COLLOID CHEMISTRY OF THE PROTEINS BOOKS ON COLLOID CHEMISTRY. By E. HATSCHEK. An Introduction to the Physics and Chemistry of Colloids. Fourth Edition. 20 Illustrations. Laboratory Manual of Elementary Colloid Chemistry. 20 Illustrations. A Handbook of Colloid Chemistry. By Dr. W. OSTWALD. Second Edition. Translated by Dr. MARTIN FISCHER. 93 Illustrations. The Formation of Colloids. By Prof. THE SVEDBERG. With 22 Illustrations. J. & A. CHURCHILL. COLLOID CHEMISTRY OF THE PROTEINS- \ BY PROF. DR. WOLFGANG PAULI DIRECTOR OF THE LABORATORY FOR PHYSICO-CHEMICAL BIOLOGY, UNIVERSITY OF VIENNA TRANSLATED BY P. C. L. THORNE, M.A. (CANTAB.), A.I.C., SIR JOHN CASS TECHNICAL INSTITUTE, LONI1ON PART I WHH 27 DIAGRAMS AND NUMEROUS TABLES PHILADELPHIA P. BLAKISTON'S SON & CO. 1012, WALNUT STREET 1922 v/. Printed in Great Britain. AUTHOR'S PREFACE THE small volume of which Part I. is here presented has been developed from lectures delivered in the winter term of 1912-13. It by no means exhausts the materials of the colloid chemistry of the proteins even up to that date. In response to many requests, it is proposed to collect in perspective the investigations carried out by the author and his co-workers in the course of many years, prominence being given to the most important results. Such fundamental work of other authors as is relevant has also been included in an attempt to round off the presentation, but for complete data the reader is referred to the well-known works of O. Cohnheim, R. H. A. Plimmer (translated into German by J. Matula), and T. B. Robertson. The second part, to appear within a year, will include the relations of the proteins to neutral salts and to the salts of the heavy metals, to colloids and to ampholytes, the properties of the albumin gels, and, finally, the physical chemistry of the purest albumin so far prepared. I take this opportunity of thanking most heartily Dr. Mona Adolf for her careful and critical reading of the proofs, and also my assistant and collaborator of many years, Dr. Joh. Matula, for drawing the figures. WOLFGANG PAULI. VIENNA. 480500 TRANSLATOR'S NOTE THE great importance of Professor Pauli's work on the proteins is ample justification for an English translation of this monograph, in which the work carried out by him and his collaborators is summarised and presented in relation to previous and concurrent research by other workers. By the application of the quantitative methods of physical and colloid chemistry a consistent theory of the behaviour of proteins, particularly in acid and alkaline solution, has been established. In the translation a number of typographical errors have been corrected, a few slight alterations suggested by Professor Pauli have been made, and the indexes have been added. Otherwise no attempt has been made to do more than give the substance of the original in English. The way of translators is hard : in this case it has been made much easier by the constant advice of Mr. Emil Hatschek, F.Inst.P., who has been good enough to read the whole of the translation in manuscript. For this great advantage I wish to record my gratitude to him. P. C. L. THORNE. SIR JOHN CASS TECHNICAL INSTITUTE, LONDON. TABLE OF CONTENTS CHAPTER I PAGE COLLOID CHEMISTRY AND THE GENERAL CHEMISTRY OF THE PROTEINS . . . . . . . . . i General development of colloid chemistry. A sharp demar- cation originally supposed to exist between colloidal and molecular disperse systems. Increase in our knowledge of transitional stages. The colloid chemistry and general physi- cal chemistry of the proteins form an inseparable whole. Connection between these aspects and structural chemistry. CHAPTER II CONDITIONS OF STABILITY IN PROTEIN SOLUTIONS . . . 10 The bearing on stability of the colloid particles of (i.) hydra - tion, (ii.) electric charge. Lyophobe and lyophile colloids. Connection between electric charge and hydration of the particles. Origin of the electric charge. Nature of the pre- cipitation process. Proteins with hydrophobe and those with hydrated neutral particles. Classification of the alterations in state in colloid systems. Reversible and irreversible trans- formations ; homodrome and heterodrome reversible changes. CHAPTER III THE ELECTRIC CHARGE ON NATURAL SOLUBLE ALBUMIN . . 19 Determination of the iso-electric reaction with the electro- phoresis apparatus. Amphoteric electrolytes. Hydrion regu- lators. Dissociation constants of amphoteric electrolytes. Chemical constitution and magnitude of dissociation con- stants. Theory of the iso-electric reaction. Equality in con- centration of albumin anions and cations ; maximum concen- tration of neutral protein particles at the iso-electric point. CHAPTER IV PROPERTIES OF PROTEINS IN ISO-ELECTRIC REACTION . . 37 Neutral and ionised albumin difference in their properties. Determination of the iso-electric point from the maximum of neutral albumin particles as found by precipitation by alcohol, by viscosity measurements, by osmotic pressure, and by imbibition. Dependence of the iso-electric reaction on the concentration of albumin. Difference in behaviour with weak and strong acids. Sorensen's new method of determining the iso-electric point. x TABLE OF CONTENTS CHAPTER V SALTS OF ALBUMIN AND ACIDS ...... Older methods of determining the extent of combination of albumin and acids. Electrometric procedure. Researches of Bugarsky and Liebermann, Manabe and Matula. Relation between concentration of protein and of acid to the extent of combination. Viscosity of acid albumin. Parallelism of ionisation and viscosity ; precipitation by alcohol ; depres- sion of the freezing point. Combination of albumin with different acids. Optical rotation of the albumin salts. Optical behaviour in various acids. The acid-albumin of Adolf and Spiegel. Precipitation of albumin by acids. CHAPTER VI SALTS OF ALBUMIN AND ACIDS (continued) . . . .80 Hydrolysis of albumin salts. Determination of mean degree of hydrolysis and of mean basic dissociation constant of albumin. Role of the terminal amino-groups and of the imino-group of the peptide linkage. Properties of desamino- glutin and of acid-albumin. Combination of albumoses and peptone with acids. CHAPTER VII SALTS OF ALBUMIN AND BASES . .:... . . . .93 Electrometric measurement of the combination of albumin with bases. Classification of combination with various strong bases according to their dissociation constants. Parallelism of viscosity, precipitation by alcohol, and ionisation of albu- minates. Precipitation of albumin by concentrated alkalis. Alkali caseinates. Equivalent conductivity of sodium, potas- sium and ammonium caseinates. Detection of normal ionisa- tion into metal ions. Determination of degree of dissociation and of mobility of caseinate ions. Valency of caseinate ions ascertained from the conductivity curve. Electrometric determination of the combination of casein and alkali. Existence of a casein-saturated complex and of a simple caseinate, both with a trivalent casein ion. Molecular weight of casein. CHAPTER VIII ALTERATIONS IN STATE OF THE ALKALI PROTEINS WITH LAPSE OF TIME . " .. . . . .no Alterations with time exhibited by viscosity, electrical con- ductivity, and extent of combination in the alkali proteins. Explanation on the basis of a rearrangement of the peptide linkage from the lactam to the lactim form. Relation to other rearrangements. Optical rotation of the alkali proteins. TABLE OF CONTENTS xi CHAPTER IX PAGE SALTS OF THE GLOBULINS : MIGRATION VELOCITY OF THE PRO- TEIN IONS . * . . . . . .121 Hardy's researches on combination of proteins with acids and alkalis. Direct determination of the mobility of globulin ions by migration experiments. Its calculation from conductivity data. Agreement of the values thus obtained. Effect of valency on the mobility of protein ions. Determination of mobility of protein ions by Pauli and Sven Oden, also by cir- cuits containing albumin salts. Robertson's work on the electro-chemical equivalent of casein. Agreement with the constitution of the caseinates previously set forth. COLLOID CHEMISTRY OF THE PROTEINS CHAPTER I COLLOID CHEMISTRY AND THE GENERAL CHEMISTRY OF THE PROTEINS IT would be superfluous to discuss which of the constituents of the living cell are most important in vital processes. Proteins, lipoids, and certain inorganic salts are alike indispensable, and have a very intimate relation, both physical and chemical, one to another. There is, however, no doubt as to the central position of the proteins in the organisation of living matter. Apparently, they occur in nature in close connection with vital processes ; in the living cell they are completely irreplaceable ; and, above all, they alone display the specific properties of living matter. In consequence, the distinctions observed, not only between different kinds of organisms, but even between individuals of the same kind, reappear on chemical investigation as variations in the respective proteins. Again, the proteins are capable of showing diversity and fine gradation, both in chemical structure and physical modification, to an extent which is lacking in any other class of substances. On account of the high molecular weight of even the simpler proteins, physical changes are brought about by minute quan- tities of substances of low molecular weight. Electrolytes in particular cause such changes very easily, owing to the amphoteric nature of the proteins. The biologist attempts to arrive at a general expression for the profound and diverse phenomena of life, and finds that the rich variety of the reactions of the proteins confronts him as one of his greatest difficulties. Such progress as has been made has occurred mostly in recent years and has been largely due to the application of physical chemistry, and of the youngest branch >2 \\^\; ^COLLOID CHEMISTRY AND THE of that subject colloid chemistry. The relations of this special branch of knowledge to the general chemistry of the proteins is so definite that a discussion of the position is desirable. An independent branch of science arises when a number of observations are in strong contrast to previous records. The differences must be so striking that investigators are spurred on to an intensive study of the most glaring peculiarities, and the contrast between the new and the old knowledge serves to define sharply the bounds of the new discipline. As time goes on and the new knowledge increases, previous discoveries, which appeared to have little in common with it, are seen in a new light. Consequently, there is, in the first place, a period of detachment from the main stream of knowledge, followed by a second phase of reunion. When the new development is rich in observations and laws peculiar to itself, it will maintain a distinctive position even after the period of coalescence has begun, particularly when it contributes new methods of inquiry. In this way colloid chemistry has achieved its autonomous position. Although there w r ere distinguished workers in the field before him, Thomas Graham (1851) must be acclaimed as the father of colloid chemistry. Not only was he the first to set forth the most important properties of colloids, but he further emphasised the fundamental distinction between colloids and crystalloids a distinction as wide as that between an organised substance and a lifeless mineral. Indeed, so vivid was this distinction to Graham and his immediate successors in research that any connection between the properties of crystalloids and colloids remained far in the background. Such a connection is, however, very plain in the considerations which formed the starting point of Graham's work. For he submitted the following line of thought : all substances show a greater or less difference in the rate or ease with which they pass from the solid or liquid state into the state of vapour ; in many cases these differences are sufficiently pronounced to form the basis of a method of % separation in fractional distillation. The differences in the rate of diffusion in a liquid are not less pronounced than in the rate GENERAL CHEMISTRY OF THE PROTEINS 3 of dispersion into a gaseous phase. Is it not possible, therefore, to build up a process of separation and purification of dissolved substances on these differences ? Graham's observation that substances which diffused with difficulty were more or less held back by parchment paper or by animal membranes indicated the method of purifying substances by dialysis, as he called the process. In this way salts and other substances which diffuse easily are removed without fundamental changes or chemical complications. It is well known how important dialysis has become, particularly in biochemical investigation. In Graham's hands it became the first general method of preparing pure colloids, a name which he gave to those substances which are distinguished by the following three criteria : I. They diffuse very slowly or too slowly for the rate to be measured. II. They are unable to penetrate certain membranes, particularly some of animal origin. III. They lack the power to appear in the crystalline form. To these a fourth has since been added. G. Bredig, in particular, has shown that the work required to be done* to separate the colloid from the solvent is very small or practically nil, as is shown by measurements on the evaporation or freezing out of the solvent. We shall see that all these criteria depend on one single property, which now appears to us as the significant characteristic of the colloid state. The greatest progress which has been made since the time of Graham lies in the following straightening-out of ideas. Graham divided substances themselves into colloids and crystalloids. In which of these forms it appeared would thus seem to depend on the chemical nature of a substance. We now know, however, that the colloid or crystalloid state is no more than a physical manifestation of a dependent condition of matter. The same chemical substance can be obtained, according to circumstances, with colloid or crystalloid characteristics. This development is, in the first place, a result of improvements in methods of preparation. It is possible to obtain in the colloidal state all kinds of substances of low molecular weight, e.g., metals, salts - of alkalis and of the alkaline earths, etc. It has, moreover, been shown that for the production of the colloidal condition a state of fine subdivision is necessary, but not division into single 4 COLLOID CHEMISTRY AND THE molecules. As all substances dissolve up to their solubility- value, that is, they become subdivided to molecular size, a colloidal dispersion only becomes possible when the solubility value is exceeded. So that difficultly soluble substances lend themselves most readily for transformation into the colloidal state (e.g., metals, many metallic sulphides and hydroxides, fats and resins, with water as dispersion medium). If, on the other hand, it is desired to obtain salts or other substances soluble in water in the colloidal state, methyl alcohol, acetone, etc., must be used as dispersion medium. Of course, it is also essential that the division of the particles must be sufficiently fine for the particles to remain free swimming and evenly distributed throughout the bulk of the liquid ; otherwise we are dealing with a slime, a coarse suspension, or an emulsion. Such fine dispersions, or, as we can now call them, colloidal solutions, pass easily through a filter. Their real nature and the difference between them and true molecular solutions was first made clear by the use of the ultramicroscope of Zsigmondy and Siedentopf, which made the degree of dispersion optically apparent and permitted a closer determination of the size of the particles. According to R. Zsigmondy, we can distinguish by their mean linear dimensions coarse suspensions with particles of o-i/z diameter, and colloidal solutions with par- ticles of o-ijLtft diameter. The latter pass without a break into the various molecular solutions as the size of particle decreases. Wolfgang Ostwald is the author of the very suitable terms dispersion medium (continuous phase) and disperse phase for the component parts of a colloidal system. The degree of dispersion (dispersity) is the quotient of the total surface and the total volume of the dispersed particles. It is easy to see that there are fundamentally two general methods of preparing a colloidal solution of a substance : (i) dispersion methods, in which large particles or aggregates are broken up, as in electrical dispersion, partial solution of coarse precipitates or peptisation ; and (2) condensation methods, in which a molecular solution is caused to become super-saturated, either by sufficient dilution or the addition of certain substances GENERAL CHEMISTRY OF THE PROTEINS 5 until formation of a finely divided disperse phase occurs. The theory of the main methods of formation of the colloidal state of all substances and the general formulation of the conditions of their existence has been developed by P. P. von Weimarn. Finally, solutions of colloidal character are obtained when substances of very high molecular weight are dissolved in water. The tendency of the large molecules to associate and polymerise can still further increase the colloidal properties of these solu- tions. Albumin, starch, glycogen, etc., are included in this group of substances, whose main characteristic is that they occur exclusively in the colloidal condition. Let us suppose that, instead of a dilute solution of a substance of low molecular weight, the same total quantity of material is present as much larger particles, which, in consequence, are fewer in number. Clearly such properties as depend on the number and size of the particles will in consequence be con- siderably altered. The osmotic pressure would fall to a small value ; the power of penetrating membranes, as also the rate of diffusion in general, would become minute ; while the directive force which orientates particLss for formation of crystals becomes very weak or can be no longer developed. Accord- ingly, no qualitative difference between typical dilute solutions, as of sugar or salts, and colloidal solutions is to be expected, but merely a gradual transition from the one to the other. In the second place, the characteristics of the latter solutions would appear. First, whereas in a very dilute solution of a substance of low molecular weight, the volume of the solid can, as a rule, be neglected, in a colloidal solution the volume of the particles is appreciable in consequence of their size. Secondly, the particles are often so large as to be visible when refined optical methods are used, e.g., by observing with the naked eye at right angles the fog produced by a bright beam of incident light (Tyndall's phenomenon], or the diffraction images in the microscope (ultramicroscope) . The disperse phase develops physical surfaces in the medium of dispersion, so that the most varied reactions of the two phases can be fixed or modified according to the physical properties of these surfaces. 6 COLLOID CHEMISTRY AND THE The work, in particular, of J. Perrin and of The. Svedberg * on colloids has demonstrated that in microscopically visible systems the properties of fine dispersions can, in certain directions, be brought into quantitative relation to those of typical solutions. We must here consider in rather more detail the work of Perrin, the upshot of which was that it is possible directly to ascertain the gas constant, and, above all, Avoga- dro's number for dispersions (as, for instance, of gamboge or mastic) with astounding accuracy. The law of Boyle and Gay Lussac pv = RT was expanded by Avogadro into his incredibly fertile hypothesis that for the same conditions of pressure and temperature the same volume of any given gas contains the same number of molecules. This development depended on the discovery that the weights of equal volumes of two gases under the same temperature and pressure conditions are proportional to the respective mole- cular weights. Therefore the gram molecule (the molecular weight in grams) of different gases contains the same number of molecules. i Van t'Hoff f has shown that the same law holds for dilute solutions, provided the osmotic pressure of the solution against the pure solvent is substituted for the gas pressure. The constant R has the same value as for gases, and the solution of gram molecules of different substances (provided no association or polymerisation occurs) gives here once again the rule of Avogadro that in each solution the same number of molecules is present. This number is a well-known constant, and will be indicated as Avogadro' s number N. The kinetic theory of gases, largely the work of Clausius and Maxwell, has for its fundamental assumption the idea of move- ment of the molecules. These movements increase with a rise in temperature, and cease altogether at absolute zero. Avogadro's hypothesis can be deduced from the gas laws by * " Die Existenz der Molekiile." Leipzig. 1912. f It is extraordinary that the fact that Graham used the analogy between solutions and gases as the basis of his work has not been emphasised. Of course, he only used the comparison for a special case, and it was left to Van t'Hoff to develop it in a general form and apply it quantitatively to dilute solutions. GENERAL CHEMISTRY OF THE PROTEINS 7 applying the laws of mechanics. For a gram molecule of a gas (or dissolved substance) the simple formula of the kinetic theory reads : pv = - Nw = RT, where N is Avogadro's number and w the mean kinetic energy of one molecule of gas or solute. The kinetic theory of gases has, moreover, caused the behaviour of gases under high pressure to be compared with that of liquids, with the result that the apparent difference in molecular condition in the two cases is not found to exist.* J. Perrin | has applied the conclusions of Van t'Hoff for solutions to the cases both of pure liquids and of suspensions. To quote his own clear summary : " Van t'Hoff 's law states that a molecule of ethyl alcohol has the same energy in an aqueous solution as when it is vaporised therefrom. It would also possess the same energy in solution in chloroform, that is, when surrounded by chloroform molecules, and similarly in solution in methyl or propyl alcohol. It may therefore be supposed that its energy is also the same in ethyl alcohol solu- tion ; in other words, when it is a constituent molecule of pure ethyl alcohol. So that, by admitting that the molecular energy is the same in the liquid as in the gaseous state, we can now state that at the same temperature the mean kinetic energy of all molecules of every liquid is the same, and is proportional to the absolute temperature. " The general hypothesis can, however, be still further deve- loped. From what has been stated above, the heavy sugar molecules in a sugar solution have the same mean energy as the mobile molecules of water. Now the sugar molecule contains 45 atoms ; that of quinine sulphate more than 100. One can enumerate still heavier and more complex molecules, to all of which the law of Van t'Hoff is applicable, for no limit is placed to the size of the molecule. Consider, now, a particle made up of several molecules, say a dust particle. Is it likely it will conform to a different law under the bombardment of the * See W. Nernst, " Theor. Chemie.," 1907, p. 226. | See Roll. Chem. Beihefte, 1910, 1, 221, for complete literature. 