FROM/THE LIBRARY OP LEONARD WILLIAM * BUCK* UNIVERSITY QF CALIFORNIA MEDICAL CENTER LIBRARY SAN FRANCISCO LEONARD W. BUCK, M.D. rag ' GENERAL CHEMISTRY OF THE ENZYMES BY HANS EULER Professor of Chemistry in tlie University of Stockholm TRANSLATED FROM THE REVISED AND ENLARGED GERMAN EDITION BY THOMAS H. POPE FIRST EDITION FIRST THOUSAND NEW YORK JOHN WILEY & SONS LONDON: CHAPMAN & HALL, LIMITED 1912 152651 Copyright, 1912 BY THOMAS H. POPE SCIENTIFIC PRESS ROBERT DRUMMOND AND COMPANY BROOKLYN, N. Y. PREFACE TO THE GERMAN EDITION As the title of this book indicates, the author has attempted to review the more important facts of enzymology from a general standpoint and to fit them, so far as is possible, into their proper places in the fabric of general and physical chemistry. The aim has not been to give a complete synopsis of our know- ledge of the enzymes, for already several such summa'ries are available. It may perhaps be asked: Is the time yet ripe for giving a representation of the physical chemistry of the enzymes? The author feels that this question must be answered in the affirmative, although it is evident that extensive regions and important problems in the subject are still entirely untouched. The period during which the marshalling of facts was the most essential task was followed by one in which it was sought to harmonize the somewhat crude and imperfect experimental data with the laws of theoretical chemistry. The deviations from theory seemed to be wide and the peculiarities of enzymic reactions numerous. Only in the most recent times has the need for experimental revision of the quantitative data made itself felt. Improvements have been effected in the practical methods, while the factors participating in the reactions have become more clearly understood and are hence more fully taken into account. It is now being found that the results obtained from these more exact and comprehensive investigations corre- spond more closely with those required to satisfy physico-chemical theories. At the stage which has thus been reached in the development of enzymology a review such as that now pub- lished does seem to be justified. The author has therefore decided to allow the two reports on this subject which appeared in the " Ergebnisse der Physiologic " in 1907 and 1910, to be iii iv PREFACE TO THE GERMAN EDITION arranged and issued in book-form, despite the fact that many problems still call for fuller treatment. Although this monograph is intended more especially as an aid to scientific research in enzymology, yet the author trusts that it will be found useful by those concerned with the prac- tical applications of enzymic actions. Thus an understanding of the dynamics of enzyme reactions is indispensable for the rational estimation of enzymic activities, such as that of pepsin in the gastric juice or that of diastases in malt, and these examples serve to show how theory may be of value to the physician and to the technical worker. An appendix to the book contains a short sketch of experi- mental methods, more especially of those for which the original literature is not readily accessible. Professor Bertrand has kindly permitted the insertion of the tables prepared by him for use with his admirable method of estimating reducing sugars. A considerable part of the labour involved in preparing this monograph has been undertaken by Miss Beth af Ugglas, Assistant in the Biochemical section of the Chemical Laboratory here and to her I wish to express my sincere thanks. H. EULER. STOCKHOLM, January, 1910. PREFACE TO THE ENGLISH EDITION ALTHOUGH only two years have elapsed since the first publication of this work in the German language, the great energy with which the study of enzyme chemistry is being prose- cuted has rendered necessary numerous additions and alterations. In view of the results of recent investigations, some of the sections, e.g., those concerned with the glucosides and the fermentation enzymes, have indeed been entirely rewritten. To Mr. Pope's request to allow of the issue of an English edition of the book the author acceded the more readily because of the great success which has for a long time past attended enzymological research in English-speaking countries. At the present time, when various different paths have become clearly marked in general enzymic chemistry, the opportunity is wel- comed of laying the author's views before English workers in this field. To Mr. Pope the author is indebted, not only for a careful translation of his book, but also for certain improvements and additions in