reactions, the equivalent of one-half its volume of 
 N / 100 HC1. The part played by the casein of milk in main- 
 taining the neutrality of this tissue-fluid must therefore be of 
 very considerable importance. In the tissues, not only are the 
 proteins more concentrated than they are in the tissue-fluids, 
 but, owing to the predominance of nucleo-proteins which have 
 a high combining-capacity for bases, their power of maintaining 
 the neutrality of the solutions is not improbably even higher 
 than that of the majority of the proteins which occur in the 
 tissue-fluids. We are not, at present, in the possession of any 
 data which would render an estimate of the relative importance 
 of these factors, for the maintenance of the neutrality of the 
 tissues themselves, in the slightest degree reliable. 
 
 LITERATURE CITED 
 
 (1) Abegg, R., and Cummings, A. C., Zeit. f. elekt. Chem. 13 (1907), 
 
 p. 17. 
 
 (2) L'Agostino, E., and Quagliariello, G., Nernst Festschr. (1912), p. 27. 
 
 (3) Bjerrum, N., Zeit. f. physikal. Chem. 53 (1905), p. 428. 
 
 (4) Blasel, L., and Matula, J., Biochem. Zeitschr. 58 (1914), p. 417. 
 
 (5) Bugarszky, S., and Liebermann, L., Arch. f. d. ges. Physiol. 72 (1898), 
 
 p. 51. 
 
 (6) von Bunge, G., Zeit. f. Biol. 12 (1876), p. 191. 
 
 (7) von Bunge, G., "Lehrbuch der physiol. und pathol. Chem.," Leipzig, 
 
 1889. 
 
 (8) de Corral, J. M., Biochem. Zeitschr. 72 (1915), p. 1. 
 
 (9) Erb, W., Zeit. f. Biol. 41 (1901), p. 309. 
 
 (10) Farkas, G., Math. Estermeszettud. Ertesito. 21, p. 45; cited after 
 Maly's Jahresber. f. Tierchem. (1902), p. 274. 
 
218 ELECTROCHEMISTRY 
 
 (11) Farkas, G., Arch. f. (Anat. und) Physiol. (1903), p. 517. 
 
 (12) Farkas, G., Arch. f. d. ges. Physiol. 98 (1903), p. 517. 
 
 (13) Farkas, G., and Scipiades, E., Arch, f . d. ges. Physiol. 98 (1903), p. 577. 
 
 (14) Fernet, E., Ann. d. Sc. Nat. Paris 4 Ser. 8 (1857), p. 160. 
 
 (15) Foa, C., C. R. Soc. Biol. Paris 58 (1905), pp. 865, 867, and 1000; 59 
 
 (1905), pp. 51 and 185. 
 
 (16) Foa, C., Archivio di Fisiologia 3 (1906), p. 369. 
 
 (17) Foa, C., Arch. f. d. ges. Physiol. 115 (1906), p. 626. 
 
 (18) Fraenkel, P., Arch. f. d. ges. Physiol. 96 (1903), p. 601. 
 
 (19) Friedenthal, H., Zeit. f. aUgem. Physiol. 1 (1902), p. 56. 
 
 (20) Friendenthal, H., Zeit. f. allgem. Physiol. 4 (1904), p. 44. 
 
 (21) Hamburger, H. J., "Osmotische Druck und lonenlehre," Wiesbaden, 
 
 (1904), Bd. 2, pp. 330 and ff. 
 
 (22) Hardy, W. B., Journ. of Physiol. 33 (1905), p. 251. 
 
 (23) Hasselbalch, K. A., Biochem. Zeitschr. 30 (1910), p. 317; 49 (1913), 
 
 p. 451. 
 
 (24) Hasselbalch, K. A., and Lundsgaard, C., Biochem. Zeitschr. 38 (1912), 
 
 Leipzig, p. 77. 
 
 (25) Heidenhain, R., and Mayer, L., Ann. d. Chem. und Pharm. (1862-63), 
 
 Suppl. 2, p. 157. 
 
 (26) Heidenhain, R., and Mayer, L., Stud. d. physiol. Inst. zu Breslau, 
 
 1863, Heft 2. 
 
 (27) Henderson, L. J., Amer. Journ. Physiol. 15 (1906), p. 257; 21 (1908), 
 
 pp. 169, 173 and 427. 
 
 (28) Henderson, L. J., Journ. Biol. Chem. 7 (1909), p. 29. 
 
 (29) Henderson, L. J., and Black, O. F., Amer. Journ. Physiol. 18 (1907), 
 
 p. 250; 21 (1908), p. 420. 
 
 (30) Henderson, L. J., and Webster, H. R., Journ. Med. Research 16 (1907), 
 
 p. 1. 
 
 (31) Hoeber, R., Arch, f . d. ges. Physiol. 81 (1900), p. 522; 99 (1903), p. 572. 
 
 (32) Hoppe-Seyler, F., "Physiologische Chemie," Berlin, 1879, Bd. 3, p. 502. 
 
 (33) Kohlrausch, F., and Heydweiller, A., Wied. Ann. 53 (1894), p. 209. 
 
 (34) Lacqueur, E., and Sackur, O., Beitr. z. chem. Physiol. und Pathol. 3 
 
 (1903), p. 196. 
 
 (35) Ley, H., Zeit. f. physiol. Chem. 30 (1899), p. 193. 
 
 (36) Loeb, Jacques, Arch. f. d. ges. Physiol. 101 (1904), p. 340; 103 (1904), 
 
 p. 503. 
 
 (37) Loeb, Jacques, "The Dynamics of Living Matter," New York (1906), 
 
 p. 95. 
 
 (38) Manabe, K., and Matula, J., Biochem. Zeit. 52 (1913), p. 369. 
 
 (39) Marshall, J. A., Amer. Journ. Physiol. 43 (1917), p. 212. 
 
 (40) Michaelis, L., Biochem. Zeit. 19 (1909), p. 181. 
 
 (41) Michaelis, L., "Die Wasserstoffionenkonzentration; ihre Bedeutung 
 
 fur die Biologie und die Methoden ihrer Messung," Berlin, 1914. 
 
 (42) Michaelis, L., and Bien, Z., Biochem. Zeit. 67 (1914), p. 198. 
 
 (43) Michaelis, L., and Grineff, W., Biochem. Zeit. 41 (1912), p. 373. 
 
 (44) Michaelis, L., and Mostyusky, H., Biochem. Zeit. 25 (1910), p. 401. 
 
 (45) Michaelis, L., and Pechstein, H., Biochem. Zeit. 47 (1912), p. 260. 
 
LITERATURE CITED 219 
 
 (46) Mroczkovsky, L, Centr. f. d. med. Wiss. Berlin (1878), p. 356. 
 
 (47) Nernst, W., Zeit. f. physik. Chem. 2 (1887), p. 613; 4 (1889), p. 129. 
 
 (48) Nernst, W., Wied. Ann. 45 (1892), p. 360. 
 
 (49) Pauli, W., and Hirschfeld, M., Biochem. Zeit. 62 (1914), p. 245. 
 
 (50) Pauli, W., and Wagner, R., Biochem. Zeit. 27 (1910), p. 296. 
 
 (51) von Rhorer, L., Arch. f. d. ges. Physiol 90 (1902), p. 368. 
 
 (52) Ringer, W. E., Zeit. f. physiol. Chem. 95 (1915), p. 195. 
 
 (53) Robertson, T. Brailsford, Journ. physical Chem. 10 (1906), p. 524. 
 
 (54) Robertson, T. Brailsford, Journ. physical Chem. 11 (1907), p. 437. 
 
 (55) Robertson, T. Brailsford, Journ. Biol. Chem. 6 (1909), p. 313. 
 
 (56) Robertson, T. Brailsford, "The Proteins," Univ. of California, Publ. 
 
 Physiol. (1909). 
 
 (57) Robertson, T. Brailsford, Ergeb. d. Physiol. 10 (1910), p. 293. 
 
 (58) Robertson, T. Brailsford, Journ. Biol. Chem. 7 (1910), p. 351. 
 
 (59) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 528. 
 
 (60) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 709. 
 
 (61) Rohonyi, H., Biochem. Zeit. 44 (1912), p. 165. 
 
 (62) Salm, E., Zeit. f. physikal. Chem. 57 (1906), p. 471. 
 
 (63) Schmidt, C. L. A., Journ. Biol. Chem. 25 (1916), p. 63. 
 
 (64) Schmidt, C. L. A., Univ. of California Publ. Pathol. 2 (1916), p. 157. 
 
 (65) Sertoli, E., Hoppe-Seyler's Med. Chem. Untersuch. Berlin (1868),. 
 
 Heft 3, p. 350. 
 
 (66) Soldner, F., Landw. Versuchsstat. 35 (1888), p. 351. 
 
 (67) Sorensen, S. P. L., Ergeb. d. physiol. 12 (1912), p. 393. 
 
 (68) Spiro, K.,'and Pemsel, W., Zeit. f. physiol. Chem. 26 (1898), p. 233. 
 
 (69) von Szily, A., Orvosihetilap (1903), Nr. 32; cited after Maly's Jah- 
 
 resber. f. Tierchem. 33 (1903), p. 179. 
 
 (70) von Szily, A., Arch. f. d. ges. Physiol. 115 (1906), pp. 72 and 82. 
 
 (71) Van Slyke, L. L., and Hart, E. B., Amer. Chem. Journ. 33 (1905), p. 
 
 461. 
 
 (72) Zuntz, N., Hermann's "Handbuch der Physiologic," Leipzig (1880), 
 
 Bd. 4 T. 2. 
 
 (73) Zuntz, N., Arch. f. (Anat. und) Physiol. (1893), p. 556. 
 
CHAPTER X 
 
 THE ELECTRICAL CONDUCTIVITY OF SOLUTIONS OF PROTEIN 
 
 SALTS 
 
 1. The Influence of Dilution upon the Conductivity of Solu- 
 tions of Protein Salts. The Ostwald dilution-law for a binary 
 electrolyte is usually formulated as follows : 
 
 (-) 
 
 where K is the dissociation-constant, fi v is the molecular con- 
 ductivity at dilution V and /^ is the molecular conductivity 
 at infinite dilution, that is: 96.44 (u + v) where u and v are 
 average equivalent migration velocities in centimetres per 
 second under unit potential gradient, of the cations and anions 
 respectively. 
 
 Now fjL v = , where x is the specific conductivity in reciprocal 
 
 ohms, and m is the equivalent concentration, i.e., y ; hence from 
 the above equation we have 
 
 _/- 1.037 X 10- 2 \ /1.037 X 10- 2 V 
 
 K[l 7 ; r-X] = mi 7 ; r X] 
 
 \ m (u + v) I \ m (u + v) / 
 
 which reduces to 
 
 1.037 X 10- 2 . 1.075 X 10- 4 2 ,.. 
 
 m = ; X H ^r-f : rr~ X*. (l) 
 
 U + V K(U + V) Z 
 
 The same formula can be derived as follows (8); let c be the 
 equivalent concentration of the ions, m and K having the same 
 significance as before; then, applying the mass law, we have 
 
 C 2 = k (m - c). 
 
 Now 1.037 X lO- 2 x = (u + v) c; hence 
 _ 1.037 X 10- 2 
 
 u + v 
 
 substituting in the above equation we regain equation (i). 
 
 220 
 
DILUTION 221 
 
 From this mode of deriving the formula it is clear that the 
 total equivalent concentration of the electrolyte may be expressed 
 in terms of the electric conductivity of its solution as the sum 
 of two factors, the first, directly proportional to the conductiv- 
 ity, being the equivalent concentration of the dissociated part 
 of the electrolyte and the second, directly proportional to an 
 exponent of the electrical conductivity (in this instance 2), be- 
 ing the undissociated part of the electrolyte. 
 
 The salts which the proteins form with inorganic bases and 
 acids obey the Ostwald dilution-law for a binary electrolyte (7) 
 (8) (10) (12) (15) (16) (17) (19), that is, the relationship between 
 the equivalent concentration of the protein salt and the con- 
 ductivity (in reciprocal ohms per cubic centimetre) is that indi- 
 cated by formula (i). It is to be observed, however, that without 
 additional assumptions we do not know what is the equivalent 
 concentration of a protein salt. If we assume, however, that m, 
 the equivalent concentration of the base or acid which is neu- 
 tralized by the protein, bears a constant proportion to the true 
 equivalent concentration of the protein salt, then (i) becomes 
 
 1.037 X lO- 2 1.075 X 10~ 4 , 
 m = x _|_ _^_^ X 2 ( u ) 
 
 p (u + v) Kp (u + v) 2 
 
 in which m is now the known equivalent concentration of the 
 base or acid which is bound by the protein and p is the number 
 of equivalents of protein salt to which each equivalent of neutralized 
 add or base gives rise. This equation may be written: 
 
 m = Ax + Bx 2 , (iii) 
 
 in which A and B are constants, respectively equal to 
 1.037 X 10-2 1.075 X 1Q- 4 
 
 P (u + v) Kp (u + *) 2 ' 
 
 The fact that the dependence of the conductivity of solutions 
 of protein salts upon their dilution obeys the formula (iii), there- 
 fore, shows that for a given salt p is constant; in other words, 
 for a given combination of acid or base with protein, containing 
 a given proportion of the acid or base, the number of equivalents 
 of protein salt to which one equivalent of neutralized acid or 
 base gives rise is independent of the dilution. 
 
 The validity of equation (iii) as applied to solutions of protein 
 salts may be gathered from the accompanying tables. In each 
 case the most probable values of the constants A and B are com- 
 
222 
 
 ELECTROCHEMISTRY 
 
 puted from all of the experimental data by Gauss' method of 
 least squares. Inserting these values of the constants and the 
 experimental values of x in equation (iii), the " calculated" 
 values of ra are computed. In the third column of the tables 
 is given the ''Degree of Dissociation" of the salt calculated from 
 the ratio of Ax in equation 3 to the " calculated" value of ra. 
 
 The conductivity of the distilled water (usually not in excess 
 of 4 X 10~ 6 ) is subtracted from each of the observed conductivities. 
 
 TABLE I 
 
 Sodium Caseinate. Approx. neutral to Litmus. Approx. 50 X 
 Equivalents NaOH per gram. Temperature 25 degrees 
 A = 19.51 B = 9611 
 
 mX10 
 (experimental) 
 
 mX10< 
 (calculated) 
 
 Degree of dissocia- 
 tion (per cent) 
 
 200 
 
 201 
 
 73 
 
 100 
 
 98 
 
 83 
 
 68 
 
 66 
 
 88 
 
 50 
 
 51 
 
 89 
 
 40 
 
 42 
 
 90 
 
 28 
 
 29 
 
 94 
 
 20 
 
 22 
 
 93 
 
 10 
 
 17 
 
 98 
 
 TABLE II* 
 
 Ammonium Caseinate. Approx. neutral to Litmus. Approx. 50 X 10~ 5 
 Equivalents NH 4 OH per gram. Temperature 25 degrees 
 A = 16.27 B = 6188 
 
 mX 10* 
 (experimental) 
 
 m X 10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 200 
 
 201 
 
 74 
 
 100 
 
 99 
 
 84 
 
 68 
 
 67 
 
 88 
 
 50 
 
 50 
 
 90 
 
 40 
 
 40 
 
 93 
 
 28 
 
 30 
 
 93 
 
 20 
 
 24 
 
 96 
 
 10 
 
 11 
 
 99 
 
 * T. Brailsford Robertson (8). The exact proportion of base to casein in these experiments is 
 not certain, since the original solution which was diluted to yield the remainder was made up by 
 shaking up an . 02 N solution of the base with excess of casein under the erroneous belief (Cf . T. 
 Brailsford Robertson (6) (8) (11)) that the maximum amount of casein which a given concentra- 
 tion of a base would dissolve, when shaken up with excess of casein, was that with which it will 
 unite to form the "neutral" caseinates; i.e., containing 50 X 1Q- 5 equivalents of base per gram. 
 Since, however, casein dissolves rapidly up to this point and very slowly afterwards the composi- 
 tion of the salt formed was probably very nearly that indicated at the head of the tables. More- 
 over the solutions were tested and found to be neutral to litmus. The last observation in Table I 
 and the last but one in Table II are omitted in computing the values of the constants. 
 
DILUTION 
 
 223 
 
 TABLE III* 
 
 Sodium Caseinate. Neutral to Phenolphthalein. 80 X 10~ 5 Equiva- 
 lents NaOH per gram. Temperature 25 degrees 
 A = 15.76 B = 4979 
 
 mX 10* 
 (experimental) 
 
 m X 10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 250 
 
 250 
 
 73 
 
 125 
 
 132 
 
 77 
 
 63 
 
 62 
 
 89 
 
 31 
 
 33 
 
 94 
 
 16 
 
 18 
 
 96 
 
 TABLE IV* 
 
 Ammonium Caseinate. Neutral to Phenolphthalein. 80 X 10~ 5 Equiva- 
 lents NH 4 OH per gram. Temperature 25 degrees 
 
 A = 12.60 5 = 3978 
 
 OT X 10* 
 
 (experimental) 
 
 m X 10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 180 
 
 181 
 
 75 
 
 90 
 
 88 
 
 84 
 
 45 
 
 44 
 
 91 
 
 23 
 
 23 
 
 96 
 
 11 
 
 11 
 
 100 
 
 * The calculations in Tables III and IV are based upon the experimental data of Sackur (21). 
 Cf. T. Brailsford Robertson (10). 
 
 TABLE V 
 
 Potassium Caseinate. Neutral to Phenolphthalein. 80 X 10~ 5 Equiva- 
 lents KOH per gram. Temperature 30 degrees 
 
 A = 12 .41 B = 1666 
 
 m X 10* 
 (experimental) 
 
 m X 10 
 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 250 
 
 250 
 
 85 
 
 125 
 
 124 
 
 89 
 
 63 
 
 64 
 
 94 
 
 31 
 
 33 
 
 97 
 
 16 
 
 17 
 
 100 
 
224 
 
 ELECTROCHEMISTRY 
 
 TABLE VI 
 
 Calcium Casemate. Neutral to Phenolphthalein. 80X10" 5 Equiva- 
 lents Ca(OH) 2 per gram. Temperature 30 degrees 
 
 A = 27.89 B = 26,700 
 
 mX 10* 
 (experimental) 
 
 m X 10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 320 
 
 320 
 
 26 
 
 240 
 
 243 
 
 29 
 
 160 
 
 151 
 
 35 
 
 120 
 
 115 
 
 39 
 
 80 
 
 81 
 
 44 
 
 60 
 
 66 
 
 48 
 
 40 
 
 47 
 
 53 
 
 20 
 
 24 
 
 67 
 
 TABLE VII 
 
 Strontium Caseinate. Neutral to Phenolphthalein. 80 X10~ 5 Equiva- 
 lents Sr(OH) 2 per gram. Temperature 30 degrees 
 
 A =32. 65 5 = 4930 
 
 m X 10* 
 
 (experimental) 
 
 m X 10 4 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 224 
 
 226 
 
 61 
 
 168 
 
 167 
 
 66 
 
 112 
 
 106 
 
 74 
 
 84 
 
 86 
 
 77 
 
 56 
 
 58 
 
 .83 
 
 42 
 
 47 
 
 85 
 
 TABLE VIII* 
 
 Barium Caseinate. Neutral to Phenolphthalein. 80X10" 5 Equiva- 
 lents Ba(OH) 2 per gram. Temperature 30 degrees 
 
 A =23. 73 5 = 23,400 
 
 m X 10* 
 (experimental) 
 
 m X 10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 320 
 
 326 
 
 24 
 
 160 
 
 147 
 
 33 
 
 80 
 
 76 
 
 42 
 
 40 
 
 41 
 
 54 
 
 20 
 
 22 
 
 64 
 
 * For data used in Tables V, VI, VII and VIII, Cf. T. Brailsford Robertson (15) (17). 
 
DILUTION 
 
 225 
 
 TABLE IX* 
 
 Potassium Caseinate. 90X10~ 6 Equivalents KOH per gram. Tem- 
 perature 30 degrees 
 A = 11.59 B = 1739 
 
 mXlO* 
 (experimental) 
 
 mXlO* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 180 
 
 180 
 
 83 
 
 135 
 
 135 
 
 87 
 
 90 
 
 90 
 
 90 
 
 45 
 
 46 
 
 96 
 
 23 
 
 25 
 
 96 
 
 * The conductivity of the trace of free KOH was in no case more than 2 per cent of the observed 
 conductivity; the observed conductivities were not corrected for this (18). 
 
 Potassium Caseinate. 
 
 TABLE X* 
 100X10- 6 Equivalents KOH per gram. 
 
 perature 30 degrees 
 A = 9.57 B = 1242 
 
 Tern- 
 
 mXlO 4 
 (experimental) 
 
 TOX10* 
 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 300 
 
 300 
 
 76 
 
 200 
 
 201 
 
 82 
 
 150 
 
 149 
 
 85 
 
 100 
 
 100 
 
 89 
 
 50 
 
 53 
 
 93 
 
 * Calculated for solutions containing small amounts of KC1 according to the method described 
 in the previous chapter (section 1) and, therefore, corrected for the conductivity of the free 
 KOH as determined by gas-chain measurements. T. Brailsford Robertson (14). 
 
 TABLE XI* 
 
 Potassium Paranucleinate. Neutral to Phenolphthalein. 36X10" 5 
 
 Equivalents KOH per gram. Temperature 25 degrees 
 
 A = 12.38 B = 3784 
 
 mXlO 4 
 (experimental) 
 
 mX10< 
 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 92 
 
 93 
 
 85 
 
 46 
 
 46 
 
 93 
 
 23 
 
 23 
 
 96 
 
 12 
 
 13 
 
 100 
 
 6 
 
 7 
 
 100 
 
 * T. Brailsford Robertson (12). 
 
226 
 
 ELECTROCHEMISTRY 
 
 TABLE XII* 
 
 Sodium Serum-globulinate. Neutral to Litmus. 9.75X10~ 5 Equiv- 
 alents NaOH per gram. Temperature 18 degrees 
 
 A = 17.65 B = 36,200 
 
 m 
 (experimental) 
 
 m 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 657 
 
 657 
 
 66 
 
 329 
 
 302 
 
 78 
 
 164 
 
 155 
 
 87 
 
 82 
 
 83 
 
 92 
 
 41 
 
 44 
 
 96 
 
 21 
 
 29 
 
 97 
 
 * Calculated from experimental data obtained by W. B. Hardy (2). 
 
 The constants determined by least squares (A = 22.23; B = 17,391) do not give, for the smaller 
 values of m, so good an agreement as the above. From the evidence afforded by the numerous 
 data set forth in these tables there is strong presumptive reason for believing that, notwithstanding 
 this lack of agreement, the dilution-law holds good in this case also, and that experimental errors 
 are responsible for the deviations. A very probable source of error in these determinations is, as 
 Hardy points out, the conductivity of the undissolved suspension of globulin, a quantity of very 
 uncertain magnitude and meaning. This conductivity was very appreciable with the globulin 
 employed by Hardy (precipitated by acetic acid), while it was unappreciable with the globulin 
 employed in my experiments (precipitated by CO2). Since the method of least squares fails to 
 yield constants which satisfy the experimental results, I have taken A for this salt to be the same 
 as A for the salt containing 18 X 10~ 5 equivalents NaOH per gram. B is estimated from the first 
 observation. It is evident that the order of agreement between the calculated and observed 
 values is tolerably good. 
 
 TABLE XIII* 
 
 Sodium Serum-globulinate. Neutral to Phenolphthalein. 18X10" 8 
 Equivalents NaOH per gram. Temperature 18 degrees 
 
 A = 17.65 B = 11,500 
 
 mX10 
 (experimental) 
 
 mX10< 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 313 
 
 310 
 
 59 
 
 156 
 
 156 
 
 71 
 
 78 
 
 77 
 
 81 
 
 39 
 
 38 
 
 88 
 
 10 
 
 10 
 
 95 
 
 * Calculated from the experimental data of W. B. Hardy (2). 
 
DILUTION 
 
 227 
 
 TABLE XIV* 
 
 Serum-globulin Chloride. 9.318X10~ 5 Equivalents HC1 per gram. 
 Temperature 18 degrees 
 
 A = 7.42 B = 36,964 
 
 m 
 (experimental) 
 
 m 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 933 
 
 926 
 
 33 
 
 461 
 
 484 
 
 42 
 
 231 
 
 219 
 
 55 
 
 115 
 
 105 
 
 68 
 
 58 
 
 54 
 
 78 
 
 29 
 
 30 
 
 83 
 
 * Calculated from the experimental data of W. B. Hardy (2). 
 
 TABLE XV 
 
 Potassium Serum-globulinate. Neutral to Phenolphthalein. 20X10" 5 
 Equivalents KOH per gram. Temperature 30 degrees 
 
 A = 19.62 B = 51,900 
 
 m X 105 
 (experimental) 
 
 TO X 106 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 296 
 
 297 
 
 77 
 
 148 
 
 147 
 
 85 
 
 ' 74 
 
 70 
 
 92 
 
 37 
 
 37 
 
 95 
 
 18 
 
 18 
 
 98 
 
 TABLE XVI* 
 
 Calcium Serum-globulinate. Neutral to Phenolphthalein. 20X10" 5 
 Equivalents Ca(OH) 2 per gram. Temperature 30 degrees 
 
 A = 43.25 B = 502,000 
 
 TO X 10* 
 (experimental) 
 
 TOX10 5 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 296 
 
 296 
 
 66 
 
 148 
 
 146 
 
 77 
 
 74 
 
 76 
 
 85 
 
 37 
 
 43 
 
 91 
 
 18 
 
 18 
 
 96 
 
 * Fourth observation omitted in computing the constants. 
 
228 
 
 ELECTROCHEMISTRY 
 
 TABLE XVII 
 
 Strontium Serum-globulinate. Neutral to Phenolphthalein. 20X10" 5 
 Equivalents Sr(OH) 2 per gram. Temperature 30 degrees 
 
 A = 32.85 B = 628,000 
 
 mXlQs 
 (experimental) 
 
 TO X 10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 74 
 
 74 
 
 76 
 
 37 
 
 36 
 
 82 
 
 18 
 
 19 
 
 87 
 
 TABLE XVIII* 
 
 Barium Serum-globulinate. Neutral to Phenolphthalein. 20X10" 6 
 Equivalents Ba(OH) 2 per gram. Temperature 30 degrees 
 
 A = 42.61 B = 369,000 
 
 TO X 105 
 (experimental) 
 
 TO X 105 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 296 
 
 297 
 
 70 
 
 140 
 
 142 
 
 81 
 
 74 
 
 78 
 
 87 
 
 37 
 
 41 
 
 93 
 
 18 
 
 18 
 
 94 
 
 * For the data utilized in Tables XV, XVI, XVII, XVIII, Cf. T. Brailsford Robertson (17). 
 
 TABLE XIX* 
 
 Ovomucoid Chloride. 45X10" 5 Equivalents HC1 per gram. Tem- 
 perature 30 degrees 
 
 A = 5.103 B = 525 
 
 TO X 10* 
 
 (experimental) 
 
 m X10* 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 180 
 
 181 
 
 78 
 
 90 
 
 89 
 
 87 
 
 45 
 
 44 
 
 93 
 
 23 - 
 
 22 
 
 96 
 
 11 
 
 11 
 
 97 
 
 * Free HC1 = less than 0.5 X 10~ 4 for the 1 per cent solution; observed conductivity not cor- 
 rected for this. Data utilized in this and the following table from T. Brailsford Robertson (16). 
 
DILUTION 
 
 229 
 
 Ovomucoid Sulphate. 
 
 TABLE XX 
 
 45X10" 5 Equivalents H 2 S0 4 per gram. 
 perature 30 degrees 
 
 A = 5.488 B = 986 
 
 Tem- 
 
 m X 10 4 
 
 (experimental) 
 
 mXlO 4 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 180 
 
 180 
 
 71 
 
 90 
 
 89 
 
 81 
 
 45 
 
 45 
 
 89 
 
 23 
 
 23 
 
 96 
 
 11 
 
 12 
 
 96 
 
 TABLE XXI 
 
 Protamin (Salmin) Sulphate. 424X10~ 6 Equivalents H 2 S0 4 per gram. 
 Temperature 30 degrees 
 
 A = 9.09 B = 6408 
 
 mXlO* 
 (experimental) 
 
 mX10< 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 105.3 
 
 105.7 
 
 65 
 
 84.2 
 
 83.4 
 
 69 
 
 52.6 
 
 52.7 
 
 76 
 
 26.3 
 
 26.7 
 
 85 
 
 13.2 
 
 13.7 
 
 94 
 
 6.6 
 
 7.1 
 
 100 
 
 3.3 
 
 3.5 
 
 100 
 
 TABLE XXII 
 
 Protamin (Salmin) Chloride. 424X10~ 5 Equivalents HC1 per gram. 
 Temperature 30 degrees 
 
 A = 6.92 B = 1781 
 
 mXlO 4 
 (experimental) 
 
 mXlO 4 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 55.8 
 
 55.9 
 
 85 
 
 27.9 
 
 27.6 
 
 92 
 
 13.9 
 
 14.1 
 
 95 
 
 7.0 
 
 7.1 
 
 97 
 
 3.5 
 
 3.6 
 
 100 
 
230 
 
 ELECTROCHEMISTRY 
 
 It will be observed that as a rule the agreement between the 
 calculated and the observed values of m is excellent and we may 
 therefore conclude that equation (ii) (or (iii)) represents the 
 dependence of conductivity upon dilution, for the salts of the 
 proteins, with very satisfactory fidelity. Recollecting the mode 
 of derivation of equation (ii) this fact might be held to prove that 
 the protein salts, in all of the solutions investigated, dissociate 
 into two ions. This deduction is not altogether a safe one, how- 
 ever. The generalized form of equation (iii) for electrolytes which 
 dissociate into n ions is:* 
 
 m = Ax + Bx n (iv) 
 
 and, provided the departure of equation (iv) from the linear 
 form is small, the factor Bx n may be represented, within the 
 experimental error, by Bx 2 even if n be in reality a higher ex- 
 ponent than 2, while vice versa, Bx 2 may be approximated by 
 Bx n , where n is a higher exponent than 2. For example, in the 
 following table the values of m for calcium caseinate solutions 
 (Cf. Table VI), calculated from formula (iii) and from the formula 
 
 m = A& + iz 3 , (v) 
 
 are compared, f 
 
 TABLE XXIII 
 
 rnXlO* 
 (experimental) 
 
 mX 10 3 
 (calculated from 
 formula (iii)) 
 
 mX10 
 (calculated from 
 formula (v)) 
 
 32 
 24 
 16 
 12 
 
 8 
 6 
 4 
 2 
 
 32 
 24 
 15 
 
 12 
 8 
 7 
 5 
 2 
 
 33 
 23 
 14 
 11 
 
 8 
 7 
 5 
 3 
 
 SA= +1 
 
 SA = 
 
 * If the electrolyte dissociates partly into 2, partly into 3, partly into 4, etc., 
 ions the equation becomes: 
 
 m = Ax + Bx* + Cz 3 + Nx n . 
 
 t Sutherland (22) (23), urging the applicability of his theory of ionization 
 to the salts of proteins, has pointed out that the results of Hardy (cited above, 
 Table XIV) obtained with HCl-globulin containing 9.32 X10~ 6 equivalents- of 
 acid per gram of protein may be expressed by the equation: 
 
 - = 0.00026 + 0.009 tr*, 
 M 
 
 in which /t is the molecular conductivity of the salt, calculated in terms of 
 HCl-concentrations, and v is the volume of solution containing one equivalent 
 of Cl. 
 
DILUTION 231 
 
 There is little to choose between the two formulae, although, 
 on the whole, the agreement between the values calculated from 
 formula (iii) and the experimental values is somewhat closer 
 than that between the values calculated from formula (v) and 
 the experimental values. As we shall see, however, the internal 
 evidence which is afforded by the numerical values of the con- 
 stants demonstrates that the true relationship between dilution 
 and conductivity for solutions of the salts which proteins form 
 with bases is that which is expressed by equation (iii); namely 
 that which is characteristic for an electrolyte which yields two 
 ions per molecule. 
 
 As regards the salts which proteins form with acids, the binary 
 formula expressed by equation (iii) yields demonstrably better 
 agreement with the experimental data obtained with salmin 
 sulphate and chloride than the ternary formula expressed by 
 equation (v). The salts which protamins form with acids would 
 therefore appear to yield only two ions, irrespective of the valency 
 of the acid anion with which the protein is combined. On the 
 other hand, the relationship of conductivity to dilution which is 
 displayed by the salts which ovomucoid forms with hydrochloric 
 acid is more adequately expressed by the ternary formula. 
 
 It will be recollected that the constants A and B in equation 
 (iii) represent, respectively, the quantities 
 
 1.037 X 10- 2 1.075 X 10 4 
 
 where u -\- v is the sum of the equivalent migration velocities of 
 the ions in centimeters per second under a potential gradient 
 of 1 volt per centimeter, p is the number of equivalents of the 
 protein salt which is yielded by the neutralization of one equiva- 
 lent of inorganic base or acid, and K is the dissociation constant 
 
 of the salt. It will be observed that ^ = We can, therefore, 
 
 K p 
 
 from the data enumerated in the above tables, compute p (u + v) 
 jr 
 
 and for each of the salts investigated. The values of these 
 constants follow. 
 
232 
 
 ELECTROCHEMISTRY 
 
 TABLE XXIV. 
 
 CASEINATES OF INORGANIC BASES 
 
 Caseinates of monacid bases 
 
 Nature of the base and proportion of base 
 to 1 gram protein 
 
 Temp., 
 degrees 
 
 p(u+v) 
 
 K 
 P 
 
 50X10" 5 NaOH per gram 
 
 25 
 
 53X10~ 5 
 
 40X1Q-3 
 
 80X10" 5 NaOH per gram 
 
 25 
 
 66X10~ 5 
 
 50X10- 3 
 
 50XKT 5 NH 4 OH per gram 
 
 25 
 
 63X10~ 5 
 
 44X10- 3 
 
 80X10~ 5 NH 4 OH per gram 
 
 25 
 
 82X10" 5 
 
 40X1Q-3 
 
 80X10" 5 KOH per gram . 
 
 30 
 
 84X10" 5 
 
 91 X 10" 3 
 
 90X10" 5 KOH per gram. . . 
 
 30 
 
 89X10~ 5 
 
 77X10" 3 
 
 100 X 10~ 5 KOH per gram 
 
 30 
 
 108X10" 5 
 
 74X10" 3 
 
 
 
 
 
 Caseinates of diacid bases 
 
 80X10" 5 Ca(OH) per gram. 
 
 30 
 
 37X10~ 5 
 
 2 9X10~3 
 
 80X10" 5 Sr(OH) 2 per gram 
 
 30 
 
 32X10~ 5 
 
 21 6X10" 3 
 
 80X10~ 5 Ba(OH) 2 per gram 
 
 30 
 
 44X10- 5 
 
 2.4X10- 3 
 
 TABLE XXV. 
 
 SERUM GLOBULINATES OF INORGANIC BASES 
 
 Globulinates of monacid bases 
 
 Nature of the base and the proportion of 
 base to 1 gram protein 
 
 Temp., 
 degrees 
 
 p(u + v) 
 
 K 
 
 P 
 
 9.75X10~ 5 NaOH per gram 
 18.0X10~ 5 NaOH per gram 
 20. OX10~ 5 NaOH per gram 
 
 18 
 18 
 30 
 
 59X10- 5 
 59X10- 6 
 53X10~ 5 
 
 8.6X10-3 
 27.0X10-3 
 7 4X10~ 3 
 
 
 
 
 
 Globulinates of diacid bases 
 
 20X10~ 5 Ca(OH) 2 per gram 
 
 30 
 
 24X10- 5 
 
 3.7X10-3 
 
 20X10- 5 Sr(OH) 2 per gram 
 20X10~ 5 Ba(OH) 2 per gram 
 
 30 
 30 
 
 32X10- 5 
 24X10~ 5 
 
 1.7X10-3 
 4.9X10- 3 
 
 
 
 
 
 TABLE XXVI. COMPOUNDS OF PROTEINS WITH ACIDS 
 
 Monobasic acids 
 
 Nature of the acid and protein and the pro- 
 portion of acid to 1 gram protein 
 
 Temp., 
 degrees 
 
 p(u-HO 
 
 K 
 P 
 
 9.318X10~ 5 HC1 per gram serum 
 globulin 
 
 18 
 
 140X10" 5 
 
 1.5X10- 3 
 
 45X10~ 5 HC1 per gram ovomucoid. . 
 442X10~ 5 HC1 per gram salmin 
 
 30 
 30 
 
 203X10- 5 
 150X10- 5 
 
 50 X10-3 
 26.9X10-3 
 
 Dibasic acids 
 
 45X10- 5 H 2 SO 4 
 coid 
 
 per gram ovomu- 
 
 30 
 
 189X10~ 5 
 
 31 X 10-3 
 
 442X10- 5 H 2 SO 4 
 
 per gram salmin . . 
 
 30 
 
 114X10- 5 
 
 12.9X10-3 
 
 For potassium paranucleinate, containing 36 X 10~ 5 equivalents of KOH per gram, the value 
 
 of p(u + 1>) is 84 X 10-6 and that of - is 41 X 10~ 8 . 
 P 
 
DILUTION 233 
 
 On surveying these tables several striking facts are immedi- 
 ately revealed. In the first place the ratio of the value of p (u + v) 
 for monacid bases to its value for diacid bases is, in every case 
 (Cf. Tables XXIV and XXV) exceedingly close to the ratio 2:1. 
 This may be interpreted in either of two ways; either the equiva- 
 lent velocity of the protein ions which are yielded by the salts 
 of monacid bases is twice as great as that of the ions which are 
 yielded by the salts of diacid bases, or else one equivalent of a 
 monacid base gives rise to twice as many equivalents of protein 
 salt as one equivalent of a diacid base. We have seen that the 
 protein salts dissociate into two heavy protein ions and, as Bredig 
 has shown (1), the equivalent velocities of heavy ions approach 
 a constant minimum. The former of the above alternatives may 
 therefore be dismissed, and we must conclude that p, the number 
 of equivalents of protein salts to which one equivalent of base 
 gives rise, is twice as great for the monacid as for the diacid bases. 
 
 Reverting to the hypothesis developed in the preceding chap- 
 ters, it appears that the monacid bases form salts with proteins 
 in the following way : 
 
 H 
 
 ++ I 
 
 -COH.N- + NaOH = - CONa + ^N- 
 
 I 
 OH 
 
 from which it is evident that for the salts of the monacid bases 
 p must be 2, i.e., one equivalent of a monacid base, when united 
 with protein, yields 2 equivalents of the protein salt. We must 
 conclude, then, that one equivalent of diacid base gives rise to 
 only one equivalent of protein salt. The most natural assumption 
 regarding the mode of combination of the proteins with diacid 
 bases, pursuing the above hypothesis, would be to suppose that 
 it takes place in accordance with the equation: 
 
 H 
 
 -CO++ I 
 
 2-COH.N- +Ca(OH) 2 = ;)Ca + 2^N- 
 
 -C0++ x I 
 
 OH 
 
 upon which supposition each equivalent of the diacid base would 
 give rise to just the same number of equivalents of protein salt 
 as an equivalent of a monacid base, namely 2. This is evidently 
 
234 ELECTROCHEMISTRY 
 
 not the case, and we must assume that two of the positive valen- 
 cies of the cation are neutralized and at the same time that two 
 of the negative valencies which the anions supply are also neu- 
 tralized. This immediately suggests that one of the two anions 
 neutralizes two of the valencies of the cation and that the true 
 constitution of the cation is either 
 
 -CO++ v -CO+, H 
 
 )Ca I \ I 
 
 -CO.N- or Ca >N- 
 
 /\ I / \ 
 
 H OH -CO +/ OH 
 
 or either of these formulae less the elements of water which are 
 attached to the nitrogen atom. If this be so then we must con- 
 clude that each molecule, of a diacid base gives rise to exactly 
 the same number of ions (apart from the question of the degree 
 of dissociation of the salt) and equivalents of protein salt as a 
 molecule of a monacid base, and that the molecule of a protein 
 salt of a diacid base must be twice as heavy as a molecule of a protein 
 salt of a monacid base. In this connection the following differ- 
 ences between the physical behavior of solutions of the caseinates 
 of the alkalies and those of the caseinates of the alkaline earths 
 may be recalled to the reader: The aqueous solutions of the 
 " neutral" or " basic" caseinates of the alkalies and ammonium 
 are clear, do not (except lithium caseinate) show any increase in 
 turbidity on warming, and are not precipitated by the addition 
 of finely divided insoluble substances or by passing through a 
 clay filter, while the aqueous solutions of the caseinates of the 
 alkaline earths are opalescent, show a marked turbidity on heat- 
 ing their solutions to 35-45 C., which disappears on cooling, 
 and are precipitated from their solutions by the addition of 
 finely divided insoluble substances or by passage through a clay 
 filter (5). Equally concentrated solutions of the hydroxides of 
 the alkalies and ammonium dissolve casein at approximately 
 the same rate. Solutions of the hydroxides of the alkaline earths 
 dissolve casein much more slowly (9) (13). 
 
 The salts which casein forms with monacid bases at "satu- 
 ration" of the base with casein, that is, when the proportion of 
 base to casein is 11.4 X 10~ 5 equivalents per gram, are soluble 
 in water, while the corresponding salts which casein forms with 
 
DILUTION 235 
 
 diacid bases are insoluble in water (11) (25). All of these differ- 
 ences, it will be observed, suggest more or less strongly that the 
 molecules of the caseinates of the alkaline earths are of greater 
 size and more ponderous than those of the casemates of the 
 alkalies. 
 
 Assuming, therefore, that p = 2 f or the salts of the alkalies 
 and 1 for the salts of the alkaline earths we find that the numer- 
 ical values of p (u -\- v) in Tables XXIV and XXV accord very 
 well with our knowledge of the mobilities of heavy ions. The 
 average value of u + v at 30 degrees for potassium caseinate 
 containing 80 X 10~ 5 equivalents of KOH per gram is 42 X 10~ 5 
 cm. per sec., while for the salts of the alkaline earths at the same 
 temperature the average value of u -\- v is 38 X 10~ 5 . Assuming 
 the migration velocities of the anions and cations to be equal 
 (since, as we have seen, their masses are nearly equal) the equiv- 
 alent velocity of a protein ion at 30 degrees would appear to be 
 about 20 X 10~ 5 cm. per sec. under a potential gradient of 1 volt 
 per centimeter. Now Bredig (1) has shown that the equivalent 
 velocities of heavy ions tend to attain, with increasing weight, 
 a constant minimum value of about 15 X 10~ 5 cm. per sec. at 
 15 degrees. At 30 degrees this velocity would be increased by 
 about 30 per cent, i.e., would become 20 X 10~ 5 cm. per sec. 
 
 Referring to Table XXVI it is evident that p (u + 0) is much 
 larger for the salts which proteins form with acids than it is 
 for the salts which they form with bases. It is about twice as 
 large, in the case of serum globulin, as it is for the salts of diacid 
 bases. We can hardly assume that the migration-velocity of 
 protein ions is twice as great in acid as in alkaline solution and 
 we must therefore adopt the alternative assumption that p is 
 twice as great in acid as in alkaline solutions. This might arise 
 by the splitting of double COH.N bonds, thus: 
 
 H Cl 
 -COH.N, -COH 
 
 - COH.N -COH 
 
 / \ 
 H OH 
 
 which indicates that, as Kossel has suggested (3) the active 
 agents in bringing about the neutralization of acids by proteins 
 
236 ELECTROCHEMISTRY 
 
 are the diamino radicals (hexone bases) which are contained in 
 the protein molecule. As we shall see in the succeeding section 
 of this chapter, the neutralization of bases by the proteins is 
 analogously accomplished, at any rate for the greater part, by 
 the dicarboxylic radicals which they contain; only the evidence 
 which we have so far discussed does not reveal this fact because 
 salts of the type 
 
 H OH 
 
 .COK 
 
 R' ++ + 
 
 X COH 
 
 / \ 
 H OH 
 
 do not appear to exist, but only salts of the type 
 
 H OH 
 
 COK 
 
 / V 
 
 H OH 
 
 in which the ratio of the valencies of the cation to the number 
 of equivalents of base included in the molecule of the protein 
 salt is 2; the same as it would be for a salt formed by the open- 
 ing up of a single COH.N bond. 
 
 The results obtained with solutions of ovomucoid in dilute 
 acids are complicated by the possible presence of free ovomucoid 
 which is itself ionized and therefore contributes to the conduc- 
 tivity of these solutions. It is evident, however (Cf. Tables XXI 
 and XXII), that the conductivity of a solution of ovomucoid 
 sulphate is practically identical with that of a solution of ovo- 
 mucoid chloride containing the same equivalent concentration 
 (45 X 10~ 5 equivalents per gram) of neutralized HC1. Hence it 
 follows that in the formation of these salts each equivalent of 
 a dibasic acid must give rise to the same number of equivalents 
 of protein salt that an equivalent of a monobasic acid produces. 
 This conclusion also applies to the salts which the protamin, 
 salmin, forms with acids (19). 
 
 2. The Depression of the Freezing-point of Water which is 
 Caused by Dissolved Protein Salts, and the Stoichiometry of 
 
DEPRESSION OF THE FREEZING-POINT 237 
 
 Protein Salts. We have hitherto found it convenient in dis- 
 cussing the mode of formation of the salts which the proteins 
 form with bases, only to consider the consequences which arise 
 out of the opening up of a single COH.N bond, without 
 involving a consideration of the question whether this COH.N 
 bond is supplied by a dicarboxylic acid group (such as glutamic 
 acid), in which case two such bonds must be opened up before 
 actual electrolytic dissociation can occur, or whether it is supplied 
 by a mono-carboxylic acid group (such as glycocoll) in which 
 case dissociation could occur directly one such group was opened 
 up. We have been able to do this because, in the formation of 
 salts with bases, all of the COH.N groups which are opened 
 up suffer the introduction of the basic radical and the equiva- 
 lence between the protein salt and the base which gives rise to 
 it is the same as that which would subsist were the salt formed 
 by the opening up of only one COH.N group. All of the 
 preceding considerations and theoretical deductions, therefore, 
 remain equally valid whether the COH.N groups under 
 consideration in each separate case are derived from a dicarboxylic 
 acid radical or from a mono-carboxylic acid radical. Only, so 
 far as our experimental knowledge goes, when we investigate 
 the freezing-point depression (i.e., the osmotic pressure) of solu- 
 tions of protein salts do we encounter facts which are definitely 
 irreconcilable with the view that the protein salts with bases 
 are formed through the agency of mono-carboxylic acid radicals 
 and which point unmistakably to the dicarboxylic add radicals 
 as the active agents in accomplishing these unions. 
 
 It has been shown by Robertson and Burnett (20) that whether 
 the caseinate formed is neutral to litmus and contains 50 X 10~ 5 
 equivalents of base per gram, or neutral to phenolphthalein and 
 containing 80 X 10~ 5 equivalents of base per gram, the freezing- 
 point depression which is brought about by the dissolved protein salt 
 bears the same proportion to the concentration of the neutralized base. 
 
 The significance of this observation has been alluded to in 
 the latter part of Chap. I. If we prepare a number of solutions 
 all containing the same amount of a base and add to these vary- 
 ing amounts of a polybasic acid R(COOH) n , such that in the 
 first solution only one, in the second two, etc., COOH groups 
 are neutralized, then, if all these salts are highly dissociated, 
 calling the osmotic pressure of the first 1, that of the second 
 
238 ELECTROCHEMISTRY 
 
 will be f, that of the third f, etc. But if, to solutions which 
 contain the same amount of a base we add varying amounts 
 of casein, the osmotic pressures of all of these solutions, as indi- 
 cated by their freezing-points are the same. Evidently, therefore, 
 a given quantity of base always gives rise to the same number 
 of protein ions, whether the base is combined with more or with 
 less protein. This obviously corresponds with the view that 
 each equivalent of base opens up a given number of COH.N 
 groups, and not at all with the view that it neutralizes terminal 
 -COOH groups. 
 
 The experimental data which were obtained by Robertson 
 and Burnett are enumerated in Tables I and II in Chap. XIII 
 (p. 333). On referring to the results which are therein cited 
 it will be observed that the depression of the freezing-point which 
 is brought about by the dissolved caseinate of the alkalies is in 
 every case very nearly that which would be observed in a solu- 
 tion of the same molecular concentration as that of the alkali 
 which is neutralized by the casein. Now these solutions are 
 quite extensively (over 80 per cent, Cf. Tables, section 1) dissoci- 
 ated and it therefore follows that each molecule of alkali gives rise 
 to one ion of caseinate. That the same is true for the alkaline 
 earths is readily seen when allowance is made for their compara- 
 tively slight degree of dissociation (Cf. Tables VI, VII and VIII). 
 Now in the formation of a salt by the splitting of a single 
 COH.N bond each molecule of neutralized base would give 
 rise to two ions of caseinate, while in the formation of a salt by 
 the splitting of a double COH.N bond one molecule of 
 neutralized base might give rise to only one ion of caseinate. 
 This will be clear from the following equations: 
 
 H 
 
 ++ I 
 
 -COH.N- + NaOH = -CONa+ ^N- 
 
 I 
 OH 
 
 H OH 
 COH.N. CONa 
 
 COH.N CONa 
 
 / \ 
 H OH 
 
DEPRESSION OF THE FREEZING-POINT 239 
 
 Evidently, therefore, it is the latter alternative which repre- 
 sents the true mode of formation of these salts, and we must 
 conclude that in the neutralization of bases the dicarboxylic 
 radicals play a leading part, just as, in the neutralization of 
 acids, the diamino radicals play a leading part. 
 
 We have seen that in the formation of ovomucoid chloride, 
 containing 45 X 10~ 5 equivalents of acid per gram, only one of 
 the COH.N bonds which is opened up suffers the introduction 
 of an HC1 molecule. In this case, therefore, if the above reason- 
 ing has been correct, the freezing-point depression due to the dis- 
 solved salt should be either twice or three times that of a solution 
 of the molecular concentration of the neutralized acid according 
 as to whether the formation of the salt is represented by the 
 equation: 
 
 H Cl 
 
 ++ \ / 
 
 , COH.N, /COH 
 
 R' ^R + HC1 + H 2 = R' ++ 
 
 x COH.N / X COH 
 
 / \ 
 H OH 
 or by the equation: 
 
 H C 
 \ / 
 R-COH.N. ^N. x 
 
 ^ R + HC1 + H 2 = 2 R.COH+++ ^ R 
 R-COH.N X ^N x 
 
 / \ 
 H OH 
 
 that is, according as to whether the diamino acid radicals in the 
 ovomucoid molecule are directly united with both carboxyls of 
 dicarboxylic acid radicals, or with only one or with monocar- 
 boxylic acid radicals. The experimental fact is that the freezing- 
 point-depression of a 0.018 m. solution of HC1 containing 4 per 
 cent of ovomucoid (1 gram per 45 X 10~ 5 equivalents) is 0.09 
 0.005 degree, corresponding to a 0.055 m. solution, or almost 
 exactly three times the molecular concentration of the neutralized 
 acid. 
 
 Hence we may conclude that the formation of this salt is repre- 
 sented by the latter of the above two equations and that in ovo- 
 mucoid the diamino radicals are not directly attached to both carboxyls 
 of dicarboxylic radicals. 
 
240 
 
 ELECTROCHEMISTRY 
 
 Continuing the addition of acid (HC1) to the above solution 
 of ovomucoid a remarkable phenomenon is observed, namely, 
 that doubling the amount of acid in the solution does not appreciably 
 alter its freezing-point * and, consequently does not alter the total 
 number of ions per cubic centimeter of the solution. Evidently, 
 upon further addition of acid, the remaining nitrogen atom of the 
 diamino radical becomes neutralized and the ion 
 
 H Cl 
 \ / 
 
 \ 
 
 R 
 
 / \ 
 
 is formed. H C1 
 
 The form of the dilution-law which we should apply to these 
 solutions is therefore (Cf. equation (v)) : 
 
 m 
 
 1.037 X 10~ 2 
 
 1.115 X 1Q- 6 
 
 x*. 
 
 P (u + v) 
 
 Applying this equation to the results enumerated in Table 
 XIX we obtain m = 5.68 x + 117,000 x 3 . 
 
 In the accompanying table the experimental and calculated values 
 of m are compared. In the third column is given the degree of 
 dissociation of the salt, estimated as the ratio of the calculated 
 value of 5.63 x to the calculated value of m. 
 
 TABLE XXVII. OVOMUCOID CHLORIDE 
 ( (45X10~ 5 equivalents HC1 per gram) 
 
 mXlO* 
 (experimental) 
 
 mXlO 4 
 (calculated) 
 
 Degree of dissocia- 
 tion, per cent 
 
 180 
 
 180 
 
 86 
 
 90 
 
 89 
 
 96 
 
 45 
 
 46 
 
 98 
 
 23 
 
 24 
 
 100 
 
 11 
 
 12 
 
 100 
 
 The same equation applies so nearly to the experimental results 
 obtained with ovomucoid sulphate (Table XX) that it is not 
 necessary to recompute the constants for this salt. 
 
 * The freezing-point-depression due to free ovomucoid is only one-third of 
 that of the above solution, namely, 0.03 0.005 degree for a 4 per cent 
 solution. 
 
DEPRESSION OF THE FREEZING-POINT 241 
 
 1 037 X 10~ 2 
 
 From the value of - -, ; r for the ovomucoid chloride 
 P (u + v) 
 
 we obtain: P (u + v) = 134 X lO" 5 , 
 
 whence, if p = 4, u + v = 46 X 10~ 5 , which agrees very well 
 with the values obtained with other protein salts for this constant 
 (Tables XXIV and XXV; Cf. also p. 235). 
 
 A one-half per cent solution of salmin hydrochloride freezes 
 at a temperature of 0.04 degrees lower than distilled water. 
 This corresponds to the freezing-point depression of a solution 
 
 in which the molecular + ionic concentration is -^ . Now the 
 
 46 
 
 equivalent concentration of HC1 neutralized by salmin in a J 
 
 AM 
 
 per cent solution of salmin hydrochloride is j=. Each molecule 
 
 of combined hydrochloric acid yields, therefore, one molecule 
 or one ion of salmin hydrochloride. Salmin hydrochloride in 
 | per cent solution is very highly ionized, since dilution only 
 increases its equivalent conductivity to a small extent and we 
 must infer, therefore, since the salt exists in solution largely in 
 the form of ions, that each molecule of combined acid yields 
 one ion of the protein salt. One molecule of salmin hydrochloride 
 contains at least four molecules of combined hydrochloric acid 
 (3) (24), hence one molecule of salmin hydrochloride must yield 
 four ions or a multiple thereof. From the value of the constant 
 p (u + v) in Table XXVI we must infer that the valency of each 
 of these ions is 4. These phenomena obviously correspond with 
 those which would be exhibited by a salt which forms and dis- 
 sociates in accordance with equations of the type: 
 
 H Cl 
 
 .COH.N. .COH++ 
 
 v 
 
 X COH.N X N COH++ 
 
 / \ 
 H Cl 
 
 and 
 
 COH.N v .COH++ . 
 
 )^ + H 2 S0 4 = R/ +S0 4 
 
 COH.N X N COH++ 
 
242 ELECTROCHEMISTRY 
 
 which are in satisfactory accord with the high diamino-acid 
 content of the salmin molecule (4). 
 
 Summing up these results, therefore, we see that in ike forma- 
 tion of the salts of proteins with acids diamino radicals are primarily 
 concerned, while in the formation of the salts of protein with bases 
 dicarboxylic radicals are primarily concerned. The correspondence 
 between the diamino-acid content of a protein and its binding 
 capacity for acids, to which Kossel has drawn attention (loc. cit.), 
 and that between the dicarboxylic acid content and the com- 
 bining capacity for bases to which I have drawn attention in 
 the latter part of the first chapter is therefore seen to be not 
 accidental but an expression of an essential feature of the union 
 between proteins and inorganic acids and bases. 
 I 3. The Dependence of the Electrical Conductivity of Solutions 
 of Protein Salts upon the Proportion of Inorganic Acid or Base 
 which the Salt Contains. In preceding paragraphs we have 
 discussed the effects of dilution upon the conductivities of solu- 
 tions of protein salts; that is, of altering the ratio j while 
 
 keeping the composition of the salt; i.e., the proportion of protein 
 to base or acid constant. We have found that these effects 
 admit of interpretation in the light of the well-known laws of 
 electrolytic dissociation and of the view that the dissociation 
 of protein salts is accomplished by the splitting of COH.N 
 bonds. We must now take up the consideration of the effects 
 of altering, not only the total concentration of the system, but 
 also the proportion of protein to base or acid. 
 
 If we add protein to dilute solutions of an acid or base there 
 usually results a depression, which we shall designate by X, of 
 the conductivity of the solution. I find that when 1 per cent of 
 casein is added to varying concentrations of KOH this depression 
 is connected with the concentration of the KOH solution (= 61) 
 to which the protein is added by a very simple relation. This 
 relation is of the form 
 
 X X 10- 5 = a&i - /36i 2 - 7 (vi) 
 
 in which a, /? and 7 are constants (14). 
 
 Applying this formula to observations in which 1 per cent of 
 casein was dissolved in varying concentrations of KOH * and the 
 
 * These solutions were the same as those employed in the gas-chain measure- 
 ments cited in the previous chapter (1 per cent casein, second series), and, 
 
PROPORTION OF INORGANIC RADICAL 
 
 243 
 
 conductivities of the solvent and of the solution were determined 
 at 30 degrees, and computing the most probable values of the 
 constants a, /3 and 7 from all of the observations by the method 
 of least squares, we obtain : 
 
 X X 10 5 = 26880 bi - 475800 bj - 28.98. 
 
 In the accompanying table (Table XXVIII) the experimental 
 values of X X 10~ 5 for 1 per cent solutions of casein in KOH of 
 various concentrations (= &i) and those calculated from the 
 above formula are compared. In the first column are given the 
 alkalinities of the solutions to which casein was added (= 61); 
 in the second are given the values of X X 10 5 experimentally 
 ascertained; in the third the calculated values of X X 10 5 ; in the 
 fourth the difference (= A) between the experimental and the 
 calculated values of X X 10 5 ; and in the fifth the possible metrical 
 error (= e) in the experimental determination of X X 10 5 . 
 
 TABLE XXVIII 
 
 61 
 
 XX10 5 
 
 (experimental) 
 
 XX 105 
 (calculated) 
 
 A 
 
 
 
 0.03000 
 
 348.8 
 
 349.3 
 
 +0.5 
 
 8.0 
 
 0.02500 
 
 343.0 
 
 345.6 
 
 +2.6 
 
 6.5 
 
 0.02000 
 
 322.1 
 
 318.3 
 
 -3.8 
 
 5.5 
 
 0.01750 
 
 299.7 
 
 295.9 
 
 -3.8 
 
 5.1 
 
 0.01500 
 
 268.2 
 
 267.2 
 
 -1.0 
 
 4.7 
 
 0.01250 
 
 230.1 
 
 232.8 
 
 +2.7 
 
 4.4 
 
 0.01000 
 
 186.8 
 
 192.3 
 
 +5.5 
 
 4.1 
 
 0.00750 
 
 141.6 
 
 145.8 
 
 +4.2 
 
 4.0 
 
 0.00500 
 
 92.1 
 
 93.5 
 
 +1-4 
 
 3.8 
 
 0.00250 
 
 43.9 
 
 35.4 
 
 -8.5 
 
 3.7 
 
 
 
 
 SA = -0.2 
 
 
 It will be seen that the deviations of the calculated from the 
 experimental values of X X 10 5 are nearly always less than the 
 possible error, due to instrumental sources alone, in the experi- 
 mental determination of X X 10 5 , while the algebraic sum of these 
 deviations is negligible. The formula therefore represents, in a 
 highly satisfactory manner, the relation between 61 and X for 
 1 per cent solutions of casein in KOH-solutions. 
 
 therefore, contained varying amounts of KC1 (Cf. Appendix). The fact that 
 the irregularity in KCl-content does not disturb the regularity of the relation 
 between X and 61 is further proof (Cf . Chap. VIII, 1), that KC1, in moderate 
 concentrations, does not appreciably influence the conductivity of potassium 
 caseinate solutions. 
 
244 
 
 ELECTROCHEMISTRY 
 
 Investigating the similar relations which obtain in solutions 
 containing different percentages of casein we find that the rela- 
 tion between X and 61 for all of the concentrations of casein in- 
 vestigated can be represented by the more general formula : 
 
 X X 10 5 
 
 (vii) 
 
 where C is the percentage of casein and a, (3 and y are constants, 
 the values of which we have already determined for 1 per cent 
 casein. For a 0.5 per cent solution of casein this equation, 
 therefore becomes: 
 
 X X 10 5 = 26,880 61 - 951,600 6i 2 - 14.49. 
 
 In the following table the experimental and calculated values of 
 X X 10 5 are compared the symbols have the same meaning 
 as in Table XXVIII. 
 
 TABLE XXIX 
 
 r 
 
 XX10* 
 
 XX10* 
 
 
 
 01 
 
 (experimental) 
 
 (calculated) 
 
 A 
 
 6 
 
 0.01000 
 
 149.9 
 
 159.2 
 
 + 9.3 
 
 2.3 
 
 0.00750 
 
 123.5 
 
 133.6 
 
 +10.1 
 
 2.0 
 
 0.00500 
 
 88.2 
 
 96.1 
 
 + 7.9 
 
 1.8 
 
 0.00250 
 
 40.6 
 
 37.6 
 
 - 3.0 
 
 1.8 
 
 0.00150 
 
 23.3 
 
 24.2 
 
 + 0.9 
 
 1.8 
 
 For a 1.5 per cent solution of casein equation (vii) becomes: 
 X X 10 5 = 26,880 61 - 317,200 bf - 43.47. 
 
 In the following table the experimental and calculated values 
 of X X 10 5 are compared: 
 
 TABLE XXX 
 
 61 
 
 XX10 6 
 (experimental) 
 
 XX ID* 
 (calculated) 
 
 A 
 
 6 
 
 0.03000 
 
 476.4 
 
 477.5 
 
 + 1.1 
 
 7.0 
 
 0.02000 
 
 366.5 
 
 367.3 
 
 + 0.8 
 
 5.1 
 
 0.01500 
 
 283.5 
 
 288.4 
 
 + 4.9 
 
 4.5 
 
 0.01000 
 
 188.5 
 
 193.6 
 
 + 5.1 
 
 4.1 
 
 0.00750 
 
 145.0 
 
 140.4 
 
 - 4.6 
 
 3.9 
 
 0.00500 
 
 95.4 
 
 83.0 
 
 -12.4 
 
 3.8 
 
PROPORTION OF INORGANIC RADICAL 
 
 245 
 
 For a 2.0 per cent solution of casein equation (vii) becomes: 
 X X 10 5 = 26,880 61 - 237,900 6 X 2 - 57.96. 
 
 In the following table the experimental and calculated values 
 of X X 10 5 are compared: 
 
 TABLE XXXI 
 
 &1 
 
 xxio* 
 
 (experimental) 
 
 xxio* 
 
 (calculated) 
 
 A 
 
 
 
 0.05000 
 
 661.6 
 
 691.4 
 
 +29.8 
 
 18.5 
 
 0.03000 
 
 539.3 
 
 534.4 
 
 - 4.9 
 
 11.5 
 
 0.02000 
 
 380.5 
 
 384.5 
 
 4- 4.0 
 
 7.6 
 
 0.01500 
 
 286.2 
 
 291.7 
 
 4- 5.5 
 
 7.1 
 
 0.01000 
 
 189.3 
 
 187.1 
 
 - 2.2 
 
 6.7 
 
 0.00500 
 
 95.7 
 
 70.5 
 
 -25.2 
 
 6.2 
 
 For a 3 per cent solution of casein equation (vii) becomes: 
 X X 10 5 = 26,880 bi - 158,600 6i 2 - 86.94. 
 
 In the following table the experimental and calculated values 
 of X X 10 5 are compared: 
 
 TABLE XXXII 
 
 bi 
 
 XX10 6 
 (experimental) 
 
 XXlQs 
 (calculated) 
 
 A 
 
 . 
 
 0.05000 
 
 873.2 
 
 860.7 
 
 -12.5 
 
 16.4 
 
 0.03000 
 
 579.7 
 
 576.8 
 
 - 2.9 
 
 9.0 
 
 0.02500 
 
 488.9 
 
 486.0 
 
 - 2.9 
 
 8.4 
 
 0.02000 
 
 391.7 
 
 387.3 
 
 - 4.4 
 
 7.6 
 
 0.01500 
 
 296.9 
 
 280.6 
 
 -16.3 
 
 7.2 
 
 0.01000 
 
 206.6 
 
 166.1 
 
 -40.5 
 
 6.8 
 
 The same relation holds good for 1.5 per cent solutions of 
 casein in Ca(OH) 2 solutions of varying concentration (equivalent 
 concentration = 61) (17). 
 
 To 50 cc. portions of a 3 per cent solution of casein in 0.024 
 N Ca(OH)2 (neutral to phenolphthalein) were added 0, 2, 4, 6, 
 or 12 cc. of 0.048 N Ca(OH) 2 and each mixture was diluted to 
 100 cc. Exactly similar solutions were made up, containing 
 however, no casein. The conductivities of each of these solu- 
 tions were then determined at 30 degrees. Applying equation 
 
246 
 
 ELECTROCHEMISTRY 
 
 (vii) to these results and computing the constants from the first, 
 fourth and sixth observations,* we obtain: 
 
 X X 10 5 = 30,830 61 - 488,000 &i 2 - 53.4. 
 In the following table the experimental and calculated values 
 of X X 10 5 are compared: 
 
 TABLE XXXIII 
 
 61 
 
 XX1Q5 
 (experimental) 
 
 XX 105 
 (calculated) 
 
 0.01200 
 
 246.3 
 
 246.3 
 
 0.01296 
 
 266.9 
 
 264.2 
 
 0.01392 
 
 281.8 
 
 281.1 
 
 0.01488 
 
 297.5 
 
 297.5 
 
 0.01584 
 
 312.6 
 
 312.5 
 
 0.01776 
 
 340.4 
 
 340.4 
 
 Formula (vii) also applies to 1 per cent solutions of ovomucoid 
 in varying concentrations of KOH and HC1, only in the add 
 solutions we must replace &i by a i} the acidity of the solution 
 to which the ovomucoid is added. For 1 per cent ovomucoid 
 in KOH solutions, computing the constants by the method of 
 least squares, we obtain: 
 
 X X 10 5 = 20,850 61 - 1,250,000 6i 2 - 35.6. 
 
 In the following table the experimental and calculated values 
 of X X 10 5 are compared: 
 
 TABLE XXXIV 
 
 61 
 
 XX 105 
 (experimental) 
 
 XXlQs 
 (calculated) 
 
 0.0005 
 
 -26.8 
 
 -25.5 
 
 0.0010 
 
 -16.0 
 
 -16.0 
 
 0.0020 
 
 + 1.1 
 
 + 1.1 
 
 0.0030 
 
 +15.7 
 
 +15.7 
 
 0.0050 
 
 +38.2 
 
 +38.2 
 
 For 1 per cent ovomucoid in HC1 solutions, computing the 
 constants by the method of least squares, we obtain: 
 X X 10 5 = 41,990 ai - 852,700 ai 2 - 41.4. 
 
 * These results do not lend themselves to determination of the constants by 
 the method of least squares, since the normal equations are nearly identical, 
 the factors differing only by magnitudes commensurate with the experimental 
 error. 
 
PROPORTION OF INORGANIC RADICAL 
 
 247 
 
 In the following table the experimental and calculated values 
 of X X 10 5 are compared: 
 
 TABLE XXXV 
 
 fll 
 
 xxio 5 
 
 (experimental) 
 
 XX1Q8 
 (calculated) 
 
 0.0005 
 
 - 17.7 
 
 - 20.6 
 
 0.0010 
 
 + 2.0 
 
 - 0.3 
 
 0.0020 
 
 + 37.7 
 
 + 39.2 
 
 0.0030 
 
 + 74.5 
 
 + 76.9 
 
 0.0040 
 
 +111.1 
 
 +113.0 
 
 0.0050 
 
 +146.2 
 
 +147.2 
 
 0.0075 
 
 +228.9 
 
 +225.5 
 
 0.0100 
 
 +291.8 
 
 +293.2 
 
 The relation does not, however, hold good for solutions of 
 casein in HC1. Doubtless it would hold good for these solutions 
 also were it possible to obtain solutions of casein in dilute HC1, 
 without at the same time introducing the chloride of the base 
 employed to dissolve the dry casein. But, as I have explained 
 in Chap. V, dry casein is not readily dissolved by dilute acids, 
 and in order to secure a solution of casein in acid it is necessary 
 to first dissolve the casein in dilute alkali, and then add acid in 
 excess of that necessary to neutralize the base and sufficient 
 to redissolve the wet, freshly precipitated casein. Now casein, 
 dissolved in acids is very markedly influenced in its solubility 
 by the presence of salts; the departure of the conductivities of 
 these solutions, therefore, from the regularity indicated in equa- 
 tion (vii) is sufficiently explicable. 
 
 The physico-chemical significance of this relation is, at the 
 present stage of our knowledge, not at all clear. It is possible 
 that equation (vii) is merely an interpolation-equation and that 
 the relation is of purely empirical significance.* It is chiefly of 
 use, at present, as we shall see, in throwing light upon questions 
 
 * It should here be noted that any relationship of this type must involve 
 the mutual dependence of the following factors: 
 
 (i) The variation of m, the amount of acid or base bound by the protein 
 with variation in ai or 61, the concentration of acid or alkali in which 
 the protein is dissolved. 
 
 (ii) The variation of K (the dissociation-constant of the salt) and possibly 
 of (u + t>) with the quantity of acid or alkali bound by the protein, 
 (iii) The effect of dilution upon the conductivities of the protein salts. 
 
248 ELECTROCHEMISTRY 
 
 involving the minimal combining capacity of proteins, ' such as 
 casein, which are insoluble in the free condition. 
 
 4. The Solubility and Minimal Combining Capacity of 
 Casein and of Serum Globulin in Solutions of Bases. We have 
 seen that the relation between 61, the concentration of a KOH- 
 solution in which casein is dissolved, and X, the depression of 
 its conductivity which is caused by the casein, are connected 
 by the relation 
 
 X X 10* = 26,880 61 - 475 ' 80( V - 28.98 C; 
 
 o 
 
 putting X equal to zero we obtain 
 
 61 = 0.00114 C or 0.05536 C. 
 
 Considering, for the present, only the smaller value of 61, we 
 see that when X = 0, that is, when the change in the conduc- 
 tivity of an alkaline solution which is brought about by dissolving 
 a given percentage of casein is zero, then the proportion of alkali 
 to casein is such that one gram of casein is combined with 
 11.4 X 10~ 5 equivalents of alkali. This is precisely the combining 
 capacity of casein at "saturation" of the base with casein, that is, 
 when the base has dissolved the maximum amount of casein which 
 it will dissolve. 
 
 The exact coincidence of the two numerical values, especially 
 when we consider that the above is computed by least squares 
 from a number of determinations which are apparently not 
 connected with estimates of the solubility of casein, is surprising, 
 and leaves no room for doubt that the magnitude of the two 
 quantities is determined by identical factors. This result is 
 probably to be interpreted as follows: The conductivity (= Zi) 
 of the solution of base to which casein is added is /u&i, where 
 fj, is the equivalent molecular conductivity of the base and 61 
 the equivalent concentration of the base. The conductivity 
 (= x) of the solution containing casein is vb + 96.43 (u + 0) z, 
 where b is the equivalent concentration of the unneutralized 
 base, u + v is the sum of the equivalent mobilities of the ions 
 and z is the equivalent- concentration of protein ions. Assuming 
 that at the concentrations employed the casein salt is tolerably 
 completely dissociated z = pm, where m is the equivalent con- 
 centration of base neutralized by the protein and p the number 
 
SOLUBILITY AND COMBINING CAPACITY 249 
 
 of equivalents of protein salt to which one equivalent of base 
 gives rise. Hence: 
 
 X = xi x - i*m 96.43 p (u + v) m. 
 
 When m = 0, therefore, that is, when the quantity of base 
 is insufficient to combine with the given mass of casein, X must 
 be 0. Hence the value of 61 when X = is a true measure of the 
 minimum combining capacity of casein. 
 
 For 1.5 per cent solutions of casein in calcium hydrate solutions 
 we found that 
 
 X X 10- 5 = 30,8306! - 488,000 &i 2 - 53.4; 
 putting X = and estimating 61 we find that 
 
 61 = 0.001782; 
 
 dividing this by 1.5, the percentage of casein, we find that X = 
 when the equivalence between the casein and the calcium hydrate 
 
 18 1 gram = 11.9 X 10~ 5 equivalents; 
 
 a value so near that (11.4 X 10~ 5 ) obtained for the alkalies that 
 they may be regarded as being, within the experimental error, 
 identical. 
 
 In the previous (German) edition of this work I stated that 
 we were justified in the light of these results in tentatively 
 assuming, pending more direct evidence, that the various bases 
 dissolve casein in equivalent-molecular proportions. Since then 
 Van Slyke and Bosworth have shown (25), by direct analytical 
 determinations, employing dialysis to remove the chloride formed 
 on neutralizing the excess of base with hydrochloric acid, that 
 the minimal proportion of a diacid base which will dissolve casein 
 is about 23 X 10~ 5 equivalents but that an insoluble compound 
 is formed containing 11.3 equivalents of the base. In other 
 words the minimal equivalent combining capacity of casein for 
 bases is the same for diacid as monacid bases. 
 
 We have seen (1) that each COH.N bond of casein which 
 is opened up by the entrance of a KOH molecule reacts with 
 the base in accordance with the equation : 
 
 H 
 
 -COH.N- + KOH = COK + ^N- 
 
 I 
 OH 
 
250 ELECTROCHEMISTRY 
 
 while in reacting with diacid bases two COH.N groups are 
 involved, part of the valencies of the ions which are formed suffer- 
 ing internal neutralization thus: 
 
 -CO++ H 
 
 2 -COH.N- + Ca(OH) 2 = /Ca + ^N- 
 
 / I 
 
 -CO.N- OH 
 
 / \ 
 H OH 
 
 If this view be correct, then one molecule of calcium hydrate 
 must obviously bind twice the weight of casein that is bound 
 by one molecule of KOH. Hence di- and monacid bases unite 
 with casein in equivalent-molecular proportions, but at "satu- 
 ration" of the base with casein, when the mass of the protein 
 ions is at its maximum, the cations formed as a result of the 
 union with diacid bases, being much more bulky than those 
 formed by the union with monacid bases, fail to pass into solu- 
 tion. Hardy (2) has found that bases dissolve serum-globulin, 
 not in equivalent molecular but in molecular proportions. It is 
 probable that in this case also an insoluble salt is formed with 
 the minimal proportion of diacid base. 
 