8 COLLOID CHEMISTRY AND THE surrounding molecules ? Will it not rather behave like a large molecule, so that its mean energy is that of a single molecule ? ' ' The movement of suspended particles was first described by the botanist Brown in 1827. Various later workers (Wiener, M. Gouy) confirmed its existence, and traced its origin to the movements of the molecules. Perrin was able to produce gamboge suspensions of particles of the same size by fractional centrifuging, and, by counting the number of particles at various depths in a cell under the microscope, was able to show that the distribution under the force of gravity followed the same exponential law as is found, for instance, in the distribution of the air in the atmosphere. Thus a difference of lop, in depth in the cell gave the same decrease in number of particles as occurs in a height of 6 kilometres in the atmosphere. The value of the constant N, calculated from the distribution equation of various emulsions is 70 X io 22 , a value for Avo- gadro's constant which agrees exceedingly well with that obtained by Van der Waals. From the above and from other demonstrations of the applicability of the kinetic theory of gases to colloid systems (see The. Svedberg), we can perceive no fundamental distinction between the behaviour of particles of molecular dimensions and I that of colloid particles up to those of a coarse suspension. We shall see further that a similar transition occurs in electro- chemical properties. ' Under these circumstances, there can be no question of a sharp demarcation between the general chemistry of substances of high molecular weight, such as proteins, and their colloid chemistry. The difference is certainly not a matter of size of particles, but rather 41 matter of structure. If- a metal or a ' simple compound is brought to the colloid state, the dispersed particles are always aggregates of simpler but similar smaller portions. If, on the other hand, the particles are those of a non-associated substance of high molecular weight, of the same degree of dispersity as the above, we have particles formed of a combination of several heterogeneous groups of atoms (for instance, albumin, which is formed of various amino-acids) . We have so far absolutely no knowledge of the distribution of GENERAL CHEMISTRY OF THE PROTEINS 9 these groups on the surface or inside the molecule. At first sight it appears that the second example is more complex than the case of the colloidal metal ; but one must not overlook the fact that the various groups which build up the complex com- pound often retain their characteristic properties, and stamp the whole molecule with their physico-chemical behaviour. These properties of compounds of high molecular weight can therefore, in favourable circumstances, throw light on many general colloidal phenomena, as happens in many respects with the proteins. Although we regard the colloid chemistry and the physical chemistry of the proteins as inseparable, we must at the same time emphasise every tendency to a relation between their colloid chemistry and their structure. This connection was the object of our own researches, and is one which will undoubtedly be largely developed in the near future. io CONDITIONS OF STABILITY CHAPTER II CONDITIONS OF STABILITY IN PROTEIN SOLUTIONS THE definition of a colloid system as a dispersion within certain limits of subdivision is satisfactory in emphasising a general property ; it does not, however, suffice to define special cases when certain hypotheses become necessary to complete the description. These serve to subdivide colloids into broad classes. Thus Wo. Ostwald has devised a classification based on the state of aggregation of the disperse phase. Colloids with solid disperse phase are suspensoids, those with liquid particles emulsoids. If the dispersion in such systems is reduced so that the disperse phase coalesces into macroscopic masses, the suspensoids give solid coagula, which settle out of the liquid. The emulsoid particles, in contrast, flow together into drops, which frequently unite to form a separate liquid layer. " If a io per cent, gelatin solution is precipitated at 30 by addition of a neutral salt (e.g., sodium sulphate) and allowed to stand for some hours at the same temperature, the gelatin is found in both layers, the upper more liquid one containing but little gelatin, the lower layer being rich in gelatin (Wo. Pauli and P. Rona*). A similar effect has been observed and further studied by K. Sptro f when casein is coagulated by heat." The classification of colloids from another point of view is important in characterising the physical properties of the pro- teins. Both in the case of typical solutions and in that of colloidal solutions, a greater or less combination between the particles and the solvent or continuous phase has been shown to exist. Crystalloids are said to be hydrated (or, in general, solvated) in solution when water is attached to the dissolved * Beitr. z. chem. Physiol. u. Path., 1902, 2, i. f Beitr, z. c)ve.m. Physiol. u. Path., 1904, 4, 300, IN PROTEIN SOLUTIONS n /* molecules in a stoichiometric proportion. In some instances this hydra tion is sharply defined (as for sulphuric acid and ferric chloride) , but in other cases the hydration is variable and shows a continuous alteration with dilution and with change of tem- perature. We can describe such phenomena with more accu- racy as addition of water (or formation of envelopes) by the particles, rather than as combinations in the more restricted sense of the word. Such an addition of the medium occurs on the particles of many colloids, and is described as hydration in the wider sense, and as imbibition when the adding of water occurs together with penetration of water into a large aggregate. The indications of hydration of a disperse phase are a disproportionately great viscosity of the solution, the decreased activity of the move- ment of the particles as displayed in diffusion or in an electric field, and an alteration in volume or density in the direction of compression of the medium. The latter is made obvious by a comparison of the volume with that calculated from the volume of the particles plus that of the medium, or by the increase in the effective concentration of a substance in solution, as also by anomalous behaviour when the solvent is removed by freezing or evaporation, owing to a stronger partial combination. In some cases there is an evolution of heat on solution in the medium. We shall see that hydration of particles of albumin solutions has been demonstrated, and, consequently, dissolved proteins are classed among hydrated (solvated) colloids. These are called hydrophile by J. Perrin, and in general ly ophite by H. Freundlich. Colloids with non-solvated particles are named hydrophobe (lyophobe), and although the definition of sus- pensoids and emulsoids is on a different basis, in practice the two classifications approximately coincide. The embarrassing use of the double classification according to the state of aggrega- tion or solvation of the disperse phase is strongly to be depre- cated. With water as continuous phase the obvious notation hydro- and anhydro-colloid is useful. Hydrated colloids are usually more stable in solution than anhydro-colloids. The Yole of solvation in the stability of various colloid 12 CONDITIONS OF STABILITY systems is brought out most clearly when the effect of the electric charge of the particles on the equilibrium of the system is investigated at the same time. When the behaviour of colloids in an electric field is explored, a movement of the particles towards one of the poles can ordinarily be demon- strated. If electro-negative, they wander to the anode (e.g., metals, metallic sulphides, mastic suspensions, silicic acid, etc.), or, if positive, to the cathode (e.g., metallic hydroxides). The /work of H. Schultze, H. Picton and S. E. Linder, W. B. Hardy, W. Biltz, J. Billiter, H. Freundlich, and others has shown that the electric charge on the particles of these colloids is essential for their stability in solution. The removal of the charge on the particles leads to a cessation of movement in an electric field, and also to precipitation. This fact was first noted and precisely stated by W. B. Hardy. Later researches show that even an incomplete discharge of the particles (for oil-emulsions + 0-03 volt) leads to coagulation (R. Ellis * and F. Powis f). This discharge and precipitation of the colloidal solution can be brought about in various ways : I. By addition of electrolytes, in which case relatively low concentrations of the ions are sufficient. Positive ions are effective in precipitating negative sols and negatiVe ions for positive sols (Hardy's Rule). The potency of the ion increases disproportionately with its valency, polyvalent being more effective than monovalentv^.and of the latter H- and OH-ions are the most powerful (H. Schultze, W. B. Hardy, H. Freundlich) . II. Oppositely charged colloids neutralise each other, and are mutually precipitated. Excess of one of them makes precipitation incomplete or may prevent it altogether (W. Biltz, J. Billiter). III. If secondary reactions are neglected, precipitation occurs on the oppositely charged pole when a colloid is subjected to an electric field. IV. The charge on the particles is decreased, even to the * Zeitsch. physikal. Chem., 1914, 89, 145. f Zeitsch. physikal. Chem., 1914, 89, 186. IN PROTEIN SOLUTIONS 13 point of coagulation, when the colloid is subjected to electrical radiations. For instance, as can be fore- seen, the /S-rays of radium precipitate a positive colloid, e.g., ferric hydroxide (W. B. Hardy, V. Henry and A. Mayer, A. Fernau and Wo. Pauli *). Let us next consider the connection between these phenomena and those displayed by a typical true solution. Here also is a stability dependent on charged particles, the ions. We know no means of causing a notable separation of the ions from solutions ; but neutral particles can be deposited from the solution in the form of a precipitate. The property discussed above gives a suitable analogy to the behaviour of normal electrolytes, if we so choose the latter that the limit of solubility of the neutral particles formed is exceeded. The precipitation of barium as sulphate, the electrolytic deposition of a metal from its salt, and similar reactions can be expressed in a form analogous to the precipitation of colloids. We can state, in general, that whenever the neutral particles of a colloid are not stable in solution it is precipitated by removal of the electric charge. There are, however, colloids in which the neutral particles are stable, and thus electrical neutralisation does not lead to precipitation. This observation was first made with natural albumin (Wo. Pauli), and it holds also for starch, gelatin, agar, gum arabic, some forms of silicic acid, etc. The first group of colloids, with neutral particles, which are unstable in solution, includes the hydro- (or lyo-) phobe, or anhydro-colloids (the suspensoids of Wo. Ostwald and P. P. von Weimarn), while the second group, with stable neutral particles, includes without exception hydrated or solvated colloids. We must now consider a more important factor. For a colloid to be stable in an electrically neutral state, it is necessary for the discharged particles formed to be sufficiently hydrated. As we shall see, many proteins (casein, globulin, boiled albumin, etc.) form heavily hydrated particles in solution when electri- * Biochem. Zeitsch., 1915, 70, 426 ; Kolloid Zeitsch., 1917, 20, 20, for the literature. 14 CONDITIONS OF STABILITY cally charged, but precipitate in the electrically neutral state, as the particles lose their water of hydra tion when discharged. The importance of solvation and electric charge of the particles in determining the stability of colloids is shown by numerous researches. The questions of the origin of the electric charge and of the mechanism of discharge and precipita- tion of the colloid particles lead us, on the other hand, into a field of various conflicting theoretical speculations. It would appear impossible to suggest a single source of the electric charge which would be applicable to all colloid systems. But in the case of the proteins, as we shall show, all experience points to the conclusion that those which behave as electrolytes owe their charge to typical ionisation processes. Similarly, silicic acid, stannic acid, tungstic acid, etc., which give positive H-ions, form particles which are charged negatively, as also do colloidal solutions of the noble metals, when positive metal ions are produced. Colloidal metallic hydroxides, on the other hand, can be regarded as complex salts of the metallic oxide with the salt from which they are prepared by hydrolysis and dialysis (see the recent work of Wo. Pauli and J. Matula*). These complex salts consist of a positive colloidal ion and a simple anion. For example, a ferric hydroxide sol prepared from ferric chloride contains the ions #Fe(OH) 3 . yFe'" and Difficulties crop up when this purely ionic conception is applied to colloids in which at present no notable ionisation has been detected : for example, emulsion of resins, and such like bodies. But even here the ionisation resulting from slight saponification at the surface of the particles is not excluded. The theory of the charging of colloid particles as an ionic process in which one ion is very large has been developed by J. Billiter. The particles have the charac- teristics of an ion with the charge of the electrode which repels them. This view of the origin of the charge on the particles of colloids leads to a theory of the discharging processes which is most simply regarded as an ionic reaction at the surface of * Kolloid Zeitsch., 1917, 21, 49 (literature). IN PROTEIN SOLUTIONS 15 the disperse phase. This conception is quantitatively worked out by H. Freundlich in his theory of adsorption of ions. When a complex salt is dealt with (as, for example, most metallic hydroxide sols), two kinds of electric neutralisation by electrolytes take place : (i) suppression of ionisation and reduction in solubility due to a considerable quantity of added ions, and (2) low solubility product of one ion, which precipi- tates in very low concentrations. The action of chlorides, nitrates, etc., on ferric hydroxide sol or eerie hydroxide sol is an example of the first, while that of oxalates, tartrates, sul- phates, etc., is an example of the second method of neutralisa- tion.* Analogous behaviour is found with proteins. Bredig's theory of the process of precipitation is most widely known. According to this view, neutralisation removes the electrostatic repulsion between the particles, and owing to the tendency of surface energy to sink to a minimum, a reduction in surface occurs by the particles joining together. It cannot be doubted that this is an important factor in the process of precipitation. In particular, the fact that there is a power of association between the neutral particles in a molecular solution must not be overlooked. It comes into play in the formation of precipitates and in crystallisation, and hence can scarcely be neglected in the case of colloids. But our knowledge in this direction is very slightly developed, and any precise connec- tion between surface tension and molecular association is lacking, though both depend on chemical constitution. Develop- ments in this border-line field are likely to come in large part from the study of colloid chemistry. Zsigmondy, again, considers the question of whether a dried colloid is again dispersed by mere addition of the dispersion medium as a basis of classification. Colloids which redisperse he calls resoluble ; those that do not, irresoluble. This criterion is also useful up to a point in facilitating the general compre- hension of colloidal behaviour. The extreme lyophobe colloids are irresoluble, while typical hydro-colloids are resoluble. The redispersion of a dried colloid is conditioned by the ionising power of the continuous medium, and the tendency of- the * See Wo. Pauli and J. Matula, loc. cit. 16 CONDITIONS OF STABILITY colloid to form ions, as well as by the solvation and aggregating power of the disperse particles due to their varied molecular association and surface tension.* Having cleared the way by this discussion, we will now briefly collect the facts which bear on the stability of proteins. Some proteins are hydrophobe, some have hydrated particles ; and the conditions under which they are stable are different in the two cases. Proteins of the first group exhibit Hardy's law of instability at the neutral point when treated with electrolytes. To this group belong heat-coagulated albumin, casein, salt-free globulin, acid-albumin, etc. On the other hand, glutin, natural albumin, and the other proteins of the second group are stable in solution, even when the particles are not electrically charged. The electric charge is, however, quite an important factor in the stability of the lyophile or hydrated proteins, for it is an accepted fact that only neutral particles are precipitated from solution by electrolytes. Accordingly, all means of displacing albumins from solution will show an optimum at the point of electric neutrality, a condition which will be dealt with in detail later on. Generally, the difference between lyophobe and lyophile albumins lies in the fact that in the first case mere discharge of the.particles (e.g., by an electric field) leads to precipitation, while in the second case discharge must be combined with effects which lead to an alteration in the relation of the particles to the solvent, so that their lyophile character is reduced or lost altogether. Saturation of the solution with certain alkali salts, the addition of alcohol or phenols, etc., brings about the latter conditions. Such variations in stability as are indicated finally by a visible coagulation form yet another class of changes to which colloids are prone. There is no fixed nomenclature for these changes, which lie on the border line of the physical and the chemical, and they are termed in general the alteration of state of colloids. Such changes are classed as reversible or irreversible coagula- * Some irresoluble colloids, after precipitation by excessive dialysis, can be redispersed by addition of some electrolyte. This effect, often called peptisation, really depends on the formation of complex salts, as Zsigmondy has shown in the case of stannic acid gel. IN PROTEIN SOLUTIONS 17 tions. The process is termed reversible when a simple reversal of the procedure which led to coagulation causes redispersion. For instance, a jelly which has set on cooling melts again when heat is applied ; whereas a protein which has been coagulated by heating does not redissolve on cooling, and is therefore said to have undergone an irreversible coagulation. If, when we cause a coagulation to pass again into the sus- pension which originally existed, we proceed in this reversal by the same path as that traversed in the coagulation process, only in the opposite direction, so that the same state is produced by the procedure, independently of the order of the procedure, such a process is called a homodrome reversible change. If, however, 2, +-J FIG. i. Homodrome and heterodrome changes of state. the reversal must go by a path different from the original one, so that similar procedure gives rise to various states of existence in the colloid, the process is heterodrome reversible. Homodrome reversibility is only realised in practice in a very restricted range and in a gradual course of reversion of a colloid.* Another property of colloids which was noticed even by Graham is connected with their reversible coagulation ; that is, their tendency to alter with age, which gives them the qualities of a more or less unfinished product. The reactions and alterations incidental to the preparation of a colloid do not, in general, terminate with the appearance of the colloidal character, but gradually proceed further after this point is reached. Lyophobe colloids, in particular, show these changes, * Homodrome reversible changes, in the strict sense, do not occur in nature, but merely form an ideal case. Nevertheless, the above classi- fication has its practical uses. See Wo. Pauli, Naturw. Rdsch., 1902, 17, Nos. 25, 26, 27. i8 CONDITIONS OF STABILITY mostly in an alteration from a state of high dispersion to one of lesser subdivision and of slight stability which in the end brings the colloid or crystalloid impurities in the solution into activity, so that part of the disperse phase is precipitated. This effect is known as the ageing of the colloid ; and the fact that the response of the colloid to any action often continues to appear for some time after the action has ceased, the hysteresis of the colloid. This effect occurs equally with a stabilising or destabilising influence. Sols of metallic hydroxides, such as ferric hydroxide sol or eerie hydroxide sol, show on ageing a continuous decrease in viscosity and an increase in electrical conductivity.* As no change in the concentration of electrolytes can be demonstrated, a gradual decrease in the hydration of the particles must be assumed to occur with lapse of time. This effect is also noticed in another research on hydroxide gels.f Ageing effects are very important when dealing with proteins. Serum, which has been freed from insoluble globulins by pro- longed dialysis and subsequent nitration, must be allowed to stand for months until the further precipitation of globulin, which invariably occurs, is complete, and the serum is quite clear. The effect of storage and the changes in activity which depend on it are particularly important in the colloidal toxins and antitoxins. Graham used the term sol for a colloid system in the liquid state, while the name gel is applied when a partial or complete separation of the disperse phase has occurred, or when the colloid has set as a whole to a jelly. The term gel is, however, very difficult to define accurately, and is used in many different senses in the literature. A closer consideration of the gels, which we would differentiate as systems with a structure from the jellies, in which a demonstrable structure is not assumed and which correspond to the sols (Lottermoser), will follow later. In the following chapters we shall again make use of the views on the classification and alteration of state of colloids which we have explained above. * A. Fernau and .Wo. Pauli, Kolloid Zeitsch., 1917, 20, 20. t Wo. Pauli and J. Matula, Kolloid Zeitsch., 1917, 21, 49. CHAPTER III THE ELECTRIC CHARGE ON NATURAL SOLUBLE ALBUMIN: THE ISO-ELECTRIC REACTION WHILE the general experience of the behaviour of lyophobe colloids paves the way to a realisation of the connection between the electric charge and the stability of the disperse phase, it is obviously necessary to obtain definite proof that albumins behave in a similar way. A direct method of showing the charge on protein particles is that of investigating the direction of their motion in an electric *- s A M FIG. 2. Electrophoresis of albumin. field (electrophoresis). This investigation was first undertaken to find out the effect of the addition of various substances to the protein solution. A simple transport apparatus in which that portion of the colloid solution which is in the neighbourhood of the poles can be abstracted for analysis serves the purpose. Thus Wo. Pauli,* in the original investigation of natural pro- teins, used a series of three small beakers connected by syphons filled with water. Platinum electrodes were put into the two outer beakers, and after the current had been passed for some time the syphons were withdrawn and the protein content of the * Beitr. z. Chem. Physiol. u. Path., 1906, 7, 531. 22 20 ELECTRIC CHARGE ON NATURAL SOLUBLE beakers determined (e.g., by a nitrogen estimation by Kjeldahl's method). In this way it was shown that serum albumin, after the removal of electrolytes by dialysis for some weeks, tends to move but slightly in an electric field. This fact was only obvious on pro- longed electrophoresis. The addition of alkali or of alkaline salts causes a marked wandering to the positive pole, while added acid or acid salts promote a movement towards the nega- tive pole. Neutral salts of the alkali or alkaline earth metals do not have a noticeable effect on the direction of movement of the protein. When, however, electrolysis has produced acid at the anode and alkali at the cathode, these secondary products cause the albumin to become charged, so that positive albumin is produced in the acid, and negative albumin in the alkali. In consequence the protein is repelled from the corresponding pole and forced towards the centre. It is therefore necessary, in using the above apparatus, to control this electrical repulsion by determining the content of the central beaker. In the follow- ing tables several examples of results are given. The albumin content is measured by the volume of N/4 acid required to neutralise the ammonia formed in a Kjeldahl determination. (A = anode cell ; M = middle cell ; C = cathode cell.) Table i. Horse-serum dialysed for nearly Seven Weeks. 250 volts and 2 x io~ 5 amps. (At the end of the experiment there was no appreciable alteration in reaction in the three vessels.} Time of Electrophoresis. 3 hours. 6 hours. 24 hours. 48 hours. * A. M. C. A. M. C. A. M. C. A. M. C. 3-7 3-8 3-85 4- 3-85 3-95 4-1.5 4-05 3'8 4-03 4-8 3-9 3-85 3'75 3'75 4-0 3-95 4-0 4-55 4'i 3'8 4-03 4-8 3-9 To the anode, Indifferent. Indifferent. To the anode. with repulsion towards the central cell. ALBUMIN : THE ISO-ELECTRIC REACTION 21 Table 2. Effect of Addition of Acids and Bases. (Time 6 hours in all cases.) Horse-serum. + o'oiN acetic acid. Ox-scrum. + O'oiN hydrochloric acid. Horse-serum. + O'oiN sodium hydroxide. A. M. C. 7-05 7'55 8-20 7-00 7-45 8-25 To the cathode. A. M. C. 7-4 8-05 9-1 7'45 8-05 9-05 To the cathode. A. M. C. 8-3 7-65 6-8 8-3 7-60 6-8 To the anode. Table 3. Effect of Addition of Salts. (Time 6 hours.) Horse-serum. + o-oiN NaCl. Horse-serum. + o-oiN CaCl-2. Ox-serum. + o-oiN NaHCOo. Ox-serum. + o-oiN NaH 2 PO.i. A. M. C. 7-6 7-9 7-55 7'5 7'9 7'55 Indifferent, some repulsion. A. M. C. 7'4 7'5 7'4 7'4 7'6 7'5 Indifferent, slight repulsion. A. M. C. 10-15 8-25 6-25 10-00 8-25 6-2 To the anode. A. M. C. 7'95 7-85 8 '05 7-8 7-85 8-05 To the cathode. The investigation was made more sensitive later on by the construction of a more perfect apparatus * for detecting move- ment in the electric field. We will now describe this apparatus, as it is capable of wide application. A large U-tube is provided with a tap in the middle of each limb, and near the top of each limb two other U- tubes, about half as big as the central one, are fused on as shown. These two tubes are joined at their lowest points by a tube also provided with a tap. The large U-tube can be fitted from the bottom at C. It is charged in this way with the colloid under examination * C. Landsteiner and Wo. Pauli, " 25 Kongr. f. Innere Medizin, 1908." 22 ELECTRIC CHARGE ON NATURAL SOLUBLE i i until it is full to a level just above the two open taps. These are then closed, avoiding the inclusion of air bubbles. The rest of the apparatus, after cleaning, is then filled with a liquid of the same conductivity, e.g., a solution of KC1 or similar salt which is in equilibrium with the colloidal solution. This liquid is levelled by opening tap III., which is then closed, and I. and II. opened. The liquid lying above the colloid must be less dense than the colloid, and a clean line of division is thus obtained. When the cur- rent is switched on, negative colloids rise up through I., positive colloids through II. With a neutral sol no movement on either side occurs. At the end of the electrophoresis taps I. and II. are closed and the con- tents of the parts of the U-tube above the taps removed by a fine pipette and tested. The apparatus can also be used as a null instrument, for instance, to find the point when no migration of the albumin occurs, for which purpose filling with trichloracetic acid gives a high degree of FIG. 3. Improved electrophoresis apparatus. sensitiveness (Wo. Pauli and Samec). L. Michaelis introduces crystals of sulphosalicylic acid for the same purpose. This apparatus can further be used for the direct measurement of the rate of motion of charged colloid particles in the electric field, provided the distance which the boundary line moves in a given time is determined with a known potential gradient. By the use of this apparatus, it can be shown that proteins, such as albumin and glutin, regularly show an electro-negative ALBUMIN : THE ISO-ELECTRIC REACTION 23 charge when in the natural condition (Landsteiner and Pauli). The same fact was noted a little later and independently by F. Botazzi * and by L. Michaelis.t The latter used Landsteiner and Pauli 's apparatus, and, by employing non-polarisable electrodes, was able to work with colloids to which greater quantities of electrolytes had been added. An anode of silver in potassium chloride, and a cathode of silver in silver nitrate, or of copper in copper sulphate, or similar combinations, are useful for this purpose. It is always important to avoid potential differences at the junctions of the separating liquids by the use of solutions of as nearly as possible the same conductivity. The ions of the electrolytes should not differ greatly in mobility. In the apparatus described above, the short electrodes are situated so far from the surface of the colloidal solution that with o-oi N solutions no disturbances due to electrolysis occur if electrodes of platinum wire are used and the application of the current is not too prolonged. As at first albumin particles move slightly to the anode owing to their negative charge, and on addition of increasing quantities of acid become positively charged, it is clear that at a certain acid concentration an indifferent point in the sign of the charge will occur. Under these circumstances albumin shows no electrophoresis in either direction. Hardy called a colloid which showed no potential difference between the particles and the medium an iso-electric colloid. The application of the iso- electric reaction of proteins as an important method of differen- tiating between them has been emphasised in many cases by L. Michaelis, who has also devoted much time with most fruitful results to the theory and practical determination of the iso-electric point. The variation in electrical properties of the proteins on addition of acid or alkali is connected with the property they display of behaving as bases towards acids, and as acids towards bases. This property has been well known for a long time, and substances which combined the properties of a weak acid with those of a weak base were called amphoteric electrolytes by * Atti. R. Accad. Lincei, 1908, 17, 49-57. t Biochem. Zeitsch., 1908, 16, 81. 24 ELECTRIC CHARGE ON NATURAL SOLUBLE Bredig. The amphoteric electrolytes are generally asymmetrical with respect to their power of dissociating into H- or OH' ions, for either the acidity or the basicit}^ is predominant. Only water, which dissociates H 2 O < > H' -f OH' is symmetrically amphoteric. The application of the law of mass action to amphoteric electrolytes by G. Bredig, J. Walker and H. Lunden * has resulted in the development of a theory applicable to them which has proved particularly fruitful in dealing with the proteins. We will next give some applications of the mass action law to electrolytes, which we shall have to use repeatedly. / The dissociation of an acid, e.g., the weak and slightly ionised acetic acid CH 3 . COOH : " H- + CH 3 . COO' is expressed by the equation C H . X C C H a coo' = K X C C H S COOH, where C is the molecular concentration of the various substances, and K is the dissociation constant of acetic acid. The value of K for a weak electrolyte is largely independent of dilution (W. Ostwald). This equation holds for every solution, provided the proper molecular values are inserted, in this case those for the acetation, hydrion and undissociated acetic' acid. If the product on the left hand of the equation is increased by a rise in the concentration of acetations or hydrions, the proportion of undissociated acetic acid molecules is increased, as its ionisa- tion is suppressed. As the acetates of the alkali metals are ionised to a great extent in solution, the concentration of acetate ions can be increased by adding them to the solution ; while the hydrion concentration can be made larger by the addition of strong acids, such as hydrochloric acid. In general, the ionisation of a weak electrolyte is reduced by adding a strong electrolyte with a common ion. Compared with it, the converse effect of the weak electrolyte on the ionisation of the strong electrolyte completely disappears. Such mixtures of a weak electrolyte with a corresponding strong electrolyte with a common ion, e.g., acetic acid acetate mixture, or the combination ammonium hydroxide ammonium chloride, have an important distinctive property which is * H. Lunden, " Affinitatsmessungen an schwachen Siiuren u. Basen." Stuttgart. F. Enke. 1908. (Literature.) ALBUMIN : THE ISO-ELECTRIC REACTION 25 brought out clearly by solving the dissociation equation as follows : 'H- = K ^ r CCH..COOH In an acetate acetic acid mixture, the ionisation of the acetic acid, which is low to begin with, is so far suppressed that the acetate ions in solution can be regarded in practice as derived solely from the acetate. As the acetate is ionised to more than 90 per cent, in solutions below o-oi N, the concentration of the acetate can be written instead of that of the ion. Further, the concentration of the un-ionised molecules of acetic acid is practically identical with that of the acetic acid actually used. So that p TT ^acetic acid f ^acetate and similarly for the base, p -IT- , ^ammonia T xx Tr\ = 1*0 X 10 / _ ~r .MI-, v m 5~1 - I 77 x I0 J- ^ammon .chloride It is easy to see that the mere proportion of acid (or base) and salt determines the concentration of H- or OH' ions ; and as this proportion is independent of dilution over a considerable range, mixtures of weak acids and bases with highly dissociated salts provide a simple means of preparing in practice media of known H- or OH' concentration. Such solutions are readily reproducible, and proof against reasonable impurities or alterations in volume. They are called regulators or buffer solutions. The table on p. 26 gives an example of the ion concentrations in such solutions. This relation between weak and strong electrolytes with a common ion is of importance in the determination of the iso-electric reaction of albumin by means of regulators (L. Michaelis). Further, such solutions are useful for finding out how much of a weak acid or base is combined with albumin, and for estimating the hydrolysis of the salt-like protein compounds formed in this way (Wo. Pauli and Hirschfeld, see Chapter V.). 26 ELECTRIC CHARGE ON NATURAL SOLUBLE Table 4. (Valid for N/ioo and more dilute solutions.] Acetic acid: ace- tate. C H- Acetic acid : acetate. C H- NH.C1 NH 4 OH C*.(i8> NH 4 C1 NH 4 OH C*. (18) 32/1 16/1 8/1 2/1 5-76x10- 2-88x10- 1-44 X io- 0-72 X IO~ 0-36 X 10- T .Q -y jf) 5 1/2 1/8 1/16 1/32 T lf\A 0-9 X io~ 5 0-45 X IO~ 5 0-22 X IO- 5 O-II X TO" 5 0-56 X I0~ 6 32/1 16/1 8/1 2/1 I-O2 X IO~ 8 0-51 X IO- 8 O-26 X IQ- 8 0-13 X I0~ 8 0-64 X IQ- 9 I/I 1/2 1/4 1/8 1/16 0-32 X I0- 9 O-l6 X IO~ 9 0-8 xio- 10 0-4 x io~ 10 0-2 XIO- 10 I/I 1/04 * From the dissociation equation of water C R . X C QH , K^ (K^ = 0-58 X io- 14 at 1 8). The proteins, as we have emphasised above, are amphoteric electrolytes, behaving both as acids and as bases. They have both an acid dissociation constant K a and a basic dissociation con- stant K 6 , or, should they be poly-basic acids or poly-acid bases, more than one dissociation constant of each kind. We will not here discuss the observations so far made, but these show that if certain conditions for the determination of the iso-electric reaction are observed, a single mean constant K a can be taken as an index of the acid strength of the protein, and similarly K & as a measure of the basic character, without encountering any difficulty. This effect is in complete accordance with the general be- haviour of polyvalent acids or bases which dissociate in several stages ; for, as a rule, the first stage is large compared with the later ones, as indicated by the greatest development of H- or OH' ions. Most of the natural proteins that have been studied, whether simple or compound (albumin, globulin, glutin, casein, haemo- globin), show a stronger acid than basic character. As, theite- - fore, the .dissociation into hydrion predominates, so does the electro-negative character of the colloidal portion of the protein, as is shown by the marked anodic migration of the pure natural substance. It can be predicted from the fact that these proteins are built up of amino-acids, which themselves are more acid ALBUMIN : THE ISO-ELECTRIC REACTION 27 than basic in character, that such bodies will be amphoteric electrolytes with K a )> K b . The following table gives the dissociation constants of some amino-acids and di-peptides, and, for comparison, those of a few weak acids and bases which are occasionally used : Table 5. Dissociation Constants at 25 (when not otherwise stated). Substance. *. K b /- K.K, Glycin . o-Alanin 1-8 xio- 10 1-9 xio- 10 2-7 X IO~ 12 5-1 XIO- 12 2-6 X io- 1-9 X io- 4-86 XIO- 22 9-96 xio- 22 Leucin . . 1-8 xio- 10 2-3 X IO- 12 2-9 x io- 4-14 x io- 25 Tyrosin . 4-0 X io- 2-6 xio- 12 3-9 X io- 1-04 X lO"' 20 Histidin 2-2 X 10- 5-7 XIO- 9 6-2 X io- 1-25 Xio- 17 Aspartic acid 5 Xio- 1-2 XIO~ 12 i-i X io- 1-8 xio- 16 fi-i asparagin . 35x10- 1-53 Xio- 12 2-07 X io- 21 Methylglycin . 2 XIO- i -7 xio- 12 2-04 X io~ 22 Dimethylglycin Phenylalanin 3 XIO- 5 X IQ- 9-8 xio- 13 1-3 xio- 12 4-4 Xio- 6 1-27 XIO- 22 3-25 xio- 21 Glycylglycin . 8 xio- 2-0 xio- 11 3-0 X 10 ~ 6 3-60 X IO- 19 Alanylglycin . 8 xio- 8 2-1 XIO- 11 3-0 X io- 6 3-60 x io~ 19 Leucylglycin . *5 X io~ 8 3-1 XIO- 11 2-2 X IO~ 6 4-5 X io- 19 Lysin . . ; 2-O X IO~ 12 Succinimide . 2-8 xio- 11 Betain (trimethyl- glycin) 7-0 X io- 13 Urea . 1-5 XIO- 14 Guanin . 8-35 xio- 12 (40 C.) Creatine i -87 xio- 11 (40 C.) Aniline . . 4-6 x io- 10 Ammonium hydrox- ide . 1-87 XIO- 5 Acetic acid 1-86 xio- 5 Lactic acid 1-38x10-* Succinic acid . 6-7 XIO- 5 Without treating the question exhaustively we can discuss on the basis of this table the relation of the dissociation con- stants to the constitution of the amino-acids, which is not without importance for the proteins. Glycin, alanin, and leucin have the same K , and their slightly different basic dissociation constants are of the same 28 ELECTRIC CHARGE ON NATURAL SOLUBLE order of magnitude. We can conclude, after inspecting their constitutional formulae, that, with the NH 2 -group in the oc-position, the length or ramifications of the carbon chain have but little influence on the properties of these amphoteric electrolytes. CH 2 (NH 2 ) CH 3 CH 3 CH OOH CH(NH 2 ) CH Glycin. COOH CH 2 Alanin. CH (NH 2 ) COOH Leucin. The introduction of a methyl group in place of the hydrogen of the amino-group causes a slight weakening both of K a and of K & in the case of glycocoll. On the other hand, in the series alanin, phenylalanin, ^-hydroxyphenylalanin (tyrosin), an obvious increase in the acid character can be seen, while the basic properties are but little affected. The formation of a dipeptide from a simple amino-acid results in a large increase of K ff (by io 2 ), and a decrease of K & (by one power of ten only). The combination of either alanin or leucin with glycin to produce a diglycin produces identical properties in the product. It is improbable that the separation of the amino- and carboxyl-groups, which adjoin in the simple a-amino-acids, is sufficient to account for the alterations in ionisation, but it is quite possible that the acid character of the peptide linkage, especially in the lactim form, may play an essential part. This is in agreement with our experience of its function in the formation of protein salts. The distance between the NH 2 and the COOH -groups is the same for glycyl-, leucyl-, and alanyl-glycin, while the rest of the carbon chains, of varied length, become branched. The latter, both in these cases and in the simple amino-acids we have con- sidered, appear to have no influence on the dissociation. A comparison of the amino-mono- and the amino-di-car- ALBUMIN : THE ISO-ELECTRIC REACTION 29 boxylic acids with the corresponding acids of the normal aliphatic series is of the highest interest. Aspartic acid is related to succinic acid in the same way as glycocoll is related to acetic acid. The relationship becomes still clearer by com- parison with the corresponding hydroxy-acids : CH 3 COOH -* HO . CH 2 . COOH > NH 2 . CH 2 . COOH Acetic acid. Glycollic acid. Glycocoll. CH 2 .COOH HO. CH COOH NH 2 .CH.COOH CH 2 . COOH CH 2 COOH CH 2 COOH Succinic acid. Malic acid. Aspartic acid. The introduction of NH 2 into the acetic acid molecule causes a vast decrease in K a , which drops from 1/86 x io~ 5 to 1*8 x io- 10 . On the other hand, on forming the amino-acid from succinic acid the acid dissociation constant rises from 6-7 x io- 5 to 1-5 x io~ 4 . The basic dissociation due to the amino- group in glycocoll and in aspartic acid does not, however, differ greatly in the two cases. If the carboxyl-group in aspartic acid is transformed into an amido-group asparagin is formed, and K a falls by io 5 to about the same order of magnitude as that of the amino-monocarboxylic acids. The basic dissocia- tion is not, however, increased, but remains rather below that of the simple amino-monocarboxylic acids. The basic character of the amino-group introduced is almost completely nullified by the acid amide linkage. From these examples it can be seen that alterations in K a and Kb are very varied and relatively independent of known constitutive alterations. Further, it is impossible to conclude that because two amphoteric electrolytes have the same K a and K 6 their constitutions correspond to any great extent. In order to understand the alterations in state of the proteins, it is of great importance to have an accurate knowledge of their dissociation when in the iso-electric state. The latter state is indicated by a lack of motion in either direction in the electric field, and shows that an equal number of electro- negative and electro-positive particles are present. The following considerations were originally brought forward by L. Michaelis, and further developed from one point of view by 30 ELECTRIC CHARGE ON NATURAL SOLUBLE S. P. L. Sorensen, and in applying them to the iso-electric state a restriction must be observed which has not so far been suffi- ciently emphasised. Two kinds only of ionised protein particles (negative and positive), which have originated exclusively from a dissociation into either H- or OH' ions, must exist in the solution. The importance of this restriction in theory and practical work will appear later on. For a given concentration of protein (or other amphoteric electrolyte) let K a and K& be the acidic and basic dissociation constants, C A - the concentration of anions, C K ' of cations, while C H - and C H' are the concentrations of H' and OH' respectively, and x that of the undissociated portion ; then the equilibrium equations are : C A .C H = K.* .......... (I.) anci C K . CQH = K 6 . x ......... (II.) At the iso-electric point C A = C K by definition, and by division I. and II. give for the dissociation equilibrium : The behaviour of the undissociated portion at the iso-electric point is of great practical interest. This portion (x) equals the total concentration (n) less the concentrations of the protein ions, thus : x = n CA C K ; substituting from I. and II., hence and x i .; *-"-77g+ The undissociated fraction, p, is the ratio of the concentration ALBUMIN : THE ISO-ELECTRIC REACTION 31 of neutral particles to the total concentration, and is also the relative measure of the undissociated part of the amphoteric electrolyte. The dissociation equation of water (of which the normality then remains constant at 1000/18) can be applied to aqueous solutions when the concentration is not too high : CH.COH-K* . . . , >\ . . . . (IV.) T Where K, = i-i X io~ 14 at 25, and, substituting -~ for C n, ^H we get P = ~~V K ?*~ ! + K, + K_CH CH r^w in which p is merely a function of C H and is at a maximum when the expression x + K + K_CH = ^ CH Aviv c has a minimum value. Differentiating, we get du K a K 6 rfC^ = ~C^ 2 " I "K W ' whence the condition for a minimum value, -W- = o, leads to K 6 K the equation ^- ^ ^. Substituting from IV., K" r * J^a ^H Thus p has a maximum value at the iso-electric point (see equation III.). The undissociated fraction, that is, the ratio of the number of electrically neutral particles to the total concentration, is at a maximum at the iso-electric point. If values of p and P H are plotted on a graph, this relation is clearly seen. The low concentrations of hydrions are plotted in negative powers of ten, i.e., C H = IO~ PH . The number P H (= logioC H ) is called the hydrion exponent by Sorensen, * For comparisons with the same protein, the above equations are valid both for molecular and for weight percentage concentrations. 32 ELECTRIC CHARGE ON NATURAL SOLUBLE and the use of this notation simplifies both calculations and the construction of the graph. In Fig. 4, the abscissae are the hydrion exponents, P H , while the ordinates are the values of p, the undissociated fraction. As the highest value of the con- centration of neutral particles (x) is when this coincides with the total concentration of the dissolved substance, the maximum value of p is i. For this value all the particles are electrically neutral, and the dissociation is nil. It is clear that for amphoteric electrolytes with the same value of the product K fl . K b , the p P H curves will be identical ; only the position of the curves will be displaced to the left 01 ^v d 56 7 8 9 10 11 f* *3 W FIG. 4. Curves of the undissociated fraction (p) for = no x 10-14. with increasing K , and to the right with increasing K b . Curves II. and V. are those for amphoteric electrolytes with product K fl . K b = io~ 16 . In curve II. K a = K 6 = io~ 8 , while in curve V. K a = io~ 3 and K 6 = io- 13 . The maximum propor- tion of neutral particles, i.e., the iso-electric point, is in the first case (II.) at P H = 7 (C H = io- 7 ), and in the second case (V.) the increase in K a shifts it to the left at P H = 2. The dependence of the form of the curve on the value of the product K a . I. II. III. IV. is clearly seen in curves I. IV. K a = K b = io- 7 K a = K b == io- 8 K fl = K 6 = io- 9 K a = K b - io- 11 K a Kfi = io- ] K a Ka K 6 = io- 16 K b = io- 18 K fe = io- 22 K a /K 6 is constant for curves I. to IV. and P H has thus the ALBUMIN : THE ISO-ELECTRIC REACTION 33 same value. The smaller the product K a . K 6 the higher and the more flattened the crest of the curve. When K a . K 6 = io- 18 , the highest value of the ordinate, p = i, is attained, and amphoteric electrolytes with K a . K 6 < io- 18 give no sharp iso-electric point, but a more or less wide iso-electric zone, for no definite movement in the electric field occurs over a con- siderable range of variation of P H . When, on the other hand, the value of K a . K b increases, the curve rises to a lesser height, and, for a value of K a . K 6 = io- 12 , reaches a value p = o. That is, neutral particles are no longer found, as the amphoteric electrolyte is completely dissociated. Consequently, to -obtain a sharply defined iso-electric point, it is necessary to confine the value of the product K . K b between the limits io~ 18 and The mechanism of the iso- electric reaction on the basis H OH of our deductions and the necessary restrictions can be illustrated as follows. Let x be the neutral particles and H OH A" and A+ the anions and FIG. 5. Diagram o the iso-electric , / i i j j reaction in buffer solutions. cations of an ampholyte due to the H+ and OH~ ions present. In the case of egg albumin A~ > A+ and the conditions are represented by diagram I. (Fig. 5)- On addition of acid, the ionisation represented by A~H+ is suppressed, until at the iso-electric point A" = A+, resulting in the effect shown by diagram II. The iso-electric condition is thus characterised by a decrease in the negative ionisation of the albumin A~ and an increase in the number of neutral particles x. The considerations which now follow are valid only for this mechanism of reaction, which, as we shall see later, is not the only possible one. The determination of the hydrion concentration at the iso- electric point leads directly only to a knowledge of the value T of . The aim of a complete electro-chemical characterisation 34 ELECTRIC CHARGE ON NATURAL SOLUBLE of natural proteins is, however, the evaluation of the values of both K and K ft . The methods employed for measuring these two constants in simpler amphoteric electrolytes, especially those directed to the determination of the dissociation equili- brium of their salts, are scarcely applicable to natural proteins, for reasons which will appear below. More recent attempts to determine K a and K 6 directly by a special method are not yet concluded. TT The methods hitherto employed for determining ~ depend ft-fc on two principles : I. The direct discovery of the iso-electric condition of a protein by means of the application of an electric field in an electrophoresis apparatus, the hydrion content of the solution being varied. II. The determination of the hydrion concentration which shows, by various tests, a maximum of neutral particles. We must next make some observations on the first method of determining the iso-electric point by electrophoresis, the discussion of the second method being deferred to the following chapter.* The technical difficulty of this determination lies mostly in the fact that even with the most precise procedure the change from movement to the anode to movement to the cathode does not occur at a sharply defined point. It more frequently happens that the movement is in both directions (or, less frequently, no movement occurs) over a wide range of hydrion concentration. Sometimes, indeed, in different observations the direction is uncertain. One is therefore forced to take the mean of the limiting values which show definite migration in either sense. The following facts are important when considering migration in both directions. If the electrophoresis apparatus is filled and left to itself with the taps open, traces of albumin can be * A third and recent method of determining the hydrion concentra- tion which remains unaltered on varied additions of ampholyte is due to Soiensen, and will be dealt with at the end of the following chapter. ALBUMIN : THE ISO-ELECTRIC REACTION 35 detected above the taps after half an hour, even when no current is applied. This diffusion is more noticeable in concentrated solutions of protein. There is thus a definite tendency for the protein to migrate in both directions, which is affected by an electric field when the protein is charged. The diffusion in one direction is then increased, and in the opposite direction it is decreased. As the iso-electric point is approached, the directive influence of the electric field continually decreases, until conditions finally approximate to those which obtain when no current is passed through the apparatus. The presence of notable quantities of the ions A~ and A+ in approxi- mately equivalent proportions also has an important effect in the region of the iso-electric point, and may possibly cause migration to a slight extent towards both electrodes. The products of electrolysis are a frequent source of error when higher concentrations of electrolytes are employed. This disturbing influence can be avoided by the use of unpolarisable electrodes (L. Michaelis) , or even in specially favourable cases by preserving a considerable space between the electrodes and the surface of the colloid. A sufficiently large space round the electrodes (Landsteiner and Pauli) and the restriction of the time of passage of the current also decrease the electrolytic effect. Finally, it is necessary, in order to reduce the develop- ment of breaks of potential and secondary electrodes in the liquid, to use as connecting liquids solutions of practically the same conductivity and with ions which differ but little in mobility (Pauli and Flecker *) . Such secondary electrodes are the chief causes of fluctuations in the sign of the charge in the iso-electric zone, in which, naturally, discharge of the colloid most easily takes place. The following table gives some values for hydrion concentrations (C H ) at the iso-electric point deter- mined by electrophoresis. All the results refer to room tem- perature (18). The work on albumin and glutin has been repeated by Pauli and Samec, with identical results. In all cases hydrion regulators, mostly acetic acid acetate mixtures, were used, but for haemoglobin, phosphate mixtures and caco- dylic acid sodium salt buffers were also employed. * Biochem. Zeitsch., 1912, 41, 995. 3-2 THE ISO-ELECTRIC REACTION Table 6. Protein. C H (found). C H (mean). Author. Albumin (about i-i 4-2 X io~ 5 2-O X IO~ 5 L. Michaelis and H. Davidsohn, 0-6 per cent.)- Biochem. Zeitsch., 1911, 33, 456. Glutin (0-5 per i'6 3'5 X io- 5 2-5 XIO- 5 L. Michaelis and W. Grineff, cent.). Biochem. Zeitsch., 1912, 41, 373- Casein (0-2 per 1-29 4-9 X io- 6 2-5 X I0~ 5 L. Michaelis and H. Pechstein, cent.). Biochem. Zeitsch., 1912, 47, 260. Haemoglobin . 