 LITERATURE CITED 
 
 (1) Bredig, G., Zeit. f. physik. Chem. 13 (1894), p. 191. 
 
 (2) Hardy, W. B., Journ of Physiol. 33 (1905), p. 251. 
 
 (3) Kossel, A., Zeit. f. physiol. Chem. 25 (1898), p. 165. 
 
 (4) Kossel, A., and Dakin, H. D., Zeit. f. physiol. Chem. 41 (1904), p. 412. 
 
 (5) Osborne, W. A., Journ. of Physiol. 27 (1901), p. 389. 
 
 (6) Robertson, T. Brailsford, Journ. Biol. Chem. 2 (1907), p. 337. 
 
 (7) Robertson, T. Brailsford, Journ. physical Chem. 11 (1907), p. 437. 
 
 (8) Robertson, T. Brailsford, Journ. physical Chem. 11 (1907), p. 542. 
 
 (9) Robertson, T. Brailsford, Journ. Biol. Chem. 5 (1908), p. 147. 
 
 (10) Robertson, T. Brailsford, Journ. physical Chem. 12 (1908), p. 473. 
 
 (11) Robertson, T. Brailsford, Journ. physical Chem. 13 (1909), p. 469. 
 
 (12) Robertson, T. Brailsford, "The Proteins," Univ. of California, Publ. 
 
 Physiol. 3 (1909), p. 115. 
 
 (13) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 377. 
 
 (14) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 528. 
 
 (15) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 601. 
 
 (16) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 709. 
 
 (17) Robertson, T. Brailsford, Journ. physical Chem. 15 (1911), p. 166. 
 
LITERATURE CITED 251 
 
 (18) Robertson, T. Brailsford, "Die physikalische Chemie der Proteine," 
 
 Dresden (1912). 
 
 (19) Robertson, T. Brailsford, Journ. physical Chem. 16 (1912), p. 382. 
 
 (20) Robertson, T. Brailsford, and Burnett, T. C., Journ. Biol. Chem. 6 
 
 (1910), p/105. 
 
 (21) Sackur, O., Zeit. f. physik. Chem. 41 (1902), p. 672. 
 
 (22) Sutherland, W., Proc. Roy. Soc. London, 79 B (1907), p. 130. 
 
 (23) Sutherland, W., Phil. Mag. 6 Ser. 14 (1907), p. 1.. 
 
 (24) Taylor, A. E., Univ. of California Publ. Pathol. 1 (1904), p. 7. 
 
 (25) Van Slyke, L. L., and Bosworth, A. W., Journ. Biol. Chem. 1 (1913), 
 
 p. 211. 
 
CHAPTER XI 
 THE ELECTROCHEMISTRY OF COAGULATION 
 
 1. The Coagulation of the Casemates by Alcohol. If one- 
 half a cubic centimeter of a 0.0125 N solution of KOH, neutralized 
 either to phenolphthalein or to litmus by the addition of casein, 
 be added to 10 cc., i.e., to 20 volumes of 99.8 per cent alcohol, 
 no coagulation of the protein occurs, although the solution which 
 is thus obtained is appreciably more opalescent than a solution 
 of equal concentration in water containing no alcohol. Even 
 if, instead of employing a solution of potassium caseinate in water, 
 we employ a 0.0125 N solution of KOH in 75 per cent alcohol, 
 containing 1.6 or 2.5 per cent of casein, adding J cc. of this to 
 10 volumes of 99.8 per cent alcohol, still no coagulation of the 
 caseinate occurs, although it is now dissolved or forms a stable 
 suspension in a 98.6 per cent solution of alcohol. The caseinate 
 can, however, be readily coagulated by adding to this mixture 
 an equal volume of ether and allowing it to stand for a few hours. 
 
 Very different is the behavior of the caseinates of the alkaline 
 earths. If to 10 cc. each of 60, 70, 75 per cent, etc., solutions of 
 alcohol we add J cc. of a 0.012 N solution of Ca(OH) 2 , neutralized 
 to phenolphthalein by casein, distinct coagulation of the casein- 
 ates occurs, on shaking, when the final concentration of alcohol 
 in the mixture is about 55 per cent. The same is true for an 
 equally concentrated solution of barium hydroxide neutralized 
 to phenolphthalein by casein. For strontium caseinate the 
 limiting concentration of alcohol at which coagulation occurs is 
 much higher, about 70 per cent. In all cases coagulation is much 
 accelerated by energetic shaking of the mixture. 
 
 It is of interest to compare the marked difference between the 
 behavior of the caseinates of the alkalies and of the alkaline earths 
 upon the addition of alcohol to their aqueous solutions, with the 
 numerous other differences which subsist between the caseinates 
 of the alkalies and those of the alkaline earths. 
 
 Thus the aqueous solutions of the "neutral" or "basic" casein- 
 
 252 
 
COAGULATION BY ALCOHOL 253 
 
 ates of the alkalies and ammonium are clear, do not, except 
 lithium caseinate, show any increase in turbidity on warming, 
 and are not precipitated by the addition of finely divided in- 
 soluble substances or by passing through a clay filter; while 
 the aqueous solutions of the caseinates of the alkaline earths 
 are opalescent, show marked increase in turbidity on heating 
 their solutions to 35-45 C. which disappears on cooling, and are 
 precipitated from their solutions by the addition of finely divided 
 insoluble substances or by passage through a clay filter (4). 
 
 The " neutral" or " basic" casemates of the alkalies, upon 
 solution in water, depress its freezing point and the amount 
 of the depression is that of a solution of the same molecular 
 concentration as the alkaline solution which is employed as 
 solvent. The depression produced by calcium caseinate is much 
 less, being less than half as great (10). 
 
 Equally concentrated solutions of the hydroxides of the alka- 
 lies and ammonium dissolve casein at approximately the same 
 rate. Solutions of the hydroxides of the alkaline earths dissolve 
 casein much more slowly (8). The rate of the solution of casein 
 by solutions of the hydroxides of the alkalies is accelerated by 
 raising the temperature above 36 degrees; the rate of the solu- 
 tion of casein by solutions of calcium hydroxide is materially 
 diminished by a similar rise in temperature (6). 
 
 The caseinates of the alkaline earths are precipitated from their 
 solutions by the addition of small concentrations of the chloride 
 of the corresponding alkaline earth (7); the caseinates of the 
 alkalies are not precipitated by the addition of similar quan- 
 tities of the chlorides of the alkalies. 
 
 I have alluded to the fact that the limiting concentration of 
 alcohol at which strontium caseinate is coagulated is consider- 
 ably higher than that at which calcium and barium caseinates 
 are coagulated. This is curious because we should expect, as 
 a rule, to find the salts of strontium possessed of properties in- 
 termediate between those of calcium and barium. It is therefore 
 the more interesting to observe that this is not the only respect 
 in which the caseinates of strontium exhibit behavior differing 
 from that of the caseinates of calcium and barium and approach- 
 ing the behavior of the caseinates of the alkalies; for casein 
 dissolves much more rapidly in solutions of strontium hydroxide 
 than in solutions of other alkaline earth hydroxides (8); the 
 
254 ELECTROCHEMISTRY 
 
 solutions of strontium caseinate are less opalescent than equally 
 concentrated solutions of calcium or barium caseinate, and the 
 dissociation-constant of "basic" strontium caseinate is much 
 larger than those of barium or calcium caseinate, and is inter- 
 mediate in magnitude between these and the dissociation con- 
 stants of the " basic" caseinates of sodium and ammonium. 
 This, however, is not a general phenomenon where protein salts 
 of the alkaline earths are concerned, because the dissociation 
 constant for the strontium salt of "insoluble" serum globulin 
 is not very appreciably different from those of the calcium and 
 barium salts (Cf. previous chapter). 
 
 The caseinates of the alkalies and alkaline earths dissolved in 
 various concentrations of alcohol afford, therefore, very favor- 
 able material for the investigation of the intimate nature of the 
 process of coagulation, since different caseinates, in the presence 
 of the same quantity of alcohol, may be, to all appearance com- 
 pletely soluble, or completely coagulated, and as we shall see, a 
 comparison of the behavior of these different caseinates towards 
 the presence of varying percentages of alcohol in their solutions 
 leads to a very considerable insight into the chemical mechanics of 
 protein-coagulation by alcohol. 
 
 2. The Applicability of the Ostwald Dilution-Law to Solutions 
 of the Casemates in Alcohol-Water Mixtures. In the preceding 
 chapters it has been shown that the Ostwald dilution-law for a 
 binary electrolyte written in the form 
 
 1.037 X 10- 2 . 1.075 X 10- 4 2 ,., 
 
 m = / i \~ x H TTI i \r x (0 
 
 p (u + v) P K (u + v) 2 
 
 is applicable to many aqueous solutions of the salts which pro- 
 teins form with inorganic acids and bases, m being the equivalent 
 molecular concentration of the acid or base which is combined 
 with the protein, p the number of equivalents of protein salt 
 to which one equivalent of neutralized acid or base gives rise, 
 x the conductivity of the solution in reciprocal ohms per cubic 
 centimeter, K the dissociation constant of the salt and u + v 
 the sum of the equivalent specific migration-velocities of the 
 ions at infinite dilution. The same law applies to solutions of 
 potassium caseinate (80 X 10~ 5 equivalents KOH per gram) in 
 alcohol-water mixtures which contain up to and less than 60 
 per cent alcohol, as the following experimental results show (9). 
 
DILUTION-LAW 255 
 
 In five hundred cubic centimeters of N/ 10 KOH were dissolved 
 62.5 grams of casein, thus forming a solution of potassium casein- 
 ate, neutral to phenolphthalein. To 50 cc. portions of this 
 solution were added, respectively, 0, 20, 40, etc., cubic centi- 
 meters of alcohol, and the mixtures were diluted to 200 cc. with 
 water. Each of these solutions was then diluted, by the addition 
 of alcohol of the same concentration, to the desired concentra- 
 tions of casein. All alcohol percentages are percentages of 99.8 
 per cent alcohol by volume. The solutions in "75 per cent 
 alcohol" were made up by adding 99.8 per cent alcohol to 50 cc. 
 of the original solution of caseinate until the volume was 200 cc., 
 and diluting with a 75 per cent by volume solution of 99.8 per 
 cent alcohol. The conductivity determinations were made at 
 30 C. The conductivity of the distilled water (= 4 X lO" 6 ) 
 has been subtracted from each of the tabulated conductivities. 
 The results are given in Table I on the following page. 
 
 Applying the above equation to these results and computing 
 
 1.037 X 10- 2 , 1.075 X 10- 5 . 
 
 the constants -, : r and ^-, : ^ for each concen- 
 p (u + v) pK (u + v) z 
 
 tration of alcohol from all of the results by the method of least 
 squares, we obtain: 
 
 For the solutions containing per cent of alcohol: 
 
 m = 12 .41 x + 1666 x 2 . (ii) 
 
 For the solutions containing 10 per cent of alcohol: 
 
 m = 15.97 x + 3325 x 2 . (iii) 
 
 For the solutions containing 20 per cent of alcohol: 
 
 m = 21.14z + 5880z 2 . (iv) 
 
 For the solutions containing 30 per cent of alcohol: 
 
 m = 27.23 x + 10,858 x 2 . (v) 
 
 For the solutions containing 40 per cent of alcohol: 
 
 m = 34.63 x + 18,931 x\ (vi) 
 
 For the solutions containing 50 per cent of alcohol: 
 
 m = 42.91 x + 35,937z 2 . (vii) 
 
 For the solutions containing 60 per cent of alcohol: 
 
 m = 53.49 x + 78,335 x 2 . (viii) 
 
 For the solutions containing 75 per cent of alcohol : 
 
 m = 29.95 x + 1,219,800 x 9 . (ix) 
 
256 
 
 ELECTROCHEMISTRY 
 
 T= a 
 
 XXXXX 
 
 i-H O5 i I i I 
 
 10 co i i co 
 C<1 CO CO I-H 
 
 ddddd 
 
 -H a-i I 
 
 nil 
 
 1181 
 
 xxxxx 
 
 CO Oi O CO <N 
 
 O O CO T-H CO 
 
 dddbd 
 
 1 
 
 XXXXX 
 
 ^^ tO CO ^~^ CO 
 
 ddddd 
 
 XXXXX 
 
 O lO 00 T ( CO 
 
 ddddd 
 
 XXXXX 
 
 I-H l- CX3 Oi l> 
 
 ^ t^ co o> o 
 
 o *o co i H co 
 ddddd 
 
 XXXXX 
 
 O IO CO r-t CO 
 
 ddddd 
 
 XXXXX 
 
 ^D lO CO ^^ CO 
 
 ddddd 
 
 XXXXX 
 
 o 10 co I-H co 
 ddddd 
 
CONCENTRATION OF ALCOHOL 257 
 
 Inserting the observed values of x in equations (ii) to (ix) , we can 
 compute the "theoretical" values of m, that is, the equivalent 
 concentration of KOH neutralized by the casein which should, 
 provided the dilution-law holds good, correspond to the observed 
 conductivities. In Table II, on the following page, the observed 
 and calculated values of m are compared. 
 
 It is evident that the correspondence between the experimental 
 results and those which are indicated by the Ostwald dilution- 
 law for a binary electrolyte is all that could be desired for the 
 solutions containing from to 60 per cent of alcohol. For the 
 solutions containing 75 per cent of alcohol the divergences be- 
 tween theory and experiment are, however, considerably greater 
 than could be accounted for by experimental error and probably 
 indicate that the law does not hold good for these solutions. The 
 sudden alteration in the relative values of the constants which 
 occurs when the percentage of alcohol attains this magnitude 
 indicates, whether the Ostwald law be considered as holding 
 good or not, a profound change in the molecular and ionic con- 
 dition of the protein. 
 
 It would appear, however, that we are justified in concluding 
 that potassium caseinate dissociates in alcohol-water mixtures 
 containing from to 60 per cent of alcohol in the same manner 
 as it does in aqueous solutions. 
 
 3. The Influence of the Concentration of Alcohol in the Solvent 
 upon the Conductivities of Solutions of Potassium Caseinate. 
 I find (9) that the conductivity (= x v ) of a solution containing 
 any given concentration of casein combined with KOH in the 
 proportion requisite to secure neutrality to phenolphthalein, and 
 dissolved in an alcohol-water mixture containing y per cent of 
 alcohol by volume is connected with the conductivity (=z ) 
 of a similar solution in water by the formula 
 
 XQ 
 
 where A is a constant which varies but slightly, if at all, with the 
 concentration of the caseinate. This constant is determined in 
 the following manner: If the logarithms to base 10 of x corre- 
 sponding to y = 0, 10, 20, 30, 40, 50, and 60 per cent be tabulated 
 in that order and each successive value of logo; be subtracted 
 from the one lying immediately above it, the differences thus 
 
258 
 
 ELECTROCHEMISTRY 
 
 1 ++ 
 
 si 
 
 oco I-H co 
 
 (MCOCOT-H 
 
 xi 
 
 8*1 
 
 CD C^ OO T-H 
 co CO C<l T-I 
 
 w 
 
 "5 CO T-H CO 
 <M COCOT-i 
 
 TH 0$ TH C<1 TH 
 
 \ \ \++ 
 
 38S8TH 
 
 si 
 
 xl 
 
 rH r-H r ! CO r 1 
 
 4- 1 1 ++ 
 
 
 - 
 
 3 
 
 3 
 
 C<J 
 
 CO rH CO 
 
 COCO TH 
 
 >OCO i-H CO 
 (M COCO T-I 
 
 1 ++ 
 
 + 
 
 ;^s^2 
 
 O TH 1 ICO TH 
 
 1 1 ++ 
 
 >o co 
 
 OQ CO 
 
 si 
 
 OC^OCOrH 
 
 I ++ 
 
 SS5S^5 
 
 i I CO 
 
 ^OCO TH CO 
 1NCOCOTH 
 
CONCENTRATION OF ALCOHOL 
 
 259 
 
 computed are observed to be appreciably constant and equal 
 to ten times the log to base 10 of A in the above equation. The 
 average value of A for all of the determinations enumerated in 
 Table I (omitting, in the computation, the solutions containing 
 75 per cent alcohol) is 1.0265. For the various concentrations 
 of potassium caseinate the values of A are as follow: 
 
 TABLE III 
 
 Concentration of KOH 
 
 L neutralized by casein 
 
 A 
 
 0.0250 
 0.0125 
 0.0063 
 0.0031 
 0.0016 
 
 1.0279 
 1.0270 
 1.0265 
 1.0252 
 1.0257 
 
 Average 1.0265 
 
 In the accompanying table (Table IV) the experimental values 
 of x and those computed from the formula 
 
 XQ A" 
 
 are compared. Two "calculated" values of x y are given, the 
 first calculated value of x y being computed from the average 
 value of A for the given concentration of caseinate, the second 
 being computed from the average value of A for all the deter- 
 minations enumerated in Table I, with the omission, of course, 
 of those for the solvent containing 75 per cent of alcohol. 
 
 The same relation also holds good for solutions of " neutral" 
 potassium caseinate, that is, for solutions which contain 50 X 10~ 5 
 equivalents of base per gram and are neutral to litmus instead 
 of ; as in the experiments cited above, 80 X 10~ 5 equivalents of 
 base per gram (neutral to phenolphthalein). Two and a half 
 per cent solutions of casein in 0.0125 N KOH were prepared, 
 containing 0, 10, 20, 50, and 75 per cent of alcohol respectively 
 The relation as we shall see, does not hold good for the solution 
 containing 75 per cent alcohol and so, in computing the value 
 of A for these solutions, we must omit the determination for the 
 solution containing 75 per cent alcohol. The average difference 
 in logio x y for 10 per cent increase in y is computed by adding 
 
260 
 
 ELECTROCHEMISTRY 
 
 II 
 
 9.J3 
 
 & . 
 
 ecooocoiococcc^i 
 
 E a 
 O'S 
 
 MI 
 
 
 s 
 
 P 
 
 B* 
 
 X| 
 
 coooecoooo^o 
 
 "50iOrH 
 
 iO O >O C 
 
 x 
 
 xi 
 
 x 
 
CONCENTRATION OF ALCOHOL 
 
 261 
 
 together the three observed differences (between x v corresponding 
 to y = and 10 per cent; 10 and 20 per cent; 20 and 50 per 
 cent) and dividing their sum by 5. In this way A was ascer- 
 tained to possess, for these solutions (at 30 degrees) the average 
 value 1.0248. In the following table the actual and computed 
 values of x v are compared : 
 
 TABLE V. CONCENTRATION OF KOH NEUTRALIZED BY 
 CASEIN 0.0125 N 
 
 V 
 
 ZyXlO 8 
 
 (observed) 
 
 z-XlO* 
 (calculated) 
 
 
 
 87 
 
 87 
 
 10 
 
 66 
 
 68 
 
 20 
 
 51 
 
 53 
 
 50 
 
 25 
 
 25 
 
 75 
 
 8 
 
 14 
 
 It is evident that the law holds good for solutions containing 
 from to 60 per cent of alcohol inclusive, but that it does not 
 hold good for solutions containing 75 per cent alcohol, since for 
 these solutions the calculated and observed values of x v are 
 widely divergent. Moreover the law does not hold good for 
 solutions of the caseinates of the alkaline earths in alcohol-water 
 mixtures; this is evidenced by the lack of constancy in the first 
 differences of logic z corresponding to equal increments of y. In 
 the accompanying table are given the values of logio # correspond- 
 ing to various values of y for solutions of calcium caseinate at 
 30 degrees, containing 1.5 per cent of casein combined with 
 80 X 10~ 6 equivalents of base per gram. 
 
 TABLE VI 
 
 y = per cent of alco- 
 hol by volume 
 
 log, * X 10 
 
 Differences 
 
 
 10 
 20 
 30 
 40 
 50 
 
 1.24797 
 0.87506 
 0.56820 
 0.34242 
 0.20412 
 0.07918 
 
 0.37291 
 0.30686 
 0.22578 
 0.13830 
 0.12494 
 
 In the following table are given the corresponding data for solu- 
 tions of strontium caseinate at 30 degrees, containing 1.5 per 
 
262 
 
 ELECTROCHEMISTRY 
 
 cent of casein combined with 80 X 10~ 5 equivalents of base per 
 gram i 
 
 TABLE VII 
 
 y = per cent of alco- 
 hol by volume 
 
 logio x X 108 
 
 Differences 
 
 
 10 
 20 
 30 
 40 
 50 
 
 1.39967 
 1.09342 
 0.79934 
 0.62325 
 0.50515 
 0.36173 
 
 0.30625 
 0.29408 
 0.17609 
 0.11810 
 0.14342 
 
 It is perhaps not without significance that in the above table 
 the first two differences are much more alike than the succeeding 
 differences. In solutions containing to 20 per cent of alcohol, 
 strontium caseinate is further from coagulation than correspond- 
 ing solutions of calcium caseinate and, as we have seen, A, in 
 
 >y 
 
 the equation x y = -j-, approaches constancy. Having regard to 
 
 the observed failure of the solutions of potassium caseinate in 
 75 per cent alcohol to conform to the law, and to the fact that 
 at an alcohol-concentration lying between 60 and 75 per cent, 
 solutions of potassium caseinate undergo a great and relatively 
 sudden increase in opacity, we may, I think, conclude that the 
 coagulation of a caseinate by alcohol is heralded by a failure of the 
 
 solution to conform to the law x y = ~^, connecting the percentage of 
 alcohol in the solution with its conductivity. 
 
 4. The Interpretation of the Law x v = ^ . As we have 
 
 observed (Table III) the value of A is not only constant for all 
 dilutions of potassium caseinate, but also is very nearly constant 
 for all of the proportions of alcohol to water employed as solvents. 
 Provided A were rigorously constant we would obviously obtain 
 the relation: 
 
 Sale. H 2 O 
 
 constant 
 
 for any given proportion of alcohol to water. This is the rela- 
 tion, suggested by Cohen (2), connecting the conductivities of 
 inorganic salts in mixtures of alcohol and water. Roth (12), 
 however, found that this rule does not strictly hold good for 
 
INTERPRETATION 263 
 
 solutions of KC1 in mixtures of alcohol and water, but that the 
 
 quotient decreases somewhat with increasing dilution. 
 
 Zalc.H 8 
 
 In the accompanying table (page 264) the values of 2^ are 
 
 Zalc. H 2 
 
 given for solutions of potassium caseinate (containing 80 X 10~ 5 
 equivalents of base per gram, Cf. Table I) containing each of the 
 proportions of alcohol to H 2 O employed. 
 
 It will be observed that the values of -^- are appreciably 
 
 Zalc. H,0 
 
 constant, for each proportion of alcohol to water, for the solu- 
 tions containing to 60 per cent of alcohol, but that in the solu- 
 tions containing 75 per cent of alcohol the value of this ratio 
 does not even approximate to constancy but decreases rapidly. 
 There is, it is true, a slight but regular diminution in the value 
 of this ratio even in the solutions containing to 60 per cent of 
 alcohol, in passing from an equivalent concentration of neutral- 
 ized KOH of 0.0250 to 0.0016, but, except in the solutions con- 
 taining 75 per cent of alcohol, the diminution is not more than 
 a few per cent. 
 
 We have seen (equations (ii) to (ix)) that the Ostwald dilution- 
 law, in the form expressed in equation (i) holds good, at any 
 rate for solutions of potassium caseinate in alcohol-water mix- 
 tures containing to 60 per cent of alcohol. 
 
 From the symmetry of the equation: 
 
 1.037 X lO- 2 . 1.075 X 10~ 4 
 
 m - 7 - i - \ x i -- TT~i - i - \T~ x t 
 
 p(u-\-v) P K (u + t?) 2 
 
 it would follow, were A s'trictly constant for all dilutions, that 
 the proportions of alcohol to water employed as solvent for the 
 caseinate affects only u + v, i.e., the migration-velocities of the 
 ions, and not the dissociation-constant (= K) or the number of 
 equivalents of caseinate resulting from the neutralization of one 
 molecule of KOH (= p).* Since the alteration in A with dilu- 
 tion of the potassium caseinate is so small for solvents containing 
 
 * For, since x appears in one term of the equation as the first power, and 
 
 in the second as the square, if the ratio of _ i s constant any other 
 
 x alc. H,O 
 
 factor in the equation which is affected by the presence of alcohol must also 
 appear in the one term of the equation as the first power and in the other as 
 the square. 
 
264 
 
 ELECTROCHEMISTRY 
 
 ncen 
 raliz 
 in 
 
 a 
 ^W 
 
 I 
 
 bo'do'd 
 
 ft 
 
 gM 
 
 I 
 
 J* 
 
 o icco i i co 
 ddodd 
 
 CO >-H i i O OS 
 CO CO CO CO C3 
 
 OjOCOj-HCO 
 iO (N CO CO i i 
 CO T-H O O O 
 
 ddddd 
 
 a 
 SW>> 
 
 111 
 
 ill 
 
 32S 
 
 O I s - Oi 00 O 
 
 I>T^ T-H OiOi 
 
 CQ OS GO D CO 
 
 o *o co I-H co 
 
 00000 
 
 O -^ Tt< rt< rj< 
 
 oo'ooo 
 
 E|l 
 
 IS s 
 
 co co co co co 
 
 SSS88 
 
 ddddd 
 
 ddddd 
 
INTERPRETATION 
 
 265 
 
 to 60 per cent of alcohol, we may conclude that in these solu- 
 tions it is for the greater part the migration-velocity of the 
 caseinate ions which is affected by the percentage of alcohol 
 in the solvent. From the values of the constants in equations 
 (iii) to (ix) it is apparent that the migration-velocities of the 
 casein-ions are progressively diminished by increasing alcohol- 
 concentration from to 60 per cent. From the facts that the 
 
 law x v = ~ no longer holds good for the solutions containing 
 
 Ay 
 
 75 per cent alcohol and the ratio ^*_ i s no t constant we may 
 
 Zalc.H,O 
 
 conclude that in these solutions the degree of dissociation of the 
 caseinate or the number of equivalents of casein produced by 
 one equivalent of KOH or both undergo a profound modification. 
 The values of the constants in equation (x) indicate an increase 
 in the magnitude of u + v and a very great decrease in the mag- 
 nitude of the dissociation-constant. In the following table the 
 
 Tr 
 
 values of p (u + v) and of corresponding to the equations (iii) 
 
 $ P 
 
 to (x) are enumerated; in the fourth column is given the per- 
 centage dissociation of the caseinate in the solutions containing 
 0.025 N KOH bound by casein (at 30 degrees). 
 
 TABLE IX 
 
 Potassium Caseinate. 80X10" 5 Equivalents of KOH per gram 
 
 Per cent of alcohol 
 by volume 
 
 "+< 
 
 K 
 
 p 
 
 Per cent of caseinate 
 dissociated in solutions 
 containing 0.025 equiv- 
 alent of potassium 
 
 
 10 
 20 
 30 
 40 
 50 
 60 
 75 
 
 83.6X10- 6 
 64.9XHT 6 , 
 49.1X10-* 
 38.0X10" 6 
 29.9X10- 6 
 24.2XHT 6 
 19.4X10- 6 
 34.6X10- 8 
 
 0.0923 
 0.0767 
 0.0760 
 0.0682 
 0.0633 
 0.0513 
 0.0365 
 0.0007 
 
 82 
 80 
 79 
 78 
 77 
 74 
 68 
 17 
 
 The degree of dissociation of the caseinate in the solutions con- 
 taining 60 per cent and less than 60 per cent of alcohol is evidently 
 but little affected by the percentage of alcohol in the solvent, the major 
 part of the effect of the alcohol upon the conductivity being attri- 
 butable to the decreased mobility of the caseinate ions. 
 
266 ELECTROCHEMISTRY 
 
 5. The Viscosities of Solutions of Potassium Caseinate in 
 Alcohol-Water Mixtures. The striking resemblance between 
 
 the formula = -j- and the Arrhenius-Euler formula, = a n , 
 
 XQ A. y TJQ 
 
 for the dependence of the viscosity of a solution upon its con- 
 centration (1), where t\ n is the viscosity of the solution, 770 that of 
 the solvent, n the concentration of the solution and a is constant, 
 forcibly suggests that the decrease in the conductivity of potas- 
 sium caseinate solutions due to the addition of alcohol between 
 alcohol-percentages of to 60 may be attributable for the greater 
 part to the hampering of the protein ions by the increased in- 
 ternal friction of the solvent.* 
 
 Accordingly the following determinations were made: 
 
 31.25 grams of casein were dissolved in 250 cc. of AT/10 KOH. 
 To 25 cc. portions of this were added 0, 10, 20, etc. cc. of alcohol, 
 the volume of each mixture being made up to 100 cc., the solution 
 containing 75 per cent alcohol being made up by adding 99.8 per 
 cent alcohol until the volume of the mixture was 100 cc. 
 
 A series of exactly similar solutions were made up in which, 
 however, 25 cc. of N/1Q KOH were employed instead of 25 cc. 
 of caseinate solution. 
 
 The viscosities of these solutions were determined at 30 degrees 
 in an Ostwald viscometer (5), for which the time of outflow, for 
 water, was 90 seconds. The times of flow were read with a stop 
 watch. The viscosities were calculated from the formula 
 
 = - , where rjo is the viscosity of distilled water, t\ that of the 
 
 solution under investigation, s and t are the density and the 
 time of outflow, respectively, of distilled water and S and t are 
 the density and time of outflow, respectively, of the solution. 
 The densities of the solutions were determined by means of a 
 normal hydrometer, reading the density to within 0.002. 
 
 Taking the viscosity of water at 30 degrees to be 0.00798 
 dyne per cubic centimeter (13) and its density as 0.996, the 
 following were the results obtained : 
 
 * In this connection the work of H. C. Jones and collaborators on the con- 
 ductivities of solutions of inorganic salts in alcohol-water and acetone-water 
 mixtures should be consulted (3). Cf. also Walden (14). 
 
VISCOSITY 
 
 267 
 
 TABLE X 
 
 Concentration of KOH neutralized to phenolphthalein by 
 Casein =0.0250 N 
 
 Per cent of alcohol 
 by volume 
 
 H in dynes per cc. for 
 solvent 
 
 H in dynes per cc. for 
 solution 
 
 Difference due to 
 caseinate 
 
 
 
 0.00816 
 
 0.01668 
 
 0.00852 
 
 10 
 
 0.01065 
 
 0.02049 
 
 0.00984 
 
 20 
 
 0.01376 
 
 0.02484 
 
 0.01108 
 
 30 
 
 0.01694 
 
 0.02814 
 
 0.01120 
 
 40 
 
 0.01913 
 
 0.03237 
 
 0.01324 
 
 50 
 
 0.02064 
 
 0.03400 
 
 0.01336 
 
 60 
 
 0.02011 
 
 0.03200 
 
 0.01189 
 
 75 
 
 0.01735 
 
 0.02345 
 
 0.00610 
 
 Walden (loc. cit.) found that for solutions of tetraethylammo- 
 nium iodide in thirty organic solvents the product of the molec- 
 ular conductivity of the salt at infinite dilution ( = u + v or, 
 the sum of the migration-velocities of its ions) and the viscosity 
 of its infinitely dilute solution is nearly constant, indicating an 
 inverse proportionality between the viscosity of the solution 
 (or what comes to the same thing at infinite dilution, the solvent) 
 and the migration-velocities of the ions which it contains. In 
 the accompanying table are given the values of p (u + v) X f? sol- 
 vent, for the various solutions investigated. 
 
 TABLE XI 
 
 Per cent of alcohol 
 
 p ( + f).i7 solvent 
 
 Per cent of alcohol 
 
 p (u + v).t) solvent 
 
 
 10 
 20 
 30 
 
 0.68X10- 5 
 0.69X10- 5 
 0.68X10- 5 
 0.64X10- 5 
 
 40 
 50 
 60 
 75 
 
 0.57X10- 5 
 0.50X10- 6 
 0.39X10- 6 
 0.60X10- 5 
 
 It is evident that the product of the ionic mobility of potas- 
 sium caseinate ions at infinite dilution and the viscosity of the 
 solvent varies very much less than either of these quantities 
 taken alone, indicating in a qualitative sense at all events, that 
 the ionic mobilities of the caseinate ions are in the main determined 
 by the viscosity of the solvent. The observed increase in the ionic 
 mobility of the caseinate ions on increasing the alcohol content 
 of the solvent from 60 to 75 per cent, in view of the fact that the 
 viscosity of the solvents passes through a maximum at from 50 
 
268 ELECTROCHEMISTRY 
 
 to 60 per cent alcohol and rapidly decreases between 60 and 
 75 per cent alcohol, is especially confirmatory of this view. 
 
 It will be noted that the viscosity of the solvent is notably 
 increased by the addition of the concentrations of casein em- 
 ployed, and this effect, of course, must vary very considerably 
 with the dilution; yet the Ostwald law holds good, and despite the 
 fact that the dissolved substance itself very materially affects the 
 viscosity of the solutions, it is the viscosity of the solvent alone which 
 determines the mobility of the protein ions. We can only conclude 
 from this that that portion of the viscosity of the solution which 
 is attributable to the caseinate itself does not in any appreciable 
 degree interfere with the mobility of the protein ions. The signif- 
 icance of this fact will be more fully discussed in Chap. XIII. 
 
 6. The Molecular Condition of Potassium Caseinate in 75 
 Per Cent Alcohol. We have seen that the behavior, optical,* 
 and electrical, of potassium caseinate dissolved in alcohol-water 
 mixtures, undergoes an abrupt change in passing from 60 to 
 75 per cent alcohol-content. The degree of dissociation, which 
 is but little affected by lower concentrations of alcohol, under- 
 goes a profound diminution in 75 per cent alcohol, and the 
 opacity of the solution undergoes a concurrent increase. The 
 phenomena suggest the possibility that this concentration of 
 alcohol leads not only to the formation of anhydrides of the 
 protein and therefore, as explained in Chaps. I and VI, to a dimi- 
 nution of the degree of dissociation, but also to a polymerization 
 of the protein, as outlined in equations (i) to (iv) in Chap. VI. 
 This polymerization might, very conceivably, be accompanied 
 by an increase in the weight of casein involved in the transport 
 of one atomic charge. Accordingly, experiments were under- 
 taken with a view to ascertaining the effect of increasing alcohol- 
 content of the solvent upon the electrochemical equivalent of 
 casein. 
 
 The experiments were carried out in the manner described 
 in Chap. VIII, upon solutions containing 50 and 75 per cent of 
 alcohol, made up in the manner described above. The altera- 
 tions in the percentage casein-content of the solutions due to 
 deposition of the casein upon the anode, was estimated from the 
 
 * Not only is the opalescence of the solution greatly increased but also the 
 change in the refractive index of the solvent due to the introduction of one 
 gram of casein per 100 c.c. is considerably diminished (Cf. Chap. XIV). 
 
MOLECULAR CONDITION 
 
 269 
 
 change in the refractive indices of the solutions, the proportion 
 between the percentage of casein in these solvents and the change 
 in the refractive index of the solvent, due to the protein, having 
 been previously determined (Cf. Chap. XIV). The volume of the 
 solution employed being always 25 cc., the actual amount of 
 casein deposited by the current was obtained by dividing the 
 alteration in percentage by 4. 
 
 The results obtained are tabulated below, the figures in the 
 6th column being obtained from those in the 5th column through 
 multiplication by the Faraday constant. 
 
 TABLE XII* 
 
 50 X 10-* Equivalents of KOH per Gram of Casein. Concentration of 
 KOH neutralized by Casein = 0.015 N 
 
 Per 
 cent of 
 alcohol 
 
 Current in 
 amperes 
 
 Time of passing 
 
 Grams casein 
 lost from solution 
 
 Electrochemical 
 equivalent in 
 grams per cou- 
 lomb 
 
 Grams of casein 
 carrying one ionic 
 charge 
 
 
 
 
 
 
 
 2336 183 
 
 50 
 75 
 
 6.88 x 10- 
 1.94 x!0- 
 
 3 hrs. 25 min. 
 4 hrs. min. 
 
 0.2550 0.0170 
 0.1260 0.0200 
 
 0.0301 0.0020. 
 0.0452 0.0072 
 
 2906 193 
 4363 695 
 
 * The weight of casein, in grams, which carries one atomic charge, in aqueous solution, is cited 
 after the determinations enumerated in Chap. VIII. The amount of casein lost from the anode 
 through resolution in electrolysis is not allowed for in the above estimates. In solutions con- 
 taining alcohol it is probably very small, since the rate of solution of casein in alkaline solvents 
 is much diminished by alcohol. 
 
 The smallness of the current employed in the electrolysis of the solution containing 75 per cent 
 alcohol was due to the high resistance of the caseinate solution. The precipitate from the solution 
 containing alcohol is slimy and not spongy as it is from aqueous solution. Nor, in 75 per cent 
 alcohol, does it adhere well to the anode so that precipitation appears to take place in the body of 
 the anodal portion of the fluid, and the solution had to be filtered to remove the casein precipitated 
 by the electrolysis. 
 
 There is evidently a very marked increase in the weight, or 
 decrease in the valency of casein ions in solution of potassium 
 caseinate when the added alcohol attains 75 per cent, leading 
 to a doubling of the weight of casein required to transport one 
 atomic charge of electricity. 
 
 From these facts and from the failure of Ostwald's dilution- 
 law to adequately represent the behavior towards dilution of 
 solutions of potassium caseinate in 75 per cent alcohol, and the 
 opalescence of these solutions, it appears probable that they par- 
 take rather of the character of suspensions than of true solutions, 
 and that the transport of electricity by the caseinate in 75 per 
 
270 ELECTROCHEMISTRY 
 
 cent alcohol is a phenomenon of "electric endosmose" rather than 
 of true electrolytic conduction through the migration of ions. 
 
 7. The Chemical Mechanics of the Coagulation of Proteins 
 by Alcohol. We have seen that when potassium caseinate is 
 dissolved in varying concentrations of alcohol, the opalescence of 
 the solution undergoes an abrupt increase in the neighborhood of 
 70 per cent, indicating the beginning of coagulation, which, 
 however, does not proceed far enough, even on further addition 
 of alcohol, to lead to the actual separation of flocculi. We have 
 also seen that for solutions containing 60 per cent and less of 
 
 alcohol the law = constant holds good and that this 
 
 Sale. H 2 
 
 implies that almost the sole effect of the alcohol in these con- 
 centrations is to modify the ionic mobility of the casein ions, 
 leaving the degree of dissociation of the caseinate comparatively 
 unaffected. In solutions containing 75 per cent of alcohol, 
 however, the degree of dissociation of the caseinate is profoundly 
 
 diminished and, consequently, the law - = constant no 
 
 Sale. H 2 O 
 
 longer holds' good. On the basis of the hypothesis and experi- 
 mental data outlined in preceding chapters this phenomenon 
 can readily be interpreted. We have seen that the protein 
 salts dissociate, not at terminal NH 2 or COOH groups but 
 at COH.N groups within the molecule. If, however, the 
 terminal NH 2 and COOH groups of the molecule are bound 
 together, as they are in anhydrides of the type HN.RCOH.NR.CO, 
 
 | _ I 
 
 then this dissociation can no longer occur. We have already 
 (Chap. VI) seen strong reason for concluding that the coagula- 
 tion of proteins is accomplished by dehydration of the protein; 
 the electrochemical behavior of caseinates dissolved in alcohol- 
 water mixture is clearly in harmony with this view. 
 
 As explained in Chap. VI, section 6, equations (i) to (iv), how- 
 ever, not merely "internal neutralization" of the protein molecule 
 may result from the dehydration of its terminal NH2 and 
 COOH groups, but also polymerization. The high electro- 
 chemical equivalent of potassium caseinate, dissolved in 75 per 
 cent alcohol, and the opacity of its solution favor the view that 
 this phenomenon also occurs in these solutions. 
 
 From the effects of alcohol upon the rate of solution of casein 
 
CHEMICAL MECHANICS OF COAGULATION 271 
 
 by alkalies Robertson and Miyake (11) have also been led to 
 infer that alcohol induces polymerization of the casein molecules 
 even in solutions of sodium caseinate in 20 to 30 per cent alcohol. 
 From the above results, however, we must infer that if in such 
 solutions the mass of the casein ions is doubled their valency 
 must also be doubled, for otherwise p would be halved and the 
 
 observed constancy of the ratio XH * in these solutions would 
 
 #H,O.alc. 
 
 not be obtained. 
 
 Incidentally, the profound diminution in the degree of disso- 
 ciation of the caseinate which results when the alcohol-concen- 
 tration attains a certain value explains, independently of any 
 theory as to the mode of dissociation of the protein salts in solution, 
 the observed fact that alcohol precipitates protein salts as such 
 from their solutions (Cf. Chap. IV, 2) and not the uncombined 
 proteins, since, previously to their precipitation, the combined 
 base or acid is bound up in an undissociated molecule. 
 
 LITERATURE CITED 
 
 (1) Arrhenius, S., Zeit. f. physik. Chem. 1 (1887), p. 285. 
 
 (2) Cohen, E., Zeit. f. physik. Chem. 25 (1898), p. 31. 
 
 (3) Jones, H. C., and Collaborators, Amer. Chem. Journ. 28 (1902), p. 
 
 329; 32 (1904), p. 521; 34 (1905), p. 481; 36 (1906), pp. 325 and 
 427; 37 (1907), p. 405; 41 (1909), p. 433; 42 (1909), p. 37. Zeit. f. 
 physik. Chem. 61 (1908), p. 641; 62 (1908), p. 41, 
 
 (4) Osborne, W. A., Journ. Physiol. 27 (1901), p. 398. 
 
 (5) Ostwald, W., and Luther, R., Phys.-chem. Messungen, 2 Aufl. p. 260. 
 
 (6) Robertson, T. Brailsford, Journ. Biol. Chem. 5 (1908), p. 147. 
 
 (7) Robertson, T. Brailsford, Journ. physical Chem. 13 (1909), p. 469. 
 
 (8) Robertson, T. Brailsford, Journ. physical Chem. 14 (1910), p. 377. 
 
 (9) Robertson, T. Brailsford, Journ. physical Chem. 15 (1911), p. 387. 
 
 (10) Robertson, T. Brailsford, and Burnett, T. C., Journ. Biol. Chem. 6 
 
 (1909), p. 105. 
 
 (11) Robertson, T. Brailsford, and Miyake, K, Journ. Biol. Chem. 26 (1916), 
 
 p. 129. 
 
 (12) Roth, W. A., Zeit. f. physik. Chem. 42 (1903), p. 209. 
 
 (13) Thorpe, T. E., and Rodger, J. W., Phil. Trans. Roy. Soc. London, 
 
 185A (1894), p. 397. 
 
 (14) Walden, P., Zeit. f. physik. Chem. 55 (1906), p. 209. 
 
PART III 
 THE PHYSICAL PROPERTIES OF PROTEIN SYSTEMS 
 
CHAPTER XII 
 
 THE PHENOMENA WHICH ACCOMPANY CHANGES IN THE 
 STATE OF AGGREGATION OF PROTEINS 
 
 1. The Passage of Dry Protein into Solution. The question 
 whether or not the proteins as a class possess a definite solubility 
 in water is one which has not so far admitted of any satisfactory 
 solution. True, certain sparingly soluble salts of the proteins 
 undoubtedly are soluble only to a definite extent in water, as 
 for example the researches of Galeotti, cited in Chap. VI, reveal; 
 when this limit of solubility is overstepped the protein salt is 
 precipitated; then other forms of protein, coagulated proteins, 
 " denatured" proteins, and so forth are definitely insoluble in 
 water under the conditions which accompany their formation. But 
 the very proteins which display these phenomena under certain 
 conditions, under somewhat different conditions, either in the 
 form of free protein or of a salt, appear to be indefinitely soluble 
 in water; miscible with it in all proportions. The phenomena 
 of " solution," chemical combination with water, and of the 
 hygroscopic, possibly purely physical, retention of water, become, 
 in these cases and so far as our knowledge at present extends, 
 inextricably confused. 
 
 On the whole it appears very probable that such proteins and 
 protein salts as gelatin and the caseinates are not only miscible 
 with water in all proportions, but also capable of entering into 
 chemical combination with water in many different proportions. 
 To this view Pauli (72), in contradistinction to Hardy (34) (35), 
 also inclines. 
 
 Base- and acid-free casein is definitely insoluble in distilled 
 water; but in water containing a base its solubility would appear 
 to be limited only by the quantity of base which the water con- 
 tains and not by the volume of water. The casein continues 
 to dissolve until, as explained in Chaps. V and X, 11.4 X 10~ 5 
 equivalents of base are combined with each gram of casein and 
 solution of the casein stops merely because the power of the base 
 to neutralize casein is exhausted, and not because the solvent 
 
 275 
 
276 PHYSICAL PROPERTIES 
 
 will hold no more of the caseinate. Addition of alkali to the 
 mixture results in the passage of more casein into solution; 
 addition of acid in the throwing of uncombined casein out of 
 solution. Apparently casemates, of the alkalies at all events, 
 are indefinitely soluble in water, although in practice we are 
 limited in the preparation of strong solutions, not only of the 
 caseinates but of other proteins, by the viscous and glairy nature 
 of such solutions, and the consequent difficulty of ensuring their 
 homogeneous character. Corresponding to this fact we find 
 that it is very difficult to prepare the caseinates of the alkalies 
 in the form of dry powders. I have precipitated lithium casein- 
 ate from 90 per cent alcohol by the addition of ether and en- 
 deavored to dry this product by washing with ether and standing 
 over sulphuric acid at 30 degrees. The result is merely a soft 
 gelatinous mass; on prolonged standing over sulphuric acid 
 (a matter of weeks) it becomes horny; at no definite time can 
 we state that the product has become anhydrous; it simply 
 exhibits a continual change in consistency with the continual 
 withdrawal of water, just as gelatin does. On the other hand, 
 proteins which are insoluble in water and certain other proteins 
 and protein salts are readily rendered anhydrous by treatment 
 with alcohol and can be prepared through this means in the 
 form of dry powders (Cf. Chap. II). The affinity of a pro- 
 tein for water, therefore, is a function which varies, not only 
 with the character of the protein, but with the character of any 
 base or acid which may be combined with it. In all cases it 
 would appear to be of considerable magnitude, however, the 
 difficulty which histologists experience in thoroughly dehy- 
 drating tissues, for example, being eloquent testimony of this 
 fact. 
 
 It has been shown by Noyes and Whitney (61) (10) that under 
 conditions involving no chemical interaction (except the pos- 
 sible formation of "solvates" (Cf. Chap. VI)), the rate of solution 
 of a crystalloid in water is at each moment proportional to the 
 difference between its concentration at that moment and its 
 concentration at saturation of the solvent. They conclude from 
 this that at the boundary of the crystals and the solution there 
 exists a film of solution, which is always saturated and, for a 
 constant rate of stirring, of constant thickness. The velocity 
 of solution is thus determined by the rate of diffusion of the 
 
PASSAGE INTO SOLUTION 277 
 
 dissolved crystalloid out of this film into the body of the fluid. 
 This leads to the equation: 
 
 log -^_ = S A*, (i) 
 
 where a is the concentration of a saturated solution of the crys- 
 talloid, x the concentration of the solution at time t, s the area 
 of the surface of the crystalloid which is exposed to the action 
 of the solvent and k a constant which varies with the rate of 
 stirring and with the rate of diffusion of the crystalloid (11), 
 and, consequently, with the temperature. 
 
 On the other hand, Boguski (4) (5) and Veley (104) (105) 
 have shown that the rate of solution of basic substances such 
 as marble or zinc in acids, a process involving chemical inter- 
 action, is proportional at each instant to the concentration of 
 the unneutralized acid and to the surface of the solid. Upon 
 the supposition that the surface remains appreciably constant 
 in area during the period occupied by the experiment (a readily 
 realizable condition) this leads to the equation : 
 
 log -r^-- = fat, (ii) 
 
 Cl ~~ */ 
 
 which is identical with the above, save that a is, in this case, 
 the initial concentration of the acid. In this case also Nernst 
 believes (60) the velocity of solution is determined by a diffusion- 
 velocity, namely the velocity with which the acid diffuses into 
 the film in contact with the solid. Naturally, were the velocity 
 of the chemical interaction low in comparison with the velocity 
 of diffusion of the acid (or any reagent playing a similar part) 
 the determining velocity would be a chemical and not a diffusion 
 velocity. An examination of this case in the light of the mass 
 law shows, however, that the form of the relation between time 
 and mass dissolved would be the same as that expressed in 
 equation (ii).* 
 
 In the light of what has been said concerning the indefinite 
 solubility of the caseinates in water, it might be anticipated 
 that equation (i) would not apply to the dissolving of solid casein 
 in alkaline water. It is quite imaginable, however, that equa- 
 tion (ii) might apply, a, in that equation, being the number of 
 
 * Provided only one molecule of acid (or other dissolved reagent) took part 
 in the reaction. 
 
278 PHYSICAL PROPERTIES 
 
 equivalents of base in the water employed to dissolve the casein. 
 The experimental fact is, however, that neither of these equations 
 expresses the relationship between the time for which a mixture 
 of solid casein and alkaline water is stirred and the mass of 
 casein dissolved (86). . 
 
 In the experiments about to be described the casein which 
 was employed was especially prepared so as to readily sink in 
 water (Cf. Chap. II, 2), that is, it was not absolutely anhydrous 
 but contained a trace of water insufficient to disturb the accu- 
 racy of the results, but very material to the success of the ex- 
 periments. Since the presence and amount of associated traces 
 of water play such an important part in determining the readi- 
 ness with which casein is wetted by a solvent, it proved to be 
 necessary, in obtaining comparable experimental results, always 
 to employ the same preparation of casein, since no two samples 
 of incompletely anhydrous casein can be relied upon to contain 
 exactly the same proportion of water. 
 
 A measured amount (usually 100 cc.) of the fluid employed 
 as solvent was placed in a beaker of squat form and 400 cc. 
 capacity and was agitated by a flattened glass rod which was 
 bent at right angles, the plane of the horizontal arm being some- 
 what inclined to the vertical, so as to communicate an upward 
 thrust to the rotating liquid. The horizontal arm of the stirrer 
 was about 2| cm. long and as near as possible to the bottom of 
 the beaker; this was rotated at an approximately constant rate 
 of about 1600 revolutions per minute by a small motor. While 
 stirring, a weighed amount of the casein was dropped into the 
 fluid. At stated intervals samples of the mixture were almost 
 instantaneously abstracted by means of a 10 cc. pipette which 
 was provided with a rubber bulb. The samples were then very 
 rapidly filtered through lightly packed glass wool. The time 
 occupied in filtration was never more than 30 seconds for the 
 5 and 10 minute samples, or more than 1 minute for the later 
 samples. The refractive index of the filtrate from each sample 
 was then determined. Denoting the refractive index of any 
 given sample by n and that of the pure solvent by wi, the quotient 
 n ~ Ul is the number of grams of casein dissolved in 100 cc. 
 
 U.UUlO^w 
 
 of the solvent at the moment when the sample was extracted 
 (Cf. Chap. XIV). 
 
PASSAGE INTO SOLUTION 
 
 279 
 
 The type of relation which was found to subsist between the 
 time which elapsed after the introduction of the casein and the 
 number of grams of casein dissolved in 100 cc. of solvent is shown 
 diagrammatically in the accompanying figure, in which the 
 abscissae represent minutes and the ordinates the number of 
 grams of casein dissolved in 100 cc. of solvent. It will be ob- 
 served that the rate of solution is at first very great, but that 
 it very rapidly falls off. It does not fall to zero, however, that 
 is, the curve does not appear to approach an asymptote, but 
 is rather of a parabolic form. Nor does this appear strange 
 when we observe that although, after two hours of stirring, the 
 
 Minutes 
 
 rate of solution of the casein is very small, yet the solvent is 
 still very far from being " saturated" with casein. The alkali- 
 equivalent of 1 gram of casein is, as we have seen (Chaps. V, 
 IX and X), 11.4 X 10~ 5 equivalent gram molecules, but the 
 proportion of the casein actually dissolved to the amount of 
 base present in the solvent was always, even after two hours 
 of stirring at room temperatures, very much less than this.* 
 
 * It might be imagined that the solutions of the casemates which contain 
 only 11.4 X 10~ 5 equivalents of base per gram of casein, and which are pre- 
 pared by neutralizing the excess of base which is employed to dissolve the 
 casein by the addition of a strong acid, are "supersaturated" with respect 
 to casein. Two facts speak very strongly against this view, however. The 
 first is that the electro-chemical behavior of casein in much more alkaline 
 solutions already foreshadows the fact that the least proportion of alkali 
 which will hold one gram of casein in solution is 11.4 X 10~ 5 equivalents (Cf. 
 Chap. X). The second is that if a 3 per cent solution of casein in 0.0043 
 N KOH (1 gram of casein to 14 X 10~ 5 equivalents of base) be prepared by 
 dissolving casein hi excess of base and then neutralizing the excess with acid, 
 it can be kept in a sealed glass vessel, in the presence of excess of toluol for 
 6 months without any deposition of casein occurring. At the end of from 
 10 months to a year a very bulky white precipitate is deposited leaving the 
 
280 PHYSICAL PROPERTIES 
 
 The relation between the time of stirring and the quantity 
 of casein dissolved does not obey either of the above cited equa- 
 tions; it does not appear to obey any of the ordinary chemical 
 reaction or solution velocity formulae. The rate at which the 
 velocity of solution falls off; the negative acceleration of the 
 process; is far too great to permit of representation by either 
 of the formulae (i) and (ii). Nor is any better agreement ob- 
 tained if we insert, for the value of a in these equations, the 
 actual number of grams of casein present in the mixture, or if, 
 allowing for the diminution in the surface of the casein exposed 
 to the action of the solvent, as solution proceeds, we endeavor 
 to apply the relation 
 
 g = K(A - x) (B - x), (iii)* 
 
 where A is the number of grams of casein which the amount of 
 alkali present in the solvent is capable of holding in solution, 
 B is the number of grams of casein actually present in the mix- 
 ture, x is the amount of casein dissolved at any given moment 
 and K is a constant. The relation between the time of stirring 
 and the amount of casein dissolved, however, does obey, very 
 accurately, the relation 
 
 x = Kt m , (iv) 
 
 where x is the amount dissolved after time t, and K and m are 
 constants which vary with the nature and concentration of the 
 alkali solution employed as solvent and with the total mass of 
 casein present in the mixture. 
 The following tables enumerate illustrative results, f The 
 
 supernatant fluid very nearly clear. It must be recollected, however, that 
 when periods of time of the order of a year are concerned no protein solution 
 can be regarded as being in equilibrium. Even in absolutely neutral solutions 
 the H+and OH' ions of the water lead to a measure of "autohydrolysis" (Cf. 
 Chap. XVI) and the above solution was distinctly acid (Cf. Chaps. V and IX). 
 Hydrolysis of caseinates of the bases leads to the formation of paranucleins 
 which are insoluble in neutral and faintly acid solutions, and to substances 
 which bind bases and therefore tend to abstract them from the casein (Cf. 
 Chap. XVI) and consequently, when the total amount of base present is so 
 very little more than enough to hold the casein in solution, to indirectly pre- 
 cipitate the casein itself. 
 
 * In its integrated form: log^ ( /l ~ X \ = Kt. 
 
 A (n X) 
 
 t For additional experimental results the reader is referred to my original 
 communication (86). 
 
PASSAGE INTO SOLUTION 
 
 281 
 
 TABLE I 
 
 Solvent: 0.00870 N KOH. Temperature 18-20 degrees 
 # = 1.48 m = 0.146 
 
 Time in minutes 
 
 Grams casein dissolved in 100 cc. solvent 
 
 A 
 
 Found 
 
 Calculated 
 
 5 
 10 
 30 
 60 
 120 
 
 1.84 
 2.11 
 2.43 
 2.70 
 2.96 
 
 1.87 
 
 2.07 
 2.43 
 2.69 
 2.98 
 
 +0.03 
 -0.04 
 0.00 
 -0.01 
 +0.02 
 
 SA = 0.00 
 
 TABLE II 
 
 Solvent: 0.00870 N NaOH. Temperature 22-23 
 K = 1.33 m = 0.163 
 
 Time in minutes 
 
 Grams casein dissolved in 100 cc. solvent 
 
 A 
 
 Found 
 
 Calculated 
 
 5 
 
 10 
 30 
 60 
 120 
 
 1.71 
 1.97 
 2.30 
 2.63 
 2.89 
 
 1.73 
 1.94 
 2.32 
 2.60 
 2.91 
 
 +0.02 
 -0.03 
 +0.02 
 -0.03 
 +0.02 
 
 SA = 0.00 
 
 TABLE III 
 
 Solvent: 0.01740 N Ba(OH) 2 . Temperature 20 degrees 
 K = 1.34 m = 0.194 
 
 Time in minutes 
 
 Grams casein dissolved in 100 cc. solvent 
 
 A 
 
 Found 
 
 Calculated 
 
 5 
 10 
 30 
 60 
 120 
 
 1.85 
 2.05 
 2.64 
 3.03 
 3.36 
 
 1.84 
 2.10 
 2.60 
 2.98 
 3.40 
 
 -0.01 
 +0.05 
 -0.04 
 -0.05 
 +0.04 
 
 SA = -0.01 
 
282 PHYSICAL PROPERTIES 
 
 quantity of solvent employed was 100 cc. and the number of 
 grams of casein initially added to it was 5. For a reason which 
 will shortly appear no especial effort was made to maintain a 
 constant temperature during the progress of an experiment, but 
 at the head of each table are given the temperatures of the mix- 
 ture at the beginning and at the end of the experiment. The 
 temperature at the beginning of the experiment is placed first. 
 In the column headed " calculated" are given the values of x 
 calculated from the above formula, the constants K and m being 
 determined from all of the observations by the method of least 
 squares, employing for this purpose the form: 
 
 logio x = m logio t + logio K. 
 
 The possible experimental error in the determination of the 
 concentration of a casein solution by means of its refractive 
 index is always 0.07 gram per 100 cc. It will be seen that the 
 differences (= A) between the observed and calculated values of 
 x are usually considerably less than the possible error in the 
 determination of the concentration of the casein in the filtrates. 
 
 Equally concentrated solutions of KOH, NaOH, and NH 4 OH 
 dissolve casein with about equal rapidity, while solutions of the 
 hydroxides of the alkaline earths dissolve casein much more 
 slowly, Sr(OH) 2 dissolving the casein most rapidly, Ca(OH) 2 
 more slowly and Ba(OH) 2 more slowly still. As I have pointed 
 out in Chaps. X and XI this fact is of significance when viewed 
 in the light of the facts that solutions of the caseinates of the 
 alkaline earths become opalescent on heating, while those of the 
 caseinates of the alkalies and ammonium do not (63), that the 
 caseinates of the alkaline earths will not pass through the pores 
 of a clay filter, while those of the alkalies and ammonium readily 
 do so (63), and that in these and in other ways the caseinates 
 of the alkaline earths give evidence of being present, in their 
 solutions, in the form of more bulky molecules than those of 
 the caseinates of the alkalies and of ammonium under equivalent 
 conditions. It appears probable that when casein is suspended 
 in solutions of the hydroxides of the alkaline earths the soluble 
 caseinate is just as readily formed as it is in solutions of the 
 alkalies, but that it is hindered in passing out of the casein par- 
 ticles, through their capillary pores, into the solution, just as it 
 is hindered in passing through the pores of a clay filter. We 
 
PASSAGE INTO SOLUTION 
 
 283 
 
 have had occasion to comment upon the fact (Cf. Chap. V) 
 that dry casein will not pass into solution in dilute acids, although 
 wet, flocculent, freshly precipitated casein will do so; never- 
 theless, acid is taken up from the solution and bound by the 
 casein (103). It appears probable, as I indicated in Chap. V, 
 that in this case soluble casein salts are formed but that they 
 are prevented, by the grossness of their molecules, from passing 
 out through the pores of the casein particles into the body of 
 the solvent. 
 
 The influence of neutral salts upon the rate of solution of 
 casein by dilute sodium hydroxide has been investigated by 
 Robertson and Miyake (89) who find that even in the presence 
 of high concentrations of these salts the relationship between 
 the time and the amount of casein dissolved is expressed by the 
 relation x = Kt m . The presence of the salts retards the solu- 
 tion of the casein, however, the retardation due to alkaline 
 earth chlorides being about one hundred times as great as that 
 which is brought about by chlorides of the alkalies. The degree 
 of retardation increases with increase of the concentration of 
 salt employed. 
 
 The amount of casein which is dissolved, in a given period of 
 time, by a solution of KOH is, within the limits of accuracy of 
 the determinations, directly proportional to the concentration of 
 the KOH solution. This is very clearly shown in Table IV, in 
 which r denotes the ratio of the number of grams of casein dis- 
 solved to the number of equivalent gram molecules (multiplied 
 by 100) of KOH present in 100 cc. of solvent employed. It will 
 
 TABLE IV 
 
 
 Concentration of the KOH solution employed as solvent 
 
 Time in 
 
 
 minutes 
 
 
 
 
 
 
 
 
 
 
 0. 00218 N 
 
 0.00435 N 
 
 0.00653 N 
 
 0. 00870 tf 
 
 0.01088 N 
 
 0. 01305 N 
 
 0. 01523 N 
 
 0.01740 AT 
 
 
 r 
 
 r 
 
 r 
 
 r* 
 
 r 
 
 r 
 
 r 
 
 r 
 
 5 
 
 21 
 
 18 
 
 16 
 
 20 
 
 19 
 
 21 
 
 20 
 
 19 
 
 10 
 
 23 
 
 21 
 
 18 
 
 23 
 
 23 
 
 24 
 
 22 
 
 21 
 
 30 
 
 29 
 
 26 
 
 26 
 
 28 
 
 27 
 
 28 
 
 26 
 
 26 
 
 60 
 
 33 
 
 29 
 
 29 
 
 31 
 
 30 
 
 31 
 
 28 
 
 28 
 
 120 
 
 33 
 
 31 
 
 31 
 
 34 
 
 33 
 
 33 
 
 30 
 
 
 * Average of two determinations at 26.5 degrees and 18 to 20 degrees, respectively. 
 
284 PHYSICAL PROPERTIES 
 
 be observed that this ratio, for any of the given periods of time, 
 is very nearly constant.* 
 
 The temperature-coefficient of the velocity of solution is very 
 small. The difference between the amounts of casein dissolved, 
 after a given time, at 18-20 degrees and at 26 degrees are only 
 slightly greater than or equal to the possible error of the deter- 
 minations. So far as the accuracy of the method employed 
 enables us to decide, the temperature-coefficient of the rate of 
 solution, between the temperatures of 20 and 36 degrees, is prac- 
 tically zero.f At higher temperatures the rate of solution in 
 solutions of the hydroxides of the alkalies is increased and the 
 rate of solution in solutions of the hydroxides of the alkaline 
 earths is very materially diminished. At these temperatures, 
 also, solutions of the caseinates of the alkaline earths, which are 
 neutral to phenolphthalein, become alkaline to phenolphthalein, 
 while solutions of the caseinates of the alkalies which are neutral 
 to phenolphthalein do not (63). I have sought to account for 
 these facts by supposing that temperatures above 36 degrees 
 lead to a polymerization of the protein moiety of the caseinates 
 of the alkaline earths (83). 
 
 The low temperature-coefficient would in itself lead us to 
 suspect that the process which determines the rate of solution 
 of casein in solutions of bases is not chemical in nature. 
 
 That the rate of solution of the casein is not determined by 
 the velocity of a chemical reaction occurring exclusively in the 
 liquid phase is also shown by the fact that the rate of solution 
 of the casein is dependent upon the mass of casein initially in- 
 troduced into the mixture. Were the rate of solution of the 
 casein dependent solely upon the velocity of a reaction between 
 casein and the alkali, taking place in the liquid phase, then since, 
 in the presence of undissolved casein, the liquid would always 
 be saturated with casein, the rate of solution should be inde- 
 pendent of the mass of undissolved casein. We are led to con- 
 clude, therefore, that the processes which determine the velocity 
 of solution occur, in part at all events, either within or at the 
 surfaces of the suspended particles of undissolved casein. 
 
 * At "saturation" of the alkali with casein the numerical value of this 
 ratio would be 91. 
 
 t It was for this reason, of course, that no special effort was made to main- 
 tain a constant temperature during the progress of the experiments. 
 
PASSAGE INTO SOLUTION 
 
 285 
 
 TABLE V 
 
 Grams casein 
 
 
 
 
 
 
 
 added to 200 cc. 
 of solvent 
 
 2.5 
 
 5.0 
 
 7.5 
 
 10.0 
 
 12.5 
 
 15.0 
 
 (0.010 N KOH) 
 
 
 
 
 
 
 
 Time in 
 
 Grams 
 
 Grams 
 
 Grams 
 
 Grams 
 
 Grams 
 
 Grams 
 
 minutes 
 
 dissolved 
 
 dissolved 
 
 dissolved 
 
 dissolved 
 
 dissolved 
 
 dissolved 
 
 5 
 
 2.24 
 
 2.76 
 
 3.28 
 
 3.68 
 
 3.68 
 
 3.82 
 
 10 
 
 2.36 
 
 3.28 
 
 3.82 
 
 4.22 
 
 4.22 
 
 4.60 
 
 30 
 
 (2.50)* 
 
 3.96 
 
 4.86 
 
 5.40 
 
 5.40 
 
 5.66 
 
 60 
 
 
 4.60 
 
 5.40 
 
 5.92 
 
 5.92 
 
 6.18 
 
 * That is to say, at some undetermined time previous to this all of the casein introduced into 
 the solvent had been dissolved. 
 
 The relation between the amount of casein dissolved in a given 
 time and the mass of casein initially added to the solvent is 
 shown in Table V and graphically in accompanying figure. The 
 temperature of the mixtures was, in all of these experiments, 
 
 6Q infantes 
 
 Grams Casein in Mixture 
 
 20 degrees. It will be seen that the rate of solution" increases, 
 at first somewhat rapidly with the mass of casein added to the 
 solvent, later more slowly. 
 
 Reverting to equation (iv), the relation x = Kt m is, it is of 
 interest to observe, the same as that found by Cameron and 
 Bell (14) (15) and later confirmed by Ostwald from the investi- 
 gations of Goppelsroeder (66), to subsist between the amount 
 of fluid absorbed by a column of sand or a strip of filter-paper 
 and the time during which the fluid has remained in contact 
 with a portion of its surface.* 
 
 The values of the constants are also of the same order of mag- 
 nitude as those found in these investigations. It is possible, 
 therefore, that the rate of solution of the casein is primarily 
 
 * According to Cameron and Bell (14), this formula can be derived from 
 the formula of Poiseuille for the flow of liquids through capillary spaces. 
 
286 PHYSICAL PROPERTIES 
 
 determined by the rate at which the particles are penetrated 
 and wetted by the solvent, the process of chemical reaction 
 between the alkali and the casein taking place at a relatively 
 great velocity. 
 
 It is, perhaps, not surprising that the factor which determines 
 the rate of solution of casein should be the velocity with which 
 it is wetted by the solvent, while that which determines the rate 
 of solution for a crystalloid is the velocity with which the dis- 
 solved substance diffuses out of a thin layer of saturated solu- 
 tion in immediate contact with the surfaces of the crystals. A 
 crystal is only wetted upon its external surface, and the wetting, 
 naturally, takes place instantly. A particle of casein (or, in 
 general, of the solid phase of any colloid) is, however, comparable 
 in structure with a sponge; the surface which may be wetted by 
 the solvent is, per unit volume, very much larger than that of 
 a crystal, and the solvent must, in wetting this surface, traverse 
 a relatively immense network of minute capillary pores. Under 
 such conditions the time occupied in wetting the surfaces of the 
 particles may well be great compared with the time required for 
 the dissolved substance to diffuse from these surfaces into the 
 solvent, or with the time required for the accomplishment of 
 the union between the protein and the alkali in the solvent. 
 
 As might be expected, similar relationships are encountered in 
 the extraction of a protein from desiccated and finely divided 
 fragments of tissue. The rate of extraction of the protamin 
 salmin by dilute hydrochloric acid from the dried spermatozoa 
 of the salmon is determined primarily by capillary forces (87) 
 (88). The accompanying chemical phenomena (decomposition 
 of compounds of salmin within the tissue, formation of salmin 
 hydrochloride, etc.) occur at a relatively very great velocity and 
 hence do not affect the rate of extraction. 
 
 The rate of extraction or passage of a protein from a colloidal 
 phase into a surrounding solvent may be determined either by 
 the rate of passage of the soluble protein compound from within 
 the colloid particles into the surrounding solvent, or by the rate 
 of penetration of the solvent into the colloid particles, or by 
 both of these processes. That the latter process, namely the 
 penetration of the colloid, is of very great importance in deter- 
 mining the observed time-relations may be inferred from the 
 fact that the absorption of acid from dilute acid solutions by 
 
PASSAGE INTO SOLUTION 287 
 
 suspended particles of casein takes place in accordance with the 
 equation x = Kt m , although in this case either no soluble com- 
 pound of casein is formed or, which is more probable, the soluble 
 compound which is formed is unable to issue forth from the 
 colloid particles within which it arises (88) (89). 
 Differentiating the equation: 
 
 x = Kt" 
 we find 
 
 in other words, the product Km, which has been termed by 
 Robertson and Miyake the coefficient of penetration, expresses 
 the constant proportionality between the velocity of solution and 
 an exponent (peculiar to each solvent) of the time during which 
 the protein has been exposed to the action of the solvent. The 
 coefficient of penetration is, in the case of the solution of casein 
 by dilute sodium hydroxide, very characteristically affected by 
 the presence in the solvent of inorganic salts. The magnitude 
 of the coefficient of penetration decreases with increasing con- 
 centrations of NaCl, KC1, CaCl 2 , SrCl2 or BaCl 2 , the acceleration 
 of the decrease being positive in the cases of NaCl and KC1 and 
 negative in the cases of CaCl 2 , SrCl 2 or BaCl 2 . Lithium chloride, 
 on the other hand, increases the value of the coefficient of pene- 
 tration with a negative acceleration, so that at concentrations 
 of this salt lying above 0.33 normal the algebraic sum of these 
 two opposite effects results in a decrease of the value of the 
 coefficient of penetration (89). 
 
 If the rate of solution of casein by alkalies were primarily 
 determined by the rate of penetration of the capillary pores in 
 the suspended colloid particles by the solvent, then the addition 
 to the solvent of any substance which markedly reduces the 
 tension of a solid-water interface should retard the rate of 
 penetration and, consequently, the rate of solution of the casein. 
 It has been found by Robertson and Miyake (90) that alcohol 
 and glycerol, both of which reduce the tension of a solid-water 
 interface, also retard the solution of casein by dilute sodium 
 hydroxide. The penetration-formula x Kt m expresses the re- 
 lationship between the quantity of casein dissolved and the 
 time of stirring of the mixture in all mixtures of glycerol and water 
 
288 PHYSICAL PROPERTIES 
 
 and in alcohol-water mixtures which contain less than 4.5 mol. or 
 more than 7 mol. of alcohol. The formula most decidedly fails 
 to apply, however, to the rate of solution of casein in dilute alkali 
 containing between 4 and 8 mol. of alcohol, for in these mix- 
 tures the extremely curious phenomenon is observed of partial re- 
 precipitation, on continued stirring, of the casein which is initially 
 dissolved, so that after 2 hours of stirring, the casein dissolved 
 in these mixtures may actually be considerably less than after 
 only I hour of stirring. The penetration-formula obviously im- 
 plies a continuous and irreversible process, while in the presence 
 of the above-mentioned concentrations of alcohol the process of 
 solution is evidently partially reversible. Above and below this 
 critical zone of concentrations the relation between the time of 
 stirring and the concentration of casein dissolved is adequately 
 expressed by the penetration-formula. 
 
 On examining the effect of alcohol upon the magnitude of the 
 coefficient of penetration it becomes evident that in the zone of 
 
 Coefficient of Penetration 
 
 0.3 
 
 0.2 
 
 \ 
 
 Concentration of 
 Alcohol 
 
 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M. 
 
 concentrations lying between 4 and 8 mol. the character of the 
 relationship between the concentration of alcohol and the mag- 
 nitude of the coefficient of penetration is undergoing a transition. 
 For concentrations of alcohol lying below 4.5 mol., the coefficient 
 of penetration decreases with negative acceleration as the con- 
 centration of alcohol increases. In mixtures containing over 
 7 mol. alcohol, however, the coefficient of penetration decreases 
 with positive acceleration, as the concentration of alcohol in- 
 
PASSAGE INTO SOLUTION 289 
 
 creases. In mixtures containing concentrations of alcohol lying 
 between 4.5 and 7 mol. inclusive, the character of the relation- 
 ship is indeterminate. These relationships are illustrated by 
 the accompanying figure in which the broken lines indicate the 
 calculated continuation of the continuous lines drawn through 
 the experimentally determined points, the formula used for the 
 calculated exterpolations being: 
 
 XiMi - KM = ac + 0C 2 , 
 
 in which KiMi KM represents the decrease in the coefficient 
 of penetration, c represents the concentration of alcohol, a is a 
 constant which is positive when c lies below 4.5 mol. and nega- 
 tive when c lies above 8 mol., and /? is a constant which is nega- 
 tive when c lies below 4.5 mol. and positive when c lies above 
 8 mol. 
 
 The only explanation of these curious phenomena which offers 
 itself lies in the fact that alcohol is a coagulant of proteins, and 
 therefore brings about dehydration and consequent polymeri- 
 zation of protein molecules. At low concentrations of alcohol 
 we have to deal with the rate of solution of " single" molecules 
 of sodium caseinate, at higher concentrations with the rate of 
 solution of polymerized molecules which we may for the sake of 
 brevity term "double" molecules, while at intermediate con- 
 centrations we have to deal with a rate of solution which is com- 
 pounded of the separate rates of solution of the two types of 
 molecules. With increasing concentration of alcohol the rate of 
 solution of the single molecules is progressively diminished, but 
 that of the double molecules is increased, owing to the fact that 
 the proportion of double to single molecules coming off from the 
 internal surfaces of the casein particles is being increased suffi- 
 ciently rapidly to more than compensate for the retardation of 
 the total rate of solution by the alcohol. At 8 mol. concentra- 
 tion, so large a proportion of the molecules coming off are of the 
 double type that thereafter the retarding effects of increasing 
 concentration of alcohol upon the total rate of solution or pene- 
 tration more than compensates for any further increase in the 
 proportion of double molecules. 
 
 At intermediate concentrations the quantity of casein dis- 
 solved at first increases and thereafter diminishes with time, 
 casein which is initially dissolved being later reprecipitated. 
 
290 PHYSICAL PROPERTIES 
 
 This can only be interpreted by supposing that the condition of 
 the bulk of the solution outside the casein particles differs, at 
 any rate for some portion of the time occupied in solution, from 
 the condition of that portion of the solvent which has actually 
 penetrated the protein particles. We know that any substance 
 such as alcohol, which reduces the tension of a solid-water inter- 
 face, tends to become concentrated at such an interface (102). 
 Hence at the surfaces of the casein particles the concentration 
 of alcohol will be greater than in the bulk of the fluid. Suppose 
 that the concentration of alcohol in the bulk of the fluid be such 
 that at equilibrium the proportion of double molecules is 50 
 per cent. If, now, the concentration of alcohol at the surface 
 of the casein particles be such as to lead to the formation of 60 
 per cent of double molecules, then out of every thirty molecules 
 coming off from the interior surfaces of the casein particles 
 eighteen will be of the double type and only twelve of the single 
 type. As these come out into the bulk of the liquid, equilibrium 
 will tend to be re-established; resulting in the formation of sixteen 
 double and sixteen single molecules, with an increase in the total 
 number of molecules to thirty-two. This equilibrium may be 
 supposed to be slowly established, and meanwhile the rate of 
 solution of the casein has fallen very low; that is, the fall in 
 concentration or concentration gradient from within the casein 
 particles to the bulk of the fluid outside is very small. By this 
 time, however, as a result of depolymerization, the molecular 
 concentration of casein in the outer fluid has actually become 
 greater than that in the saturated solution which fills the interior 
 spaces of the casein particles. Thus the concentration gradient 
 has become negative, and as the more concentrated casein solu- 
 tion diffuses back into the less concentrated but nevertheless 
 saturated solution filling the interspaces of the sponge-like par- 
 ticles of casein, the excess of casein must be reprecipitated. When 
 the concentration of alcohol is sufficiently great, so that the 
 molecular condition of the casern is practically the same within 
 and without the particles, a negative concentration gradient can 
 never arise and the curve of solution reassumes the normal form, 
 representing now, however, the curve of solution of double mole- 
 cules instead of single molecules as at lower concentrations of 
 alcohol. 
 This interpretation of the above facts finds further confirma- 
 
PASSAGE INTO SOLUTION 291 
 
 tion in the effect of glycerol upon the rate of solution of casein 
 in dilute alkali. In glycerol we have a substance which, like 
 alcohol, reduces the tension of a solid-water interface but which, 
 unlike alcohol, does not coagulate (polymerize) proteins in solu- 
 tion. Accordingly we find that glycerol decreases the rate of 
 solution of casein progressively as its concentration increases. 
 No sign of reprecipitation of dissolved casein is observed in any 
 of the mixtures. The penetration formula x = Kt m applies to 
 the rate of solution in all of the mixtures employed and the value 
 of the coefficient of penetration progressively decreases in a 
 smooth curve with decreasing acceleration as the concentration 
 of glycerol increases. 
 
 These results illustrate the part which may be played by capil- 
 lary phenomena in heterogeneous systems which contain proteins, 
 the importance of which has, of recent years, been especially 
 insisted upon by, among others, Wo. Ostwald and H. Freund- 
 lich (67). 
 
 It is of interest to estimate, by exterpolation from the pene- 
 tration formula, the amount of time which would be required to 
 "saturate" an alkaline solution with casein by stirring up excess 
 of undissolved casein in it. We have seen that the relation be- 
 tween the percentage ( = x) of casein dissolved in a given solution 
 of alkali and the time ( = t) of stirring is expressed by the equation 
 x = Kt m . For a mixture of 5 grams of solid casein with 100 cc. 
 of 0.00870 N KOH the values of K and m at 18-20 degrees are, 
 respectively, 1.48 and 0.146. The quantity of casein required 
 to " saturate" 100 cc. of this solution would be 7.63 grams. 
 Calculating the value of t corresponding to this value of x we 
 find that it would take no less than thirty-one years to fully 
 "saturate" the solution with casein, at the rate of stirring em- 
 ployed.* This very clearly indicates the great importance which 
 the time factor may assume in heterogeneous systems which 
 contain proteins. 
 
 * For more dilute solutions of alkali mixed with the same number (5) of 
 grams of casein per 100 cc. the time required for "saturation" would appear 
 to be shorter, thus for 0.00435 N KOH, calculating as above, it is only about 
 17 days. This is because not only the concentration of alkali but also the 
 mass of casein, as we have seen, plays a part in determining the rate of solu- 
 tion. The ratio of casein to alkali was of course greater in the more dilute 
 alkaline solutions in inverse proportion to the concentration of the alkali. 
 
292 PHYSICAL PROPERTIES 
 
 2. The Swelling of Protein Jellies. A phenomenon which is 
 doubtless very closely allied to that of the solution of solid pro- 
 teins is the swelling or taking up of water which protein jellies 
 undergo when immersed in water or in certain watery solutions. 
 
 When a plate of water-poor gelatin is immersed in water, 
 especially if the water contains a little added acid or alkali, the 
 plate takes up a very considerable quantity of water, many 
 times its own weight, and at the same time swells to relatively 
 enormous dimensions. 
 
 It was first pointed out by Quincke (80) that the swelling of 
 gelatin is accompanied by a volume contraction, that is, the 
 volume of the swollen jelly is less than the sum of the volumes 
 of the original unswollen jelly and the absorbed water, and it 
 was further pointed out by Wiedemann and Ludeking (107) 
 that this process is accompanied by a disengagement of heat. 
 Careful measurements of the heat liberated and the volume 
 contraction during the swelling of casein in water have been 
 made by Katz (46) who finds that the relationship between the 
 heat liberated (= W) and the amount of water (= i grams) 
 taken up by one gram of protein is expressed by the equation: 
 
 in which A and B are constants. This implies that at the initial 
 moment of the swelling process, the heat liberated by one gram 
 
 of protein per gram of water absorbed will be equal to the ratio 
 
 ^ 
 
 ^, which in this case equalled 265 Cal. The volume-contraction 
 
 > 
 
 follows an analogous relationship: 
 
 . 
 Jf/M 
 
 in which / and g are constants and we may similarly calculate, 
 from the ratio - the value of C , or the volume contraction per 
 
 gram of protein per gram of water absorbed at the moment of 
 initiation of the process, and this calculation yields the value 
 0.3 cc. 
 
 The time-relations and equilibria in the process of the swelling 
 of gelatin have been especially studied by Hofmeister (40), Pauli 
 
SWELLING 293 
 
 (70) (71) (72), Wo. Ostwald (64) (65), Chiari (16), Ehrenberg 
 (23), Procter (74) (75) (76) (77) (78), and Lenk (50). 
 
 Hofmeister found that the swelling of gelatin plates proceeds, 
 at first rapidly and then more slowly, until it attains a maximum 
 which is a function of the thickness and weight of the plate. 
 After the attainment of this maximum the superficial layers of 
 the plate tend to go into solution, especially if the water be acid 
 or alkaline, and the plate, in consequence loses weight. Desig- 
 nating by the symbol W the weight of water which unit weight 
 of the gelatin absorbs from water or a watery solution in t 
 minutes, Hofmeister found that the empirical equation: 
 
 W-P 1- l \ (v) 
 
 applies with tolerable accuracy, P being the maximum amount 
 of water which unit weight of the gelatin plate will imbibe (the 
 swelling-maximum), c a constant and d the thickness in milli- 
 meters of the plate at its maximal degree of swelling. This 
 leads to the conclusion that the initial velocity of swelling is 
 proportional to the amount of swelling which the plate is able 
 to undergo and that it therefore decreases regularly as the degree 
 of swelling approaches more and more nearly the maximum. 
 
 Pauli assumed that each particle of the gelatin takes up water 
 from every neighboring, more fully swollen particle at a velocity 
 proportional to the difference between their water contents. This 
 leads to the equation: 
 
 v 1 i M - Q 
 
 where Q is the quantity of water taken up by unit weight of 
 gelatin in time t, M is the maximum degree of swelling which 
 the gelatin attains, Qi is the quantity of water taken up by unit 
 weight of gelatin in time ti and K is a constant which varies 
 inversely with the thickness of the plate. 
 
 Hofmeister's formula leads to the conclusion that the velocity 
 of swelling is at every instant proportional to the square of the 
 swelling which the plate has yet to undergo, Pauli's to the con- 
 clusion that the velocity of swelling is at every instant propor- 
 tional to the first power of the same quantity (termed by Pauli 
 the " swelling deficit")- Both formulae lead to the conclusion 
 
294 PHYSICAL PROPERTIES 
 
 that the velocity of swelling is at a maximum at the instant of 
 immersion and therefore decreases with time. This corresponds 
 to the experimental facts. 
 
 As we shall see in the next chapter, the proteins exert a small, 
 but definite, osmotic pressure. They are at the same time not 
 diffusible through colloids, or only very slightly so. Any crystal- 
 loids which may be present in the external fluid which bathes 
 the gelatin can penetrate the gelatin albeit, possibly, more slowly 
 than the water. The gelatin plate, therefore, acts like an osmom- 
 eter which provides its own membrane which is permeable to 
 crystalloids and not to colloids. Hence osmotic forces must 
 play a part in the taking up of water by protein jellies. A phe- 
 nomenon in the domain of crystalloids which presents some 
 analogies to this aspect of the swelling of colloids is the following:* 
 If we place at the bottom .of a column of distilled water a layer 
 of phenol and introduce below this a layer of a saturated solution 
 of KC1 in water and now allow the system to stand at constant 
 temperature the layer of phenol gradually moves up the column 
 of water; in other words the layer of solution below the phenol 
 "swells." The solvent, water, being soluble in phenol, the phenol 
 is permeable to it, while the KC1 being insoluble in phenol, cannot 
 pass through the layer of phenol. 
 
 Not only osmotic, but also chemical phenomena must, however, 
 play a part in the swelling of protein jellies. As we have seen in 
 Chaps. VI and XI the passage of a protein into solution involves 
 the addition of the elements of water to terminal NH 2 and 
 COOH groups and also, possibly, to internal N.HOC 
 groups, resulting in the depolymerization of the protein. Not 
 only osmotic phenomena but hydration of the gelatin must there- 
 fore occur in the process of swelling. In evidence of the correctness 
 of this view Pauli points to the fact that, according to Weidemann 
 and Liideking (loc. cit.), the swelling of gelatin is accompanied 
 by a disengagement of heat, while the solution of gelatin is ac- 
 companied by an absorption of heat. Evidently the processes of 
 solution and swelling are each composed of two factors, one 
 leading to a disengagement and the other to the absorption of 
 heat. The former process is, Pauli believes, the chemical binding 
 of water by the protein, the latter the passage of the hydrated 
 
 * To which my attention was drawn, in this connection, by my colleague 
 Dr. F. G. Cottrell. 
 
SWELLING 295 
 
 protein into solution (or, in swelling, the osmotic inhibition of 
 water). In swelling the chemical heat-effect predominates; in 
 the dissolving of the gelatin, the heat-effect of solution. 
 
 That a part, at least, of the water in swollen gelatin is chemi- 
 cally bound by the protein is shown by the following experiment 
 (74). If two gelatin plates be brought to the same degree of 
 swelling, the one in neutral and the other in acid water, and if 
 they be then immersed in absolute alcohol, the alcohol will ab- 
 stract all of the water from the plate which has been swollen in 
 neutral water, but not from the plate which has been swollen 
 in acid water. Now, as we have seen in Chap. V, the fact that 
 a constituent of a system can be completely removed from it by 
 washing out with an appropriate solvent affords no valid proof 
 that the constituent in question did not exist, within the system, 
 in a state of chemical combination. But if, on the contrary, it 
 should prove impossible or exceptionally difficult to remove the 
 constituent by washing with a fluid in which it is very soluble, 
 then the prima facie evidence that it exists in the system in a 
 condition of chemical union is very strong. We may conclude, 
 therefore, that when gelatin is swollen in acid water a part of 
 the water which is taken up by the gelatin is chemically bound 
 by it; and we have no valid reason for supposing that the water 
 taken up from neutral solutions is not similarly bound although 
 less firmly. 
 
 The equilibria attained in. the swelling of gelatin in solutions 
 of acids have recently been very thoroughly investigated by 
 Procter. This investigator has found that gelatin absorbs both 
 acid and water from acid solutions, but absorbs the acid in excess, 
 so that the proportion of acid in the surrounding fluid diminishes. 
 If the initial concentration of acid in the external fluid lies be- 
 tween 0.01 and 0.25 N, then assuming that at the end of the process 
 (attainment of maximal swelling) the concentration of free acid 
 is the same within and without the jelly, the amount of acid 
 which is "bound" by the gelatin is 0.7 to 0.8 X 10~ 3 (= 70 to 
 80 X 10~ 5 ) equivalents per gram. The equivalence, at the 
 attainment of maximal swelling, is the same for all strong acids 
 but falls below this value for weak acids. While the proportion 
 of acid which is "bound" by the gelatin varies but slightly with 
 the concentration of the acid in the surrounding fluid, this is not 
 true of the degree of swelling attained, which in strongly acid 
 
296 PHYSICAL PROPERTIES 
 
 solutions attains its maximum at a dilution below that required 
 for complete fixation of the acid by the gelatin, and then falls in 
 a continuous curve with increasing concentration of the external 
 acid solution. Procter interprets this by supposing that the 
 swelling of the gelatin is determined by two opposing forces, the 
 osmotic pressure of the acid-gelatin compound imprisoned by 
 its indiffusibility within the jelly being balanced by the osmotic 
 pressure of the acid outside. That the acid in the outer fluid 
 does exert an osmotic pressure at the surface of the jelly Procter 
 infers from the fact that concentrated neutral salts contract the 
 jelly to a horny consistency and expel the associated (imbibed 
 or combined) water without affecting the acid-gelatin compound. 
 In view of the considerations set forth in Chap. VI we can see, 
 however, that the assumption that the external acid exerts an 
 osmotic pressure is an unnecessary one and, indeed, inconsistent 
 with the fact that the acid freely penetrates the gelatin and 
 combines with it. In view of the known dependence of coagula- 
 tion upon phenomena of dehydration (Cf. Chap. VI) and the fact 
 that protein salts may be coagulated as such and without decom- 
 position, we may infer that the shrinkage of gelatin jellies upon 
 addition of concentrated salts to the external medium is not 
 due to the osmotic pressure of the salts, since gelatin is known 
 to be permeable to inorganic ions, but to the competition between 
 the inorganic salt and the gelatin salt for water. 
 
 Procter has taken advantage of the fact that the acid-gelatin 
 compound is coagulated without decomposition by concentrated 
 salt solutions to determine the quantity of acid " bound" by 
 gelatin in acid solutions of varying concentration. He finds 
 that the proportion of acid which is bound per unit mass of 
 gelatin does not agree with the requirements of the Ostwald 
 dilution-law for a binary electrolyte, no values of the hydrolytic 
 dissociation-constant and the molecular weight yielding calcu- 
 lated results which are consistent with the initial rapid and 
 subsequent slow rise in combining capacity with increasing con- 
 centration of acid in the surrounding medium. Better agree- 
 ment between theory and experiment is attained by assuming 
 that gelatin behaves as a diacid base having a molecular weight 
 of 839, which agrees with the molecular weight estimated by 
 Paal from freezing- and boiling-point measurements (68). 
 
 The taking up of water by gelatin from acid solutions is ac- 
 
SWELLING 297 
 
 counted for by Procter in much the same manner as that out- 
 lined above (76). He pictures the gelatin acid-compound as a 
 coherent mass from which the gelatin molecules cannot diffuse or 
 separate and which in most respects behaves like a single enor- 
 mous complex molecule. It is reasonable, he considers, to visual- 
 ize it as a felted mass of amino-acid chains held to each other by 
 attractions which possibly attach only their ends, but freely 
 admitting the passage of liquid between them. He assumes, in 
 accordance with our former conceptions of the mode of forma- 
 tion and ionization of protein salts, that the compound yields 
 acid anions, but these anions, although diffusible, are held within 
 the mass by electrostatic forces, since they cannot pass beyond 
 the sphere of attraction of their companion colloid ions which 
 form the jelly and which are therefore immobilized. The only 
 way, therefore, in which the osmotic pressure of the anions can 
 take effect is, not by their movement but by the movement of 
 water, resulting in the swelling of the entire jelly mass and its 
 dilution by admixture with the outside solution. 
 
 Two very serious objections attach to this interpretation of 
 the phenomena. In the first place, as Procter himself has pointed 
 out, were this the actual mechanism of swelling, then the opera- 
 tive force compelling movement of the water would, in ultimate 
 analysis, be the electrostatic tension which prevents the acid 
 anions from moving outwards into the surrounding solvent. 
 There should thus be a measurable potential difference between 
 the gelatin jelly and the external medium. This potential differ- 
 ence has been sought for by Ehrenberg who was unable to detect 
 any measurable potential between the interior of a jelly and the 
 external medium (23). In the second place, as Procter also 
 points out, another difficulty lies in the fact that the condition 
 which would thus arise would offer no equilibrium, since the 
 acid anions and the free acid itself could not simultaneously 
 be equal in concentration within and without the jelly. Our 
 more recent views regarding the mode of formation and ionization 
 of protein salts reconcile both these difficulties, however, for, 
 since we may assume that no inorganic ions, or at most a very 
 small proportion, are yielded by the protein-acid compound, the 
 swelling of the jelly must be due, just as it is in the case of gel- 
 atin immersed in neutral water, to the osmotic pressure of the 
 colloid particles themselves, which, being unable to penetrate 
 
298 PHYSICAL PROPERTIES 
 
 the colloid network in which they are entangled necessarily 
 compel the compensating migration of water. No electro- 
 static tension between the jelly and the external solution need 
 be assumed and since no acid anions are yielded by the protein 
 salts, simultaneous equality of concentration of the uncombined 
 acid and the acid anions within and without the jelly will be 
 assured by their normal and equal diffusion into the jelly. The 
 increased swelling capacity of gelatin in solutions of acids or 
 alkalies is merely the expression of the fact that the ionization 
 of the protein salt leads to an increase in the number of colloid 
 particles per unit volume of the jelly and possibly also in part 
 of the fact that protein ions have a greater affinity for water 
 than undissociated protein molecules. 
 
 This conception of the process of swelling would still yield 
 no equilibrium or swelling-maximum were there no compensating 
 force acting in an opposite sense to the osmotic pressure of the 
 gelatin. Since gelatin plates when immersed in water do not 
 swell indefinitely until swelling merges insensibly into solution 
 but, on the contrary, display a more or less well-marked swelling- 
 maximum, the osmotic pressure exerted by the colloid particles 
 within the jelly must, upon attainment of this maximum, be 
 balanced by an equal opposing force which Procter interprets 
 as the tension of the elastic colloid network (77). Applying 
 Hooke's law,' Procter finds that this tension (= E) is defined 
 by the equation : 
 
 TjJ C 1 I V - 
 
 \ sp. gr. of gelatin/ ' 
 
 where E is the stress inducing swelling, V is the volume attained 
 by one gram of gelatin, and C is the modulus of elasticity. He 
 finds that C = 0.00125 at 7 degrees; 0.00055 at 15 degrees; and 
 0.00021 at 18 degrees. The value of E at first increases with 
 acidity to a maximum and then decreases, slowly approaching 
 zero. 
 
 It has been shown by Ostwald (64) and Chiari (16) that gelatin 
 in very faintly acid media displays a well-marked minimum of 
 swelling-capacity. This minimum occurs at a hydrogen ion con- 
 centration almost exactly coinciding with the hydrogen ion 
 concentration of gelatin solutions in which the gelatin is "iso- 
 eleetric," i.e., does not wander in an electrical field. In other 
 
GELATINIZATION AND COAGULATION 299 
 
 words minimal combining capacity and ionization of the jelly 
 is accompanied by minimal swelling capacity. 
 
 Hofmeister has studied the effects of salts, acids and bases 
 upon the rate and magnitude of the swelling which gelatin plates 
 undergo when immersed in their solutions; we have had occasion 
 to comment upon the significance and interpretation of his results 
 in Chap. VI. Wo. Ostwald (65) has investigated the influence 
 of the concentration of added salts upon the swelling of gelatin 
 plates in water. He asserts that the degree of swelling attained 
 after a given period does not vary continuously in a definite 
 sense as the concentration of the salt is increased, the curve 
 displaying the influence of the concentrations of the salt upon the 
 degree of swelling exhibiting very marked maxima and minima. 
 We shall have occasion to further dwell upon the significance 
 of these results in the next chapter. 
 
 The phenomena which accompany and the processes which 
 underlie the taking up or loss of water by living tissues have been 
 investigated by Loeb (52) (53), Cooke (21), Bottazzi and Scalinci 
 (6), Beutner (3), and Korosy (47). Bottazzi and Scalinci have 
 drawn attention to the remarkable fact, previously pointed out 
 by von Schroeder in the case of gelatin (93), that the crystalline 
 lens swells very markedly in distilled water or physiological 
 saline solution, but progressively loses water when suspended in 
 saturated water vapor at the same temperature. This phe- 
 nomenon undoubtedly involves a departure from the second law 
 of thermodynamics, realizing the possibility which was depicted 
 figuratively by "Maxwell's demon." It is not surprising, perhaps, 
 that the exception to the rule should have been first encountered 
 in the domain of colloid jellies in which definite structures of 
 molecular dimensions play so pronounced a r61e in determining 
 their behavior. 
 
 The significance of the phenomena of swelling in certain aspects 
 of clinical medicine has been especially insisted upon by M. H. 
 Fischer and discussed by him at length in his work upon cedema 
 (26). Exception has, however, been taken by Moore to certain as- 
 pects of Fischer's interpretation of the phenomena of cedema (58). 
 
 3. The Gelatinization and Coagulation of Proteins. If an 
 insoluble gel, such as white of egg coagulated by fixatives, the 
 gel of collodion produced by the action of chloroform upon an 
 ether solution, common black india-rubber, or the hydrogel of 
 
300 PHYSICAL PROPERTIES 
 
 silica, be examined under high magnification they can all be 
 demonstrated, Hardy states (33) (34), to possess a fine open 
 spongy structure. When, for example, a 13 per cent solution 
 of egg-white is fixed with sublimate, sections are found to show 
 a sponge or net structure. Staining the section with iron- 
 hsematoxylin, with saturated solutions of acid and basic dyes or 
 even by evaporation to dryness in solutions of such dyes, failed 
 to produce the staining of any substance within the meshes of 
 the net, while pressure applied to the gel resulted in the squeezing 
 of fluid out of these interstices. The structure of the gel is 
 therefore that of an open sponge-work of solid, containing fluid 
 within its meshes. Direct experimentation with agar showed 
 that in a gel containing 1 per cent agar the solid framework is 
 a solution of water in agar, while the fluid contained in the inter- 
 stices is a dilute solution of agar in water; upon the heating the 
 system the two components become miscible in each other and 
 we obtain a homogeneous solution. Upon the basis of these 
 facts Hardy draws a far-reaching analogy between this system 
 (and other jellies which are heat-reversible) and the system 
 phenol-water, which, if it contains more than 71 per cent or less 
 than 76 per cent of phenol separates, at temperatures below 
 80 degrees, into two phases, the one a solution of phenol in water, 
 the other a solution of water in phenol. According to the view 
 developed by Hardy, the two cases differ only in the fact that 
 upon separation of the two phases in the agar-water system the 
 system retains a structure, while in the phenol-water system no 
 structure is retained. Essentially, he believes, the difference 
 between the two systems consists in this, that when the phenol- 
 water system separates into two phases the phases become sepa- 
 rated by the minimum possible surface, namely a plane; while 
 when the agar-water system separates into two phases they 
 remain in contact over an area far larger than the minimum. In 
 the latter case it would appear that the surface tension at the 
 surface of separation of the two phases is very low, so that the 
 force leading to a diminution of surface is indefinitely small. 
 
 Pauli and Rona (72) object to the use which Hardy has made 
 of the term "phase" in. this connection. They point out that 
 the fluid which may be pressed out of an agar jelly may contain 
 from to 0.14 per cent of agar according to the magnitude and 
 mode of application of the pressure, and they urge, in considera- 
 
GELATINIZATION AND COAGULATION 301 
 
 tion of this fact, that there can be no sharp separation into phases 
 within an agar, and, presumably, within a gelatin jelly. They 
 believe that a sharp distinction should be drawn between the 
 coagulation of a protein by dehydrating agents and the gelatin- 
 ization of its solution, since in the former case a sharp separation 
 into two phases occurs while in the latter it does not. The 
 experimental basis of this objection to Hardy's view is, however, 
 not altogether a sufficient one, since if any two parts of a chemical 
 system, which are of more than molecular thickness, are sepa- 
 rated by a surface, they constitute, in the sense of the phase-rule, 
 separate phases. The observation of Pauli and Rona shows, 
 however, that, at least in agar jellies, if separate phases exist 
 they are not of definite or constant composition. 
 
 The fact that in protein jellies which do consist of two phases 
 the phases are not of constant composition is also very clearly 
 revealed by Hardy's own results, of which a description follows: 
 
 The manner in which the structure of an insoluble gel is built 
 up can, according to Hardy, be readily observed in the ternary 
 mixture alcohol, gelatin, and water. If 13.5 grams of gelatin 
 are mixed with 50 cc. of water and 50 cc. of absolute alcohol, 
 a mixture is formed which is optically homogeneous at 17 to 20 
 degrees but which separates into two phases at temperatures 
 below this. "As the temperature falls below the limit a clouding 
 occurs which I find to be due to the appearance of fluid droplets 
 which gradually increase in size until they measure 3 ju. On 
 cooling further, these fluid droplets become solid and they begin 
 to adhere to one another.* The framework is therefore an open 
 structure which holds the fluid phase in its interstices." " When 
 once formed the phases have considerable stability. If the drop- 
 lets are composed of a solid solution one may, by the addition 
 of water, cause them to increase to relatively vast dimensions 
 without their being destroyed; as they increase in size their 
 refractive index approximates more and more to that of the 
 external phase until t ley are finally lost sight of. The addition 
 of alcohol, however, once more brings them into view and causes 
 them to shrink. Owing to this stability, once a configuration 
 has been established, one has to far overstep the conditions of 
 its formation in order to destroy it. This would account for 
 
 * The formation of similar droplets has been observed by Pauli and Rona 
 (72) in the coagulation of gelatin by salts. 
 
302 PHYSICAL PROPERTIES 
 
 the remarkable hysteresis observed in reversible gels. . . . When 
 water is added to a ternary mixture so as to considerably swell 
 the droplets the system is unstable and the two phases mix at once 
 when it is mechanically agitated" (34). 
 
 In gels of this type which are dilute with respect to the colloid, 
 therefore, the structure is that of an open sponge- work; the 
 meshes being filled with water or a water-rich solution of the sub- 
 stance forming the gel, while the frame- work of the sponge con- 
 sists of anastomosing threads composed of linearly arranged 
 globules of the water-poor phase. In such gels, therefore, the sur- 
 face of the water-poor phase is convex while that of the water-rich 
 phase is concave; in other words, the water-poor phase is internal 
 to the water-rich or external phase. If, however, to a ternary 
 mixture of gelatin, alcohol and water which forms such a gel 
 as that described above, more gelatin be added, the character 
 of the gel changes entirely, the water-poor phase becomes con- 
 cave and the water-rich phase instead of being, as formerly, 
 concave becomes convex to it. On cooling such a mixture to a 
 temperature below that at which it forms an optically homo- 
 geneous solution, droplets separate out which are poor in gelatin, 
 while the interstitial portion of the system, which is rich in 
 gelatin, solidifies. Thus the gel comes to possess a honeycomb 
 structure, the droplets being poor in gelatin, rich in water. This 
 is very clearly shown in the following determinations of Hardy. 
 
 TEMPERATURE OF THE MIXTURE 15 DEGREES 
 
 (Equal parts of water and alcohol) 
 
 Per cent gelatin in 
 mixture 
 
 Per cent gelatin in 
 droplets (internal) 
 phase) 
 
 Per cent gelatin in 
 interstices (external 
 phase) 
 
 6.7 
 13.5 
 36.5 
 
 17.0 
 18.0 
 8.5 
 
 2.0 
 5.5 
 40.0 
 
 From these determinations it is also clear, as I have said, 
 that the two phases are -not of constant composition, but may, 
 under different conditions of total concentration, etc., vary widely 
 in their relative and absolute gelatin and water content. This 
 system differs, therefore, from the system phenol-water not only 
 
GELATINIZATION AND COAGULATION 303 
 
 in the extent of the surface which separates the phases, but also 
 in the variability of the composition of its phases, in this respect 
 resembling rather the system hydrated silica-water, investigated 
 by van Bemmelen (1). 
 
 An inversion of the external and internal phases of a diphasic 
 system, similar to that observed by Hardy on increasing the con- 
 centration of gelatin in the system gelatin-alcohol-water, may also 
 be observed in the system olive oil-alkaline water, on increasing 
 the proportion of oil to water (84) (85), and is here manifestly 
 due to the inability of a limited quantity of alkaline water to 
 surround and envelop an unlimited quantity of oil. 
 
 The question has been raised whether the jelly which is formed 
 by gelatin dissolved in water (instead of alcohol-water mixtures) 
 really possesses a structure analogous to that observed by Hardy 
 in ternary systems. It has been urged that this structure is 
 an artefact arising out of partial coagulation of the protein, 
 since it is not directly visible in binary systems. The action of 
 coagulants upon jellies which already possess a structure of this 
 type, however, is not to otherwise alter but merely to coarsen 
 the structure. This is due to loss of water on the part of the 
 colloid-rich droplets with a consequent diminution of the volume 
 of the colloid-rich phase and an increase in the volume of the 
 more fluid interstices. This can be shown, not only by direct 
 observation, but also by the relative ease with which water can 
 be expressed from the jelly before and after " fixation." From 
 Poiseuilles' law for the outflow of liquids from capillary tubes, 
 it follows that the pressure required to express the fluid from the 
 interstices of a gel at a given rate must vary approximately as 
 the inverse fourth power of the diameter of the meshes, although, 
 of course, the variable viscosity of the expressed fluid will be a 
 factor introducing departures from this simple law. Now a 
 hydrogel containing 13 per cent of pure gelatin at a tempera- 
 ture of 15 degrees will endure a pressure of 400 pounds to the 
 square inch without expression of water; after fixation with 
 formalin or corrosive sublimate, however, the fluid can be ex- 
 pressed from the gel like water from a sponge, with simple hand- 
 pressure (33). 
 
 Since more complete coagulation does not alter the type of 
 structure possessed by jellies of partially coagulated protein, 
 but merely coarsens it, it is a fair inference that jellies which 
 
304 PHYSICAL PROPERTIES 
 
 have undergone no measure of coagulation also possess the type 
 of structure outlined by Hardy, but that owing to its fineness the 
 details of this structure are not visible. 
 
 The existence of a structure in jellies formed by the solution 
 of gelatin in water is also objectively demonstrated by the ob- 
 servation of Liesegang (51) that when silver nitrate diffuses 
 into gelatin which is impregnated with potassium bichromate, 
 the precipitation of insoluble silver bichromate does not occur 
 indifferently in all parts of the area of diffusion but in concentric 
 circles. It has also been shown by Rohonyi (91) that when 
 thin films of gelatin are frozen the ice crystals are formed in 
 concentric rings. It is difficult to clearly conceive any mecha- 
 nism which would permit this in a perfectly homogeneous medium. 
 The theory that crystallization is inhibited by the gelatin until 
 a certain degree of super-saturation is attained might account 
 for failure of precipitation or crystallization at certain points, 
 but, provided the jelly is strictly homogeneous and structureless 
 it fails to account for its appearance at other points. That the 
 distinction between gelation and coagulation is merely a dis- 
 tinction of degree and not of kind has been shown by Buglia (12). 
 
 The experiments of Hardy show that on adding water to the 
 system alcohol-water-gelatin, the gelatin-rich phase progressively 
 imbibes water until it passes by a series of insensible transitions 
 into a solution of gelatin. Having regard to this fact and to the 
 probability, which will be indicated in the following chapter, that 
 the structure of a jelly of uncoagulated protein is merely the con- 
 tinuation of a structure which pre-exists in the solutions which 
 become gelatinized, it appears highly probable that in uncoagulated 
 protein jellies the structure is of molecular dimensions, so that the 
 constituents of the jelly are not separated from one another by any 
 definite interface. This conception does not in the least militate 
 against the view that the structure of a coagulated or partially 
 coagulated jelly merely results from the coarsening of a pre-exist- 
 ing molecular structure. It is probable that the coagulation of a 
 protein solution, without passing through the intermediate stage 
 of gelatinization, is similarly accompanied and possibly (from 
 a mechanical standpoint) accomplished by the coarsening of a 
 pre-existing molecular structure. 
 
 4. The Coagulation of Proteins by Heat, Light and Hydro- 
 static Pressure. Several aspects of the phenomenon of heat- 
 
HEAT, LIGHT AND PRESSURE 305 
 
 coagulation have been discussed at some length in Chap. VI, and 
 it has been pointed out in that chapter, and incidentally, in 
 Section 1 of this chapter that the application of heat to proteins 
 in solution results, probably, not only in the abstraction of the 
 elements of water from the protein, with the formation of anhyd- 
 rides, but also in its polymerization. 
 
 Under definite conditions of concentration, reaction and salt- 
 content of its solution, etc., the coagulation-temperature of * a 
 protein is tolerably constant, and Fredericque (27), Halliburton 
 (29) and others have utilized this, property very extensively in 
 the endeavor to separate and characterize different proteins. 
 
 The concentration of protein, and especially the presence of 
 other substances in the solution, very markedly modifies the 
 coagulation-temperature, however. The influence of inorganic 
 salts, upon the "temperature of coagulation" has been inci- 
 dentally discussed in Chap. VI. The influence of a variety 
 of salts in acid and alkaline solutions upon the coagulation- 
 temperature of proteins has been very extensively studied by 
 Pauli and his pupils (32), and found to be in satisfactory accord- 
 ance with the view that heat-coagulation, like salt- or alcohol- 
 coagulation is accomplished through the dehydration of the 
 protein molecules. 
 
 The very important observation has been made by Chick 
 and Martin (17) that the heat-coagulation of haemoglobin and 
 egg albumin (in solutions of crystallized preparations of these 
 substances) is not an instantaneous process, but that it proceeds 
 with a definite velocity which decreases as the protein becomes 
 coagulated and increases very markedly with rise in temperature. 
 The relation between time of exposure to a temperature suffi- 
 cient to bring about coagulation and the quantity of protein 
 coagulated is, for haemoglobin, that which is characteristic for 
 the occurrence of a monomolecular chemical reaction, namely: 
 
 log ^ = Ktt 
 
 Lt 
 
 where C is the initial concentration of the substance (protein), 
 C t its concentration at time t and K is a constant (the velocity- 
 constant of the reaction). The following are illustrative of their 
 results : 
 
306 
 
 PHYSICAL PROPERTIES 
 
 COAGULATION OF HAEMOGLOBIN, 3 PER CENT SOLUTION 
 
 Experi- 
 
 Temp, 
 
 Time in 
 
 Concentration of 
 haemoglobin (first 
 
 loem C 
 
 \og w C -\og w C t 
 
 ment 
 
 degrees 
 
 minutes 
 
 sample = 100 = C ) 
 
 M -'&10 
 
 t 
 
 1 
 
 60 
 
 
 
 100 
 
 2 000 
 
 
 
 
 30 
 
 54 
 
 1.732 
 
 0.0090 
 
 
 
 90 
 
 13.5 
 
 1.130 
 
 0.0097 
 
 
 
 
 
 
 Mean 0.0094 
 
 2 
 
 62.6 
 
 
 
 100 
 
 2.000 
 
 
 
 
 20 
 
 42 
 
 1.623 
 
 O.m 
 
 
 
 45 
 
 12 
 
 1.079 
 
 0.020 
 
 
 
 70 
 
 4.8 
 
 0.681 
 
 0.019 
 
 
 
 
 
 
 Mean 0.019 
 
 3 
 
 65.6 
 
 
 
 100 
 
 2.000 
 
 f 
 
 
 
 10 
 
 35.5 
 
 1.550 
 
 Q. 045* 
 
 
 
 20 
 
 11.0 
 
 1.041 
 
 0.048 
 
 
 
 30 
 
 5.0 
 
 0.699 
 
 0.043 
 
 
 
 
 
 
 Mean 0.045 
 
 4 
 
 67.6 
 
 
 
 100 
 
 2.000 
 
 
 
 
 3 
 
 61.4 
 
 1.788 
 
 6'07i' 
 
 
 
 6 
 
 34.8 
 
 1.542 
 
 0.076 
 
 
 
 9 
 
 24.9 
 
 1.396 
 
 0.067 
 
 
 
 
 
 
 Mean 0.071 
 
 5 
 
 70.4 
 
 
 
 100 
 
 2.000 
 
 
 
 
 2 
 
 52.5 
 
 1.720 
 
 o'.ii " 
 
 
 
 4 
 
 25.3 
 
 1.404 
 
 0.15 
 
 
 
 6 
 
 14.1 
 
 1.150 
 
 0.15 
 
 
 
 7.5 
 
 7.6 
 
 0.886 
 
 0.15 
 
 
 
 
 
 
 Mean 15 
 
 
 
 
 
 
 
 The temperature-coefficient of the process is seen to be very 
 high, the velocity-constant being multiplied about 15 times by 
 10 degrees rise in temperature (from 60-70.4 degrees). The 
 relationship between the temperature and the value of K is very 
 satisfactorily represented by the Arrhenius equation : 
 
 a relation which, although not strictly deducible from the gas 
 laws (60) is analogous in form to the van't Hoff equation con- 
 necting the value of the equilibrium-constant with the tempera- 
 ture and which is deducible from the gas laws. We are probably 
 justified in concluding, therefore, as we did in the analogous 
 case afforded by the variation in the dissociation-constant of 
 
HEAT, LIGHT AND PRESSURE 307 
 
 oxy haemoglobin with temperature,* that immediately prior to its 
 coagulation in the above experiments the haemoglobin existed 
 in the solution in a molecularly dispersed condition, obeying the 
 law of Avogadro. 
 
 The high temperature-coefficient of the process explains, as 
 Chick and Martin point out, the apparent, but unreal constancy 
 of the "coagulation-temperature" which was so much insisted 
 upon by the earlier investigators quoted above. With rising 
 temperature a point is reached at which the reaction is so rapid 
 as to appear almost instantaneous. 
 
 It is obvious that the monomolecular reaction-formula would 
 apply either to the dehydration of protein according to the formula 
 
 H 2 N.R.COH.N.R.COOH = HN.R.COH.N.R.CO + H 2 
 
 I I 
 
 or to its hydration according to a formula of the type 
 Protein + H 2 O = coagulated protein, 
 
 since in the latter case, the mass of water being very much greater 
 than that of the protein, the active mass of water would be appre- 
 ciably constant throughout the reaction. 
 
 From the fact that dry protein, heated to high temperatures 
 does not undergo typical heat-coagulation, i.e., does not lose its 
 solubility in water (24) Chick and Martin conclude that the heat- 
 coagulation of protein is a process of hydration. From the results 
 of Pauli and myself, cited in Chap. VI, it is evident, however, 
 that the process of heat-coagulation is not one of hydration but 
 of dehydration of the protein, f according to some or all of the 
 equations (i) to (iv) in Chap. VI, section 6. From the fact that 
 the base-combining capacity of casein diminishes with rising tem- 
 perature, and also the solubility of some of the caseinates, we 
 have concluded { that with rising temperature this protein, at 
 all events, undergoes some measure of polymerization on heating 
 through the dehydration of the end NH 2 and COOH groups 
 of adjacent molecules. If this were a general phenomenon 
 
 * Cf. Chap. VI. f Cf. also Michailow (56) and Starke (98). 
 
 t Cf. Chap. VI also 1 of this chapter and T. Brailsford Robertson (83). 
 
 Mann (55) states that in his opinion heat coagul tion is brought about 
 by one portion of the albumin molecule precipitating the remainder, a view 
 which is essentially similar to that expressed above. Sutherland (99) has also 
 expressed the view that coagulation of a protein is the result of polymerization 
 through the neutralization of "Valencies which are usually latent." 
 
308 PHYSICAL PROPERTIES 
 
 then it would follow, from van't HofFs "Principle of Mobile 
 Equilibrium" that the hydrolysis of protein (hydration) is ac- 
 companied by an evolution of heat, which conclusion is in com- 
 plete accord with experimental observation.* The apparent 
 discrepancy between the conclusion reached by Chick and Martin 
 and those which have been formulated by other investigators 
 in this field may therefore be due to the fact that dry protein 
 has been deprived of the elements of water in its end NH 2 
 and COOH groups, and that these, consequently, cannot react 
 to form polymers of the protein. f It is evident that in the process 
 of dehydrating proteins by heat not only the reaction: 
 
 H 2 N.R.COH.N.R.COOH = HN.R.COH.N.R.CO + H 2 
 
 occurs, but also reactions of the type 
 
 2 H 2 N.R.COH.N.R.COOH 
 
 = H 2 N.R.COH.N.R.COH.N.R.COH.N.R.COOH + H 2 0, 
 
 and it is the formation of these polymers which leads to the 
 apparently irreversible character of the process; apparently and 
 not actually irreversible because as Corin and Ansiaux (22) 
 have shown, if a solution of protein be cooled and vigorously 
 
 * Cf . 1 and the results of Wiedemann and Ltldeking cited in this chap- 
 ter. The heat of reaction of protein hydrolysis is extremely small; observers 
 using the usual indirect methods of determination either failed to detect 
 any change in the heat-content of the system or else have observed a very 
 slight disengagement of heat (101), (49), (36). Henderson and Ryder, however, 
 using the beautiful and excessively sensitive isothermal method of calorimetry 
 of T. W. Richards, have demonstrated that the tryptic hydrolysis of casein 
 is accomplished by a progressive evolution of heat (37). It should be clearly 
 borne in mind in this connection that the effect of raising the temperature upon 
 a chemical reaction is always twofold. It shifts the station of equilibrium in 
 one sense or in the opposite and, always, it accelerates the reaction in either 
 sense to a greater or less degree (i.e., it magnifies both velocity constants, but 
 unequally). The action of heat upon proteins must always be to shift the 
 station of equilibrium in the direction of polymerization (i.e., condensation), 
 since the reaction of hydrolysis is exothermic. But the fact that fairly com- 
 plete hydrolysis will occur at the temperature of steam shows that the shift in 
 equilibrium between the lower protein complexes and the amino-acids which 
 are the products of their hydrolysis is not so great as to extinguish the reaction 
 of hydrolysis. 
 
 t It should also be recollected, in this connection, that reactions occurring 
 in solid systems are notoriously extremely slow, owing to the high internal 
 friction of the system and the consequent hampering of molecular motion. 
 
HEAT, LIGHT AND PRESSURE 309 
 
 shaken just as the first traces of heat coagulation appear the 
 incipient coagula will again pass into solution. The apparent 
 irreversibility of the later stages of heat-coagulation is probably 
 attributable to the high internal friction of the floccula which 
 are formed, leading to extremely slow molecular movement and 
 the introduction of a time-element of very considerable magnitude. 
 
 This deduction receives decided support from the discovery 
 by Chick and Martin (18) (19) (20) that the heat-coagulation of 
 proteins consists of two processes which they severally term 
 ^denaturation " and " agglutination." In acid solutions the 
 process of agglutination or aggregation takes place at a rate 
 very greatly in excess of denaturation and therefore the rate of 
 formation of flocculated protein is primarily determined by the 
 rate of denaturation, i.e., by the slower process. In alkaline 
 solutions, however, aggregation does not occur, but may be 
 induced by subsequent acidification or by saturation with sodium 
 chloride. In acid solutions the rate of denaturation is acceler- 
 ated by H + ions and these decrease during the reaction through 
 the binding of acid by the denatured protein. If the concen- 
 tration of H + ions be kept constant the reaction follows the 
 monomolecular reaction-formula, but if this precaution be omit- 
 ted then the reaction velocity falls off more quickly than would 
 be anticipated from the monomolecular formula (18) (100). 
 Similarly in alkaline solutions of egg-albumin the OH' ion con- 
 centration decreases during denaturation, but if the OH' concen- 
 tration be kept constant the reaction is of the first order. 
 
 The diminution in the acidity of acid solutions of protein 
 during heat-coagulation has also been observed and quanti- 
 tatively determined by Sorensen and Jurgensen (97) and Quagli- 
 ariello (79). 
 
 We may conclude, therefore, that the results of Chick and 
 Martin are in satisfactory harmony with the view of Hofmeister 
 and Pauli that coagulation, including the heat-coagulation of 
 protein, is essentially a phenomenon of dehydration of which 
 the first stage, that of internal neutralization through the loss 
 of the elements of water from end NH 2 and COOH groups, 
 probably corresponds to the phenomenon of "denaturation" 
 while the subsequent or simultaneous polymerization of these 
 anhydrides leads to the formation of particles so large as to 
 assume the properties of matter in mass, i.e., flocculi. 
 
310 PHYSICAL PROPERTIES 
 
 These facts undoubtedly underlie and explain the apparent 
 transformation of serum albumin into serum globulins observed 
 by Moll on heating blood serum to which alkali had been added 
 (57). As Gibson (28) and Schmidt (92) have pointed out, a 
 transformation of serum albumin into globulin would involve a 
 synthesis of glycocoll, which amino-acid is not contained in the 
 albumin molecule. There can be little doubt that the protein 
 which Moll regards as globulin produced from albumin is in 
 reality " denatured" albumin which acidification will flocculate, 
 just as it flocculates a globulin. 
 
 The reversible character of " denaturation " (presumably 
 anhydride-formation without polymerization) is indicated by 
 the observation of Berczeller (2) that while the surface tension 
 of a protein solution which is so salt-free as not to coagulate 
 on heating, nevertheless diminishes on heating, this diminution 
 is reversible and spontaneously disappears on standing. 
 
 It has been shown by Bovie that protein solutions in quartz 
 vessels are coagulated by exposure to ultraviolet light (7). This 
 type of coagulation, like heat-coagulation, demonstrably con- 
 sists of two separate processes. The first process, that of denatu- 
 ration, has, like other photochemical processes, a very low 
 temperature-coefficient and takes place almost as rapidly at 
 C. as at room temperature. The second process, that of 
 agglutination or flocculation, has a high temperature-coefficient 
 indicating that it is not primarily a photochemical process, but 
 a spontaneous consequence of the photochemical denaturation 
 which precedes it. 
 
 Fernan and Pauli (25) have shown that exposure to the radia- 
 tions from radium leads to coagulation of protein (serum albumin) 
 in acid or alkaline solutions. Unlike heat-coagulation the co- 
 agulation in acid solutions by radium radiations is accompanied 
 by no diminution of their H + concentration. 
 
 It has been shown by Bridgman (9) that the application of 
 very great hydrostatic pressures results in the coagulation of 
 white of egg. The pressure is applied very slowly to avoid any 
 rise in temperature due to the compression and that the effect 
 is not due to heat is further demonstrated by the fact that it is 
 more easily elicited at degrees than at 20 degrees. The applica- 
 tion of five thousand atmospheres produces stiffening of the 
 white of egg; six thousand atmospheres compression, applied for 
 
CRYSTALLIZATION 31 1 
 
 thirty minutes, produces an appearance of the white resembling 
 that of curdled milk, while seven thousand atmospheres pressure 
 brings about complete gelation. 
 
 5. The Crystallization of Proteins. A number of proteins, 
 particularly the vegetable proteins and haemoglobin, are readily 
 obtainable in the form of crystals. Other proteins, such as 
 egg- and serum-albumin only yield crystals with considerable 
 difficulty. For the crystallization of these latter proteins mere 
 concentration of their solutions is insufficient; inorganic salts 
 in very high concentration must also be present (39) (41) (42) 
 (48) (94) (73). Thus egg-albumin may be prepared in a crys- 
 talline condition by adding to its solution an equal volume of 
 saturated ammonium sulphate solution and acidifying with 
 acetic acid. The coagulum which forms becomes crystalline on 
 standing. It would appear that the crystalline product is not 
 the uncombined protein but a salt of the protein, formed with 
 an acid and also, probably, with the ammonium sulphate (Cf. 
 Chap. VI) (59). 
 
 The protein crystals are optically true crystals. According to 
 Wichmann all albumin-crystals are either crystallographically 
 identical or else isomorphic. They very readily absorb impuri- 
 ties (106) (45) (95), and the circumstances of their preparation 
 involve the occlusion of a considerable quantity of the mother 
 liquor. Hence repeated recrystallization is required to remove 
 from them even colloidal impurities (95). Contamination by 
 crystalloidal substances, combined or physically associated, 
 obviously cannot be avoided, and these must be subsequently 
 got rid of by very prolonged dialysis of the dissolved crystals. 
 
 A monumental and most fundamentally important contribu- 
 tion to our knowledge of the crystallography of proteins has 
 been furnished by the investigations of Reichert and Brown (82) 
 on the relationship of the morphology of haemoglobin crystals 
 to the biological classification of the species from which it is 
 derived. From an enormous number of measurements of crystal 
 angles, etc., conducted upon haemoglobins derived from a large 
 variety of species, these investigators conclude in the first place 
 that the crystals of the species of any genus belong to the same 
 crystallographic system and generally to the same crystallo- 
 graphic group, and they have approximately the same axial 
 ratios, or their ratios are in simple relation with each other. In 
 
312 PHYSICAL PROPERTIES 
 
 other words the haemoglobin crystals of any genus are isomorphous. 
 In some cases this isomorphism may be extended to include 
 several genera, but this is usually not the case unless, as in the 
 case of the dogs and foxes, for example, the genera are very 
 closely related. On the other hand the oxyhaemoglobin obtained 
 from the same species always crystallizes in the same form, al- 
 though often with different habit when obtained by different 
 methods of preparation. But upon comparing the haemoglobins 
 from different species of a genus it is found that they differ from 
 one another to a greater or less degree in angles or axial ratio, 
 in optical characters and particularly in those characters com- 
 prised under the general term " crystal habit," so that one species 
 can usually be distinguished from another by the form of its 
 haemoglobin crystals. But these differences, within the limits 
 of a given genus, are not such as to preclude the crystals from 
 all species of that genus being placed in an isomorphous series. 
 
 A clear relationship is thus seen to subsist between the physico- 
 chemical behavior of a constituent of organisms and their place 
 in the phylogenetic scale of relationships as established by their 
 gross morphology, and a long stride has thus been taken toward 
 the establishment of a physico-chemical basis for morphological 
 distinctions. The further and entirely independent question now 
 arises, however, as to the chemical interpretation of the observed 
 physico-chemical phenomena. 
 
 Our experience with the crystallography of inorganic and the 
 simpler organic substances has led us to infer with a considerable 
 degree of confidence that substances which show differences in 
 crystallographic structure are different chemical substances. 
 Crystal form is affected even by isomeric modifications which 
 analysis, unaided by other methods of investigation, fails to 
 reveal. Now the enormous number of atoms in a protein mole- 
 cule encourages, at first sight, the supposition that an enormous 
 and indeed, for all practical purposes, an infinite number of 
 isomerides are possible between which the most refined methods 
 of analysis would not enable us to distinguish, but which would 
 very probably differ from one another in the morphology of 
 their crystals. In point of fact, however, the available number 
 of isomers would be very greatly restricted by the necessity of 
 maintaining unaffected the amino-acid groupings of the protein 
 moiety which could not differ materially in different species 
 
CRYSTALLIZATION 313 
 
 without leading to decided differences in the chemical behavior 
 of the haemoglobins which have not been observed by any in- 
 vestigator. Further doubt is thrown upon this interpretation 
 of the facts observed by Reichert and Brown by the observation 
 of Hiifner, recently confirmed with the utmost precision by 
 Butterfield (13), Heubner and Rosenberg (38), and Schumm (96) 
 that the characteristic absorption bands and the ratio of the 
 absorption of light in different parts of the spectrum of haemo- 
 globin is absolutely identical in species so far removed from one 
 another as the horse and man (Schumm) or the rabbit, sheep 
 and hog (Heubner and Rosenberg). Now these are properties 
 which we should anticipate might be materially affected by 
 internal differences of atomic arrangement. 
 
 Further reason for doubting the correctness of referring the 
 differences of crystal structure displayed by the haemoglobins of 
 different animals to internal differences in the molecules of the 
 haemoglobins is supplied by the observation of Loeb and Brown 
 (54) that the crystal-form of the haemoglobin of the mule is inter- 
 mediate in character between that of the horse and that of the donkey. 
 For if we assume that each different crystal form represents a 
 different internal atomic arrangement of the haemoglobin molecule, 
 then the number of such arrangements, even if very great, must 
 nevertheless be limited. The number of possible forms of crystals 
 must therefore also be limited and moreover the possible modi- 
 fications of forms must be discontinuous, i.e., there must exist 
 forms between which no intermediate forms are possible. This 
 being the case it would be very remarkable indeed were the hybrid- 
 ization of two closely related species to lead to the synthesis 
 of a new isomeric variety of haemoglobin not yet appropriated 
 by any existing species of animal and in addition lying between 
 the haemoglobins of the parent species. If analogous phenomena 
 should be displayed by all hybrids and by all varieties and mu- 
 tations that might have arisen or might conceivably arise in the 
 future we would have to admit that the haemoglobins already 
 recognizable as differing from one another in crystal form are 
 only a small proportion of those which are realizable. 
 
 A much more reasonable supposition is that embodied in the 
 view that the differences in crystal form observed by Reichert 
 and Brown are attributable, not to the internal variation of atomic 
 grouping in the haemoglobin molecules but to external varia- 
 
314 PHYSICAL PROPERTIES 
 
 tions in the milieu from which they are crystallized. The tech- 
 nique adopted by Reichert and Brown was to induce crystal- 
 lization directly in the laked blood. Now we know from the 
 observations of the immunologists that the blood plasma from 
 any species of animal differs antigenically from that derived 
 from any other species (62) and, since all known antigens are 
 proteins, we infer that the proteins, or, more probably, the com- 
 pound protein complexes (Cf. Chap. VII) in blood plasmas 
 derived from different species are in certain definite respects 
 different from each other. The crystals of each species studied 
 by Reichert and Brown were therefore deposited from a different 
 medium and it is not improbable that the observed differences 
 in the crystals are attributable to these known differences in 
 the media in which they were formed. It is well known that 
 crystal-habit is modified by alterations of the medium from which 
 the crystals are deposited. That modifications of this origin so 
 great as to preclude inclusion of the crystals formed in different 
 media in the same isomorphous series have not hitherto been 
 observed in the domain of inorganic chemistry is not improbably 
 attributable to the simpler character of the conditions accom- 
 panying crystallization in inorganic or non-colloidal media. We 
 have seen that there are many reasons for supposing that proteins, 
 even in solution, are disposed in a certain reticular structure 
 and if, as the facts dwelt upon in Chap. VII would seem to in- 
 dicate, characteristic protein complexes, formed by the union 
 in differing proportions of a relatively small number of simpler 
 protein components, exist in each type of blood plasma we may 
 well suppose that the reticular structures of the solutions com- 
 prising these plasma would likewise differ from one another. 
 Having regard to the markedly cohesive properties of proteins, 
 crystallization within the meshes of such a reticulum might 
 very conceivably, through external strains imposed by points 
 of attachment to the reticulum, modify the effects of the internal 
 strains which find their expression in crystal form. 
 
 This hypothesis finds decided support in the fact first observed 
 by Halliburton (39) (31) and confirmed by Reichert (81) that 
 the crystal form of oxyhaemoglobin derived from a given species 
 may be profoundly modified by admixture with the blood of 
 another species. The following are illustrative results obtained 
 by Halliburton, the " normal" form of rat haemoglobin crystals 
 
CRYSTALLIZATION 
 
 315 
 
 being rhombic, those obtained from guinea-pigs being normally 
 tetragonal and those from squirrel's blood hexagonal. 
 
 Blood of 
 
 Mixed with that of 
 
 Form of haemoglobin crystals deposited from the mixture 
 
 Rat 
 
 Squirrel 
 
 Both rhombic prisms and hexagons present. 
 
 Rat 
 
 Guinea-pig 
 
 No rhombic prisms of the shape usually seen 
 in rat's blood present. No tetrahedra. 
 Crystals are all rhombic prisms with hexa- 
 gonal habit. 
 
 Squirrel 
 
 Guinea-pig 
 
 Hexagonal plates and tetrahedra both pres- 
 ent. Many tetrahedra imperfect. The 
 tetrahedra all reduced to about half the 
 size of those prepared from the unmixed 
 blood of the same guinea-pigs. 
 
 Dog 
 
 Squirrel 
 
 Fine rhombic needles and hexagonal plates 
 both present in abundance. 
 
 Dog 
 
 Guinea-pig 
 
 The greater number of the crystals formed 
 are very small tetrahedra about a quarter 
 the size of those prepared from the blood of 
 the same guinea-pigs. The optical prop- 
 erties are, however, the same. Rhombic 
 prisms, very slender, like those of dog's 
 blood, are also seen. 
 
 According to Reichert the degree of modification of crystal 
 form induced by admixture of two bloods depends very greatly 
 upon the proportion in which they are mixed. 
 
 In view of these facts there can be little doubt that the nature 
 of the milieu in which crystallization occurs does play an im- 
 portant part in determining the form of the crystals which are 
 deposited, and having regard to the known individuality of the 
 plasma from different biological species it would appear unneces- 
 sary to seek further for the origin of the differences in crystal 
 form of the oxyhaemoglobins derived from blood of different 
 species of animals. 
 
 We are now in a position, also, to interpret the changes in 
 crystal-form which result from repeated re-crystallization of 
 haemoglobin (Halliburton (30) (31)), for as Wichmann (106) and, 
 more recently, Katz (45) have shown, the crystalline proteins 
 swell in or absorb the surrounding fluid menstruum in a manner 
 analogous to the swelling of jellies. A number of re-crystal- 
 lizations are therefore required to remove completely traces of 
 the original menstruum in which crystallization occurred. 
 
316 PHYSICAL PROPERTIES 
 
 Bradley and Sansum (8) believe that the haemoglobins from 
 different animals are antigenically different because guinea-pigs 
 sensitized to ox or dog haemoglobin failed to display anaphylactic 
 shock or reacted but slightly to haemoglobins of other origins, 
 while they reacted strongly to the haemoglobin with which they 
 were sensitized. As the haemoglobin preparations employed by 
 Bradley and Sansum were admittedly (with the exception, 
 they believe, of the dog haemoglobin) not free from contamina- 
 tion by serum the interpretation of these results is open to serious 
 question. Doubt is especially thrown upon this evidence for 
 the specificity of haemoglobins from different species by the fact 
 that the animals sensitized to the purest preparation of haemo- 
 globin employed, that of the dog, reacted strongly not only to 
 to dog haemoglobin but also to dog serum. Having regard to the 
 investigations of Wichmann and Katz, cited above, revealing 
 the marked ability of crystalline proteins to absorb the menstruum 
 from which they are deposited, and to the observation of Schulz 
 and Zsigmondy (95) that egg albumin must be recrystallized 
 from 3 to 6 times in order to remove appreciable contamination 
 by other proteins, we may infer that in all probability the speci- 
 ficities demonstrated by Bradley and Sansum are serum-speci- 
 ficities and not haemoglobin-specificities. 
 
 According to Howell (43), fibrin must now be added to our 
 list of crystallizable proteins. He finds that when fibrinogen is 
 precipitated by thrombin, the fibrin, in media of normal H + 
 concentrations, separates out in crystalline needles readily recog- 
 nizable as such under the ultramicroscope. They vary in length 
 from 10 to 30 microns and form a close mesh- work. The normal 
 blood-clot is therefore a crystalline gel. The blood of inverte- 
 brates, however, yields a non-crystalline gel and a similar gel is 
 yielded by mammalian fibrin in alkaline media. Such non- 
 crystalline gels, however, fail to display the spontaneous con- 
 traction or "synaeresis" which is so characteristic of normal 
 blood-clots. 
 
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 (99) Sutherland, W., Proc. Roy. Soc. London, 79B (1907), p. 130. 
 
 (100) Sutherland, W., Journ. Physiol., Proc., 42 (1911), p. vii. 
 
 (101) Tangl, F., Arch. f. d. ges. Physiol., 115 (1906), p. 1. 
 
 (102) Thomson, J. J., "Application of Dynamics to Physics and Chemis- 
 
 try," London (1888), p. 190. 
 
 (103) Van Slyke, L. L., and Van Slyke, D. D., Amer. Chem. Journ., 38 
 
 (1907), p. 383. 
 
 (104) Veley, V. H., Journ. Chem. Soc., London, 55 (1889), p. 361. 
 
 (105) Veley, V. H., Phil. Trans. Roy. Soc. London, 182 (1891), p. 79. 
 
 (106) Wichmann, A., Zeit. f. physiol. Chem., 27 (1899), p. 575. 
 
 (107) Wiedemann, E., and Ludeking, C., Wied. Ann. der Physik., N. F., 25 
 
 (1885), p. 145. 
 
 (108) Zeynek, R., Arch. f. (Anat. und) physiol. (1899), p. 460. 
 
CHAPTER XIII 
 
 CERTAIN PHYSICAL PROPERTIES OF PROTEIN SOLUTIONS, ETC. 
 
 1. The Viscosity of Protein Solutions. Protein solutions are 
 usually characterized by the possession of a high viscosity. The 
 question has been raised whether the viscosity is similar in nature 
 to that of a solution of a crystalloid substance, or whether it is 
 not, rather, comparable with the viscosity of a suspension of solid 
 particles (40). In support of this latter view it has been pointed 
 out that Bottazzi's measurements of the influence of concen- 
 tration upon the viscosity of protein solutions indicate that the 
 viscosity of these solutions does not change continuously with 
 change in concentration, as it does in solutions of crystalloids, but 
 changes per saltum (12). The measurements of Bottazzi were 
 made upon relatively few and widely separated concentrations, 
 however, and subsequent observers have not confirmed his results 
 (108) (59). Thus Sackur finds that the viscosity of solutions of 
 the "basic" casemates of sodium and ammonium (i.e., containing 
 about 80xlO~ 5 equivalents of base per gram and neutral to 
 phenolphthalein) varies with the concentration according to the 
 
 Arrhenius-Euler formula = A n , where rj is the viscosity of the 
 
 *7o 
 
 solution, T]Q that of the solvent, n is the concentration of the solu- 
 tion and A is a constant, the numerical value of which depends 
 upon the nature of the dissolved substance and upon the tempera- 
 ture (2). The following are Sackur's results: 
 
 n (in equivalent) 
 sodium) 
 
 -2 (15 degrees) 
 
 70 
 
 log A 
 
 0.01830 
 
 1.870 
 
 14.8 
 
 0.01370 
 
 1.581 
 
 14.5 
 
 0.00915 
 
 1.363 
 
 14.3 
 
 0.00547 
 
 1.202 
 
 14.6 
 
 0.00458 
 
 1.165 
 
 14.5 
 
 A remarkable feature of these results is the extraordinarily high 
 value of A, involving a very rapid increase in viscosity with 
 
 320 
 
VISCOSITY 
 
 321 
 
 increasing concentration. For the majority of crystalloids the 
 value of A is not greatly in excess of unity, while for sodium 
 casemate, as we see, it is of the order of 10 14 . This fact alone 
 would lead us to suspect that the mechanism which produces 
 the viscosity of these solutions is different in nature from that 
 which produces the viscosity of solutions of crystalloids. Sackur 
 has endeavored to ascertain which constituent of the solutions of 
 sodium caseinate plays the greater part in determining their 
 viscosity. He arrived at a conclusion the correctness of which 
 more recent investigations have fully established, by a process of 
 reasoning, however, which recent investigations have shown to 
 be in some respects fallacious. He argued that the viscosity of 
 these solutions might be attributable, primarily, to undissociated 
 sodium caseinate, or the product of its hydrolytic dissociation,* 
 i.e., free casein, or to caseinate ions. Hydrolytic dissociation 
 would, he believed, be diminished by the addition of alkali and 
 increased by the addition of acid. In the former case, according 
 to his view, the number of caseinate ions should be increased, in 
 the latter decreased. He found that the addition of alkali in- 
 creased and the addition of acid diminished the viscosity of a neutral 
 (to phenolphthalein) solution of sodium caseinate; the following 
 are among his results: 
 
 Per cent of casein 
 
 Normality of total 
 sodium 
 
 - (15 degrees) 
 fo 
 
 0.716 
 
 0.0063 
 
 1.24 
 
 0.716 
 
 0.0126 
 
 1.36 
 
 0.716 
 
 0.0189 
 
 1.38 
 
 0.716 
 
 0.0252 
 
 1.34 
 
 hence, he argued, the viscosity of these solutions is primarily 
 attributable to caseinate ions. 
 
 We have seen, however (Chaps. IV and VIII), that the casemates 
 do not undergo hydrolytic dissociation in solution. In fact the 
 initial solution to which Sackur added alkali in order to suppress 
 hydrolytic dissociation was neutral to phenolphthalein and there- 
 fore could not have contained more than 10~ 5 N NaOH derived 
 from hydrolytic dissociation of the sodium caseinate. We have 
 also seen (Chap. IX) that on adding alkali to a solution of a casein- 
 
 * Since free casein is insoluble this possibility may be dismissed. 
 
322 PHYSICAL PROPERTIES 
 
 ate which is neutral to phenolphthalein the added alkali does not 
 remain unneutralized, as Sackur assumes, but, on the contrary, 
 is partially bound by the casein until 180xlO~ 5 equivalents are 
 combined with every gram of casein. But we have also seen 
 (Chap. IX) that each successive equivalent of combined alkali 
 splits another NHOC bond in the casein molecule and con- 
 sequently gives rise to another pair of ions. With increasing 
 content of combined base, therefore, the caseinate, apart from 
 slight modifications of its degree of dissociation, yields a corre- 
 sponding proportion of ions. The fact that the viscosity of 
 caseinate solutions markedly increases with alkalinity is therefore 
 in strong support of Sackur's thesis that the viscosity of these 
 solutions is primarily attributable to protein ions, although not 
 for the reasons which he advances. The fact that the increase in 
 viscosity with increasing alkalinity attains a maximum at just 
 about the same time that the combining capacity of the casein 
 attains a maximum (Cf . above table and the tables and diagram 
 in Chap. IX) lends very striking support to this hypothesis. 
 
 The view that the viscosity of protein solutions is in a remark- 
 ably high degree dependent upon the protein ions which they 
 contain has also been advanced, with substantial experimental 
 support, by W. B. Hardy (41) and by Bottazzi (14). The latter 
 observer has shown that the viscosity of protein solutions is at a 
 minimum when ionic protein is absent, when the protein is un- 
 combined with base or acid, and that on adding either acids or 
 bases to this solution the viscosity increases. 
 
 Now a very little consideration suffices to show that the viscosity 
 of protein solutions is of a different type from the viscosity, for 
 example, of solutions of sugar or glycerol in water. Apart from 
 the extraordinary magnitude of A, alluded to above, the type of 
 viscosity exhibited by solutions of proteins differs from the vis- 
 cousness of a glycerol-water mixture in that it affords no hindrance, 
 or very slight hindrance, to the motion of ions and of crystalloid 
 molecules. The properties of agar are, in this respect, very similar 
 to those of protein. Thus Graham (35) showed that the velocity 
 with which crystalloids diffuse through gelatin jellies is remarkably 
 near to that with which they diffuse through water, and Voigt- 
 lander (126) has confirmed this result for agar jellies. Similar 
 results have been obtained by Hiif ner (47) . According to Bechhold 
 and Ziegler (6) the rate of diffusion of (at any rate concentrated) 
 
VISCOSITY 323 
 
 crystalloids is diminished by gelatin jellies, and the degree of 
 hindrance is materially modified by the presence, within the jelly, 
 of other dissolved substances, but the hindrance is extremely small 
 in comparison with the enormous viscosity of the jellies. 
 
 Similarly Reformatzky (90) has shown that the velocity with 
 which methyl acetate is decomposed by acids in agar jelly (i.e., the 
 number of molecular collisions per second) is within 1 per cent of 
 its value in pure water. 
 
 Lodge (65), Whetham (129) (130) and Masson (69) have shown 
 that the specific mobilities of the majority of inorganic ions is the 
 same in agar jellies as it is in water. Dumanski (28) has shown 
 that if allowance be made for the diminution in the cross-section 
 
 of the conducting field, due to the presence of gelatin ( = (-) 
 where g is the number of grams of gelatin per gram of solution and 
 c the specific gravity of gelatin) the conductivities of inorganic 
 
 salt solutions in gelatin jellies are very slightly less than those of 
 equally concentrated solutions in pure water. 
 
 We have seen* that the dependence of the conductivity of 
 protein solutions upon their dilution is of perfectly normal char- 
 acter, resembling the dependence of the conductivity of a crystal- 
 loidal electrolyte upon dilution, despite the fact that the protein 
 solutions, as the above-cited results of Sackur reveal, vary enor- 
 mously in their viscosity with dilution. 
 
 On the other hand, the intimate dependence of the conductivity 
 of solutions of electrolytes upon the ordinary type of viscosity has 
 been commented upon and quantitatively estimated by a host of 
 observers, among whom Walden may be especially mentioned 
 (127) (51) (37). Viscosities, very much less than those of the 
 dilutest jellies, profoundly diminish the conductive power of 
 electrolytes. Nor must it be imagined that the viscosity of a 
 protein jelly is essentially different, in any respect save magnitude, 
 from that of a protein solution, for, as von Schroeder (110) has 
 shown, the viscosity of a solution of gelatin, cooled below the 
 gelation-point, increases progressively and regularly with time, 
 until the extremely viscous solution passes insensibly into a jelly. 
 
 Not only inorganic, but also protein ions are profoundly in- 
 fluenced in their mobilities by the type of viscousness which 
 
 * Cf. Chap. X, also W. B. Hardy (41). 
 
324 PHYSICAL PROPERTIES 
 
 alcohol- or glycerol-water mixtures exhibit. Thus we have seen, 
 in Chapter XI, that the mobilities of caseinate ions in alcohol- 
 water mixtures are almost exactly in inverse proportion to the 
 viscosity of the solvent, and this despite the fact that the viscosity 
 of the solvent, measured by the time which it takes to run through 
 a capillary tube, is so profoundly influenced by the introduction of 
 the caseinate itself as to be in many cases, doubled in magnitude. 
 Solutions of KC1 in alcohol-water mixtures are, comparatively 
 speaking, unaltered in their viscousness by the KC1, yet the 
 dependence of the conductivities of solutions of potassium casein- 
 ate upon the percentage (between and 60 per cent) of alcohol 
 which they contain obeys exactly the same law as that which 
 applies to the conductivity of KC1 solutions in alcohol-water 
 mixtures, a law which implicitly involves the conclusion that the 
 total effect is due solely to the alteration in the mobilities of the 
 ions attributable to the viscousness of the solvent. To make 
 the matter clear by reference to a numerical example: Referring to 
 the tables in Chapter XI we see that the viscosity of JV/10 KOH 
 (dilute alkali) is doubled by the introduction of 3.125 per cent of 
 casein; halving the concentration of the solution, according to 
 Sackur's results cited above, must more than halve the effect of the 
 caseinate upon its viscousness; yet the mobilities of the caseinate 
 ions are unaltered by this. The viscosity of the solvent can be 
 also doubled by the introduction of 40 per cent alcohol and this, 
 on the contrary, halves the mobility of the caseinate ions. The 
 alcohol considerably modifies the degree to which the caseinate 
 alters the viscousness of the solvent, yet this fact does not at all 
 disturb the regularity of the relation between the viscosity of the 
 solvent and the mobility of the caseinate ions which are dissolved 
 therein. In estimating the influence of viscosity upon the mobilities 
 of caseinate ions we can entirely disregard that portion of the viscosity 
 of the solution which, although comparable in magnitude with the 
 viscosity of the solvent, is attributable to the caseinate itself. 
 
 Bearing in mind the possibility, which was indicated in the 
 previous chapter, that protein solutions may contain a pre- 
 formed molecular structure analogous to that of the jellies or 
 coagula which they can form, we are strongly impelled towards 
 the belief that the type of viscosity which solutions of proteins 
 exhibit may in some manner owe its existence to this structure, 
 and not to the type of internal friction which hinders molecular 
 
VISCOSITY 325 
 
 and ionic motion. Thus a netlike structure, such as a tennis net, 
 will offer no hindrance to the passage through it of a quickly 
 moving body which is smaller than its meshes, other than that 
 which is due to the fact that the material which composes the net 
 occupies a small fraction of the area which the body must traverse, 
 but to any force which involves deformation of the structure, for 
 instance, a force which seeks to drag it through a small tube, it will 
 offer a very considerable resistance. On the other hand the 
 resistance which is offered to a small moving body by a viscous 
 liquid (viscous, that is, in the ordinary sense) is accurately meas- 
 ured by the resistance which the liquid offers to passage through 
 a tube. Now the direct methods which we employ to measure 
 the viscosity of fluids are all such (rotation of a relatively large 
 body within the fluid; passage of the liquid through a capillary 
 tube, etc.) as would involve the deformation of any molecular 
 structure within the fluid. Our direct methods of estimating 
 viscosity do not enable us to distinguish between that type of 
 viscosity which is attributable to a structure within the fluid and 
 the other type of viscosity which we may term "true" internal 
 friction. The indirect measurement which the conductivity esti- 
 mations afford, however, only reveals the latter type of viscosity 
 and, as we have seen, when we employ this method of estimation 
 the presence of protein is found to leave the viscosity of the solvent 
 unaltered.* 
 
 If we admit, however, that the viscosity of protein solutions is 
 due to a molecular net-structure within them, then we are forced 
 to a conclusion which, in the light of our present inadequate 
 knowledge of the mechanics of ionization, appears very curious. 
 We have seen (Cf. above) that the viscosity of protein solutions 
 must be attributable, primarily, to the protein ions which they 
 contain. We are led to conclude, therefore, that the net-structure 
 
 * Save to the degree involved by the fact that the material composing the 
 net-structure occupies a certain proportion of the cross-area of the conducting 
 field. As we have seen above, the results of Dumanski show that if allowance 
 be made for this diminution in cross-area the conductivities of solutions of 
 electrolytes in gelatin jellies are nearly identical with those of equally concen- 
 trated solutions in water. The slight deviations which Dumanski observed 
 are probably attributable to the fact that the calculation of this diminution in 
 area involved the specific gravity of the gelatin in its dissolved condition, and 
 this is not necessarily the same as that of unhydrated gelatin, i.e., gelatin in 
 its undissolved condition. 
 
326 PHYSICAL PROPERTIES 
 
 within protein solutions is built up out of protein ions.* Such 
 a conclusion is totally out of harmony with the prevailing view 
 that ions, in solutions of electrolytes, are mutually independent 
 and physically discrete bodies, and would appear to invite a dis- 
 tinction between the mode of ionization of ordinary electrolytes 
 and that of protein salts. It is very questionable, however, 
 whether such a distinction would be valid. Many modern in- 
 vestigations point to the existence of some modification of the 
 familiar " Grotthus-chain " in solutions of electrolytes, most 
 impressive among which is possibly the fact that in watery solu- 
 tions the most rapidly-moving ions are those which, by their 
 combination, give rise to water itself, i.e., hydrogen and hydroxyl, 
 while in other solvents hydrogen and hydroxyl are not the most 
 rapidly moving, but those which, by their combination, give rise to 
 the solvent (24) (125) (25) (60). The fact that the viscosities of 
 solutions of crystalloid electrolytes have not so far revealed the 
 presence of a net-structure within them is possibly attributable to 
 the tenuity of the net and to the fineness of its framework; to 
 revert to the analogy employed above, a net of the finest and most 
 flexible silk will readily pass without appreciable resistance 
 through a tube which would offer a considerable resistance to the 
 passage of a net of coarse thread. The phenomena to which I 
 have drawn attention may quite conceivably be of very general 
 occurrence, and of greater physical magnitude in solutions of 
 proteins simply because of the greater size of the protein mole- 
 cules. In this connection the views regarding the nature of 
 ionization which have been put forward by Wm. Sutherland (118) 
 offer extremely tempting suggestions. 
 