0-92 2-8 X io~ 7 1-8 x io- 7 L. Michaelis and H. Davidsohn, Biochem. Zeitsch., 1912, 41, 102. CHAPTER IV THE PROPERTIES OF PROTEINS IN ISO-ELECTRIC REACTION THE alterations in properties which natural proteins display in the iso-electric region can, generally speaking, be explained by the difference in physico-chemical behaviour between neutral and ionised protein particles. The relation between the alterations in natural proteins and their electric charge has been worked out in a series of researches by Pauli and his collaborators, with which we shall now deal in detail. We propose briefly to anticipate here such results as appear neces- sary for the understanding of the discussions which follow. Neutral albumin shows lower hydration or imbibition, lower viscosity and lower osmotic pressure than ionised albumin. In contrast with electrically charged albumin, it is coagulated both by heat and by the addition of alcohol, and, similarly, only neutral particles form the substratum of coagulation by salts, acids and bases. A small addition of neutral salts stabilises neutral albumin against both spontaneous coagulation and that caused by alcohol or by heating ; whereas it causes ionised albumin to be discharged and dehydrated, and to become sensitive to coagulating agents. The methods which employ the properties of neutral albumin for the determination of iso-electric behaviour work most simply for those natural proteins of which the neutral -particles are not stable in solution owing to their lyophobe character. Casein and the globulins are the most important representatives of this class, and in consequence they show a maximum of precipitation at the iso-electric point. The location of this maximum in mixtures of regulators of varied hydrion concen- tration is easily carried out. The values given in the following table were obtained in this way. THE PROPERTIES OF PROTEINS Table 7. Maxima of Precipitation. Substance (under i per cent.) C H (found). C H (mean). K a K 6 Author. Serum globulin . 2-9 4-6 X IO- 8 3-6 X lo- 6 2-2 X IO-* L. Michaelis and P. Rona.* Edestin . ca. 1-3 x io~ 7 1-4 X io~ 7 2-8 X 100 L. Michaelis and P. Rona. Gliadin . 5 -8 7-5 xio- 10 6-0 xio- 10 6-0 x io-' L. Michaelis and P. Rona. Casein (from milk) 2-0 2-8 X I0~ l 2-4 x io- L. Michaelis and H. Pechstein-t * Biochem. Zeitsch., 1910, 28, 193. f Biochem. Zeitsch., 1912, 47, 260. The iso-electric point for hydrated natural proteins like albumin and glutin can be found by a greater variety of methods depending on the properties of the neutral particles. The simplest of these is the method of precipitation by alcohol, as worked out by Fault in conjunction with J. Matula and M. Samec. By suitable alteration of the protein concen- tration and of the quantity of alcohol added, an extremely sharp location of the maximum of precipitation is possible. The following table gives examples from such investigations * Table ya. Ox-serum Albumin. (1-8 c.c. alcohol to 3 c.c. mixture.) Acetic acid: acetate. C H 0-2 % albumin. albumin. i-o % albumin. i % albumin. 30 c.c. albumin + 10 c.c. alcohol, milligrams precipitated i 3 0-6 X io- A A z 1 6- 1 (no addition). I 2 0-9 x io- db i 17-25 I I 1-8 X .10- * * 4* 39-22 2 I 3 i 3-6 x io- 5-4 X io- V V 29-22 19-43 4 ! 8-2 x io- ~ + + * Unpublished results of Pauli and Samec, 1912-13. IN ISO-ELECTRIC REACTION 39 on albumin (from ox-serum) and on glutin, both carefully dialysed for many weeks. Table 8. Glutin A (" deutsche Goldmarke"). i per cent, final concentration N/ioo acetate. C H (acetic acid acetate). Relative co-efficient of viscosity. Precipitation by alcohol. 0-6 X IQ- 4954 0-9 X io- 4885 1-8 X io- 4769 * * 3-6 x io- 4977 5'4 X io- 5509 7-2 X io- 5 5996 Acetic acid acetate mixtures were used as hydrion regulators ; the degree of flocculation is indicated by crosses.* Experience has shown, fur- ther, that the maximum of neutral particles is also ex- pressed in a minimum value of the viscosity, corresponding to the general difference of viscosity between neutral and ionised protein particles. Fig. 6 (from a research by Pauli and Matula f ) shows the depression in viscosity at the iso-electric point in the > 6. Viscosity of glutin at the iso-electric point. case of a i per cent, glutin at * r ^ 35, with acetic acid acetate mixture. There is good agreement in this instance, as in similar investigations (e.g., with serum containing globulin), between the viscosity minimum and the * slight, deeper, very deep turbidity. The signs + > + -K + + + , indicate visible formation of precipitate of the same degrees of opacity. f Kolloid Zeiisch., 1913, 12, 222. 40 THE PROPERTIES OF PROTEINS maximum of precipitation by alcohol. Further, the P H corresponding to these points agrees satisfactorily with the iso-electric point determined by electrophoresis. Finally, the osmotic pressure of a protein in the presence of a buffer solution shows a clear and easily reproducible minimum at the iso-electric point. As the ionic dissociation of the protein particles produces an increase of the osmotically effective concentration under the ordinary conditions of experi- ment, so the formation of neutral particles, at the expense of the ionised particles, causes a diminution in the observed osmotic pressure. As the protein is ionised with liberation of H', metal, or acid ions these kinds of ions are retained by the electrostatic attraction of the colloidal protein ions ; the latter cannot penetrate the osmotic membrane. The imbibition of glutin discs is analogous to the osmotic behaviour. Table 9. Glutin 0-5 per cent., N/ioo Acetate. Acetic acid : acetate. Migration. Osmotic pressure in millimetre water. Precipitation by alcohol. O To anode 106 o : i ,, . . . . 73 i : 4 ,, 5 1 ' 3 a 4- i : 2 i : i Changing amphoteric : To cathode 44 * 49 * db 2 : i > ... 3 ' i ... Imbibition of 5 per cent, gelatin. Acetic acid : acetate. Dialysed for three days. Per cent, water taken up. j 3 16-9 i 2 9'5* i I 10-5 2 I 36-8 IN ISO-ELECTRIC REACTION 41 The theory of the iso-electric reaction due to L. Michaelis * implies, as already emphasised, the assumption that only two kinds of ions, A+ and A~, are present, arising from the dissocia- tion of OH~ or H+ respectively. The following equations then hold good : K 0= [Al]-CH and Kft = [A+L COH- T At the iso-electric point A+ = A- and C OH = r^> so that the ^H hydrion concentration is _ Consequently, the hydrion concentration at the iso-electric point should, at a given temperature, be a constant, quite independent of the dilution of the albumin. The work of Michaelis and of Pauli and Samec on electrophoresis, as also that of the two latter on precipitation by alcohol, does actually show that the hydrion concentration is independent of the content of protein over a considerable range (Table 7a). An experimental proof of this rule exists, however, only when acetic acid acetate mixture is used, whereas the theory of the iso-electric reaction was extended by its author to the case of any acid, regarding the hydrion concentration present as alone decisive. Sorensen | has formulated this view as follows : " To produce in an aqueous solution of an ampholyte the hydrion concentration corresponding to the iso-electric point, so much acid must be added to it (if K a > K 6 ) that the concentra- tion of acid equals the difference between hydrion and hydroxyl ion concentrations at the iso-electric point. The quantity of acid is therefore independent of the ampholyte concentration, and is the same as the amount required to impart to pure water the hydrion concentration existing at the iso-electric point of the substance in question." L. Michaelis { states quite generally: " If a soluble ampholyte is added to a solution of definite hydrion concentra- tion, it behaves like any acid when the [H*] is less than that at * See " Handbuch der Biochem." Supplement, p. 24 ff (Jena, 1913), and S. P. L. Sorensen, Ergebn. der PhysioL, 1912, 12, 503. t Ergebn. der PhysioL, 1912, 12, 503. J Biochem. Zeitsch., 1912, 47, 251. 42 THE PROPERTIES OF PROTEINS the iso-electric point, and as a base if the [H-] is greater than that value. In other words, the ampholyte, like any acid, increases [H-] in the first case quoted ; in the second, like any base, it reduces the [H-]. If, however, the original [H-] is just equal to that at the iso-electric point the added ampholyte pro- duces no change in its value." It remains now to be shown that these considerations do not hold good for strong acids and albumins free from salts (Pauli and Samec*). These authors found, in fact, that on addition of a strong acid a concentration was reached at which a maximum of neutral particles was present, which could be demonstrated both by viscosity measurements and by the alcohol precipitation. This point, however, unlike that obtained by the use of buffer solutions, is largely dependent on the albumin concentration, and in less degree on the nature of the acid. The examples given below and on p. 43 demonstrate this. The determination of the osmotic pressure also shows the existence of a minimum value. The osmotic pressure is mea- sured against an aqueous solution of the same acid content as the original mixture of acid and albumin put into the osmotic cell. During the twenty-four hours required for the pressure to become constant, acid enters the cell from the outer vessel, Table 10. Viscosity of Glutin + Sulphuric Acid at 35. t _ Time of flow of the solution t f . Time of flow of water Concentration of acid. Glutin 1-09%. io~ 4 was found to obtain.* * In a later research of Pauli and T. Oryng (Biochem, Zeitsch., 1915, 76, 373) it is shown that using i per cent, serum albumin in a calomel electrode for determining the concentration of chlorions, a chlorine concentration of about 3 x io- 4 normal is developed compared with the solubility of mercurous chloride in water, i x io- 5 N. This increase in [Cl 7 ] is due to formation of complex chlorides by the calomel and the albumin. On addition of acid, the mercurous ion is displaced from the albumin, and hydrion takes its place. In the above case we should equate the chlorion concentration, which arises from the salt formation between the HC1 and the protein, to the normality of the acid added. For the hydrion is practically completely combined, while the IN ISO-ELECTRIC REACTION 45 It is certain from this work by Pauli and Samec that when strong acids are added to albumin or glutin these proteins show a number of properties which agree with those found by means of buffer solutions at the iso-electric point. The hydrion con- centration at which this effect occurs is, however, far below that determined by acetic acid acetate mixtures for the iso- electric point. This is accompanied by a relatively large quantity of acid entering into combination for i per cent, albumin or glutin it is of the order of io~ 4 N/i. The equation of electrical neutrality in which, as is well known, the sum of positive ionic charges is equal to the sum of negative ionic charges in any given mixture of electrolytes, for the point of charge in precipitation by alcohol and in viscosity found in the above investigations is as follows : CQ- + COH- + C A - = C K + + C H + or, inserting the values in powers of ten which have been determined : io- (CIO + io- (OH') + C A - = C A + -f io- 6 (HO- In any case, at this point the concentration of negatively charged albumin must be much reduced compared with the positively charged portion. In fact, in electrophoresis experi- ments in this region, migration to the cathode is exclusively observed (Table 15). With strong acids, therefore, the maxi- mum of neutral particles does not coincide with the point of iso- electric migration. Apparently, with strong acids, in lower concentration than the P H of the iso-electric point found by buffer solutions, a regular combination with the albumin occurs. This com- bination increases with further addition of acid with formation of new positive albumin ions. One can conclude theoretically that in this case also a parti- cular acid concentration must exist at which the originally anodic migration changes to a cathodic migration, giving rise chlorion remains completely dissociated not only at such concentrations, but even with much higher content of HC1 in a 5 per cent, albumin solution. In the proof appended of the extinction of the positive albumin ions, the chief emphasis must be laid on the observed migration to the positive pole rather than on the excess of chlorine ions present. 4 6 THE PROPERTIES OF PROTEINS to iso-electric effects. This change point can also be demon- strated in the electrophoresis apparatus. For glutin the con- centration of added acid which produces this effect lies between 7 x io- 5 and 10 X io~ 5 N/i HC1. The following table gives some collected results for glutin B. Table 15. Electrophoresis of Glutin (i per cent.). (75 volts for one hour at 35.) N . HCl. o 2-5 X IO~ 5 5 X IO- 6 7-5 X IO- 5 10 X io- 5 Migration. To anode. To cathode. For the same sample of glutin the maximum number of neutral particles occurs in i x io- 3 N.HC1. Table 16. Precipitation and Viscosity of 0-5 per cent. Glutin. N . HCl. Relative Viscosity at 35. Saturation with toluene. io c.c. solution + 3 c.c. alcohol. Depth of the centri- fuged precipitate in millimetres. O 1-285 O 0-5 x io~ 3 r-245 A A negligibly small. I X IO~ S I-2I3 + + * * 35 * 1-25 X io- 3 1-204 * V + V 24 1-5 X io- 8 I-2I5 + + 21 2 X IO- 3 I-23I 19 At the point of iso-electric migration the chlorion concentra- tion is certainly from 7 io X io- 5 N. This is only a mini- mum value, in view of the possibility of slight diffusion into the albumin from the acid of the same dilution which lies above it in the electrophoresis apparatus. In this protein-acid mixture the hydrion concentration is at most io- 6 N. In any case IN ISO-ELECTRIC REACTION 47 there occurs at the change point in electrophoresis a consider- able combination of acid and consequent formation of electro- positive albumin ions. So that a much larger addition of acid is necessary than the iso-electric reaction found by Sorensen and Michaelis would lead us to expect. The considerations developed by these authors for ampholytes in general, therefore, do not hold for proteins and strong acids. The relatively simple results obtained by the use of hydrion regulators con- taining weak acids exhibit only a particularly favourable limiting case, which varies progressively. Any theoretical expression of the reactions of albumin with strong acids in low concentration must accordingly take into account the following facts : 1. Addition of strong acids to albumin gives a point of symmetrical electrophoresis and apparently iso-electric behaviour. 2. In these mixtures the concentration of hydrion is very low, and much smaller than that which obtains at the iso-electric point as found by acetic acid acetate buffer solutions. Thus for i per cent, glutin and albumin it is of the order of io~ 6 . 3. The negative ion of the acid is abundantly present and free in this range of concentration, and there is an excess of electro-positively charged albumin over that which is negatively charged. 4. Further addition of acid produces a maximum of neutral particles, as shown by maximum precipitation by alcohol and minimum viscosity, the electrophoresis being entirely to the cathode in this region. 5. The quantity of acid necessary to produce symmetrical electrophoresis or maximum of neutral particles in- creases with a rise in the albumin content. A comprehensive view of the complicated relations governing such cases may be gathered from the following considerations. The strong combination of acid with the protein is certainly demonstrated and must be due to development of activity of basic valencies of the protein which do not function in the pre- sence of very weak acids such as exist in acetic acid acetate 48 THE PROPERTIES OF PROTEINS mixtures. In other words, the mean basic dissociation constant Kb is increased. The hydrion concentration necessary for the development of iso-electric behaviour is decreased by this increase in K&. Such an effect is actually observed in the results given above for the effect of adding strong acids. The so-called iso-electric behaviour culminates in the migra- tion of equal quantities of albumin in both directions, or by symmetrical repulsion, when an electric field is applied. This result, however, only proves an equal volume concentration of positively and negatively charged quantities of protein pro- vided that the electro-positive and electro-negative protein particles transported have the same valency. For, suppose the positive particles are w-valent, and the negative ones mono- valent, then the same weight contains n positive charges, but only one negative charge, so that when one unit of elec- tricity is transferred, the quantity of albumin which migrates towards the negative pole is -th of the quantity which goes towards the positive pole. The quantity of albumin trans- ported in each direction is the same only when n times as many positive charges are brought by the albumin to the cathode as there are negative charges brought to the anode. We can, by this time, follow the main lines of the complex mechanism of the reaction of albumin with strong acids if three principles which are in agreement with all previous investigations are grasped. 1. When a strong acid is added it reacts with the basic part of the albumin A+OH in accordance with the equa- tion, for example : A+OH + HC1 A+C1 -|- H 2 0. If the acid is strong enough, it reacts with further basic valencies of the protein when more acid is added, thus : A+C1 + HC1 > A++C1 2 , forming polyvalent ions. (See the next Chapter.) 2. Increase in [H-] represses the ionisation of the acid portion of the protein A~H with formation of neutral particles, which can, however, react by means of their basic valencies when more acid is added. IN ISO-ELECTRIC REACTION 49 H Cl A ** A n 3. The ions formed from pure albumin and weak acids and bases can be considered monovalent in contra-distinc- tion to those of the protein salts. The addition of strong acids to albumin gives rise to four different effects in the solution which are shown by the points a, b, c, d (Fig. 7). In illustration of these we shall adopt the scheme previously employed, and, to simplify notation, choose the reaction with hydro- chloric acid as an example. a. The first point which is reached when acid is gradually added is due to the reaction of A+OH with HC1 *- U 717 *" and a slight repression of the ionisation of A~H, so that hydrions and chlorions are present in equal num- bers, as also are the charges on the negative and positive albumin.* This is a true iso-electric point which, how- ever, can only be found by an electrometric determina- tion of [H-] and [Cl']. It occurs at such a low con- centration of acid that the small concentration of at Cl FIG. 7. a, true iso-electric point. b, point of iso-molar migration. c, the maximum of neutral particles. d, maximum combination with acid. chlorions cannot be measured. Under these conditions the protein migrates to the anode. b. As more acid is added a further point is reached, when the albumin changes its direction of migration and moves towards the cathode instead of towards the anode. This change point in electrophoresis is only apparently iso-electric, as proved by electrometric measurements, which show an excess of negative Cr ions over the positive H- ions, the latter having a concen- tration of 2 x io- 6 the former of 07 i-o X io~ 4 . One is * The OH' ions which are present according to the equation CH COH ~ K w are here omitted. 50 THE PROPERTIES OF PROTEINS forced to the conclusion that the same mass of albumin carries many more positive than negative charges, so that the valency of the positive albumin ion must be assumed to be relatively high. This point can therefore be described as iso-molar with reference to electric transport, and as aniso-electric with respect to the albumin ions. c. Still further addition of acid produces purely cathodic migration of the albumin, but at the same time the number of neutral particles reaches a maximum, as is shown by a maxi- mum in the quantity precipitated by alcohol, and a minimum of viscosity, which occurs at approximately the same point. The [Cr] now exceeds i-o x io~ 3 , but the [H>] is still 2 x io- 6 . Taking into consideration the equation of electrical neutrality and also the strong migration to the cathode, we conclude that the negative ionisation of the albumin is repressed almost to extinction owing to the formation of neutral particles, which indicate their existence by their precipitation by alcohol and by the decrease in viscosity. The minimum osmotic pressure, however, does not occur at the point c of maximum neutral particles, but at an acid concentration which lies between b and c, at which [Cl'] for i per cent, glutin is 3-8 x io~ 4 and for albumin 57 X io~ 4 (see Tables 13 and 14). The explanation of this lies in the fact that the ionisation of A n +. nCl gives rise to an increase in osmotic pressure which counteracts, and finally exceeds, the decrease caused by the formation of neutral particles. In consequence of this effect, the pressure minimum lies at an acid concentration below that represented by the point c. d. Above the point c, further quantities of acid convert the remaining neutral albumin into acid albumin up to the point of saturation of the albumin with acid, when a maximum of ionisation represented by d is reached. The properties of the acid albumin in this zone will be discussed in the next chapter. The rough diagrams of Fig. 7 serve to illustrate the points at which the properties of acid albumin undergo change. It will be easily understood from them that, with this mechanism of reaction, the quantity of acid required to reach one of these points depends on the albumin concentration ; it will be IN ISO-ELECTRIC REACTION 51 equally clear that, owing to the almost complete combination with the acid which occurs, the free H' ions, over a wide range preserve a low concentration, which fluctuates only between narrow limits. On account of the differing valency of the negative groups present, and of the newly-formed positive albumin ions, points which are apparently iso-electric appear when considerable combination with acid occurs. These points have many pro- perties in common with the true iso-electric points found by using hydrion regulators, but they must be clearly distinguished therefrom. The main difference between the two cases lies in the fact that the first three points (a, b, c) are merged into a single point when regulators are used, whereas with strong acids they lie wide apart. Nevertheless, the difference must not be considered absolute. With regulator mixtures, which greatly reduce the ionisation of the acid employed, the forma- tion of polyvalent albumin ions is largely prevented. Conse- quently there is to some extent a satisfactory agreement between the properties of albumin and those required by the theory of Michaelis, which holds strictly only for amphoytes of simple structure.* S. P. L. Sorensenf has quite recently made use of the rule that the quantities of acid or base required to make a solution iso-electric are independent of the concentration of ampholyte as a means of determining the iso-electric H* ion concentration. To this end a concentration of H- ion was sought which remained unaltered on addition of any arbitrary quantity of amphoteric electrolyte. A close examination of Sorensen's conditions of work is necessary in order to test how far his results can be reconciled with the quite contradictory ones obtained in the case of strong acids. Sorensen used for his experiments egg * From this point of view, the importance of the fact that slight variations in the behaviour of the components of the buffer mixture produce small divergences from the point of change of the properties of the protein is brought out. These variations show a certain conformity to those larger differences shown when strong acids are used. Moreover, the hydrion concentration which causes minimum viscosity is regularly slightly greater than that at which the precipitation by alcohol shows a maximum. f Zeitsch. physiol. Ghent., 1918, 103, 192. See also A. Hasselbalch, Biochem. Zeitsch., 1916, 78, 129. 42 52 THE PROPERTIES OF PROTEINS albumin, purified with special care by repeated recrystallisation and dialysis. Consider a series of mixtures with the same content of ammonium sulphate, but containing increasing quantities of sulphuric acid. We get from these a curve in which the free H' ion content (h, the abscissas) increases with the excess of sulphuric acid present (t, the ordinates). Suppose we now choose such a mixture that the curve (e = O, in Fig. 8) contains the iso-electric concentration of H- ions for egg albumin. At 60 3:20 ^> $ 40 60 16 18 FIG. 8. Ammonium sulphate concentration = 0-36 N in all cases. e = milligram equivalents of protein nitrogen in 100 c.c. this point, the hydrion concentration produced by the same concentration of ammonium sulphate and the same excess of acid should remain unaltered by the presence of egg albumin of any concentration (expressed as e milligram equivalents of protein nitrogen). We find instead that the curves obtained for various concentrations of protein intersect the pure ammo- nium sulphate sulphuric acid curve, within the limits of IN ISO-ELECTRIC REACTION 53 experimental error, at a point representing the same excess of acid and the same hydrion concentration. Sorensen has made a large number of such determinations with egg albumin, and deduces as a mean value from twenty sets of results, 1574 X io~ 6 as the iso-electric point of egg albumin. These determinations are undeniably the most accurate investigations of the iso-electric point so far carried out. From our point of view the following points must be noted : This work of Sorensen is another investigation carried out with the use of a weak acid. For, by the presence of ammonium sulphate in the relatively high concentration used in the research quoted above, the strength of sulphuric acid is reduced to one-eighth of its ordinary value. Sorensen himself regards such solutions as mixtures of the acid H[NH 4 SO 4 ] with the salt NH 4 [NH 4 S0 4 ]. This work is, therefore, a valuable proof of the fact that if a sufficiently weak acid is used, the iso-electric point is largely independent of the ampholyte concentration. This had already been demonstrated for acetic acid acetate mixtures by Michaelis, using the method of electrophoresis, and by Pauli by the use of alcohol precipitation and viscornetry. The behaviour of proteins when strong acids are employed in the absence of their salts comprises, however, a complex of pheno- mena quite by itself, as has been previously emphasised. In pure albumin, as in weak acids and bases in general, a notable dissociation by steps is not to be expected. Conse- quently it is enough to assume that a single K and K 6 functions in the buffer solutions. The action of one of the strongest of the acidic and basic groups is sufficient in the first instance to decide the acidic and basic behaviour of the substance. It is easy to understand, in these circumstances, that all differences in constitution do not find expression in the values of K a and K 6 , as has already been seen in the case of the simplest amino- acids and dipep tides. Consequently the iso-electric reaction of various proteins with buffer solutions is only partly affected by differences in structure. Consistent with this view is the scarcely surprising fact that proteins of such widely differing 54 PROTEINS IN ISO-ELECTRIC REACTION structure as glutin, albumin and casein show such a very close agreement in the value of the iso-electric concentration (2 X lo- 5 ) in acetic acid acetate mixtures. The supposition that, on further combination with acid, a newly-formed posi- tive polyvalent protein ion appears is necessitated by the behaviour of the salts of albumin with acids described in the following chapter. CHAPTER V SALTS OF ALBUMIN AND ACIDS THE reaction of albumin with acid, in a I per cent, solution of the former, undergoes a change when the acid concentration exceeds io- 3 N. Below that concentration, the evidence for combination is complex, but with greater quantities of acid the process becomes more simple, and reveals itself as the mere saturation of the basic valencies of the protein. The plain fact that proteins combine both with acids and bases has been known for a long time. On the other hand, quantitative measurements of the extent of combination awaited the clear conception of the terms acid and base, which only physical chemistry could furnish. The results of the application of titrimetric methods to the determination of the capacity of proteins for combination with acids and bases (Spiro and Pemsel) were only made clear by the progressive understanding of the function of indicators (W. Ostwald, H. Friedenthal, E. Salm and others). Certain proteins, such as casein, have been shown to form approximately neutral salts when titrated with strong alkalis, using phenol phthalein as indicator, the point of colour change in this instance being near the true point of neutrality in the physico-chemical sense (Laqueur and Sackur) . Many other indicators, such as phloro- glucin vanillin, give an idea of the maximum combination of acids with albumin, which, although only rough, is sufficient for many purposes (J. Christiansen). The work of S. P. L. Sorensen,* in particular, has shown that, in o'rder to arrive at useful conclusions as to hydrion concentrations by the use of indicators, special precautions must be taken when proteins * Compt. rend. lab. Carlsberg, 1907, 7, i; 1909, 8, i; Biochem. Zeitsch., 1907, 7, 45 ; 1909, 21, 131 ; 1909, 22, 352. For summary and literature, see Ergebn. d. Physiol., 1912, 12, 303 ; and L. Michaelis, " Die Wasserstomonenkonzentrat'ion." 1914. Berlin. J. Springer. 56 COLLOID CHEMISTRY OF THE PROTEINS are present. Our exposition of the combination of albumin with acids, and of the physico-chemical properties of the pro- ducts formed thereby, will be based mostly on recent researches, which, without disturbing the equilibrium between protein and acid, have led, on the basis of direct determination of hydrion concentrations, to a pretty comprehensive view of the pheno- mena which occur. Measurements of electrical conductivity, as undertaken by J. Sjoqvist* in the first instance, give a limited insight into the behaviour of proteins with acids. As long as combination occurs, the conductivity shows a considerable decrease owing to the disappearance of the rapidly-moving hydrogen ions. This, however, is an indirect and uncertain way of arriving at the capacity of the albumin for combination with acid, because, as we shall see, the mobility of the charged albumin particles is in no wise independent of the quantity of acid combined therewith, and, moreover, on account of the high mobility of the hydrogen ion, a slight excess of uncombined acid makes a considerable difference to the conductivity of the mixture. The important work of F. A. Hofmann and O. Cohnheim,f in which the acceleration of the inversion of cane sugar by hydrogen ions was applied to estimate the hydrion content of mixtures of proteins and acids, only fails on account of the slight sensitiveness of the method in such cases. The work of St. Bugarsky and L. Liebermann J marks a stage of notable progress in the investigation of the matter under discussion. These authors introduced the measurement of H and Cl ions by the electrometric method to determine the combination of acids, bases, and common salt with proteins. Until recently, this work was one of the main supports of the view of the salt-like nature of the acid and alkali albumins which was commonly accepted. The products were therefore considered to be subject in typical fashion to the laws of electrolytic dissociation and of hydrolysis, which were conse- quently applied to them. * Skand. Arch. PhysioL, 1894, 5, 277 ; 1895, 6 255. f O. Cohnheim, Zeitsch. Bid., 1896, 33, 489 ; see also W. B. Hardy, /. PhysioL, 1905, 33, 251, J Arch. $es. PhysioL, 1898, 72, 51. SALTS OF ALBUMIN AND ACIDS 57 Bugarsky and Liebermann worked on egg albumin dialysed free from salts, and on mixtures of albumoses purified in a similar way. Their measurements of the hydrion concentra- tion are, however, more troublesome and less reliable than those more recently carried out by S. P. L. Sorensen and Hasselbalch with much improved technique. The following table gives an example of the results of Bugarsky and Lieber- mann : Table 17. (g = gm. albumin to 100 cc. 0-05 N Hydrochloric Acid.) g. % H combined. % Cl combined. 0-4 0-8 9-0 18-9 10-7 2O-2 1-6 33'3 38-0 3-2 60-2 64*0 6-4 96-56 76-0 12-8 99-67 These figures, which are displayed graphically in Fig. 9 show that on addition of more than 6 gm. albumin to 100 c.c. of 0-05 N HCl, the acid combines pletely with it. 100 com- Also, with 3 gm. albumin and lesser quantities, the H- ions and Cl' ions are taken "jip in equal number, but above that concentra- tion of protein, the H* ion is more completely combined than the Cl' ion. The authors deduce from this the II v- Y c of^rotei FIG. 9 (Table 17). constitution of the compound of albumin and acid : " The compound albumin HCl dissociates in water to give Cl' ions and albumin H- ions (the latter might be named ' albuminium ') ; 58 COLLOID CHEMISTRY OF THE PROTEINS hence its constitution is analogous to that of ammonium chloride." Bugarsky and Liebermann attribute to the albuminium chloride a smaller dissociation than that of ammonium chloride, and hence, as soon as a sufficient excess of free hydrochloric acid is present, as is the case with a lower albumin content, the compound is but little ionised. When, however, much albumin is in solution, and the acid almost completely combined with it, the dissociation into Cl' ions, which in lower concentration is suppressed, comes into play, giving thus many more Cl' than H> ions. This thoroughly plausible conclusion has been accepted by most authorities, although it is based solely on the single experiment quoted above. Indeed, it rests entirely on the difference in combination of H - ions and Q/ ions observed once only at an albumin concentration of 6-4 gm. This view was controverted by T. B. Robertson,* who pointed out the quite inadequate experimental evidence for it, and pronounced the single experiment relied on by Bugarsky and Liebermann as doubtful. Robertson, on his part, supposes that when albumin combines with acids, complete combination with both ions always ensues ; and, as the conductivities of such acid protein mixtures assume such high values, that not merely the small balance of free acid but also the acid albumin must contribute to it. Robertson therefore assumes that fresh positive and negative protein ions are formed, of which the former should contain the H- ion and the latter the Cl' ion.f This hypothesis, which is put forward as quite general, we have proved to be certainly inapplicable to the combination of albumin or glutin with acids. It is, in the first instance, contrary to the fundamental fact that cataphoresis experiments * " The Physical Chemistry of the Proteins." Longman. 1918. f According to Robertson, albumin reacts with HC1 by the operation of the peptide linkage : CO NH > C(OH) = N Thus . . . C(OH) = N . . . -f HC1 > . . . COH ++ . . . + N" .. . Cl SALTS OF ALBUMIN AND ACIDS 59 undertaken with every precaution show that movement to the anode alone occurs in acid albumin, indicating the presence of only positive albumin ions. The ionisation of albumin-acid compounds has been made quite clear by the careful measurements of H* and Cl' ion concentrations by the potentiometer method undertaken by K. Manabe and J. Matula.* Their results for an albumin made from ox-blood serum are given below. The arrange- ment is much clearer than that of Bugarsky and Liebermann, for in this case the albumin concentration (about i per cent.) was kept constant, the acid alone being varied. Table 18 (Fig. 10). Ox-serum Albumin (end concentration 1*09 per cent.) + HC1. A. Hydrion Concentrations. Final concentration oHCl(x N). P H. C H. H combined. 0-005 3-410 3-89 X IO- 4 4-48 X IO- 8 0-007 3-3I3 4-75 x io- 6-32 X I0~ 8 O-OI 3-085 8-22 X IO~ 4 8-84 X IO- 8 O-O2 2-2Q5 5-07 X IO~ 3 1-41 x io- 2 0-03 1-52 X io- 2 (extrap.) O-O5 I-536 2-91 X IO~ S 1-79 x io- 2 B. Chlorion Concentrations. HCl. P C1. C C1. Cl combined. O-OO5 2-366 4-31 X IO~ 8 5-6l X 10- 0-007 O-OI 2-340 2-085 4-57 X io- 8 8-22 X IO~ 8 2-23 X 10- 1-39 X 10- 0-02 1-863 1-37 x io- 3 5-44 X io- 0-03 1-678 2-10 X IO- 2 7-54 X io- O-O5 1-517 3-04 X IO- 2 1-66 x io- It can be seen from these results that in lower concentrations of acid practically all the H- ions are combined. With an increase in the quantity of acid the combination tends to a * Biochem. Zeitsch., 1913, 52, 369. 6o COLLOID CHEMISTRY OF THE PROTEINS VUJ O'OZ 0-01 i ^ K* ;** ^ H* - s' tr" y^ ^' ^^ :^ --<; r/ 0,0 f OVZ O'Od 0\ FIG. 10 (Table 18). maximum, which amounts in this case to i-S X io~ 2 N per litre in 1-09 per cent, albumin solution. The Cl' ions, on the other hand, remain to a large extent free in the beginning, and only above a concentration of 0-02 N acid does the combina- tion of the chlorine increase rapidly. If, now, the difference of the combined H- and Cl' ions is plotted as ordinates (Fig. 10), a direct view of the behaviour of the ioni- sation in acid albumin is obtained (the fine dotted curve) . The ionisation increases to a maximum in about 0-02 N HC1, and then falls off, until at a concentration of 0-05 N it is almost completely suppressed. This phenomenon indicates that the free acid which accumulates on further acidification, after con- siderable saturation - of the protein finally suppresses almost completely the ionisa- ^ tion of the albumin chloride. It is only this work and similar G01 experiments with serum albumin which proved that the theory of the ionisation of albumin chloride for- Hut in / ___ 0'01 MZ P05 (HtynCl FIG. ii. Glutin and hydrochloric acid. mulated by Bugarsky and Liebermann was based on facts, whereas the alternative conception of T. B. Robertson is definitely refuted. Experiments with glutin confirm the main result obtained in the work on albumin, that is, that ionisation of the acid- protein occurs with splitting off of chlorine ions, indeed, in this case the fraction dissociated is still larger than with serum SALTS OF ALBUMIN AND ACIDS 61 albumin. The strong tendency to ionisation of the glutin chlorides is indicated by the fact that even with an acid con- centration of 0-05 N the dissociation of the protein salt is not notably suppressed. Glutin chloride thus approaches some- what closely to ammonium chloride in its properties. Table 19 (Fig. u). Glutin (i per cent.) + HC1. Final concentration of HC1. P H" H combined. p ci- Cl combined. 0-003 N 4'74 2-9 x io- 3 2-50 _ 0-005 N 3-97 4-8 X IO- 3 2-32 4-0 X IO- 5 0-007 N 3-80 6-6 X IO- 3 2-IQ 3-4 X io-* o-oi N 3-01 8-7 X IO- 3 2-OO 0-015 N 2-44 10-8 x io~ 3 I-8 9 1-8 x io- 3 0-02 N 2-IO II-I X IO- 2 1-74 i-i X io- 3 0-03 N i-53 0-05 N _ 1-31 The results of varying the protein content with a constant concentration of acid can now be better understood. It is clear that, at a low concentration of albumin, with a given content of acid, relatively more H* ions will be combined than when more albumin is present, owing to the fact that up to a point excess of acid favours the combination of the H- ions (curve H, Fig. 12). Further, as the combination of the Cl' ions always lags behind that of the H' ions, the absolute quantity of combined Cl' ions gradually rises to a maximum with increasing concentrations of protein, and then rapidly decreases (see curve II., Fig. io). Table 20 (Fig. 12). Serum Albumin (various concentrations) + 0*02 N HC1. Concentration of albumin (%). H combined. Cl combined. 0-46 0-92 i'37 i-73 7-26 X IO- 3 II-3 X IO~ 3 13-5 X IO- 3 16-4 X IO- 8 3-31 x io- 3 5-30 x io- 3 7-32 x io- 3 5-95 x io- 3 62 COLLOID CHEMISTRY OF THE PROTEINS 0-01 The natural explanation of these relations is that with low content of albumin at constant acidity the behaviour of the H- and Cl' ions approaches that which obtains in presence of excess of acid, whereas when much albumin is present the behaviour is akin to that of complete combination of the acid. When the same series of determinations is made at higher con- centrations of acid, the divergence of the curves is displaced in the direction of the higher concentration of albumin (Fig. 12) . At the same time these investigations lead to the observation that the measurements of Bugarsky and Liebermann give too high a value for the free H- ions, and consequently the figures for combined acid are too low (compare Fig. 12 and Fig. 9). The maximum ionisa- tion of albumin chloride implies a maximum of positively charged albu- min particles, and an investigation of the migration of acid f/rvrei'4 albumin by Pauli and Samec * goes to prove this. With constant difference of potential the quantity of albumin moving towards the cathode falls off again when the concentration of acid rises above o -05 N. Previous to the more recent electrometric measurements given above, the behaviour of acid albumin on ionisation and the existence of a maximum value for the ionisation were deduced from observations on the viscosity of such mixtures. The work of Pauli and Handovsky f showed that when hydrochloric acid is added to albumin (e.g., a i per cent, serum albumin solution), a considerable rise in viscosity occurs. A maximum value is obtained between 0-017 N and 0-02 N acidity, further addition of acid causing a decrease in viscosity (Table 21, Fig. 13). * Not yet published. | Biochem. Zeitsch., 1909, 18, 340. OW5 - concen tration c FIG. 12. Combination with variable protein concentration. SALTS OF ALBUMIN AND ACIDS E. Laqueur and O. Sackur * were the first to observe the increase in viscosity on salt-formation by proteins. Such an increase was found in sodium caseinate, obtained by dis- solving casein in caustic soda solution, when, with rising concentration of alkali, a maximum and then a falling off in viscosity was noted. They attributed the increase in viscosity to the free casein ions. W. B. Hardy f found a similar increase in viscosity on dissolving globulin in acids or bases. Laqueur and Sackur concluded that the decrease viscosity with excess of 00j Wt OVfnHCt FIG. 13. Viscosity of albumin in hydrochloric acid. in viscosity witn excess alkali was due to the effect of the common ion of the sodium hydroxide in repressing the ionisation of the protein salt, as sodium chloride also produces this result. It is easy to show, 0-01 0-02 0'03 OW O'OSnHCt FIG. 14. Viscosity and difference in ion concentration. however, that neutral salts of varied cation affect the viscosity of sodium caseinate in the same way. It is, in fact, an effect produced by salts in general, as we shall see later, and thus the * Beitr. z. Chem. PhysioL u. Path., 1903, 3, 193. t /. PhysioL, 1905, 33, 251. 64 COLLOID CHEMISTRY OF THE PROTEINS Table 21 (Fig. 13). Concentration of HCl . o.o 0*005 o - oi 0-015 0*017 0-02 0-03 0-04 0-05 N 1-041 1-083 1-166 1-243 i'243 1-232 1-165 1*136 i-i2i observations of Laqueur and Sackur on the effect of salts lose their validity as an explanation of the alkalinity-viscosity curve of casein. Nevertheless, their view, that in excess of alkali the ionisation of the casemate is suppressed, has proved to be correct. Pauli and his co-workers have explained the course of the viscosity curve of hydrochloric acid and albumin by the same ionic conceptions as those employed by Laqueur and Sackur. The work of Manabe and Matula has finally settled the paral- lelism between the viscosity curve and the ionisation curve as determined by the electrometric method (Table 2iA, Fig. 14). Table 21 a. Ox-serum (1-61%). Ox-serum (0-8%). Concentration of HCl. I/to. H- - Cl'. tlto. H- - Cl'. , 1-077 1-035 3 X io- 3 1-83 X io- 3 5-65 X IO- 4 4 X io- 3 1-079 5 X io- 3 3-15 X IO- 3 1-51 x io- 3 6 X io- 3 I-I 33 7 X io- 3 4-64 X IO~ 3 3-01 x io- s X io- 2 5-8l X IO- 3 5-03 X io- 2 1-204 2 X io- 2 - 3-94 X IO- 3 I-2I2 5 X io- 2 6 X io- 2 7 X io- 2 6-72 X IO- 3 1-343 1-383 2-94 X TO- 3 I-2O4 2 X io- 2 i-oi x io~ 2 i-39i 2-74 X IO- 3 I-I86 2-2 X IO- 2 1-385 3 x io- 2 6-06 X IO~ 3 1-289 5 X io- 2 8-5 X io- 4 6-0 x io- 4 I-IO7 We give next of the viscosity by Pauli and R a very complete series of careful measurements of horse-serum albumin (free from salts) made . Wagner,* which include the initial decrease. * Biochem. Zeitsch., 1910, 27, 297. SALTS OF ALBUMIN AND ACIDS They accordingly embrace the two change points c and d of acid albumin referred to in the preceding chapter. Table 22. Albumin (i per cent.). N.HCl. Relative viscosity. N.HCl. Relative viscosity. 0-0 I -088 0-02 487 * o-ooi I -086 O-O24 451 0-0016 1-084 O-O3 -426 O-OO2 1-084 * 0-04 353 0-0024 1-086 O-O5 284 0-004 I-I02 0-06 268 0-006 1-128 0-08 228 O'OI 1-322 O-I 199 0-016 1-402 Glutin gives similar results to those obtained in viscosity measurements on albumin, and is particularly useful in deter- mining the effect of temperature on viscosity. With a falling temperature a disproportionate increase in viscosity takes place in the region of the maximum value, whereas the concen- tration of acid which gives the maximum viscosity is practi- cally independent of temperature for a given content of glutin. As might be expected from the electrometric results, the maxi- mum viscosity moves in the direction of greater acidity when the concentration of glutin is raised. All these relations can be seen from the results of Pauli and 0. Falek * given in Table 23. Table 23. Concentration Relative viscosity 0-3% glutin. i% glutin. of HC1. 35- 30. 35. 40. O 1236 1-1069 1-0934 1-5540 0-002 N 3018 I-I964 1-6644 0-0025 1-2970 0-003 3905 I-3423 I-2938 1-8598 0-004 4077 1-3743 I-3463 I-9563 0-005 -4302 * 1-3850 * I-3683 * 2-0988 o-oi 3291 1-2889 I-282I 2-7632 * O'O2 I-22O2 2-5I49 * Biochem. Zeitsch., 1912, 47, 269. 66 COLLOID CHEMISTRY OF THE PROTEINS In connection with the above viscometric measurements, C. Schorr * has made the important observation that the quantity of the precipitate produced by alcohol in acid albumin solution passes through a minimum value when increasing quantities of acid are added. The point of minimum precipitate coincides with the maxima of viscosity and of ionisation, and still larger quantities of acid cause the quantity precipitated to increase again. The following table gives the results of work on a i-i per cent, ox-serum. The mixture of albumin and acid was precipitated by the addition of ten times its volume of alcohol. Table 24. Concentration ( x N^. Hydrochloric acid. Trichloracetic acid. Acetic acid. o-ooi + .+ + + + + + + + O OO2 + + + + + -j- -j- + 0-003 + -f + + 0-005 + + + O-OI -^ -j- -j- 0-02 + 0-025 0-04 i O-O5 ~F -|- o-i =F 0-2 -j- db 0-3 Measurements of the depression of the freezing point, due to Bugarsky and Liebermann,t lead to the result that addition of albumin to solutions of hydrochloric acid causes a decrease in the lowering of freezing point, showing that the molecular concentration has become less. The molecules of the albumin salt formed on combination of the albumin with the acid cause a diminution in the total number of molecules of albu- min plus acid. Further, by working out the results of these authors it is clear that when increasing quantities of albumin are added to a constant quantity of acid, the depression caused per gram of albumin first of all increases and then falls off * Biochem. Zeitsch., 1911, 37, 424. Loc..cit. SALTS OF ALBUMIN AND ACIDS 67 again. As we can now see, this is due to the appearance of free Cl' ions in high concentrations of albumin, which take part in the lowering of the freezing point. With lower albumin content the excess of acid present represses the ionisation of the albumin chloride. The actual results are shown in Table 25, in which A is the observed depression, D the depression calculated from the sum of acid and albumin molecules, and ence produced per gram of albumin. D g the differ- Table 25. (g = grams egg albumin ; 0-05 N HCl in all cases. ^ D A. D - A & g o 0-186 O 0-2 0-184 0-004 0-4 0-181 0-007 17-5 0-8 0-172 O-O2 25-0 1-6 0-146 0-049 30-0 3'2 O-IOI O-IOI 31-8 6-4 0-087 O-I2I 19-0 We can now summarise the information supplied by these various researches on the behaviour of albumin and glutin in the presence of hydrochloric acid as follows. On adding acid to a given weight of protein, combination occurs to an extent which increases with increase of acid up to a maximum. Thus, for i gm. serum albumin the maximum combination is with 1-66 x io- 3 gm. mol. HCl or 60-59 m - HCl. ; for i gm. glutin the value is 1*5 X io~ 3 gm. mol. HCl or 54 mg. HCl. When combination occurs, the hydrogen of the acid forms with the albumin an electro-positive albumin ion, while the chlorine ions largely remain unattached. With excess of acid, the ionisation of the protein chloride is suppressed, as is shown by a rapid increase in the combination of the chlorine ions with the pro- tein, and also by a decrease in the quantity of albumin trans- ported in the electric field. The hydration of the protein particles, moreover, increases as the combination with acid 52 68 COLLOID CHEMISTRY OF THE PROTEINS and the consequent ionisation proceeds, as is shown by the in- crease in viscosity, which, however, decreases once again when excess of acid has reduced the ionisation, and thus increased the number of neutral particles. The precipitation by alcohol also is reduced to practically nil as the ionisation of the acid protein increases, but reappears again when the ionisation is reduced. The difference in properties between ionised and neutral albumin, which has already been sketched in the previous chapter, and to which we shall return later, is reflected in the behaviour of albumin chloride. But, whereas the behaviour of albiimin in the iso-electric region (in the presence of buffer solutions) is a question of the existence of ionic and of neutral particles of natural albumin (or of the polyamino-acids of E. Fischer), we have here to deal with the ionic and neutral particles of the albumin salts formed when the protein combines with acids. The structure of these salts must now be discussed, and first of all the salts formed with different acids must be considered. The ideas so far presented have been based on the salts of proteins with hydrochloric acid, on account of the ease with which the behaviour on dissociation can be investigated by measurements of the H- and Cl' ion concentrations. Corre- sponding measurements with bromide and iodide have also been possible, but no remarkable differences between these salts and the chloride have appeared. On the other hand, electrometric measurements with polyvalent anions such as S0 4 " and P0 4 '" fail because the accuracy of such measurements decreases considerably with increasing valency of the anion. It is insufficient even for SO 4 , according to experiments made by Pauli and Hirschfeld.