 The hypothesis that gelatin solutions, even those which do not 
 gelatinize, contain a structure has also been put forward by Wo. 
 Ostwald (80). The influence of added inorganic salts upon the 
 viscosity of gelatin solutions has been studied by von Schroeder 
 (loc. cit.); this observer finds that the chlorides and nitrates of 
 the alkalies diminish the viscosity of gelatin solutions while sul- 
 phates increase it. Moreover the alteration in viscosity induced 
 by the added salts does not vary continuously in a definite sense 
 
 * Indeed, whether we assume that the viscosity of protein solutions is due 
 to protein ions or not, it would be difficult to avoid this conclusion in the light 
 of the fact that many highly viscous solutions of casemates are over 90 per cent 
 dissociated (Cf. Chap. X). 
 
VISCOSITY 327 
 
 as more of the salt is added, but the curve displaying the influence 
 of the concentration of the salt upon the viscosity of the solution 
 exhibits very marked maxima and minima. Now Ostwald has 
 found that the influence of added salts upon the swelling of gelatin 
 plates in water (Cf. Chap. XII) is of a similar character to their 
 influence upon the viscosity of gelatin solutions; that the degree 
 of swelling runs closely parallel with the diminution in the viscosity 
 of solutions, as determined by von Schroeder, since the concen- 
 trations of salts at which maxima of swelling occur are nearly identi- 
 cal with those at which minima in the viscosity of solutions are 
 observed. From this Ostwald argues that the passage of gelatin 
 into solution does not destroy the structure of the gel but that this 
 structure persists in solution. 
 
 Sutherland (119) in developing the theories to which I have 
 referred above, has also expressed the opinion that protein solu- 
 tions possess a structure.* 
 
 Of great interest in this connection is the observation which a 
 number of investigators have made (110) (59) (34) (109) (33) that 
 the viscosities of gelatin solutions, insufficiently concentrated to 
 gelatinize, change somewhat on standing. Garrett has also 
 observed (33) that the logarithmic decrement of a disc oscillating 
 in gelatin or albumin solutions is not a constant, as it is in water 
 or other homogeneous fluids, but, on the contrary, increases as a 
 linear function of the time. This was traced to adhesion between 
 the disc and the protein; if the disc was taken out of the fluid and 
 washed the initial value of the decrement was always the same. 
 This is obviously to be explained, on the basis of the above 
 hypothesis, by the adhesion of portions of the net-structure to 
 the oscillating disc. Garrett has further observed that very 
 slight mechanical disturbance of a gelatin solution produces con- 
 siderable alteration in the magnitude of the oscillation-decrement. 
 
 Preliminary heating diminishes the viscosity of gelatin solutions 
 and the magnitude of the effect is a function of the time of expo- 
 sure to the higher temperature (110) (33). Garrett attributes this 
 phenomenon to a partial destruction by heating of a structure 
 within the fluid. Ostwald (79) has found that preliminary 
 heating of gelatin increases the rapidity with which it swells in 
 water. He also interprets this by supposing that the structure of 
 
 * Cf. also C. O. Weber (128). 
 
328 PHYSICAL PROPERTIES 
 
 the gelatin is partially destroyed by heating. Garrett has also 
 found that in a solution of gelatin which had been boiled the 
 logarithmic decrement of an oscillating disc does not increase with 
 time. 
 
 According to Lichtwrtz and Renner (61) the viscosity of serum 
 albumin solutions falls with rising temperature from 15 to 60 C. 
 in the same proportion as the viscosity of water. Chick and 
 Lubrzynski (20) have also shown that the influence of tempera- 
 ture upon the viscosity of egg-albumin solutions containing up to 
 20 per cent of protein is slight and a linear function of temperature 
 being similar to the influence of temperature upon the viscosity 
 of a solution of a crystalloid. In solutions containing a higher 
 percentage of protein, however, the relationship between viscosity 
 and temperature is curvilinear, the decrease being most rapid at 
 low temperatures. From these results it would appear reasonable 
 to infer that a change of temperature at temperatures lying below 
 those necessary to induce coagulation affects the viscosity of a 
 protein solution chiefly through altering the viscosity of the solvent, 
 a deduction which is in harmony with the view expressed above 
 that proteins in solution are not in any large proportion present 
 therein in the form of independently mobile particles. 
 
 It has been shown by Christiansen (22) that acid protein solu- 
 tions attain a maximum of viscosity when a slight excess of free 
 acid is present, presumably coinciding approximately with the 
 attainment of maximum combining capacity for the acid. The 
 acidity which produces maximum viscosity is also the optimum 
 acidity for the digestive action of pepsin. The viscosity of the 
 solution decreases during digestion, a fact which had previously 
 been demonstrated by Spriggs (116). 
 
 2. The Cohesiveness of Protein Solutions. Closely allied 
 to the property of viscosity is the property which may be somewhat 
 vaguely designated "cohesiveness." The influence of a variety of 
 aqueous solutions of acids, bases and salts upon the cohesiveness of 
 gluten has been investigated by Wood and Hardy (132). These 
 observers prepared gluten by washing flour in a stream of water 
 to remove the starch. The protein residue is a coherent stringy 
 mass, insoluble in water and consisting essentially of a mixture of 
 gliadin and glutenin (Cf. Chap. II). The action of solutions upon 
 the cohesiveness of the gluten was estimated quantitatively by 
 suspending a small mass of gluten on a bent glass rod in a beaker 
 
ELASTICITY 329 
 
 containing 120 cc. of the solution. It was found that dilute acids 
 very quickly led to a very marked diminution in cohesiveness, 
 allowing the drop to fall off the rod and disperse, forming a cloudy 
 solution. More concentrated acid, however, maintains the cohe- 
 sion; in other words, with increasing concentration of acid the 
 cohesiveness of the gluten is first lowered, until it reaches a mini- 
 mum, and then raised until it regains its value in water. There 
 is no simple relationship between the concentration of an acid in 
 which cohesiveness is retained just sufficiently to hold the drop to 
 the rod and the concentration of hydrogen ions in these soluti6ns 
 as indicated by their conductivities, although higher concentra- 
 tions of weak than of strong acids are as a rule required. Salts 
 lessen the power which acids and alkalies possess of weakening the 
 cohesion of gluten, and the concentration of salt required to nullify 
 the dispersive action of the various acids varies with the concen- 
 tration of acid in a very characteristic manner which is illus- 
 trated by numerous curves in Wood and Hardy's paper. These 
 authors interpret their results on the supposition that diminu- 
 tion in the cohesiveness of the gluten is brought about by the 
 appearance of electric double layers upon the surfaces of the 
 gluten particles. 
 
 3. The Elasticity of Protein Solutions. The elasticity of 
 protein solutions and jellies has been investigated by Rankine 
 (89), Rohloff and Shin jo (98), Reiger (94), Leick (58) and Hay- 
 craft (42). They find that even dilute solutions of gelatin resist 
 deformation to a slight extent, a fact which would appear to point 
 to the existence within these solutions of some species of molecular 
 (or ionic) structure such as that indicated in section 1. This 
 " form-elasticity " of gelatin solutions is diminished by heating. 
 
 The form-elasticity of a freshly prepared gelatin jelly increases 
 with time until it attains a maximum. According to Hay craft, 
 gelatin jellies obey Hooke's law, that is to say, for moderate 
 (linear) strains the deformation which is produced is directly pro- 
 portional to the applied force, and the recovery, when the strain 
 is removed, is rapid and complete. 
 
 According to Henderson and Brink (43) the compressibility of 
 gelatin solutions is somewhat less than the compressibility of water 
 and lower the greater the concentration of gelatin in the solution. 
 With varying compression the compressibility varies in much the 
 same way as the compressibilities of other solutions. 
 
330 
 
 PHYSICAL PROPERTIES 
 
 4. The Diffusion of Proteins in Solution. It was pointed out 
 by Graham in his classic memoirs on diffusion, that the proteins, 
 like other colloids, are very sparingly diffusible. Nevertheless 
 they diffuse through water, albeit with the extreme slowness 
 indicated by their relatively enormous molecular weight. From 
 the law of Fick (10) (30) we have: 
 
 where ds is the quantity of diffusing substance which passes in the 
 time dz through a diffusion cylinder having the cross-section q, 
 c is the concentration of the substance in the whole cross-section 
 at the point x, c + dc is the same quantity at the point x + dx 
 and D is a constant peculiar to the substance, expressive of its 
 diffusibility, and known as the "diffusion coefficient." 
 
 The following are the diffusion coefficients of certain proteins, 
 estimated by the observers named and cited after Wo. Ostwald 
 (81) (45) (46). For the purpose of comparison the value of D 
 for sodium chloride is placed at the head of the list. 
 
 Substance 
 
 Observer 
 
 D 
 
 Temperature, 
 degrees 
 
 Sodium chloride 
 
 Voigtlander 
 
 1 040 
 
 20 
 
 Clupein sulphate 
 
 Herzog 
 
 074 
 
 18 
 
 Egg-albumin 
 
 Herzog 
 
 059 
 
 18 
 
 Ovomucoid 
 
 Herzog 
 
 0.044 
 
 18 
 
 Albumin 
 
 Graham-Stefan 
 
 063 
 
 13 
 
 
 
 
 
 Now it has been shown by Nernst (73) and Planck (82) that the 
 driving force which produces diffusion is osmotic pressure. Hence 
 we can conclude that the proteins, in solution, exert a definite, 
 although small osmotic pressure. 
 
 From the diffusion coefficient of egg-albumin, Herzog and 
 Kasarnovsky (46) have calculated that it exerts an osmotic 
 pressure corresponding to a molecular weight of about 20,000. 
 That proteins in solution exert a definite osmotic pressure is also 
 shown by cryoscopic, ebullition, and direct measurements, of 
 which a brief description will be found below. 
 
 It has been found by Dabrovsky (23) that the diffusion-coeffi- 
 cient of crystallized egg-albumin is decidedly greater when the 
 diffusion takes place into a solution of ammonium sulphate than 
 
FREEZING- AND BOILING-POINTS 331 
 
 when it takes place into distilled water. From the value of the 
 coefficient of diffusion and the viscosity of the liquid into which 
 diffusion occurs, it is possible, employing the formula of Einstein, 
 to compute the relative volumes occupied by the particles of egg- 
 albumin when diffusing into different solvents. Applying this 
 method of computation, Dabrovsky finds that the volume occu- 
 pied by the egg-albumin molecules when dissolved in distilled 
 water is no less than six times as great as that which they occupy 
 when dissolved in a 3.6 per cent solution of ammonium sulphate. 
 The extraordinary diminution of molecular volume which am- 
 monium sulphate brings about must undoubtedly be connected 
 with the high coagulating power of this salt and is probably to be 
 attributed to dehydration of the protein, i.e., to abstraction of a 
 galaxy of associated water-molecules (Cf. Chap. VI, section 6, 
 also this Chapter, section 6). 
 
 5. The Freezing- and Boiling-points of Protein Solutions. 
 As is well known, the depression of the freezing-point of a solvent 
 which is produced by a dissolved substance is proportional to the 
 osmotic pressure which the dissolved substance exerts in the solu- 
 tion. Similarly, the elevation of the boiling-point of the solvent 
 is proportional to the osmotic pressure of the dissolved substance. 
 Corresponding to the extremely high molecular weights of proteins 
 the osmotic pressures of their solutions (containing practical per- 
 centages of protein) are low, and the depression of the freezing- 
 point and elevation of the boiling-point of water which are brought 
 about by the introduction of protein are small. 
 
 Consequently Sabanejev (112) found that the depression of 
 the freezing-point which is brought about by egg-albumin could 
 be almost entirely accounted for by the (estimated) pressure of 
 the inorganic constituents of the protein, and Sebanejev and 
 Alexandrov (114) estimated from their determinations that the 
 molecular weight of albumin must be at least 14,000 in order to 
 account for the extremely slight lowering of the freezing-point 
 which could be attributed to the protein alone. The proteoses, 
 Sebanejev estimated, also from cryoscopic determinations, to 
 possess a molecular weight of from 2000 to 3000, while the molec- 
 ular weight of the peptones was estimated to be 400 or less (113). 
 
 Tamman (120) measured the difference between the lowering of 
 the freezing-point of the serum of the horse before and after coagu- 
 lating the proteins by heat, and removing them, and found that 
 
332 PHYSICAL PROPERTIES 
 
 the difference amounted to only 0.006 degrees, the experimental 
 error of the cryoscopic method being about 0.005 degrees. In 
 connection with experiments such as these it should be recollected, 
 however, that heating induces many modifications in protein- 
 containing solutions which might conceivably affect the osmotic 
 pressure (freezing-point) in a variety of diverse ways (92) . Liide- 
 king found that even 40 per cent of gelatin in solution did not 
 perceptibly alter the boiling-point (67) and Krafft and Wiglow 
 (55) confirmed his results. Bugarszky and Liebermann (17) 
 estimated the depression of the freezing-point due to dissolved 
 egg-albumin, albumose and pepsin, and their ash, separately, and 
 deducted the latter from the former. They estimated in this 
 manner the molecular (or, in reality mean molecular and ionic) 
 weight of egg-albumin to be 6400, that of albumose to be 2400 and 
 that of pepsin to be 760. They also found that if egg-albumin, 
 albumose or pepsin be added to solutions of acids or alkalies, the 
 cryoscopic depression of the resultant solution is less than the sum 
 of the depressions due to the acid or alkali and the proteins dis- 
 solved separately; while if the egg-albumin, albumose or pepsin 
 were dissolved in salt solutions the cryoscopic depression of the 
 mixture was found to be (within the limits of experimental error) 
 identical with the sum of the cryoscopic depressions due to the 
 protein and the salt dissolved separately. The significance of 
 these latter results has already been commented upon in Chap. IV. 
 Bugarszky and Tangl (18) carried out an extended series of 
 investigations aiming at the determination of the cryoscopic 
 depression due to the non-electrolytes of the blood, among which 
 they included the proteins. They determined the chlorine con- 
 tent of the blood and, from that, deduced the equivalent molec- 
 ular concentration of the sodium chloride in the blood and its 
 conductivity; they then measured the conductivity of the blood 
 and subtracting from it that due to sodium chloride, considered 
 the remainder as due to sodium carbonate and estimated there- 
 from the molecular concentration of the sodium carbonate. They 
 then subtracted the cryoscopic depression due to the sodium 
 chloride and sodium carbonate contents thus estimated, from the 
 observed cryoscopic depression of the blood; the difference they 
 ascribed to the non-electrolytes and proteins. In this way they 
 estimated the concentration of non-electrolytes in horse's blood 
 to be about 0.056 mol. per litre. The accuracy of this determina- 
 
FREEZING- AND BOILING-POINTS 
 
 333 
 
 tion is, however, very questionable, since it depends upon the 
 assumption that the proteins in blood are non-electrolytes. While 
 it is probable as Hardy's results indicate (Cf. Chap. VII, section 6), 
 that the proteins in normal blood are non-ionic, the possibility 
 must not be lost sight of that they may participate, as isolated 
 water soluble proteins frequently do, in the conduction of a cur- 
 rent through their solutions (Cf. Chaps. VIII and X). 
 
 The majority of the above estimates were not carried out upon 
 pure proteins. Robertson and Burnett (106), however, have 
 investigated the cryoscopic depression due to dissolved casemates 
 of the alkalies and alkaline earths, employing pure casein. The 
 following were the results obtained. 
 
 TABLE I 
 
 Caseinates containing 50X10~ 5 equivalents of base per gram 
 (Experimental error of determination 0.0025 degrees) 
 
 Base 
 
 Concentration of 
 base neutralized by 
 casein 
 
 A = depression of 
 the freezing-point 
 of water 
 
 Indicating a molec- 
 ular plus ionic 
 concentration of 
 
 NH 4 OH 
 
 m 
 
 045 
 
 m 
 
 NH 4 OH... 
 
 50 
 m 
 
 035 
 
 41 
 m 
 
 NH 4 OH 
 
 50 
 m 
 
 055 
 
 53 
 
 m 
 
 NH 4 OH... 
 
 33.3 
 
 m 
 
 055 
 
 34 
 
 m 
 
 NH 4 OH 
 
 33.3 
 m 
 
 07 
 
 34 
 m 
 
 NH 4 OH 
 
 20 
 m 
 
 095 
 
 26 
 m 
 
 KOH . 
 
 15 
 m 
 
 0325 
 
 19 
 
 m 
 
 KOH 
 
 50 
 
 m 
 
 0375 
 
 57 
 m 
 
 KOH 
 
 50 
 
 m 
 
 0425 
 
 49 
 m 
 
 KOH 
 
 33.3 
 m 
 
 0475 
 
 44 
 m 
 
 KOH 
 
 33.3 
 
 m 
 
 05 
 
 39 
 m 
 
 KOH . 
 
 20 
 m 
 
 007^ 
 
 37 
 
 m 
 
 
 20 
 
 
 25 
 
334 
 
 PHYSICAL PROPERTIES 
 TABLE L (Continued) 
 
 Base 
 
 Concentration of 
 base neutralized 
 by casein 
 
 A = depression of 
 the freezing-point 
 of water 
 
 Indicating a molec- 
 ular plus ionic 
 concentration of 
 
 KOH 
 
 m 
 
 10 
 
 m 
 
 LiOH 
 
 15 
 m 
 
 03 
 
 18.5 
 m 
 
 LiOH 
 LiOH 
 
 59.5 
 m 
 40 
 
 m 
 
 0.045 
 07 
 
 62 
 m 
 11 
 m 
 
 LiOH 
 
 24 
 
 m 
 
 08 
 
 26 
 
 m 
 
 Ca(OH) 2 
 
 18 
 m 
 
 015 
 
 20 
 
 m 
 
 Ca(OH) 2 
 
 91 
 
 m 
 
 015 
 
 120 
 m 
 
 Ca(OH) 2 
 
 91 
 
 m 
 
 0175 
 
 120 
 
 m 
 
 Ca(OH) 2 
 
 91 
 m 
 
 02 
 
 101 
 
 m 
 
 Ca(OH) 2 .. .. 
 
 61 
 
 m 
 
 02 
 
 92.5 
 m 
 
 Ca(OH) 2 
 
 61 
 
 m 
 
 02 
 
 92.5 
 m 
 
 Ca(OH) 2 
 
 61 
 m 
 
 0.025 
 
 92.5 
 m 
 
 Ca(OH) 2 
 
 45.5 
 m 
 
 0.025 
 
 74 
 m 
 
 Ca(OH) 2 
 
 45.5 
 m 
 
 025 
 
 74 
 m 
 
 
 45.5 
 
 
 74 
 
FREEZING- AND BOILING-POINTS 
 
 335 
 
 TABLE II 
 
 Caseinates containing 80X10" 5 equivalents of base per gram 
 (Experimental error of determination 0.0025 degrees) 
 
 Base 
 
 Concentration of 
 base neutralized 
 by casein 
 
 A = depression of 
 the freezing-point 
 of water 
 
 Indicating a molec- 
 ular plus ionic 
 concentration of 
 
 NH 4 OH 
 
 m 
 
 0.04 
 
 m 
 
 NH 4 OH 
 
 36 
 
 m 
 
 0.04 
 
 46 
 
 m 
 
 NH 4 OH 
 
 34 
 
 m 
 
 0.0475 
 
 46 
 m 
 
 NH 4 OH 
 
 27 
 m 
 
 0.05 
 
 39 
 
 m 
 
 NH 4 OH 
 
 25 
 
 m 
 
 0.07 
 
 37 
 m 
 
 NH 4 OH 
 
 20 
 m 
 
 0.055 
 
 26.5 
 m 
 
 NH 4 OH 
 
 18 
 m 
 
 0.075 
 
 34 
 
 m 
 
 NH 4 OH . ... 
 
 17 
 
 m 
 
 0.10 
 
 25 
 
 m 
 
 KOH 
 
 15 
 
 m 
 
 0.035 
 
 18.5 
 m 
 
 KOH 
 
 50 
 
 m 
 
 0.04 
 
 53 
 
 m 
 
 KOH 
 
 33 
 
 m 
 
 0.055 
 
 46 
 m 
 
 KOH 
 
 33 
 
 m 
 
 0.06 
 
 34 
 
 m 
 
 KOH 
 
 25 
 
 m 
 
 0.07 
 
 31 
 
 m 
 
 KOH 
 
 20 
 
 m 
 
 0.0875 
 
 26.5 
 m 
 
 KOH 
 
 17 
 
 m 
 
 0.095 
 
 20 
 m 
 
 LiOH 
 
 15 
 
 m 
 
 0.04 
 
 19 
 
 m 
 
 LiOH 
 
 40 
 m 
 
 0.05 
 
 46 
 m 
 
 LiOH 
 
 30 
 
 m 
 
 0.07 
 
 37 
 
 m 
 
 Ca(OH) 2 
 
 20 
 
 m 
 
 015 
 
 26 
 m 
 
 Ca(OH) 2 
 
 91 
 
 m 
 
 02 
 
 120 
 m 
 
 Ca(OH) 2 
 
 68 
 m 
 
 0225 
 
 92.5 
 m 
 
 
 45.5 
 
 
 80 
 
336 PHYSICAL PROPERTIES 
 
 The significance of these results and of results which I have 
 obtained with ovomucoid is commented upon in Chap. X. It may 
 further be remarked here that the observed depressions could not 
 have been due to impurities associated with the casein for the 
 following reasons: 
 
 (1) Keeping the concentration of base constant it is evident 
 that increasing the quantity of casein dissolved in it in the pro- 
 portion of 8 to 5 does not alter the observed depression in any 
 appreciable degree, whereas, if this were due to impurities associ- 
 ated with the casein, it should result in a proportionate increase 
 in the observed depression. 
 
 (2) Keeping the concentration of casein constant and increasing 
 the concentration of base bound by it in the proportion of 8 to 5 
 results in a proportionate increase in the observed depression. 
 
 The cryoscopic depression is, therefore, obviously conditioned 
 primarily by the combined alkali. This might be interpreted to 
 indicate that the depression is in reality due to the base and not to 
 the protein. This, however, is not the case, since, as we have seen 
 (Chaps. V and IX), these solutions were respectively neutral to 
 litmus and to phenolphthalein and therefore contained no, or only 
 a trace of, free base. Nor were the depressions due to inorganic 
 ions, since, as we have seen (Chap. VIII), such solutions contain no 
 inorganic ions. As I have explained in Chap. X, the observed 
 depressions must be attributed primarily to protein ions, each 
 equivalent of combined base yielding the same number of protein 
 ions, derived through the splitting of successive N.HOC 
 bonds. 
 
 One fact should be especially commented upon here, and that 
 is that the observed depressions in the two sets of solutions ex- 
 amined stood in direct proportion to the concentration of combined 
 base. If this were so for other concentrations, then at zero con- 
 centration of combined base, if casein were soluble under such 
 conditions, the freezing-point depression due to dissolved casein 
 would be zero. In other words the possibility is indicated that 
 base- and acid-free protein may exert an immeasurably small 
 osmotic pressure. I have elsewhere attributed this to polymeri- 
 zation of the protein as the uncombined protein is set free (104) 
 (106). 
 
 6. The Osmotic Pressure of Proteins in Solution. The 
 direct determination of the osmotic pressure of protein solutions is 
 
OSMOTIC PRESSURE 337 
 
 a task fraught with immense difficulties, on account of the difficulty 
 of preparing ideally pure proteins. The original investigations 
 of Graham (35) appeared to indicate that colloids in general exert 
 a high osmotic pressure. Subsequent investigators, however, 
 attributed these results to an admixture of crystalloids. Starling 
 endeavored to measure directly the osmotic pressure of the pro- 
 teins in blood serum by using for his osmometer a membrane 
 permeable to salts but impermeable to proteins (117) and this 
 method has since then been employed in all accurate work upon 
 the subject, since, as Reid has pointed out (92) it is the only method 
 of procedure which is applicable to the problem. We have no 
 assurance that any given protein preparation is totally free from 
 (not necessarily conductive) impurities which may influence the 
 direct measurement of osmotic pressure; it is, therefore, essential 
 to employ a membrane which is permeable to such impurities and 
 thus, if time be allowed for the system to come to equilibrium, 
 differentiates between protein and non-protein constituents of the 
 solution investigated. For this purpose Reid employs a mem- 
 brane of vegetable parchment, which, as he has shown, is per- 
 meable even to nucleic acid although it is impermeable to the 
 proteins employed by him in his investigations. By extremely 
 prolonged purification Reid has succeeded in obtaining prepara- 
 tions of egg-albumin which exhibit no measurable osmotic pressure 
 when examined by this method. In subsequent investigations 
 (93), however, he obtained osmotic pressures, due to dissolved 
 haemoglobin, of perfectly constant value and such as to indicate 
 a molecular weight of about 48,000. Barcroft and Hill (4) have, 
 however, demonstrated by thermodynamical methods that in 
 solutions containing haemoglobin prepared by less prolonged 
 dialysis the molecular weight of this substance is about 16,669. 
 Roaf (96) employing the differential osmotic method just de- 
 scribed, finds that the molecular weight of haemoglobin, dissolved 
 in distilled water, is about 32,000, while in sodium carbonate 
 solutions it is 16,000. These results appear to confirm the view 
 (Cf. also Barcroft and Hill loc. cit.) that non-ionic protein is 
 polymerized and so exerts a considerably smaller osmotic pressure 
 than ionic protein. 
 
 Benj. Moore and Roaf and collaborators (70) (71) (72) (95) (97) 
 and R. S. Lillie (63) have made the extremely interesting discovery 
 that the osmotic pressure which is exerted by proteins (deter- 
 
338 
 
 PHYSICAL PROPERTIES 
 
 mined differentially, as described) varies very pronouncedly with 
 the nature of the inorganic acids, bases or salts which their solu- 
 tions contain.* 
 
 The following are among Lillie's results, obtained when dilute 
 acids and alkalies were employed as solvents: 
 
 TABLE III 
 1.5 Per Cent Gelatin in Dilute HC1 Solutions 
 
 Solvent 
 
 Osmotic pressure of 
 protein in mm. Hg. 
 
 Water 
 ra/3100 HC1 
 m/2050 HC1 
 m/1550HCl.- 
 ra/1025HCl 
 ra/770 HC1 
 
 8.2 
 6.8 
 12.3 
 17.9 
 26.5 
 32.4 
 
 ra/620 HC1 
 ra/412 HC1 
 
 34.9 
 39.3 
 
 
 
 TABLE IV 
 1.5 Per Cent Gelatin in Dilute KOH Solutions 
 
 Solvent 
 
 Osmotic pressure of 
 protein in mm. Hg. 
 
 Water 
 
 7.9 
 
 w/3100 KOH 
 
 14.1 
 
 m/620 KOH 
 
 23.7 
 
 w/412 KOH 
 
 25.1 
 
 m/310 KOH 
 
 29.0 
 
 
 
 In Lillie's words, "In the presence of either acid or alkali the 
 osmotic pressure of gelatin thus shows a marked increase, which, 
 within the above range of concentrations, exhibits a certain pro- 
 portionality to the quantity of acid or alkali added. For equiva- 
 lent concentrations, acid produces a somewhat greater increase 
 than alkali. The change in osmotic properties is to be attributed 
 to a finer subdivision of the colloid particles and a consequent 
 increase in the surface of intersection between colloid particles and 
 
 * In this connection it is of interest to note that von Wittich (131), von 
 Regeczy (91), Oker-Blom (76) and others have shown that proteins diffuse 
 mote rapidly into sodium chloride solutions than they do into distilled water. 
 Cf. also Dabrovsky (23). 
 
OSMOTIC PRESSURE 339 
 
 medium." These results are obviously in complete agreement 
 with results obtained by Robertson and Burnett, employing the 
 cryoscopic method, which are commented on above. The decrease 
 in pressure observed in very dilute acid may be attributable to the 
 neutralization by the acid of base bound by the protein prepara- 
 tion employed. Lillie draws an analogy between his results and 
 those obtained by Ostwald (78) (79) in investigating the swelling 
 of gelatin plates in dilute acid and bases. Egg-albumin, how- 
 ever, exhibits the opposite behavior, namely, the osmotic pressure 
 which it exerts is slightly diminished by acids and bases. 
 
 The osmotic pressure of gelatin and egg-albumin is unaffected 
 by the addition of non-electrolytes such as cane-sugar, dextrose, 
 glycerol and urea, but is considerably affected by the addition of 
 inorganic salts being (and this is true both for gelatin and egg- 
 albumin) depressed thereby. According to Lillie the depression 
 of the osmotic pressure exerted by this protein is a function of the 
 nature of both the anion and cation of the added salt. It increases 
 in the order alkali metals < alkaline earths < heavy metals (for 
 cations) ; and CNS < I < Br < N0 3 < Cl < F < plurivalent 
 anions, S0 4 , tartrate, citrate, phosphate (for anions). This 
 observation is of extraordinary significance when we recollect that 
 this is the order in which, according to Hofmeister and Pauli, the 
 various ions bring about the dehydration and coagulation of the 
 salts which proteins form with bases (Cf. Chap. VI).* 
 
 Very remarkable phenomena are displayed by the solutions of 
 soluble chitin which have been prepared by Alsberg and Hedblom 
 (1) from the chitin of Limulus polyphemus by prolonged treatment 
 with weak hydrochloric acid. The chitin when subjected to this 
 treatment forms at first a gelatinous mass and later a colloidal 
 solution. The analytical figures obtained for this "soluble 
 chitin" and the gelatinized chitin obtained by adding KOH and 
 then reneutralizing with HC1 are best explained, according to the 
 authors, by assuming that the chitin, on passing into colloidal 
 solution, unites with water. Soluble chitin depresses the freezing 
 point but slightly, so that its molecular weight is probably very 
 high. It passes through collodion and parchment paper, but has 
 the extraordinary property of carrying the water in which it is 
 
 * Moore and Roaf (72) have sought to explain the coagulation of proteins 
 by salts by the formation of large aggregates, basing their argument upon data 
 similar to these obtained by Lillie. 
 
340 PHYSICAL PROPERTIES 
 
 dissolved through the membrane so that the space within the 
 latter may become nearly empty. It is probable that these phe- 
 nomena depend rather upon the extreme affinity of this colloid for 
 water than upon ordinary osmotic forces. 
 
 C. J. Martin (68) and W. M. Bayliss (5) find that the os- 
 motic pressures of egg-albumin and of haemoglobin solutions vary 
 directly with the absolute temperature, thus obeying the law of 
 Gay-Lussac. 
 
 7. The Nature of Protein Solutions. The view has been held 
 by many investigators that the proteins, and colloids generally, do 
 not enter into true solution, forming molecularly dispersed systems 
 which obey the law of Avogadro, but are to be regarded, on the 
 contrary, as forming stable suspensions when they appear to pass 
 into solution in water; a view which reaches its most extreme 
 expression in the statement of Duclaux (26) that "Les Colloids 
 doivent etre consideres comme etant, dans Peau, d'une insolubilite 
 absolue." Others, arguing* from the a priori assumption that 
 "solutions" of colloids are necessarily, in reality, suspensions, have 
 objected to the application of theoretical principles to them which 
 involve the law of Avogadro (such as the mass-law, etc.). It is 
 obvious that this is a reversion of the customary procedure of 
 science; the applicability of a law is in the first place a question in 
 itself, independent of any other generalizations, and, in the second 
 place, the applicability of a law involving Avogadro's law may in 
 itself be regarded as presumptive evidence that the law of Avo- 
 gadro applies as well. 
 
 From what has been said above, it is evident that under certain 
 conditions, particularly in the form of their salts, the proteins 
 diffuse in water and exert a definite osmotic pressure. Hence 
 Avogadro's rule must hold good, although the time required for 
 the protein molecules to distribute themselves uniformly through- 
 out any given volume of fluid may conceivably, in certain instances, 
 be very great. 
 
 Apart altogether from direct observation, however, the posses- 
 sion of a definite osmotic pressure by proteins and certain other 
 colloids in solutions is directly deducible from the fact that chemi- 
 cal reactions which involve them attain definite equilibria (101). 
 The work which is performed in the transformation of a molecule 
 of substance is the sum of two factors, the one the chemical work 
 * For example, W. Nernst (75). 
 
NATURE OF PROTEIN SOLUTIONS 341 
 
 which is involved in the transformation, the other the work per- 
 formed in bringing the molecule to the pressure of the system (56) ; 
 the latter factor is, of course, dependent upon the concentration of 
 the substances within the system, while the former, equally 
 obviously, is not. If the reacting components in a system exerted 
 no osmotic pressure whatever, the expression for the work done in 
 the transformation of a given mass of the components would, 
 therefore, be a constant one (depending only upon temperature), 
 and, consequently, at any stage of the reaction the work done in 
 transforming unit mass would be the same, whatever the concen- 
 tration of the reacting components. Under these conditions the 
 reaction would always proceed to an end in one direction or the 
 opposite; since the work performed at every stage of the reaction 
 would be unaffected by the concentration of the reacting com- 
 ponents. Now it has been shown that the reactions between 
 toxins and anti-toxins, lysins, and anti-lysins, etc., attain definite 
 equilibria (3) and hence these bodies must, in solution, exert 
 definite osmotic pressure. The probable protein nature of these 
 bodies has been commented upon in a previous chapter (Chap. 
 VII, section 7). Moreover, it has been shown that a definite 
 equilibrium is attained between proteins and the antibodies which 
 are produced in the serum when these proteins are injected into 
 the circulation (3), and definite equilibria are attained between 
 different proteins in solution (121) and between proteins and in- 
 organic acids and bases (Cf. Chap. IX) and in protein digests (122) 
 (123) (99) (102) (107). Many of these equilibria, it has been 
 shown, can be approached from either direction, so that they are 
 not " false" equilibria (27) attributable to the internal molecular 
 friction or hysteresis of the systems. We may, therefore, safely 
 conclude that in many instances, and especially in the form of 
 their salts, proteins and bodies allied to proteins exert, when in 
 solution, definite osmotic pressures; and are distributed in molec- 
 ular dispersion throughout their solutions in accordance with the 
 law of Avogadro. 
 
 It is, of course, not for a moment contended that this is the case 
 in every apparent "solution" of protein. Proteins, like other 
 bodies, and more easily than the majority of crystalloids, can also 
 be obtained in a suspended condition. Under such circumstances 
 they may form apparently stable suspensions, simulating true 
 solutions in certain respects. An illustration of such suspension 
 
342 PHYSICAL PROPERTIES 
 
 is afforded by potassium caseinate in 75 per cent alcohol (Cf. 
 Chap. XI). 
 
 Certain authors, following Graham, regard all colloidal solu- 
 tions, including protein solutions, as being " supersaturated." 
 The above considerations dispose of this view also. Linebarger 
 (64) has also shown, by direct experimentation on the rate of coagu- 
 lation, that solutions of proteins cannot be correctly regarded as 
 supersaturated. Very numerous observations, detailed in previous 
 chapters, will occur to the reader which also conflict with this view, 
 for example, the constancy of the proportion between base and 
 casein at " saturation" of the former with the latter. 
 
 8. The Opalescence of Protein Solutions; the Tyndall Effect. 
 The majority of protein solutions are decidedly opalescent, 
 i.e., they contain particles of sufficient size to scatter and reflect a 
 proportion of transmitted light. Even when the opalescence is 
 very small, when the solution is viewed at right angles to the 
 direction of a pencil of light traversing it, a certain degree of 
 scattering (the Tyndall effect) is observed. Upon this fact many 
 investigators have based the view that proteins in solution form 
 heterogeneous and not molecularly dispersed systems. 
 
 It has, however, been pointed out by Konovalov (53) that the 
 dust particles, which render it so difficult to obtain even the purest 
 water in an "optically void" condition, may act as nuclei, under 
 conditions in which the expenditure of energy necessary for an 
 alteration in concentration is very small (i.e., when the osmotic 
 pressure of the dissolved substance is very small), attracting 
 layers of the dissolved substance and so producing opalescence. 
 In this way it is possible that the observed opalescence in such 
 solutions is due only to a minute proportion of the dissolved sub- 
 stance condensed by capillary action around accidental insoluble 
 contaminations. Arrhenius (3) has advanced a similar view. "It 
 may, however, be urged that the presence of some submicroscopic 
 particles does not prove at all that the whole of the quantity of 
 protein present, or even a considerable part of it, is in this state 
 of pseudo-solution." 
 
 It is, however, not at all inconceivable that the opalescence of 
 
 many colloidal solutions may be due to the grossness of the colloid 
 
 molecules themselves,* but it is unlikely that this is the case in 
 
 solutions of proteins. It has been shown by Lobry de Bruyn (16) 
 
 * C. O. Weber (128), p. 75. 
 
OPALESCENCE 343 
 
 that the smallest particles which are capable of scattering light are 
 from fifty to a hundred times smaller than the mean wave length 
 of light at 0.5 /*, the diameter of such particles could be not less 
 than from 5 to 10 MM- Now we have seen in Chaps. VIII and IX, 
 that the molecular weight of casein in solutions containing 11.4 X 
 10~ 5 equivalents of base per gram of protein, is probably about 
 17,600 (estimated from electrochemical data). From the analysis 
 of Hammarsten (39) it appears that if casein be possessed of this 
 molecular weight, then its empirical formula must be, approxi- 
 mately: 
 
 Now it has been shown by Kopp (54) that the relative molecular 
 volumes of organic substances can, with certain marked exceptions, 
 be calculated as an additive function of their constituent atoms.* 
 The mode in which the valencies of the atoms are distributed 
 affects their contribution to the total volume, however, "carbonyl" 
 oxygen contributing an amount differing from that contributed by 
 hydroxyl oxygen. From the analysis of Abderhalden and others it 
 would appear that if casein possesses the above molecular weight 
 it must contain about four molecules of tyrosin, and one each of the 
 other hydroxy-acids which it is known to contain, namely, serin 
 and caseinic acid, or in all, neglecting the necessarily few terminal 
 COOH groups (Cf. Chap. I) it would appear to contain only 
 about 8 atoms of hydroxyl oxygen out of the 250 atoms of oxygen 
 which are present. But it will be recollected (Chap. I) that there 
 is strong reason to suspect that in many of the COHN groups 
 within the protein molecule the oxygen is in the hydroxy condition. 
 We must, therefore, make two estimates of the molecular volume, 
 one on the supposition that the molecule contains only carbonyl 
 oxygen and the other on the supposition that it contains only 
 hydroxyl oxygen. The true volume will, it is probable, lie be- 
 tween the two. Now the difference in molecular volume due to 
 carbon is 11, that due to hydrogen is 5.5, to nitrogen (as in am- 
 monia) 2.3, to carbonyl oxygen 12.2, to sulphur 22.6, to phos- 
 phorus 25.5, yielding, for the first estimate of the relative molecular 
 volume of casein: 
 
 777 X 11 + 1241 X 5.5 + 197 X 2.3 + 250 X 12.2 + 4 X 22.6 
 + 5 X 25.5 = 19,060; 
 
 * The figures employed in the calculation which follows are cited after 
 Ostwald (77). 
 
344 PHYSICAL PROPERTIES 
 
 a second estimate, regarding the oxygen as hydroxyl oxygen 
 (difference due to one atom of hydroxyl oxygen = 7.8), yields the 
 molecular volume 17,960. The volume, estimated in this way, 
 therefore, lies between 18,000 and 19,000. The absolute volume of 
 
 i i -n +u 't r u 18,000 , 19,000 , 
 
 the molecule will therefore he between - and or about 
 
 o.o o.o 
 
 3400 times that of a hydrogen molecule, and about 523 times that 
 of a molecule of carbon dioxide. The diameter of the molecule 
 will, therefore, be approximately v^523 = about 8 times that of a 
 molecule of carbon dioxide. Now the diameter of a molecule of 
 C02 is, according to Nernst (74), 0.3 w, hence that of a molecule 
 of casein must be about 2.4 /*/*, or about one-half the diameter of 
 the smallest particle which will scatter transmitted light. Ad- 
 mitting the great uncertainty which, in our present state of 
 incomplete knowledge, attaches to such estimations as these, we 
 must yet admit that it is improbable that the opalescence of solu- 
 tions of proteins such as casein is directly attributable to the 
 protein molecules themselves; and it appears the more improbable 
 when we recollect that in pronouncedly opalescent solutions casein 
 salts, and many other protein salts are, as we have seen (Chaps. 
 VIII-X), quite extensively ionized, that is, split up into particles 
 which occupy, themselves, a fraction of the volume computed 
 above. When we recollect that the non-filterable character of 
 many proteins in solution (Cf. Chap. VII, section 6) and the vis- 
 cosity of protein solutions (Cf . the earlier part of this chapter) are 
 alike attributable to ionic protein, the suggestion offers itself that 
 the opalescence of protein solutions (as distinguished from that of 
 suspensions of partially coagulated protein) may possibly be 
 attributable to the peculiar characteristics of ionic protein. It is 
 possible that the non-filterable character of ionic protein and its 
 power of scattering transmitted light are alike due to a bulky 
 water-complex, which is associated with each of the protein ions 
 (Cf. Chap. VI, section 6 and Chap. VII, section 6). In support of 
 this view may be mentioned the remarkable fact that the addition 
 of alcohol or of acetone, in quantities insufficient to initiate coagu- 
 lation, to solutions of the caseinates leads to a very decided de- 
 crease in their opalescence. On the other hand, it is possible that 
 the net-like structure which we have seen reason to believe that 
 protein ions form in solution (section 1, this chapter) is account- 
 able, not only for the viscosity of solutions of ionic protein, but 
 
SURFACE TENSION 345 
 
 also for the non-filterability of ionic protein and the opalescence 
 of its solutions. Between these alternatives, if indeed they are 
 alternatives, our data do not, as yet, suffice for us to decide. 
 
 The opalescence of protein suspensions has been made by Kober 
 (52) the basis of a method of determining proteins quantitatively 
 (Cf. Chap. III). 
 
 9. The Surface Tension of Protein Solutions. The influence 
 of dissolved protein upon the tension of air-water surfaces has been 
 investigated by Quincke (85) (86), Zlobicki (134), Frei (31), Buglia 
 (19), Iscovesco (59), Shorter (115), Bottazzi (13) (15), and Berc- 
 zeller (9). Gelatin, egg-globulin and haemoglobin diminish the 
 air- water tension.* The diminution is greater the higher the 
 temperature (between and 24.4 degrees, Zlobicki). The surface- 
 tension of gelatin solutions and blood serum is increased by the 
 addition of small quantities of alkali, and diminished by the 
 
 'addition of small quantities of acid (Buglia, Frei). 
 
 According to Bottazzi (13) (15) the surface tension of serum 
 albumin solutions is at a maximum when their ionization is at a 
 minimum and at a minimum when their ionization is at a maxi- 
 mum. The maximum depression of the surface tension of water 
 by serum albumin occurs at a reaction just on the acid side of 
 neutrality. Undissolved protein does not affect the surface ten- 
 sion of water when shaken up with it (9). 
 
 According to Berczeller (9), the surface tension of a protein 
 solution which is so electrolyte-free as not to coagulate on heating 
 nevertheless diminishes on heating. This reduction of surface 
 tension is reversible, for on standing for some time at ordinary 
 temperature the solution regains its original surface tension. 
 
 The surface tension of protein solutions diminishes during 
 digestion (9). 
 
 10. The Formation of Surface-films by Dissolved Proteins. 
 Proteins, when dissolved in water, would appear, as a rule, to 
 diminish the surface tension at surfaces bounded by substances 
 other than air, since they tend to become concentrated at such 
 surfaces and form films of insoluble protein there, and, as Willard 
 Gibbs pointed out in his classic memoir on equilibrium in hetero- 
 geneous systems, such a tendency is indicative of a diminution in 
 the free energy of the surface at which such concentration occurs. 
 
 * According to Iscovesco, however, pure egg-albumin raises the air-water 
 tension. 
 
346 PHYSICAL PROPERTIES 
 
 This phenomenon has received extended consideration at the hands 
 of Hermann (44), Berthold (11), Ramsden (87) (88), and Shorter 
 (115), who have shown that protein solutions can be wholly 
 or partially coagulated by mere mechanical agitation, or by the 
 purely surface-action of fine powders, such as burnt clay or char- 
 coal. Ramsden also explains the formation of a film at the surface 
 of heated milk and other protein solutions in the same way.* 
 
 The formation of these protein films by surface-action can be 
 very conveniently illustrated by the following experiments (100) 
 (103). If chloroform be shaken up with casein, gelatin or pro- 
 tamin solutions, it settles in fine particles or droplets, which, if 
 numerous, form a milky layer at the bottom of the vessel; by 
 transmitted light, however, they appear perfectly clear. These 
 droplets are extraordinarily stable, and do not coalesce, however 
 long they stand in contact; they may be repeatedly washed in 
 water until all traces of protein have been removed from the super- 
 natant fluid, and they still remain perfectly stable and distinct 
 from one another; they may be shaken up in chloroform itself or 
 treated with JV/10 potassium hydroxide without impairing their 
 form or stability. If, however, they be heated to nearly the 
 
 * The non-filterability of many proteins, that is, their inability to pass 
 through the pores of a clay filter has, by some authors, also been attributed to 
 surface-action. This factor alone, however, is insufficient to explain the total 
 non-filterability of these proteins through certain filters. True, when solutions 
 of substances which reduce the surface-tension at the surfaces of insoluble 
 powders are filtered through such powders, the first portion of the filtrate 
 which filters through contains less of the dissolved substance than the original 
 solution (Cf. J. J. Thomson (124)); for example, when potassium perman- 
 ganate solution is filtered through fine quartz sand the first portions of the 
 nitrate may be colorless. But subsequent portions of the nitrate contain 
 a greater proportion of the solute and, finally, the unaltered solution filters 
 through, owing to the fact that the surfaces of the pores of the filter are 
 now fully coated by a film of more than molecular thickness. If this were 
 the only factor concerned in holding back dissolved proteins from passing 
 through clay filters, the protein would sooner or later pass through the filter, 
 which, in many cases it does not (Cf. F. W. Zahn (133), J. Lehmann (57)). 
 True, it is possible that the first portions of the protein which are de- 
 posited upon the walls of the pores, partially clog up the pores and narrow 
 them, thus assisting the filter to hold the protein back; but even if this be the 
 case, the ultimate factor which determines the impermeability of such filters 
 for proteins must either be the grossness of the protein particles themselves, 
 or of their associated water-complexes, or else the existence within the protein 
 solution of a structure connecting together the protein particles. 
 
SURFACE-FILMS 347 
 
 boiling point of chloroform, under a layer of water, the droplets 
 burst and coalesce, forming a homogeneous layer of chloroform. 
 If treated with alcohol they immediately dissolve, leaving a fine 
 membraneous precipitate of protein floating in water. Thus, if we 
 shake up chloroform with about twice its volume of a 1 per cent 
 solution of protamin sulphate, and, after standing, separate the 
 chloroform droplets by pouring off the supernatant liquid, and 
 then, after repeatedly washing the droplets in water by decanta- 
 tion, add to the small amount of supernatant water about an equal 
 volume of alcohol and gently agitate, the droplets which are thus 
 stirred up into the alcohol-water layer can be seen to swell up 
 rapidly and burst, and the fine membranes which surround them 
 can be seen falling down through the alcohol-water. If we now 
 add several volumes of alcohol and shake up the liquid, the chloro- 
 form droplets all disappear, and what we now have is a clear, 
 homogeneous solution in which innumerable minute membranes 
 can be clearly seen floating.* 
 
 That these surface films possess quite different properties from 
 the protein in the body of the fluid is evident; this is shown by 
 their great insolubility and by the fact that they are not readily 
 acted upon by acids and alkalies. Now many investigators have 
 pointed out that at the surface separating two phases of a system 
 marked changes in chemical equilibrium frequently occur (74) 
 (62) (48), owing to the alteration in chemical potential which 
 occurs at such surfaces (124). It appears probable that at surfaces 
 within protein solutions similar changes in equilibrium occur, 
 leading to the formation of polymers or anhydrides of the protein.f 
 These alterations in equilibrium are very slowly reversed upon 
 removal of the protein from the surface which caused the change 
 in equilibrium; this is shown by the insolubility of the coagula 
 which are produced in this way. Evidently some phenomenon 
 analogous to hysteresis prevents or greatly delays complete return 
 
 * The formation of these films also explains the power which proteins 
 possess to render many emulsions stable, which are not stable in pure water 
 (Cf., for example, Jamison and Hertz (50)). It also explains the tendency of 
 protein solutions to form foams. For the analogous part which is played by 
 soap films Cf. Quincke (84) and T. Brailsford Robertson (104). 
 
 t In this connection it should be noted that the increase in the concentra- 
 tion of proteins at such surfaces, due to the diminution in the surface energy 
 which the protein causes, must be accompanied by a corresponding diminution 
 of the active mass of water at these surfaces. 
 
348 PHYSICAL PROPERTIES 
 
 to the equilibrium which was disturbed. The occurrence of such 
 phenomena of hysteresis in heterogeneous systems containing 
 proteins, and their probable dependence upon the internal friction 
 of the solid phase have been commented upon in connection with 
 the heat-coagulation of proteins (Cf. previous chapter). 
 
 I have shown that the films which are formed by gelatin around 
 chloroform droplets are permeable to alcohol and also, but less, 
 permeable to chloroform. The membranes are permeable to 
 various substances in the following order, those to which they are 
 most permeable being placed first, alcohol, ether, ethyl acetate, 
 scharlach R., chloroform, toluol, xylol, carbon bisulphide. 
 
 The interesting observation has been made by Shorter (115) 
 that the surface-elasticity of freshly prepared protein solutions 
 undergoes a progressive increase with time. These increases are 
 not uniform but step-like, and represent, with irregular fluctua- 
 tion, the deposition of successive layers of molecular thickness. 
 Equilibrium is only attained with extreme slowness, a phenome- 
 non which Shorter attributes to the fact that in solutions an in- 
 crease in concentration of the protein at any point has a large 
 effect upon surface-tension but only a small effect upon the osmotic 
 pressure of the underlying layers of solution. In concentrated so- 
 lutions of protein, after a very prolonged period, it was found in 
 some cases that the progressive thickening of the surface-membrane 
 was suceeded by thinning. It is not certain, however, where such 
 long periods of time are concerned, that autohydrolysis of the pro- 
 tein may not have contributed toward modification of the results. 
 
 The possible biological significance of protein films of this char- 
 acter has been discussed at some length in my communications 
 referred to above. 
 
 11. The Specific Gravities of Protein Solutions. We have 
 seen (Chap. XII) that the taking up of water by concentrated 
 proteins (gelatin) is accompanied by a volume-contraction (67); 
 hence in concentrated protein solutions the specific gravity cannot 
 be calculated directly from the specific gravities of the solvent and 
 solute respectively. 
 
 The specific gravities of dilute protein solutions have been in- 
 vestigated by Haebler (38), Lehnstein (66) and others. According 
 to the latter author, the change in the specific gravity of a sodium 
 hydroxide solution due to the introduction of protein is directly 
 proportional to the percentage of protein introduced. 
 
SPECIFIC GRAVITY 349 
 
 Chick and Martin (21) have compared the densities of proteins 
 in the dissolved and in the dry conditions and find invariably an 
 increase in density of the dissolved protein corresponding to the 
 volume-contraction which accompanies the passage of proteins 
 into solution. Thus dry casein suspended in benzol has a specific 
 gravity of 1.318, while a 7.85 per cent solution of casein in dilute 
 sodium hydroxide has a specific gravity of 1.0240, whence the 
 density of the dissolved sodium caseinate = 1.42, while correcting 
 for the effect due to sodium hydroxide, the dissolved casein itself 
 has a density of 1.39. 
 
 The following table summarizes the results obtained with 
 various proteins: 
 
 Protein 
 
 Density in solution 
 
 Density in the dry 
 state 
 
 Casein 
 
 1.390 
 
 1.318 
 
 Crystallized egg-albumin 
 
 1.359 
 
 1.269 
 
 Crystallized serum-albumin 
 
 1.378 
 
 1.275 
 
 Serum globulin 
 
 1 374 
 
 1 293 
 
 
 
 
 The more dilute a solution of sodium caseinate, the greater its 
 apparent density was found to be. This was not the case in solu- 
 tions of serum globulin, or albumin. 
 
 12. The Magnetic Properties of the Proteins. It was shown 
 in 1845 by Faraday (29) and later by Pliicker (83) that haemo- 
 globin contains iron; it is, nevertheless, like the other constituents 
 of living tissues, decidedly diamagnetic. This observation has 
 been confirmed in an extended series of investigations by Gamgee 
 (32), who finds that not only oxyhsemoglobin but also CO-haemo- 
 globin and methsemoglobin are strongly diamagnetic bodies. The 
 iron-containing radical, hsematin, and its derivative hsemin are, 
 on the contrary, as might be expected from their content of iron, 
 strongly magnetic bodies. 
 
 Benedicenti (7) (8) finds that the presence of protein inhibits 
 the lowering of the diamagnetic constant of water by the addition 
 of dissolved ferric chloride or finely pulverized iron. 
 
 13. "The Gold-Number" of Proteins. It was found by 
 Zsigmondy (135) that colloidal gold is precipitated from its solution 
 (or suspension) by the addition of sodium chloride, but that this 
 precipitation can be prevented by the addition to the solution of 
 many other colloids, among which may be reckoned the proteins. 
 
350 PHYSICAL PROPERTIES 
 
 This phenomenon was investigated more fully by Schulz and 
 Zsigmondy (10), who define the number of milligrams of a colloid 
 which is just insufficient to prevent 10 cc. of a colloidal gold solu- 
 tion from showing a change in color after the addition of 1 cc. 
 of 10 per cent NaCl solutions as the " gold-number " of the colloid. 
 It appears to be highly characteristic for certain proteins. The 
 following are the values determined by Schulz and Zsigmondy for 
 the various proteins contained in or derived from egg-white: 
 
 Globulin 0.02-0.05 
 
 Ovomucoid . 04-0 . 08 
 
 Crystallized egg-albumin 2 . 00-8 . 00 
 
 Other (con-) albumin . 03-0 . 05 
 
 Alkali-albuminate 0.006-0.04 
 
 The great difference between the crystalline albumin " gold- 
 number" and that of the other proteins in egg-white made it 
 possible to recognize the presence of very small quantities of 
 colloidal contamination in the egg-albumin. Schulz and Zsig- 
 mondy were able to show by this method that egg-albumin must 
 be recrystallized from three to six times to remove appreciable 
 quantities of such contaminations. 
 
 The determination of the " gold-number " of cerebro-spinal 
 fluid has been found to be of very great value in the differential 
 diagnosis of the various forms of syphilitic sequelae, the modifi- 
 cations observed being attributable to the marked increase in 
 globulin-content which accompanies and characterizes certain of 
 these conditions. 
 
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CHAPTER XIV 
 
 OPTICAL PROPERTIES OF PROTEIN SOLUTIONS* 
 
 1. The Specific Rotatory Power of the Proteins. It was 
 
 observed by Hoppe-Seyler (16) (18) that the majority of animal 
 proteins, in aqueous solutions, rotate the plane of polarized light 
 to the left, and he measured the specific rotatory powers of several 
 of the animal proteins. These investigations were continued by 
 Fredericque (7) and Ktihne (26) who proposed employing the 
 specific rotatory power of a protein as a means of characterizing 
 it and establishing its identity. The results which were obtained 
 by these observers have been greatly extended and amplified by 
 a large number of observers who have investigated this physical 
 property of a variety of proteins. It appears that all of the 
 naturally occurring proteins, excepting the nucleo-proteins and 
 haemoglobin, rotate the plane of polarized light to the left. The 
 following are among the results which have been obtained by 
 observers who have worked with proteins of animal origin: 
 
 TABLE I 
 
 Protein 
 
 Observer 
 
 Nature of solution 
 
 Degrees 
 
 Serum albumin 
 Serum albumin 
 
 Hoppe-Seyler (20) 
 Fredericque (7) 
 
 in water 
 
 -56.00 
 57 30 
 
 Serum albumin 
 Serum albumin 
 
 Serum albumin 
 
 Starke (62) 
 Sebelien (59) 
 
 Maximovitsch (31) 
 
 dialyzed solution 
 in distilled water 
 
 -60.05 
 -60. 10 to -62. 60 
 
 -47.47 
 
 Egg albumin 
 
 F. G. Hopkins (15) 
 
 dilute ammonium 
 
 -30.70 
 
 Egg albumin 
 
 Osborne and Camp- 
 
 sulphate 
 water 
 
 28 60 to 30 80 
 
 Lact-albumin .' 
 
 bell (38) 
 Sebelein (59) 
 
 dialyzed solution in 
 
 -36. 40 to 36. 98 
 
 a-crystallin from the crystalline 
 lens . 
 
 Morner (33) 
 
 distilled water 
 neutral solution in 
 
 -46 90 
 
 
 
 dilute ammonia 
 
 
 * The opalescence of protein solutions has already been discussed (Chap 
 XIII, 8). 
 
 355 
 
356 
 
 PHYSICAL PROPERTIES 
 
 TABLE I. (Continued) 
 
 Protein 
 
 Observer 
 
 Nature of solution 
 
 Degrees 
 
 /3-crystallin from the crystalline 
 lens . .. . 
 
 Morner (33) 
 
 dialyzed solution 
 
 43 30 
 
 Fibrinogen 
 Casein 
 Casein 
 
 Mittelbach (32) 
 Hoppe-Seyler (20) 
 Hoppe-Seyler (20) 
 
 in dilute acetic 
 acid 
 dilute salt solution 
 neutral solution 
 faintly alkaline 
 
 -52.50 
 -80.00 
 76 00 
 
 Casein 
 
 Hoppe-Seyler (20) 
 
 strongly alkaline 
 
 91 00 
 
 Serum globulin 
 
 Fredericque (7) 
 
 dilute saline solu- 
 
 47 80 
 
 Ovomucoid 
 
 Osborne and Camp- 
 
 tion of mixed 
 globulins 
 
 61 10 to 61 38 
 
 Alkali-albumose from egg-albu- 
 min 
 Pepsin-peptone from fibrin 
 Pepsin-peptone from fibrin 
 
 Pepsin-glutin peptone 
 
 bell (38) 
 
 Maas (30) 
 Borkel (3) 
 Borkel (3) 
 
 Scheermesser (55) 
 
 water 
 12.5 per cent am- 
 monia 
 
 -49.40 
 -36.36 
 -39.42 
 
 -77. 08 to -77. 81 
 
 a-antipeptone, by tryptic diges- 
 tion from fibrin 
 
 Muller (34) 
 
 water 
 
 24 50 
 
 0-antipptone by tryptic diges- 
 tion from fibrin 
 a-antipeptone out of Witte's pep- 
 tone 
 Antipeptone from gelatin 
 Globin from haemoglobin 
 
 Muller (34) 
 
 Siegfried (60) 
 Kruger (25) 
 Gamgee and Hill (9) 
 
 water 
 
 water 
 water 
 weakly acid watery 
 
 -32.40 
 
 -18. 45 to -19.69 
 -100.80 
 -65.50 
 
 Globin from hsemoglobin 
 Haemoglobin 
 
 GamgeeandHill(9) 
 Gamgee and Hill (9) 
 
 alcoholic solution 
 solution in dilute 
 acetic acid 
 water 
 
 -54.20 (red light) 
 +10 40 (red light) 
 
 Nucleo-histone from thymus 
 ct-nucleo-protein from pancreas . . 
 Nuclein from pancreas 
 
 Nucleo-protein of suprarenal 
 glands 
 
 Gamgee and Jones 
 (10) 
 Gamgee and Jones 
 (10) 
 Gamgee and Jones 
 (10) 
 Gamgee and Jones 
 (10) 
 
 dilute ammonia 
 dilute ammonia 
 water 
 water 
 
 +37.50 (red light) 
 
 +37. 50 to +38. 10 
 (red light) 
 +64.40 (red light) 
 
 +48. 10 (red light) 
 
 
 
 
 
ROTATORY POWER 
 
 357 
 
 The following results * are those which have been obtained by 
 observers who have worked with vegetable proteins. 
 
 TABLE II 
 
 Protein 
 
 Observer 
 
 Nature of solution 
 
 Degrees 
 
 Edestin, hemp-seed 
 Edestin, hemp-seed 
 Edestin, hemp-seed 
 Excelsin, Brazil-nut 
 
 Chittenden and Mendel 
 Alexander 
 Osborne and Harris 
 Alexander 
 
 10% NaCl 
 10% NaCl 
 10% NaCl 
 10% NaCl 
 
 - 43.48 
 - 41.5 
 - 41.7 
 - 40 5 
 
 Excelsin, Brazil-nut 
 Globulin flax-seed 
 
 Osborne and Harris 
 Alexander 
 
 10% NaCl 
 10% NaCl 
 
 - 42.94 
 38 5 
 
 Globulin, flax-seed 
 
 Osborne and Harris 
 
 10% NaCl 
 
 43 53 
 
 Globulin, squash-seed 
 
 Osborne and Harris 
 
 10% NaCl 
 
 38 32 
 
 Amandin, almond 
 
 Osborne and Harris 
 
 10% NaCl 
 
 56 44 
 
 Corylin, hazel-nut . . .... 
 
 Osborne and Harris 
 
 10% NaCl 
 
 43 09 
 
 Juglansin, English walnut 
 Juglansin, American black 
 walnut 
 
 Osborne and Harris 
 Osborne and Harris 
 
 10% NaCl 
 10% NaOl 
 
 - 45.21 
 44 43 
 
 Juglansin, butternut 
 Phaseolin, kidney-bean 
 
 Osborne and Harris 
 Osborne and Harris 
 
 10% NaCl 
 10% NaCl 
 
 - 45.40 
 41.46 
 
 Legumin, horse-bean 
 
 Osborne and Harris 
 
 10% NaCl 
 
 44.09 
 
 Ricin castor-bean 
 
 Osborne, Mendel and Harris 
 
 water 
 
 28 85 
 
 Gliadin wheat 
 
 Osborne and Harris 
 
 80% alcohol 
 
 92 28 
 
 dliadin, wheat 
 
 Kjeldahl 
 
 55% alcohol 
 
 92 
 
 Gliadin, wheat. .. . . 
 
 Kjeldahl 
 
 glacial acetic acid 
 
 81 f 
 
 Gliadin, wheat 
 
 Kjeldahl 
 
 5.1% acetic acid 
 
 111 
 
 Gliadin, wheat 
 <iliadin, wheat 
 Gliadin, wheat 
 Gliadin, wheat 
 
 Kjeldahl 
 Mathewson 
 Mathewson 
 Mathewson 
 
 phenol 
 70% methyl alcohol 
 70% ethyl alcohol 
 60% ethyl alcohol 
 
 -130.0 
 - 95.65 
 - 91.95 
 96 66 
 
 Gliadin wheat 
 
 Mathewson 
 
 50% ethyl alcohol 
 
 98 45 
 
 Gliadin, wheat 
 
 Mathewson 
 
 60% propyl alcohol 
 
 101 10 
 
 Gliadin, wheat 
 
 Mathewson 
 
 70% phenol 
 
 123 15 
 
 Gliadin, wheat 
 
 Mathewson 
 
 phenol, anhydrous 
 
 131 77 
 
 Gliadin, wheat ... . 
 
 Mathewson 
 
 paracresol 
 
 121 00 
 
 Gliadin, wheat 
 Gliadin, wheat 
 
 Mathewson 
 Mathewson 
 
 benzyl alcohol 
 glacial acetic acid 
 
 - 53.10 
 78 60 
 
 Gliadin a, wheat 
 
 Lindet and Ammann 
 
 70% alcohol 
 
 81 6 
 
 Gliadin ft, wheat 
 
 Lindet and Ammann 
 
 70% alcohol 
 
 95 
 
 Gliadin, rye 
 
 Kjeldahl 
 
 55% alcohol 
 
 121 
 
 Gliadin, rye 
 
 Kjeldahl 
 
 "dilute acid" 
 
 144 
 
 Gliadin rye 
 
 Kjeldahl 
 
 glacial acetic acid 
 
 105 
 
 Gliadin, rye 
 
 Kjeldahl 
 
 phenol 
 
 157 
 
 Gliadin, rye 
 
 Lindet and Ammann 
 
 70% alcohol 
 
 87 8 
 
 Gliadin, barley 
 
 Lindet and Ammann 
 
 70% alcohol 
 
 87 8 
 
 Hordein, barley 
 Hordein, rye 
 Zein a, maize 
 Zein /3 maize 
 
 Lindet and Ammann 
 Lindet and Ammann 
 Lindet and Ammann 
 Lindet and Ammann 
 
 70% alcohol 
 70% alcohol 
 70% alcohol 
 70% alcohol 
 
 -137.5 
 -137.5 
 - 29.6 
 40 
 
 Zein, maize 
 
 Osborne and Harris 
 
 90% alcohol 
 
 28 
 
 Zein, maize 
 
 Kjeldahl 
 
 75% alcohol 
 
 35 
 
 Zein, maize 
 
 Kjeldahl 
 
 glacial acetic acid 
 
 28 
 
 
 
 
 
 * Cited after T. B. Osborne (37), which consult for references to the 
 literature. 
 
358 PHYSICAL PROPERTIES 
 
 From these results it is clear that the nature of the solvent plays 
 a very considerable part in determining the rotatory power of pro- 
 teins. Furthermore, it would appear that as a rule the salts which 
 proteins form with acids and bases differ very markedly in rotatory 
 power from the free protein (3) (20) (60) (1) (4) (6) (39). In an 
 analogous manner the rotatory powers of the salts which the simple 
 amino-acids form with acids and bases differ very markedly from 
 those of the free acids (40). As a rule too little attention has 
 been paid to this fact by investigators who have sought to char- 
 acterize the proteins by their rotatory powers. It is evident that 
 the specific rotatory power of a protein cannot be regarded as 
 characteristic of it until the nature of the protein salt and of the 
 solvent employed are rigidly defined. 
 
 According to Alexander (1) the rotatory power of a dissolved 
 protein is considerably affected by exposure of the solution to a 
 high temperature for a period preceding the measurement. It 
 must not be forgotten, however, that partial hydrolysis of the 
 protein may play a very considerable part in bringing about this 
 change. 
 
 2. The Absorption of Light by Protein Solutions. The 
 peculiar absorption-spectra of solutions of haemoglobin and its 
 colored derivatives have been very extensively studied (17) (19) 
 (63) (21) (22) (36) (5) (58) (14). From the results of these in- 
 vestigations it is evident that the absorption-bands (a and 0) in 
 the visible spectrum which are caused by these solutions are 
 primarily attributable to the haematin- and not to the distinctively 
 protein moiety of the haemoglobin molecule. In addition to 
 these bands, however, the absorption-spectrum of haemoglobin 
 and its derivatives reveals another absorption-band on the extreme 
 edge of the violet end of the visible spectrum (61) (2) (8). Ac- 
 cording to Gamgee (8) this absorption-band is given not only by 
 solutions of haemoglobin but also by solutions of haematin; he 
 therefore attributes it to the haematin, and not to the protein 
 moiety of the haemoglobin molecule. According to Soret, how- 
 ever, an absorption-band on the extreme edge of the ultra-violet 
 can be demonstrated, by employing a fluorescent screen, in the 
 absorption-spectra of solutions of a large variety of proteins, 
 among which may be mentioned egg-albumin, mucin (from 
 snails), casein, globulin and syntonin. This band is followed, on 
 the ultra-violet side, by a region of especial transparency. Accord- 
 
REFRACTIVE INDICES 359 
 
 ing to Soret it is unaffected by acids, but on adding NaOH or 
 ammonia to the protein solution the region of transparency in 
 the ultraviolet disappears completely and the band of absorption 
 is displaced towards the less refrangible end of the spectrum. On 
 neutralizing the solution with acid the original absorption-spectrum 
 is restored. A similar absorption-spectrum is yielded by solutions 
 of tyrosin and it is affected in a similar manner by alkalies. Soret 
 therefore attributes the absorption-band in the absorption spectra 
 of protein solutions to the tyrosin radical which the proteins 
 contain. 
 
 Kober (23) has recently carried out a spectrographic examina- 
 tion of solutions of a number of amino-acids and polyamino-acids. 
 He finds that the aliphatic amino-acids display only general 
 absorption in the extreme ultraviolet. The aromatic acids, 
 tyrosin and phenylalanin do, however, show definite absorption- 
 bands in the ultra-violet spectrum. The absorption-band in the 
 ultraviolet which is displayed by proteins in solution is therefore 
 attributable not only to tyrosin radicals, as Soret supposed, but 
 also in some measure to phenylalanin radicals. 
 
 Since the occurrence of any photochemical reaction is dependent 
 upon the absorption of the chemically active rays by some con- 
 stituent or constituents of the reacting system it would appear 
 possible that the markedly toxic action of ultraviolet light upon 
 many unicellular organisms may be dependent upon the absorption 
 of the ultraviolet light-rays by the proteins of the organisms 
 affected. Strong confirmation of this view is afforded by the 
 experiments of Harris and Hoyt (12) who have shown that if ultra- 
 violet light be passed through a thin layer of gelatin or peptone 
 solution its toxicity for paramoeda is diminished to a much 
 greater extent than by passage through a similar layer of distilled 
 water. Solutions of urea, sugar or alanin do not confer this pro- 
 tection, while solutions of the aromatic amino-acids protect the 
 organisms very markedly. Leucin likewise confers a very marked 
 protection, but this amino-acid is rapidly decomposed by ultra- 
 violet light, yielding colored products, and the decomposition is ac- 
 companied by an increase in the protective power of the solution. 
 
 3. The Refractive Indices of Protein Solutions. The in- 
 fluence of dissolved proteins upon the refractive indices of various 
 solvents has been especially studied by Reiss (41) (43), Herlitzka 
 (13), Schmidt (56) and myself (43-54). 
 
360 PHYSICAL PROPERTIES 
 
 Reiss measured the change in the refractive indices of dilute 
 salt solutions which results from the introduction of varying 
 amounts of the " pseudoglobulins " of blood serum. His pseudo- 
 globulins were prepared by fractional coagulation with ammonium 
 sulphate and purified by prolonged dialysis. Fraction I (pseudo- 
 globulin I) was coagulated at 32 to 36 per cent saturation with 
 ammonium sulphate, fraction II (pseudoglobulin II) was coagu- 
 lated at 36 to- 39 per cent saturation. The change in the refractive 
 index of the solvent due to the introduction of these proteins was 
 found to be directly proportional to the quantity of protein dis- 
 solved in it; the change due to the introduction of 1 per cent of 
 the "pseudoglobulin I " being 0.00224 and that due to the introduc- 
 tion of 1 per cent of the " pseudoglobulin II" 0.00230. The 
 difference between these determinations is not sufficient to con- 
 stitute a basis for distinction between the two globulin-fractions, 
 since it is not greater than that which might have arisen through 
 experimental error. Reiss also measured the influence of other 
 constituents of serum (especially crystallized and amorphous 
 serum albumin) upon the refractive indices of dilute saline solu- 
 tions. 
 
 I have amplified and confirmed these results of Reiss, employing 
 for this purpose a variety of proteins (casein, paranuclein, ovo- 
 mucoid, ovovitellin, serum globulin, gliadin, etc.) and not only 
 aqueous solvents but alcohol-water mixtures, acetone-water mix- 
 tures, etc. A large number of determinations upon casein in 
 aqueous solutions, between the concentrations which it is feasible 
 to employ* show that the change in the refractive indices of 
 aqueous solvents due to the introduction of this protein is very 
 accurately proportional to its concentration (43) (56). Accord- 
 ingly the refractive index of a solution of this protein is given by: 
 
 n HI = a X c 
 
 where n is the refractive index of the solution, n\ that of the solvent, 
 c the percentage of casein in the solution and a a constant which 
 is characteristic of the protein (for example, casein) which is 
 
 * If the concentration of protein be too small (less than about 0.5 per cent) 
 the experimental error of the determination becomes a significant proportion 
 of the observed difference in the refractive index of the solvent, due to the 
 introduction of the protein. If the concentration of protein be too large then 
 the solutions are too opaque to enable an exact reading to be obtained. 
 
REFRACTIVE INDICES 
 
 361 
 
 employed and represents the change in the refractive index of the 
 solvent which is brought about by dissolving one gram of the 
 protein in 100 cc. 
 The following results are illustrative:* 
 
 TABLE III 
 SolventO.lOATNaOH 
 
 Concentration 
 casein per cent 
 
 Refractive index 
 
 a for nj = 1.33444 
 
 0.5 
 
 1.3352 
 
 0.00152 
 
 1.0 
 
 1.3360 
 
 0.00156 
 
 1.5 
 
 1.3368 
 
 0.00157 
 
 2.0 
 
 1.3375 
 
 0.00153 
 
 2.5 
 
 1.3383 
 
 0.00154 
 
 3.0 
 
 1.3390 
 
 0.00152 
 
 4.0 
 
 1.3405 
 
 0.00152 
 
 5.0 
 
 1.3420 
 
 0.00151 
 
 6.0 
 
 1.3436 
 
 0.00153 
 
 In the first column of the table is given the amount of casein in 
 grams which was dissolved in 100 cc. of the solution. In the 
 second column is given the refractive index of the solution meas- 
 ured at 20 C. In the third is given the value of the constant a 
 calculated from the above formula for the given value of the 
 constant wi (i.e., the refractive index of the solvent, previously 
 determined at the same temperature). 
 
 The equation n HI = a X c also holds good for solutions of 
 ovomucoid in water (44), of paranuclein in JV/10 KOH (45), of 
 potassium serum globulinate (46), of potassium casemate in 
 alcohol- water mixtures (47), of gliadin in various solvents (54) and 
 of edestin in aqueous solvents (56). 
 
 The change in the refractive indices of aqueous solvents which 
 is brought about by a given percentage of casein is independent of 
 the nature of the acid or base with which the casein is combined. 
 This is shown by the fact that this change is the same whether the 
 casein be dissolved in dilute KOH, NaOH, LiOH, NH 4 OH, 
 Sr(OH) 2 , Ba(OH) 2 , Ca(OH) 2 or HC1 solutions. Also, between 
 0.01 N and 0.1 N it is independent of the concentration of alkali 
 
 * In all of these determinations a Pulfrich refractometer was employed, 
 reading the angle of total reflection accurately to within 1', and a sodium flame 
 was employed as the source of light. 
 
362 PHYSICAL PROPERTIES 
 
 which is employed as solvent. Similarly, the value of a, in the 
 above equation, is the same for " insoluble" serum globulin 
 whether this protein be dissolved in TV/IO KOH or in N/40 HC1. 
 The influence which a protein exerts upon the refractive index of 
 its solution is therefore independent of the nature or proportion of 
 acid or base which is combined with it, differing in this respect very 
 strikingly from the optical rotatory power of dissolved protein, 
 which, as we have seen, is very intimately dependent upon the 
 nature and proportion of combined inorganic acid or base. The 
 reason for this fact is readily perceived when we reflect that the' 
 power of dissolved protein to refract light is a function of the space 
 which is occupied by the protein molecules. Now the molecular 
 volume, and, indeed, the molecular refractivity is an additive 
 function of the atomic volumes (or refractivities) of the atoms 
 which together make up the molecule. In a molecule which 
 contains over a thousand atoms, as a protein molecule does, the 
 substitution of even several H atoms by K atoms or the addition 
 of a few H or Cl atoms or OH groups might be expected not to 
 exert an appreciable influence upon the volume or refractive power 
 of the whole molecule. The refractive index of a protein solution 
 also remains unaltered by hydrolysis, upon which fact I have 
 based a method of determining the comparative activities of 
 trypsin solutions (50). 
 
 From the investigations of Gladstone and Dale (11) and of 
 
 N 1 
 Landolt (27) it appears that the expression -, , where N is the 
 
 refractive index of a substance and d its density, which is known 
 as the specific refractivity, is very constant, being only slightly 
 dependent upon the temperature. Moreover .each particular 
 substance in a mixture preserves its own specific refractivity 
 nearly unchanged; hence the refractive index of a mixture or of 
 a solution can generally be readily calculated from the refractive 
 indices of its components. Now the density of a dilute protein 
 solution is always very nearly that of water, that is 1, so that the 
 specific refractivity of a dilute protein solution may, with a toler- 
 able approach to accuracy, be taken as n 1, where n is the re- 
 fractive index of the solution. Suppose c per cent of protein were 
 to be dissolved in a solvent of specific refractivity Ni I (for 
 instance, a dilute acid or alkali, which is nearly equal in density to 
 pure water), then, if Gladstone's law holds good, and the density 
 
REFRACTIVE INDICES 363 
 
 of the dissolved protein itself be d (Cf. Chap. XIII, section 11) 
 the refractive index of the mixture should be given by: 
 
 whence 
 and 
 
 (ri n _ W-l)(100-c) (N-l)c 1 
 
 100 100 "V 
 
 100 (n -1) = 100 (Ni - 1) - c (l - i - Ni + %} 
 
 \ a a/ 
 
 n 
 
 100 
 
 or T /- 1 TIT , -AT\ v 
 
 n-A^fl --_#!+_)_, (j) 
 
 which is identical with the relation n HI = a X C which, as we 
 have already seen, subsists between the refractive index of a pro- 
 tein solution and its percentage concentration.* Hence we may 
 conclude that for solutions of proteins in aqueous solvents Gladstone's 
 law of mixtures holds good. The constant a is obviously given by 
 
 y^. (1 -3 Ni + -T j . For solutions of casein containing be- 
 tween 0.25 and 6.00 per cent the average value of a is 0.00125. 
 Hence, by exterpolation, the refractive index of the pure protein 
 should be 1.675, taking the refractive index of water as 1.333 and 
 the density of dissolved casein as 1.39 (Cf. Chap. XIII, section 11). 
 For serum globulin this constant is 1.774. The refractive index 
 of the pure protein does not readily admit of direct determination 
 in these cases. The protamin salts, clupein sulphate and salmin 
 sulphate can, however, readily be obtained in a fluid (oily) con- 
 dition. Kossel (24) has determined the refractive indices of these 
 fluids and finds them to be, respectively, 1.443 and 1.442, figures 
 which are of the same order of magnitude as the above estimates. 
 If the density of the solvent be d' instead of 1 and c be small, so 
 that the density of the solution is very nearly that of the solvent, 
 then equation (i) becomes: 
 
 n ~ Ni = ( 1 -j- Ni+ j N )m < H > 
 
 * From this it would appear that the value of a should vary with the re- 
 fractive index even of a dilute solution of a base or acid which is not exactly 
 the same as that of water, that is, with the nature and concentration of the 
 (dilute) acid or base employed as a solvent. It will be observed, however, 
 that in the experimental values of a only a one-hundredth part of the difference 
 between the refractive index of the acid or alkali solution and that of water 
 appears. This quantity, for the solutions employed, is far too small to be 
 detectable. 
 
364 PHYSICAL PROPERTIES 
 
 which is the same form as (i) . Obviously, however, in a solution of 
 a protein in a solvent the density or refractivity of which departs 
 at all widely from those of water, although the law n n\ = a X c 
 might be expected to hold good, the constant a would not have 
 the same value as in aqueous solvents. In other words, in non- 
 aqueous solvents the law n n\ = a X c should hold good, but, 
 for a given protein the value of a should vary with the nature of 
 the solvent depending upon its refractivity and its density. In 
 satisfactory correspondence with this deduction we find that the 
 law n HI = a X c holds good for solutions of potassium casein- 
 ate in alcohol-water mixtures containing from to 75 per cent of 
 alcohol, for solutions of serum-globulin in alcohol-water mixtures, 
 and for solutions of gliadin in a variety of solvents. As we shall 
 see, however, although the value of a for a given protein varies 
 with the nature of the solvent in the sense demanded by the above 
 theory, this variation cannot be quantitatively expressed by the 
 
 formula 100 a = N ^ -Ni + 1 - ~ 
 a a 
 
 In estimating the accuracy with which a can be determined in 
 the equation n n\ = a X c, it is essential to recollect that the 
 experimental error in determining the angle of total reflection with 
 the Pulfrich Refractometer is 1'. This, for the instruments 
 which I have employed, corresponds to an error of 0.00009 in 
 the determination of the refractive index of a given solution. Now 
 it is obvious that since the absolute error in the determination of 
 n H! is the same, the error in the determination of a must be 
 less in proportion to the magnitude of c. 
 
 In order to assign to each determination its due weight in the 
 estimation of the mean value of a for any solvent we must therefore 
 add together all of the observed values of n n\ and divide this 
 sum by the sum of the concentrations employed. This procedure 
 has been adopted in my own estimates, cited below. The follow- 
 ing are the values of a for various proteins and solvents which 
 have so far been ascertained. The influence of temperature upon 
 the magnitude of a appears to be very slight. In fact for casein I 
 have found the value of a to be the same, within the experimental 
 error, at 40 degrees as it is at 20 degrees. Hence the temperatures 
 at which the observations were made are not specified in the 
 accompanying table; they lay, in every case, however, between 
 ordinary room-temperatures and 24 degrees. 
 
REFRACTIVE INDICES 
 
 365 
 
 iO"OiOOiOiOiOtO 
 
 OOOOOOOO 
 
 . fl d d S3 d 
 i-HOOOOO 
 o> 02 IB 02 2 
 
 Hill 
 
 OOOOOOO 
 
 OO 
 
 
 8 1 
 
 ooooo 
 
 -H-H-H-H-H -H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H-H 
 
 05 C3 05 t- CO TJH 
 
 oooooooooooooooooooooooooooo 
 
 > V 
 
 00 _|| 
 
 
 _ 03 
 
 o fl 
 
 O 
 
 
 nr 
 
 o o 
 
 |lllHll|l|o|l 
 
 d a a a 
 
 s a a 
 
 llllll=JJJ fl - 
 
 
 
 
 
 
 
 
 
 
 
 
 <1 
 
 is ; ; ; ; .s.a 
 
 
 
 
 
 " 
 
 : : S i i ; '"ii'i. 
 
 S'd'S^??^???? ~ 
 
 *rr MGc3c3c3c30gTOC3 
 
366 PHYSICAL PROPERTIES 
 
 Calculating the theoretical values of a, on the assumption that 
 Gladstone's law of mixtures holds good for solutions of protein in 
 any solvent we at once find that although a varies with the nature 
 of the solvent in the sense demanded by Gladstone's law, yet the 
 law does not hold good, for different solvents, with any approach 
 to quantitative exactitude. Thus the calculated value of a for 
 solutions of casein in 50 per cent alcohol is 0.00095, assuming the 
 density of casein dissolved in 50 per cent alcohol to be the same 
 as that of casein dissolved in water, while if we assume the 
 density of casein dissolved in 50 per cent alcohol to be that of un- 
 dissolved casein (Cf. Chap. XIII, section 11) the calculated value 
 of a becomes 0.00121; the experimental value, however, is 0.00149; 
 the calculated value of a for insoluble serum-globulin in 50 per cent 
 alcohol is 0.00169, the experimental value is 0.00119. Hence Glad- 
 stone's law of mixtures holds good for solutions of protein in a 
 specified solvent, but no longer holds good if we vary the nature 
 
 N 1 
 of the solvent. Now it is a characteristic of the quantity -= 
 
 a 
 
 that although it is independent of temperature and of concentra- 
 tion, it varies when the state of aggregation varies (35). We may 
 conclude, therefore, as we have had occasion to indicate before, 
 that the physical condition (number of associated water molecules, 
 degree of dispersion, etc.) of proteins in alcohol-water mixtures and 
 other non-aqueous solvents is not the same as it is in water.* 
 
 Comparing the above cited determinations by Robertson and 
 Greaves, of the refractive indices of gliadin in various solvents, 
 with Mathewson's determination of the rotatory power of gliadin 
 in various solvents, it is evident that, as might have been expected, 
 there is no correspondence between the effects of different solvents 
 upon these two physical properties of dissolved protein. 
 
 It will be observed that the value of a for solutions of gliadin in 
 75 per cent phenol is negative. This is due to the fact that the 
 refractive index of 75 per cent phenol is nearly equal to that of 
 gliadin itself, and its density is greater so that a mixture of the two 
 substances has a refractive index which is less than that of the 75 
 per cent phenol employed as solvent. 
 
 The influence of temperature upon the refractive indices of 
 protein solutions has been determined by myself and by Herlitzka 
 (13). As stated above, I find the effect of temperature upon the 
 
 * Cf . Chaps. I and XIII. 
 
REFRACTIVE INDICES 367 
 
 value of a for solutions of caseinates in water to be very small. In 
 fact, if allowance be made for the alteration in the refractive index 
 of the water with varying temperature, I have been unable to 
 observe any alteration in a, distinctly greater than the possible 
 experimental error, between 20 and 40 degrees. According to 
 Herlitzka, however, if egg-albumin be employed, a distinct in- 
 fluence of temperature upon the refractivity of the protein itself 
 can be observed. He interprets his results in the light of the 
 Lorentz-Lorentz modification of Gladstone's law of mixtures (28) 
 (29). 
 
 The utilization of the refractometric method for determining 
 the concentrations of various proteins in solutions and in body- 
 fluids has been discussed in Chap. III. 
 
 LITERATURE CITED 
 
 (1) Alexander, A. C., Journ. Exper. Med., 1 (1896), p. 304. 
 
 (2) d'Arsonval, A., Arch, de physiol. Norm et Path. Ser. (5), 2 (1890), p. 
 
 340. 
 
 (3) Borkel, C., Zeit. f. physiol. Chem., 38 (1903), p. 289. 
 
 (4) Billow, K., Arch. f. d. ges. Physiol., 58 (1894), p. 207. 
 
 (5) Butterfield, E. E., Zeit. f. physiol. Chem., 79 (1912), p. 439. 
 
 (6) Framm, F., Arch. f. d. ges. Physiol., 68 (1897), p. 144. 
 
 (7) Fredericque, L., Arch, de Biol. 1 (1880), p. 457; 2 (1881), p. 379. 
 
 (8) Gamgee, A., Proc. Roy. Soc. London, 59 (1896), p. 276. 
 
 (9) Gamgee, A., and Hill, Croft, Beitr. z. chem. Physiol. und Path., 4 
 
 (1903), p. 1. 
 
 (10) Gamgee, A., and Jones, W., Beitr. z. chem. Physiol. und Path. 4 (1903), 
 
 p. 10. 
 
 (11) Gladstone, J. H., and Dale, T. P., Phil. Trans. Roy. Soc. London, 148 
 
 (1858), p. 8; 153 (1863), p. 316. 
 
 (12) Harris, F. I., and Hoyt, H. S., "Science," N. S., 46 (1917), p. 318. 
 
 (13) Herlitzka, A., Zeit. f. chem. und der Kolloide, 7 (1910), p. 251. 
 
 (14) Heubner, W., and Rosenberg, H., Biochem. Zeit., 38 (1912), p. 345. 
 
 (15) Hopkins, F. Gowland, Journ. of Physiol., 25 (1900), p. 306. 
 
 (16) Hoppe-Seyler, F., Virchow's Arch., 11 (1857), p. 552. 
 
 (17) Hoppe-Seyler, F., Virchow's Arch., 33 (1862), p. 446. 
 
 (18) Hoppe-Seyler, F., Zeit. f. chem. und Pharm., 7 (1864), p. 737. 
 
 (19) Hoppe-Seyler, F., Med.-Chem. Untersuch. (1867), p. 169. 
 
 (20) Hoppe-Seyler, F., Handb. d. physiol. und pathol. Chem., 7 Aufl. Berlin 
 
 (1903), p. 368. 
 
 (21) Hufner, G., Journ. f. prakt. Chem. (2), 22 (1880), p. 362. 
 
 (22) Hufner, G., Arch. f. (Anat. und) Physiol. (1890), p. 1; (1894), pp. 130 
 
 and 209; (1895), p. 213; (1901), Suppl., p. 187. 
 
 (23) Kober, P. A., Journ. Biol. Chem., 22 (1915), p. 433. 
 
368 PHYSICAL PROPERTIES 
 
 (24) Kossel, A., Zeit. f. physiol. Chem., 25 (1898), p. 165. 
 
 (25) Kruger, T. H., Zeit. f. physiol. Chem., 38 (1903), p. 320. 
 
 (26) Ktthne, W., and Chittenden, R. H., Zeit. f. Biol., 20 (1884), p. 11. 
 
 (27) Landolt, H., Pogg. Ann., 123 (1864), p. 595. 
 
 (28) Lorentz, H. A., Weid. Ann., 9 (1880), p. 641. 
 
 (29) Lorentz, L., Weid. Ann., 11 (1880), p. 70. 
 
 (30) Maas, O., Zeit. f. physiol. Chem., 30 (1900), p. 61. 
 
 (31) Maximovitsch, S., Journ. Russ. phys. Chem. Soc., 6 (1901), p. 460; 
 
 cited after Maly's Jahresbr. f. Tierchem., 31, p. 35. 
 
 (32) Mittelbach, F., Zeit. f. physiol. Chem., 19 (1894), p. 289. 
 
 (33) Morner, C. T., Zeit. f. physiol. Chem., 18 (1893), p. 61. 
 
 (34) Miiller, F., Zeit. f. physiol. Chem., 38 (1903), p. 265. 
 
 (35) Nernst, W., "Theoretical Chemistry," Transl. 4th German Edn., 
 
 New York (1904), p. 307. 
 
 (36) von Noorden, C., Zeit. f. physiol. Chem., 4 (1879), p. 9. 
 
 (37) Osborne, T. B., "The Vegetable Proteins," London, 1909. 
 
 (38) Osborne, T. B., and Campbell, G. F., Journ. Amer. Chem. Soc., 22 
 
 (1900), p. 422. 
 
 (39) Pauli, W., Samec, M., and Strauss, E., Biochem. Zeit., 59 (1914), p. 
 
 470. 
 
 (40) Plimmer, R. H. A., "The Chemical Constitution of the Proteins," 
 
 London (1908), Part I, p. 79. 
 
 (41) Reiss, E., Arch. f. exper. Path, und Pharm., 51 (1903), p. 18. 
 
 (42) Reiss, E., Beitr. z. chem. Physiol. und Path., 4 (1904), p. 150. 
 
 (43) Robertson, T. Brailsford, Journ. Physical Chem., 13 (1909), p. 469. 
 
 (44) Robertson, T. Brailsford, Journ. Biol. Chem., 7 (1910), p. 359. 
 
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 (46) Robertson, T. Brailsford, Journ. Biol. Chem., 8 (1910), p. 441. 
 
 (47) Robertson, T. Brailsford, Journ. Biol. Chem., 8 (1910), p. 507. 
 
 (48) Robertson, T. Brailsford, Journ. Biol. Chem., 11 (1912), p. 179. 
 
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 (51) Robertson, T. Brailsford, Journ. Biol. Chem., 13 (1912), p. 325. 
 
 (52) Robertson, T. Brailsford, Journ. Biol. Chem., 13 (1913), p. 455. 
 
 (53) Robertson, T. Brailsford, Journ. Biol. Chem., 13 (1913), p. 499. 
 
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 (1911), p. 181. 
 
 (55) Scheermesser, W., Zeit. f. physiol. Chem., 37 (1903), p. 363. 
 
 (56) Schmidt, C. L. A., Journ. Biol. Chem., 23 (1915), p. 487. 
 
 (57) Schorer, G., Aus. der. Med. Klinik. der Universitat Bern (1908). 
 
 (58) Schumm, O., Zeit. f. physiol. Chem., 83 (1913), p. 1. 
 
 (59) Sebelien, J., Zeit. f. physiol. Chem., 9 (1885), p. 445. 
 
 (60) Siegfried, M., Zeit. f. physiol. Chem., 35 (1902), p. 164. 
 
 (61) Soret, J. L., C. R. de 1'Acad. des. Sc., 97 (1883), pp. 642 and 1269. 
 
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 Maly's Jahresber. f. Tierchem., 11 (1881), p. 17. 
 
 (63) Stokes, G. G., Phil. Mag. 4th Ser., 27 (1864), p. 388. 
 
PART IV 
 CHEMICAL DYNAMICS IN PROTEIN SYSTEMS* 
 
 * Any survey of this subject must, of course, involve an incidental exposi- 
 tion of the properties and behavior of the enzymes, to which reference must 
 necessarily be made. Nevertheless any attempt to enter fully into a discussion 
 of the properties of enzymes would, of course, lead us into regions quite for- 
 eign to the scope of this work. For exhaustive treatments of the general 
 subject of enzyme-action the reader is referred to the works of Oppenheimer 
 (15), Bredig (8), Taylor (19), Vernon (20), Euler (9) and Bayliss (7). 
 
CHAPTER XV 
 THE HYDROLYSIS OF THE POLYPEPTIDS 
 
 1. The Hydrolysis of Polypeptids by Proteolytic Enzymes. 
 
 By the aid of the various hydrolysing agencies the synthetic poly- 
 peptids are capable of being split, with successive additions of 
 the elements of water, into their constituent amino-acids. Among 
 these hydrolysing agencies must be reckoned a number of the 
 protein-splitting enzymes which are found in a variety of tissues, 
 tissue-extracts and secretions. 
 
 The action of the enzymes of the pancreas (trypsin and, pre- 
 sumably, others) upon the dipeptids was first investigated by 
 Fischer and Bergell (12) who acted upon solutions of various 
 dipeptids with pancreas-extract. They found that: 
 
 glycyl-glycin 
 
 /3-naphthalenesnlphoglycyl-d-alanin 
 /3-naphthalenesulpho-d-alanyl-glycin 
 Di-/3-naphthalenesulphotyrosyl-dl-leucin 
 while 
 
 j8-naphthalenesulphoglycyl-l-tyrosin 
 
 /3-naphthaleneglycyl-dl-leucin 
 
 carboxethyl-glycyl-dl-leucin 
 
 glycyl-1-tyrosin 
 
 leucyl-alanin 
 
 alanyl-leucin 
 
 leucyl-leucin 
 
 were not hydrolysed. 
 
 were hydrolysed. 
 
 In the course of these investigations the exceedingly interest- 
 ing fact was encountered that the racemic compounds are hydro- 
 lysed asymmetrically by the pancreas extract; thus only 1-leucin 
 was obtained by the action of the extract upon carboxethyl- 
 glycyl-d-1-leucin, the remainder (containing d-leucin) not being 
 acted upon. Now l-leucin is the naturally occurring variety, the 
 form, that is, which is found in the proteins. 
 
 These investigations were greatly extended by Fischer and 
 Abderhalden (11) who employed a pancreatic juice prepared 
 by Pavlov from a pancreatic fistula and activated by entero- 
 
 371 
 
372 
 
 CHEMICAL DYNAMICS 
 
 kinase obtained from the succus entericus of the duodenum. 
 The result of these investigations was to show that the synthetic 
 polypeptids can be divided into two distinct classes, members 
 of the one class being hydrolysed or (if racemic) partially hydro- 
 lysed by the trypsin; members of the other class not being 
 hydrolysed by trypsin. The various polypeptids employed by 
 Fischer and Abderhalden were distributed between these two 
 classes as follows: 
 
 Hydrolysed 
 
 * Alanyl-glycin 
 
 * Alanyl-alanin 
 
 * Alanyl-leucin A 
 
 * Leucyl-isoserin A 
 Glycyl-1-tyrosin 
 Leucyl-1-tyrosin 
 
 * Alanyl-glycyl-glycin 
 
 * Leucyl-glycyl-glycin 
 
 * Glycyl-leucyl-alanin 
 
 Not Hydrolysed 
 Glycyl-alanin 
 Glycyl-glycin 
 Alanyl-leucin B 
 Leucyl-alariin 
 Leucyl-glycin 
 Leucyl-leucin 
 Aminobutyryl-glycin 
 Aminobutyryl-aminobutyric acid A 
 Aminobutyryl-aminobutyric acid B 
 Valyl-glycin 
 
 Alanyl-leucyl-glycin 
 
 Dialanyl-cystin 
 
 Dileucyl-cystin 
 
 Tetraglycyl-glycin 
 
 Triglycyl-glycin ester 
 
 d-alanyl-d-alanin 
 
 d-alanyl-1-leucin 
 
 1-leucyl-l-leucin 
 
 1-leucyl-d-glutamic acid 
 
 Glycyl-phenylalanin 
 
 Leucyl-prolin 
 
 Diglycyl-glycin 
 
 Triglycyl-glycin 
 
 Dileucyl-glycyl-glycin 
 
 d-alanyl-1-alanin 
 
 1-alanyl-d-alanin 
 
 1-leucyl-d-leucin 
 
 d-leucyl-1-leucin 
 
 The compounds which are marked by asterisks were racemic. 
 Of all these the hydrolysis proved to be asymmetric and only the 
 naturally-occurring stereo-isomer was attacked. 
 
 There is thus a remarkable correlation between the ability of 
 these enzymes to attack certain amino-acid unions and the occur- 
 rence of these unions among the natural proteins which these 
 enzymes attack in the ordinary course of events. As we shall 
 see in the following chapter there are many excellent reasons 
 for believing that the action of the proteolytic enzymes, like 
 that of many of the inorganic catalysors, is dependent upon the 
 formation of intermediate compounds of the enzyme with the 
 substrate. It appears highly probable, therefore, that the enzyme 
 
HYDROLYSIS OF POLYPEPTIDS 373 
 
 is itself possessed of an optical structure which fits it to unite 
 with certain stereo-isomers and only with them. To pursue 
 Emil Fischer's classical illustration, the enzyme and these par- 
 ticular stereo-isomeric unions fit one another as a key does a lock. 
 The position is therefore this, that the proteolytic enzymes are 
 only adapted to "fit" certain modes of union between amino- 
 acids and that these are precisely the modes of union which occur 
 in the proteins which it is their function to digest. To a certain 
 type of mind, a teleological "explanation" of this fact would 
 doubtless prove very inviting. But the biochemist demands 
 something more satisfactory than teleology, or even than the 
 conveniently comprehensive nee-Darwinian generalization of 
 "adaptation through survival of the fittest." Our physico- 
 chemical view-point inclines us to suspect the existence of some 
 physico-chemical mechanism which, in the building up of these 
 bodies has necessarily brought about their mutual adaptation to 
 and dependence upon one another. The nature of this mechanism 
 is, I believe, not far to seek. In Chap. XVII we shall see that 
 not only the hydrolysis but also the synthesis of the proteins is 
 brought about through the agency of enzymes, and that the 
 enzymes which bring about the synthesis of the proteins are, 
 in all probability, but slight modifications (probably anhydrides) 
 of the enzymes which bring about their hydrolysis. Synthesis, 
 just as hydrolysis, must involve the formation of intermediate 
 compounds between the enzyme and the substrate (in this in- 
 stance the constituent amino-acids out of which the protein is 
 to be built up). Since the enzyme is only adapted to unite with 
 certain stereo-isomers of the amino-acids only these stereo- 
 isomers can be transported into the structure which the enzyme 
 is engaged in building up. In other words, the stereo-isomeric 
 structure of the naturally occurring proteolytic enzymes neces- 
 sarily determines the stereo-isomeric structure of all of the natu- 
 rally occurring proteins, and for the same reason that these enzymes 
 can only split certain amino-acid unions, they can only cause 
 these unions to arise. Hence the occurrence of only those stereo- 
 isomeric groups within the protein molecule which are open to 
 attack by the proteolytic enzymes may be regarded as a conse- 
 quence of their power to react with the digestive enzymes (17). 
 
 From the above-cited results of Fischer and Abderhalden it 
 is evident that not only the stereo-isomeric structure of the 
 
374 CHEMICAL DYNAMICS 
 
 amino-acid radicals in a polypeptid is of great importance in 
 determining the susceptibility of the polypeptids to hydrolyis 
 by trypsin, but also the order in which the amino-acid groups 
 are combined and the nature of these groups themselves, apart 
 altogether from their optical configuration. Thus alanyl-glycin, 
 CH 3 .CH 2 NH 2 .CO.NH.CH 2 .COOH, is readily hydrolysed; while 
 the isomeric glycyl-alanin, NH 2 .CH 2 .CO.NH.CH.CH 3 .COOH, is 
 not. When alanin is the acyl radical, as in alanyl-glycin, alanyl- 
 alanin and alanyl-leucin, hydrolysis occurs, but it does not when 
 leucin, valin or aminobutyric acid are the acyl radicals (for in- 
 stance, leucyl-alanin, leucyl-glycin and leucyl-prolin). 
 
 The number of amino-acid groups within the molecule is also 
 of great importance. Thus tetraglycyl-glycin is hydrolysed in 
 the presence of trypsin, while glycyl-glycin, diglycyl-glycin and 
 triglycyl-glycin are not; other things being equal it is evident 
 that mere length of the peptid chain per se confers upon it greater 
 susceptibility to attack by this enzyme. 
 
 It will be observed, on surveying the above results, that a 
 number of polypeptids are not hydrolysable by pure trypsin 
 which nevertheless are built up out of naturally occurring radicals. 
 Particularly resistant to hydrolysis are those peptids which con- 
 tain a preponderance of glycin, phenylalanin or pyrrolidin- 
 carboxylic acid.* In the light of the theory presented above, 
 therefore, the question forces itself upon us, how can such peptid 
 groups come to form, as they do, constituent parts of the mole- 
 cules of many proteins? Obviously, if they cannot be digested 
 by trypsin and our theory is valid then they cannot have been 
 introduced into the protein molecule through the agency of trypsin ; 
 they must have been introduced through the agency of some 
 other enzyme or enzymes. 
 
 In this connection the very significant fact will be observed 
 that whereas Fischer and Bergell succeeded in hydrolysing leucyl- 
 alanin through the agency of pancreas-extract, Fischer and Abder- 
 halden were unable to secure the hydrolysis of this substance 
 through the agency of pure trypsin. This pointed to the exist- 
 ence in the extract of enzymes other than trypsin which, unlike 
 
 * Emil Fischer and E. Abderhalden (10). The connection between these 
 results and the theory of Kuhne that the proteins are built up of hemi- and 
 anti-groups, respectively, hydrolysable and non-hydrolysable by pepsin and 
 trypsin is sufficiently evident to require no further comment here. 
 
HYDROLYSIS OF POLYPEPTIDS 375 
 
 trypsin, are able to bring about the hydrolysis of leucyl-alanm. 
 The possibility was thus clearly indicated that a variety of enr 
 zymes might be found to exist in the various tissues and tissue 
 fluids, distinguishable from one another in then: power to hydro- 
 lyse various polypeptids, but only with difficulty distinguishable 
 from one another in their action upon proteins, since every pro- 
 tein, it might be anticipated, contains unions susceptible to attack 
 by any one of these enzymes. 
 
 The possibility thus indicated has been rendered a certainty, 
 thanks to the extensive researches of Abderhalden and his col- 
 laborators (2). These investigators have employed the extracts 
 and press-juices of various organs, prepared by Buchner's method 
 of grinding up with sand and kieselguhr and pressing out at a 
 pressure of 100 to 300 atmospheres, which contain a number of 
 different enzymes capable of bringing about the hydrolysis of 
 various polypeptids. These enzymes are not so selective in their 
 action as pure trypsin, they can hydrolyse polypeptids which 
 trypsin cannot, and the press-juices of different organs exhibit 
 characteristic differences in regard to the polypeptids which 
 they can and those which they cannot attack. Most striking 
 differences were observed between the enzymatic activities of 
 the red blood-corpuscles and those of the plasma which bathes 
 them. The red corpuscles of the horse (but not those of the ox) 
 hydrolyse glycyl-1-tyrosin, which is not hydrolysed by white 
 corpuscles nor by plasma or serum; they also hydrolyse diglycyl- 
 glycin which, it will be recollected, is not hydrolysed by trypsin. 
 Plasma and serum both hydrolyse d-1-alanyl-glycin, diglycyl 
 glycin and triglycyl glycin; hence the enzymatic activity of the 
 serum is not attributable to trypsin or erepsin absorbed from 
 the intestinal wall. 
 
 Pure pepsin, prepared by Pavlov, does not act upon any of 
 the polypeptids, whereas it splits the natural proteins into some 
 half-dozen peptones and proteoses, although it does not split 
 these substances (which are in reality polypeptids) any further, 
 and hence yields no amino-acids. Of this fact two alternative 
 explanations are offered (16). Either the proteins contain cer- 
 tain types of union (such as ether-like combinations and so forth) 
 which are not present in the synthetic polypeptids and which 
 are the only points of union which pepsin can attack; or else 
 the great length of the amino-acid chain in the proteins confers 
 
376 CHEMICAL DYNAMICS 
 
 upon it greater susceptibility to pepsin than the shorter poly- 
 peptid chains possess. In support of the former view it is pointed 
 out that although erepsin, obtained from the succus entericus, 
 is able to hydrolyse peptones and polypeptids, yet it cannot 
 hydrolyse proteins, a fact which would appear to point towards 
 the existence of a few linkages in proteins which are not pres- 
 ent in polypeptids. In support of the latter view it is pointed 
 out that polypeptid-chains of greater length are more susceptible 
 to attack by other enzymes (i.e., trypsin) than the shorter chains, 
 so that the possibility cannot be overlooked that the suscepti- 
 bility of proteins to attack by pepsin may merely be attributable 
 to their great complexity, i.e., to the extreme length of the 
 polypeptid-chain. 
 
 2. The Kinetics of the Hydrolysis of Polypeptids by Proteo- 
 lytic Enzymes. The progressive hydrolysis of diglycyl-tyrosin 
 in the presence of trypsin was followed by Taylor (19). He 
 states that the results which he obtained were irregular and un- 
 satisfactory, but he regarded these irregularities as being attrib- 
 utable to analytical errors. 
 
 The optically active dipeptids, d-alanyl-d-alanin, d-alanyl- 
 1-leucin and glycyl-1-tyrosin, have been employed by Abderhalden 
 and Koelker, Abderhalden and Michaelis and Abderhalden and 
 Gigon (3) (4) (6) (14) in investigating the time relations of their 
 hydrolysis by trypsin. The degree of hydrolysis at any moment 
 can readily be followed by observing the optical rotation of the 
 solution, the rotation due to d-alanyl-d-alanin, for example, being 
 negative (a D 20 = 21.2), while that of the products of complete 
 hydrolysis is positive. The change in the optical rotation is, of 
 course, directly proportional to the degree of hydrolysis.* 
 
 The relation between the time and the extent of hydrolysis 
 is, as Abderhalden and Michaelis have shown, susceptible of 
 fairly simple formulation. The form of relation which is char- 
 acteristic for a monomolecular reaction (that is, a transformation 
 which involves only one species of molecule) is expressed by the 
 differential equation: 
 
 * Subject to a slight correction due to the fact that the specific rotatory 
 powers of the dipeptids are not absolutely independent of their concentrations. 
 Cf. Koelker (14). 
 
KINETICS OF HYDROLYSIS 
 
 377 
 
 in which x is the amount transformed after time t, a is the initial 
 quantity of substrate and k is the velocity-constant of the reaction. 
 When integrated this yields the equation: 
 
 log 
 
 a x 
 
 = kt. 
 
 G) 
 
 In the derivation of this equation it is to be noted that the 
 velocity of the reverse reaction is regarded as being negligible, 
 so that no station of equilibrium is reached until the transfor- 
 mation is practically complete. This condition is tolerably well 
 fulfilled in the reaction under consideration, for the hydrolysis 
 of the polypeptid does not cease until the quantity of unde- 
 composed polypeptid is practically unappreciable. In the fol- 
 lowing table are given the values of the constant k for the 
 hydrolysis of d-alanyl-d-alanin by liver extract at 37 degrees 
 calculated by Abderhalden and Michaelis from the experimental 
 values of a x and t obtained by Abderhalden and Koelker. 
 In every case the initial concentration of the dipeptid was such 
 that 1.45 grams of the substance were dissolved in 6 cc. of the 
 digest. 
 
 Time 
 in 
 min- 
 utes 
 
 1.45 units 
 of dipeptid 
 + 6 cc* of 
 ferment so- 
 lution k = 
 
 Time 
 in 
 min- 
 utes 
 
 1.45 units 
 of dipeptid 
 -f- 4 cc. of 
 ferment solu- 
 tion k = 
 
 Time 
 in 
 min- 
 utes 
 
 1.45 units 
 of dipeptid 
 + 3 cc. of fer- 
 ment solution 
 * = 
 
 Time 
 in 
 min- 
 utes 
 
 1.45 units 
 of dipeptid 
 + 2 cc. of fer- 
 ment solution 
 fc- 
 
 3 
 
 0.0453 
 
 3 
 
 0.0192 
 
 5 
 
 0.0125 
 
 5 
 
 0.00710 
 
 7 
 
 0.0390 
 
 7 
 
 0.0183 
 
 6.5 
 
 0.0132 
 
 11 
 
 0.00847 
 
 11 
 
 0.0342 
 
 10 
 
 0.0202 
 
 7.5 
 
 0.0134 
 
 15 
 
 0.00834 
 
 13 
 
 0.0336 
 
 11 
 
 0.0197 
 
 16 
 
 0.0142 
 
 23 
 
 0.00901 
 
 18 
 
 0.0380 
 
 16 
 
 0.0214 
 
 22 
 
 0.0161 
 
 31 
 
 0.01020 
 
 20 
 
 0.0373 
 
 17 
 
 0.0203 
 
 23 
 
 0.0163 
 
 41 
 
 0.01216 
 
 24 
 
 0.0399 
 
 25 
 
 0.0268 
 
 28 
 
 0.0192 
 
 53 
 
 0.01474 
 
 27 
 
 0.0451 
 
 30 
 
 0.0294 
 
 29 
 
 0.0196 
 
 65 
 
 0.01920 
 
 34 
 
 0.0430 
 
 34 
 
 0.0342 
 
 30 
 
 0.0209 
 
 80 
 
 0.01810 
 
 
 
 35 
 
 0.0359 
 
 38 
 
 0232 
 
 
 
 
 
 
 
 45 
 
 0.0265 
 
 
 
 
 
 
 
 
 
 
 
 It is evident that, within the experimental error, k calculated from 
 formula (i) is tolerably constant for the higher concentrations of 
 ferment solution (6 cc. of ferment solution); for lower ferment- 
 concentrations, however, the value of k does not even approxi- 
 mate to constancy, but rises markedly as hydrolysis proceeds; 
 in other words the velocity of the transformation does not fall 
 
378 
 
 CHEMICAL DYNAMICS 
 
 off so rapidly as it should were it a simple monomolecular re- 
 action of which the velocity constant is simply enhanced in 
 magnitude by the presence of the ferment. 
 
 In the derivation of equation (i) it is assumed that the velocity 
 of transformation at any moment is proportional to the mass of 
 substrate which is at that moment undergoing change. The 
 theoretical velocity therefore falls off in direct proportion as the 
 substrate is used up. The next simplest assumption, taking 
 cognizance of the fact that the velocity of transformation does 
 not decrease with the rapidity demanded by the monomolecular 
 formula, is that the velocity of transformation is independent 
 of the mass of the substrate, and is therefore expressed by the 
 equation : 
 
 dx 
 
 which, when integrated, yields 
 
 x = kit. (ii) 
 
 Calculating the values of ki from this formula, for the above 
 four series of experimental data Abderhalden and Michaelis 
 obtained the following results : 
 
 1 . 45 units of 
 
 1.45 units of 
 
 1 . 45 units of 
 
 1.45 units of 
 
 dipeptid + 6 cc. 
 of ferment solution 
 
 dipeptid + 4 cc. 
 of ferment solution 
 
 dipeptid + 3 cc. 
 of ferment solution 
 
 dipeptid + 2 cc. 
 of ferment solution 
 
 0.1300 
 
 0.0600 
 
 0.0380 
 
 0.0240 
 
 0.0843 
 
 0.0529 
 
 0.0400 
 
 0.0255 
 
 0.0713 
 
 0.0540 
 
 0.0400 
 
 0.0240 
 
 0.0708 
 
 0.0518 
 
 0.0369 
 
 0.0239 
 
 0.0639 
 
 0.0494 
 
 0.0368 
 
 0.0242 
 
 0.0595 
 
 0.0494 
 
 0.0365 
 
 0.0241 
 
 0.0538 
 
 0.0456 
 
 0.0368 
 
 0.0228 
 
 0.0504 
 
 0.0420 
 
 0.0366 
 
 0.0212 
 
 0.0411 
 
 0.0397 
 
 0.0314 
 
 0.0180 
 
 
 
 0332 
 
 
 
 
 0302 
 
 
 
 
 
 
 It is evident that k 1} calculated from formula (ii), tends to 
 approximate constancy for the lower ferment concentrations (2 cc. 
 and 3 cc.), while it altogether fails to do so for the higher ferment- 
 concentrations. To the latter solutions, as we have seen, equa- 
 tion (i) applies tolerably well. Neither equation, therefore, 
 taken by itself, represents the entire process; each applies under 
 
KINETICS OF HYDROLYSIS 
 
 379 
 
 certain limiting conditions. The possibility was thus indicated 
 that a combination of the two formulae might be found to ade- 
 quately represent all of the phenomena. Obviously the process 
 represented by equation (i) plays a predominant part when the 
 ferment-concentration is high; that represented by equation 
 (ii) plays a predominant part when the ferment-concentration 
 is low. We must therefore introduce into the combined equation 
 some factor e which will denote the proportionality between 
 the two processes, and which will be dependent upon the mass 
 of ferment. We thus obtain the equation : 
 
 lognat. 
 
 a x 
 
 ex 
 
 k z t; 
 
 (iii) 
 
 introducing the modulus of the natural logarithms (0.4343) we 
 obtain 
 
 a 
 
 logic 
 
 a x 
 
 which may be written 
 logi 
 
 + 0.4343 ex = 0.4343 fcrf, 
 
 + 0.4343 ex = 
 
 a x 
 
 (iv) 
 
 The following are the values of k z calculated by Abderhalden 
 and Michaelis from equation (iv) for the four series of deter- 
 minations cited above: 
 
 1.45 units of 
 
 1 . 45 units of 
 
 1 . 45 units of 
 
 1.45 units of 
 
 dipeptid + 6 cc. 
 of ferment solution 
 
 dipeptid + 4 cc. 
 of ferment solution 
 
 dipeptid + 3 cc. 
 of ferment solution 
 
 dipeptid + 2 cc. 
 of ferment solution 
 
 e = 0.7 
 
 e = 1.5 
 
 6 = 3.0 
 
 e = 10.0 
 
 100 k t = 
 
 100*3 = 
 
 100A 3 = 
 
 100 k t = 
 
 (8.49) 
 
 5.74 
 
 6.20 
 
 11.1 
 
 6.63 
 
 6.52 
 
 6.54 
 
 11.9 
 
 5.49 
 
 5.90 
 
 6.56 
 
 11.2 
 
 5.52 
 
 5.51 
 
 6.22 
 
 11.3 
 
 5.68 
 
 5.25 
 
 6.41 
 
 11.5 
 
 5.54 
 
 5.91 
 
 6.40 
 
 11.7 
 
 5.63 
 
 5.91 
 
 6.72 
 
 11.1 
 
 6.04 
 
 6.15 
 
 6.53 
 
 11.1 
 
 5.55 
 
 6.17 
 
 6.17 
 
 (9.6) 
 
 
 
 6.64 
 
 
 
 
 6.58 
 
 
 
 
 
 
 With the exception of the two values enclosed in brackets, 
 which are obviously influenced by experimental errors, the value 
 of & 3 for each set of determinations is evidently, within the ex- 
 
380 CHEMICAL DYNAMICS 
 
 perimental error, constant. The value of e, it is evident, rises 
 with decreasing ferment-concentration, but not in direct pro- 
 portion. 
 
 It is of great interest to observe that equation (iii) and (iv) 
 is of the same form as that which Henri (13) (6) has found to 
 hold good for the inversion of cane sugar by invertase. The 
 theoretical foundation of Henri's equation, therefore, calls for 
 consideration in this connection.* 
 
 Henri started from the point of view that the ferment, in the 
 presence of a substrate which is undergoing digestion, may con- 
 ceivably exist in three modifications, namely, in combination 
 with the substrate (concentration = F^, in combination with 
 the products (concentration = F p ), and in the free condition 
 (concentration = Ff). Calling the total concentration of ferment 
 F, we obviously have: 
 
 F = F. + F 9 + Ff.' (v) 
 
 Assuming that in the formation of the ferment-substrate 
 compound one molecule of ferment unites with one molecule 
 of substrate we have, from the mass-law: 
 
 F,(a-x)=F 8 , (vi) 
 
 in which is the equilibrium-constant of the reaction. 
 
 Similarly assuming that in the formation of the ferment- 
 products compound one molecule of ferment unites with one 
 molecule of the hydrolysis-products we have: 
 
 F>* = l ~F f , (vii) 
 
 in which - is the equilibrium-constant of the reaction. 
 n 
 
 Combining equations (v), (vi) and (vii) we obtain: 
 
 F 
 
 (viii) 
 
 l + m(a-x)+nx 
 and 
 
 j? mF ( fl ~ *) f^\ 
 
 '" ' 
 
 * The derivation of Henri's equation which follows is, essentially, quoted 
 from Taylor (19). 
 
KINETICS OF HYDROLYSIS 381 
 
 According to Henri, the velocity of hydrolysis might con- 
 ceivably be directly proportional to the mass of the ferment- 
 substrate compound (= F a ) or to the mass of the free ferment 
 and that of the substrate. In the former case we obtain: 
 
 dx kmF(a x) . . 
 
 ~j~4 ~ i i _ ( \ i ' W 
 
 at 1 ~r m (a x ) -r nx 
 
 in which k is the velocity-constant of the reaction. In the latter 
 case we have: 
 
 dx = kF(a - x) ( .. 
 
 dt 1 + m (a x) -f nx 
 
 Obviously both equations are identical in form; the former 
 when integrated, leads to the equation: 
 
 (1 + na) log h (m n) x = kjt, (xii) 
 
 a ~~ x 
 
 in which k s is a constant which is directly proportional to the 
 total concentration of ferment, and n and m are independent 
 of the mass of ferment or substrate, but are obviously dependent 
 upon the temperature and the conditions (reaction, etc.) of the 
 experiment. Putting 
 
 1 +na 
 
 we obviously regain the equation of Abderhalden and Michaelis. 
 The condition that m and n should be independent of the mass 
 of ferment, however, is obviously not fulfilled by Abderhalden 
 and Michaelis' results, since varies notably with the concen- 
 tration of the ferment; while k s does not approximate to direct 
 proportionality to the mass of ferment. 
 
 In the derivation of Henri's equation it will be evident that ' 
 many simplifying assumptions are made which are not justified 
 by anything save the fact that they afford the simplest con- 
 ception of the relations. Thus it is assumed that the active 
 mass of the ferment in so far as hydrolysis is concerned is directly 
 proportional to its actual mass, that one molecule of ferment 
 reacts with one molecule of substrate, that one molecule of fer- 
 ment reacts with one molecule of the products of hydrolysis, 
 that the concentration of the ferment-substrate compound is 
 
382 
 
 CHEMICAL DYNAMICS 
 
 evanescent in comparison with that of the substrate and so forth. 
 If we try to generalize Henri's equation by removing these 
 simplifying assumptions we obtain equations which are, mathe- 
 matically speaking, unmanageable. The surprising thing is, 
 therefore, not that Henri's equation fails to adequately repre- 
 sent Abderhalden and Koelker's results, but that, if the con- 
 ditions in the system are as complex as those which Henri depicts, 
 any simple relation can be found which will express the progress 
 of the reaction with any approach to fidelity. 
 
 Computing the average values of 100 & 3 calculated from the 
 equation (iv) for the various ferment-concentrations employed 
 by Abderhalden and Koelker we obtain: 
 
 cc. of ferment solu- 
 tion in 6 cc. of digest 
 
 100*3 
 
 cc. of ferment solu- 
 tion in 6 cc. of digest 
 
 lOOifca 
 
 2 
 3 
 
 11.6 
 
 6.45 
 
 4 
 6 
 
 6.01 
 5.76 
 
 from which it is evident that the velocity-constant of hydrolysis, 
 when computed from Henri's equation, far from being directly 
 proportional to the ferment-mass is actually greater the more 
 dilute the ferment. It appears distinctly within the bounds of 
 possibility that the intensity of the hydrolysing activity of the 
 ferment is relatively greater the more dilute its solution. This, 
 in turn, suggests the possibility that the ferment may exist both 
 in an active and inactive form (active and inactive, that is, with 
 respect to the acceleration of hydrolysis). The proportion of in- 
 active ferment would, of course, depend, not only upon the total 
 mass of ferment in the system, but upon the mass of uncombined 
 active ferment. If this were the case then k and e in Abder- 
 halden and Michaelis' equation, accepting Henri's derivation of 
 the equation, might be expected to be functions, not only of the 
 total ferment mass and of the relation between the mass of free 
 active ferment and that of the substrate and products respec- 
 tively, but also of the relation between the proportion of ferment 
 in the inactive condition and the mass of the uncombined active 
 ferment (Cf. Chap. XVII). 
 
 On the other hand, it is possible that the coincidence between 
 Abderhalden and Michaelis' equation and that of Henri is merely 
 formal, that, as Abderhalden and Michaelis suggest, the true 
 
KINETICS OF HYDROLYSIS 
 
 383 
 
 equation expressing the course of hydrolysis is in reality equation 
 (i), but that as hydrolysis proceeds the reaction is subject to an 
 acceleration, possibly attributable, either directly or indirectly, 
 to the products of the reaction. 
 
 Euler (9) has studied the hydrolysis of glycin by erepsin, 
 obtained from the intestinal wall. The method employed was 
 that of following the hydrolysis by measuring the progressive 
 alteration of the electrical conductivity of the solution, direct 
 proportionality between the alteration in conductivity and that 
 of the dipeptid-concentration having previously been established. 
 He finds that the velocity of hydrolysis is very intimately de- 
 pendent upon the alkalinity of the solution, thus: 
 
 1/10 N GLYCYL-GLYCIN. 5 G. EREPSIN [POWDER IN 100 CC. 
 
 Alkali-concentration N 0.04 0.05 0.075 0.10 
 
 Reaction-velocity constant X 1000 0.05 7.0 6.2 2.6 0.2 
 
 For a given initial NaOH, dipeptid and erepsin concentration 
 the monomolecular formula represents the progress of this re- 
 action with tolerable fidelity, as the following results show: 
 
 0.10 N GLYCYL GLYCIN. 5 GRAMS EREPSIN IN 100 CC. 
 
 0.04 AT Na 
 
 Minutes 
 
 1000 (a - x) 
 
 1000 k 
 
 Minutes 
 
 1000 (a - z) 
 
 1000 k 
 
 o 
 
 920 
 
 
 o 
 
 935 
 
 
 7 
 
 819 
 
 7.20 
 
 10 
 
 806 
 
 6.46 
 
 15 
 
 721 
 
 7.08 
 
 17 
 
 739 
 
 6.00 
 
 22 
 
 649 
 
 6.88 
 
 25 
 
 654 
 
 6.18 
 
 30 
 
 579 
 
 6.70 
 
 30 
 
 622 
 
 6.90 
 
 0.05 N Na 
 
 Within certain limits the reaction-velocity is independent of 
 the initial concentration of this dipeptid, but this holds good 
 only within certain limits of the proportion enzyme : substrate. 
 If the enzyme-concentration is small, for a given Na-concentra- 
 tion the reaction-velocity rises with increasing concentration of 
 glycyl-glycin. Euler attributes this fact to neutralization of 
 injurious excess of NaOH by the additional glycyl-glycin. The 
 progressive change in the reaction-constant as hydrolysis proceeds 
 may doubtless be attributed to the fact that the substrate and 
 products are possessed of different combining capacities for bases, 
 so that the proportion of free base alters as the reaction proceeds. 
 
384 
 
 CHEMICAL DYNAMICS 
 
 Euler has arrived at the conclusion that in solutions of glycyl- 
 glycin which contain a base, only the salt of the dipeptid under- 
 goes hydrolysis. In terms of recent theories of catalysis (18) 
 this may be held to indicate that the substance which actually 
 undergoes hydrolysis is the dipeptid ion. 
 
 For high ferment-concentrations the velocity of the hydrolysis 
 of glycyl-glycin is, according to Euler, directly proportional to 
 the concentration of erepsin. If, however, the ferment concen- 
 tration is low, then the value of k, calculated from the mono- 
 molecular formula, increases much more rapidly than the con- 
 centration of erepsin. 
 
 The influence of various added substances (salts, ammo-acids, 
 and so forth) upon the rate of hydrolysis of dipeptids by liver- 
 extract has been studied by Abderhalden and Gigon (3). They 
 find that dilute solutions of KCN accelerate while strong solu- 
 tions greatly retard the hydrolysis of d-1-leucyl-glycin; sodium 
 fluoride strongly retards the hydrolysis of d-1-leucyl-glycin and 
 of glycyl-1-tyrosin; "physiological salt solution" has no effect 
 upon the rate of hydrolysis. In high concentrations both mag- 
 nesium chloride and sulphate depress the rate of hydrolysis; 
 calcium chloride accelerates the hydrolysis and strontium chlo- 
 ride is indifferent, the substrates being d-1-leucyl-leucin and glycyl- 
 1-tyrosin. 
 
 The effect of amino-acids upon the rate of hydrolysis is of 
 surpassing interest since these are the products of the reaction, 
 and if they exert an influence upon the rate of hydrolysis they will 
 induce more or less marked deviations in the relation between 
 the duration and the extent of hydrolysis from that which would 
 be indicated by the monomolecular law or by Henri's modifi- 
 cation of the monomolecular law, described above. Employing 
 glycyl-1-tyrosin they find that : 
 
 The hydrolysis is 
 accelerated by 
 
 The hydrolysis is 
 unaffected by 
 
 The hydrolysis is 
 slightly retarded by 
 
 The hydrolysis is 
 strongly retarded by 
 
 1-alanin 
 
 glycocoll 
 d-leucin 
 
 d-1-alanin 
 d-valin 
 
 d-alanin 
 d-1-valin 
 
 
 
 d-1-leucin 
 
 1-leucin 
 
 
 
 d-1-serin 
 
 1-serin 
 
 
 
 
 1-tyrosin 
 
 
 
 d-isoserin 
 
 d-1-isoserin 
 
 
 
 
 1-isoserin 
 
 
 
 
 
ORDER IN WHICH AMINO-ACIDS ARE SPLIT 385 
 
 In addition it was observed that d-glutamic acid, d-tryptophane, 
 1-diamino-trioxydodecanic acid, d-1-aminobutyric acid and phenyl- 
 alanin depress the rate of hydrolysis. 
 
 In every case it will be observed that the effects of optical 
 antipodes are considerably different, in quantity or even, in the 
 case of alanin, in sense, from one another and that the racemic 
 body affects the hydrolysis in an intermediate manner. Of 
 great significance is the fact that 1-tyrosin, which is itself a prod- 
 uct of the hydrolysis of glycyl-1-tyrosin, strongly retards the 
 hydrolysis, although glycocoll, which is the other product, does 
 not. Abderhalden and Gigon attribute this to a binding of the 
 ferment by the tyrosin. The affinity between glycocoll and the 
 ferment is slight and so this product does not retard hydrolysis 
 so markedly. It is possible that the low degree of affinity be- 
 tween glycocoll and the majority of the proteolytic ferments is 
 responsible for the difficultly digestible character of many of the 
 peptids which are rich in glycocoll. 
 
 The influence of temperature upon the rate of hydrolysis of 
 glycyl-1-tyrosin and d-1-leucyl-glycin by liver and pancreas- 
 extracts has also been studied by Abderhalden, Caemmerer and 
 Pincussohn (2). In each case, within certain limits, the rate of 
 hydrolysis is very greatly accelerated by a rise in temperature. 
 Very high temperatures, naturally, delay or prevent hydrolysis 
 by destruction of the enzyme. For liver extract, working upon 
 these substrates, the temperature-optimum proved to be about 
 55 degrees; for pancreas-extract between 45 and 50 degrees. 
 
 3. The Order in which Amino-acids are Split off from Poly- 
 peptids by Proteolytic Ferments. The optical properties of 
 certain polypeptids and of their possible decomposition-products 
 have been utilized by Abderhalden and Koelker (4), Abderhalden 
 and Brahm (1) and Abderhalden, Koelker and Medigreceanu (5) 
 in the attempt to ascertain which point in a tri- or tetra-peptid 
 is first attacked by a proteolytic ferment. Thus 1-leucyl-glycyl- 
 d-alanin has a molecular rotation * of +52 degrees. It might 
 conceivably yield in the first place either 1-leucyl-glycin (+172 
 degrees) and d-alanin (+2 degrees), or else glycyl-d-alanin (73 
 degrees) and 1-leucyl ( 13 degrees), or else, through simultaneous 
 splitting of both NHOC bonds, all three of the products 
 
 * The rotations given are 1/100 of the molecular rotations. Cf. Koelker 
 (14). 
 
386 CHEMICAL DYNAMICS 
 
 1-leucyl (13 degrees), glycin (0 degrees) and d-alanin (+2 
 degrees). If the first link to be broken, therefore, is that be- 
 tween the glycyl and alanyl groups the positive rotation of the 
 original solution should increase until it reaches a maximum. 
 If this link were the only one attacked then this maximum rota- 
 tion should be that of a mixture of 1-leucyl-glycin and d-alanin, 
 i.e., over three times that of the original solution. If the first 
 link to be broken were that between the leucyl- and the glycyl- 
 groups the original positive rotation should decrease and finally 
 become negative. If the two links were split simultaneously 
 the positive rotations should decrease until it became very slightly 
 negative. These possibilities are made clear by the following 
 diagram: 
 
 1-leueyl-glycyl-d-alanin 
 
 
 + 172 
 
 -73 
 
 The experimental result, employing trypsin, is that the rota- 
 tion at first increases to an extent of about 40 per cent. Hence 
 1-leucyl-glycin must be liberated and the point of first attack 
 (or, at least, most rapid attack) must be the bond between the 
 glycyl and the alanyl groups. After attaining a maximum 
 positive rotation only 40 per cent in excess of that of the original 
 solution, however, the rotation declines again, owing to the 
 hydrolysis of the 1-leucyl-glycin. Hence the enzyme does not 
 complete the decomposition of the one bond before attacking 
 the other. In other words the two reactions proceed side by 
 side but at different velocities, that of the splitting of the glycyl- 
 alaniri bond being the most rapid. The leucyl-glycin bond, 
 however, does not appear to be attacked at all while it is bound 
 up in the tripeptid molecule, there being no evidence of the 
 formation of glycyl-d-alanin. The progress of the reaction is 
 therefore, it would appear, the following, first the glycyl-alanin 
 link is broken and 1-leucyl-glycin and alanin are split off. The 
 v.elocity of this reaction is so much greater than that of the other 
 possible reactions (splitting of the leucyl-glycyl link) that the 
 latter reaction does not occur to any appreciable extent. The 
 1-leucyl-glycin which is thus formed is immediately attacked by 
 
ORDER IN WHICH AMINO-ACIDS ARE SPLIT 387 
 
 the ferment. The substrate of this second reaction being, however, 
 a product of the first, its velocity is at first low, since the sub- 
 strate concentration is low; but as velocity of the first reaction 
 declines, owing to the consumption of the substrate, that of 
 the second reaction increases, owing to the increase in the mass 
 of its substrate. 
 
 When however, instead of trypsin, yeast endotryptase is em- 
 ployed as the ferment, not the glycyl-alanin bond but the leucyl- 
 glycyl bond is first attacked. There is thus no question but 
 that the mode of action of yeast endotryptase is quite different 
 from that of trypsin and a means of sharply distinguishing be- 
 tween different enzymes is clearly indicated. It will also be 
 evident that in digests containing a mixture of proteolytic enyzmes, 
 such as occurs in tissue-extracts, etc., the conditions of hydrolysis 
 must be much more complex than they are in digests containing 
 only a single enzyme; since under such circumstances two or 
 more parallel reactions may be occurring, possessed of different 
 specific velocities, at each step in the progressive hydrolysis of 
 of the peptid or protein. 
 
 Glycyl-d-alanyl-glycin is first attacked by trypsin at the glycyl- 
 d-alanyl junction, by yeast endotryptase at the alanyl-glycin 
 junction; d-alanyl-glycyl-glycyl-glycin is first attacked by trypsin 
 at the glycyl-glycin junction. 
 
 LITERATURE CITED 
 
 (1) Abderhalden, E., and Brahm, C., Zeit. f. physiol. Chem., 57 (1908), 
 
 p. 342. 
 
 (2) Abderhalden, E., and Collaborators (Babkin, Bergell, Bloch, Damm- 
 
 hahn, Deetjen, Gigon, Heise, Hunter, Kautsch, Koelker, Lussana, 
 Manwaring, McLester, Medigreceanu, Michaelis, Oppler, Pilliet, 
 Pincussohn, Pringsheim, Rona, Samuely, Schittenhelm, Teruuchi, 
 Walther, Weichardt). Zeit. f. physiol. Chem., 39 (1903), p. 9; 
 46 (1905), pp. 176 and 187; 47 (1906), pp. 159, 346, 359, 391, 466; 
 48 (1906), pp. 537, 557; 49 (1906), pp. 1, 21, 26, 31; 51 (1907), pp. 
 294, 308, 334; 54 (1908), p. 363; 55 (1908), pp. 371, 377, 384, 390, 
 395, 416; 57 (1908), p. 332; 59 (1909), p. 249; 60 (1909), p. 415; 61 
 (1909), p. 200; 62 (1909), pp. 120, 136, 145, 243; 66 (1910), pp. 265, 
 277; 68 (1910), p. 471. 
 
 (3) Abderhalden, E., and Gigon, A., Zeit. f. physioL Chem., 53 (1907), 
 
 p. 251. 
 
 (4) Abderhalden, E., and Koelker, A. H., Zeit. f . physiol. Chem., 51 (1907), 
 
 p. 294. 
 
388 CHEMICAL DYNAMICS 
 
 (5) Abderhalden, E., and Koelker, A. H., and Medigreceanu, F., Zeit. f. 
 
 physiol. Chem., 62 1909), p. 145. 
 
 (6) Abderhalden, E., and Michaelis, L., Zeit. f. physiol. Chem., 52 (1907), 
 
 p. 326. 
 
 (7) Bayliss, W. M., "The Nature of Enzyme Action," London, 1908. 
 . (8) Bredig, G., Ergeb. d. Physiol., 1 Abt. 1 (1902), p. 134. 
 
 (9) Euler, H., "Allgemeine Chemie der Enzyme," Weisbaden, 1910. 
 
 (10) Fischer, E., and Abderhalden, E., Zeit. f . physiol. Chem., 39 (1903), p. 
 
 81. 
 
 (11) Fischer, E., and Abderhalden, E., Zeit. f. physiol. Chem., 46 (1905), p. 
 
 52; 51 (1907), p 264. 
 
 (12) Fischer, E., and Bergell, P., Ber. d. d. chem. Ges., 36 (1903), p. 2592; 
 
 37 (1904), p. 3103. 
 
 (13) Henri, V., "Lois generales de Faction des Diastases," Paris (1903), p. 
 
 92. 
 
 (14) Koelker, A. H., Journ. Biol. Chem., 8 (1910), p. 145. 
 
 (15) Oppenheimer, C., "Ferments and their Actions," Erlangen (1901), 
 
 English trans., London (1901). 
 
 (16) Plimmer, R. H. A., "The Chemical Constitution of the Proteins," 
 
 London (1908), Pt. 2, p. 50. 
 
 (17) Robertson, T. Brailsford, "The Proteins," Univ. of California Publ. 
 
 Physiol., 3 (1909), p. 123. 
 
 (18) Stieglitz, J., Amer. Chem. Journ., 39 (1908), p. 29. 
 
 (19) Taylor, A. E., "On Fermentation," Univ. of California Publ. Pathol. 
 
 1 (1907), p. 87. 
 
 (20) Vernon, H. M., " Intracellular Enzymes," London, 1908. 
 
CHAPTER XVI 
 THE HYDROLYSIS OF THE PROTEINS 
 
 1. The Proteolytic Enzymes as Catalysors. The fact that 
 proteins, in the presence of water, can be hydrolysed to ammo- 
 acids by prolonged heating, e.g., by superheated steam (126) 
 (80) (81) (76) (69) (70) (87) (115) without the addition to the 
 system of any acid, alkali or ferment, indicates that the process 
 of hydrolysis is occurring, although slowly, at all temperatures 
 and in the absence of catalysors other than, possibly, the hydrogen 
 or hydroxyl ions of water itself. The fact that, at ordinary 
 temperatures, catalysors can bring about the hydrolysis of pro- 
 teins shows that even at these temperatures the proteins are not 
 in equilibrium with their products. The influence of rising tem- 
 perature upon a chemical reaction is always twofold; it shifts 
 the station of equilibrium in one sense or in the opposite and, 
 always, it accelerates the reaction to a greater or less degree 
 (i.e., magnifies the velocity-constant). The action of heat upon 
 proteins must be in all cases to shift the station of equilibrium 
 in the direction of polymerization (i.e., condensation) since the 
 reaction of hydrolysis is faintly exothermic, but the fact that 
 fairly complete hydrolysis occurs at temperatures above 100 
 degrees shows that the shift in the equilibrium between the lower 
 protein complexes and the amino-acids which are the products of 
 their hydrolysis is not so great as to extinguish the reaction of 
 hydrolysis. The effect of high temperatures in accelerating the 
 auto-hydrolysis of proteins is to be looked upon, therefore, as 
 that of rendering readily detectable, through acceleration, a 
 reaction which occurs, although slowly, at all temperatures (133). 
 It is possible to demonstrate directly, however, and without 
 appeal to inference, that the hydrolysis of proteins in neutral 
 watery solutions does occur at normal temperatures; the velocity 
 of hydrolysis is, however, usually very low under these conditions. 
 Taylor has shown that an appreciable proportion of pure sterile 
 globulin kept in distilled water at ordinary temperatures for 
 18 months is hydrolysed to proteoses; he has also found that 
 
 389 
 
390 CHEMICAL DYNAMICS 
 
 leucin may be recovered from a sterile suspension of casein in 
 pure water and that arginin may be recovered from a solution 
 of protamin sulphate in pure water, both after the lapse of a 
 year or more (133); other examples of the slow autohydrolysis 
 of proteins in pure water might be adduced (133) (131). In a 
 few cases, however, the autohydrolysis occurs at a readily meas- 
 ureable velocity. I have found (110) that the velocity-constant 
 of the hydrolysis of casein in milk at 36 degrees (in the presence 
 of excess of toluol) is 0.000546, common logarithms being em- 
 ployed and the time expressed in hours (using the monomolecular 
 formula, Cf. previous chapter equation (i)). For a 2.8 per cent 
 solution of casein in NaOH, carefully neutralized to litmus and 
 therefore containing H + and OH' ions in the concentrations in 
 which they exist in pure water, the velocity-constant at 36 de- 
 grees, similarly estimated, proved to be 0.000518. In the first 
 experiment the extent of hydrolysis was estimated by deter- 
 mining the residue of undigested casein after 32 days, in the 
 second experiment the undigested residue was determined after 
 20 days. Now in the second experiment, at all events, no fer- 
 ments were present and in both experiments the solutions were 
 almost exactly neutral. We must therefore regard the observed 
 hydrolysis as being not due to catalysors but to the action of the 
 solvent water itself or of its ions.* Expressed in numerical 
 terms, the above-cited results mean that in absolutely neutral 
 solution, in the absence of any proteolytic enzymes, one-half of 
 a caseinate of sodium or calcium is hydrolysed in about 24 days. 
 
 The hydrolysis of neutral caseinates by trypsin or pepsin 
 affords, therefore, an unusually favorable example, among pro- 
 tein reactions, of the action of an enzyme in accelerating an 
 already progressing reaction. 
 
 * Tt might be inferred from these results that normal milk contains no 
 proteolytic enzymes. This inference would not be altogether a safe one, how- 
 ever, since the milk which was employed was obtained in the open market and 
 there is reason to suspect that it had been manipulated in a manner which, it 
 is possible, may have destroyed pre-existing enzymes. No difficulty was en- 
 countered in keeping this milk sterile, throughout the course of the experi- 
 ments, by the simple addition of toluol. When milk obtained from the 
 University Experimental Farm was employed, however, which had not been 
 pasteurized, it was found impossible to keep the milk sterile, for the length 
 of time required, without employing, as sterilizing agents, substances which 
 might conceivably destroy or injure proteolytic enzymes. 
 
PROTEOLYTIC ENZYMES AS CATALYSORS 391 
 
 The autohydrolysis of- the casemates in neutral and faintly 
 alkaline solutions has been more extensively studied by E. H. 
 Walters (147) who has shown that the neutral caseinates of 
 lithium, sodium and potassium in sterile solution undergo com- 
 paratively rapid autohydrolysis, approximately five per cent of 
 the protein being hydrolysed in ninety-six hours at 37.5 C. 
 The "basic" caseinates (neutral to phenolphthalein) of the same 
 bases undergo autohydrolysis at a slightly higher velocity. The 
 velocity of the autohydrolysis of the "basic" casemates of cal- 
 cium and barium is about three times as great as that of the 
 autohydrolysis of the caseinates of lithium, sodium or potassium, 
 indicating very clearly that some factor other than the H+ or 
 OH' ions plays a part in determining the velocity of the 
 autohydrolysis. 
 
 The proteolytic enzymes have long been regarded, by the 
 majority of investigators * as true catalysors; but the evidence 
 which has been brought forward in support of or in opposition 
 to this view has varied very much in character from time to 
 time. With the rapid gain in exact knowledge in the field of 
 physical chemistry which characterized the scientific advance 
 in the latter part of the last century, the characteristic features 
 of many catalytic processes became very thoroughly known and 
 certain catalytic reactions came to be regarded as "typical" 
 for no better reason than that they were the best studied and 
 therefore the best known. Now these so-called "typical" in- 
 stances of catalysis had in the first place attracted the attention 
 of investigators simply because of the marked peculiarities which 
 they exhibited and which appeared to differentiate them sharply 
 from other chemical reactions, and thus, by a not unnatural 
 process of circular reasoning these peculiarities came to be re- 
 garded as "typical" and diagnostic of "true" catalysis. Our 
 conceptions and definitions of "catalysis" liave therefore altered 
 and enlarged as the number of "typical" instances has grown, 
 and, correspondingly, features in the mode of action of ferments 
 have been brought forward, at one time in support of the thesis 
 that the ferments are not true catalysors, and at another time 
 in support of the exact converse of this view. Unquestionably 
 attempts have been made by many authors to shape the phe- 
 nomena of fermentation into accord with prematurely rigid 
 * Starting with Berzelius and Liebig. Cf. Jacob Berzelius (8). 
 
392 CHEMICAL DYNAMICS 
 
 conceptions of catalysis based upon insufficiently extended inves- 
 tigation. 
 
 A phenomenon which at a very early date greatly impressed 
 the investigators in this field was the extraordinarily small quan- 
 tity of a catalytic agent which, in " typical" instances of catalysis, 
 was found sufficient to bring about the chemical alteration of 
 enormous quantities of material. Thus T % of a milligram of 
 colloidal platinum will bring about the combination of the hydro- 
 gen and oxygen in no less than 10 litres of gas, without the least 
 reduction in its efficiency as a catalysor (25); 0.000001 grams of 
 potassium permanganate in 10 cc. of solution notably accelerates 
 the reduction of mercuric chloride by oxalic acid (64); the rate 
 of oxidation of an aqueous solution of sodium sulphite is per- 
 ceptibly accelerated by the presence of 0.0000000000001 N CuSO 4 , 
 or even by merely dipping a strip of clean metallic copper in 
 the water for less than a minute (136). Were any appreciable 
 proportion of the catalyst used up during the process of catalysis 
 it is obvious that quantities so minute as these would be incapable 
 of bringing about the conversion of such enormous quantities of 
 material, and, in fact, we find in many cases, even when relatively 
 large quantities of catalysor are employed, the catalysor is not 
 appreciably used up during the progress of the reaction; thus 
 a solution of cane sugar contains the same amount of acid after 
 hydrolysis as it did before (19) (67). But if the catalysor is 
 unaltered at the end of the reaction which it accelerates then it 
 must have accomplished this acceleration without adding any energy 
 to or subtracting any energy from the reacting system, in other 
 words it cannot in any way have affected the final equilibrium 
 of the system, but only hastened the attainment of that equi- 
 librium, since any shift in equilibrium must in general be ac- 
 companied either by an expenditure or an absorption of energy. 
 This theoretical deduction has been confirmed in a great variety 
 of instances in the field of non-fermentative catalysis*; in the 
 field of fermentations data bearing upon this deduction are either 
 lacking or fail to establish its validity (27) (14). 
 
 If, however, the station of equilibrium in any reaction is un- 
 affected by the presence of a given catalysor, then that catalysor 
 
 * Cf. especially Turbuba (137) (138), who has shown that the equilibrium 
 between aldehyde and paraldehyde is the same whether sulphur dioxide, zinc 
 sulphate, hydrochloric acid, oxalic acid or phosphoric acid is used as catalysor. 
 
PROTEOLYTIC ENZYMES AS CATALYSORS 393 
 
 must accelerate, and accelerate equally, the attainment of that 
 equilibrium from either side; it must in other words accelerate 
 the forward and the reverse reactions equally. This is readily 
 seen when we reflect that equilibrium in the reacting system is 
 attained when the velocity of the forward is equal to that of the 
 reverse reaction. If the velocity of the forward reaction is 
 increased by any agency, therefore, and the station of equi- 
 librium is unaffected, the velocity of the reverse reaction must 
 also have been increased and in the same proportion. The 
 correctness of this theoretical deduction has also been established 
 in a number of "typical" instances of non-fermentative catalyses, 
 but although it has been shown that yeast (58) (59), kephirlactase 
 (28), diastase (22), emulsin (24), lipase (7) (65) (55) (9) (130) 
 (132) (98) (99), trypsin (135) and pepsin (108) (109) (111) (112) 
 (35) will not only accelerate the hydrolysis of maltose, lactose, 
 glycogen, amygdalin, fats, protamin and paranuclein, respec- 
 tively, but also the synthesis of these substances from the prod- 
 ucts of their hydrolysis, yet it has in no instance been shown 
 that the velocity of the reverse reaction is accelerated in the 
 same proportion as that of the forward reaction; indeed such 
 evidence as exists tends to show that this is not the case (14) 
 
 CUD. 
 
 From the study of a limited number of instances of non- 
 fermentative catalysors, however, it appeared, as I have said, 
 that the catalysors are substances which remain unchanged at 
 the end of the reaction which they accelerate and which do not 
 shift the station of equilibrium and therefore cannot initiate, 
 but only accelerate reactions (89) (90) (91) (92) (93). 
 
 Recent investigations, particularly those of Stieglitz (127) (128) 
 and Euler (27), have shown that the above definition of catalysis 
 is too rigid and that the cases which it adequately covers are 
 merely extreme instances of a much more general phenomenon. 
 
 It has long been known that in many instances of unquestioned 
 catalysis the acceleration of the reaction in question is accom- 
 plished, or at least accompanied by, the formation of inter- 
 mediate compounds between the substrate and the catalysor. 
 The classic illustration of a catalysed reaction of this type is 
 that afforded by the "continuous etherification process" for the 
 production of ether from alcohol. In this process sulphuric 
 acid is employed as catalysor and, as is well known, the sulphuric 
 
394 CHEMICAL DYNAMICS 
 
 acid first combines with the alcohol, forming a substance "which 
 can be isolated, namely ethyl-sulphuric acid and this compound 
 reacts with another molecule of alcohol, forming ether and re- 
 generating the sulphuric acid, in accordance with the equations 
 
 HO X C 2 H 5 0. 
 
 C 2 H 5 OH + S0 2 = H.OH + S0 2 
 
 HO V 
 S0 2 + C 2 H 5 OH = S0 2 + C 2 H 5 - O - C 2 H 5 
 
 and, at the end of the reaction, the sulphuric acid molecule which 
 is set free is unaltered and can carry another molecule of alcohol 
 through the same series of transformations. Hence, as in the 
 "typical" cases of catalysis cited above, the only things which 
 prevent the reaction from proceeding indefinitely, with the con- 
 version of an indefinite quantity of alcohol through the agency 
 of a limited quantity of sulphuric acid, are the failure of the 
 supply of alcohol, or the continued accumulation of the products; 
 thus, if the water which is formed in the above reaction be allowed 
 to remain in the system the sulphuric acid becomes, finally, so 
 highly diluted that the velocity of the transformation sinks to 
 a negligible magnitude, i.e., for practical purposes, the reaction 
 ceases. 
 
 Another example of the same kind is the use of nitric oxide 
 as a "carrier" of oxygen in the manufacture of sulphuric acid. 
 The first reaction is the combination of NO with O to form 
 
 nitrogen peroxide: 
 
 2 NO + O 2 = 2 NO 2 . 
 
 This is brought into contact with SO 2 and steam, sulphuric acid 
 is formed and nitric oxide regenerated : 
 
 S0 2 + H 2 + NO 2 = H 2 S0 4 + NO 
 
 and the NO is now free again to "carry" oxygen into the sul- 
 phurous, converting it into sulphuric acid. 
 