* Hence, it is necessary to rely entirely on measurements of H* ion concentrations when in- vestigating the combination of proteins with acids other than the halogen hydracids. The fraction of the acid which com- bines with the protein can therefore only be determined in this way in the case of strong acids or of weak acids of known dissociation constant. These methods depend on the assump- tion which applies to all salts, that the protein salts are con- * Biochem. Zeitsch., 1914, 62, 245. SALTS OF ALBUMIN AND ACIDS 69 siderably dissociated and to an extent independent of the strength of the acid. According to Arrhenius, if a strong acid is partly neutralised by an alkali (by a protein in the case we are considering), the portion of the acid which remains free has the same degree of dissociation as that of the original acid in the same volume. Thus, a mixture containing 0*05 N H 2 S0 4 and 0*05 N Na 2 SO 4 is dissociated to the same extent as 0*1 N H 2 S0 4 . If a is the degree of dissociation of an acid in concentration n, and after partial neutralisation without alteration in volume the con- centration of free acid is n', the portion of the acid dissociated is riv.. A measurement of the hydrion concentration gives this value C H = Woe, and thus the combined (or neutralised) part of n CH the acid, n n ' = - . a In this way it is possible to obtain a value for the combination of albumin with strong acids from a knowledge of the original concentration of acid, n t the corresponding degree of dissocia- tion, a, and the hydrion concentration, C H , measured after addi- tion of the protein. Thus Pauli and Hirschfeld found that the quantity of sulphuric acid which entered into combination with albumin and glutin was nearly the same as for hydrochloric acid. It is, however, rather less than the latter in high con- centrations. Table 26 (Fig. 15). Horse-serum Albumin (i'26 per cent.). Hydrochloric acid. Sulphuric acid. Concentration of acid. C H- Combined per litre. C H- Combined per litre. o-ooi N _ _ I-O2 X IO~ 5 N 3-89 x io- 4 N 0-002 1-77x10- N 1-982 x io~ 3 N 0-003 6-68x10- 2-93 X io- 3 - 0-005 2-15x10- 4-779 XIO- 3 1-77 x io~ 4 4-792 X I0~ s 0-OOy 2-85 x io- 4 6-655 xio- 3 o-oi 8-31 x io~ 8-95 X 10- 002 3-24x10- 1-661 x io- 2 3-4 xio- 1-539 X io- O-O25 5-7 xio- 1-902 x io- 2 5-32x10- 1-761 x io~ 0-04 1-81 x io- 2-08 x io~ 2 1-48 x io- 1-82 X 10- 0-05 2-45 X io- 2-39 XIO- 2 2-04 X 10- 1-87 x io- 70 COLLOID CHEMISTRY OF THE PROTEINS The degree of combination of weak acids can be derived from the values of CH on the basis of the law of mass action. The method of calculation is shown by the following example, as worked out by the above authors for the acetic acid acetate mixtures which they used. When an acetate is added to acetic acid the dissociation isotherm of the acid K . CCH S .COOH = CH is transformed into the expression K . CCHS.COOH = CH (Cn + CcHsCOo) (i) where K is the dissociation constant of acetic acid (1-8 x io- 5 ), C H the concentration of free hydrions, and C CH3 coo the concen- tration of the acetate ions which have their origin in the com- bination with albumin. We can equate the latter value to the hydrogen combined with the albumin, or to the acid combined therewith, as the albumin salt is highly dissociated in the dilute solution. The number of acetate ions arising from the free acid must be equal to C H . Further, if n is the normality of the total acetic acid employed in the reaction, the following relation holds between it and the undissociated part of the acid CCHSCOOH ' CCH S COOH = n C H CCH S COO Substituting in equation i K( C H CcH 3 coo) = C H (C H + CCH S COO), so that the acid combined with the protein is K( - C H ) -- CH , } CCH.COO - K + CH In this way the combination of acetic acid and albumin can be studied, and, indeed, the expression is capable of general application provided the dissociation constant of the acid is known, and the salts formed with albumin are considerably dissociated (see Table 27, Fig. 15). The measurements of Pauli and Hirschfeld given below show that the combination of albumin with acetic acid falls con- siderably short of that which occurs with strong acids. This SALTS 0F ALBUMIN AND ACIDS is, however, only true if acids of the same normal concentration are compared ; if solutions of the same hydrion concentration are compared, then the quantity of the weaker acetic acid which O'Oi 0-0* MS 0*0* FIG. 15. Combination with various acids. combines is notably greater. Thus a 0-2 N acetic acid corre- sponds to a 0-002 N hydrochloric acid ; but nearly 9 x io~ 3 N acid is combined in the former case or more than four times as much as in the hydrochloric acid solution. The same result is met with in the swelling of gelatin in various acids. Table 27 (Fig. 15). Horse-serum Albumin (1*26 per cent.) + Acetic Acid. Concentration of acid. a of the acetic acid. H- ion concentration of the acetic acid. C H of the mixture. Cembined acid. 0-005 N 0-0569 2-85Xio~ 4 N 1-31x10- N 3-00 x io- N O'O2 0-0296 5-92 X io- 4 4-97x10- 5-24x10- 0-04 O-O2II 8-44 x io- 4 1-02 X 10- 6-00 x io~ 0-05 0-0184 9-20 X io- 4 1-22 X IQ- 6-47 x io- 0-1 0-OI3I 1-31 X io- 2-27XIO- 7-11 xio- 0-2 O'O9O 1-8 Xio- 8 3-9IXIO- 8-6 xio- 0-4 0-0066 2-64 x io-* 6-88x10- 9-47X10- o-5 O-OO572 2-86XIO-* 9-79x10- (?) 9-84 Xio- In the work of Manabe and Matula already quoted, in which the relation between combination with acid and concentration of albumin was studied, a relative increase in the extent of combination as the content of albumin decreased was observed. 72 COLLOID CHEMISTRY OF THE PROTEINS A similar effect was noticed with acetic acid. Thus in Table 28 the quantity of combined acid in a half and a quarter of the original albumin content is distinctly greater than a half and a quarter of that found in the original concentration. This result is due to the suppression of hydrolysis by the relative excess of acid in the lower concentrations of albumin (see below). It does not represent, however, the maximum com- bination, which takes place only in considerable excess of acid. Table 28. Acetic Acid -j- Ox-serum Albumin of various Concentrations. Concentration of acid. Acid combined with albumin of concentration. 1-26%. 0-63%. 0-315%. 0-005 N o-oi 0-05 0-1 3-00 x io- 3 N 6-47 X IO- 3 7-II X IO- 3 3-12 x io- 3 N 6-97 x io- 3 2-206 X io-*N 4-96 X io- 3 On the other hand, in excess of acid, and when the maximum combination has been attained, the quantity of combined acid 0-01 A dote a Gluttn+HCl Aeict FIG. 1 6. Glutin and hydrochloric acid. is proportional to the albumin content, and consequently this value is specific for the protein concerned. Fig. 16, from the SALTS OF ALBUMIN AND ACIDS 73 work of Pauli and Hirschfeld, shows the behaviour of 1*0, 0-5 and 0-25 per cent, glutin very clearly. The studies of Pauli with H. Handovsky, 0. Falek and M. Hirschfeld on the relations of viscosity to the combination of albumin with various acids are particularly interesting. Table 29. Relative Viscosities of Ox-serum Albumin with different Acids. (Relative viscosity of the serum albumin = 1-041.) Concentration Hydrochloric Sulphuric Trichloracetic Citric Acetic of acid. acid. acid. acid. acid. acid. o-oi N 166 061 1-073 044 1-046 O-O2 232 061 1-059 066 1-052 0-03 I6 5 060 1-053 IOO I -066 0-04 I 3 6 064 1-056 III 1-075 0-05 121 066 I -060 I 4 I 1-091 [ Let us consider first the viscosity curves of albumin chloride and acetate. The former rises steeply to a maximum, while the latter shows a gentle slope upwards. The difference is satisfactorily explained by the lower strength of the acetic acid, which leads to a less extensive formation of albumin ions, but not to a repression of the dissociation of the strongly ionised albumin acetate. This conception, however, fails to account for the behaviour of the strong trichloracetic and sulphuric acids, which display a much smaller increase in viscosity than either acetic acid or even much weaker acids, such as mono- chloracetic, oxalic and citric acids, which give a larger increase in the viscosity of the protein. ' As the electrometric measure- ments give no basis for the supposition that differences in degree of combination occur with equally strong acids, the variation in viscosity in this case can only be due to differences in hydra- tion caused in some other way. Such differences in hydration can be accounted for either by varied ionisation of the individual protein salts, which when less ionised would be less viscous, or by the difference in structure of the positive albumin ions of 1*5 74 COLLOID CHEMISTRY OF THE PROTEINS different protein salts, in which case variations in hydration of the ions and of the neutral particles can have a combined effect. The latter alternative can be regarded at once as improbable, and, in analogy with typical metallic salts, we may assume the identity of the albumin ions of the protein salts in the same way that the same cations are found to be given by the salts of a metal with different acids. Accurate electro- metric measurements on albumin chloride show, however, that a small portion of the acid combines with the protein in a manner which appears to be molecular, both the ions taking part. Considerable differences in the properties of the albumin ions of different protein salts may be caused by this portion of the acid ; and that small Oxalic Act 'c quantities of substances can effect considerable changes in the properties of the ions of acid albumin is shown by investigations of the com- bination of acid albumin and neutral salts (see later). The molecular reaction of the uncombined part of the acid FIG. 17. Viscosity curves with various mus t also be considered, and it is probably no mere accident that the marked anomaly of sulphuric and trichlor- acetic acids is coincident with their considerable power of dehydration. As, however, the relations in ionisation of the various protein salts, as determined by free anions, are at the moment far from clear, a decision as to the ongm of the differing hydration of these salts must be sought in other ways. In this respect the researches of Pauli and Samec* on the optical rotation of protein salts provide some useful suggestions. These authors found that even in low concentrations both acids and bases give rise to an increase in the optical rotation of albumin. The table and * Biochem. Zeitsch., 1914. 59, 470. HCl O'OSn SALTS OF ALBUMIN AND ACIDS 75 figure following give the results of investigations on serum albumin. Table 30. (a of the pure albumin = 1-02 ; ox-serum albumin concentration = 1-07 per cent.} Concentration of acid. HC1. H 2 S0 4 . COOH. I COOH. CH 3 . COOH. CH 2 C1. COOH. CC1 3 . COOH. 0-005 N i'i3 1-07 06 1-025 7 1-04 O-OI 1-25 1-09 12 1-03 13 1-05 0-02 1-38 I-I2 18 1-04 18 1-07 0-04 i-39 I-I7 30 1-30 20 I -08 0-05 i-39 1-19 34 I -08 22 I -08 These and other similar experiments show that for an increase in rotation it is not the strength of the acid that is decisive. Thus, oxalic acid is more effective than the much stronger trichloracetic acid or sulphuric acid, and acids as different in strength as trichlor- acetic and acetic acids have much the same effect on the optical rotation. In this respect there is a notable agreement between the curves for viscosity and for optical rotation of the protein salts of the various acids. On the other hand, the optical rotation fails to give the maximum value and subsequent decrease that is so characteristic of the viscosity, e.g., of hydrochloric acid and proteins. The rotation tends rather to remain constant after reaching the maximum. It is thus the general formation of salts of albumin, and not FIG. 1 8. Rotation curves with various acids. 76 COLLOID CHEMISTRY OF THE PROTEINS their ionisation, which is important in determining the optical rotation. We must therefore seek the explanation of the differences in rotation of the protein salts in constitutive differences of the protein portion of these salts rather than in variations in ionisation with a common anion. It is extremely probable that the widespread agreement between rotation and viscosity in the series of albumin salts with various acids is due to the same or, at least, similar constitutive differences exert- ing their influence on the viscosity of the acid proteins. The very recent work of M. Adolf and E. Spiegel * on an acid-albumin made by boiling dialysed ox-serum albumin with dilute hydrochloric acid has shown with certainty that, when the precipitated acid albumin is dissolved in increasing quan- tities of hydrochloric acid, the rotation passes through a maximum. Unlike natural albumin, however, of which the rotation remains at the maximum level on further addition of acid, with this material the value decreases in more acid solu- tions until it finally sinks to the original value. Table 31 gives some of these results. The values of the viscosity for increasing concentrations of acid are added for comparison, showing the characteristic course of the curve with a sharp peak at the maximum point due to repression of the ionisation in excess of acid. Table 31. (Final concentration of acid-albumin, 0-62 per cent.) Concentration of hydrochloric acid. Rotation a. Concentration of hydrochloric acid. Relative viscosity. 0-008 N 0-29 0-008 N 154 O-O28 0-33 0-013 I 9 6 0-058 0-49 0-018 -I 5 8 0-079 0-33 O-O28 135 O-IO8 0-29 0-048 103 0-058 103 If, on the one hand, the viscosity (Table 31) and the extent of combination of acid-albumin (Table 39) are compared with * Biochem. Zeitsch., 1920, 104, 175. SALTS OF ALBUMIN AND ACIDS 77 the variation in optical activity with increasing addition of acid on the other hand, it is clear that not only the viscosity maximum, but also the combination with the greatest quantity of the acid employed and the ionisation maximum are coinci- dent. The peak of the rotation curve does not coincide but lies rather on the part representing the general decrease in the viscosity. It is not, apparently, a difference in the rotation of the ions and the neutral molecules of the protein salts, but rather an effect of higher acid concentration on the constitu- tion of the acid-albumin that here determines the characteristic course of the curve for rotation. C. Schorr's * work on the precipitation by alcohol of the acid albumins of different acids gives the same interesting difference, which, for hydrochloric acid and acetic acid, lies entirely in the comparison with the viscosity curves in the two cases. In the first instance the maximum of viscosity corre- sponds to one of precipitation ; in the second case the increase in the alcohol precipitate only appears in higher concentrations of the acid. With albumin sulphate low viscosity corresponds well with greater precipitation by alcohol, so much so that to show the variation in precipitation decrease and subsequent increase with continuously increasing acid concentration it is necessary to employ a 45 per cent, alcohol in place of the 96 per cent, alcohol used in the work with hydrochloric acid. The ioni- sation effect is then clearly seen. Albumin trichloracetate falls quite outside any connection between viscosity and alcohol precipitation, as it is entirely precipitated in analogy with albumin chloride. The viscosity in this case indicates a great difference in the hydra tion (Table 24). Here the strength of the acid is clearly expressed in a corresponding difference of ionisation of the albumin salt, as shown by the effect of addi- tion of alcohol. In general, the precipitation by alcohol, as shown by the researches which have been quoted, is an indi- cator of the dissociative properties of the salt examined in this way. A comparison of the viscosity values in concentration of acid of 0-05 N and the precipitation of the albumin by acids is * Biochem. Zeitsch., 1911, 37, 424. 78 COLLOID CHEMISTRY OF THE PROTEINS particularly instructive. It is well known that in high acid concentrations albumin is precipitated with formation of a coagulum, which is not dispersed again on dilution. If this irreversible change is regarded as a secondary effect, one can reasonably suppose that this precipitation is only effective on the neutral particles, for only such can be deposited out of a solution. It can further be supposed that neutral particles are more soluble the more they are hydrated. The viscosity of the acid proteins after the maximum has been passed is a measure of the number of neutral particles, and therefore of their hydra- tion ; consequently a relation between viscosity and precipi- tation by acids is to be expected in the sense that low viscosity and low precipitation will be coincident. This has actually been found by Pauli and R. Wagner,* who measured the con- centration of various acids required to precipitate a serum albumin free from salts (Table 32). Table 32. Acid 0-05 N. Relative viscosity (25), i % albumin + 0-05 N acid. Concentration (of the acid required H' ion concentration to precipitate at 18). CC1 3 COOH 1-0603 ,\ 0-04 N 0-0356 N jl CC1 2 HCOOH O-OI 0-0981 l\ H 2 S0 4 1-0656 M 0-2 0-1163 1 CH 3 COOH 1-0906 II-2 0-1411 HN0 3 ' 0-2 0-1813 HC1 1-1206 / 1 0-4 0-35I7 CH 2 C1COOH 1-2156 ! \ i-5 0-4108 / 1 The agreement in this table is very satisfactory, particularly when one considers that the final products of the acid precipita- tion exhibit considerable differences in properties (e.g., solubi- lity in excess of acid) . The reaction of nitric acid with albumin does not permit of viscosity measurements at 25 when more than o-o i N acid is present. Even better agreement is obtained when the series of hydrion concentrations, corresponding to the * " Anzeiger Akad. Wissensch. Wien." 1910. No. IX. Biochem. Zeitsch., 1920, 104, 190. SALTS OF ALBUMIN AND ACIDS 79 concentration of acid which produces precipitation, is com- pared with the viscosity. It increases with increasing viscosity (i.e., hydration). These results become comprehensible if the precipitation of the acid albumin by excess of acid is considered as the action of the common ion of the acid, in the same way that common salt is precipitated by hydrochloric acid. In this way an excess of dissociated acid is required to precipitate the acid protein, which is the greater when the number of neutral particles is less, and when they are heavily hydrated. This conception makes the precipitation of albumin in high acid concentrations intelligible as a displacement of the albu- min salt from solution. The completeness of the precipitation, the varied behaviour with excess of acid, etc., are, however, secondary effects which depend on the acid used, and will not be further entered into at this point. 8o COLLOID CHEMISTRY OF THE PROTEINS CHAPTER VI SALTS OF ALBUMIN AND ACIDS (continued) THE question of the hydrolytic dissociation of the albumin salts is very important for a complete understanding of these substances. It is well known that the salts of weak acids or of weak bases have the property of reacting in aqueous solution with formation of notable quantities of free acid and base. In accordance with the equation : BS + H 2 =; SH + BOH . . . (i) if the acid is the stronger the hydrion concentration is increased owing to the dissociation : SH=^S' + H-, or, if the base is stronger than the acid, the greater ionisation of the former gives rise to more OH' ions. In the presence of excess of one of the products of . hydrolysis, the hydrolytic dissociation is repressed in accordance with the law of mass action, so that in equation i the reaction is reversed in the direction of the upper arrow, i.e. from right to left. The fact that the combination of albumin with acids or alkalis increases with excess of acid or of alkali lends support to the view that the protein functions as a weak base or weak acid under these conditions, and so the addition of free acid or base respectively opposes the hydrolytic dissociation of the albumin salt. This view, notwithstanding the lack of convincing quantitative measurements, held the field until recently (for literature see Cohnheim, " Chemie der Eiweisskorper," 3 e Auflage). Robertson (loc. cit.), after his work on casein, opposed this conception and decried altogether the hydrolytic dissociation of the albumin salts. As a matter of fact, the behaviour throughout is not nearly as simple as would appear from the usual descriptions of it, and on account of the importance of the matter it will now be further considered. SALTS OF ALBUMIN AND ACIDS 81 Experience shows that albumin mixed with acid at low con- centration (up to 0-05 N), if it is not warmed above room temperature and not kept for more than a few days, can be freed from the acid by dialysis without any change in its original properties.* On the basis of what we know of albumin salts it can be stated without a doubt that here the dialysis operates by hydrolytic elimination of the acid at the bounding membrane and subsequent exosmosis of the acid. This experi- ence can, moreover, be expressed and confirmed experimentally by saying that the curve of acid combination (Table 18, Fig. 10) for decreasing acid concentration coincides point for point with that obtained for increasing concentrations of acid. This indicates a homodrome reversible change of state. Let us now consider rather more closely the work of Pauli and Hirschfeld (loc. cit., p. 68) as shown in Table 33 and Fig. 19. Table 33 (Fig. 19). Horse-serum Albumin (1*26 per cent.). Concentration of acid. HC1 combined. HaSOi combined. CH 3 COOH combined. 0-002 1-90 X I0~ s _ _ 0-005 4-78 X io- 8 4-79 X io- 8 30 X IO- 8 O-OI 8-95 X io~ s 0-02 1-66 X io- 2 1-54 x io- s 5-24 x io- 8 0-04 2-08 X io~ a 1-82 x io- 1 6-00 x io- 8 0-05 1-87 x io~ 2 6-47 X IO- 8 o-i 7-11 X io- 3 O-2 8-6 X io- 8 0-4 9-47 x io- 8 The 1-26 per cent, serum albumin employed is capable of combining with a maximum quantity of 2-1 X io- 2 N acid per litre. This limiting value, however, only occurs in a con- siderable excess of acid (0-05 N). If, now, 2-1 x io- 2 N acid is added to albumin, instead of AC, only AB (Fig. 19) represents the combined acid so that the degree of hydrolysis, that is, the fraction of the total concentration which is free, is BC/AC. In * Such an albumin is called natural or genuine ; whereas one of which the properties (e.g., solubility) have been altered by irreversible trans- formation without complete decomposition is termed denatured. 82 COLLOID CHEMISTRY OF THE PROTEINS a similar way the degree of hydrolysis with increasing addition of acid can be read off within OA, as the corresponding quantity of acid which remains free is included in the area OBC. Further, the course of the curve OB shows that the degree of hydrolysis steadily increases up to the value of the com- bined acid represented by AB. Beyond that point the excess of acid rapidly increases, and the hydrolysis is suppressed, as is easily seen by the form of the area BCD. At D the hydrolysis of the protein salt is practically nil. The tabular statement which follows, giving in the usual way the mean hydrolysis of the serum albumin used with increasing acid concentration, demon- strates the same variation. Table 34. 0'01 O'OZ. 003 00* n FIG. 19. Hydrolysis of acid albumin. N.HC1. Hydrolysis per cent. K b (v 1000). 0-005 5-o 2-36 x io~ 8 O-OI 7'5 1-03 x io- f 0-024 18-0 1-57 x io- 10 O-O25 8-57 0-03 4-76 0-04 i-i Our observations, therefore, support the view that the com- bination with acid increases proportionally with the content of albumin. The value of maximum combination of 2-1 X io- 2 N HC1 per litre for 1-26 per cent albumin gives for a con- centration of I per cent, albumin the value 1-66 X io~ 2 N HC1. K. Manabe and J. Matula * obtained as a maximum value 1-8 X io~ 2 N. acid for a 1-09 per cent, solution of serum albumin, which is equal to 1-65 X io~ 2 N HC1 for a i per cent, solution. * Biochem. Zeitsch., 1913, 52, 385. SALTS OF ALBUMIN AND ACIDS Pauli and Hirschfeld found for a i per cent, solution of the purest serum albumin the value 1-66 x io~ 2 N HC1. This I per cent albumin gave, in a solution of hydrochloric acid of 1-66 x io- 2 N strength, a hydrolysis of 187 per cent. The proportionality between optimum combination with acid and content of protein is also shown in an experiment of Pauli and Hirschfeld on glutin (see Fig. 16). In a solution which contains i per cent, glutin in acid of concentration i -5 X io~ 2 N HC1, (the maximum quantity combined at that concentration of the protein) , the hydrolysis is 25 per cent. We have now every evidence for the view, as opposed to that developed by Robert- Table 35- Dilution. Content of Hydrolysis per cent. Hydrolysis calculated from Vv. Protein per cent. HCl. c 1-26 0-022 N 18-7 _ Albumin \ i : i 0-63 O-OII 30-4 ( i:3 0-3I5 0-00505 38-4 37-4 ( 1*6 0-015 N 25-00 Glutin -4 ' < i : i o-5 0-0075 29-00 i i:3 0-25 0-00375 40-00 50*0 son, that albumin reacts as a polyacid base. We know from the electrometric measurements which have been quoted and which are typical, that the anion of the added acid gives a dissociated salt, and we must conclude, on the other hand, from the extent of the combination with acid, that several molecules of acid react with one molecule of albumin. For, as the maximum acid combined in i per cent, (i.e., io gm. per litre) of albumin is 1-66 X io- 2 N HCl, so for i gm. per litre the acid concentration would be 1-66 x io- 3 N ; and hence, if it is supposed that only one molecule of acid combines with each molecule of albumin, the molecular weight of the latter works out at less than 600. If, however, the albumin reacts as a polyacid base, forming salts with polyvalent ions, for which there is manifold proof, then the behaviour on hydrolytic dissociation becomes intelligible. fr 2 84 COLLOID CHEMISTRY OF THE PROTEINS Such a compound would first react with acids with its strongest basic valency, and, when that was saturated, the weaker ones would begin to come into play ; the hydrolysis also would be very small in low acid concentrations, and would rise to a maxi- mum with increasing addition of acid. The equilibrium of hydrolysis shows that hydrolytic dissocia- tion increases with dilution. The table on p. 83 shows such an increase in hydrolysis of the protein salt when it is subjected to increasing dilution. Having thus found the degree of hydrolysis we have a way of determining the mean * basic dissociation constant K 6 of the albumin in the protein salt in question. The expression for the hydrolysis constant is : TT _ X K H2 Q X and in the case of the salt of a weak base and a strong acid the dissociation constants of the salt and of the acid are practically the same, hence KB If x is the degree of hydrolysis, then i x is the non-hydrolysed fraction, and the corresponding concentrations in a volume v */ -r / are - and . Accordingly, the equilibrium equation of hydrolysis is TT I X _ X X V ~ V ' V v K H2 o x 2 or K = -JpS? == - r KB (i x) . v and hence K B - K HO(I~*)P (l) Jv * Albumin as a polyvalent base must exhibit a corresponding number of basic dissociation constants. The values of these constants decrease rapidly from that of the first, but it is not possible at present to deter- mine the separate values. The mean value of K B for an albumin in its combination with strong acid may be defined as that constant which a univalent base would have which displays in like circumstances the same hydrolytic dissociation as the collective available basic valencies of the reacting protein. SALTS OF ALBUMIN AND ACIDS 85 At 18 the dissociation constant of water K H2 o 0-62XIQ- 14 , and if we vary the molecular weight of albumin between the limits 1,000 and 10,000, for a i per cent, solution, v will vary correspondingly between 100 and 1,000. The mean basic dissociation constant K B of the albumin, calculated on a degree of hydrolysis of 0-18 from equation i, gives the values 1-57 X lo- 11 and 1-57 x io- 10 . Of these two figures the latter probably approaches more nearly to the true value as the molecular weight of the protein is nearer 10,000. The mean value of KB thus obtained corresponds quite well in its order of magnitude with what we should expect for a high poly- peptide, for the value increases from 2-3 5-1 X io~ 12 for a monoamino-acid (K. Winkelblech) to 2 3 X io- 11 for the simplest dipeptide (H. Euler). In the formation of albumin salts with acids, the basic valencies of the amino-group operate first according to the equation previously proposed by W. B. Hardy and Wo. Pauli : X NH 2 /NHjjHCl R/ + HC1 > R< \COOH XX)OH The more recently propounded views of Robertson have failed to hold the field in spite of many valuable suggestions, such as that of the role of peptide linkage, or the possibility of reaction in the enol-form, as no practical proof can be given. On the other hand, it was necessary to decide the question whether the terminal amino-groups, particularly in the diamino-acids, lysin and arginin, are the sole places of combination with acids. This point can be tested by the desamino-proteins which have been deprived of these amino-groups. When acted on by nitrous acid (sodium nitrite + glacial acetic acid) the amino- groups react according to the equation : CH 2 NH 2 CH 2 OH ' + HN0 2 - -* | -f N 2 + H 2 0, COOH COOH a reaction which has also been successfully applied for the determination of amino-nitrogen in proteins. Of the desamino- proteins the most useful for the study of physico-chemical 86 COLLOID CHEMISTRY OF THE PROTEINS behaviour is desamino-glutin, which is easily soluble in water (L. Blasel and J. Matula).* With the removal of amino-groups glutin loses completely its characteristic property of gelatinisa- tion. It gives a clear solution of distinctly acid character, as might be expected. It reddens litmus and in 0-75 per cent, solution gives a hydrion concentration of 179 x io~ 5 N. It is remarkable to note, however, that desamino-glutin still shows a considerable power of combining with acids. Table 36. 0*75 per cent. Desamino-glutin + HC1. NHCl. C H' Comhined acid. 0-005 8-93 x io- 4-09 X IO~ 3 0-01 3-44 x io- 6-44 X IO- 3 O-O2 1-14 x io- 8-II X IO-* 0-03 2-13 X IO- 7-63 X IO- O-O5 3-90 X 10- 8-50 X IO- 3 Whereas we can estimate the maximum combining capacity of a 0-75 per cent, desamino-glutin solution as 9 x io~ 3 N HC1 per litre, a glutin solution of equal con- centration takes up on the average 1-15 x io- 2 N HC1. Thus, after removal of amino - groups, about 78 per cent, of the power of combining with acids remains. The chlorion of the combined acid is at C OV1 0-01 002. 003 FIG. 20. Desamino-glatin and hydrochloric acid. H first completely dissociated (see Fig. 20) up to an acid concen- tration of 0-015 N HC1, and then in excess of acid its ionisation is suppressed. Hence desamino-glutin also forms a salt ionised like a typical chloride, and, further, a consideration of the * Biochem. Zeitsch., 1914, 58, 417. SALTS OF ALBUMIN AND ACIDS 87 quantity of acid combined shows that several molecules of acid have reacted with one molecule of the protein. Hausmann finds for the amino-nitrogen of gelatin the value of 35-8 per cent, of the total nitrogen, which is far too high ; in more complete later determinations about 26 per cent, amino-nitrogen was found. Electrometric measurements show that the basic valency is reduced by 22 per cent, by removal of the amino- groups. The NH-group of the peptide linkage is the first obvious direction in which to look for the origin of the combination of the desamino-proteins with acids. The acid can be added on at this point in just the same way as on to a terminal amino- group. C N + HC1 >... C NHC1 U the value calculated for i per cent, casein- ate solutions, the degree of dissociation being 0-66, is A K .c as ^Na . cas = 13 '9- Direct experiment gives the value 14-13 (Pauli, loc. cit., p. 499). These results are undeniable evidence of the existence of alkali caseinates, which dissociate into alkali metal ions and caseinate ions. The mobility of the caseinate ion, calculated from the A a of the alkali caseinate, is v Cas =30-1. This value is entirely within the range of mobility shown by numerous organic ions. We shall return later to the importance of high valency and large atomicity in regard to the mobility of the ion. With a knowledge of the equivalent conductivity of the caseinates and of their regular behaviour, it is possible to * The pertinent observation of Pauli (loc. cit.) that a practically complete dissociation of the caseinate occurs is justified by a con- sideration of the degree of dissociation. 104 COLLOID CHEMISTRY OF THE PROTEINS evaluate the valency of the caseinate ion. Taking the result of Laqueur and Sackur that i gm. of casein is neutralised by 0-881 millimols of sodium hydroxide, the equivalent weight of 1,000 casein is 0-881 = 1,135. This value, in the light of more recent work (see below), appears to be somewhat too high. The mole- cular weight of casein is certainly greater than this, but will be an integral multiple of this number. From consideration of a large number of mono- and poly- basic organic acids (up to a basicity of five), W. Ostwald has deduced the rule that the increase in conductivity when equiva- lent weights of the sodium salts are diluted is proportional to the valency of the anions. The relation is particularly simple if the increase in conductivity at 25 for volumes of 32 litres and 1,024 litres is considered. The difference A 1024 A 32 = n X 10, where n is the valency of the anion. The following table by W. Ostwald shows over what a wide range the rule holds good. Table 52. Sodium salts of A A 1024 32- Quinolinic acid . . . . IQ-8 = 2 x 9'9 \ Phenyl pyridine carboxylic acid 18-1 = 2 X 9'I 1 Pyridine tricarboxylic acid [i, 2, 3] 31-0 = 3 X 10-3 } Pyridine tricarboxylic acid [i, 2, 4] 29-4 = 3 X 9-8 { Methyl pyridine tricarboxylic acid . Pseudo-aconitic acid .... 30-8 29-6 = 3 X 10-3 ( = 3 X 9'9 J Pyridine tetracarboxylic acid . 40-4 = 4 X io-i Propargylene tetracarboxylic acid . Pyridine pentacarboxylic acid 41-8 50-1 = 4 x 10-45 f = 5 X io-o For sodium caseinate, the values A 32 == 45, and A 1024 = 75, are obtained graphically when v and A v are used as co-ordinates in place of i/V and A v . The difference is 30 or 3 X 10, arid thus in this salt the caseinate is by Ostwald's rule tribasic. The mole- cular weight itself therefore becomes 3,400. Laqueur and Sackur believed the casein ions to have a low mobility, and the use. of the relative decrease in conductivity, (A x A 2 )/A 2 , to be essential when calculating the basicity, and therefore estimated the basicity of the casein to be 4 6. The normal behaviour SALTS OF ALBUMIN WITH BASES 105 of casein with regard to the equivalent conductivity, however, leaves no doubt that this is an ordinary case for the application of Ostwald's rule. Further, the relation emphasised by Bodlander and Storbeck that the degree of dissociation of a salt of (n -f i) ions is the wth power of the degree of dissociation of a salt of (i + T ) ions, although of restricted application, gives the value n = 3 for the valency of the caseinate ions when sodium caseinate is compared with sodium bromide and sodium chloride. The experimental determination of the valency of the casein- ate ion by the osmotic pressure presents no mathematical difficulty when the degree of dissociation is known. It is, however, rendered impossible by disturbances due to hydro- lysis which, in conse- quence of the constant dialysis of even small quantities of alkali, increases so much that usually after twelve hours a separation and even precipitation of casein set free by hydrolysis makes its appearance. Under these conditions, the molecular weight calculated from the osmotic pressure is invariably too high. Electrometric measurements have shown that of the proteins thus investigated casein is distinguished by a quite extra- ordinary capacity for combining with alkali in excess of the alkaline solution. A value of 10 milligram equivalents of alkali hydroxide per gram of casein can be attained, corresponding to a value of 100 for the equivalent of the casein ; and so, as a valency of three is deduced from the experiments in which a gram of casein combines with 0-9 millimols of sodium hydroxide, the maximum valency displayed when saturated with alkali is, in round figures, thirty. This high value for the combining capacity of casein is obtained from the work of Pauli and F. Kryz quoted below, in which, however, there is some uncertainty in the values for 4 69 FIG. 24 (Table 53) . 10 JO io6 COLLOID CHEMISTRY OF THE PROTEINS concentrations of more than 0-06 N alkali. It can be seen from Fig. 24 that a range of practically complete combination follows on increasing alkali content, as new acid valencies of the casein come into play almost in proportion to the quantity of alkali present. Table 53 (Fig. 24). Stock Solution : 4*5 gm. Dry Casein in 500 c.c. o-oi NNaOH. (Electrometric determination made one hour after mixing.} 0-45 per cent, casein. 0-225 per cent, casein. 0-1125 per cent, casein. N NaOH. N NaOH. N NaOH. Added. Combined. Added. Combined. Added. Combined. 0-025 0-045 0-065 0-085 0-105 0-125 0-0124 0-0189 0-0257 0-0307 0-0376 0-041 0-0425 0-0625 0-0825 O-IO25 0-0085 O-OII* 0-139 0-167 0-04125 0-06125 0-08125 0-IOI25 O-I2I25 0-00425 0-0047 0-0051 0-0064 0-00778 The original neutral alkali caseinate made by Laqueur and Sackur by titration with phenol phthalein contained 0-9 (or accurately 0-881) millimols of alkali per gram of dry casein ; whereas the work of Pauli and J. Matula gives the result that when excess of casein is shaken at 20 in o-oi N sodium hydroxide about 2 gm. of casein dissolve in each 100 c.c. The fairly clear and stable solution obtained in this way contains only 0-5 millimols of alkali per gram of casein. This somewhat complex behaviour becomes much clearer if we start from this caseinate saturated with casein. In the first place it is possible to dilute the above solution with half its volume of alkali of the same concentration without any consi- derable displacement of the H ion concentration from the neutral point. A series of such results is given in Table 54 on p. 107. It follows from these experiments that all caseinates contain- ing between I and 2 gm. of casein per millimol of alkali behave SALTS OF ALBUMIN WITH BASES 107 Table 54. (Measurements at 20 ; content of alkali o-oi N in all cases.) Per cent, casein. PH- C H or C OH - 1-92 6-308 4-92 X io- 7 N H- 0-82 7-964 7-92 x io- 7 N OH' 2-01 6-387 4-00 x io- 7 N H- i-33 7' 1 43 1-20 x io~ 7 N OH' I-OI 7-771 5-10 x io- 7 N OH 7 0-905 7-294 1-70 x io- 7 N OH' as neutral salts, and thus the caseinate obtained by Laqueur and Sackur on titrating casein in presence of phenol phthalein represents only one arbitrarily selected member of this con- tinuous series. The particular case they investigated certainly lies near that end of the series where the concentration of Table 55 (Fig. 25, Curve II.). (t = 20, C H = 4-92 X io- 7 .) % casein. K. V. \. 5-76 _ 33'3 40-7 * 1-92 50-6 X io- 100 50-6 0-96 27-7 X 10- 200 55'4 0-48 14-9 X io- 400 59'6 0-24 8-4 X io- 800 67-2 O'I2 4-6 X io- I6OO 73-6 ~ ~~ 00 79-0 Table 56 (Fig. 25, Curve III.). = 25. % casein. K. V. A. 2-88 33-3 44-1 * 0-96 52-7 x io- IOO 52-7 0-48 28-7 X io- 200 57'4 0-24 15-7 X io- 4OO 64-8 0-12 9-0 X io- 800 72-0 O-O6 4-9 X io- 1600 00 83^4 io8 COLLOID CHEMISTRY OF THE PROTEINS 110 iOO i.i goo w zoo protein is least. An increase in alkali content at this point leads to increasing quantities of it remaining in the free state. The results shown in Tables 55 and 56 give the conductivity data for increasing dilution of the casein-saturated caseinate with water. The original solution was made by shaking with 0-03 N sodium hydroxide. Similar figures are given for such a solution diluted with an equal volume of the alkali, forming a caseinate half saturated with casein. The conductivity curves (Fig. 25) run parallel to each other. The A a of the salt saturated with protein has the value 79, corresponding to Vcaseinate = 28 ; the A^ of the half saturated caseinate has the value 83 84, which leads to a value for mobility of the caseinate ion of 32 -5 . Thus the mobility is 14 per cent, less in the saturated solu- tion than it is in the latter case. The application of Ostwald's rule gives A 1024 A 32 = 3 X 10-1, or a tri- valent casein ion, in both cases. Hence when one passes from the half saturation to the almost complete saturation of the alkali salt with casein there is no change in valency, and only a moderate decrease in mobility of the ions. Of course an increase in valency would cause a great rise in the ionic conductivity (see p. 125). We should expect such an increase in valency from the curves showing the course of combination of alkali and casein, when the continued addition of alkali causes new acid valencies of the casein to become active. If to a solution of alkali caseinate saturated with casein vu 58 56 5*t & on a v ^ N, -+ n lafi- >eCo ncen trah 04 06 0# 10 FIG. 26. SALTS OF ALBUMIN WITH BASES 109 increasing quantities of the same solution of base are added, there is a moderate increase in conductivity up to a dilution of one half. Beyond that point the equivalent conductivity increases rapidly owing to increasing valency and consequent increase in mobility of the ions. This is shown by the following results, in which the free alkali is taken into consideration. The corresponding curve (Fig. 26), which must be read from right to left, shows a sharp break when the dilution becomes greater than one half (between 0-4 and 0-6). Table 57 (Fig. 26). 2-01 per cent, dry casein in 100 c.c. o-oi N NaOH, progressively diluted with o-oi N NaOH. % casein. Relative concentration. A. P H- C H or0 OH . 2'OI I'OO 5 2'6 6-396 4'10 X io- 7 NH- x '33 0-66 54' 1 6-91 1-2 X io- 7 NOH' I'OO '5 7-771 5' 1 X io- 7 N OH' 0-90 0-45 56-4 [55'94] * 8-3 1-7 X io-NOH' 0-80 0-4 60-3 [59-7 6 ] * 9-698 2-0 X io- 5 N OH' 0-50 0-25 78-6 " * Corrected values. These results all become explicable without difficulty when the following view is adopted. A neutral salt exists, composed of approximately i millimol of alkali and i gm. of casein, the ion being trivalent, viz. : Na 3 . (Casemate)' 77 (I.) This salt can form a typical complex salt by addition of a molecule of casein : Na 3 . (Casemate)'". (Casein) (II.) the valency remaining unaltered. Addition of alkali converts salt (II.) into salt (I.) with moderate increase in conductivity, the valency remaining constant. The equivalent weight of casein calculated from these results is 1,000, somewhat lower than the value obtained by Laqueur and Sackur, giving a molecular weight of 3,000. Probably the alkali salts of the globulins and of the acid-albumins behave in a similar way to that of casein, which, however, is particularly suitable for experiment owing to the abundance of its acid valencies. CHAPTER VIII ALTERATIONS IN STATE OF THE ALKALI PROTEINS WITH LAPSE OF TIME MORE detailed experiments have soon shown that the physico- chemical properties of alkali protein solutions alter with lapse of time in a remarkable manner. This occurs even at room temperature and on addition of quite low concentrations of alkali when no question of breaking down of the protein can arise. The compounds of acids and proteins, on the other hand, are remarkably stable under the same conditions. These changes of state with time were first investigated in a detailed manner by C. Schorr * at this Institute. He found that the viscosity of an approximately i per cent, serum albumin in alkali concentrations of 0-04 N upwards rose with time to a maximum value and then decreased again. The following table gives typical results for the viscosity at 24-5. Table 58. Time. N NaOH. Time. N NaOH. Time. N NaOH. 0-083. 0-25. 0-083. 0-25. 0-083. 0-25. Original value (extra- polated) 1-351 I-378 - 28' 25" I-456 2hr. 3 7' - 595 5' 35" 1-374 - 30' - 2hr. 51' 30" 612 7' 30" I-45I - 31' 40" 1-471 3 hr. 5' 594 8' 20" 1-386 - 33' 30" 594 3hr. 4 i' 586 n' 10" 1-397 - 36' 30" 624 4hr. 13' - 580 1-465 i 3' 30" 1-508 - 37' 40" 1-488 4 hr. 45' i '45 1 14' - 1-411 - 40' 30" 607 5hr. 20' - ^- I-425 14' 30" I-5I7 hr. 9' 30" 1-542 581 5hr. 55' - 1-412 16' 45" 1-418 1-539 hr. 27' 30" 1-567 24 hr. - - 1-512 i 9' 40" 1-430 1-554 hr. 30' 567 33 hr. - -- 1-318 2I / 30" 1-563 hr. 48' 30" 557 46 hr. - - 22' 35" 1-441 hr. 54 1-578 27' 30" 1-583 2hr. 22' - 1-588 * Biochem. Zeitsch., 1911, 37, 424. ALTERATIONS IN ALKALI PROTEINS in A lower temperature delays the alteration in viscosity con- siderably and increases the maximum value, probably owing to inhibition of the processes which find their expression in the decrease of viscosity. The variations in viscosity correspond to a change in con- ductivity with time ; this property suffers a continuous decrease, and no point when the decrease is reversed corresponding to the alteration in viscosity can be recognised, as the following table shows. Table 59. Specific Conductivity of Alkali Albumin (K X io 6 ). Time after mixing (extrapolated). 0-0125 N NaOH. 0-0463 N NaOH. 0-125 N NaOH. - o' 1,465 7,188 2,3258 - 12' 7,i656 13' 2,3196 ~ l< 7,i6i 4 1,4586 2,317-2 17' i,455i 2,3163 18' 7,i52 6 19' - 20' 2,3144 23' 1,454,'i 2,3129 - 25' 2,3125 - 27' 7,!326 2,3115 30; 7,1297 36' 39' 7,n8 2 2,3o6 fi 42' 2,3059 44' 7,0983 - 45' 1,4481 - 56' 60' i hr. 10' 1,4424 i hr. 19' ' i hr. 24' 2 hr. 4' 3 hr. 6' M34o 6 hr.. 25' 6,9327 2,273s 7 hr. 35' 6,7583 16 hr. 35' 16 hr. 48' 20 hr. - 1,338! 24 hr. - 6,38o 6 2,241 29 hr. - 6,2903 51 hr. - 1,250-2 55 hr. - ~ ~ 112 ALTERATIONS IN STATE OF THE The 1-18 per cent, serum albumin used by Schorr shows a minimum in the quantity precipitated by alcohol (Table 45), together with a maximum of viscosity and of conductivity in an alkali concentration of approximately 0-05 N. The following tables show this property. Table 60. 7^ = viscosity of the NaOH ; 77 viscosity of ox-serum albumin -\- NaOH ; rj ca i c . = calculated viscosity of NaOH -\- albumin. NaOH. TI I ~n- Irtlc. 1 'Jcalc. o-o N o-ooo I-072 072 o-ooo 0-005 I-OOI 086 073 0-013 0-04 I-OI 'US 082 0-033 0:05 I-OI2 2 7 084 0-186 o-i I-O24 26 5 096 0-169 o-5 I-I09 320 181 0-139 Table 61. Specific Conductivity of Alkali Albumin at 25 (x io 6 ). t of the NaOH ; K, of the ox-serum albumin -f- NaOH (observed] Kcaic., of the albumin + NaOH (calculated}. NaOH. K]. K. K ca,c. K ca,c. - K - o-o N 142 142 o 0-005 Illg 417 I26i 84 0-0125 2739 1465 288i 142 0-037 7837 5425 7979 255 0-0463 9655 7l8 8 979i 261 o-i 2,0165 1,833 2,031 198 0-125 2,491 2,326 2,505 179 0-25 4 ,872 4,806 4,886 80 This maximum of albumin ions corresponds with the fact that the increase in viscosity with time also reaches a maximum in this concentration of alkali. The same holds good of the decrease in conductivity of alkali albumin mixtures with lapse of time. Compare, for example, the following table with the preceding one. ALKALI PROTEINS WITH LAPSE OF TIME 113 Table 62. Diminution of Conductivity with Time as related to concen- tration of Alkali (i*i per cent. Ox-serum Albumin). NaOH. Extrapolated original value KO x io 6 . Conductivity after 7 hr., K X 106. Percentage diminution KO- K -^ x 100. 0-005 N 4 1 ? 4 I6 o-o % 0-0125 1.465 1,412 3-6 0-037 5,425 5,129 5'6 0-0463 7,l88 6,763 5-9 o-i 18,330 17,630 3-8 0-125 23,260 22,690 2-5 0-25 48,060 47,650 0-9 The variation in viscosity and conductivity of a i per cent, ox-serum plus 0-125 N sodium hydroxide is most marked in the first six hours. Schorr was unable to discover during this time any difference in molecular concentration by determining the depression of the freezing point. This method, however, is inadequate to detect the small changes which might occur. The changes in viscosity with time lend considerable pro- bability to the conclusion that the hydration of the albumin ions and perhaps also of the neutral particles of alkali protein first increases with time, and then decreases again. The fact that a relative maximum occurs, with increasing alkali content, at a point corresponding to the maximum ionisation of the alkali protein, shows that the ions are principally concerned in this effect. The rise in hydration of the particles of albumin accounts for the decrease in electrical conductivity as the mobility of the protein ions is thereby diminished. Apart from the fact that a parallel between viscosity and conductivity is missing over a considerable range, other evidence is all in favour of the possibility of an increasing combination of the protein with the alkali as time goes on. This process leads to the disappearance of the particularly mobile hydroxyl ion, and hence to a continuous decrease in conductivity. It is only necessary now to demonstrate that the quantity of alkali which enters into combination actually increases as time proceeds, and this is shown by the following experiments of Pauli and Spitzer (loc. cit.). ii4 ALTERATIONS IN STATE OF THE Table 63. Variation in combination of Alkali and Albumin with Lapse of Time. After time. P H- OH' C combined per litre. O'OiN NaOH ; 1-26 % serum albumin. 15' 80' 280' 425' 25 hr. 12-003 II-987 11-941 II-899 II-83 4 2-137 2-153 2-199 2-24I * 2-306 203 X 10- 232 X 10- 309 X 10- 373 X io- 460 X IO~ o-o$N NaOH ; 1-26 % serum albumin. 15' 1 60' 400' 24 hr. 12-600 12-553 12-499 12-435 1-540 I-587 I-64I I-705 1-672 X 10- 2-Ol8 X 10- 2-366 X 10- 2-726 X IO- The alkali caseinates also show changes in physico-chemical properties with lapse of time, the viscosity variations being particularly interesting. Laqueur and Sackur made the far-reaching generalisation, which later work has shown to be unexceptionable, that the ions of the protein are respon- sible for high viscosity. It is based on two observations : first, the increase in viscosity. of caseinates on addition of alkali, and secondly, the decrease when sodium chloride is added to sodium caseinate. These authors explained the latter effect by the action of the common ion of the sodium chloride in suppressing the ionisation of the sodium caseinate. This loses its cogency since it has been shown by Pauli and Handovsky (loc. cit.) that any salt, whether it contains a common ion or not, produces this result with alkali proteins. Further, investigation has also shown the viscosity of the alkali caseinates to be less simple than this, and in particular to be complicated by changes with lapse of time. The experimental results * in Table 64 show this behaviour clearly. The viscosity of the caseinate actually passes through a maximum value as the concentration of alkali is increased, but the high viscosity falls off with progress of time, and in five to ten * Biochem. Zeitsch., 1915, JO, 489. ALKALI PROTEINS WITH LAPSE OF TIME 115 Table 64. Sodium Hydroxide + 0*5 per cent. Casein at 25. Concentrations of NaOH (X N). 0-0045. 0-0055. 0-0095. 0-0145. 0-0185. 0-0195. Immediately 1-1185 1849 1-2028 2064 1939 1885 After 5' 1723 1-2028 2064 1939 1885 10' 1-1023 1454 I-I975 1993 1939 1885 15' 1-1849 1939 1939 1885 20' 1383 1777 1849 1939 1885 30' 1-0933 1310 1759 -1849 40' 1221 1723 1795 50' 1149 1634 1705 66' 1149 1580 1508 70' 1490 1-1490 After After After After After 17 hr. 5hr. 8 hr. 45 hr. 30 hr. 1-1041 1-1041 1-1039 1-1039 1-0956 hours reaches a fairly constant value which is independent of the alkali concentration. An increase in viscosity preceding the decrease, such as appears when time observations are made on alkali albumins, is not noticeable in the case of casein. On the other hand the rapid decrease in electrical conductivity is also displayed by casein ; for example, with 0-5 per cent, caseinate plus 0-025 N sodium hydroxide, it falls to half the original value after forty-eight hours (see the same paper, Table 4) . The increase with time of the quantity of alkali which enters into combination is also very clear with alkali caseinates, as the following table shows. Table 65. Temperature 18 21. 0-5 per cent, casein in all cases. Time in days. 0-02 N NaOH combined. 0-04 N NaOH combined. 0-08 N NaOH combined. 