 In a large number of instances in which the intermediate 
 reactions have not been defined and their occurrence established, 
 it has nevertheless been shown that the catalysor forms com- 
 pounds with the substrate or with modifications of the substrate, 
 
PROTEOLYTIC ENZYMES AS CATALYSORS 395 
 
 of such a character as to strongly suggest that they play an im- 
 portant part in determining the course of the reaction. Thus 
 in the presence of aluminium chloride, sulphuryl chloride and 
 sulphur chloride react thus : 
 
 S0 2 C1 2 + S 2 C1 2 = S0 2 + 2 SC1 2 
 
 and it appears that the aluminium chloride aids the reaction 
 through the formation of double compounds with the various 
 molecular species which participate in the reaction, for many 
 such compounds are known, for example A1 2 C1 2 2 S0 2 , AlClsSCU, 
 and so forth (20). 
 
 A very decisive series of instances of catalysis through the 
 formation of] intermediate compounds has been established by 
 the painstaking and extensive researches of Stieglitz (1) (127) 
 (128). In the presence of acids the iminoesters (in aqueous 
 solution) are decomposed into ammonia and the corresponding, 
 sparingly ionized organic acid. It has been shown by Stieglitz, 
 with the utmost quantitative precision, that the catalytic action 
 of the mineral acid, in this reaction, is accomplished through 
 the formation of salts of the catalysing acid with the ester, since 
 the molecular species which actually undergoes hydrolysis in 
 this system is the ester-ion and the salts of the ester are much more 
 completely electrolytically dissociated than the ester itself, so that 
 the active mass of the substrate is increased by the presence of the 
 catalysor. Similarly, in the catalytic decomposition of methyl 
 acetate by acids an oxonium salt of the ester with the acid is 
 formed and it is the positive ion of this salt which undergoes 
 hydrolysis. Now this latter is a "typical" instance of catalysis. 
 The catalytic agent is not used up during the reaction, it can be 
 recovered from the system unaltered when the reaction is com- 
 plete, and the equilibrium of the reaction is not measurably 
 disturbed by the presence of the catalysor. Nevertheless, in a 
 comprehensive mathematical analysis of the conditions which 
 must actually obtain in such a system, Stieglitz has shown that 
 the point of equilibrium must be shifted by the catalysor, albeit 
 to an immeasurably slight degree, and that in the specific instance 
 under consideration the equilibrium is only inappreciably shifted 
 simply because the quantity of the compound which is formed 
 at any moment, between the catalysor and the substrate, is 
 evanescently small, since these salts are subject to very extensive 
 
396 CHEMICAL DYNAMICS 
 
 hydrolytic dissociation. When the salt formation is at any 
 instant extensive then, as Stieglitz points out, although in every 
 respect the mechanism of the acceleration of the hydrolysis by 
 the catalysing acid is the same, yet a definite shift in equilibrium 
 results from the presence of the catalysor and, concurrently, the 
 catalysor is in some measure used up during the progress of the 
 reaction and some energy has to be expended in order to recover 
 it, unaltered, from the system in its final condition of equilibrium. 
 I quote from Stieglitz' article : * 
 
 "In accordance with the results, our views concerning catalytic 
 action must be modified in regard to all three of the commonly 
 assumed fundamental characteristics of catalytic action, (1) that 
 the acceleration must be proportionate to the concentration of 
 the catalytic agent present; (2) that the agent must not appear 
 to combine with any of the substances undergoing change; and 
 (3) that the ultimate condition of equilibrium must not be 
 measurably modified by the presence of a catalysor. These 
 characteristics are practically true only for limiting cases where 
 the amount of salt-formation is so small as to be beyond the 
 scope of our measurements. None of them is absolutely true 
 under any conditions. When the amount of salt-formation 
 becomes measurable, as for iminoesters, they need not hold even 
 approximately, and still the fundamental mechanism and mode 
 of the catalysis is the same in these cases as in the others. The 
 one vital fact, then, of an acceleration due to an increase in the 
 active mass or concentration of a reacting component in a cata- 
 lytic action is the only fundamental fact common to all catalytic 
 actions. " 
 
 With this enlarged conception of catalysis in mind we need 
 no longer have scruples in regarding the fermentative reactions 
 as instances of true catalysis. They exhibit the essential phe- 
 nomenon of acceleration through the presence of a chemical 
 agency which is added to the system, in other words the ferment. 
 Our task is therefore, not to compare the fermentative reactions 
 with a special and arbitrarily chosen class of catalytic reactions 
 but, as in all instances of catalysis, to attempt to unravel the 
 chemical mechanism of the observed acceleration. 
 
 In a system so complex as that afforded by a protein mixed 
 with an enzyme in aqueous solution several possibilities present 
 * Julius Stieglitz (127). Cf. also Hans Euler (26). 
 
PROTEOLYTIC ENZYMES AS CATALYSORS 397 
 
 themselves, which are too frequently overlooked, and which 
 doubtless play a part in determining the complexity of the 
 phenomena which are experimentally observed. 
 
 In the first place, we have seen that certain of the proteins, 
 or protein salts, undergo hydrolysis at a measureable rate in 
 the absence of proteolytic enzymes. Now, from the principle of 
 the mutual independence of different reactions (21) (53) (82) 
 it follows that when the proteins are acted upon by proteolytic 
 enzymes both the catalysed and the uncatalysed reactions must 
 be proceeding side by side, albeit the latter, possibly, at a re- 
 duced velocity.* Hence if the catalysed reaction is not over- 
 whelmingly more rapid than the uncatalysed reaction the prog- 
 ress of the latter must disturb the time- and mass-relations of the 
 former. The velocity-constant which is actually measured will 
 be the sum of the constants for the catalysed and the uncatalysed 
 reactions; only the former constant will bear a specific relation 
 to the mass of the catalysor; the sum of the constants, which 
 would be the quantity actually measured, would not, therefore 
 bear this relation to the mass of the catalysor, but exhibit de- 
 partures from it of greater or less magnitude according to the 
 magnitude of the constant for the uncatalysed reaction. 
 
 In the second place, we have seen, in discussing the hydrolysis 
 of the polypeptids, that a tripeptid may undergo hydrolysis by 
 splitting at either linkage, and that the hydrolysis due to the 
 splitting of the one linkage may be accelerated by one proteo- 
 lytic enzyme, and that due to the splitting of the other linkage 
 by another enzyme. In so complex a peptid as a protein many 
 linkages vulnerable to the enzyme under consideration must 
 exist and it is entirely within the bounds of probability that any 
 given enzyme shares its attack between two or more vulnerable 
 linkages and that, consequently, what we directly observe is not 
 the progress of one definite reaction, or even a catenary series 
 of reactions, but a number of parallel chains of successive hydrol- 
 yses. On the other hand, we know that the vulnerability of the 
 various linkages in the protein molecule to various proteoclastic 
 enzymes, differs so that one group of linkages will be preferen- 
 tially attacked by one enzyme, and another group by yet another 
 enzyme, while no enzyme or group of enzymes will open all of the 
 
 * Owing to reduction of the active mass of the free protein through the 
 formation of a protein-ferment compound. 
 
398 CHEMICAL DYNAMICS 
 
 COHN linkages which are susceptible to hydrolysis by acids 
 (29) (114) (56) (2). Hence if the enzyme-preparation which we 
 employ is impure, i.e., contains an admixture of proteolytic en- 
 zymes, then the number of possible parallel reactions and number 
 of simultaneous points of attack in the protein molecule must be 
 greatly enhanced. It is probably to this fact that we must in 
 part attribute the much greater activity of organ-extracts and of 
 organs and tissues in situ than of isolated ferments in bringing 
 about the hydrolysis of proteins (133). 
 
 In the third place if the proteolytic enzymes are proteins or 
 peptids, as in many cases seems highly probable, they must be 
 subject to hydration and dehydration, just as the proteins are, 
 and in any case we know that they are thermolabile and subject 
 in aqueous solutions to loss of activity, which is usually attributed 
 to hydrolysis, and to modifications leading to precipitation fol- 
 lowing dehydration by concentrated inorganic salts. The pos- 
 sibility is thus indicated that the proteolytic ferments may exist 
 in two or more modifications, nor can we assume that only one 
 of these modifications is capable of reacting with the substrate, 
 or its products, and influencing the velocity with which they 
 undergo modification. The activity of the ferment in acceler- 
 ating either hydrolysis or synthesis of protein may therefore 
 vary with the conditions under which it acts and among these 
 conditions must be reckoned both the relative and the absolute con- 
 centrations of the substrate and products of the reaction, since they 
 must modify, through combination with them, the equilibrium 
 between any different forms of the enzyme which may exist 
 in the system. In quite general terms, however, and without 
 assuming the existence of more than one form of the ferment, 
 it may be stated that if the ferment enters, at any stage of the 
 reaction of protein-hydrolysis, into combination with the sub- 
 strate to any appreciable extent (and we shall see that in many 
 instances it does), since the final equilibrium of the reaction must 
 therefore be affected by the ferment, the ferment must to some 
 extent be used up in the hydrolysis and the activity of the ferment 
 must vary as the reaction proceeds. 
 
 2. The Evidence for- the Existence of Intermediate Com- 
 pounds between the Proteolytic Ferments and their Substrates. 
 
 The existence of compounds between proteins and proteolytic 
 enzymes has been established in a number of cases by a variety 
 
INTERMEDIATE COMPOUNDS 399 
 
 of observers. Thus Vernon (145) has shown that serum albumin, 
 paraglobulin and particularly egg-albumin, when added to a 
 tryptic digest, markedly delay the hydrolysis of another protein 
 by the trypsin. Obviously, if the enzyme did not enter into 
 combination with its substrates, the rate at which it attacks 
 one substrate should not be influenced by the presence of another. 
 Hedin (46) has shown that the power of egg-albumin to delay the 
 hydrolysis of another protein by trypsin is much greater if the 
 egg-albumin and the trypsin are mixed together before the trypsin 
 is added to the digest than after. Evidently the egg-albumin- 
 trypsin compound is but slowly reversible; when none of the 
 trypsin is bound by another substrate the active mass of free 
 trypsin is greater and the proportion bound by albumin is there- 
 fore greater. Once the trypsin is bound by albumin it is only very 
 slowly abstracted from the combination by another substrate.* 
 
 Dauwe (23) has shown that trypsin can be extracted from its 
 solution by coagulated egg-white or by fibrin, and the ferment 
 can be regained from the compound by prolonged washing with 
 water. From what has been said in Chap. V, section 3, it will be 
 clearly realized that this latter fact does not in the least militate 
 against the view that the combination between the ferment and 
 the substrate is chemical in character. 
 
 A very striking instance of the way in which the formation 
 of compounds of this type may interfere with the time- and mass- 
 relations in the main hydrolysis under observation has been 
 discovered by Hedin (49). When trypsin acts upon casein, the 
 time required for the attainment of a given degree of hydrolysis 
 (= is inversely proportional to the concentration of ferment 
 (= p). In other words pt = constant. But, if egg-albumin be 
 present, a proportion of the trypsin is bound by the albumin 
 and this proportion is greater the more dilute the ferment, so that 
 the quantity of ferment which is free to act upon the trypsin 
 is no longer proportionate to its concentration and the law no 
 longer holds good. From this it is clear that if we wish to obtain 
 readily interpretable time- and mass-relations in a protein digest 
 we must employ only pure proteins. We have seen, also, that we 
 
 * Hedin has also shown that a greater proportion of trypsin is bound at 
 higher than at lower temperatures. But if excess of trypsin is attached to the 
 albumin by temporarily raising the temperature, on lowering the temperature 
 the trypsin is not given up again. 
 
400 CHEMICAL DYNAMICS 
 
 must employ only a single enzyme. Anyone who possesses an 
 extensive knowledge of the literature on protein hydrolysis by 
 enzymes will readily admit that these conditions have very rarely 
 been realized. 
 
 3. The Kinetics of Protein Hydrolysis by Enzymes. In 
 several instances it has been found that very small quantities 
 indeed of proteolytic enzyme will suffice to convert large quan- 
 tities of protein; thus one part of rennet will curdle from 400,000 
 to 800,000 parts of milk (42) and a pepsin powder has been pre- 
 pared which in seven hours dissolved 500,000 times its weight 
 of fibrin. From these facts it has been urged by many observers 
 that the fermentative splitting of proteins is an instance of 
 "typical" catalysis, in the limited sense of the term described 
 in section 1. The justification for this view is totally inadequate, 
 for it has not been shown that at the end of extensive hydrolysis 
 the enzyme can be recovered from the digest, wholly unaltered 
 in efficiency, without the expenditure or absorption of any 
 energy. In fact, in many cases it is known that the activity of 
 the enzyme is impaired during the hydrolysis. Since this im- 
 pairment of activity is even more pronounced when proteins 
 are not present, it is usually conceded that autohydrolysis of 
 the enzyme itself suffices to account for the whole of this effect; 
 this is clearly not necessarily the case; while autohydrolysis of 
 the enzyme renders it very difficult to ascertain whether the 
 enzyme is actually exhausted by the hydrolysis or not, yet it does 
 not exclude the possibility that such exhaustion occurs. Now 
 it must be constantly borne in mind that the proteins are very 
 nearly thermoneutral substances; their hydrolysis (Cf. section 1) 
 is accompanied by the disengagement of heat, it is true, but this 
 disengagement of heat is very minute. Hence the energy change 
 which is involved in a shift of the equilibrium between protein 
 and the products of its hydrolysis must be very minute and a 
 shift in the equilibrium of a correspondingly minute quantity 
 of a less thermoneutral substance might very well suffice to 
 provide or absorb the requisite energy to bring about a very 
 decided shift in the equilibrium between protein and its prod- 
 ucts. We cannot assume, therefore, from the smallness of the 
 amount of enzyme which will transform large quantities of pro- 
 tein, that the enzyme does not exert any influence upon the 
 equilibrium which the protein finally attains with its products. 
 
KINETICS OF PROTEIN HYDROLYSIS 401 
 
 If we assume, however, (i) that the ferment does not enter 
 appreciably into combination with the substrate or products 
 and hence does not shift the equilibrium between the protein 
 and its products, (ii) that the products of the reaction do not 
 appreciably depress the velocity of hydrolysis, and (iii) that 
 the reaction of the digest either does not affect the velocity of 
 hydrolysis or is unaltered by its progress, then for a certain period 
 of the hydrolysis, we may regard the transformation as concerning, 
 on the whole, only one molecular species, and the equation 
 
 log^ = fc (i) 
 
 should hold good. 
 
 It will be well, before proceeding further, to clearly understand 
 what is implied in the statement that this equation will hold 
 good for a certain period of the hydrolysis. When the first 
 COH.N bond in the protein molecule splits (considering, 
 for the present, only one of the possible parallel reactions, since, 
 in a system of parallel reactions of the same order, the reaction- 
 constants of the different reactions simply add themselves to- 
 gether to produce the gross resultant) a product p, is the result. 
 This product, however, now becomes the substrate for a second 
 reaction involving the splitting of another COH.N bond 
 and the production of a second substance pz. Two possibilities 
 now exist. The transformation pi * p z may be specifically more 
 rapid than the transformation p > p or it may be specifically 
 less rapid. In either case it will initially be in an absolute sense 
 less rapid than the primary reaction since its substrate-concen- 
 tration is initially zero and, consequently, the absolute velocity of 
 the second reaction will rise until it attains a maximum value, and 
 the combined reactions will then proceed with the velocity and 
 time-relations of the specifically slowest reaction. If this should 
 chance to be the primary reaction then equation (i) will hold 
 throughout; if it should chance to be the secondary reaction 
 then equation (i) will hold after the secondary reaction has attained 
 its maximum velocity but not before. 
 
 The meaning and derivation of equation (i) will be clear when 
 it is recollected that the reaction of protein hydrolysis consists 
 in the addition of a molecule of water to a COH.N bond 
 in the protein molecule. In dilute solutions the active mass of 
 
402 CHEMICAL DYNAMICS 
 
 water will not be appreciably affected by the inclusion of a 
 minute proportion of the water in the products of hydrolysis. 
 Only one molecular species is appreciably changing in concen- 
 tration, therefore, namely the protein. The number of molec- 
 ular collisions per second between the water and the protein 
 molecules will therefore be proportionate, at any instant, to the 
 concentration of unaltered protein at that instant. If the initial 
 concentration is a and the amount hydrolysed after time t 
 
 is x, the velocity of hydrolysis f-^-J is given by -r = k (a x), 
 
 in which k is the velocity-constant (specific velocity, or velocity 
 per unit concentration) of the reaction. Integrating this, and 
 recollecting that when t = 0, x = we obtain equation (i) . 
 
 The simplifying assumptions which we have been compelled 
 to make in deriving this formula are very numerous and very 
 many of them devoid of either theoretical or experimental justi- 
 fication. Hence it is not surprising that it has not often been 
 found to hold good by those who have followed the time-relations 
 of protein hydrolysis. 
 
 Victor Henri and Larguier des Bancels (54) studied the di- 
 gestion of gelatin and casein by trypsin, following the hydrolysis 
 by observing the increase in the conductivity of the digest, under 
 the assumption that each molecule of hydrolysed protein con- 
 tributes equally to the observed increase in conductivity. They 
 found that the values of 
 
 7 1 i a 
 
 k = - log 
 
 t 6 a x 
 
 were tolerably constant for brief periods of digestion and for 
 varying values of a ( initial substrate-concentration), in none 
 of their experiments, however, did they approach the stage of 
 complete digestion, and Arrhenius (3) has shown that their 
 results are equally well expressed by the formula: 
 
 x = kiVt (ii) 
 
 which is the well-known "rule of Schutz" to the effect that the 
 quantity of protein which is digested by a given quantity of 
 proteolytic ferment is proportional to the square root of the 
 period occupied in digestion. 
 
 Bayliss, also employing the conductivity method (5), studied 
 the hydrolysis of casein by trypsin. He found that the constant, 
 
KINETICS OF PROTEIN HYDROLYSIS 403 
 
 calculated from the monomolecular formula, falls off rather 
 rapidly as digestion proceeds. The decrease in the value of the 
 constant is not attributable wholly to autodestruction of the 
 ferment. Arrhenius has, moreover, shown that Bayliss' results 
 are also in satisfactory accord with the rule of Schtitz. 
 
 Taylor (133), studying the hydrolysis of protamin sulphate 
 by trypsin, found a tolerable accord with the monomolecular 
 law: 
 
 Substrate 0.150. Vol. 100. Ferment 0.001. Temp. 34 degrees, 
 time = 15 30 45 60 75 90 105 150 
 
 k X 10 4 68 69 69 76 66 64 62 60 Average = 67 
 
 Substrate 0.100. Vol. 100. Ferment 0.001. Temp. 34 degrees, 
 time = 15 30 45 60 75 90 105 150 
 
 k X 10 4 79 84 80 76 73 70 64 68 Average = 74 
 
 Substrate 0.0075. Vol. 100. Ferment 0.001. Temp. 34 degrees, 
 time = 15 30 45 60 75 90 105 150 
 
 k X 10 4 90 97 92 88 83 88 79 74 Average = 86 
 
 but it will be observed that the constant rises in value as the 
 substrate-concentration decreases. Therefore, as Taylor points 
 out, in a physical sense the direct proportionality between the 
 velocity of hydrolysis and the concentration of unhydrolysed 
 protein "is only spurious, since it holds but for each particular 
 system." 
 
 On the other hand, Samojlov (116), Walter (148), Sjoquist (125), 
 Schiitz and Huppert (124), Gross (41) and Meyer (79), working 
 with pepsin and employing a variety of substrates, have con- 
 curred in finding that for a considerable period of digestion the 
 Schiitz rule holds good; Borissov (11), employing trypsin. has 
 also confirmed the Schiitz rule and, as we have seen, Arrhenius 
 has shown that the results of Bayliss and of Henri and des Bancels 
 are in accord with this rule. Weis (150) working with the 
 enzyme contained in malt-extract, with wheat-protein as sub- 
 strate, has also confirmed the Schiitz rule, although in very 
 dilute solutions of ferment the exponent of the time tended to 
 rise and approach unity, i.e., the relation tends to become 
 
 x = k s t. (iii) 
 
 Arrhenius (loc. cit.) has shown, from the results of Weis, 
 that the quantity of protein hydrolysed by a given quantity of 
 
404 CHEMICAL DYNAMICS 
 
 ferment in a given time is directly proportional to the initial 
 concentration of the substrate, i.e., the true form of equation (ii) is 
 
 a result which had previously been established by Schiitz and 
 Huppert (loc. cit.) 
 
 Arrhenius (loc. cit., p. 64) has pointed out that the Schiitz 
 rule may be derived from the monomolecular formula provided 
 we assume that the catalysor in the system enters into combi- 
 nation with and so is inactivated by the products of the reaction. 
 This derivation is, however, only valid for the early stages of 
 hydrolysis, during which x is small in comparison with a. 
 
 Regarding the dependence of the velocity of hydrolysis upon 
 the ferment-concentration, Taylor (loc. cit.) finds that the time 
 required to hydrolyse a given quantity of protamin sulphate to 
 a given degree is inversely proportional to the quantity of fer- 
 ment present. I have found (107) that the velocity-constant 
 of the hydrolysis of calcium and barium caseinate in dilute 
 solutions, also calculated from the monomolecular formula, is 
 directly proportional to the concentration of trypsin, but at 
 higher substrate concentrations (A^/400 Ca(OH) 2 neutralized by 
 casein) the ratio k/F increases with increasing ferment-concen- 
 tration. Hedin (47) (48) (49) has studied the hydrolysis of 
 casein by trypsin very carefully from this standpoint and finds 
 that the relation 
 
 Ft = constant, (iv) 
 
 where F is the mass of ferment and t the time required to attain 
 a certain degree of hydrolysis, holds good over a wide range of 
 ferment-concentrations. 
 
 Employing the method of observing the rate of solution of 
 coagulated protein enclosed in capillary tubes Schiitz found 
 that the rate of digestion is proportional to the square root 
 of the ferment (pepsin) -concentration. Taylor (loc. cit.) and 
 Arrhenius (loc. cit.) have, however, pointed out that if this 
 method of measurement be employed, processes of diffusion and 
 solution are liable to be confused with the process of hydrolysis. 
 From the results of Hedin, however, which are cited above, 
 since Ft = constant and the rule of Schiitz (x = k V7) holds 
 good in a large number of instances of protein hydrolysis, it 
 
KINETICS OF PROTEIN HYDROLYSIS 405 
 
 would appear that in these instances, at least, the extent of 
 hydrolysis after a given time must be proportional to the square 
 root of the ferment-concentration (41). 
 
 The influence of the formation of compounds between the 
 substrate and the enzyme upon the kinetics of hydrolysis is very 
 clearly revealed by the experiments of Bogddndy (10) who has 
 shown that when a large excess of substrate is present the velocity 
 of protein hydrolysis by pepsin depends only upon the mass of 
 the ferment, while, when a large excess of ferment is present, 
 it depends only upon the mass of the substrate. This is obviously 
 what we should expect to be the case were the actual substance 
 undergoing hydrolysis a compound of the substrate and the 
 enzyme. 
 
 The hydrolysis of caseinates by trypsin has been exhaustively 
 investigated by Walters (146) who has shown that the relation 
 between the time of hydrolysis and the amount of sodium casein- 
 ate hydrolysed is, for all stages of the reaction, almost exactly 
 what would be expected from the monomolecular formula. More- 
 over the velocity of hydrolysis is directly proportional to the 
 concentration of trypsin and there is a less exact proportionality 
 between the initial concentration of the substrate and the velocity 
 of hydrolysis, the velocity-constant decreasing slightly as the 
 concentration of the substrate increases. The nature of the 
 base combined with the casein has little or no influence upon 
 the process of hydrolysis by trypsin (although it has a decided 
 influence upon the velocity of autohydrolysis (147)) from which 
 we may infer, since caseinates of the alkalies and of the alkaline 
 earths are hydrolysed with equal velocity, that the degree of 
 electrolytic dissociation of the caseinate has little or no effect 
 upon the velocity of its hydrolysis by trypsin. 
 
 We thus see that under different conditions of enzyme- and 
 substrate-concentration and with different substrates very dif- 
 ferent laws are found to express the relationship between time 
 and the extent of hydrolysis of the proteins and polypeptids. 
 None of these relations holds good outside certain definite limits 
 of substrate- and enzyme-concentration and each of them only 
 holds good for a limited portion of the hydrolysis. We are in- 
 clined to suspect that each of these relations is but part of some 
 more general relation which, it is possible, holds good for all 
 of the cases cited above and for all stages of the hydrolysis. 
 
406 CHEMICAL DYNAMICS 
 
 The task of seeking for such a relation, when we recall all of the 
 factors which may very possibly enter into it, might well seem, 
 for purposes of practical utility, well-nigh hopeless, were it not 
 for the fact that the limiting cases of this general law have 
 already been ascertained to hold good over quite extensive and 
 practically attainable ranges of time and concentration. Evi- 
 dently the number of the factors which play an appreciable part 
 in determining the phenomena observed is not so great as we 
 might be inclined to anticipate. 
 
 We have seen that Henri's law does not suffice; it is not 
 sufficiently general to cover all of the time- and mass-relations 
 in the hydrolysis of the polypeptids (Cf. the previous chapter, 
 section 2) ; we are led to suspect that some important factor has 
 been omitted in the derivation of Henri's equation. 
 
 In the following chapter we shall see that pepsin is capable 
 of exerting synthetic activity under conditions such that its 
 hydrolytic activity is absent. We have already had occasion 
 to indicate that the proteolytic ferments may exist in more than 
 one modification. In the light of these facts we shall make the 
 following assumptions regarding the mode of action of the pro- 
 teolytic ferments upon the proteins. 
 
 Let us suppose that the proteolytic ferments act as " carriers" 
 of water into the protein molecule, just as nitric oxide, in the 
 manufacture of sulphuric acid, acts as a " carrier" of oxygen into 
 sulphurous acid. We must therefore assume that these ferments 
 can exist both in a hydrated and in a dehydrated form, just as 
 nitric acid can exist in the reduced form of nitrous oxide. More- 
 over we must conclude that at the splitting of each COH.N 
 bond in the protein molecule some such reactions as the following 
 
 occur : 
 
 HF 
 I 
 
 (A) -COH.N- + HFFOH = - COH.N - 
 
 (hydrated I 
 
 * FOH 
 
 \ ferment-substrate compound) 
 
 HF 
 
 (B) - COH.N - - - COOH + H 2 N - + FF 
 
 I (products of hydrolysis) (dehydrated 
 
 FOH 
 
 (ferment-substrate 
 compound) 
 
KINETICS OF PROTEIN HYDROLYSIS 407 
 
 So far we have, in essentials, made only two assumptions, the 
 one the original assumption that the ferment acts as a ''carrier" 
 of water, the other that one molecule of ferment reacts with one 
 -COH.N- bond. 
 Combining the above two equations we obtain: 
 
 (C) -COH.N- + HFFOH = -COOH + H 2 N- + FF 
 
 from which it is evident that the point of equilibrium in the 
 reaction : 
 
 (D) FF + H 2 <= HFFOH 
 
 must be shifted in some measure towards the left by the presence 
 of the substrate and the extent of this shift must bear a constant 
 proportion (a) to (a x). But this equilibrium must also be 
 shifted towards the right by the presence of the products of the 
 hydrolysis of the protein, and this shift must bear a constant 
 proportion (0) to x 2 . 
 
 Let us now analyse the physical meaning of Henri's equation. 
 This equation, as we have seen in Chap. XV, may be written: 
 
 log ^ + ax = kFt, (v) 
 
 tt X 
 
 in which a and k are constants and F is the total mass of ferment 
 present in the system. 
 Differentiating this equation we obtain: 
 
 dx F 7 / \ / -\ 
 
 37 = 1-1 1 \ k(a x), (vi) 
 
 dt I + a (a x) 
 
 which means that the actual "active mass" of the proteolytic 
 ferment, that proportion, namely, which accelerates the hydrol- 
 ysis by multiplying the velocity-constant, is not F but 
 
 F 
 
 l + a(a-x)' 
 
 In other words, the process of combination between the ferment 
 and the substrate and its products which Henri depicts, results 
 in the inactivation of a certain constant proportion of the ferment 
 by each molecule of the substrate. The mechanism of this will 
 be clear from equation (C); by the same equation it will also 
 be clear that a proportion of the ferment is at the same time 
 activated (rendered available for the acceleration of the hydrolysis). 
 The quantity of ferment thus activated must evidently bear a 
 
408 CHEMICAL DYNAMICS 
 
 constant proportion /3 to x 2 . Introducing this factor into equa- 
 tion (vi) we obtain: 
 
 dx F 
 
 / -x 
 (vn) 
 
 Integrating, we obtain: 
 
 (1 - 0a 2 ) log + (a + 0a) x + ^x* = kFt. (viii) 
 
 a x 
 
 In the derivation of Henri's equation it is assumed either that 
 the ferment is unaltered during the processes which accomplish 
 the introduction of the elements of water into the protein mole- 
 cule or else that the equilibrium in equation (D) lies so far to the 
 right that for all practical purposes the ferment is present wholly 
 in the hydrated, proteolytically active form. Equation (viii) 
 is Henri's equation generalized to the extent of including any 
 species of equilibrium in equation (D). 
 Equation (viii) may be written : 
 
 a , a -f /So |8 kFt ,. . 
 
 log J^ + nr^* + 2(i-^) x (T=W) 
 
 from which it is evident that it contains three mutually inde- 
 pendent constants. This fact deprives it of any great utility for 
 the purpose of comparing numerical data with the theoretical 
 deductions, since an equation containing three mutually inde- 
 pendent constants may be made to fit with tolerable exactitude 
 any continuous curve by an appropriate choice of constants. 
 In the absence of very numerous and very exact data, a numerical 
 comparison between theory and experiment involving this 
 equation, therefore, would be devoid of utility. It is of interest 
 to observe, however, that all the relationships between x, F and 
 t found by the various observers, whose investigations we have 
 cited above and in the previous chapter, are especial cases of the 
 general relation which is expressed in equation (ix). Thus if 
 /3 be small, that is, if the shift in the equilibrium between the 
 hydrated and unhydrated forms of the enzyme due to unit mass 
 of the products of the hydrolysis is very small, or if the equi- 
 librium in equation (D) . lies far to the right, then we regain 
 Henri's equation. If not only but a is small, i.e., if the part 
 played by the protein and its products in determining the equi- 
 librium between the different forms of ferment is small, then we 
 
KINETICS OF PROTEIN HYDROLYSIS 
 
 409 
 
 regain the monomolecular formula. If jSa 2 is large in comparison 
 with 1 and a is small then we obtain the relation 
 
 a . x . x 2 kF . 
 
 which, when x is small, yields the relation: 
 - = kFt (equation (iii)). 
 
 It is obvious from equation (vii) that if the velocity constant 
 k were to be calculated from the monomolecular formula through- 
 out the reaction of hydrolysis, k would tend to fall off as hydrolysis 
 proceeded, i.e., as x increased, and also, it will be evident, even 
 for small values of x the constant would decrease with increasing 
 initial substrate-concentration. This obviously accords with the 
 facts observed. 
 
 The way in which the relationship expressed in equation (ix) 
 may simulate the monomolecular formula, the Schiitz rule, etc., 
 under certain conditions, is very well shown by the following 
 table. The values of t corresponding to various values of x 
 are calculated from formula (ix) on the assumption that a = 10 
 and that a \ a a Q j^p 
 
 = . =: 1_^ 
 
 1 /3( ~ 
 
 2 (1 - /3a 2 ) ( 
 
 From these values of t and the given values of x are calculated 
 the constants corresponding to the law of direct proportionality 
 between the quantity digested and the time, to the Schiitz rule 
 (t = kx 2 ) and to the monomolecular formula: 
 
 X 
 
 t 
 
 H 
 
 *=^ 
 
 ""-'rh 
 
 0.1 
 
 0.1144 
 
 1.144 
 
 11.44 
 
 26.0 
 
 0.2 
 
 0.2488 
 
 1.244 
 
 6.22 
 
 28.3 
 
 0.5 
 
 0.7723 
 
 1.545 
 
 3.09 
 
 34.6 
 
 1.0 
 
 2.046 
 
 2.046 
 
 2.05 
 
 44.5 
 
 2.0 
 
 6.097 
 
 3.049 
 
 1.52 
 
 62.9 
 
 3.0 
 
 12.155 
 
 4.051 
 
 1.35 
 
 78.4 
 
 4.0 
 
 20.222 
 
 5.055 
 
 1.26 
 
 91.1 
 
 5.0 
 
 30.301 
 
 6.060 
 
 1.21 
 
 100.7 
 
 6.0 
 
 42.398 
 
 7.066 
 
 1.18 
 
 106.5 
 
 7.0 
 
 56.523 
 
 8.075 
 
 1.15 
 
 108.1 
 
 8.0 
 
 . 72.699 
 
 9.087 
 
 1.14 
 
 104.0 
 
 9.0 
 
 89.000 
 
 9.889 
 
 1.21 
 
 89.0 
 
410 CHEMICAL DYNAMICS 
 
 It will be seen that from x = 0.1 to x = 0.5 the law of direct 
 proportionality very nearly holds good; from x = 3.0 to x = 9.0 
 the Schtitz rule very nearly holds good, while from x = 4.0 to 
 x = 8.0 the monomolecular formula adequately expresses the 
 relationship between x and t. 
 
 4. The Influence of Acid and Alkali upon the Rate of Protein 
 Hydrolysis by Enzymes. Of great importance in determining 
 the rate of hydrolysis of proteins by proteolytic enzymes is the 
 reaction (H + or OH' concentration) of the solution in which 
 digestion is occurring. The activity of pepsin is greatly en- 
 hanced by a low degree of acidity, greater acidity hindering its 
 activity through destruction of the enzyme itself by the excess 
 of acid. The activity of trypsin, on the other hand, is very 
 markedly favored by a very slight alkalinity, excess of alkali 
 being even more destructive to trypsin than excess of acid is 
 to pepsin. An excess of acid very rapidly destroys the activity 
 of trypsin, while an alkaline medium leads to the somewhat 
 
 N 
 less rapid destruction of pepsin. According to Taylor -^ to 
 
 N 
 
 OH' is the optimum alkalinity for the action of trypsin 
 
 1400 
 
 upon protamin sulphate (129). 
 
 The nature of the part played by the alkali and acid in protein 
 hydrolysis by enzymes is by no means clear. Since acids and 
 alkalies (H + and OH' ions) are well-known hydrolysing agents, 
 a very natural assumption regarding their influence upon protein 
 hydrolysis by enzymes is to suppose that they play the part of 
 accessory catalysors, the catalytic action of the acid or alkali 
 being added to that of the enzyme. As regards the influence 
 of acids upon the hydrolysis of proteins by pepsin, however, 
 there are many well-known facts which speak against this view. 
 
 The influence of acids is by no means proportional to their 
 degree of dissociation as would be expected were their influence 
 due to a catalytic action of the hydrogen ion (6) (68). On the 
 contrary, hydrochloric acid has an almost specific action upon 
 the hydrolysis of proteins by pepsin, so that many observers 
 have inclined to the belief that the real ferment is in this instance 
 a compound of pepsin with hydrochloric acid (121). Loeb (74) 
 has suggested that the part played by the acid, in accelerating 
 the hydrolysis of proteins by pepsin, is analogous to the part 
 
INFLUENCE OF ACID AND ALKALI 411 
 
 played by acids in accelerating the hydrolysis of the imino- 
 esters by increasing the active mass of the substrate through salt 
 formation (Cf. Stieglitz' experiments, cited in section 1). Loeb 
 believes that the portion of the pepsin which is active in bring- 
 ing about the hydrolysis of proteins is that portion of it which 
 is ionized, and that acids increase the active mass of the ferment 
 by forming ionized salts with it. Referring to the theory of 
 protein ionization which is developed in earlier chapters of this 
 work, and the intimate relation which, as we have seen, subsists 
 between the degree of ionization and the degree of hydration 
 of a protein (and presumably of these enzymes) this view would 
 appear very probable, especially if we accept the account of the 
 mechanism of the hydrolysis of proteins by enzymes which is 
 given in the latter part (equations (A) to (D)) of the preceding 
 section. 
 
 As regards the influence of alkalies upon the activity of trypsin, 
 many of the facts appear, at first sight, to be more in harmony 
 with the view that the alkali acts as an accessory catalysor, 
 since it is stated that the hydroxides of the alkalies and alkaline 
 earths act in proportion to their degree of dissociation (63) 
 although divergencies from the proportionality of the action to 
 the degree of dissociation have been found (6). 
 
 It has frequently been pointed out that the progress of hy- 
 drolysis of proteins is accompanied by marked changes in the 
 acidity or alkalinity of the solutions in which the hydrolysis 
 occurs (39) (40) (129) (107) (113). This is to be attributed to 
 the splitting of COH.N bonds, in hydrolysis, which do not, 
 while bound up in the protein molecule, assist in the neutra- 
 lization of bases and acids,* but which, when converted into 
 COOH and H 2 N groups may be presumed to play the part 
 which such groups ordinarily play; hence any excess of acid 
 or base tends to disappear during hydrolysis. One might expect 
 that this change in hydrogen or hydroxyl concentration would 
 
 * We have seen that the COH.N groups provided by dicarboxyl- and 
 diamino-acid groups are chiefly involved in the neutralization of bases and 
 acids by proteins. In hydrolysis not only these bonds are split, but also the 
 COH.N bonds linking monocarboxyl and monoamino-acid groups. It 
 is for this reason that the combining capacity for bases of the products of 
 protein hydrolysis is greater, although only slightly greater, than that of the 
 protein from which they are derived. 
 
412 CHEMICAL DYNAMICS 
 
 lead to deviation from regularity in the relationship between 
 the degree and the time of hydrolysis which is experimentally 
 observed. Nevertheless, if the degree of alkalinity be sufficient, 
 this is not the case. Thus in Taylor's experiments upon the 
 hydrolysis of protamin sulphate the alkalinity of the system 
 changed markedly with the progress of hydrolysis, yet he ob- 
 tained results in satisfactory accord with the monomolecular law. 
 The reason for this fact is very clearly revealed by the experi- 
 ments of Robertson and Schmidt (113). We measured the rate 
 of change in the alkalinity of tryptic digests of sodium caseinate 
 and of protamin sulphate (dissolved in dilute alkali) by means 
 of the gas chain. The progressive change in hydroxyl-concen- 
 trations of these digests, it was found, can be expressed by a 
 monomolecular formula when the total OH' concentration is 
 greater than 10" 6 , but thereafter, or when the OH' concentration 
 initially is less than this, the progressive change in alkalinity 
 is no longer to be expressed by a monomolecular formula, but 
 is represented by the formula 
 
 kt = 
 
 B(B- 
 
 (where t is the time since the gas-electrodes first came into equi- 
 librium with the solution and B is the OH' concentration when 
 t = 0), which is characteristic of a bimolecular reaction. For a 
 short range of intermediate alkalinities the order of the reaction 
 is indeterminate. These facts are illustrated by the tables on 
 the following page. 
 
 The point at which the order of the reaction changed is indi- 
 cated by an asterisk. It will be seen that it corresponds tolerably 
 closely with COH' = 10" 6 ; and it will also be observed that it is 
 the same for substrates so very different as casein and protamin 
 sulphate. This indicates that the change in the order of the 
 reaction at a certain OH' concentration depends upon an alter- 
 ation in some relationship between the alkali and the ferment 
 and not upon the alteration of such a relationship between 
 the substrate and the ferment. 
 
 The most probable interpretation to be placed upon these 
 results is that the active agent in bringing about the hydrolysis 
 is not uncombined trypsin but a compound of trypsin with the 
 base which the solution contains. The efficiency of bases in 
 
INFLUENCE OF ACID AND ALKALI 
 
 413 
 
 TABLE I. CASEIN 
 
 To AT/100 NaOH containing about 2 per cent of casein was added an 
 equal volume of AT/300 NaOH. To 50 cc. of this solution were added 
 10 milligrams of trypsin. Temperature 15 to 18 degrees. 
 
 Time in 
 minutes 
 after 
 mixing 
 
 t-t! 
 
 COH/ 
 
 z 
 
 1 
 
 (monomolecu- 
 lar) 
 
 1 
 
 (bimolecular) 
 
 51 
 
 
 28.4 X10~ 7 
 
 
 
 
 59 
 
 "6 
 
 27 3 X10~ 7 
 
 
 
 
 69 
 
 10 
 
 26.2 X10~ 7 
 
 1.1 xio- 7 
 
 180 X10~ 5 
 
 154X10~ 6 
 
 73 
 
 14 
 
 24.2 X10~ 7 
 
 3.1 X10~ 7 
 
 375 XIO- 5 
 
 336 XIO- 6 
 
 99 
 
 40 
 
 20.6 X10~ 7 
 
 6.7 X10~ 7 
 
 305X10- 5 
 
 296X10- 6 
 
 126 
 
 67 
 
 16.2 XHT 7 
 
 11.1 X10~ 7 
 
 338X10- 5 
 
 376X10- 6 
 
 153 
 
 94 
 
 12.8 X10- 7 
 
 14.5 XIO- 7 
 
 352X10- 5 
 
 445X10- 6 
 
 197 
 
 138 
 
 9.3 X10- 7 
 
 18.0 X10~ 7 
 
 340X10- 5 
 
 516X10- 6 
 
 225 
 
 166 
 
 7.6 X10- 7 
 
 19.7 XIO- 7 
 
 336X10- 5 
 
 574X10- 6 
 
 246 
 
 187 
 
 6.7 X10- 7 * 
 
 20.6 XIO- 7 
 
 326X10- 5 
 
 600X10- 6 
 
 276 
 
 217 
 
 5.95X10- 7 
 
 21.35X10- 7 
 
 305X10- 5 
 
 604X10- 6 
 
 303 
 
 244 
 
 5.45X10- 7 
 
 21.85X10~ 7 
 
 293X1Q- 5 
 
 627X10- 6 
 
 338 
 
 279 
 
 4.7 X10~ 7 
 
 22.6 XIO- 7 
 
 275 X10~ 5 
 
 635X10- 6 
 
 368 
 
 309 
 
 4.0 X10~ 7 
 
 23.3 XIO- 7 
 
 270X10- 5 
 
 690X10- 6 
 
 408 
 
 349 
 
 3.7 X10- 7 
 
 23.6 XIO- 7 
 
 250X10- 5 
 
 670X10- 6 
 
 443 
 
 384 
 
 3.5 X10~ 7 
 
 23.8 XIO- 7 
 
 232X10- 5 
 
 642 XIO- 6 
 
 470 
 
 411 
 
 3.3 X10~ 7 
 
 24.0 X10~ 7 
 
 224X10- 5 
 
 654X10- 6 
 
 547 
 
 488 
 
 2.8 X10- 7 
 
 24.5 X10~ 7 
 
 203 XIO- 5 
 
 660 X10~ 6 
 
 601 
 
 542 
 
 2.6 X10~ 7 
 
 24.7 XIO- 7 
 
 190X10- 5 
 
 656X10- 6 
 
 647 
 
 588 
 
 2.5 X10~ 7 
 
 24.8 X10~ 7 
 
 178X10- 5 
 
 627 XIO- 6 
 
 722 
 
 663 
 
 2.5 X10~ 7 
 
 24.8 XIO- 7 
 
 157 XIO- 5 
 
 556X10- 6 
 
 TABLE II. PROTAMIN SULPHATE 
 
 Ten grams of protamin sulphate in one litre of solution. Ten milligrams 
 of trypsin and 2.5 of AT/10 NaOH to 25 cc. 
 
 Time in 
 minutes 
 after 
 mixing 
 
 *-<t 
 
 COH' 
 
 X 
 
 k 
 (monomolecu- 
 far) 
 
 i 
 
 (bimolecular) 
 
 60 
 
 
 28.5X10- 7 
 
 
 
 
 85 
 
 
 
 24.3X10- 7 
 
 
 
 
 
 108 
 136 
 181 
 241 
 307 
 356 
 400 
 453 
 502 
 
 23 
 
 51 
 96 
 156 
 222 
 271 
 315 
 368 
 417 
 
 20.7X10- 7 
 15.0 XIO- 7 
 10.5X10- 7 
 7.0X10- 7 * 
 5.3X10- 7 
 4.5X10- 7 
 4.0X10' 7 
 3.5X10- 7 
 3.1X10~ 7 
 
 3.6X10- 7 
 9.3X10' 7 
 13. 8 XIO- 7 
 17. 3 XIO- 7 
 19.0X10- 7 
 19. 8 XIO- 7 
 20.3X10- 7 
 20.8X10- 7 
 21.2X10- 7 
 
 305 XIO- 6 
 411 X10~ 5 
 380X10- 5 
 346X10- 5 
 298X10- 5 
 270 XIO- 6 
 249 XIO- 5 
 227 XIO- 6 
 213X10- 5 
 
 311X10- 6 
 500X10- 6 
 564 XIO- 6 
 647 X10~ 6 
 664X10- 6 
 668X10- 6 
 664X10- 6 
 663 XIO- 6 
 676X10- 6 
 
414 CHEMICAL DYNAMICS 
 
 activating trypsin is thus readily understood. When the con- 
 centration of unneutralized base is in excess of 10 X 10~ 6 all of 
 the trypsin is combined with the base and in the form of the 
 proteolytically active salt;* but when the alkalinity falls below 
 this limit a proportion of the trypsin is no longer combined with 
 alkali and this proportion is inactive. Below this limit of OH' 
 concentration, therefore, the velocity-constant of hydrolysis 
 (calculated from the monomolecular formula) must fall off in 
 proportion as COH' decreases, in other words, the relation be- 
 tween COH' and time will be expressed by the bimolecular 
 formula. 
 
 This explanation of our results is especially supported by the 
 fact that when the velocity of hydrolysis has fallen very low, 
 owing to decreasing alkalinity, the velocity can be raised again, 
 and the order of the reaction again made monomolecular, by 
 simply increasing the alkalinity of the solution. This is shown 
 in table III in wnich the break indicates that fresh alkali was 
 added. 
 
 We may therefore conclude that in all probability pepsin and 
 trypsin are only able to exert their proteolytic activity (i.e., act 
 as carriers of water) when they are present in their solutions in 
 the form of salts with acids and bases respectively. 
 
 The above experiments establish a lower limit of hydroxyl- 
 concentration, below which the alkalinity is not sufficient to 
 secure the greatest velocity of transformation. An upper limit, 
 above which the alkalinity is too great to secure the greatest 
 velocity of transformation, is indicated by the fact that trypsin 
 undergoes autohydrolysis in solution and this autohydrolysis is 
 greatly accelerated by alkalies (144) (145); directly, therefore, 
 the rate of destruction of the trypsin by the excess of alkali 
 measurably affects the progress of the reaction an upper limit 
 of alkalinity is reached. The experiments of Taylor, cited above, 
 
 N- N 
 
 establish this upper limit at about ^ rrtr . OH'; between .. ^^ ^^ 
 jy loUU 1,UUU,UUU 
 
 and _ OH', therefore, all alkalinities are equally favorable. 
 
 * Subject, of course, to dehydration as in equations (C) and (D) in the preced- 
 ing section. In equations (viii)' and (ix) it is the value of F which is to be con- 
 sidered as being affected by the alkali, not the proportion of the hydrated to 
 the dehydrated form of the enzyme, although this may also very possibly be 
 affected by the OH' concentration, j 
 
INFLUENCE OF ADDED SUBSTANCES 
 
 415 
 
 TABLE III 
 
 Ten grams of protamin sulphate in 1 litre of solution. Ten milligrams 
 of trypsin and 2.1 cc. of AT/10 NaOH to 25 cc. 
 
 Time in 
 minutes 
 after 
 mixing 
 
 ,- 
 
 C OH < 
 
 z 
 
 I 
 
 (monomolecu- 
 lar) 
 
 1 
 
 (bimolecular) 
 
 29 
 
 
 23.3 XlO" 7 
 
 
 
 
 38 
 
 o 
 
 22.4 XlO" 7 
 
 o 
 
 
 
 63 
 
 25 
 
 16^25X10- 7 
 
 6. 15 X10~ 7 
 
 559 XIO- 5 
 
 676X10-8 
 
 78 
 
 40 
 
 13.3 X10~ 7 
 
 9.1 XIO- 7 
 
 567 XIO" 5 
 
 765X10-8 
 
 93 
 
 55 
 
 10.5 XIO- 7 
 
 11.9 X10~ 7 
 
 601 X10~ 5 
 
 920X10-8 
 
 109 
 
 71 
 
 8.9 X10~ 7 * 
 
 13.5 X10~ 7 
 
 565 XIO' 5 
 
 954X10-8 
 
 124 
 
 86 
 
 7.6 X10~ 7 
 
 14.8 X10~ 7 
 
 545 XIO- 5 
 
 943X10-8 
 
 132 
 
 94 
 
 7.0 X10~ 7 
 
 15.4 X10~ 7 
 
 537 X10~ 5 
 
 1046X10- 6 
 
 13 
 
 
 27.3 X10" 7 
 
 
 
 
 23 
 
 6 
 
 26 3 X10~ 7 
 
 o 
 
 
 
 33 
 
 10 
 
 24.3 XIO- 7 
 
 2.0 XIO- 7 
 
 334 XIO- 5 
 
 313X10-8 
 
 51 
 
 28 
 
 20.7 XIO- 7 
 
 5.6 X10~ 7 
 
 371X10- 5 
 
 367X10-8 
 
 72 
 
 49 
 
 18.3 X10~ 7 
 
 8.0 X10~ 7 
 
 323 XIO- 5 
 
 339X10-8 
 
 90 
 
 67 
 
 16.9 X10~ 7 
 
 9.4 X10~ 7 
 
 286X10- 5 
 
 316X10-8 
 
 111 
 
 88 
 
 13.8 X10~ 7 
 
 12.5 X10~ 7 
 
 317 X10~ 5 
 
 391X10-8 
 
 123 
 
 100 
 
 12.8 X10~ 7 
 
 13.5 XIO- 7 
 
 314X10- 5 
 
 401X10- 6 
 
 152 
 
 129 
 
 11.3 X10~ 7 * 
 
 15.0 X10~ 7 
 
 285 XIO- 5 
 
 391X10-8 
 
 170 
 
 147 
 
 10.5 X10~ 7 
 
 15.8 XIO- 7 
 
 272 XIO- 5 
 
 389X10-8 
 
 199 
 
 176 
 
 9.7 X10~ 7 
 
 16.6 X10~ 7 
 
 246X10- 5 
 
 369X10- 6 
 
 232 
 
 209 
 
 7.9 X10~ 7 
 
 18.4 XIO- 7 
 
 250 XIO- 5 
 
 423X10-8 
 
 251 
 
 228 
 
 7.6 XIO- 7 
 
 18.7 X10~ 7 
 
 236X10- 5 
 
 411X10-8 
 
 5. The Influence of Added Substances upon the Rate of 
 Protein Hydrolysis by Enzymes. The influence of added 
 substances upon the rapidity of the hydrolysis of proteins by 
 enzymes has been extensively studied, owing to its supposed 
 importance in connection with the digestion of protein in the 
 alimentary canal (97) (119) (51) (149) (73) (16) (96) (45) (18) 
 (151) (30) (66) (15) (100) (152) (120). Despite this extended 
 investigation, however, the results obtained by different observers 
 have been conflicting in the extreme. Investigations which I 
 have carried out on the influence of salts upon the hydrolysis 
 of caseinates by trypsin indicate the probable source of the dis- 
 crepancies between the results of different observers (107). I 
 find that the order of efficiency of salts in accelerating the hy- 
 drolysis of casein by trypsin is the order of their efficiency in 
 accelerating the solution of casein by alkalies. If this rule holds 
 good for other proteins, then one might anticipate that the 
 influence of salts upon their hydrolysis by enzymes would vary 
 
416 CHEMICAL DYNAMICS 
 
 somewhat with the nature of the protein or protein salt employed 
 as substrate. 
 
 The effect of salts in accelerating the hydrolysis of casein by 
 trypsin is, in some instances, very marked. Thus in a solution 
 of "neutral" calcium caseinate containing 143 milligrams of 
 casein in 200 cc. plus 1 cc. of a 0.025 per cent solution of Gruebler's 
 trypsin at 36.5 degrees the velocity-constant of hydrolysis was 
 11 (calculated, in arbitrary units, from the monomolecular for- 
 mula) while, upon the addition of 0.05 N NaCl the value of the 
 constant rises to 44 (in the same units). 
 
 The influence of the concentration of the added NaCl upon 
 the acceleration of the tryptic hydrolysis of caseinates which 
 it brings about is of great interest, since, so far as my observa- 
 tions have extended, it is of the same nature as its influence 
 upon the degree of swelling which gelatin plates undergo when 
 immersed in water. It will be recollected (Cf. Chap. XII) that 
 when gelatin plates are immersed in solutions of NaCl of vary- 
 ing concentration, the degree of swelling which they attain after 
 a given period does not increase continuously with the concen- 
 tration of the solution but exhibits marked maxima and minima 
 at definite concentrations (94). Exactly similar phenomena 
 occur when NaCl in varying concentration is added to a tryptic 
 digest of casein. The acceleration in hydrolysis which is pro- 
 duced by the added NaCl does not continuously increase with 
 increasing concentration of the NaCl, but exhibits marked max- 
 ima and minima. 
 
 6. The Action of the Coagulating Ferments, Rennet and 
 Thrombin. As is well known, rennet occurs, together with 
 pepsin, in the gastric juice and in the juices of many plants and 
 brings about the clotting of milk through the transformation 
 of casein into paracasein. Thrombin occurs in some part of the 
 circulating blood, possibly the leucocytes, and brings about the 
 coagulation of blood through the transformation of fibrinogen 
 into fibrin. The modes of action of these ferments have been 
 the subject of very many investigations (102) (103) (31) (84) 
 (75) (61), which cannot be dwelt upon in detail here. In brief, 
 however, it may be stated that rennet converts casein into 
 paracasein, whether calcium salts are present or not, and the 
 paracasein having been formed (which is soluble in the absence 
 of salts of the alkaline earths), it is coagulated (or precipitated; 
 
COAGULATING FERMENTS 
 
 417 
 
 Cf. Chap. VI) by calcium salts or other salts of the alkaline 
 earths. The part played by calcium in forwarding the coagu- 
 lation of blood is very different; here the dependency is one 
 of the ferment (or a pro-ferment), and not of the coagulation 
 of the substrate, upon the presence of calcium salts; it would 
 appear that calcium salts activate a pro-thrombin, converting it 
 into the ferment thrombin, which will then induce coagulation 
 whether calcium salts be present or not. The function of cal- 
 cium in these two processes is therefore very different. 
 
 Fuld (32), Gerber (37) and Madsen (77) have shown that for 
 the coagulation of milk by rennet F X t constant; i.e., the 
 velocity of coagulation is proportional to the concentration of 
 ferment. Regarding the influence of calcium salts, very inter- 
 esting results have been obtained by Reichel and Spiro (104). 
 These observers added to 8 cc. of milk different quantities (R) 
 of rennet solution and different amounts (p) of calcium chloride, 
 and diluted with water to a total volume of 100 cc. They then 
 measured the time required for coagulation at a constant tem- 
 perature. The following were their results : 
 
 CaCh in 
 
 Time of coagulation 
 
 Constant = (p + 0.6) t 
 
 P 
 
 R = 1 cc. 
 
 R = 0.5 cc. 
 
 R = 0.25cc. 
 
 R = lcc. 
 
 R = 0.5 cc. 
 
 R = 0.25 cc. 
 
 0.00 
 
 95.0 
 
 48.0 
 
 24.0 
 
 57.0 
 
 28.8 
 
 14.4 
 
 0.05 
 
 88.6 
 
 45.6 
 
 23.0 
 
 57.6 
 
 29.6 
 
 15.0 
 
 0.1 
 
 79.0 
 
 41.6 
 
 22.0 
 
 55.3 
 
 29.1 
 
 15.4 
 
 0.2 
 
 66.4 
 
 36.0 
 
 19.0 
 
 53.1 
 
 28.8 
 
 15.2 
 
 0.5 
 
 48.0 
 
 26.4 
 
 14.0 
 
 52.8 
 
 29.0 
 
 15.4 
 
 1.0 
 
 30.0 
 
 18.2 
 
 10.6 
 
 48.0 
 
 29.1 
 
 17.0 
 
 2.0 
 
 17.0 
 
 11.0 
 
 6.8 
 
 44.2 
 
 28.6 
 
 17.7 
 
 5.0 
 
 10.0 
 
 7.4 
 
 5.4 
 
 56.0 
 
 41.4 
 
 30.2 
 
 10.0 
 
 13.0 
 
 9.2 
 
 6.2 
 
 137.8 
 
 97.5 
 
 65.7 
 
 20.0 
 
 22.0 
 
 15.0 
 
 8.6 
 
 453.2 
 
 309.0 
 
 177.2 
 
 It is evident that the product (p + 0.6) t is constant for each 
 value of R, provided p does not exceed 2 per cent. The velocity 
 is also proportional to R } so that the complete relationship is 
 R (p + 0.6) t = constant. The quantity 0.6 which must be added 
 to p in the above equation is to be regarded as the (percentage) 
 concentration of calcium ions present already in milk, before 
 any calcium chloride is added. According to Reichel and Spiro 
 this indicates that only about 15 per cent of the calcium in milk 
 
418 CHEMICAL DYNAMICS 
 
 is ionic; this obviously corresponds very well with the statement 
 in Chap. VIII and elsewhere in this work that the calcium bound 
 in calcium caseinate is not electrolytically, dissociated as such. 
 
 In the opinion of Nencki and Sieber (86), Schoumov-Simanovski 
 (123), Pavlov and Parastschuk (95), Savjallov (118), Gevin (38), 
 Migay and Savitsch (83), Van Dam (139) (140) (141), Savitsch 
 (117), Herzog (57), Funk and Niemann (34), Laqueur (71) (72) 
 and Bosworth (12) (13), rennet and pepsin are identical sub- 
 stances, the transformation of casein into paracasein being the 
 first step in hydrolysis or else a side-hydrolysis (135). On the 
 other hand, Hammarsten (43) (44), Hedin (50), Rakoczy (101) 
 and others are of the opinion that rennet and pepsin are dis- 
 tinguishable from one another. The evidence for the identity 
 of the two enzymes lies in the fact that in no way, either quanti- 
 tatively or qualitatively, can the activity of rennet be separated 
 from that of pepsin, statements to the contrary effect (4) (52) 
 (135) (62) (122) (43) having been disproved or else shown to 
 rest upon experimental data obtained under conditions neces- 
 sarily involving inhibition, or apparent inhibition, of the one 
 form of action or of the other (Cf. especially Van Dam, (139) 
 (140) (141)). This is very well illustrated by the following 
 experiment of Morgenroth (85). If mixtures of calcium caseinate 
 and rennet are kept at low temperatures no coagulation occurs, 
 but the digestion process does occur; after these mixtures have 
 been held for some time at the low temperature, if they are heated 
 to 20 degrees they coagulate instantly. Thus the process which 
 underlies the clotting takes place at the lower temperature but 
 cannot be evidenced by actual clotting until the temperature has 
 risen. It has repeatedly been pointed out (105) (106) (72) that 
 the difference between the behavior of paracasein towards calcium 
 salts and that of casein is essentially this, that calcium para- 
 caseinate is coagulated by calcium salts at lower temperatures 
 than calcium caseinate. 
 
 Now it has been shown by Van Slyke and Hart (143), Geake 
 (36) and Van Slyke and Bosworth (142) that the paracasein 
 freed from its combination with calcium is analytically indis- 
 tinguishable from casein, and Van Slyke and Bosworth have 
 shown that the minimal combining-capacity of paracasein for 
 bases is exactly double that of casein. These facts admit of 
 only one interpretation, namely, that casein is hydrolytically 
 
INFLUENCE OF TEMPERATURE 419 
 
 split by rennet into two equal parts. The correctness of this 
 interpretation has been completely confirmed by Bosworth (12) 
 who has shown that in the first place no substance other than 
 paracasein results from the action of rennet upon casein, while, 
 in the second place, no matter what proteolytic enzyme (rennet 
 or trypsin) be employed to split the casein, the first step in 
 hydrolysis is the production of paracasein (13). 
 
 As regards the kinetics of the action of fibrin-ferment (throm- 
 bin) Fuld (33) finds that the Schtitz rule (velocity proportional 
 to the square root of the ferment-mass) very nearly holds good, 
 the actual relation between the velocity (v) of coagulation and 
 the mass of fibrin-ferment (E) being: 
 
 C. J. Martin finds that for the coagulation of blood by throm- 
 bin F X t = constant (78). 
 
 Fibrin-ferment undergoes marked exhaustion during the 
 process of coagulation (84) (60) . 
 
 7. The Influence of Temperature upon the Velocity of Protein 
 Hydrolysis. The influence of temperature upon the hydrolysis 
 of gelatin by trypsin has been carefully studied by Madsen and 
 Walbaum (cited after Arrhenius (3)). The results are in good 
 agreement with the formula: 
 
 where e is the base of the Napierian logarithms, Vi and v are the 
 velocities corresponding to absolute temperatures TI and T and 
 IJL is a constant, in this instance 10,570. This is the law which 
 is characteristic of the influence of temperature upon other 
 chemical reactions, and its possible significance, when it is found 
 applicable to protein systems, has been commented upon in 
 Chap. VII. 
 
 The influence of temperature upon the rate of digestion of 
 thymol-gelatin by pepsin is also capable of representation by the 
 above formula (Arrhenius, loc. cit., p. 71). In this case /z = 10,750. 
 The velocity both of pepsin and trypsin-digestion is, therefore, 
 doubled by a rise in temperature from 20 to 30 degrees. 
 
 Using different substrates it is found that, as might be expected, 
 
420 CHEMICAL DYNAMICS 
 
 the value of p for hydrolysis by a given enzyme varies with the 
 nature of the substrate. (Cf. table in Arrhenius' work, loc. cit., 
 pp. 99-98.) 
 
 The influence of temperature upon the coagulative power of 
 rennet has been studied by Fuld (32) ; here also the van't Hoff- 
 Arrhenius' rule holds good; /* is 20,650. 
 
 The interesting observation has been made by Cesana (17) in 
 studying the influence of temperature upon the ultramicroscopic 
 appearance of trypsin solutions, that the optimum temperature 
 for its proteolytic activity corresponds with that at which there 
 is a minimum of visible granules in the ultramicroscopic field 
 and a maximum dispersion of the trypsin leading to a maximum 
 frequency of collisions between trypsin particles or molecules 
 and the molecules of the substrate. 
 
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CHAPTER XVII 
 THE ENZYMATIC SYNTHESIS OF PROTEINS 
 
 1. The Reversion of the Hydrolysis of Proteins by Pepsin and 
 Trypsin. In "typical" catalysed reactions (Cf. previous Chap- 
 ter, section 1), since the equilibrium of the system is not shifted 
 by the catalysor and the point of equilibrium is determined by 
 the ratio of the velocity-constants of the forward and the opposed 
 reactions, both velocity-constants must be multiplied to an equal 
 degree by the catalysor; in other words, if under given conditions 
 a point of equilibrium exists at which an appreciable proportion 
 of the substrate remains unaltered, then, under such conditions, 
 the pure products (unmixed with substrate) are not in equilibrium 
 and must be tending to restore the substrate, and this tendency 
 must be increased by the presence of the catalysor in question. 
 
 As this fact became generally appreciated, following its ex- 
 perimental verification in a large number of non-fermentative 
 catalytic reactions, and as the belief gained ground that the 
 ferments were to be regarded as "typical" catalysors, it was 
 anticipated that fermentative reversions might be accomplished, 
 that is the synthesis of substances which are normally hydrolysed 
 (or otherwise altered) by enzymes, through acting upon the 
 products of hydrolysis with the same enzymes. This theoretical 
 anticipation was first experimentally realized by Croft Hill (31) 
 (32) in the fermentative synthesis of isomaltose (16) (2) (3) 
 from glucose, and has since been realized in the fermentative 
 synthesis of polysaccharides, fats, and proteins by various investi- 
 gators (7) (34) (26) (8) (66) (67) (69) (70) (47) (48) (9) (51) (52). 
 
 In order to make clear the conditions under which such re- 
 versions occur it will be well to consider in some detail the funda- 
 mental experiment performed by Croft Hill. 
 
 The hydrolysis of maltose (or of isomaltose) is represented 
 by the equation: 
 
 (A) Ci 2 H 2 20n + H 2 - 2 C 6 H 12 6 ; 
 
 the reversion is represented by the equation : 
 
 (B) 2 C 6 H 12 6 - CuH^On -|- H 2 0. 
 
 425 
 
426 CHEMICAL DYNAMICS 
 
 The condition of " balance" at equilibrium is therefore rep- 
 resented by the equation: 
 
 (C) Ci 2 H22On + H 2 O < 2 C 6 H 12 6 . 
 
 If mi be the mass of maltose, w 2 that of glucose and W that of 
 water, then at equilibrium we shall have, in accordance with the 
 mass-law : 
 
 miW = j*mf, (i) 
 
 in which k and & 2 are the velocity-constants of the forward and 
 the reverse reactions, respectively. If maltase is indeed a 
 "typical" catalysor, then we must assume that although ki 
 and k z are each multiplied by the presence of the catalysor their 
 ratio remains unaffected and, consequently, the relative pro- 
 portions between mi and mz and W at equilibrium are not affected 
 by the presence of maltase.* 
 
 Now the value of the ratio -^ for this reaction is such that in 
 
 ki 
 
 dilute solutions (i.e., when W in equation (i) is large) the point 
 of equilibrium in equation (C) lies so far over towards the right 
 that hydrolysis of the maltose, at the end of the reaction, is 
 practically complete, no appreciable quantity of unhydrolysed 
 maltose remaining in the system. This being the case we cannot 
 expect, through the agency of any "typical" catalysor, to secure 
 the synthesis of maltose in a dilute solution of glucose. A con- 
 siderable increase in the concentration of the system, however, 
 although it does not bring about any appreciable alteration 
 either in ki or in & 2 , nevertheless alters the proportion of maltose 
 to glucose at equilibrium very profoundly. The reason for this 
 is apparent upon inspection of equation (i). Increasing the 
 concentration of the system is equivalent to reducing the mag- 
 nitude of W and hence inducing a compensating increase in mi 
 (the mass of maltose) at equilibrium. In a concentrated system 
 we find, accordingly, that the hydrolysis of maltose is never 
 complete, whether maltase or any other catalysor be employed 
 to accelerate the process. A concentrated solution of glucose is 
 therefore not in a condition of equilibrium until a proportion 
 of maltose has been restored to the system by the occurrence of 
 
 * It should be clearly borne in mind that the verity of this statement has 
 never been proven. 
 
REVERSION OF HYDROLYSIS 427 
 
 reaction (B) . In the absence of any catalysors this reaction takes 
 place very slowly, but if any of the catalysors be present which 
 are capable of accelerating the hydrolysis of maltose, for example 
 maltase, then the synthesis of maltose or of isomaltose occurs 
 with measurable rapidity. It was by adding maltase to con- 
 centrated glucose that Croft Hill obtained a fermentative synthesis 
 of isomaltose. 
 
 In satisfactory accord with the above account of the chemical 
 mechanics of fermentative reversions, the majority of observers 
 have found that reversion of a fermentative hydrolysis is only 
 attainable when the enzyme is made to act upon the concentrated 
 products of the hydrolysis. Nevertheless reversion does not 
 always occur when it might be expected to occur; thus Bradley 
 (9) has shown that in the presence of 50 per cent of water no 
 appreciable synthesis of triolein from glycerin and oleic acid 
 can be brought about through the agency of lipase, although 
 in the presence of 50 per cent of water an appreciable proportion 
 of triolein remains unhydrolysed. The converse is also true, 
 reversion occurs when it might not be anticipated. Thus I find 
 (53) that it is possible to completely hydrolyse paranuclein to 
 products which have lost its characteristic properties and yet, 
 without any previous concentration, to bring about, through 
 simply altering the temperature and the enzyme concentration, 
 a fermentative synthesis of this substance. These apparent 
 anomalies arise from the fact that these ferments do not belong 
 to that restricted class of catalysors which, as explained in sec- 
 tion 1 of the previous chapter, has come to be regarded as typical, 
 but to what Stieglitz has shown to be the more general class of 
 catalysors, namely those which appreciably participate in the 
 reactions which they accelerate and therefore appreciably disturb 
 the final equilibrium. The significance of these phenomena, in so 
 far as the proteolytic enzymes are concerned, will be more 
 fully discussed below. 
 
 The fermentative synthesis of protamin through the action 
 of trypsin upon the concentrated products of its hydrolysis has 
 been accomplished by Taylor (69) (70) and I have described the 
 fermentative synthesis of paranuclein (51) (52) through the 
 action of pepsin upon the products of its hydrolysis. 
 
 In Taylor's experiments the mixed products of the tryptic 
 digestion of protamin sulphate were converted into carbonates 
 
428 CHEMICAL DYNAMICS 
 
 and then (the ferment having been destroyed by boiling) evapo- 
 rated until precipitation (at room temperatures) just began. 
 The solution which was thus prepared contained the products of 
 the hydrolysis of 400 grams of protamin and was free from un- 
 hydrolysed protamin, since the original solution (before evapo- 
 ration) was miscible with five volumes of acidulated alcohol 
 without any resulting opacity. To this solution were then added 
 300 cc. of a glycerin extract of the livers of the large soft-shelled 
 California clam (Schizothcerus nuttalli), which contain a proteo- 
 lytic ferment of the trypsin type which is very resistant to auto- 
 destruction and therefore especially adapted for employment in 
 experiments of long duration. After the addition of this trypsin 
 the solution was still miscible with alcohol without cloudiness. 
 Excess of toluol was then added, and the flask (containing over 
 4 litres) was set aside at room temperature. 
 
 The mixture gradually became opalescent, then cloudy, and, 
 finally, after the lapse of some months, a distinct precipitate 
 had formed. After five months the flask was opened, the con- 
 tents were heated to boiling to destroy the ferment, acidulated 
 with sulphuric acid, which dissolved the precipitate, and filtered. 
 Then four volumes of absolute alcohol were added, which resulted 
 in the throwing down of a heavy white precipitate, which, after 
 purification, was analysed. In all, about 2 grams were obtained. 
 
 The percentage formula of the protamin sulphate employed 
 (salmin sulphate prepared from the spermatozoa of Roccus 
 lineatus) had previously been determined by Taylor and found 
 to be: 
 
 H 2 S0 4 . 
 
 The analysis of the synthetical preparation yielded the following 
 results : 
 
 
 Calculated for 
 C3oHoN 17 O6.2 H 2 SO<, 
 per cent 
 
 Found, 
 per cent 
 
 c = . 
 
 37.85 
 
 37 68 
 
 H = 
 
 6.72 
 
 6 89 
 
 N - 
 
 25 13 
 
 24 90 
 
 H 2 SO 4 = 
 
 20 60 
 
 20 68 
 
 
 
 
 The substance was digestible by trypsin and not digestible by 
 pepsin. Taylor concludes that it was salmin. A concentrated 
 
REVERSION OF HYDROLYSIS 429 
 
 solution of the products of the hydrolysis of protamin, to which 
 no ferment had been added, yielded no trace of protamin after 
 standing for several months. 
 
 My experiments were conducted as follows: Four hundred cc. 
 of N/W potassium hydroxide were neutralized (or rendered 
 faintly acid) to litmus by the addition of casein and the solu- 
 tion was subjected to the action of pepsin for a considerable 
 period, fresh pepsin Q g. per litre) being added from time to time, 
 at 40 C. The digest at the end of this period, contained no 
 casein, but a considerable quantity of paranuclein had been 
 precipitated and had not undergone hydrolysis. The digest was 
 then heated to 100 degrees for about 10 or 15 minutes to destroy 
 the ferment, and filtered while hot. The solution was then 
 evaporated on a water-bath to about 70 cc. (the total concen- 
 tration of the system having been thus multiplied by about 6). 
 This concentrated solution of the products of the peptic digestion 
 of casein (and its first product of hydrolysis, paranuclein) is a 
 clear brown syrup which is strongly acid and gives no precipitate 
 or opalescence upon the addition of acetic acid, or upon the 
 addition of acetic acid in excess after previously rendering the 
 solution alkaline by the addition of KOH or NaOH. Both casein 
 and paranuclein are therefore absent from it. To 70 cc. of this 
 solution were added 30 cc. of a concentrated (approximately 
 10 per cent) solution of Gruebler's pepsin puriss. sice, which had 
 previously been filtered through rapid-filtering paper. The 
 mixture of the two solutions is a clear brown syrupy fluid which 
 yields no trace of a precipitate with acetic acid either before or 
 after neutralization with alkali. The mixture was set aside at 
 40 C. in the presence of excess of toluol to prevent bacterial 
 infection. Within two hours a thick white precipitate had formed 
 in the solution; after 48 hours the solution was filtered and the 
 precipitate thus collected on the filter was dissolved in a minimal 
 amount of sodium hydroxide and the filter so arranged that the 
 alkaline solution dropped directly into water acidified with acetic 
 acid. The precipitate thus obtained was reprecipitated twice 
 and was washed by decantation several times, employing several 
 litres of water at each decantation. Finally it was collected on 
 a hardened filter, washed with several litres of alcohol and ether, 
 and dried, first over CaCk and then over H 2 S0 4 at 60 C. In 
 this way 1.02 grams of a friable greyish-white hygroscopic powder 
 
430 CHEMICAL DYNAMICS 
 
 were obtained. This substance gave every indication of being 
 identical with paranuclein, the first product of the peptic hy- 
 drolysis of casein. Accordingly, for purposes of comparison, 
 paranuclein was prepared by partly digesting sodium caseinate 
 with pepsin, filtering off the precipitate, dissolving in sodium 
 hydroxide and precipitating with acetic acid. The paranuclein 
 was reprecipitated twice, washed with large volumes of water, 
 alcohol, and ether, and dried over CaCl2 and later over H 2 S0 4 
 at 60 degrees. A powder exactly similar in appearance to the 
 synthesized substance was thus obtained. 
 
 The synthesized substance and the paranuclein were both 
 analysed for phosphorus by Neumann's method, with the follow- 
 ing results : 
 
 Synthetic Substance Paranuclein 
 
 P 2 O 5 = 1.61 per cent P 2 O 5 = 4.18 per cent 
 
 Previous observers agree in stating that the percentage com- 
 position of paranuclein varies very greatly with the circumstances 
 under which it is prepared, the percentages of phosphorus which 
 have been found in various preparations varying from 0.88 to 
 6.86 (43). This fact leads us to suspect that the substance 
 which has been termed paranuclein is, in reality, a mixture of 
 at least two substances and the hypothesis which has suggested 
 itself to me is that during the hydrolysis of casein by pepsin a 
 substance of high phosphorus content, insoluble in dilute acids, 
 is first formed; that this substance splits off a soluble phosphorus- 
 containing moiety, leaving another insoluble substance of lower 
 phosphorus content, and that this second substance is in its turn 
 attacked and split up into soluble substances. That this expla- 
 nation is probably the correct one, although, of course, several 
 such steps may possibly be involved, is shown by the following 
 experiment. 
 
 One gram of the paranuclein containing 4.18 per cent of P 2 O 5 
 was dissolved in 400 cc. of 0.045 N Ca(OH) 2 and the mixture 
 was allowed to stand at 40 C. for 12 hours; acetic acid was then 
 added in excess and the precipitate purified, washed and dried 
 in the manner described above. This substance, which we may 
 designate paranuclein A, was analysed for phosphorus and found 
 to contain 1.51 per cent of P 2 5 , thus agreeing closely with the 
 synthetic substance. Only a little over 0.2 gram of this sub- 
 
REVERSION OF HYDROLYSIS 431 
 
 stance was obtained from the gram of paranuclein originally 
 dissolved in the lime-water. 
 
 The splitting off of phosphorus from paranuclein in alkaline solu- 
 tion has been commented on by other observers (61). 
 
 The substance which is obtained synthetically, as described 
 above, resembles paranuclein A very closely both in general prop- 
 erties and in its percentage-content of P20 & . The synthesized 
 substance is insoluble in dilute weak acids, readily soluble in 
 dilute alkali, precipitates protamin from a 1 per cent solution of 
 the sulphate at a reaction just alkaline to phenolphthalein (at 
 which reaction both the protamin and the paranuclein remain 
 in stable solution when not mixed), and a 2 per cent solution in 
 N/ 10 sodium hydroxide is precipitated by m/10 ferric ammo- 
 nium sulphate; these being all well-known properties of para- 
 nuclein (61) (42). 
 
 The synthesized substance also resembles my preparation of 
 paranuclein A in the following properties; in approximately 
 2 per cent solution in N / 100 NaOH it gives the xanthoproteic, 
 Millon's, Adamkiewicz and the biuret (violet) reactions; it is 
 precipitated by cupric chloride (1 vol. of N/l to 100) and by 
 zinc chloride, but not by mercuric chloride (5 vols.; of JV/10) 
 it is precipitated by picric and tannic acids, but the precipitate 
 redissolves on rendering the solution alkaline; it is not precipi- 
 tated by the addition of five volumes of absolute alcohol; and 
 the precipitate which is at first produced by the addition of acetic 
 acid is soluble in considerable excess of the reagent. 
 
 The change in the refractive index of JV/50 KOH which is 
 brought about by the introduction of one per cent of the synthetic 
 substance is identical with that which is brought about by the 
 introduction of one gram of paranuclein or paranuclein A (54), 
 namely, 0.00140 for sodium light. 
 
 If no pepsin be added to the concentrated solution of the 
 products of the peptic digestion of casein, prepared as described 
 above, the solution, after keeping for over a year at room-tem- 
 peratures or for several months at 40 C. remains perfectly clear 
 and homogeneous and gives no tests for paranuclein or for casein. 
 A 10 per cent filtered solution of Gruebler's pepsin, on standing 
 for many months at 40 degrees, also remains perfectly clear and 
 homogeneous. Yet these two solutions, when mixed in the 
 proportion of one volume of ferment-solution to five volumes of 
 
432 CHEMICAL DYNAMICS 
 
 the solution of the casein-products, give a voluminous precipitate 
 which remains permanent for many weeks. 
 
 The fact that paranuclein A, rather than paranuclein or casein, 
 is produced in the reversion of protein hydrolysis which pre- 
 sumably occurs in the experiments described above, is probably 
 to be interpreted as follows : 
 
 In the initial stages of the hydrolysis of casein, paranuclein 
 is produced, part of which undergoes further hydrolysis and 
 part of which escapes further hydrolysis owing to the fact that 
 it is thrown out of the sphere of action of the enzymes by pre- 
 cipitation (59) (60) (61). The proportion of the paranuclein 
 which undergoes hydrolysis passes through the intermediate 
 stage paranuclein A and then to the stage of proteoses and 
 peptones. Only that proportion of the paranuclein which under- 
 goes this further hydrolysis, therefore, yields to the filtered and 
 concentrated digest all of the substances which are necessary 
 for resynthesis. In event of synthesis occurring in a mixture 
 such as that described above, the first substance which resulted 
 which is insoluble in solutions of weak acids would necessarily 
 be thrown out of the sphere of action and the reaction would 
 terminate at this point. Hence, from what has been said above, 
 it is evident that were a member of the paranuclein group pro- 
 duced in these mixtures it would be paranuclein A, rather than 
 one of the other members of the group of higher phosphorus- 
 content. 
 
 In the above experiments, in order to secure synthesis of the 
 paranuclein, the products of its hydrolysis had to be concen- 
 trated to a considerable degree. From what has been said in 
 the beginning of this section the rationale of this procedure will 
 be evident. Later experiments, however (53), showed that 
 previous concentration of the products of the complete peptic 
 hydrolysis of paranuclein is not necessary if the synthesis be 
 carried out in the presence of a considerable excess of pepsin 
 and at a much higher temperature, namely from 60 to 70 de- 
 grees. 
 
 I made up the following mixtures in duplicate, having first 
 ascertained that the unconcentrated products of the peptic 
 hydrolysis of AT/50 alkali caseinates and 10 per cent pepsin 
 (Gruebler's puriss. sice.) can be kept separately for weeks at 
 65 degrees without a trace of precipitate forming in either solution. 
 
REVERSION OF HYDROLYSIS 433 
 
 (a) 10 cc. of unconcentrated products + 0.5 cc. of 15 per cent pepsin. 
 
 (6) 10 cc. of unconcentrated products + 1.0 cc. of 15 per cent pepsin. 
 
 (c) 10 cc. of unconcentrated products + 1.5 cc. of 15 per cent pepsin. 
 
 (d) 10 cc. of unconcentrated products + 2.0 cc. of 15 per cent pepsin. 
 
 (e) 10 cc. of unconcentrated products + 3.0 cc. of 15 per cent pepsin. 
 
 The one set was kept at 65 degrees, while the other was kept 
 at 36 degrees, both in tightly stoppered vessels containing excess 
 of toluol. After 24 hours there was no sign of any precipitate 
 or opalescence in the mixtures which had been kept at 36 degrees, 
 while in the duplicate set, which had been kept at 65 degrees, 
 (e) contained a heavy precipitate which left the supernatant 
 fluid clear, (c) and (d) contained, also, heavy precipitates which, 
 however, left the supernatant fluid strongly opalescent, and (a) 
 and (b) both contained slight precipitates. After 24 hours more, 
 no change had occurred in any of the solutions and those which 
 had been kept at 65 degrees were now returned to 36 degrees. 
 After a lapse of three weeks no trace of precipitate had appeared 
 in any of those solutions which had been at 36 degrees throughout, 
 while no further change had occurred in those which had been 
 kept at 65 degrees for 48 hours. 
 
 The probable identity of the precipitate which is thus pro- 
 duced, with that which is produced by the action of pepsin upon 
 the concentrated (=6 times) products at 36 degrees was shown 
 by the following experiments. 
 
 Thirty cubic centimeters of 15 per cent pepsin (Gruebler's 
 puriss. sice.) were added to 150 cc. of the unconcentrated prod- 
 ucts of the complete peptic hydrolysis of JV/50 sodium caseinate 
 and the mixture was kept at 65 degrees for 48 hours in the 
 presence of excess of toluol. The resulting precipitate was col- 
 lected on a hardened filter paper and washed with distilled water 
 until the washings were colorless; it was then dissolved by allow- 
 ing dilute sodium hydrate to pass through the filter and repre- 
 cipitated by allowing this filtrate to pass into a beaker contain- 
 ing excess of dilute acetic acid. The precipitate thus obtained 
 was collected on a hardened filter paper, washed with large 
 volumes of water, alcohol, and ether and dried over CaCk and 
 later over H 2 S0 4 . The product was a greyish-white, friable 
 hygroscopic powder resembling in its physical properties and 
 precipitation-reactions "paranuclein A" It was analysed for 
 phosphorus by Neumann's method and found to contain 1.65 
 
434 CHEMICAL DYNAMICS 
 
 per cent of PzO?>, thus agreeing closely with paranuclein A and 
 with the substance synthesized at 36 degrees from the concen- 
 trated products of the hydrolysis of paranuclein. 
 
 Furthermore, the change in the refractive index of AT/50 KOH 
 which is brought about by the introduction of one per cent of 
 this synthetic substance is identical with that which is brought 
 about by the introduction of one per cent of paranuclein, para- 
 nuclein A or of the substance synthesized at 36 degrees, namely 
 0.00140 for sodium light (54). 
 
 A reversion of hydrolysis can be brought about, therefore, 
 even in the diluted products of the complete peptic hydrolysis 
 of N/10 solutions of the alkali caseinates (diluted, since varying 
 amounts of pepsin solutions were added to the solution of products) 
 by the addition of 0.5 cc. of 15 per cent pepsin to 10 cc. of 
 products (final concentration of pepsin 0.75 per cent) and keeping 
 the mixture for 24 hours at 65 degrees, while it requires 15 cc. 
 of 10 per cent pepsin in 100 cc. of mixture (final concentration 
 of pepsin 1.5 per cent) to bring about, in 24 hours, reversion of 
 the hydrolysis at 36 degrees in a solution of products which has 
 been concentrated four or five times. 
 
 It is an extremely significant fact that the synthesis which 
 occurs at 65 degrees does so at a temperature from 10 to 15 de- 
 grees in excess of that at which, according to the majority of 
 authors, pepsin is rapidly and completely deprived of its proteo- 
 lytic activity.* True, the destruction even, at this temperature, 
 must be a matter of time, and one might be inclined to believe 
 that a short period of very intense action at 65 degrees produced, 
 in the above experiments, a similar result to the much more pro- 
 longed but weaker action of pepsin at 36 degrees. The facts are 
 not in favor of this view, however, since the appearance of the pre- 
 cipitate which marks a certain stage in the reversion does not occur 
 (if the pepsin is not too concentrated) until the solution has been 
 standing at 65 degrees for two or three hours, and it progressively 
 increases in amount for over 24 hours. It appears that the active 
 agent in reversion is not identical with the active agent in hydrolysis. 
 
 * Cf. Oppenheimer (45).- From the determinations of Schwarz (64) it 
 appears that concentrated (10 per cent) solutions of pepsin are deprived of 
 their power of accelerating protein hydrolysis and at the same time acquire 
 considerable power of inhibiting the activity of unaltered pepsin after having 
 been heated to 60 degrees for 5 minutes. 
 
REVERSION OF HYDROLYSIS 
 
 435 
 
 Additional experiments were undertaken by myself and Biddle 
 (55) with a view to further elucidating the relationship between 
 the paranucleins and the synthetic substances which I have 
 described. We determined their carbon, hydrogen and nitrogen 
 content, with the results tabulated below*; for the purpose of 
 comparison analyses of paranuclein by Lubavin (41) are also 
 included in the table. 
 
 
 Paranuclein 
 (Lubavin), 
 per cent 
 
 Paranuclein 
 (Robertson and 
 Biddle), 
 per cent 
 
 Paranuclein A 
 from paranu- 
 clein, 
 per cent 
 
 Substance 
 synthesized 
 36 degrees, 
 per cent* 
 
 Substance 
 synthesized 
 70 degrees, 
 per cent 
 
 c 
 
 48.5 
 
 49.98 
 
 49.47 
 
 53.39 
 
 49.99 
 
 H 
 
 7.1 
 
 7.20 
 
 6.80 
 
 7.80 
 
 7.00 
 
 N 
 
 13.3 
 
 12.80 
 
 12.50 
 
 13.00 
 
 13.10 
 
 * Three separate preparations of this substance were made, the figures given (except that for N 
 which was only determined in the first preparation) are those obtained with the preparation which 
 had been subjected to the most rigorous purification. 
 
 It is evident that the carbon, hydrogen, and nitrogen-contents 
 of paranuclein, paranuclein A and the substance synthesized 
 at 70 degrees, are so closely similar that they may be considered 
 identical; they also agree well with the original analyses of 
 Lubavin. The product obtained at 36 degrees, however, con- 
 tains a very appreciably higher percentage of carbon. Since the 
 carbon-content of this preparation was found to sink somewhat 
 during successive purifications it appears possible that the high 
 carbon is due to contamination with some impurity, possibly a 
 coagulose (Cf. below). The product which is formed at 36 
 degrees is deposited from the mixture more slowly than that 
 which is formed at 70 degrees, hence it would be more liable to 
 carry down contaminations. 
 
 The objection has been urged by Bayliss (5) and by Rohonyi 
 (57) that it is impossible to adequately identify a protein by 
 purely analytical similarities. They point out that Gruebler's 
 pepsin contains a substance which is coagulable by heating and 
 that after this substance has been removed the pepsin solution 
 no longer retains the power of causing the appearance of a pre- 
 cipitate when mixed with a solution of the products of the peptic 
 hydrolysis of casein. Merck's pepsin, on the other hand, yields 
 
 * For quantitive details of the methods employed in preparing these sub- 
 stances in bulk consult the communication just cited. 
 
436 CHEMICAL DYNAMICS 
 
 no coagulum on heating and yields no precipitate upon admixture 
 with a concentrated solution of the products of the peptic 
 hydrolysis of casein. From these facts they infer that the sub- 
 stance which I believed to have been enzymatically synthesized 
 paranuclein was, in reality, a compound of the heat-coagulable pro- 
 tein contained in Gruebler's pepsin with the caseoses contained in 
 the solution of the products of the peptic hydrolysis of casein. 
 
 While the analytical characterization and identification of a 
 protein substance is extremely difficult and inconclusive, the 
 phenomena of specific immunization, on the other hand, afford 
 us a method of identifying protein substances which, so far as 
 our knowledge at present extends, is decisive and extremely 
 sensitive. The investigations of Wells (75) and of Wells and 
 Osborne (76) have especially demonstrated the high degree of 
 specificity displayed by the immune-bodies which appear in the 
 circulation of animals as a result of repeated administration of 
 foreign proteins. It would appear to be thoroughly established 
 that only protein substances are antigenic, that protein split- 
 products below a certain degree of complexity are non-antigenic, 
 and that the immune-bodies which are developed in response 
 to immunization against a given protein will react only with 
 that protein or with a protein which contains, as an integral 
 portion of its molecule, a large fraction of the molecule of the 
 protein employed in immunization. 
 
 Advantage was taken of these facts by Gay and Robertson 
 (20) to investigate more fully the question of the identity or 
 non-identity of the enzymatically synthesized paranuclein with 
 the paranuclein which results from the partial hydrolysis of 
 casein. They found, employing both anaphylaxis and alexin- 
 fixation as indicators of the development of immune-bodies, that 
 the products of the complete peptic hydrolysis of casein are 
 toxic for normal animals. They have, however, no antigenic 
 property, nor any specific intoxicating effect upon animals sen- 
 sitized to themselves or to paranuclein. Gruebler's pepsin itself 
 is likewise non-antigenic. Paranuclein and the enzymatically 
 synthesized paranuclein, however, both yield specific antibodies 
 and these antibodies react- interchangeably with either substance. 
 These results would appear to yield unequivocal evidence of the 
 occurrence of a genuine protein synthesis under the conditions 
 outlined above. 
 
COAGULOSES AND PLASTEINS 437 
 
 The question remains as to why this synthesis is not achieved, 
 or, at least, does not result in the production of a visible pre- 
 cipitate, when Merck's pepsin is substituted for Gruebler's prep- 
 aration. While a positive answer to this question cannot as yet 
 be advanced with confidence, it would appear to offer no very 
 valid reason, especially in view of the experiments of Gay and 
 Robertson, for deciding that enzymatic synthesis does occur 
 under defined conditions in mixtures of Gruebler's pepsin and 
 solutions of the products of the complete peptic hydrolysis of 
 casein. It would appear, on the contrary, to afford additional 
 justification for the view, expressed above, that the active agent 
 in the reversion is not identical with the active agent in hydrolysis. 
 The former substance is present in Gruebler's pepsin, but is 
 absent from Merck's preparation. 
 
 The occurrence of synthetic processes (i.e., the reverse of 
 hydrolyses) in mixtures of pepsin and concentrated solutions of 
 the peptic split-products of proteins has also been demonstrated, 
 in quite a different manner, by Henriques and Gjaldbaek (27). 
 These observers have shown that if pepsin be added to an acid 
 and concentrated solution of the peptic split-products of a protein, 
 a synthetic process (union of COOH and H 2 N groups) occurs, 
 as indicated (a) by a progressive increase in the substances pre- 
 cipitable by tannic acid, and (6) by a decrease in the groups 
 (H 2 N ) which are capable of reacting with formaldehyde. The 
 process occurs at all temperatures lying between 5 and 70 degrees. 
 The more completely split the material in the digest is to begin 
 with the less complex are the products which result, but at the 
 same time their mass is greater. The most complex substances 
 which are formed in consequence of this reversion do not contain 
 much more formol-titratable nitrogen than the original proteins. 
 They have, moreover, shown (28) that the synthesis is reversible, 
 more dilution leading to a progressive increase in the formol- 
 titre. Furthermore, a decrease in the formol-titre also occurs 
 when trypsin is added to a concentrated solution of the products 
 of the peptic hydrolysis of protein. 
 
 2. The Probable Nature of the Coaguloses and Plasteins. 
 It will be observed, on referring to the analytical data obtained 
 by Robertson and Biddle (55), that the carbon content of the 
 paranucleins (49-50 per cent) is considerably lower than that 
 of the proteins (51-55 per cent). In this, and in other respects, 
 
438 CHEMICAL DYNAMICS 
 
 paranuclein differs very fundamentally from the coaguloses, for 
 the carbon-content of the coaguloses, so far from being abnor- 
 mally low, is abnormally high (58-59 per cent, Kurajev). The 
 syntheses described above are therefore not merely examples of 
 "plastein" or "coagulose" formation. 
 
 Kuhne and Chittenden (35) observed that if the portion of a 
 protein which is not acted upon by prolonged digestion with 
 pepsin be subsequently subjected to tryptic digestion a jelly 
 separates out which they termed the " anti-albumid coagulum" 
 and which is very slowly attacked by trypsin. Okunev, Lavrov, 
 Savjalov, Kurajev and Umber (44) (38) (39) (62) (63) (36) (50) 
 have prepared similar substances by acting upon peptic digests 
 with rennet, or with salts (ammonium sulphate, Cf. Umber, 
 loc. cit.), or with finely divided insoluble powders (lycopodium), 
 and have termed them "plasteins" or " coaguloses." These sub- 
 stances are albumoses and not proteins, .since they are digested 
 by erepsin (Lavrov and Salaskin). Savjalov finds that the 
 amount of plastein which is formed runs parallel with the quantity 
 of hetero-albumose which the protein yields, that is, to the 
 quantity of the ^difficultly-digestible anti-fraction. 
 
 All the circumstances of plastein formation point toward a 
 modified hydrolysis as the condition of their formation. No 
 preliminary concentration of the system is requisite, and no 
 rise in temperature. It appears probable, therefore, that plas- 
 tein formation is not a reversion of protein hydrolysis, as some 
 authors have suggested, but rather a coagulation due to partial 
 hydrolysis induced by the rennet-like action of ferments upon 
 certain albumoses or peptids,* these playing the part which is 
 played, in our more familiar experience, by calcium caseinate. 
 
 In satisfactory harmony with this view Herrmann and Chain 
 (30) have found that the antisera from rabbits immunized with 
 plastein derived from Witte's peptone, tested by the precipitin 
 reaction, react with plastein, not only from Witte peptone but 
 from other proteins, for example, edestin, serum albumin, egg- 
 albumin and the globulin from almonds. They do not react, 
 however, with Witte peptone itself nor with casein. In this 
 respect the plasteins differ very decisively from enzymatically 
 synthesized paranuclein, for the antiserum to synthetic para- 
 nuclein reacts not only with normal paranuclein but also with 
 * Regarding the probable mode of action of rennet (Cf . previous chapter, 6). 
 
CHEMICAL MECHANICS OF SYNTHESIS 439 
 
 the protein, casein, from which paranuclein is derived and of 
 which it forms a large and integral proportion. The plasteins, 
 on the other hand, would appear to form, antigenically speaking, 
 a group of substances which are very closely related to one 
 another and probably contain a large common fraction. 
 
 3. The Chemical Mechanics of the Fermentative Synthesis 
 of Proteins. We have seen in section 1 that in instances of 
 "typical" catalytic hydrolysis, although the degree of hydrolysis 
 in dilute systems, under the influence of a catalysor, may be prac- 
 tically complete, yet, if the solution of the products be concen- 
 trated, the station of equilibrium will be shifted and may be 
 shifted to such an extent that the pure products are not in 
 equilibrium and an appreciable quantity of substrate may be re- 
 stored to the system under the influence of a catalysor. Hence 
 it was natural to suppose, as Taylor did (loc. cit.) that the synthe- 
 sis of protamin, accomplished through the action of trypsin upon 
 the concentrated products of its hydrolysis, is an example of the 
 reversion of a " typical" catalysed reaction in which the catalysor 
 plays no part in determining the final equilibrium. Neverthe- 
 less it must be recollected that the validity of this view can by 
 no means be regarded as proven until it has been shown that the 
 station of equilibrium in the presence of the catalysor is definitely 
 the same as it is in its absence, and this has not been done; 
 indeed, the great technical difficulty of the problem, as it at 
 present appears to us, discourages the attempt. Moreover, 
 there are many facts, a number of which have been alluded to 
 in the previous chapter, which speak very decidedly against the 
 view that the fermentative syntheses of proteins are instances 
 of " typical" catalytic reversion. To those which have already 
 been dwelt upon may be added the following. 
 
 We have seen that the velocity of protein hydrolysis is, under 
 certain conditions, directly proportional to the concentration of 
 the ferment, under others proportional to the square root of the 
 ferment-concentration (Chap. XVI, section 3). If the proteolytic 
 ferments act as " typical" catalysors and do not in any way affect 
 the final equilibrium in the system, then the velocity of synthesis 
 must also vary directly or as the square root of the ferment- 
 concentration, for otherwise the ratio between the velocity con- 
 stants of the forward and opposed reactions would be a function 
 of the ferment-concentration and, since the station of equilibrium 
 
440 CHEMICAL DYNAMICS 
 
 is determined by this ratio, the equilibrium of the system would 
 also be a function of the ferment-concentration. The synthesis 
 of paranuclein A through the agency of pepsin affords us an 
 opportunity of determining the influence of the ferment-concen- 
 tration upon the velocity of protein synthesis, since the product 
 is rapidly formed and can readily be determined quantitatively. 
 The following was the experimental procedure: 
 
 The products of the complete peptic hydrolysis of N/ 10 
 potassium hydrate neutralized to litmus by casein* were evapo- 
 rated to one-sixth of their volume and filtered. Seventy-five 
 cubic centimeters of the clear, deep yellow filtrate were placed 
 in each of six flasks and to each, respectively 0, 5, 10, 15, 20 and 
 25 cc. of 10 per cent pepsin (puriss. sice. Gruebler's) were added, 
 and the total volume in each flask was made up to 100 cc. by the 
 addition of distilled water. After the addition of toluol, the 
 tightly-stoppered flasks were set aside at 36 degrees for 22 hours. 
 At the end of this time the flasks containing 25 and 20 cc. of 
 10 per cent pepsin, respectively, contained heavy precipitates, 
 while that containing 15 cc. contained a precipitate and those 
 containing 5 and 10 cc. had undergone no change other than a 
 slight increase in opacity. The contents of the flasks were now 
 filtered through rapid-filtering papers and the precipitates were 
 washed with distilled water until colorless filtrates were obtained; 
 in all cases the filtrates gave no precipitates or increase in opal- 
 escence upon the addition of acetic acid. The filters were then 
 washed with 10 cc. of JV/10 potassium hydrate and the filtrates 
 were collected in water containing 20 cc. of N/W acetic acid. 
 
 * Prepared as follows: To 6 litres of JV/50 sodium or potassium caseinate, 
 neutral or faintly acid to litmus, were added two gram of Gruebler's pepsin 
 puriss. sice, which had previously been dissolved in a little water. This 
 solution, after thorough mixing, was allowed to stand at 36 degrees for 10 
 days, 2 more grams of pepsin being added after the first 4 days (hi the 
 presence of toluol), and was then sterilized by steam at 100 degrees and 
 filtered through hardened filter paper. To the filtrate were then added two 
 more grams of pepsin, dissolved, as in the previous cases, in a little water, 
 toluol introduced, and the solution was again allowed to stand at 36 degrees for 
 7 to 8 days; it was then again sterilized by steam at 100 degrees and filtered 
 through hardened filter paper. The filtrate thus obtained is of a clear yellow 
 color with little or no opalescenca and gives no trace of a precipitate or 
 opalescence upon the addition of acetic acid either before or after neutralization 
 with alkali; hence both casein and paranuclein are completely absent from 
 the solution. 
 
CHEMICAL MECHANICS OF SYNTHESIS 
 
 441 
 
 The filter papers were then thoroughly macerated in dilute 
 potassium hydrate and the magma thus prepared was filtered 
 and washed with distilled water, the filtrate and washings being 
 collected in the beaker which received the first washings with 
 TV/ 10 potassium hydrate. Care was taken to prevent more than 
 a slight excess of acetic acid from being finally present in this 
 beaker. The precipitate which formed in the beaker settled rapidly 
 in small flocculi, was collected upon a soft rapid-filtering paper 
 and thoroughly washed with distilled water until the washings 
 were neutral to litmus. In all cases the filtrates and washings 
 were perfectly clear and free from protein. The papers and 
 precipitates thus obtained were macerated in water containing 
 a known quantity (10 cc.) of TV/10 potassium hydrate, the magma 
 thus obtained was diluted to about 200 cc., phenolphthalein 
 (4 drops of 2 per cent alcoholic solution) was added and the solu- 
 tions thus prepared were titrated to neutrality with N/ 10 hydro- 
 chloric acid. A weighed amount (227 milligrams) of paranuclein 
 A prepared in the manner described in section 1 was dissolved in 
 about 100 cc. of distilled water containing exactly 10 cc. of TV/10 
 potassium hydroxide, and the solution was titrated to neutrality 
 with TV/ 10 hydrochloric acid and phenolphthalein indicator; in 
 this way it was found that 1 gram of paranuclein A neutralizes 
 4.8 cc. of TV/10 potassium hydroxide*; hence 1 cc. of TV/10 alkali 
 = 0.208 gram of paranuclein A and we can estimate from the 
 determinations described above the amount of paranuclein A 
 produced in each of the mixtures by varying amounts of pepsin. 
 The following were the results obtained: 
 
 Amount of pepsin in 100 cc. of 
 solution 
 
 Milligrams of paranuclein A pro- 
 duced at the end of 22 hours 
 
 25 cc. of 10 per cent . . . 
 
 296 
 
 20 cc. of 10 per cent 
 
 210 
 
 15 cc. of 10 per cent 
 
 162 
 
 10 cc. of 10 per cent 
 
 
 
 5 cc. of 10 per cent 
 
 
 
 cc. of 10 per cent 
 
 
 
 * I have found that upon standing in the presence of excess of alkali, the 
 amount of alkali neutralized by a given quantity of paranuclein A increases 
 slightly. I have been unable to determine whether this phenomenon is due to 
 the slow dissolving of microscopic suspended particles or whether it is due to 
 
442 CHEMICAL DYNAMICS 
 
 From these results it is clear that the velocity of reversion is 
 by no means directly proportional to the concentration of pepsin, 
 nor even proportional to the square root of the ferment-concen- 
 tration. While the velocity of synthesis in the most concentrated 
 solutions (25 to 15 cc. of 10 per cent pepsin in 100 cc.) roughly 
 approximates to direct proportionality to the concentration of 
 ferment, in the more dilute solutions (10 to 5 cc. of 10 per cent 
 pepsin in 100 cc.) the velocity of synthesis falls off with extraor- 
 dinary rapidity as the concentration of the ferment diminishes. 
 Making every possible allowance for experimental error arising 
 out of loss of material during the estimation, an increase in 
 pepsin-concentration from 1.0 per cent to 1.5 per cent multiplies 
 the velocity of the synthesis over 10 times, while an increase in 
 pepsin concentration from 1.5 per cent to 2.5 per cent only 
 doubles it; these facts are obviously irreconcilable alike with 
 direct proportionality between the velocity of synthesis and the 
 concentration of ferment, and with the Schiitz rule of propor- 
 tionality to the square root of the concentration of the ferment. 
 The velocity of reversion does not bear the same relation to the 
 concentration of ferment that the velocity of hydrolysis does; 
 hence the ratio of the velocity constants of hydrolysis and re- 
 version must be dependent upon the concentration of the ferment, 
 or, in other words, the equilibrium between protein and its products 
 must, to some extent, be altered by pepsin. 
 
 We have seen that a reversion of the hydrolysis of paranuclein 
 can be brought about without any previous concentration of the 
 products of its hydrolysis, provided the temperature be raised 
 to 60-70 degrees. In the light of this fact there can be little 
 doubt that a shift in the station of equilibrium between para- 
 nuclein A and its products occurs as a result of the addition of 
 pepsin and that this shift in equilibrium is favored by a rise in 
 temperature. 
 
 That a shift in equilibrium is involved even at lower tempera- 
 hydrolysis (56). The number of cubic centimeters neutralized by one gram 
 given above is the lower figure, obtained directly after complete solution, 
 judged by the disappearance of obvious particles within the solution. I have 
 obtained figures as high as 5.2- after allowing the solution to stand for about 
 an hour at a warm temperature before titration. The titrations in the experi- 
 ment described were performed immediately after the complete disappearance 
 of obvious particles. 
 
CHEMICAL MECHANICS OF SYNTHESIS 443 
 
 tures is shown by the fact that if a sufficiency of dry pepsin* be 
 added to the unconcentrated products of the complete peptic 
 hydrolysis of N/W sodium caseinate synthesis will occur at 36 
 degrees. I have found about 2 grams of pepsin (Gruebler's 
 puriss. sice.) per hundred cubic centimeters sufficient for this 
 purpose. That the equilibrium is still further shifted by a rise 
 in temperature is shown by the following experiment : 
 
 To 300 cc. of the unconcentrated products of the peptic hy- 
 drolysis of N/ 10 sodium caseinate were added 6 grams of dry 
 pepsin. After 48 hours at 36 degrees (in the presence of excess 
 of toluol) a precipitate had formed, while the supernatant fluid 
 remained somewhat opalescent; after 6 days the supernatant 
 fluid was quite clear and a bulky precipitate lay at the bottom 
 of the flask; the clear fluid was now decanted from the precipi- 
 tate and divided into two parts; the one was kept at 36 degrees 
 and the other at 65 degrees; within 8 hours a precipitate had 
 formed in the latter, the supernatant fluid being strongly opal- 
 escent, while the part of the solution which remained at 36 
 degrees had developed no trace of precipitate or opalescence 
 after a period of two weeks. It is clear, therefore, that the 
 system had arrived at equilibrium at 36 degrees before the fluid 
 was decanted, since this fluid must have been " saturated" with 
 paranuclein A (soluble in these acid solutions only to an unde- 
 tectable extent) and any further formation of paranuclein A 
 would have resulted in an increase in opalescence if not in actual 
 precipitation. This did not occur, however, even during a period 
 of two weeks. Yet at 65 degrees a fairly abundant precipitate 
 was produced within 8 hours. 
 
 It is clear, therefore, that in the enzymatic synthesis of para- 
 nuclein A a marked shift in equilibrium is involved, and we 
 must therefore definitely abandon the view that pepsin is a 
 " typical" catalysor which does not, to an appreciable extent, 
 participate in the reactions which it catalyses. 
 
 The existence of a similar influence of trypsin upon the equi- 
 librium in protein solutions is possibly indicated by the investi- 
 gations of Walters (73), who has shown that whereas the time- 
 relations of the hydrolysis of casein* by trypsin are very accurately 
 
 * Dry, that is to say, not in solution, for otherwise water is added to the 
 system and the solution of products is actually diluted by the addition of the 
 ferment. 
 
444 CHEMICAL DYNAMICS 
 
 defined by the mono-molecular formula, terminating only when 
 hydrolysis is practically complete, the autohydrolysis, on the 
 contrary, which caseinates undergo in aqueous solution in the 
 absence of enzymes falls off in velocity with time much more 
 rapidly than would be indicated by the monomolecular formula, 
 so that the process comes to a stop when a very large proportion 
 of the protein is still unhydrolysed. The products of the tryptic 
 hydrolysis of casein exert, moreover, an almost negligible effect 
 upon the velocity of autohydrolysis, even when added in very 
 high concentration. We must infer, therefore, either that trypsin, 
 in dilute solutions, shifts the equilibrium between protein <=^ 
 products towards the right, or else that the rapid slowing-down 
 of the autohydrolysis is due to the attainment of a " false equi- 
 librium," i.e., an indefinite delay in the attainment of equi- 
 librium due to the interposition of a specifically slow reaction 
 in a series of catenary reactions, each one of which utilizes the 
 products of the preceding reaction. 
 
 4. Reciprocal Catalysis. We have seen that enzymatic 
 synthesis of paranuclein A may occur at temperatures 10 to 15 
 degrees in excess of that at which the hydrolysing activity of 
 pepsin is rapidly and completely destroyed. This invites adop- 
 tion of the view, previously expressed by Euler (17) (18) that 
 the synthesizing and hydrolysing forms of the enzyme are not 
 identical. Euler believes that two varieties of every hydrolysing 
 enzyme exist, the one accelerating, primarily, hydrolysis, the other 
 accelerating, primarily, synthesis, and that under definite condi- 
 tions a definite equilibrium exists between the two enzymes. He 
 bases this view, in the main, upon the non-constancy of the 
 velocity-constant of certain enzyme-accelerated reactions with 
 varying substrate-concentration, and upon the non-identity of 
 the synthesized product (isomaltose) derived by the action of 
 maltase upon concentrated glucose with the maltose from which 
 the glucose was derived by hydrolysis, and of the product (iso- 
 lactose) obtained by the action of lactase upon a concentrated 
 solution of galactose and glucose with the lactose from which 
 the galactose and glucose are derived.* Euler believes that the 
 " anti-trypsins " found in -serum and in egg-white are simply the 
 synthesizing form of the enzyme and that the anti-pepsin and 
 
 * It is stated, however, that even sulphuric acid, acting upon a concentrated 
 solution of glucose, gives rise, not to maltose but to isomaltose (19). 
 
RECIPROCAL CATALYSIS 445 
 
 anti-trypsin which appear in the circulation after the injection 
 of the enzymes do so as a result of the production of the synthe- 
 sizing form in restoration of the equilibrium between the synthe- 
 sizing enzyme and the hydrolysing form. 
 
 The hypothesis which I have put forward (53) in explanation 
 of the phenomena of protein hydrolysis and synthesis is a modi- 
 fication and extension of Euler's hypothesis, and involves the 
 view that these processes are examples of which I have termed 
 " reciprocal catalysis." The essential features of this hypoth- 
 esis have already been embodied in equations (A) to (D) in Chap. 
 XV, section 1. For convenience of reference they may be recap- 
 itulated here. 
 
 We know that during or preceding the hydrolysis of proteins 
 by proteolytic enzymes the ferment combines with the substrate 
 (previous chapter, 2). Furthermore, the reaction consists in the 
 introduction of the elements of water into COH.N bonds 
 of the protein molecule, each bond which is attacked yielding 
 two molecules. We may therefore represent the reaction of 
 hydrolysis as follows: 
 
 HF 
 I 
 
 (A) - COH.N - + HFFOH - - COH.N - 
 
 I 
 FOH 
 
 HF 
 
 I 
 
 (B) -COH.N -COOH + H 2 N- + FF . 
 
 I 
 FOH 
 
 while, subsequently, the dehydrated form of the enzyme (FF) 
 reacts with water to regenerate the hydrated form : 
 
 (D) FF + H 2 + HFFOH 
 
 enzymatic synthesis of a protein consisting in the reverse of 
 these reactions. 
 
 The net result of the first two reactions is the transference of the 
 elements of water from the ferment to the substrate molecule, 
 while, in the third reaction, the ferment recoups itself from the 
 medium. Provided the station of equilibrium in the reaction 
 (D) lay far enough to the right and the velocity of this reaction 
 
446 CHEMICAL DYNAMICS 
 
 measured from left to right were great compared with that of 
 either of the reactions (A) and (B) measured from right to left, 
 and provided, also, the quantity of enzyme combined at any 
 instant with the substrate were unappreciable we would obtain, 
 for the kinetics of the reaction of hydrolysis, the monomolecular 
 equation, in which the velocity constant of hydrolysis would 
 be proportional to the ferment-mass. If these conditions were 
 not fulfilled we would obtain, provided the proportion of the 
 dehydrated to the hydrated enzyme did not alter very appre- 
 ciably during the reaction, the differential equation 
 
 dx kiF (A - x) 
 
 dt l + a(A-x)-ftx 2 ' 
 or its integrated form: 
 
 A , a + 0A_ ft 
 
 (i) 
 
 4 ,.., 
 
 (Cf. Chap. XVI.) For the reversion we would obviously obtain 
 the differential equation: 
 
 _ dx = k 2 Fx* ..... 
 
 dt ~ l-a(A-x)+ftx 2 ' 
 
 From equations (A) to (D), applying the mass-law, it will be 
 obvious that the quantity of enzyme dehydrated by unit mass 
 of substrate will be dependent upon the mass of the hydrated form 
 which is present, consequently, the constant a (and similarly the 
 constant ft) will be dependent upon the mass of ferment present, 
 and will also, if an appreciable shift in equilibrium represented by 
 equation (D) occurs as a result of the hydrolysis, alter somewhat 
 in value as hydrolysis proceeds *; hence the final position of equi- 
 librium, when the velocity of reversion is equal to the velocity of 
 hydrolysis, must be dependent upon the mass of ferment. 
 
 Quite independently of the mathematical formulation of the 
 tune-relations involved in this process, it will be clearly seen that 
 the presence of the enzyme must result in a greater or smaller 
 shifting of the point of equilibrium between the protein and its 
 products when we reflect that the hydrated form HFFOH, accord- 
 ing to the above scheme., only accelerates the hydrolysis, while 
 the anhydrous form FF only accelerates the synthesis, and since 
 
 * It is for this reason that we cannot employ equations (i) and (iii) to deter- 
 mine the station of equilibrium in these systems. 
 
RECIPROCAL CATALYSIS 447 
 
 these are, in general, present in unequal concentrations, the equi- 
 librium between the forward and reverse reactions of protein 
 hydrolysis must be shifted. Since, however, for every shift in 
 equilibrium, there must be a corresponding expenditure of energy, 
 the equilibrium between the anhydrous and hydrated forms of the 
 enzyme must also be shifted by the protein; just as the enzyme 
 accelerates the hydrolysis of the protein more than its synthesis 
 when the hydrated form of the enzyme is initially present in con- 
 siderable excess of its anhydrous form, so the protein accelerates 
 the dehydration of the enzyme more than its hydration because it 
 is initially present in great excess of its products of hydrolysis. 
 This latter fact will itself lead to a slowing of the hydrolysis of the 
 protein since the hydrated (and hydrolysis accelerating) form of 
 the enzyme is thereby diminished in concentration; as the hydroly- 
 sis proceeds, however, this effect will diminish; the products of the 
 protein hydrolysis will tend to increase the proportion of the 
 hydrated form of the enzyme to the anhydrous form, and the rate 
 of hydrolysis will increase at the expense of the rate of synthesis. 
 Ultimately, it is evident that a condition of equilibrium must be 
 reached in which the station of equilibrium between the protein 
 and its products is shifted further in the direction protein > 
 products than its position in the absence of the enzyme, while the 
 station of equilibrium between the hydrated and anhydrous forms 
 of the enzyme is shifted further in the direction hydrated form 
 anhydrous form than its position in the absence of the protein. 
 This position of equilibrium will depend, obviously, upon the total 
 concentration of the enzyme and of the substrate, respectively, 
 and once attained, a further addition of substrate would reinaugu- 
 rate the hydrolysis of protein, it is true, because the active mass of 
 hydrolysable protein would thus be increased, but it would shift 
 the point of equilibrium in the direction products > protein; 
 addition of enzyme would shift the station of equilibrium in the 
 direction protein > products, as has been found by Bayliss and 
 others (4) (68). This latter statement, however, holds good only 
 while the water in the system is in great excess of the enzyme, so 
 that varying concentration of the enzyme does not appreciably 
 affect the proportion subsisting between the hydrated and anhy- 
 drous forms at equilibrium in the absence of proteins. If, however, 
 the enzyme be very concentrated, then the proportion of water to 
 enzyme (Cf. Eq. (D)) will appreciably affect the equilibrium be- 
 
448 CHEMICAL DYNAMICS 
 
 tween the hydrated and anhydrous forms of the enzyme, and there 
 will be present a relatively greater proportion of the anhydrous 
 (synthesis accelerating) form FF; hence, under these conditions, 
 a further addition of enzyme will shift the equilibrium of the 
 protein in the direction products > protein. 
 
 A remarkable feature of the syntheses of protein through 
 enzyme agency which have been accomplished by Taylor and 
 myself is the high concentration of enzyme which has to be em- 
 ployed; the reason for this is now clear; the highly concentrated 
 enzyme contains a greater proportion of the anhydrous form and 
 it shifts the equilibrium of the protein in the direction of synthesis. 
 It is now clear, also, why a sufficiently high concentration of en- 
 zyme will actually bring about synthesis of protein in the uncon- 
 centrated products of the complete hydrolysis of a solution of 
 protein, brought about by the agency of dilute enzyme. The 
 dependence of the velocity-constant of hydrolysis, calculated from 
 the monomolecular formula, upon the initial concentration of the 
 substrate (Cf . Chap. XVI, section 3) is also readily comprehended, 
 since, as pointed out above, increase in the concentration of sub- 
 strate must, in the initial stages of hydrolysis, lead to an increase 
 in the proportion of the anhydrous to the hydrated form of the 
 enzyme, and hence to a diminution of the concentration of the 
 hydrolysis-accelerating form of the enzyme; hence the velocity- 
 constant of hydrolysis diminishes with increasing substrate- 
 concentration. 
 
 6. The Influence of Temperature upon the Enzymatic Syn- 
 thesis of Proteins. The action of high temperatures in enhanc- 
 ing the synthesizing power of the enzyme while diminishing or 
 abolishing its power of accelerating hydrolysis may be interpreted 
 in either of two ways. Either the high temperature destroys the 
 hydrated form by accelerating its hydrolysis, while leaving the 
 anhydrous form unaffected, so that during the period that the 
 anhydrous is changing into the hydrated form only the synthesis 
 of the protein is being accelerated, and not its hydrolysis, or, more 
 probably, the high temperature actually shifts the equilibrium of 
 reaction (D) in the direction HFFOH FF + H 2 O, a conception 
 which, if we regard the proteolytic enzymes as being substances 
 analogous to proteins, is in good accord with our knowledge of the 
 influence of heat upon these bodies (Chap. XII). The fact that 
 heating a proteolytic enzyme leads to the formation of substances 
 
INFLUENCE OF TEMPERATURE 449 
 
 which strongly retard protein hydrolysis has been observed by a 
 number of investigators. Thus Schwarz (64) has shown that if 
 solutions of pepsin be heated to 80 degrees for some time and then 
 added to peptic digests the digestion is greatly retarded, while 
 Pollack (46) had previously obtained, by heating pancreas extracts 
 to 70 degrees, a substance which greatly retards tryptic hydrolysis 
 of proteins; he further observed that this substance is a colloid 
 since it does not pass through the membrane of a dialysor. Hensel 
 (29) has observed that if the mucous membrane of a stomach be 
 treated with acidulated water at 50 degrees, the watery extract 
 thus obtained contains an organic substance which greatly retards 
 peptic hydrolysis of proteins. Beam and Cramer (6) have found 
 that solutions of rennet which have been heated to from 56 to 
 60 degrees exert an inhibitory influence on the activity of unheated 
 rennet. Bayliss (4) has found that heated trypsin not only greatly 
 delays the tryptic hydrolysis of casein but would appear to in- 
 augurate a chemical reaction in the reverse sense, since mixtures 
 of heated trypsin and a casemate, instead of increasing in conduc- 
 tivity with time, at first decrease in conductivity, later slowly 
 increasing. Similar phenomena have been observed with other 
 enzymes (15) (6). 
 
 The anti-tryptic actions of such substances as egg-white (72) (4), 
 normal blood serum (22) (17) (49) (37) (12) (24) and extracts of 
 intestinal worms (13) and the anti-peptic and anti-tryptic actions 
 of the bodies produced in the circulation by the injection of pepsin 
 and trypsin into living animals (58) (1) (74) have usually been 
 attributed to the formation of more or less stable compounds 
 between the ferment and the anti-ferment. One would be in- 
 clined to similarly attribute the action of heated solutions of pepsin 
 and trypsin in inhibiting these ferments to the formation of com- 
 pounds between the heated and the normal ferment were it not 
 that the heated ferment, although deprived of its power to acceler- 
 ate hydrolysis of protein, markedly accelerates its synthesis. The 
 inhibitory action of the heated ferment is thus clearly seen to 
 consist in bringing about a change which is opposite in sense to 
 that which is brought about by the normal ferment.* We see, 
 
 * Bayliss, regarding the phenomenon from the standpoint of Ehrlich's 
 "side-chain" hypothesis, has advanced the opinion that the decrease in con- 
 ductivity which is observed upon the addition of heated ferment to a solu- 
 tion of protein is to be attributed to the destruction of the "zymophore," or 
 
450 CHEMICAL DYNAMICS 
 
 also, that the loss of proteolytic activity which occurs on heating 
 solutions of proteolytic ferments is to be attributed, not to auto- 
 hydrolysis of the ferment, but to the partial or complete conversion 
 of the hydrolysing enzyme into the synthesizing form. From the 
 above account of the chemical mechanics of the enzymatic hydroly- 
 sis and synthesis of proteins it would appear that the "inactiva- 
 tion" of proteolytic ferments by heat (provided actual coagulation 
 does not occur) should be a reversible process, if not in the pure 
 enzyme-solution, at any rate in the presence of products of protein 
 hydrolysis.* Owing to the fact that the enzymatic activity of 
 hydrolysing enzymes has in the past been identified with their 
 power to accelerate hydrolysis, the application of heat to enzyme 
 solutions has always been regarded as involving the " destruction " 
 of the enzymes and restoration of their activity under varying 
 conditions following a return to lower temperatures has not been 
 anticipated and therefore has not been looked for with any especial 
 care. Nevertheless, Bayliss (4) has observed that the power of 
 heated trypsin to accelerate protein hydrolysis is to some extent 
 regained after the heated enzyme has been mixed with protein for 
 some time, and Howell (33) has observed that thrombin is not 
 " destroyed" by moderate heating, but is reversibly thermolabile 
 inasmuch as it regains its activity on treatment with alkali and 
 subsequent neutralization at lower temperatures. Gramenisky 
 (21) has also observed that oxidase inactivated by heating rapidly 
 regains its activity on standing at lower temperatures.! 
 
 6. The Thermodynamics of the Enzymatic Hydrolysis and 
 Synthesis of Proteins. The thermodynamical aspect of the 
 hypothesis developed above is of interest. As I have pointed out, 
 the shift in equilibrium of the system protein + products towards 
 
 digesting group of the ferment while the "haptophore" group, by which the 
 enzyme attaches itself to the protein molecule, is unaffected. The heated 
 ferment he terms "zymoid," and he believes that the initial decrease in the 
 conductivity of mixtures of casein and heated ferment is to be attributed to 
 the formation of a compound between the "zymoid" and the protein. This 
 view, of course, fails to account for the reversion of protein hydrolysis which 
 is brought about by heated pepsin. 
 
 * Since, as we have seen in the preceding section, these will catalyse the 
 reaction FF + H 2 O -> HFFOH. 
 
 f In this connection it is of extreme significance that the oxidase employed 
 by Gramenisky behaved, at 80 degrees, no'longer as an oxidase but as a reduc- 
 tase, or reducing ferment. 
 
THERMODYNAMICS 451 
 
 the right which results from the introduction of a proteolytic 
 enzyme into the system must result in a corresponding shift in the 
 equilibrium of the system anhydrous enzyme + hydrated enzyme 
 towards the left and vice versa. Since the enzyme is usually 
 present in small concentrations compared with the protein, the 
 shift in the equilibrium between the two forms of the enzyme must 
 be large compared with that of the equilibrium between the protein 
 and its products; or else the energy expended in a shift of the enzyme- 
 equilibrium must be great compared with the energy expended in a 
 shift of the protein equilibrium. The latter appears to be the more 
 probable view; since, otherwise, the shift in the protein-equilibrium 
 would probably be in all cases too small to be observed, and, 
 moreover, we know that the reaction of protein hydrolysis is 
 only very faintly exothermic (65) (23) (25) (40) so that the energy 
 change involved in a shift in the equilibrium between protein and 
 the products of its hydrolysis is probably very small, and a slight 
 shift in the equilibrium of a relatively very small amount of enzyme 
 might suffice, if the reaction of enzyme hydration involved a 
 relatively considerable energy-change, to provide the energy for a 
 considerable change in the equilibrium of a relatively large mass of 
 protein. 
 
 It will be observed, therefore, that the processes of the enzymatic 
 hydrolysis and synthesis of proteins do not necessarily involve 
 any deviation from the laws of thermodynamics, in spite of the 
 fact that the enzyme which catalyses these processes markedly in- 
 fluences the equilibrium between the protein and its products. In 
 the analogous case which is afforded by the enzymatic synthesis 
 and hydrolysis of fats Dietz (14) assumes that because the station 
 of equilibrium is altered by the presence of the enzyme the second 
 law of thermodynamics must be violated in these systems, for, he 
 argues, since the catalysor (enzyme) can be withdrawn from the 
 system without the expenditure of any work, it would only be 
 necessary to allow the system to come to equilibrium in the pres- 
 ence of the enzyme and then withdraw the enzyme and allow it to 
 come to its normal station of equilibrium and then reintroduce the 
 enzyme, and so forth, to bring about an endless cycle of changes 
 and to secure an unlimited quantity of energy without the expendi- 
 ture of any work. But in this argument, it should be pointed out, 
 Dietz assumes the answer to the very question at issue; he assumes 
 in a word, that the enzyme in question is a "typical" catalysor, 
 
452 CHEMICAL DYNAMICS 
 
 not entering to any appreciable extent into the reaction, and there- 
 fore not requiring the expenditure of any energy to withdraw it 
 from the system. Most investigators will prefer to assume, from 
 Dietz' results, that lipase is not a " typical" catalysor, but enters 
 appreciably into the reaction which it catalyses (10) (11). 
 
 7. Biological Applications. It may here be noted that the 
 hypothesis outlined above is not inconsistent with the generally 
 expressed view that the enzyme may be recovered without appreci- 
 able loss of activity from a protein digest which has reached equi- 
 librium. Even if, in consequence of the presence of protein, the 
 station of equilibrium between the two modifications of the 
 enzyme has been shifted in such a direction as to diminish its 
 power of accelerating hydrolysis, yet upon removal from the 
 system the enzyme would regain its normal equilibrium, although, 
 of course, this process might involve a greater or a shorter period 
 of time. The energy thus apparently gained would be slowly lost 
 through the slow (because of the absence of catalysors) regaining 
 of equilibrium between the protein and its products. The protein 
 + products system would, in other words, be left " supersaturated" 
 in respect to products, or, were the enzyme in the system highly 
 concentrated, or did it, for any other reason, initially contain a 
 high proportion of anhydrous (synthesis-accelerating) form so that 
 it gained power of accelerating hydrolyses as a result of the presence 
 of protein, then the protein <F products system would be left, by 
 removal of the enzyme, " supersaturated " with respect to protein. 
 In the former case the anhydrous <=* hydrated enzyme system 
 would be left supersaturated with respect to the anhydrous 
 (synthesis-accelerating) form, in the latter, with respect to the 
 hydrated (hydrolysis-accelerating) form. By mechanical separa- 
 tion of the enzyme and substrate at various periods in living 
 tissues it is obvious that a cycle of such operations could occur, 
 the enzyme being utilized over and over again, now for hydrolysis 
 and now for synthesis. It is obvious that if such reciprocal rela- 
 tions as that outlined above find a place in living material, the 
 organism may be enabled thereby to temporarily and locally store 
 up large quantities of energy. The significance of this possibility 
 in the general interpretation of life-phenomena is patent. 
 
 Finally, it may be pointed out that the conceptions which I have 
 developed above enable us to understand how the living organism, 
 through temporary mechanical or chemical separation and localiza- 
 
LITERATURE CITED 453 
 
 tion of the synthesizing forms of these enzymes can bring about 
 protein-synthesis even in the presence of the considerable quantity 
 of water which living tissues contain. If we regard the enzymes 
 as " typical 7 ' catalysors then, as Bradley has pointed out (9), 
 enzymatic synthesis of fat (and the same may be said of the enzy- 
 matic synthesis of protein) could only occur under conditions 
 approximating to dessication, conditions which it is very difficult 
 to picture as occurring in living tissues. 
 
 LITERATURE CITED 
 
 (1) Achalme, P., Ann. de 1'Inst. Pasteur, 15 (1901), p. 737. 
 
 (2) Armstrong, E. F., Journ. Chem. Soc. London, 83 (1903), p. 1305. 
 
 (3) Armstrong, E. F., Proc. Roy. Soc. London, 76B (1905), p. 592. 
 
 (4) Bayliss, W. M., Arch. des. Sci. Biol. Petrograd, 2 Suppl. (1904), p. 261. 
 
 (5) Bayliss, W. M., Joura. of Physiol., 46 (1913), p. 236. 
 
 (6) Beam, A. R., and Cramer, W., Biochem. Journ., 2 (1907), p. 174. 
 
 (7) Berninzone, Atti. del soc. ligi di Sc. nat. e geograph. Genoa, 2 (1900), 
 
 p. 327. 
 
 (8) Bodenstein, M., and Dietz, W., Zeit. f . Elektrochem., 12 (1906), p. 605. 
 
 (9) Bradley, H. C., Journ. Biol. Chem., 8 (1910), p. 251. 
 
 (10) Bradley, H. C., Journ. Biol. Chem., 13 (1912), pp. 407 and 431. 
 
 (11) Bradley, H. C., and Kellersberger, E., Journ. Biol. Chem., 13 (1912), 
 
 pp. 419 and 425. 
 
 (12) Cathcart, E. P., Journ. of Physiol., 32 (1905), p. 299. 
 
 (13) Dastre, A., and Stessano, A., C. R. Soc. Biol. Paris, 55 (1903), p. 635. 
 
 (14) Dietz, W., Zeit. f. physiol. Chem., 52 (1907), p. 279. 
 
 (15) Donath, H., Beitr. z. chem. Physiol. und Path., 10 (1907), p. 390. 
 
 (16) Emmerling, O., Ber. d. d. chem. Ges., 34 (1901), p. 3810. 
 
 (17) Euler, H., Zeit. f. physiol. Chem., 52 (1907), pp. 146 and 152. 
 
 (18) Euler, H., "Allgemeine Chemie der Enzyme," Wiesbaden (1910). 
 
 (19) Fischer, W., cited after Taylor (68). 
 
 (20) Gay, F. P., and Robertson, T. Brailsford, Journ. Biol. Chem. 12 (1912), 
 
 p. 233. 
 
 (21) Gramenisky, M. Y., Zeit. f. physiol. Chem., 69 (1911), p. 286. 
 
 (22) Hahn, M., Ber!. klin. Wochenschr., 34 (1897), p. 499. 
 
 (23) Hari, P., Arch. f. d. ges. Physiol., 115 (1906), p. 11; 121 (1908), 
 
 p. 459. 
 
 (24) Hedin, S. G., C. R. Soc. Biol., Paris, 55 (1903), p. 132. 
 
 (25) Henderson, L. J., and Ryder, C. T., Proc. Amer. Soc. Biol. Chemists, 1 
 
 (1907), p. 26. 
 
 (26) Hanriot, M., C. R. de 1'Acad. Sci., Paris, 132 (1901), p. 212. 
 
 (27) Henriques, V., and Gjaldbaek, J. K., Zeit. f . physiol. Chem., 71 (1911), 
 
 p. 475. 
 
 (28) Henriques, V., and Gjaldbaek, J. K., Zeit. f . physiol. Chem., 81 (1912), 
 
 p. 439. 
 
454 CHEMICAL DYNAMICS 
 
 (29) Hensel, Proc. of the Public Health Assn., Petrograd, Jan. (1903), 
 
 cited after Biochem. Centralbl., 1 (1904), p. 404. 
 
 (30) Herrmann, J., and Chain, A., Zeit. f . physiol. Chem., 77 (1912), p. 289. 
 
 (31) Hill, Croft, Journ. Chem. Soc., London, 73 (1898), p. 643. 
 
 (32) Hill, Croft, Ber. d. d. chem. Ges., 34 (1901), p. 1380. 
 
 (33) Howell, W. H., Cleveland Med. Journ., 9 (1911), p. 118; cited after 
 
 Chem. Abstracts, 5 (1911), p. 145. 
 
 (34) Kastle, J. H., and Loevenhart, A. S., Amer. Chem. Journ., 24 (1900), 
 
 p. 491. 
 
 (35) Kuhne, W., and Chittenden, R. H., Zeit. f. Biol., 19 (1883), p. 159. 
 
 (36) Kurajev, D., Beitr. z. chem. Physiol. und Path., 1 (1901), p. 121; 4 
 
 (1904), p. 476. 
 
 (37) Landsteiner, K., Centr. f. Bakt., 27 (1900), p. 357. 
 
 (38) Lavrov, D., Zeit. f. physiol. Chem., 51 (1907), p. 1; 53 (1907), p. 1; 
 
 56 (1908), p. 343. 
 
 (39) Lavrov, D., and Salaskin, S., Zeit. f . physiol. Chem., 36 (1902), p. 277. 
 
 (40) von Lengyel, R., Arch. f. d. ges. Physiol., 115 (1906), p. 7. 
 
 (41) Lubavin, W., Hoppe-Seyler's Med.-Chem. Untersuch. Berlin (1866), 
 
 p. 453. 
 
 (42) Milroy, T. H., Zeit. f. physiol. Chem., 22 (1896), p. 307. 
 
 (43) von Moraczewski, W., Zeit. f. Physiol. Chem., 20 (1895), p. 28. 
 
 (44) Okunov, W. H., Diss. Petrograd, cited after Maly's Jahresber. f. 
 
 Tierchem., 25 (1895), p. 291. 
 
 (45) Oppenheimer, C., "Ferments and their Actions," transl. by Ainsworth 
 
 Mitchell, London (1901), p. 92. 
 
 (46) Pollack, L., Beitr. z. chem. Physiol. und Path., 6 (1905), p. 95. 
 
 (47) Pottevin, H., Bull. Soc. Chim., 35 (1906), p. 693. 
 
 (48) Pottevin, H., Ann. Inst. Pasteur, 20 (1906), p. 901. 
 
 (49) Pugliese and Coggi, cited after Euler (17). 
 
 (50) Rakoczy, A., Zeit. f. physiol. Chem., 75 (1911), p. 273. 
 
 (51) Robertson, T. Brailsford, Univ. of California Publ. Physiol., 3 (1907), 
 
 p. 59. 
 
 (52) Robertson, T. Brailsford, Journ. Biol. Chem., 3 (1907), p. 95. 
 
 (53) Robertson, T. Brailsford, Journ. Biol. Chem., 5 (1909), p. 493. 
 
 (54) Robertson, T. Brailsford, Journ. Biol. Chem., 8 (1910), p. 287. 
 
 (55) Robertson, T. Brailsford, and Biddle, H. C., Journ. Biol. Chem., 9 
 
 (1911), p. 295. 
 
 (56) Robertson, T. Brailsford, and Schmidt, C. L. A., Journ. Biol. Chem., 
 
 5 (1908), p. 31. 
 
 (57) Rohonyi, H., Biochem. Zeit., 53 (1913), p. 179. 
 
 (58) Sachs, H., Fortschr. d. Med., 20 (1902), p. 425. 
 
 (59) Salkowski, E., Arch. f. d. ges. Physiol., 63 (1896), p. 401. 
 
 (60) Salkowski, E., Zeit. f. physiol. Chem., 27 (1899), p. 297; 32 (1901), 
 
 p. 245. 
 
 (61) Salkowski, E., and Hahn, M., Arch, f . d. ges. Physiol., 59 (1895), p. 225. 
 
 (62) Savjalov, W. W., Arch. f. d. ges. Physiol., 85 (1901), p. 171. 
 
 (63) Savjalov, W. W., Zeit. f. physiol. Chem., 54 (1907), p. 119. 
 
 (64) Schwartz, O., Beitr. z. chem. Physiol. und Path., 6 (1905), p. 524 
 
LITERATURE CITED 455 
 
 (65) Tangl, F., Arch. f. d. ges. Physiol., 115 (1906), p. 1. 
 
 (66) Taylor, A. E., Univ. of Calif. Publ. Pathol., 1 (1904), p. 33. 
 
 (67) Taylor, A. E., Journ. Biol. Chem., 2 (1906), p. 87. 
 
 (68) Taylor, A. E., "On Fermentation," Univ. of California Publ. Pathol., 
 
 1 (1907), p. 185. 
 
 (69) Taylor, A. E., Univ. of California Publ. Pathol., 1 (1907), p. 343. 
 
 (70) Taylor, A. E., Journ. Biol. Chem., 3 (1907), p. 87. 
 
 (71) Umber, F., Zeit. f. physiol. Chem., 25 (1898), p. 258. 
 
 (72) Vernon, H. M., Journ. of Physiol., 31 (1904), p. 346. 
 
 (73) Walters, E. H., Journ. Biol. Chem., 11 (1912), p. 267; 12 (1912), p. 43. 
 
 (74) Weinlard, E., Zeit. f . Biol., 44 (1902), p. 46. 
 
 (75) Wells, H. G., Journ. Infect. Dis., 5 (1908), p. 449; 6 (1909), p. 506; 
 
 9 (1911), p. 147. 
 
 (76) Wells, H. G., and Osborne, T. B., Journ. Infect. Dis., 8 (1911), p. 66; 
 
 12 (1913), p. 341; 14 (1914), p. 377; 17 (1915), p. 259; 19 (1916), 
 p. 183. 
 
APPENDIX 
 
 THE TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 
 IN PROTEIN SYSTEMS 
 
 The carrying out of accurate electrochemical measurements in 
 solutions which contain proteins is attended by a number of diffi- 
 culties which result in part from the physical characteristics of 
 such solutions and in part from the instability of the proteins and 
 their tendency to undergo hydrolysis. The following is a descrip- 
 tion of the technique which I have found it best to employ when 
 obtaining such measurements in solutions of casein especially* but 
 also of ovomucoid and serum globulin. My experimental results 
 cited in Chaps. VIII to XI, inclusive, were obtained by these 
 methods; they are obviously applicable, with necessary or advisable 
 modifications, to aqueous solutions of the majority of proteins.! 
 
 In the gas-chain determinations two platinized platinum elec- 
 trodes saturated with hydrogen are used, the one being dipped 
 in the solution of unknown hydrogen- or hydroxyl-concentration, 
 the other in a solution of known hydrogen- or hydroxyl-concentra- 
 tion, the latter being, as a rule, the solution of acid or base in which 
 the protein is dissolved and the former the solution of protein. 
 The electrodes which I employ are of a design due to my colleague, 
 
 * Robertson, T. Brailsford, Journ. of Physical Chem., 14 (1910), p. 528. 
 t For the technique employed in determining H+ and OH' concentrations 
 and methods used in the computation of results, consult also 
 
 Bovie, W. T., Journ. Medical Research 33 (1915), p. 295. 
 
 Clark, F. W., Meyers, C. N., and Acree, S. F., Journ. Physical Chem. 20 
 
 (1916), p. 241. 
 
 Clark, W. M., Journ. Biol. Chem. 23 (1915), p. 475. 
 Clark, W. M., Journ. Biol. Chem. 25 (1916), p. 479. 
 Clark, W. M., Journ. Bacteriology 2 (1917), pp. 1, 109, 191. 
 Hildebrand, J. H., Journ. Amer. Chem. Soc. 35 (1913), p. 847. 
 Lewis, G. N., Brighton, T. B., and Sebastian, R. L., Journ. Amer. Chem. Soc. 
 
 39 (1917), p. 2245. 
 
 Michaelis, L., Die Wasserstoffionenkonzentration, Berlin (1914). 
 Schmidt, C. L. A., Univ. Calif. Publ. Physiol. 3 (1909), p. 101. 
 Sharp, L. T., and Hoagland, D. R., Journ. Agric. Research 7 (1916), p. 123. 
 Sorensen, S. P. L., Ergeb. d. Physiol. 12 (1912), p. 393. 
 
 456 
 
TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 457 
 
 Dr. F. G. Cottrell; they have been described in detail by Schmidt 
 and Finger.* 
 
 The hydrogen is generated by the electrolysis of 6 per cent (by 
 volume) sulphuric acid, in an apparatus which has previously been 
 described by Schmidt and Finger in the paper cited above. To 
 guard against the possibility of any oxygen, ozone or hydrogen 
 peroxide being carried over with the hydrogen from the generator, 
 the gas is passed through a heated glass tube which is loosely filled 
 with platinized asbestos and which has wrapped around it for a 
 distance of about 20 cm. a coil of fine resistance- wire, the internal 
 diameter of the tube being about 0.5 cm. The hydrogen is com- 
 pletely cooled before it reaches the electrodes, because, after leaving 
 the heater, it passes through a narrow glass tube, partly filled with 
 glass wool, about 70 cm. long, leading to the water-bath; since a 
 considerable portion of this tube is within the incubator which 
 holds the water-bath, the temperature of the hydrogen, when it 
 reaches the electrodes, may be considered as that of the incubator 
 itself. The coil of the heater attached to the hydrogen-generator 
 is heated by a portion of the same current (110- volt circuit) which 
 generates the hydrogen, the current being led into the generator 
 through 4 lamps in parallel, one of the lamps being connected in 
 series with the coil. In order to maintain the pressure of hydrogen 
 which is necessary to drive it through the electrodes, the oxygen 
 which comes off from the outer cylinder of the generator is carried 
 off by a tube which dips into a column of water, the depth of the 
 opening of the tube in the water being adjusted until the levels of 
 the fluid in the inner and outer cylinders of the generator are 
 approximately equal. 
 
 The complete chain is arranged as follows: The syphon-tube of 
 the " half -element " containing the fluid into which the electrode 
 dips is immersed in a beaker filled with the same fluid. Thus the 
 " half -element " containing the solution of unknown hydrogen- or 
 hydroxyl-concentration is in fluid connection with a beaker which 
 contains the same solution and the " half-element " containing the 
 solution of known hydrogen- or hydroxyl-concentration is in fluid 
 connection with a beaker filled with that solution. The two 
 beakers are then connected by a U-tube filled with agar saturated 
 
 * C. L. A. Schmidt and C. P. Finger, Journ. of Physical Chem., 12 (1908), 
 p. 406. For improvements in the original design, Cf. C. L. A. Schmidt, Univ. 
 of Calif. Publ. Pathol., 2 (1916), p. 157. 
 
458 APPENDIX 
 
 with KC1, thus effectively preventing any mixing of the two solu- 
 tions and diminishing any contact-difference of potential between 
 them.* The gas is passed through the electrode which dips into 
 the solution containing protein (the solution, that is, of unknown 
 hydrogen- or hydroxyl-concentration) at the rate of from one to 
 two large bubbles per second, and the excess of gas is allowed to 
 pass through the other electrode. The whole chain is immersed 
 in a small water-bath which is placed inside an incubator main- 
 tained at a temperature lying between 31 and 32 degrees (Cf. 
 below). It was thought necessary, at first, not to permit the 
 hydrogen to escape into the incubator, lest it should be ignited, 
 on opening the door of the incubator, by the flame beneath; con- 
 sequently the electrodes were inserted into the half-elements 
 through tightly-fitting rubber stoppers, and rubber tubes were 
 attached to the side-tubes of the half-element and carried outside 
 the incubator and the cupboard within which the incubator is set 
 up. For reasons which will shortly be described, however, this 
 procedure was, of necessity, abandoned and the gas was permitted 
 to escape into the incubator. There appears to be no danger 
 involved in this procedure. The incubator is of the usual double- 
 walled type employed by bacteriologists; its internal dimensions 
 are 45 cm. wide by 24 cm. deep by 48 cm. high. It is provided 
 with two doors, the outer of the usual double-walled type, the 
 inner a glass door through which thermometers, etc., can be read 
 without disturbing the apparatus or causing fluctuations of tem- 
 perature by currents of air. The inner chamber is provided at 
 the top with two small air-exits. 
 
 The potentials between the electrodes of the chain are measured 
 on a 100-cm. potentiometer bridge-wire (previously standardized).! 
 For the detection of the zero-point on the bridge-wire I have 
 employed a D' Arson val galvanometer provided with a damping 
 coil; this instrument gave a decided deflection with the potential 
 corresponding to 1 mm. displacement on the bridge in all of the 
 experiments described in Chap. IX. The constant fall in potential 
 from end to end of the potentiometer wire is best supplied by a 
 good storage-cell; but I have found an arrangement of four 
 Gladstone-Lalande cells (Model G-50), two in parallel and two 
 
 * N. Bjerrum, Zeit. f. physik. Chem., 53 (1905), p. 428. 
 t The potentiometer manufactured by the Leeds & Northrup Co. is, how- 
 ever, more convenient to use, and also more accurate. 
 
TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 459 
 
 in series, very satisfactory. The potential derived from these is 
 measured against a standard Weston cell just before and just after 
 every reading. The potential provided by the Gladstone-Lalande 
 cells is quite sufficiently constant during the progress of an obser- 
 vation, provided they are always short-circuited for 15 to 20 
 minutes beforehand. 
 
 The electrodes are platinized with Lummer and Kurlbaum's 
 solution. They are very carefully washed, both within and with- 
 out, between the observations, first in a stream of distilled water 
 and then in the solution in which they are about to be immersed; 
 every few days, if they are in constant use, they should also be 
 washed in chromic and sulphuric acid solution and then, after 
 thorough washing in a stream of distilled water, allowed to soak 
 for 12 hours in distilled water. From time to time they should be 
 replatinized. 
 
 Induct or iiim 
 
 Weston Cell 
 
 Gas Chain 
 
 -Hllh- 
 
 Dry Cells 
 
 Short Circuiting Key 
 
 ,1 
 
 Bridge Wire 
 
 Telephone 
 
 Gladstone-Lalande Cells 
 
 The conductivity-vessel which I employ is of the Kohlrausch- 
 Holborn type, with a thermometer dipping into the fluid between 
 the electrodes. This is immersed in the same water-bath as the 
 gas-chain, and the conductivities of the solutions are measured at 
 exactly 30 degrees. The electrodes are platinized with Lummer 
 and Kurlbaum's solution. The same bridge-wire is employed for 
 determining the conductivities and for determining potentials. A 
 telephone is employed to detect the zero-point and the alternating 
 
460 APPENDIX 
 
 current is supplied by an inductorium of the Ostwald type. The 
 rheostat reads correctly to within 0.01 per cent and supplies any 
 desired resistance between 0.1 ohm and 1000 ohms. The resist- 
 ance in the rheostat is always adjusted until the zero-point is 
 exactly in the middle of the bridge; the resistance in the rheostat 
 is then, of course, exactly equal to that of the conductivity-vessel 
 rilled with the fluid under investigation. The arrangement of the 
 wiring is represented in the preceding diagram. 
 
 The wires of the conductivity circuit are all of " bell-wire" so that 
 their resistance can be neglected. The wires of the potentiom- 
 eter-circuit are somewhat thinner. All of the wires are insulated 
 and carefully supported on glass, and are never allowed to touch 
 the table. Where it is necessary to carry wires through the table, 
 for example, they are run through glass tubes. The wire con- 
 nected with the slider on the bridge is encased in rubber tubing.* 
 
 I have pointed out in Chaps. V and XII that it is difficult to 
 obtain solutions of caseinates of much higher acidity than neutral- 
 ity to litmus by merely shaking up casein in solutions of bases, 
 not because casein will not form such solutions, but because, 
 although it dissolves at first with considerable rapidity, after the 
 excess of alkali is neutralized, further casein dissolves with extreme 
 slowness. Now the conductivities of solutions containing proteins 
 must be measured as soon as possible after the complete solution 
 of the protein, for, otherwise, the hydrolysis which the protein 
 undergoes, the more rapidly the more alkaline its solution, intro- 
 duces a serious error into the determination. Hence it is impera- 
 tive, not only that the conductivities of these solutions should 
 be determined as soon as possible after complete solution of the 
 protein, but, also, that the preparation of the solution, after 
 the introduction of the protein, should consume as little time as 
 possible. Hence all of the solutions of casein which are acid to 
 phenolphthalein are best prepared by dissolving the casein in 
 excess of alkali and then neutralizing this excess with acid. 
 This procedure, if gas-chain measurements are being made at 
 the same time, has the further advantage that the absolute 
 conductivities of the solutions thus obtained being higher, the 
 detection of the zero-point on the bridge-wire by means of a gal- 
 
 * More accurate conductance measurements may be made by employing 
 the method of E. W. Washburn. Cf . " The Measurement of Conductivity of 
 Electrolytes," Leeds and Northrup Co., Philadelphia, 1916. 
 
TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 461 
 
 variometer is rendered easier and a less sensitive galvanometer 
 can be employed than would otherwise be required. It should not 
 be forgotten, however, that although a small concentration of KC1, 
 for example, does not alter the dissociation of caseinates of the alka- 
 lies nor the combining-capacity of casein for bases (Chap. VIII) 
 yet this is certainly not the case with many other proteins. 
 
 In this connection it is to be carefully noted that if the solution 
 employed to dissolve the casein be too alkaline, little or nothing is 
 gained by the rapidity of its solution, because the rapidity of its 
 hydrolysis is also great. On the other hand, as I have said, if too 
 small a proportion of free alkali is present solution is so slow that 
 hydrolysis is extensive. Evidently an avoidance of both of these 
 extremes will yield the most satisfactory results. I have found 
 the proportion 10 cc. of N/ 10 KOH to 1 gram of casein to be the 
 most satisfactory solvent for this protein. In the experiments 
 described in Chaps. IX and X, save in the formation of solutions 
 containing more than 100 X 10~ 5 equivalents of base per gram of 
 casein, therefore, the procedure was as follows: Part of the KOH 
 was neutralized until the portion unneutralized stood in this pro- 
 portion to the mass of casein undergoing solution, the casein was 
 dissolved therein, and then the desired final proportion of KOH to 
 casein was attained by the further addition of HC1. For example, 
 it was desired to obtain a solution of 1 per cent casein in 0.005 
 N KOH. Accordingly, to 75 cc. of 0.1 N KOH were added 50 cc. 
 of 0.1 AT HC1 and in this were dissolved 2.5 grams of casein; upon 
 the attainment of complete solution and while stirring, 12.5 cc. of 
 0.1 N HC1 were added and the whole solution was made up to 250 
 cc. with distilled water; another solution was made up in precisely 
 the same way but without the introduction of the casein; the 
 conductivities of both solutions were than determined and their 
 difference (= X) estimated.* The two solutions were arranged in 
 the gas-chain in the manner described above and the potential 
 between the gas-electrodes immersed in them determined. Hence, 
 of course, the OH' concentration of the solution not containing 
 casein being known, that of the solution containing casein was 
 
 * Since the concentration of KC1 is the same in both solutions it adds the 
 same amount to both conductivities and this disappears in their difference; 
 that is, presuming that the caseinate does not combine with or decompose the 
 KC1 and that the presence of excess of K+ ions does not depress the dissociation 
 of the potassium caseinate; Cf. Chap. VIII. 
 