0-0124 N 0-0177 N 0-0307 N 2 0-0134 0-0186 0-0315 3 0-0134 0-0202 0-0335 5 0-0142 O-O2O2 , 6 0-0143 7 0-0159 O-O2I5 8 0-0152 0-0221 ~~ 82 n6 ALTERATIONS IN STATE OF THE An alteration of the freezing point depression with time can be noted in such solutions when the alkali content is high. This result corresponds to a decrease in total molecular con- centration, and, like the fall in conductivity with time, probably finds its explanation in the increased combination of alkali with casein, as proved electrometrically. In any case, the observation of a decrease in molecular concentration of the alkali casemates is evidence against the existence of any considerable hydrolytic decomposition of the protein. These observed results can be considered from the chemical point of view by dealing with the question of the structure of the salts of proteins with bases. The earlier workers, par- ticularly Liebermann and Bugarsky, held the view that proteins form typical metallic salts with bases. This conception was adhered to by W. B. Hardy and by Pauli, and expressed in the following equation, in which the free carboxyl groups react as acid valencies with formation of a negative protein ion : /NH 2 /NH 2 R< + NaOH -- > R< + H 2 O. X COOH X COO . Na T. B. Robertson, also, is entitled to the credit of emphasising the importance of the peptide linkage as the source of basic and acid valencies, and bases his theory on the following scheme, in which the peptide group reacts in its enol form : ...... COH:N ...... + NaOH -- > ... CONa++ + N" ... OH H According to this scheme, the protein is ionised by rupture at the peptide linkage with formation of two oppositely charged protein fragments. Further, according to Robertson, diamino- and dicarboxyl-groups play a leading part, so that, doubling the previous equation, the formation of two quadrivalent radicles from a double peptide linkage is seen to occur : X COH:N /COK++ N" R/ +2KOH -- >R< + X COH:N- VOK++ N" ALKALI PROTEINS WITH LAPSE OF TIME 117 Protein salts of the Robertson type, therefore, give no free metal ions, but only equal numbers of oppositely charged protein ions. When investigated in the electrophoresis appa- ratus, however, the protein salts with alkali show only negative protein ions, in the same way as when acid is added, migration to the cathode alone occurs. In this place, without going into the arguments which Robertson uses to support his theory, we will give a summary of the facts which tend to show that alkalis form regular salts with proteins, the latter behaving as polybasic I acids. 1. The protein part of the alkali protein migrates to the positive pole, while the alkali content increases at the negative pole. 2. The viscosity of solutions of the protein salt passes through a maximum value corresponding to a repression of the ionisation of the alkali protein in excess of alkali a behaviour exactly parallel to that of acid albumin in excess of acid. 3. The difference in electrical conductivity between sodium caseinate and ammonium or potassium caseinate corresponds to the difference in mobility of the sodium ion compared with the ammonium or potas- sium ion. This behaviour is compatible only with the existence of free metal ions. 4. When casein is added to sodium hydroxide solution a fall in the total molecular concentration of the latter occurs which can be demonstrated by the diminution of the freezing point depression. This effect is pro- duced by the polyacid character of the casein, as a result of which only one polyvalent casein ion appears when several OH ions of the alkali are replaced. In the proteins that have been studied the basic constant K b is large compared with the acid constant K a . In dialysis of natural albumin the alkali is the more difficult to remove, so much so that at one stage in the dialysis the protein reacts as an alkali protein. The carboxyl-groups at the end of the chains in the protein molecule primarily provide the acid valencies for combination n8 ALTERATIONS IN STATE OF THE with alkali. These are the first to be protected by methylation of the protein, but even then a marked capacity for combination with alkali persists. For, besides the terminal carboxyl groups, the peptide linkages can also react as acid valencies. There is no doubt that by transformation into the lactim form (II.), thus : I. Lactamor ~^~~^~ _ + ~f = Lactim or II. keto form ' enol form it can function as an acid group with a hydrogen atom replace- able by metals. It is quite unknown how far the peptide linkages in the various proteins are originally in form I. or form II., but there is a certain amount of evidence that the enol form of some part of the peptide complex arises only as a secondary effect of the addition of alkali. This view is supported by the changes of hydration and conductivity with time, which, ceteris paribus, distinguish the alkali proteins from the acid proteins and which appear to be essentially a conse- quence of increased combination with alkali, which in turn is accomplished by rearrangement into the lactim form. In this way albumin can be regarded as a pseudo-acid as defined by Hantzsch. The occurrence of isomerism by transformation of the peptide linkage has been recognised for some time in the case of the di- and tripeptides. H. Leuchs and W. Manasse * showed, in 1907, that the ester of carbethoxyglycylglycine on hydrolysis with alkalis passes from the lactam form I. into the lactim form II. Here by the action of alkalis, therefore, the resulting glycylglycincarboxylic acid on esterification gives an ester isomeric with the original substance. Leuchs and La Forge f a year later found the same effect with diglycylglycin ester, in which both peptide linkages undergo the change. In the supposed transformation of the peptide linkage in higher polypeptides with which we are concerned, no asym- metrical carbon atom is directly engaged, and we were in fact * Ber., 1907, 40, 3235. t Ber., 1908, 41, 2586. ALKALI PROTEINS WITH LAPSE OF TIME 119 unable to observe any alteration of the optical rotation with lapse of time in glutin solutions in concentrations of alkali up to o-i N. On the other hand, a very marked falling off of optical rotation with time has been observed as a result of the alterations of state which take place in clupein and in glutin (A. Kossel *) . An explanation has been furnished by H. D. Dakin.f It is here a question of the change from the keto to the enol form by the action of strong alkali on the group : CH 2 CH I. I ;= || .::.< . . , II. -CO -C(OH) In this case an asymmetric carbon atom is concerned, so that, as the following example (hydantoin) shows, an optically inactive form II. results : NH . CO . NH NH . CO . NH I. R C* CO ; : R C==COH II. Dakin's representation of the rearrangement shows the carbon atom next the peptide linkage behaving as an asymmetric carbon atom, but the formation of the hydroxyl group with acid properties follows as in the lactim form in amino-acid chains. For clearer comparison we give below the scheme of Leuchs of the transformation at the peptide linkage and that of Dakin of the change at neighbouring linkages in the case of a tripeptide. Leuchs. NH 2 . CH 2 . CO . NH . CH 2 . CO . NH 2 COOH ^Z I. Lactam. NH 2 . CH 2 C(OH) : N . CH 2 C(OH) : N . CH 2 COOH II. Lactim. Dakin. NH . CO . CHR . NH 2 NH . CO . CH . R . NH 2 R.C*H.CO.NH-CHR ; > RC : C . OH . NH . CH . R COOH COOH I. Keto form. II. Enol form. * Zeitsch. physiol. Chem., 1909, 59, 492 ; 1909, 60, 311 ; 1910, 63, 165. t Am. Chem. /., 1910, 44, 48 ; /, Biol. Chem., 1912, 19, 357. 120 ALTERATIONS IN ALKALI PROTEINS Actual experiments on the combination with alkali of compounds with the Dakin tautomerism have not yet appeared. The difference in optical rotation between proteins and their salts with alkali observed by Pauli and M. Samec,* in direct analogy with the optical activity of the acid salts of the proteins already discussed, must not be confused with this variation in rotation with time. It has already been mentioned that salts of the proteins with bases show a regular behaviour in their combination with alkali in increasing concentrations ; the alteration in viscosity due to hydration of the alkali salt also varies in the same manner. Further, the order for different bases corresponds to that of their dissociation constants. With addition of alkali, the optical rotation is also observed to increase to a maximum corresponding to the saturation point. This value, however, remains constant on further addition of alkali. As in the case of acid, excess of alkali gives no decrease in rotation which can be attributed to repression of the ionisation. Thus, here again it is only the formation of the alkali protein and not the ionisation which affects the rotation. Table 66. Optical Rotation 1 C 1 -f- u 2 w 2 C 2 -f .... V\ = Vjwfi -f v 2 w 2 C 2 + .... When u lt u 2 ,v lf v 2 .... are the mobilities, w^w 2 ,w lt w 2 ...., are the valencies, and C lt C 2 ...., C lf C 2 ... are the concentrations of cations and anions respectively. Pauli and Matula have carried out experiments (not yet published) with a view to determining the mobility of the protein ions in this way. Various circuits, in which albumin salts were included, were built up, the following being a typical example. Solutions of the protein salt and of hydrochloric acid were included between two normal calomel electrodes. In the second circuit given below, concentrated potassium chloride solution, which gives the equally mobile K' and Cl' ions, was interposed to eliminate the diffusion potential. Circuit I. Hg/HgCl . N KCl/o-002 N HC1 -j- i per cent, albumin/ 0-002 N HC1/N KC1 . HgCl/Hg. * Zeitsch. physikai. Chem., 1907, 59, 118 ; 1908, 63, 325. MIGRATION VELOCITY OF THE PROTEIN IONS 131 Circuit II. Hg/HgCl . N KCl/o-002 N HC1 -f i per cent. alb./KCl cone./ 0-002 NHC1/N KC1 . HgCl/Hg. T -293 E L 0-025 volt. E n . = 0-059 v ^- Diffusion potential = E n . E L = 0-0322 + 0-0005 volt. A careful analysis showed that the albumin solution contained ammonium sulphate in a concentration of 0-0046 N, owing to the purification of the albumin by the precipitation method. Hence C S o 4 = C N H 4 = 46 X io~ 5 . The hydrion concentration 2 in the albumin HC1 mixture was 5-4 X io~ 5 N, and the concentration of chlorine ions 170 X io- 5 N. In the solution of pure hydrochloric acid C H = C C i = 170 X io~ 5 N. Thus combination of hydrion with the albumin accounts for the difference, 164 X io~ 5 N, in the hydrion concentration ; and this is equal to the normality of the albumin salt. Also, UH = 330, v c i = 68, y s o 4 = 7 X > W NH 4 = 67. These various values were then substituted in Henderson's equation, which was then solved for U A ib- The value obtained is 5 8, if it is assumed that the albumin ion is monovalent in this low concentration of acid. This result, as far as order of magnitude is concerned, is in satisfactory agreement with that obtained by Pauli and Oden for the mobility in low concentra- tions of acid. The determination of migration velocity by the method of diffusion potentials has proved in practice to be a somewhat complicated matter and not at all sensitive ; but such methods have a special importance in elucidating the origin of the bio-electric current. The behaviour of salts of the proteins on electrolysis is closely connected with the valency of the protein ions. As we now know that proteins form typical metallic salts with alkalis, we should expect that when alkali proteins are electrolysed the metallic ion would appear at the cathode, and the protein ion at the anode. The former ions react with the water with formation of the original alkali, the latter in the case of a protein 9-2 132 COLLOID CHEMISTRY OF THE PROTEINS is insoluble, and is precipitated as such. The alkali caseinate described above, as it is capable of more accurate definition, is particularly suitable for investigations of this kind, as in solution the concentration of free hydroxyl ions and of alkali ions is so small as not to come into consideration during electrolysis. If a second electrolytic cell in which the products can be easily estimated, e.g., a silver voltameter, is included in the main circuit with the solution under investigation, the ratio of the weight x>f silver deposited to the quantity of protein precipi- tated can be determined. The equivalent of the latter can then be deduced from Faraday's law. Thus in the case of the two caseinates I. K 3 . (Caseinate) '" and II. K 3 . ([Caseinate]"' . Casein) when introduced into such a circuit in separate cells, the same current will precipitate at the anode twice as much casein in solution II. as in solution I. As it happens, T. B. Robertson,* inter alia, has made a number of measurements of the electro-chemical equivalents in such mixtures of casein and alkali. Solutions containing 50 X io- 5 gm. equivalents (0*5 millimol) and 100 X io~ 5 gm. equivalents (i millimol) of potassium hydroxide per gram of casein respectively correspond accurately to the caseinates mentioned above. Robertson's measurements of the electro-chemical equivalent of a protein are, so far, the only ones of their kind, and it is to be regretted that he is so much under the influence of his theory of the formation of protein ions that he has failed to make the deductions which would be warranted by the importance and accuracy of his work. His apparatus comprised a U-tube with platinum wires leading into it. The two limbs of the U-tube could be connected by a three-way stop-cock, or could be separately emptied. The anode was a spiral of platinum wire of such a length as to permit of a suitable current density, and -at the same time retain * " Physical Chemistry of the Proteins," p. 176, where the literature is quoted. MIGRATION VELOCITY OF THE PROTEIN IONS 133 the adhering precipitate of casein. The concentration of casein in the solutions at the electrodes was found by determina- tions of the alteration in refractive index with a refractometer. A silver voltameter arranged for titrimetric estimation was included in the circuit. The work was carried out at a tempera- ture of 30. The liquid at the anode always became poorer in casein but unchanged in reaction towards indicators, whereas that at the cathode also became weaker in casein, but developed a strongly alkaline reaction. This behaviour is easy to under- stand from the conception of the structure of the caseinates which we have emphasised in the foregoing pages. When, on the other hand, we apply Robertson's conception, difficulties arise at once with these results. He maintains that the alkali enters the molecule at the peptide linkage, with production of two oppositely charged protein ions : H R.N"andKO.C.R+ + OH The former would move to the anode, the latter towards the cathode. Robertson further assumes that the negative ion reacts with water at the anode with liberation of oxygen and formation of solid casein, and that the positive ion reacts with water at the cathode, hydrogen being liberated and casein formed. The casein at this pole dissolves at once in the potas- sium hydroxide which is produced at the same time. Thus he quite overlooks the fact that these protein products do not give the original casein when they react with water, but can only give a new protein product, which must be different at the two electrodes, the new substances being fractional parts of the casein. According to Robertson the secondary reaction at the cathode must always lead to the saturation of the base with protein, and thus even with varying proportion of alkali to casein in the original solution, a constant electro-chemical equivalent would be found for various caseinates. The observed values, however, show no such constancy, for with increasing content of alkali the electro-chemical equivalent decreases. 134 COLLOID CHEMISTRY OF THE PROTEINS This variation was corrected for by Robertson on the basis of the following line of reasoning. If the anode with the adhering precipitate of casein is immersed in the original caseinate solution, part of the precipitate passes into solution ; this is particularly the case in the solutions containing more alkali and less casein. This quantity is determined, taken as a loss during Table 71. 50 X io~ 5 equivalents KOH per gram of casein. Per cent, casein. Current in amperes. Time of electrolysis. Gram casein lost from the solution. Electro-chemical equivalent, milli- gram per coulomb. 6 11-51 X IO- 4 2hr. 15' 0-2155 23-1 1-9 4 9-47 X I0- 4 2 hr. o' 0-1645 in 2 4 -I 2-6 4 18-05 X 10- 2hr. 15' 0-3815 M 26-1 1-2 3 10-54 x I0 ~ 2 hr. o' 0-1810 r 9 23-8 2-3 3 2 17-36 X 10- 11-22 X IO~ 2 hr. o' 2 hr. o r 0-3125! V, o-i8io| 25-0 1-4 22-4 2-2 2 15-84 X IO- i hr. 25' 0-1678 1 2O-8 i 2-2 Mean : 23-6 2-7 Table 72. 100 X io- 5 equivalents KOH per gram of casein. Per cent, casein. Current in amperes. Time of electrolysis. Gram casein lost from the solution. Electro-chemical equivalent, milli- gram per coulomb. 3 2 12-03 X IO- 4 12-56 X IO~ 4 2hr. i hr. 0-0988 0-0175 0-0493 0-0175 Mean : II-4 2 10-9 3'9 11-15 3 electrolysis, and the weight of the precipitate corrected by this factor. This argument is, however, erroneous, for the observa- tions on the solution of the precipitate are not made under the conditions which prevail in the neighbourhood of the anode. In that area, in consequence of the -more rapid transference of the K- ions (Hittorf), the liquid rapidly becomes weak in casejn- ate and thus the quantity of the very substance to which the MIGRATION VELOCITY OF THE PROTEIN IONS 135 capacity of dissolving the casein may be attributed is diminished. Apart from the fact that Robertson's corrected values still show clearly a tendency to decrease with increasing concentration of alkali, no safe basis for the correction applied by him exists at all. We give, therefore, the uncorrected, actual experimental results of this author in the tables on p. 134. A glance at these results shows that, as a matter of fact, salt I. (Table 72) exhibits half the electro-chemical equivalent of salt II. (Table 71), as we should expect from the constitution of the caseinates. For the complex ion [casein (caseinate'")] the mean value of 23-7 mg. is obtained ; for the simple ion (caseinate) '" the value of 11-15 mg. per coulomb. If the equivalent weight be calculated therefrom, taking 96,540 as the number of coulombs required to transport i gm. equivalent of an ion, we obtain 1,076 as the equivalent weight of casein which has been taken to be about 1,000 in the preceding pages. These electro-chemical measurements, therefore, provide valuable evidence in favour of the constitution of the caseinates which we have previously assumed. INDEX OF NAMES ABBOTT, G. A., 125 Adolf, M., 76, 89 Arrhenius, S., 69 Avogadro, A., 6 BlLLITER, J., 12, 14 Biltz, W., 12 Blasel, L., 86 Bodlander, G., 105 Bottazi, F., 23 Boyle, R., 6 Bray, W. C., 125 Bredig, G., 3, 15, 24 Brown, R., 8 Bugarsky, St., 56, 57, 58, 59, 60, 62, 66, 90, 93 Burton, E. F., 128 CHRISTIANSEN, J., 55, 88, 89 Clausius, R., 6 Cohnheim, O., 56, 80 DAKIN, H. D., 119 Davidsohn, H., 36 Dukes, P., 89 ELLIS, R., 12 Euler, H., 85 FALEK, O., 65, 73, 92, 97 Faraday, M., 132 Fernau, A., 13, 1 8 Fischer, E., 68 Flecker, L., 35 Freundlich, H., n, 12, 15 Friedenthal, H., 55 GAY-LUSSAC, J. F., 6 Gouy, M., 8 Graham, T., 2, 17 Grineff, W., 36 HAMMERSTEN, O., 101 Handovsky, H., 62, 73, 97, 98, 114 Hantzsch, A., 118 Hardy, W. B., 12, 13, 16, 56, 63, 85, 121 126 Hasselbalch, A., 51, 57 Hausmann, Wa., 87 Helmholtz, H. L. F. von, 125 Henderson, L. J., 130, 131 Henri, V., 13 Hevesy, G. von, 126 Hirschfeld, M., 25, 68, 69, 70, 73, 81, 83, 90 Hittorf, W., 134 Hoff, J. H. van't, 6, 7 Hofmann, F. A., 56 KJELDAHL, J., 20 Kossel, A., 119 Kryz, F., 105 LA FORGE, F. B., 118 Landsteiner, C., 21, 35 Laqueur, E., 55, 63, 64, 101, 102, 104, 106, 107, 109, 114 Leuchs, H., 118 Liebermann, L., 56, 57, 58, 59, 60, 62, 66, 90, 93 Linder, S. E., 12 Lottermoser, C. A., 18 Lunden, H., 24 MANABE, K., 59, 64, 71, 82 Manasse, W., 118 Matula, J., 14, 18, 38, 39, 59, 62, 71, 82, 86, 101, 106, 130 Maxwell, J. C., 6 Mayer, A., 13 Michaelis, L., 22, 23, 25, 29, 35, 36, 38, 41, 47, 51, 55 NERNST, WA., 7, 129 ODEN, SVEN, 125, 127, 130, 131 Oryng, T., 44 Ostwald, Wilh., 55, 104 Ostwald, Wo., 4, 10, 13 PAULI, Wo., 10, 13, 14, 17, 18, 19, 21, 22, 25, 35, 38, 39, 41, 42, 44, 45, 62, 64, 65, 68, 69, 70, 73, 74, 78, 81, 83, 85, 90, 92, 93. 97. IOI I0 3> i5, 106, 114, 120, 127, 130, 131 INDEX OF NAMES 137 Pechstein, H., 36, 38 Pemsel, W., 55 Perrin, J. t 6, 7, 8, n Pick, E. P., 91 Picton, H., 12 Plimmer, R. H. A., 87 Powis, F., 12 ROBERTSON, T. B., 58, 60, 80, 83, 95, 116, 117, 132 135 Rona, P., 10, 38 SACKUR, O., 55, 63, 64, 101, 102, 104, 106, 107, 109, 114 Salm, E., 55 Samec, M., 22, 35, 38, 41, 42, 45, 62, 74, 120 Schorr, C., 66, 77, 96, no Schultz, H., 12 Siedentopf, H., 4 Siegfried, M., 87 Sjoqvist, J., 56 Sorensen, S. P. L., 30, 31, 34, 41, 47, 5i, 57 Spiegel, E., 76, 89 Spiro, K., 10, 55 Spitzer, A., 93 Storbeck, O., 105 Svedberg, The., 6, 8 TYNDALL, J., 5 WAALS, VAN DER, J. D., 8 Wagner, R., 64, 78, 99 Walker, J., 24 Wegscheider, R., 125 Weimarn, P. P. von, 5, 13 Whetham, W. C. D., 124, 127 Wiener, C., 8 Winkelblech, K. f 85 ZSIGMONDY, R., 4, 15, l6, 125, 128 INDEX OF SUBJECTS ACID dissociation constant of pro- teins, 26 Acid-albumin of Adolf and Spiegel : optical rotation of, 76 combination of, 89 Acids : effect on electrophoresis, 20 combination of : with albumin, 55 sqq. with desamino-proteins, 86 with globulins, 121 Adsorption of ionised albumin, 89 Ageing : of colloids, 1 8 of alkali proteins, no Albumin : and acids, 55 sqq. and acetic acid, 71 and dilute strong acids, 47 sqq. and alkalis, 93 sqq. Albuminium salts, 57 Albumoses, viscosity in acids, 91 Alcohol precipitation of proteins, 38 sqq. in acids, 66, 77 in alkalis, 96 sqq. Alkali albumin, conductivity of, 112, 113 Alkalis, combination of: with proteins, 93 sqq. with globulins, 122 Amino-acids, dissociation constants of, 27 Ammonium sulphate sulphuric acid mixture, 52 Amphoteric electrolytes, 23 Anhydro-colloids, u Aniso-electric point, 50 Avogadro's number, 7 sq. BASES. See also Alkalis. effect on electrophoresis, 20 salts of proteins with, 93 sqq. Basic dissociation constant of pro- teins, 26, 84 sqq. Brownian movement, 8 Buffer solutions, 25 CASEIN, combination with alkali, 101 sqq. Caseinate ion : mobility of, 103 valency of, 103 Casemates, properties of, 101 sq. complex, 1 08 sqq. Coagulation of colloids, 16 Colloid particles : dimensions of, 4 electric charge on, 12 Colloids : and crystalloids, 2 dispersity of, 4 hydration in, 10 mutual precipitation of, 12 preparation of, 4 Colloidal solutions, 4 and true solutions, 13 Common ion, action of, 69 sqq., 79, 114 Concentration of proteins and iso- electric reaction, 41 Conductivity : method for estimating combina- tion, 56 of alkali albumin, in of globulin salts, 123 Constitution : and acidic properties of proteins, 116 and basic properties of proteins, 85 and colloidal property, 8 and dissociation constant, 28 and iso-electric reaction, 53 DEGRADED proteins, combination with acids, 90 sqq. Dehydration on discharge of particles, M Denatured albumins, properties of, 76, 89 Depression of the freezing point : by acid albumin, 66 by alkali albumin, 113 variation with time, 116 Desamino-proteins, properties of, 85 sqq. Dialysis, 3 Diffusion potential method, 130 INDEX OF SUBJECTS 139 Dipeptides, dissociation constants of, 27 Dispersoids, 10 Dissociation : and constitution, 28 at the iso-electric point, 29 of acid proteins, 59 sqq. of alkali proteins, 99 of amino-acids, 27 of amphoteric electrolytes, 24 of dipeptides, 27 ELECTRIC charge : due to ionisation, 14 on natural albumin, 19 Electrical radiations, action on col- loids, 13 Electro-chemical equivalent of pro- teins, 132 Electrolysis of protein salts, 131 sqq. Electrolytes, action on colloids, 12 Electrometric method, application of, 56 sqq., 94 sqq. Electrophoresis : apparatus, 19, 22 of glutin in acid, 46 of albumin in acid, 58, 62 of albumin in alkali, 99 Equivalent : of casein, 135 of globulin, 123 GAS Laws, application of, 6 sqq. Gels, 1 8 Globulins, salts of, 121 sqq. Glutoses, viscosity in acids, 91 HARDY'S rule, 12 Heterodrome and homodrome changes, 17 Hydration : of colloids, 10 of protein ions, 92, 113 Hydrion concentrations in buffer solutions, 26 Hydro-colloids, n Hydrolysis : of acid proteins, 80 sqq. of caseinates, 101 Hysteresis, 18 IMBIBITION, n of gelatin in buffer solutions, 40 Inversion method, application of, 56 Ionisation. See Dissociation. Irresoluble colloids, 15 Iso-electric point, 23 dissociation at, 32 hydrion concentrations at, 36 methods of finding, 34 Sorensen's method, 51 properties of proteins at, 37 sqq. Iso-electric reaction : in buffer solutions, 33 sqq. in strong acids, 49 sqq. Isomerism in polypeptides, 118 Iso-molar transport point, 50 KINETIC Theory, 7 LACTAM and lactim forms, 87, 118 Lyophile and lyophobe colloids, n MAXIMUM of neutral particles, 31, 45 Migration velocity of protein ions, 124 sqq. Molecular weight : of albumin, 83 of casein, 109 of globulin, 123 substances of high, 5 Mutual precipitation of colloids, 12 NEGATIVE charge on natural albumin, 23 Neutral albumin, properties of, 37 OPTICAL rotation : of alkali albumin, 119 sq. of various acid albumins, 74 Osmotic pressure of proteins : in alkalis, 105 in buffer solutions, 40 in acids, 44 Ostwald's basicity rule, application of, 104 PEPSIN, action on albumin, 88 sq. Peptide linkage, 87 sqq. Peptisation, 16 Precipitation : of colloids, Bredig's theory, 15 by acids, of albumin, 77 by alcohol, 38 sq., 43, 66, 77, 96 sqq. by alkalis, 99 by phenol, 88 Protalbumose, viscosity in acids, 91 140 INDEX OF SUBJECTS Proteins : importance of, i hydrophobe and hydrophile, 16 stability of, 16 properties at iso-electric point, 37 sqq. salts of : with acids, 55 sqq. with bases, 93 sqq. Pseudo-ions, 126 REGULATORS, hydrion, 25 Resoluble colloids, 15 Reversible and irreversible coagula- tions, 1 6 SALTS : effect on electrophoresis, 20 of proteins : with acids, 55 sqq. with bases, 93 sqq. Sols, 1 8 Structural and colloid chemistry, 8 Structure. See Constitution. Suspensoids, 10 TITRIMETRIC method of estimating combination, 55, 101 UNDISSOCIATED portion at the iso- electric point, 30 VALENCY : effect on mobility of ions, 125 importance at iso-electric point, 48 of caseinate ions, 103 of globulin ions, 123 of protein ions, 126 Viscosity : at iso-electric point, 39 of proteins : in acids, 42 sqq. in alkalis, 96 sqq. variation with time, no sqq. PRINTED IN GREAT BRITAIN BV THE WHITEFRIARS PRESS, LTD., LONDON AND TONJiRIDGE ~ sssss"" OVERDUE. nrr 13 1933 UNIVERSITY OF CALIFORNIA LIBRARY