462 
 
 APPENDIX 
 
 determined. The effect of the presence of KC1 upon the dissocia- 
 tion of the KOH is negligible provided the KOH and KC1 are 
 always sufficiently dilute to be practically completely dissociated. 
 The extent of the error which is introduced into the determina- 
 tion of X by dissolving the casein in the first instance in a solution 
 of too high alkalinity may be gauged from the following results : 
 
 FINAL SOLUTION 3 PER CENT CASEIN IN 0.015 N KOH 
 
 Amount of unneutralized KOH 
 employed to dissolve 7.5 
 grams casein 
 
 XX 106, 
 
 100 cc . 
 
 305 
 
 75 cc . . . . 
 
 296 9 
 
 
 
 I have mentioned that the desired concentration of the KOH 
 unneutralized by HC1 in the solutions containing casein is attained 
 by the addition, to the solutions of the casein in excess of KOH, of 
 HC1 while stirring. This is a matter of some importance. If acid 
 be poured into a solution of a caseinate which is imperfectly mixed, 
 the casein which is precipitated in the acid portions of the fluid 
 forms bulky coagula and is only with difficulty redissolved, even 
 if the quantity of alkali still unneutralized by the acid is more than 
 sufficient to hold in solution all of the casein that may be present. 
 Consequently, the solution of the caseinate must be rapidly stirred 
 while the acid is being added. The same procedure, of course, 
 considerably enhances the rapidity with which the casein dissolves 
 in the alkali employed for its solution. I place the fluid in which 
 the casein is to be dissolved, together with the casein,* in a beaker 
 of squat form and 400 cc. capacity. The mixture is then agitated 
 by a flattened glass rod bent at right angles so that the horizontal 
 arm is about 2J cm. long and as near as possible to the bottom of 
 the beaker; this is rotated at the rate of about 1600 revolutions 
 per minute by a small motor. As soon as the casein is completely 
 dissolved the acid is delivered into solution, a few drops at a time, 
 from a pipette, the opening of which is held at some depth below the 
 surface of the liquid. In some of the earlier experiments the acid 
 was poured upon the surface of the solution, but all of these solu- 
 tions foam to a certain extent and the foam is not agitated by the 
 
 * Regarding the procedure to be followed in adding the casein to the solvent 
 Cf. Chap. XII, 1. 
 
TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 463 
 
 stirrer with the same rapidity as the fluid which lies below it, 
 consequently, if the acid is poured upon the surface, casein is 
 precipitated within the foam and is only with great difficulty 
 redissolved, hence solutions prepared in this manner yielded very 
 irregular results both in the gas-chain and in the conductivity de- 
 terminations. In delivering the acid into the solution it is very 
 necessary to avoid holding the opening of the pipette too close to 
 the side of the beaker, as in that case a film of casein is precipitated 
 on the glass and this film redissolves with great difficulty. 
 
 Since the conductivities of solutions of the caseinates must be 
 determined as soon as possible after the introduction of casein 
 into the solution employed to dissolve it, for otherwise hydrolysis 
 introduces a considerable error, it is evident that as little time as 
 possible must be consumed in bringing the temperature of the 
 solution to that at which its conductivity is to be determined. 
 This could be achieved in either of two ways: either a small 
 volume of fluid could be employed, so disposed as to take up the 
 temperature of the water-bath very quickly; or the water-bath 
 may be maintained at a somewhat higher temperature than that 
 actually desired, and the conductivity of the fluid can be measured 
 at the moment when it reaches the desired temperature. For 
 reasons which will be sufficiently obvious the latter procedure is 
 found to be more convenient. The water-bath is kept at a tem- 
 perature lying between 31.5 and 32.5 degrees, a preliminary 
 measurement of the conductivity of the solution is made at a 
 temperature between 29 and 29.5 degrees and this preliminary 
 determination is corrected at precisely 30 degrees.* 
 
 Solution of 1 per cent casein in 0.03 N KOH : 
 
 X X 10 5 determined immediately 348. 8 
 
 X X 10 5 determined after allowing solution to stand at about 
 
 30 degrees for 20 minutes 356. 3 
 
 It is obvious, however, that in this procedure the gas-chain 
 measurements are made at a temperature some 2 degrees higher 
 than the conductivity measurements, and it may be inquired to 
 
 * For maintaining constant temperatures, W. M. Clark employs an air- 
 bath; Cf. Journ. Amer. Chem. Soc., 35 (1913), p. 1889; Journ. Biol. Chem., 
 23 (1915), p. 475. C. L. A. Schmidt, Univ. of Calif. Publ. Pathol., 2 (1916), 
 p. 157, employs an oil-bath similar in construction to that described by 
 G. A. Hulett, Physical Review, 32 (1911), p. 257. 
 
464 APPENDIX 
 
 what extent this invalidates the comparison of the two sets of 
 determinations. The error which is thus introduced, for the small 
 difference of temperature concerned is, however, negligible, since 
 it was found by actual trial that the difference between the poten- 
 tials measured at 30 degrees and those measured at 34 degrees 
 could barely, with certainty, be detected upon my potentiometer- 
 bridge. Nor is this fact surprising for, exclusive of any possible 
 change in the equilibrium between protein and alkali, the potential, 
 according to the Nernst formula, varies directly as the absolute 
 temperature, and, therefore, only increases by 1 /300th per degree 
 centigrade at 30 degrees. It has been observed by W. A. Osborne* 
 and myself f that there is no evidence of an appreciable shift in the 
 equilibrium between casein and alkali, as the temperature rises, 
 until the temperature of 36 degrees is reached. 
 
 I have mentioned in describing the apparatus employed, that 
 difficulty was encountered in leading off the hydrogen, after it had 
 bubbled through the fluids, through the exit-tubes of the half- 
 element to the outside of the incubator. The difficulty was this, 
 all of the solutions containing casein foam upon passing the 
 hydrogen through them, and to a greater extent the less the excess 
 of alkali in the solutions. This foam, collecting in the exit and 
 connected rubber tubes, gave rise to a pressure which drove the 
 fluid out of the half-element and thus interrupted the continuity 
 of the chain. Accordingly, it was found necessary, not only to 
 abandon the idea of conveying the waste hydrogen out of the 
 incubator, but also to cut off short the exit-tube of the half-element 
 and permit the foam to escape freely into the water-bath (the 
 lower end of the exit-tube was, of course, well above the surface of 
 the water in the bath). After this plan had been adopted no 
 further trouble was encountered from this source. Foaming, may, 
 however, be prevented or greatly reduced by the addition of a few 
 drops of octyl or amyl alcohol. 
 
 All observers who have endeavored to measure the conductivi- 
 ties of solutions containing proteins have encountered the difficulty 
 involved in the precipitation which occurs at the electrodes, 
 particularly in the case of casein, in neutral or very faintly acid 
 solutions. Whetham and -Hardy { in order to minimize the error 
 
 * W. A. Osborne, Journ. Physiol., 27 (1901), p. 398. 
 
 t T. Brailsford Robertson, Journ. Biol. Chem., 5 (1908), p. 147. 
 
 t W. B. Hardy, Journ. of Physiol., 33 (1905), p. 251. 
 
TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 465 
 
 arising from this source, adopted the plan of heating the electrode, 
 after platinization, to a dull red, thus clumping the platinum black 
 and reducing the total surface of the electrode. 
 
 I formerly tacitly assumed this phenomenon to be connected in 
 some way with the passage of the alternating current through the 
 solution. In my own experiments, however, I speedily found that 
 in neutral or faintly acid solutions of potassium caseinate marked 
 precipitation of casein occurs at the gas electrode, without the 
 passage of any current, usually after but sometimes even before 
 the passage of the hydrogen. At first I was inclined to attribute 
 this to gases collected, in the interval between experiments, in the 
 tubes conveying the hydrogen, to impurities in the hydrogen, etc., 
 but after very careful exclusion of all of these possibilities the 
 precipitation still took place, and it occurred to me that it might 
 be due to the hydrogen ions dissolved in the platinum itself. 
 When hydrogen is passed through a platinized platinum electrode, 
 the potential measured across the chain represents, at first, a 
 higher acidity of this electrode than it does later on, when the 
 electrode has come into equilibrium with the solution; in other 
 words, the hydrogen ions dissolved in the platinum have not yet 
 come into equilibrium with those in the solution; an excess of 
 hydrogen ions is still present in the platinum. It therefore 
 appeared possible that this initial acidity of the platinum itself 
 might be responsible for the precipitation of casein at its surface, 
 and this idea found confirmation in the fact that on prolonged 
 passage of hydrogen the film of protein deposited on the electrode 
 slowly redissolved. It occurred to me that it might be possible to 
 avoid this precipitation altogether by bringing the electrode nearly 
 to equilibrium with a low concentration of hydrogen ions before 
 introducing it into the solution at all; accordingly, before making 
 a gas-chain determination with a caseinate solution of very low 
 hydroxyl concentration (neutral or acid to litmus) the electrode 
 which was to be dipped into the protein solution was immersed in 
 distilled water and gas was passed through it for an hour or more 
 it was then immediately washed in the protein solution and used. 
 The device was found to work excellently, and in all of the experi- 
 ments described herein precipitation at the electrodes was avoided 
 entirely, at least so far as the eye could perceive. A similar device 
 was found effective in avoiding precipitation at the electrodes of 
 the conductivity-vessel. The vessel was simply filled with dis- 
 
466 APPENDIX 
 
 tilled water and allowed to stand in the water-bath for some hours 
 before making a determination. 
 
 In order to ensure a correct determination of the potential of the 
 chain in these experiments it was always found necessary to pass 
 the gas for three hours and, in the neutral or faintly acid solutions, 
 for as much as six hours. If two successive readings on the bridge, 
 taken an hour apart, did not differ by more than 1 mm. the result 
 was considered correct. Difficulty in obtaining constant readings 
 is, of course, only encountered in the chains yielding the highest 
 potentials, in which, as the apparatus is arranged, 1 mm. on the 
 bridge-wire makes a difference of only about one-half per cent to 
 the calculated value of the potential. In the chains of low poten- 
 tial greater accuracy is desired, but no difficulty is encountered in 
 attaining it, since, in these, successive readings are nearly always 
 identical, or if they are not, the difference can invariably be traced 
 to some obvious source of error which may be eliminated in a 
 repetition of the experiment. 
 
 Since, however, prolonged exposure of the protein solution in 
 the gas-chain to the temperature of the water-bath is essential, the 
 question arises whether the accuracy of the determinations is not 
 invalidated by hydrolysis of the casein. The answer to this ques- 
 tion is in the negative, the change in the hydrogen- or hydroxyl- 
 concentration of a protein solution, due to hydrolysis, is negligible 
 in comparison with the change in its conductivity. Moreover, it 
 has been shown* that the displacement of the neutral point on the 
 potentiometer-wire, due to hydrolysis of the casein, in an alkaline 
 solution of a caseinate is opposed in sense to the displacement due 
 to the coming to equilibrium of the electrode with the solution in 
 which it is dipped. Were appreciable change in the hydroxyl 
 concentration of alkaline solutions of casein due to hydrolysis 
 occurring, therefore, the position of the neutral point on the bridge 
 should indicate at first a diminishing and later increasing potential. 
 I have discovered no trace of this save in one or two of the most 
 alkaline solutions which I have employed (1 per cent casein in 
 0.03 NKOH; 3 per cent casein in 0.05 N KOH), in these the 
 displacement due to hydrolysis was never more than 1 mm. and 
 the minimum value of the potential was taken as the true one. It 
 is, however, to be recollected that the hydrolysis was probably 
 
 * T. Brailsford Robertson and C. L. A. Schmidt, Journ. Biol. Chem., 5 
 (1908), p. 31. 
 
TECHNIQUE OF ELECTROCHEMICAL MEASUREMENTS 467 
 
 most rapid in the period of time preceding the attainment of this 
 minimum, so that even the minimum potential may be consider- 
 ably in error, especially as in the chains containing these solutions 
 the potential was low and 1 mm. displacement on the bridge 
 introduced a considerable error into the determination of the 
 potential. This error is diminished in its percentage magnitude 
 in the calculation therefrom of m, the amount of alkali neutralized 
 by the protein, but, nevertheless, the determination of m in the 
 solutions mentioned is not to be considered trustworthy. In less 
 alkaline solutions no effect upon the hydroxyl-concentration of 
 the caseinate solutions, due to hydrolysis, could be discovered; 
 had such an effect been present to any appreciable extent it would, 
 of course, have been detected in these solutions much more readily 
 than in those in which such an effect was detected, since the poten- 
 tials of the chains containing these solutions were higher, so that 
 a smaller change in hydroxyl-concentration of the solution would 
 have produced a greater (absolute) displacement of the neutral 
 point upon the bridge-wire. 
 
 In concluding these remarks, it may be stated that unless all of 
 the precautions which have been described are observed with the 
 utmost fidelity the results of experiments such as these, and con- 
 ducted in this manner, will be found to be wholly irregular, and, 
 save in a qualitative sense, untrustworthy. 
 
INDEX OF AUTHORS 
 
 The numbers in heavy type refer to the bibliographies. 
 
 ABDERHALDEN, E., 31, 32, 161, 387, 
 388, 15, 16, 154, 343, 371, 372, 373, 
 374, 375, 376, 377, 378, 379, 381, 
 382, 384, 385. 
 
 ABEGG, R., 217, 195. 
 
 ACHALME, P., 453, 449. 
 
 ACRES, S. F., 420, 395, 456. 
 
 ALEXANDER, A. C., 367, 357, 358. 
 
 ALEXANDROV, N., 353, 331. 
 
 ALLEMANN, O., 105, 161, 91, 148. 
 
 ALSBERG, C. L., 350, 339. 
 
 ALTMANN, R., 161, 143. 
 
 AMMANN, L., 357. 
 
 ANDERSON, H. C., 420, 398. 
 
 ANDERSON, J. A., 135, 126. 
 
 ANNENKOV, A., 33, 6. 
 
 ANSIAUX, G., 317, 308. 
 
 ARMSTRONG, E. F., 421, 453, 393, 425. 
 
 ARNY, H. V., 65, 105, 56, 97. 
 
 ARRHENIUS, S., 161, 271, 350, 420, 
 156, 189, 266, 306, 320, 341, 342, 
 402, 403, 404, 419, 420. 
 
 BABO, P., 134, 126. 
 
 BANG, I., 420, 418. 
 
 BARCROFT, J., 161, 350, 160, 161, 337. 
 
 BARTH, L., 31, 4. 
 
 BASSET, H., 134, 127. 
 
 BAYLISS, W. M., 83, 350, 388, 420, 
 
 453, 80, 340, 369, 402, 403, 435, 447, 
 
 449, 450. 
 
 BEARN, A. R., 453, 449. 
 BEATTY, W. A., 32, 16. 
 BECHAMP, A., 105, 90. 
 BECHHOLD, H., 161, 350, 143, 322. 
 BEERS, W. H., 84, 82. 
 BELL, J. M., 317, 285. 
 BENEDICENTI, A., 161, 350, 142, 349. 
 BERCZELLER, L., 161, 316, 350, 148, 
 
 310, 345. 
 
 BERG, W. N., 420, 410, 411. 
 BERGELL, P., 387, 388, 15, 371, 374. 
 BERNARD, C., 134, 108. 
 
 BERNINZONE, 420, 453, 393, 425. 
 BERTHELLOT, M. P. E., 350, 330. 
 BERTHOLD, G., 350, 346. 
 BERZELIUS, J., 420, 391. 
 BEUTNER, R., 194, 316, 193, 299. 
 BIDDLE, H. C., 423, 454, 393, 435, 437. 
 BIEN, Z., 218, 210. 
 
 BlERNACKE, E., 83, 80. 
 
 BIGLAND, D., 352, 337. 
 
 BILLITZER, J., 31, 134, 26, 108, 118. 
 
 BILTZ, W., 134, 119. 
 
 BlRCHARD, F. J., 33, 19. 
 
 BJERRUM, N., 217, 197, 458. 
 
 BLACK, O. F., 218, 214. 
 
 BLASEL, L., 31, 54, 194, 217, 23, 51, 
 
 168, 204. 
 
 BLISH, M. J., 32, 17. 
 BLUM, F., 161, 156, 157, 158. 
 BODENSTEIN, M., 420, 453, 393, 425. 
 BOEHM, R., 423, 415. 
 BOGDANDY, S., 420, 405. 
 BOGUSKY, J. G., 316, 277. 
 BOHR, C., 162, 159, 160. . 
 BONAMARTINI, G., 134, 162, 133, 140. 
 BORISSOV, P., 420, 403. 
 BORKEL, C., 367, 356, 358. 
 BOSWORTH, A. W., 54, 66, 105, 106, 
 
 251, 420, 424, 37, 42, 56, 89, 90, 91, 
 
 92, 93, 98, 101, 235, 249, 418, 419. 
 BOTTAZZI, F., 317, 350, 351, 299, 320, 
 
 322, 345. 
 
 BOTTGER, W., 83, 78. 
 BOVIE, W. T., 317, 310, 456. 
 BOWERS, W. G., 83, 78. 
 BRADLEY, H. C., 317, 420, 453, 316, 
 
 392, 393, 425, 427, 452, 453. 
 BRAEUNNING, H., 420, 415. 
 BRAHM, C., 387, 385. 
 BREDIG, G., 31, 83, 134, 194, 250, 388, 
 
 26, 67, 108, 118, 184, 233, 235, 369. 
 BRIDGMAN, P. W., 317, 310. 
 BRIGGS, R. S., 65, 65. 
 BRINK, F. N., 351, 329. 
 
 469 
 
470 
 
 INDEX OF AUTHORS 
 
 BRITO, P. S., 194, 177, 190. 
 BBOECK, C. T., 31, 30. 
 BROOK, F. W., 162, 157, 158. 
 BROWN, A. P., 55, 318, 319, 44, 311, 
 
 313, 314. 
 
 BROWNING, C. H., 162, 155. 
 BRUNER, L., 317, 276. 
 BRUNNER, E., 317, 277. 
 BRUYN, LOBRY DE, M. C. A., 351, 
 
 342. 
 
 BUBNOW, N. A., 420, 415. 
 BUCHNER, E., 375. 
 BUCK, L. W., 65, 65. 
 BUGARSZKY, S., 32, 83, 134, 194, 217, 
 
 351, 26, 75, 76, 77, 78, 134, 167, 170, 
 
 195, 332. 
 
 BUGLIA, G., 317, 351, 304, 345. 
 BULOW, K, 367, 358. 
 BUNGE, G. VON, 217, 213. 
 BURIAN, R., 105, 86. 
 BURNETT, T. C., 33, 251, 271, 353, 
 
 28, 237, 238, 253, 333, 339. 
 BURTON, D., 319, 293, 298. 
 BUTTERFIELD, E. E., 162, 317, 367, 
 
 161, 313, 358. 
 BUTTNER, C., 422, 389. 
 
 CAEMMERER, G., 387, 385. 
 CAMERON, A. T., 32, 163, 22, 158. 
 CAMERON, K. F., 317, 285. 
 CAMIS, M., 161, 160. 
 CAMPBELL, G. F., 54, 163, 368, 44, 
 
 148, 355, 356. 
 
 CATHCART, E. P., 453, 449. 
 CESANA, G., 420, 420. 
 CHAIN, A., 454, 438. 
 CHIARI, R., 54, 317, 51, 293, 298. 
 CHICK, H., 83, 134, 317, 351, 70, 108, 
 
 123, 129, 305, 307, 308, 309, 328, 
 
 349. 
 CHITTENDEN, R. H., 83, 135, 368, 420, 
 
 454, 80, 109, 355, 357, 415, 438. / 
 CHRISTIANSEN, J., 351, 328. / 
 CLAPP, S. H., 33, 16. 
 CLARK, F. W., 456. 
 CLARK, W. M., 84, 81, 456,^63. 
 CLEMENT, M., 420, 392. 
 COGGI, 454, 449. 
 COHEN, E., 271, 262. 
 COHNHEIM, O., 83, 105, 162, 71, 79, 
 
 80, 88, 138. 
 
 CONROY, J. T., 420, 395. 
 COOKE, E., 317, 299. 
 
 COPPADORE, A., 420, 397. 
 CORIN, J., 317, 308. 
 CORRAL, J. M. DE, 217, 211. 
 COTTRELL, F. G., 294, 457. 
 COURANT, G., 105, 89, 90. 
 CRAMER, M., 420, 453, 393, 449. 
 CUMMINGS, A. C., 217, 197. 
 CUMMINS, A. W., 420, 415. 
 CURTIUS, T., 32, 9. 
 
 DABROVSKY, S., 351, 330, 331, 338. 
 D'AGOSTINO, E., 83, 217, 351, 78, 204, 
 
 322 
 
 DAKIN, H. D., 32, 194, 30, 31, 190. 
 DALE, T. P., 367, 362. 
 DAM, W. VAN, 194, 424, 169, 418. 
 DANEEL, H., 351, 326. 
 D'ARSONVAL, A., 367, 358. 
 DASTRE, A., 453, 449. 
 DAUWE, F., 420, 399. 
 DEMPWOLFF, C., 351, 326. 
 DERHAM, H. G., 134, 127. 
 DBS BANCELS, L., 421, 397, 402, 403. 
 DESORMES, M., 420, 392. 
 DIETZ, W., 420, 453, 393, 425, 451. 
 DONATH, H., 453, 449. 
 DONNAN, F. G., 134, 127, 128. 
 DRECHSEL, E., 32, 162, 5, 158. 
 DUCLAUX, J., 351, 340. 
 DUDLEY, H. W., 32, 30. 
 DUHEM, P., 351, 341. 
 DUMANSKI, A., 351, 323, 325. 
 
 EDKINS, J. S., 83, 80. 
 
 EHRENBERG, R., 317, 293, 297. 
 
 EHRLICH, P., 449. 
 
 EINSTEIN, A., 331. 
 
 ELY, J. S., 83, 80. 
 
 EMMERLING, O., 421, 453, 393, 425. 
 
 ERB, W., 32, 83, 217, 21, 72, 78, 206. 
 
 ERNST, C., 421, 392. 
 
 EULER, H., 388, 421, 453, 369, 383, 
 
 384, 392, 393, 396, 444, 445, 449. 
 EVES, F., 83, 80. 
 ) 
 
 FALEK, O., 163, 148. 
 FALK, F., 83, 80. 
 FARADAY, M., 351, 349. 
 FARKAS, G., 217, 218, 211. 
 FARMER, J. B., 317, 307. 
 FEINSCHMIDT, J., 162, 148. 
 FELS, B., 83, 81, 212. 
 FERNAN, A., 317, 310. 
 
INDEX OF AUTHORS 
 
 471 
 
 FERNET, E., 218, 213. 
 
 FICK, A., 361, 330. 
 
 FIELD, C. W., 66, 58, 60. 
 
 FINDLAY, A., 134, 123. 
 
 FINGER, C. P., 78, 457. 
 
 FISCHER, E., 32, 388, 421, 5, 10, 11, 
 
 12, 13, 15, 18, 23, 371, 372, 373, 
 
 374, 393. 
 
 FISCHER, M. H., 317, 299. 
 FISCHER, W., 453, 444. 
 FOA, C., 218, 212. 
 FOURNEAU, E., 32, 10, 11. 
 FRAENKEL, P., 218, 211. 
 FRAMM, F., 367, 358. 
 FRANKEL, E. M., 421, 398. 
 FRASER, J. W., 421, 415. 
 FREDERICQUE, L., 317, 367, 305, 355, 
 
 356. 
 
 FREI, W., 361, 345. 
 FREUND, E., 54, 40. 
 FREUNDLICH, H., 134, 108, 117, 118, 
 
 291. 
 FRIEDENTHAL, H., 83, 218, 81, 212, 
 
 214. 
 FULD, E., 162, 421, 143, 416, 417, 419, 
 
 420. 
 
 FUNK, C., 421, 418. 
 FURTH, O. VON, 162, 158. 
 
 GALEOTTI, G., 134, 162, 108, 121, 124, 
 
 125, 138, 139, 275. 
 GAMGEE, A., 351, 367, 349, 356, 358. 
 GANSSER, E., 162, 160. 
 GARRETT, H., 351, 327, 328. 
 GAVRILOV, N., 32, 19. 
 GAY, F. P., 162, 421, 453, 149, 154, 
 
 155, 393, 436, 437. 
 GEAKE, A., 421, 418. 
 GERBER, C., 421, 417. 
 GEVIN, J. W. A.,. 421, 418. 
 GIBBS, J. W., 134, 123, 345. 
 GIBSON, R. B., 317, 310. 
 GIES, W. J., 164, 420, 146, 410, 411. 
 GIGON, A., 387, 376, 384, 385. 
 GILLESPIE, A. L., 421, 411. 
 GJALDBAEK, J. K., 421, 463, 398, 437. 
 GLADSTONE, J. H., 367, 362, 363, 366, 
 
 367. 
 
 GOKUM, F., 351, 327. 
 GOMBERG, M., 194, 191. 
 GORTNER, R. A., 32, 17. 
 GOTO, M., 106, 86, 87. 
 GRAHAM-STEFAN, 330. 
 
 GRAHAM, T., 134, 162, 351, 112, 142, 
 
 143, 322, 330, 337, 342. 
 GRAMENISKY, M. Y., 463, 450. 
 GRAVES, S. S., 66, 58. 
 GREAVES, J. E., 368, 48, 359, 361, 
 
 365, 366. 
 
 GREEN, W. H., 351, 323. 
 GREENWOOD, M., 421, 411. 
 GRIMEAUX, E., 32, 9. 
 GRINEFF, W., 218, 210. 
 GROSS, O., 421, 403, 405. 
 GRUEBLER, G., 54, 48. 
 GUTHE, K. E., 194, 178. 
 
 HAEBLER, M., 361, 348. 
 
 HAHN, M., 453, 454, 432, 449. 
 
 HALLIBURTON, W. D., 317, 305, 314, 
 315. 
 
 HAMBURGER, H. J., 83, 218, 76, 195. 
 
 HAMMARSTEN, O., 54, 105, 361, 421, 
 37, 40, 41, 93, 343, 400, 418. 
 
 HANDOVSKY, H., 135, 317, 122, 131, 
 305. 
 
 HANRIOT, M., 421, 453, 393, 425. 
 
 HARDNACK, E., 162, 158. 
 
 HARDY, W. B., 64, 83, 105, 134, 162, 
 194, 218, 250, 317, 351, 364, 40, 74, 
 75, 76, 80, 98, 99, 100, 107, 108, 113, 
 114, 117, 124, 131, 134, 150, 152, 
 153, 171, 176, 187, 188, 189, 190, 
 205, 226, 227, 230, 250, 275, 300, 
 301, 302, 303, 304, 320, 322, 323, 
 328, 329, 333, 464. 
 
 HARI, P., 317, 453, 308, 451. 
 
 HARRIS, F. I., 367, 359. 
 
 HARRIS, I. F., 55, 48, 357. 
 
 HARRIS, V. D., 421, 415. 
 
 HART, E. B., 65, 84, 106, 219, 424, 40, 
 69, 89, 91, 93, 95, 97, 204, 207, 418. 
 
 HARTLEY, W. N., 134, 135, 126, 127. 
 
 HASSELBALCH, K. A., 218, 211. 
 
 HAYCRAFT, J. B., 351, 329. 
 
 HAYWOOD, J. K., 105, 95. 
 
 HEDBLOM, C. A., 360, 339. 
 
 HEDIN, S. G., 32, 162, 421, 453, 5, 
 156, 399, 404, 418, 449. 
 
 HEIDENHAIN, M., 83, 162, 82, 146. 
 
 HEIDENHAIN, R., 218, 421, 213, 415. 
 
 HEKMA, E., 105, 101. 
 
 HEMMETER, J. C., 421, 418. 
 
 HENDERSON, L. J., 218, 317, 361, 
 453, 214, 215, 216, 308, 329, 451. 
 
 HENDRIX, B. M., 33, 31. 
 
472 
 
 INDEX OF AUTHORS 
 
 HENRI, V., 388, 421, 380, 382, 384, 
 
 397, 402, 403, 406, 407, 408. 
 HENEIQUES, V., 421, 453, 398, 437. 
 HENSEL, 454, 449. 
 HENZE, M., 162, 142, 157, 158. 
 HEBLITZKA, A., 367, 359, 366, 367. 
 HERMANN, L., 351, 346. 
 HERRMANN, J., 454, 438. 
 HERTZ, A. F., 352, 347. 
 HERZOG, R. O., 351, 352, 421, 330, 418. 
 HEUBNER, W., 317, 367, 313, 358. 
 HEYDWEILLER, A., 218, 204, 215. 
 HILDEBRAND, J. H., 83, 78, 456. 
 HILL, A. V., 161, 350, 160, 337. 
 HILL, CROFT, 421, 454, 356, 393, 425, 
 
 427. 
 
 HIRSCHFELD, M., 33, 219, 23, 203. 
 HIRSCHSTEIN, L., 164, 194, 140, 170. 
 HOAGLAND, D. R., 456. 
 HOBER, R., 135, 218, 120, 211. 
 HOFF, VAN'T, 123, 160, 306, 308, 420. 
 HOFMEISTER, F., 32, 135, 162, 317, 18, 
 
 21, 22, 23, 108, 109, 110, 111, 125, 
 
 157, 292, 299, 309, 311, 339. 
 HOLBORN, L., 194, 174. 
 HOLZBERG, H. L., 162, 146. 
 HOPKINS, F. G., 54, 162, 318, 367, 44, 
 
 157, 158, 311, 355. 
 HOPPE-SEYLER, F., 54, 162, 218, 367, 
 
 45, 148, 214, 355, 356, 358. 
 HOWELL, W. H., 194, 318, 421, 422, 
 
 454, 177, 190, 316, 416, 419, 450. 
 HOYT, H. S., 367, 359. 
 HUFNER, G., 32, 162, 318, 352, 367, 
 
 4, 159, 160, 313, 322. 
 HULETT, G. A., 352, 347, 463. 
 
 HUNDESCHAGEN, F., 162, 158. 
 
 HUNTER, A., 162, 387, 148, 149. 
 HUPPERT, H., 423, 403, 404. 
 HURWITZ, S. H., 65, 65. 
 
 ISCOVESCO, H., 352, 345. 
 IZAR, G., 162, 148. 
 
 JACOBY, M., 422, 418. 
 JAGER, L. DE, 105, 89. 
 JAMISON, R., 352, 347. 
 JAQUET, A., 162, 160. 
 JESSEN-HANSEN, H., 33, 19, 22. 
 JEWETT, R. M., 65, 65. 
 JOACHIM, J., 54, 40. 
 JONES, H. C., 135, 271, 352, 126, 127, 
 130, 266, 323. 
 
 JONES, W., 367, 356. 
 JURGENSEN, E., 319, 309. 
 
 KAJANDER, N., 317, 277. 
 KANITZ, A., 422, 411. 
 KASERNOVSKY, H., 351, 352, 330. 
 KASTLE, J. H., 422, 454, 393, 425. 
 KATZ, J. R., 318, 292, 311, 315, 
 
 316. 
 
 KAUDER, G., 135, 109. 
 KAUFFMANN, R., 422, 415. 
 KELLERSBERGER, E., 453, 452. 
 KENDALL, E. C., 163, 158. 
 KENNAWAY, E. L., 163, 158. 
 KING, W. O. R., 161, 160. 
 KIRSCHOFF, J., 422, 392. 
 KJELDAHL, J., 357. 
 KLUG, F., 422, 410. 
 KOBER, P. A., 32, 65, 163, 352, 367, 
 
 22, 30, 31, 58, 59, 60, 142, 345, 
 
 359. 
 KOELKER, A. H., 31, 387, 388, 15, 376, 
 
 377, 382, 385. 
 KOENIGS, E., 32, 23. 
 KOHLRAUSCH, F., 194, 218, 174, 204, 
 
 215. 
 
 KONOVALOV, D., 352, 342. 
 KOPP, H., 352, 343. 
 KOROSY, K., 318, 299. 
 KOSSEL, A., 32, 54, 105, 163, 250, 
 
 368, 5, 8, 19, 21, 22, 25, 30, 53, 54, 
 
 86, 87, 143, 148, 158, 235, 241, 242, 
 
 363. 
 
 KOSUTANY, T., 54, 49. 
 KOUKOL-YASNOPOLSKY, W., 422, 389. 
 KRAFFT, F., 352, 332. 
 KRAUSE, E., 164, 156. 
 KRIEGER, H. T., 83, 318, 71, 311. 
 KRONIG, B., 83, 82. 
 KRUGER, T. H., 368, 356. 
 KRUKENBERG, 422, 389. 
 KUHNE, W., 54, 135, 368, 454, 41, 109, 
 
 355, 374, 438. 
 KULIKOV, J., 33, 6. 
 KURAJEV, D., 105, 454, 87, 438. 
 KUTSCHER, F., 32, 163, 5, 148. 
 
 LACQUBUR, E., 54, 105, 218, 422, 40, 
 
 88, 89, 204, 207, 418. 
 LA FRANCA, S., 83, 82. 
 LANDOLT, H., 368, 362. 
 LANDSTEINER, K., 454, 449. 
 LANGLEY, J. N., 83, 80. 
 
INDEX OF AUTHORS 
 
 473 
 
 LANGSTEIN, L., 161, 154. 
 LARMOR, J., 362, 341. 
 LAVROV, D., 454, 438. 
 LEAVBNWORTH, C. S., 33, 83, 105, 163, 
 
 19, 24, 71, 103, 142. 
 LE CHATELIER, H. L., 135, 126. 
 LEHMANN, J., 352, 346. 
 LEICK, A., 352, 329. 
 LENGYEL, R. VON, 318, 454, 308, 451. 
 LENK, E., 318, 293. 
 LESCOEUR, H., 105, 95. 
 LEVENE, P. A., 32, 16. 
 LEVITES, S., 32, 352, 19, 20, 320, 
 
 327. 
 
 LEWIN, M., 135, 126. 
 LEWIS, G. N., 135, 352, 126, 127, 326, 
 
 456. 
 
 LEWITH, I., 135, 109. 
 LEY, H., 218, 206. 
 LICHTWITZ, L., 352, 328. 
 LIEBERMANN, L., 32, 83, 134, 163, 194, 
 
 217, 351, 26, 75, 76, 77, 78, 134, 
 
 143, 167, 170, 195, 332. 
 LIEBIG, J., 32, 4, 391. 
 LIEBRECHT, A., 163, 157. 
 LIEBREICH, O., 352, 347. 
 LIESEGANG, R. E., 318, 304. 
 LILLIE, R. S., 352, 337, 338, 339. 
 
 LlLIENFELD, L., 163, 146. 
 LlNDBERGER, V., 422, 415. 
 
 LINDER, S. E., 135, 112, 113, 114, 115, 
 
 116, 117. 
 LINDET, L., 357. 
 
 LlNEBARGER, C. S., 352, 342. 
 
 LIPPICH, F., 163, 141. 
 
 LODGE, O., 352, 323. 
 
 LOEB, J., 83, 135, 194, 218, 318, 422, 
 
 82, 118, 193, 212, 214, 299, 313, 
 
 410, 411. 
 
 LOEB, L., 422, 416. 
 LOEVENHART, A. S., 105, 194, 422, 
 
 454, 91, 97, 169, 393, 425. 
 LOEW, O., 163, 194, 158, 193. 
 LOHNSTEIN, T., 352, 348. 
 LOMBARDI, M., 134, 162. 
 LORENTZ, H. A., 368, 367. 
 LORENTZ, L., 368, 367. 
 LUBAVIN, N., 422, 464, 389, 435. 
 LUBRZYNSKI, E., 361, 328. 
 LUBS, H. A., 84, 81. 
 LtJDEKiNG, C., 319, 362, 292, 294, 
 
 308, 332, 348. 
 LUNDEN, H., 83, 67. 
 
 LUNDSGAARD, C., 218, 211. 
 
 LUTHER, R., 271, 266. 
 
 MAAS, O., 368, 356. 
 MADSEN, T., 83, 422, 82, 417, 419. 
 MALFATTI, H., 163, 143. 
 MANABE, K., 194, 218, 168, 204. 
 MANN, G., 32, 135, 163, 318, 21, 109, 
 
 130, 147, 307. 
 MARCUS, E., 54, 40. 
 MARSHALL, J. A., 218, 217. 
 MARTIN, C. J., 83, 134, 317, 352, 422, 
 
 70, 123, 129, 305, 307, 309, 340, 
 
 349, 419. 
 
 MASSON, O., 362, 323. 
 MATHEWS, A. P., 83, 163, 82, 146, 
 
 147. 
 
 MATHEWSON, W. E., 357, 366. 
 MATULA, J., 31, 54, 194, 217, 218, 23, 
 
 51, 168, 204. 
 
 MAXIMOVICH, S., 163, 368, 152, 355. 
 MAYER, K., 422, 403. 
 MAYER, L., 218, 213. 
 MEDIGRECEANU, F., 387, 385. 
 MEISSNER, C., 422, 389. 
 MELLANBY, J., 83, 106, 135, 75, 100, 
 
 112. 
 
 MELLOR, J. W., 422, 397. 
 MENDEL, L. B., 163, 158, 357. 
 MEYER, K. F., 65, 65. 
 MEYERS, C. N., 456. 
 MICHAELIS, L., 83, 105, 164, 218, 387, 
 
 388, 76, 91, 142, 148, 195, 210, 211, 
 
 376, 377, 378, 379, 381, 456. 
 MICHAILOV, W., 318, 307. 
 MIESCHER, F., 105, 163, 86, 87, 143, 
 
 146. 
 
 MIGAY, T. J., 422, 418. 
 MILROY, T. H., 464, 431. 
 MITTELBACH, F., 368, 356. 
 MIYAKE, K., 271, 319, 271, 283, 287. 
 MODELSKI, J. VON, 33, 20. 
 MOLL, L., 318, 310. 
 MOORE, A. R., 318, 299. 
 MOORE, B. S., 135, 194, 362, 127, 169, 
 
 337, 339. 
 
 MORACZEWSKI, W., 464, 430. 
 MORAWTTZ, P., 422, 416, 419. 
 MORGENROTH, J., 422, 418. 
 MORNER, C. T., 64, 368, 49, 355, 356. 
 MORNER, K. A. H., 163, 318, 158, 311. 
 MOSTYUSKY, H., 218, 210. 
 MROCZKOVSKY, I., 219, 213. 
 
474 
 
 INDEX OF AUTHORS 
 
 MULLER, F., 64, 368, 50, 356. 
 MYLIUS, F., 163, 143. 
 
 NAGELI, O., 194, 193. 
 
 NASSE, O., 136, 110. 
 
 NENCKI, M., 422, 418. 
 
 NERNST, W., 163, 219, 318, 352, 368, 
 
 160, 195, 196, 277, 330, 340, 344, 
 
 347, 464. 
 
 NEUMANN, J., 163, 142, 430. 
 NEUMEISTER, R., 422, 389. 
 NIEMANN, A., 421, 418. 
 NOORDEN, C. VON, 368, 358. 
 NOTES, A. A., 318, 276. 
 NURENBERG, A., 163, 157. 
 NUTTALL, G. H. F., 318, 314. 
 NYMAN, M., 83, 82. 
 
 OBER, J. E., 136, 117, 121. 
 OBERMEYER, F., 33, 19. 
 OKER-BLOM, M., 352, 338. 
 OKUNOV, W. H., 454, 438. 
 OPPENHEIMER, C., 163, 388, 422, 454 
 
 156, 369, 434. 
 ORYNG, T., 194, 169. 
 OSBORNE, T. B., 33, 64, 55, 83, 105, 
 163, 368, 455, 16, 19, 21, 24, 26, 44, 
 46, 48, 49, 71, 101, 102, 103, 133, 
 142, 148, 355, 356, 357, 436. 
 OSBORNE, W. A., 83, 105, 163, 194, 
 250, 271, 318, 27, 71, 88, 90, 148 
 169, 193, 234, 253, 282, 284, 464. 
 OSTWALD, WILH., 136, 271, 352, 422, 
 
 79, 266, 343, 393. 
 
 OSTWALD, Wo., 163, 318, 352, 422, 
 160, 285, 291, 293, 298, 299, 326 
 327, 330, 339, 416. 
 OSWALD, A., 163, 157, 158. 
 OTA, K., 135, 125. 
 OTTO, E., 32, 11. 
 
 PAAL, C., 318, 296. 
 
 PALME, H., 318, 283. 
 
 PARASTSCHUK, S. W., 422, 418. 
 
 PARKER, W. H., 352, 337. 
 
 PATTEN, A. J., 32, 21. 
 
 PAUL, T., 83, 82. 
 
 PAUL, W., 317, 310. 
 
 PAULI, W., 33, 135, 163, 194, 219, 318, 
 368, 23, 107, 108, 111, 112, 119, 
 120, 121, 122, 130, 131, 133/134, 
 148, 169, 189, 191, 203, 211, 275, 
 292, 293, 294, 300, 301, 305, 307, 
 309, 310, 339, 358. 
 
 PAVLOV, I. P., 422, 371, 375, 418. 
 PECHSTEIN, H., 218, 210. 
 PELET-JOLIVET, L., 147. 
 PEMSEL, W., 84, 219, 70, 214. 
 PFEIFFER, E., 422, 415. 
 PFEIFFER, J. A. F., 66, 58, 60. 
 PFEIFFER, P., 33, 20. 
 PICKERING, S. U., 135, 163, 126, 153. 
 PICTON, H., 135, 112, 113, 114, 115, 
 
 116, 117. 
 
 PINCUSSOHN, L., 387, 385. 
 PINKUS, S. N., 54, 162, 318, 44, 158, 
 
 311. 
 
 PLANCK, M., 362, 330. 
 PLATNER, A. E., 83, 67. 
 PLIMMER, R. H. A., 33, 55, 163, 368, 
 
 388, 6, 29, 44, 148, 358, 375. 
 PLUCKER, P., 352, 349. 
 PODOLINSKI, H., 422, 415. 
 POHL, J., 135, 163, 109, 143. 
 POLLACK, L., 454, 449. 
 
 POSTERNAK, S., 135, 120. 
 
 POTILITZIN, A., 135, 126. 
 POTTEVIN, H., 423, 454, 393, 425. 
 POYNTING, J. H., 136, 126. 
 PRATT, T. M., 65, 105, 56, 97. 
 PREYER, W. T., 55, 318, 43, 44, 311. 
 PRICE, T. M., 423, 415. 
 PROCTER, H. R., 164, 318, 319, 146 
 
 293, 295, 296, 297, 298. 
 PROST, E., 136, 112, 113, 116. 
 PROUST, M., 33, 4. 
 PUGLIESE, 454, 449. 
 
 QUAGLIARIELLO, G., 83, 217, 319, 78, 
 
 204, 309. 
 
 QUINAN, C., 65, 40. 
 QUINCKE, H., 319, 352, 292, 345, 347. 
 
 RABELLO-ALVES, S., 161, 350, 142, 
 
 349. 
 
 RAKOCZY, A., 423, 454, 418, 438. 
 RAMSDEN, W., 352, 353. 
 RANKINE, A. O., 363, 329. 
 RAUDNITZ, R. W., 423, 416. 
 REFORMATZKY, S., 353, 323. 
 REGECZY, E. N., 363, 338. 
 REICHEL, H., 423, 417. 
 REICHERT, E. T., 56, 319, 44, 311, 
 
 313, 314. 
 
 REID, W., 164, 353, 160, 332, 337. 
 REIGER, R., 353, 329. 
 REISS, E., 66, 368, 60, 359, 360, 365. 
 
INDEX OF AUTHORS 
 
 475 
 
 RENNER, A., 362, 328. 
 
 RHORER, L. VON, 83, 219, 74, 206. 
 
 RICHARDS, T. W., 194, 193, 308. 
 
 RIGHETTI, H., 66, 65. 
 
 RINGER, S., 423, 418. 
 
 RINGER, W. E., 194, 219, 168, 204. 
 
 RlTTHAUSEN, H., 55, 48. 
 
 ROAF, H. E., 194, 352, 353, 169, 337, 
 339. 
 
 ROBERTS, F., 161, 160. 
 
 ROBERTSON, T. B., 33, 55, 66, 83, 105, 
 106, 136, 162, 164, 194, 219, 250, 
 271, 319, 353, 368, 388, 421, 423, 
 453, 454, 20, 26, 27, 28, 36, 39, 44, 
 46, 49, 51, 56, 60, 65, 67, 69, 70, 73, 
 74, 82, 88, 89, 90, 92, 93, 96, 99, 
 100, 103, 127, 146, 147, 149, 152, 
 154, 155, 160, 170, 171, 177, 178, 
 179, 180, 183, 193, 195, 197, 208, 
 209, 210, 214, 215, 216, 221, 222, 
 223, 224, 225, 228, 234, 237, 238, 
 242, 245, 249, 253, 257, 271, 278, 
 280, 283, 284, 286, 287, 303, 307, 
 333, 336, 339, 340, 341, 346, 347, 
 359, 360, 361, 362, 365, 373, 390, 
 393, 404, 411, 412, 415, 425, 427, 
 428, 432, 434, 436, 437, 442, 445, 
 448, 456, 464, 466. 
 
 RODGER, J. W., 271, 266. 
 
 ROGOZINSKI, F., 164, 423, 156, 398. 
 
 ROHLOFF, C., 353, 329. 
 
 ROHMANN, F., 164, 194, 140, 170. 
 
 ROHONYI, H., 33, 194, 219, 319, 454, 
 26, 168, 204, 304, 435. 
 
 RONA, P., 105, 135, 164, 318, 387, 91, 
 142, 148, 293, 300, 301. 
 
 ROOSE, E., 164, 158. 
 
 ROOZEBOOM, B., 123, 124. 
 
 ROSE, F., 136, 122. 
 
 ROSENBERG, H., 317, 367, 313, 358. 
 
 ROSENBLOOM, J., 164, 146. 
 
 ROSENTHALER, L., 83, 80. 
 
 ROTH, W. A., 271, 262. 
 ROTHMUND, V., 136, 130. 
 ROWE, A. H., 66, 65. 
 RUSSELL, W. S., 136, 126. 
 RYDER, C. T., 317, 453, 308, 451. 
 
 SABANEJEV, A., 353, 331. 
 
 SACHS, H., 454, 449. 
 
 SACKUR, O., 54, 105, 194, 218, 251, 
 
 353, 40, 88, 89, 204, 207, 223, 320, 
 
 321, 322, 323, 324. 
 
 SAINSBURY, H., 423, 418. 
 
 SALASKIN, S., 454. 
 
 SALKOWSKI, E., 106, 423, 454, 90, 
 389, 431, 432. 
 
 SALM, E., 33, 84, 106, 219, 21, 81, 91, 
 99, 103, 201, 212. 
 
 SAMEC, M., 135, 194, 368, 130, 189, 
 191, 358. 
 
 SAMOJLOFF, A., 423, 403. 
 
 SAMUELY, F., 106, 387, 87. 
 
 SANSUM, W. D., 317, 316. 
 
 SAUNDERS, E. R., 421, 411. 
 
 SAVITSCH, W. W., 422, 423, 418. 
 
 SAVJALOV, W., 423, 454, 418, 438. 
 
 SCALINCI, N., 317, 299. 
 
 SCHAFER, E. A., 136, 112. 
 
 SCHAFER, F., 423, 415. 
 
 SCHEERMESSER, W., 368, 356. 
 
 SCHIERBECK, N. P., 423, 415. 
 
 SCHIFF, H., 33, 9, 18. 
 
 SCHITTENHELM, A., 161, 387, 154. 
 
 SCHLOSSMANN, A., 106, 97. 
 
 SCHMIDLIN, J., 32, 23. 
 
 SCHMIDT, A., 56, 41. 
 
 SCHMIDT, C., 423, 410. 
 
 SCHMIDT, C. L. A., 33, 66, 84, 164, 
 219, 319, 353, 368, 423, 454, 30, 65, 
 78, 79, 149, 150, 154, 155, 156, 195, 
 204, 310, 341, 359, 360, 361, 365, 
 411, 412, 442, 456, 457, 463, 466. 
 
 SCHMIDT, E. S., 66, 319, 65, 310. 
 
 SCHMIDT-NIELSEN, S and S., 423, 418. 
 
 SCHORER, G., 66, 368, 65, 362. 
 
 SCHORR, C., 363, 327. 
 
 SCHREINEMAKERS, F. A. H., 136, 124. 
 
 SCHROEDER, P. VON, 319, 353, 299, 
 
 323, 326, 327. 
 SCHULTZE, H., 136, 112, 114, 115, 
 
 116, 132. 
 SCHULTZE, F. N., 55, 162, 319, 353, 
 
 51, 311, 316, 350. 
 SCHUMM, O., 319, 368, 313, 358. 
 
 SCHUMOV-SlMANOVSKY, E. O., 423, 
 
 418. 
 
 SCHUTZ, E., 423, 402, 403, 404, 409, 
 419, 442. 
 
 SCHUTZENBERGER, P., 33, 9. 
 
 SCHWARTZ, O., 464, 434, 449. 
 SCIPIADES, E., 218, 211. 
 SCOTT, L., 164, 157. 
 SEBELJEN, J., 368, 355. 
 SEEMAN, T., 65, 50. 
 SERTOLI, E., 219, 213, 214. 
 
476 
 
 INDEX OF AUTHORS 
 
 SHARP, L. T., 456. 
 
 SHINJO, S., 353, 329. 
 
 SHORTER, S. A., 353, 345, 346, 348. 
 
 SIEBER, N., 422, 418. 
 
 SIEGFRIED, M., 164, 368, 144, 145, 
 
 356, 358. 
 SJOQUIST, J., 33, 84, 194, 423, 26, 74, 
 
 170, 403. 
 
 SKRAUP, H., 164, 156. 
 SLAVU, 161, 154. 
 SOLDNER, F., 56, 106, 219, 40, 88, 89, 
 
 90, 204, 207. 
 SORENSEN, S. P. L., 33, 84, 219, 319, 
 
 19, 22, 81, 195, 309, 456. 
 SORET, J. L., 368, 358, 359. 
 SPIRO, K., 84, 136, 219, 423, 70, 108, 
 
 123, 214, 417. 
 SPRIGGS, E. I., 353, 328. 
 STARKE, J., 319, 307. 
 STARKE, K. V., 368, 355. 
 STARLING, E. H., 83, 353, 80, 337. 
 STESSANO, A., 453, 449. 
 STEUDEL, H., 55, 423, 50, 389. 
 STIEGLITZ, J., 388, 423, 384, 393, 395, 
 
 396, 411, 427. 
 STILES, P. G., 84, 82. 
 STIRLING, W., 194, 177, 190. 
 STOKES, G. C., 368, 358. 
 STRAUSS, E., 164, 194, 368, 158, 189, 
 
 191, 358. 
 
 STRECKER, A., 84, 67. 
 STRONG, W. W., 135, 126. 
 SUGIURA, K., 32, 163, 22, 142. 
 SUIDA, W., 164, 148. 
 SUTHERLAND, W., 251, 319, 353, 230, 
 
 307, 309, 326, 327. 
 SZILY, A. VON, 219, 211, 212. 
 
 TAMMAN, G., 353, 331. 
 
 TANGL, F., 319, 351, 455, 308, 332, 
 451. 
 
 TAYLOR, A. E., 65, 106, 251, 363, 388, 
 423, 424, 465, 40, 54, 87, 241, 341, 
 369, 376, 380, 389, 390, 393, 398, 
 403, 404, 410, 411, 412, 414, 418, 
 425, 427, 428, 439, 447, 448. 
 
 THOMPSON, W. B., 66, 65. 
 
 THOMSON, J. J., 319, 363, 290, 346, 
 347. 
 
 THORPE, T. E., 271, 266. 
 
 TIMPE, H., 106, 89. 
 
 TITOFF, A., 424, 392. 
 
 TJIMSTRA, S. B., 363, 326, 
 
 TOLLOCZKO, S., 317, 276. 
 TOLMAN, R., 136, 125. 
 TOOTH, H. H., 421, 415. 
 TRANTER, C. L., 66, 65. 
 TRUNKEL, H., 106, 93. 
 TURBUBA, D., 424, 392. 
 
 UGGLAS, B. AF, 164, 149, 150. 
 UHL, R., 164, 159. 
 UHLER, H. S., 135, 125, 126. 
 UMBACH, T., 162, 156. 
 UMBER, F., 455, 438. 
 UNDERBILL, F. P., 33, 31. 
 
 VAN BEMMELEN, J. M., 316, 303. 
 
 VAN SLYKE, D. D., 31, 33, 55, 106, 
 319, 16, 19, 20, 46, 95, 96, 283. 
 
 VAN SLYKE, L. L., 64, 65, 66, 84, 105, 
 106, 219, 261, 319, 424, 37, 40, 46, 56, 
 69, 89, 90, 91, 92, 93, 95, 96, 97, 98, 
 101, 204, 207, 235, 249, 283, 418. 
 
 VAUBEL, W., 162, 157. 
 
 VELEY, V. H., 319, 277. 
 
 VERNON, H. M., 33, 84, 388, 424, 466, 
 23, 80, 369, 399, 414, 449. 
 
 VIRCHOW, R., 136, 108. 
 
 VOIGTLANDER, F., 364, 322, 330. 
 
 VOITINOVICI, A., 161, 154. 
 
 WAGNER, R., 135, 219, 122, 211. 
 
 WALBUM, 419. 
 
 WALDEN, P., 136, 271, 354, 266, 267, 
 
 323. 
 
 WALTER, A. A., 424, 403. 
 WALTERS, E. H., 424, 456, 391, 405, 
 
 443. 
 
 WASHBURN, E. W., 460. 
 WASILIEV, N. P., 424, 415. 
 WEBER, C. O., 354, 327, 342. 
 WEBSTER, A., 194, 169. 
 WEBSTER, H. R., 218, 214. 
 WECHSLER, E., 164, 158. 
 WEINLARD, E., 455, 449. 
 WEISS, F., 32, 106, 163, 424, 8, 22, 86, 
 
 158, 415. 
 
 WELLS, C. E., 66, 65. 
 WELLS, H. G., 465, 436. 
 WHETHAM, W. C. D., 134, 136, 364, 
 
 114, 116, 118,323, 464. 
 WHITNEY, W. R., 136, 318, 117, 121, 
 
 276. 
 
 WICHMANN, A., 319, 311, 315, 316. 
 WIEDEMANN, E., 319, 292, 294, 308. 
 
INDEX OF AUTHORS 
 
 477 
 
 WIGLOW, H., 362, 332. 
 WILHEIM, R., 32, 19. 
 WILSON, G. H., 162, 155. 
 WILSON, J. A., 319, 293. 
 
 WlNKELBLECH, K., 33, 194, 28, 
 
 191. 
 
 WINTER, O. B., 106, 93. 
 WITTICH, VON, 354, 338. 
 WOOD, T. B., 354, 328, 329. 
 WOOLSEY, J. H., 66, 65. 
 WROBLEWSKI, A., 424, 415. 
 WYRONBOFF, M. G., 136, 126. 
 
 ZAHN, F. W., 354, 346. 
 
 ZANETTI, C. U., 65, 50. 
 
 ZELINSKY, N., 33, 5, 6. 
 
 ZEYNEK, R., 319, 311. 
 
 ZIEGLER, J., 360, 322. 
 
 ZINNSER, H., 164, 154. 
 
 ZINOFFSKI, O., 164, 160. 
 
 ZLOBICKI, L., 354, 345. 
 
 ZSIGMONDY, R., 319, 363, 364, 311, 
 
 316, 349, 350. 
 ZUATTRO, G. DI, 162, 148. 
 ZUNTZ, N., 219, 214. 
 
INDEX OF SUBJECTS 
 
 Absorption of light, 358. 
 Absorption-spectrum, 30, 161, 190, 
 
 358. 
 
 Acidosis, 212, 215. 
 Acids, Compounds of proteins with, 
 
 232 
 
 Adsorption, 118, 147, 160. 
 Agglutination, 309. 
 Aggregation, State of, 275, 366. 
 Alcohol, Coagulation by, 252. 
 Alcohol, Influence on conductivity, 
 
 257. 
 Alcohol, Influence on structure of 
 
 jellies, 301. 
 
 Alcohol-soluble Proteins, 48. 
 Alcohol-water mixtures, 254. 
 Alkaloidal reagents, 145. 
 Alkaloids, 145. 
 Amandin, 357. 
 Amino-acid chains, 375. 
 Amino-acid content and properties 
 
 of proteins, 8. 
 Amino-acids, 4, 6, 9, 142. 
 Ammonium Caseinate, 222, 223. 
 Amphoteric Acids, 20, 67. 
 Amphoteric character of the proteins, 
 
 81. 
 
 Amygdalin, 393. 
 Anaphylaxis, 154, 316. 
 Anhydrides, . 10, 29, 130, 268, 309, 
 
 347. 
 
 Antibodies, 154, 156, 436. 
 Antigenic properties, 154, 436. 
 Antipepsin, 444, 449. 
 Antitrypsin, 444, 449. 
 Aromatic amino-acids, protective 
 
 action of, 359. 
 Autohydrolysis, 389, 391, 400, 405, 
 
 444. 
 Avogadro's law, 161, 307, 340. 
 
 Bacteria, 82. 
 Barium caseinate, 224. 
 Barium serum globulinate, 228. 
 Benzoyl derivatives, 156. 
 Bicarbonates, 214. 
 
 Bimolecular reaction formula, 32. 
 Biological applications, 192, 211, 452. 
 Biuret reaction, 14, 18, 142, 431. 
 Blood serum analysis, 60, 331. 
 Boiling point, elevation of, 331. 
 
 Calcium caseinate, 224, 230, 261. 
 Calcium salts in coagulation of milk, 
 
 417. 
 
 Calcium serum globulinate, 227. 
 Cane sugar, 79. 
 
 Capillary forces, 286, 303, 342. 
 Carbamino acids, 144. 
 Carbonic acid, 144. 
 Carbon monoxide, 159. 
 Casern, 22, 37, 88, 104, 127, 140, 171, 
 
 198, 204, 243, 271, 343, 346, 349, 
 
 356, 361, 365, 390, 399, 402, 413, 
 
 416, 429, 460. 
 
 Casemates, 27, 232, 237, 320, 333. 
 Casemates, Coagulation of, 252. 
 Caseinates, Hydrolysis of, 405, 415. 
 Catalysis, 79, 391. 
 Catalysors, 389, 426. 
 Cerebrospinal fluid, 60, 350. 
 Characterization of Proteins, 16. 
 Chemical dynamics, 369. 
 Chemical individuals, Proteins as, 34. 
 Chemical statics, 1. 
 Chitin, 339. 
 
 Classification, biological, 311. 
 Clupein, 53, 87. 
 Coagulating ferments, 416. 
 Coagulating power of salts, 109. 
 Coagulation, 68, 108, 119, 252, 299, 
 
 304, 342. 
 Coagulation, Chemical mechanics of, 
 
 270. 
 
 Coagulation of jellies, 303. 
 Coagulation-temperature, 111. 
 Coaguloses, 437. 
 Cobalt chloride, 128. 
 Cohesiveness, 314, 328. 
 Combining capacity, 73, 93, 195, 204, 
 
 248. 
 Combining weight, 21, 56, 180. 
 
 479 
 
480 
 
 INDEX OF SUBJECTS 
 
 Compounds of the proteins, 67, 85, 
 
 107, 137. 
 
 Compound proteins, 148. 
 Compressibility, 329. 
 Concentration-cells, 76, 138, 167. 
 Conductivity, 74, 92, 95, 171, 220, 
 
 383, 402, 449, 460. 
 Constitution of the proteins, 3. 
 Copper albuminate, 121. 
 Copper sulphate, 124, 133. 
 Cryoscopic method, 74. 
 Crystal habit, 314. 
 Crystallization of proteins, 43, 44, 47, 
 
 311. 
 
 Crystallin, 355, 356. 
 Crystalline lens, 299. 
 Cupric chloride, 128. 
 Cupric hydrate, 142. 
 
 Deaminized gelatin, 23, 51, 169, 203. 
 Decomposition of the Proteins, 4. 
 Degree of dissociation, 222, 265, 271, 
 
 322. 
 
 Dehydration, 126, 289, 296, 445. 
 Denaturation, 309. 
 Denatured protein, 70. 
 Density, 349. 
 Deuteroalbumose, 156. 
 Diacid bases, 233. 
 Dialysis, 57, 160, 169, 193, 249, 311, 
 
 449. 
 Diamino-acids, 5, 17, 25, 144, 176, 
 
 236. 
 
 Diastase, 393. 
 
 Dicarboxylic acids, 25, 176, 236. 
 Diffusion, 277, 304, 322, 330. 
 Digestibility, 18. 
 Diketopiperazines, 15, 29, 120. 
 Dilution, 201, 206, 220. 
 Dilution law, 221, 254, 263. 
 Dissociation-constant, 214, 220, 231, 
 
 263. 
 
 Dissociation, Degree of, 69. 
 Dissociation of protein salts, 167. 
 Double electrical layer, 114. 
 Dyes, 145. 
 
 Edestin, 21, 24, 71, 102, 104, 133, 357, 
 
 365. 
 Egg-albumin, 21, 44, 74, 77, 110, 113, 
 
 119, 122, 124, 133, 138, 168, 210, 
 
 305, 311, 328, 331, 337, 349, 350, 
 
 355, 399. 
 
 Egg-globulin, 350. 
 
 Elasticity, 329, 348. 
 
 Electrical condition, Influence of, 
 upon precipitation and coagula- 
 tion, 112. 
 
 Electrochemical equivalent, 178, 181, 
 182, 268. 
 
 Electrochemical measurements, 456. 
 
 Electrochemistry, 165. 
 
 Electrolysis, 113, 119, 151, 176. 
 
 Electrostatic tension, 192, 195, 297. 
 
 Emulsin, 393. 
 
 Energy storage in living organisms, 
 452. 
 
 Enzymatic synthesis, 439, 448. 
 
 Enzymes, 80. 
 
 Equilibrium, 94, 117, 123, 340, 393, 
 396, 425, 439, 447, 451. 
 
 Equivalent conductivity, 26, 118, 
 174, 248. 
 
 Erepsin, 384. 
 
 Excelsin, 357. 
 
 Fats, enzymatic synthesis of, 425. 
 
 Fatty acids, 148. 
 
 Ferments, compounds of proteins 
 
 with, 156. 
 Ferment-substrate compounds, 380, 
 
 445. 
 
 Ferric alum, 97. 
 Fibrin, 42, 101, 104, 316, 399, 400, 
 
 416. 
 
 Fibrinogen, 356, 416. 
 Formic acid, 127. 
 Formol titration, 19, 437. 
 Free radicals, 192. 
 Freezing point, Depression of, 28, 75, 
 
 236, 331. 
 
 Gas chain, 77, 456. 
 Gastric Juice, 212. 
 Gelatin, 51, 110, 203, 292, 301, 322, 
 
 323, 326, 329, 332, 346, 402. 
 Gelatinization, 299. 
 Gladstone's law, 362. 
 Gliadin, 24, 48, 328, 357, 365. 
 Globin, 51, 356, 365. 
 Globin caseinate, 149. 
 Glutin, 328. 
 Glycerol-water mixtures, Viscosity 
 
 of, 322. 
 
 Glycogen, 393. 
 Gold number, 349. 
 
INDEX OF SUBJECTS 
 
 481 
 
 Gorgonin, 158. 
 Grotthus chain, 326. 
 Guanidin, 158. 
 
 Haematin, 52, 349, 358. 
 
 Haemoglobin, 14, 43, 159, 305, 311, 
 337, 349, 356, 358. 
 
 Haemoglobin caseinate, 150. 
 
 Halogens, 12: 
 
 Halogen substitution compounds, 
 157. 
 
 Heat coagulation, 70, 305. 
 
 Heat, Liberation of, in protein hy- 
 drolysis, 400. 
 
 Heat, Liberation of, in swelling, 292. 
 
 Heavy metals, 121, 137, 159. 
 
 Hemi-albumose, 110. 
 
 Henri's equation, 380, 406. 
 
 Heterogeneous systems, 291. 
 
 Homogeneity of the protein group, 3. 
 
 Honeycomb structure of jellies, 302. 
 
 Hooke's law, 298, 329. 
 
 Hordein, 357. 
 
 Hybridization, Influence of on haemo- 
 globin crystals, 313. 
 
 Hydantoins, 30, 190. 
 
 Hydration, 108, 119, 125, 129, 411. 
 
 Hydrogen electrode, 197, 204, 456, 
 465. 
 
 Hydrolysis, 4, 14, 23, 57, 280, 371, 
 389, 460, 466. 
 
 Hydrolytic dissociation, 54, 132, 206, 
 321. 
 
 Hydrostatic pressure, 310. 
 
 Hysteresis, 341, 347. 
 
 Imino groups, 16. 
 
 Immunization, 65, 436. 
 
 Indicators, 56. 
 
 Indicator method, 81. 
 
 Individuality of the tissues and tis- 
 sue fluids, 154. 
 
 Infection, Globulins in, 65. 
 
 Instability of the proteins, 86. 
 
 Intermediate compounds in catal- 
 ysis, 393, 398, 445. 
 
 Internal salts, 28, 131, 270, 309. 
 
 Iodized proteins, 156, 157. 
 
 lonization, Mechanism of, 325. 
 
 Isoelectric condition, 209. 
 
 Isohydric solutions, 203. 
 
 Isolation of proteins, 34. 
 
 Isomaltose, Enzymatic synthesis of, 
 
 425. 
 Isomorphism, 312. 
 
 Jahn's law, 138. 
 
 Jellies, Structure of, 297, 300. 
 
 Juglansin, 357. 
 
 Kephir lactase, 393. 
 Kinetics of hydrolysis, 400. 
 
 Labile hydrogen atom, 30, 189. 
 
 Lactalbumin, 355. 
 
 Lactose, 393. 
 
 Leather, Tanning of, 146. 
 
 Legumin, 357. 
 
 Lipase, 393. 
 
 Lipoids, 148. 
 
 Magnetic properties, Masking of, 142. 
 
 Magnetic properties of proteins, 349. 
 
 Malt extract, 403. 
 
 Maltose, 393. 
 
 Masking of physiological effects, 82. 
 
 Mass law, 116, 201, 220, 340. 
 
 Maxwell's demon, 299. 
 
 Melanin nitrogen, 17. 
 
 Methyl acetate, Saponification of, 80. 
 
 Methyl derivatives, 156. 
 
 Migration velocity, 26, 184, 187, 220, 
 
 231, 235, 263, 267. 
 Milk, 59, 65, 390, 400, 416. 
 Millon's reaction, 14, 431. 
 Modulus of elasticity, 298. 
 Molecular conductivity, 74, 170. 
 Molecular volume, 331, 343, 362. 
 Molecular weight, 15, 21, 139, 296, 
 
 331, 337, 343. 
 Monoamino acids, 5. 
 Monomolecular reaction, 378, 409, 
 
 444. 
 Mucoid, 146. 
 
 Nephelometric method, 58. 
 
 Network structure in protein solu- 
 tions, 297, 300, 325. 
 
 Neutrality of the tissues and tissue 
 fluids, 211. 
 
 Neutral salts, Influence of upon rate 
 of solution, 283. 
 
 Neutral salts, Influence of upon swell- 
 ing, 299. 
 
 Nitro substitution compounds, 157. 
 
482 
 
 INDEX OF SUBJECTS 
 
 Non-dissociable inorganic radical, 
 
 167, 208. 
 
 Non-ionic protein, 133. 
 Non-proteins in blood serum, 60. 
 Nucleic acid, 86, 143. 
 Nucleo proteins, 217, 356. 
 
 (Edema, 299. 
 
 Oligodynamic action, 193. 
 
 Olive oil, Emulsions of, 303. 
 
 Opalescence, 262, 342. 
 
 Optical properties of protein solu- 
 tions, 355. 
 
 Optical structure, 373. 
 
 Optimum temperature, 420. 
 
 Osmometer, Differential, 337. 
 
 Osmotic pressure, 160, 196, 294, 330, 
 336, 340. 
 
 Ovomucoid, 49, 103, 104, 142, 202, 
 246, 336, 350, 356, 365. 
 
 Ovomucoid chloride, 228, 239. 
 
 Ovomucoid sulphate, 229. 
 
 Ovovitellin, 44, 72, 365. 
 
 Oxidase, 450. 
 
 Oxygen, Compounds of proteins with, 
 159. 
 
 Pancreatic juice, 212, 371. 
 
 Paracasein, 97, 416. 
 
 Paramoecia, Toxicity of ultra-violet 
 
 light for, 359. 
 Paranuclein, 36, 155, 365, 393, 427, 
 
 429, 440. 
 
 Penetration, Coefficient of, 287. 
 Penetration formula, 287. 
 Penetration of colloidal particles, 286. 
 Pentavalent nitrogen, 67. 
 Pepsin, 328, 375, 393, 400, 410, 429, 
 
 440. 
 
 Peptids, 15. 
 
 Peptones, 13, 15, 110, 168, 356. 
 Permeability of protein films, 348. 
 Phaseolin, 357. 
 Phase rule, 123. 
 Phenylthiocarbamid, 130. 
 Phosphoric acid, 142, 213. 
 Phosphorus, 38, 40, 430. 
 Phosphotungstic acid, 71. 
 Phylogenetic relationships, 312. 
 Physical properties, 273, 320. 
 Plasteins, 437. 
 Poisseuille's law, 303. 
 Polyamino acids, 12, 13, 189. 
 
 Polymerization, 268, 289, 309, 336, 
 347, 389. 
 
 Polypeptids, 13, 127, 371, 385, 397. 
 
 Polypeptid structure, Consequences 
 of the, 20. 
 
 Polysaccharides, Enzymatic synthe- 
 sis of, 425. 
 
 Potassium caseinate, 171, 177, 223, 
 225, 255, 262, 266. 
 
 Potassium paranucleinate, 225. 
 
 Potassium serum globulinate, 227. 
 
 Potentiometric method, 76, 93, 99, 
 167, 195, 211, 215. 
 
 Precipitating power, 112. 
 
 Precipitation and coagulation, Chem- 
 ical mechanics of, 125. 
 
 Precipitation, Indirect method of, 71. 
 
 Precipitation of proteins, 68, 79, 107. 
 
 Precipitins, 154. 
 
 Principle of mobile equilibrium, 308. 
 
 Proportion of inorganic acid or base, 
 Influence of, on conductivity, 242. 
 
 Protamin, 22, 53, 86, 346, 393, 403, 
 410, 413, 428. 
 
 Protamin caseinate, 149. 
 
 Protamin chloride, 229. 
 
 Protamin edestinate, 155. 
 
 Protamin sulphate, 229. 
 
 Protein complexes, 1 52. 
 
 Protein ions, 173, 176, 184, 321, 323, 
 344. 
 
 Purification of proteins, 34. 
 
 Quantitative estimation of proteins, 
 56. 
 
 Racemized proteins, 30. 
 
 Radium, 310. 
 
 Reaction isochore, 160. 
 
 Reaction velocity, 123, 280, 305, 377, 
 
 381, 401. 
 
 Reciprocal catalysis, 444. 
 Refractive Index, 92, 359. 
 Refractometric method, 60. 
 Rennet, 97, 400, 416, 438. 
 Reversion of hydrolysis, 425. 
 Ricin, 357. 
 Rotatory power, 30, 58, 355, 376, 386. 
 
 Saliva, 217. 
 
 Salmin, 22, 53, 86, 365, 428. 
 Salmin chloride, 229, 241. 
 Salmin sulphate, 229. 
 
INDEX OF SUBJECTS 
 
 483 
 
 Saturation of acids and bases by pro- 
 teins, 91, 207, 248. 
 Schiitz rule, 402, 409, 419, 442. 
 Scombrin, 87. 
 
 Selective action of living tissues, 192. 
 Serum albumin, 62, 109, 124, 169, 
 
 203, 311, 328, 345, 349, 355, 365, 
 
 399. 
 Serum globulin, 40, 64, 98, 104, 150, 
 
 187, 349, 356, 365. 
 Serum globulinates, 232. 
 Serum globulin chloride, 227. 
 Serum proteins, Neutralizing power 
 
 of, 215. 
 
 Silicic acid, 143. 
 Silver nitrate, 124, 138. 
 Soaps, Compounds of proteins with, 
 
 148. 
 
 Sodium caseinate, 222, 223, 412. 
 Sodium serum globulinate, 226. 
 Solubility, 248, 275. 
 Solution, Passage into, 275. 
 Solution pressure, 195. 
 Solution, Rate of, 276. 
 Solvate theory, 130. 
 Specific gravity, 348. 
 Specificity, 154, 436. 
 Specific refractivity, 362. 
 Spectrophotometric method, 159. 
 Spongework structure of jellies, 300. 
 Stoichiometry, 85, 90, 236. 
 Strontium caseinate, 224. 
 Strontium serum globulinate, 228. 
 Structure of protein solutions, 314, 
 
 324. 
 
 Sturin, 22, 53, 87. 
 Sulphur, Compounds of proteins 
 
 with, 159. 
 
 Supersaturation, 279, 304, 342. 
 Surface films, 345. 
 Surface tension, 290, 345. 
 Suspensions, 341. 
 Swelling deficit, 293. 
 
 Swelling maximum, 293. 
 
 Swelling of jellies, 110, 292, 327, 339, 
 
 416. 
 
 Synaeresis, 316. 
 Synthesis, Enzymatic, 425. 
 Synthesis of Proteins, 9. 
 Syphilis, 350. 
 
 Temperature coefficient, 160, 284, 
 
 306, 310, 419. 
 
 Tetraethylammonium iodide, 267. 
 Thermodynamics, 337, 450. 
 Thermodynamics, Second law of, 299, 
 
 451. 
 
 Thrombin, 316, 416, 450. 
 Thymus histone, 150, 356. 
 Thyreoglobulin, 158. 
 Thyroid gland, 158. 
 Tissues, Swelling of, 299. 
 Toxins, 156. 
 Triolein, 427. 
 Trivalent carbon, 192. 
 Trypsin, 371, 393, 399, 402, 405, 410, 
 
 415, 428. 
 
 Tyndall effect, 342. 
 Types of union, 17. 
 
 Ultramicroscope, 316. 
 Ultraviolet light, 310. 
 Ultraviolet light, Toxic action of, 359. 
 
 Valency, 24, 113, 120, 132, 183. 
 
 Vanadium, 142. 
 
 Vegetable proteins, 46, 101, 311, 357. 
 
 Velocities, Chemical, 117, 426. 
 
 Viscosity, 210, 266, 320. 
 
 Volume contraction, 292, 348. 
 
 Yeast, 393. 
 
 Yeast endotryptase, 387. 
 
 Zein, 48,357. 
 Zinc sulphate, 141. 
 
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