the part dealing with practical methods and for the references to the literature. In the initial treatment of so extensive a subject as enzyme chemistry omissions are scarcely avoidable, and the author would be grateful to any readers who may contribute, either by sending him copies of their papers or by any other means, to render a subsequent edition more complete. H. EULER. STOCKHOLM, March, 1912. v JOURNALS REFERRED TO BRIEFLY Biochem. Z.: Biochemische Zeitschrift (Berlin). Chem. Ber. : Berichte der deutschen chemischen Gesellschaft. C. R. : Comptes rendus de rAcademie des Sciences (Paris). H. : Hoppe-Seyler's Zeitschrift fur physiologische Chemie. Hofm. Beitr.: Hofmeister's Beitrage zur chemischen Physiologic und Pathologic. Lieb. Ann.: Justus Liebig's Annalen der Chemie. Pfliig. Arch. : Pfliiger's Archiv fiir die gesammte Physiologic. Soc. Biol.: Comptes rendus de la Socie*te* Biologique (Paris). vi CONTENTS PAOE Introduction - 1 CHAPTER I Special Chemistry of the Enzymes 5 Nomenclature 5 Classification 6 Sphere of Action of the Enzymes; Their Preparation and Puri- fication 7 Esterases 9 Enzymes of the Higher Carbohydrates 13 'Enzymes of the Glucosides and Disaccharides 18 Other Enzymes which Hydrolyse Glucosides 27 Phytase 31 Hexosephosphatase 32 Pectinase 32 Qarbamases (Proteinases) '. 33 Pepsin 33 Trypsin 36 Erepsin 38 Proteolytic Enzymes of Plants 38 Nucleases 41 Urease 43 Amidases (Desamidases) 43 Coagulating Enzymes 45 Chymosin 45 Thrombin, Fibrin-ferment 49 Enzymes of Fermentation 50 Enzymes of Alcoholic Fermentation 51 Lactic Acid Bacteria-zymase 58 Oxydases 58 Alcoholase 60 Aldehydases 61 Laccase 62 Tyrosinase 64 vi i viii CONTENTS Peroxydases 65 Catalases 67 Reducing Enzymes (Reductases; Reducase) 68 Appendix 69 CHAPTER II Physical Properties of the Enzymes 71 Adsorption 75 Solid, Neutral, Adsorption-media 81 CHAPTER III Activators (Co-enzymes), Paralysors and Poisons 90 Kinases of Unknown Composition 91 Special Organic Activators 92 Acids, Bases, and Neutral Salts 94 Protective Agents 115 Inhibiting Agents (Paralysors) 115 Inorganic Salts 116 Organic Poisons and Inhibiting Agents 118 CHAPTER IV Chemical Dynamics of Enzyme Reactions 124 Theoretical Principles of Enzymic Dynamics 125 Catalysis 127 Reversible Reactions 137 Experimental Data on the Course of Enzyme Reactions 146 Esterases and Lipases 146 Amylase 155 Invertase 158 Maltase 166 Lactase 168 Enzymes of Emulsin 171 Proteolytic Enzymes 175 Rennet (Chymosin) 200 Fibrin-ferment 205 Zymase 206 Catalases 215 Oxydases 219 Peroxydases 223 Tyrosinase 22S Oxidation of Xanthine . . .230 CONTENTS ix CHAPTER V PAGE Influence of Temperature and Radiation on Enzymic Reactions . 231 Influence of Radiation . 245 CHAPTER VI Chemical Statics in Enzyme Reactions 251 Equilibria 252 End-states and Stationary States 255 CHAPTER VII Enzymic Syntheses 261 CHAPTER VIII Specificity of Enzyme Action 274 Conclusion 283 APPENDIX Practical Methods 286 Tables for the Estimation of Sugars by Bertrand's Method 306 Index of Authors 313 Index of Subjects 321 GENERAL CHEMISTRY OF THE ENZYMES INTRODUCTION THE name enzymes or unorganised ferments is given to animal or vegetable substances of unknown composition and constitution which, in the organism itself or even independently of the organ or cells in which they arise, are able to accelerate chemical reactions. The term enzyme is thus included in the much more general term, catalyst. By catalyst we understand a substance which, without being required by the accelerated reaction or appearing among the final products, alters the velocity with which a chemical system strives to attain its final condition. But enzymes are at least with the degree of purity in which they have as yet been obtained rarely ideal catalysts. Only with difficulty, however, can a limit be set between these substances and ideal catalysts, this being greatly dependent on the experimental conditions. The literature of the last few years shows, indeed, that a certain limitation in the meaning of the term enzyme is desirable ; and, as a rule, those substances which are required in stoichio- metric proportions by the reactions in which they participate are not regarded as enzymes. With non-enzymic catalyses the quantity of the accelerating substance is mostly small compared with that of the substance acted on indeed, an ideal catalyst should accelerate the trans- formation of unlimited amounts of " substrate." Even in the most minute quantities some enzymes certainly exert very con- siderable amounts of action; but usually their activity becomes limited with lapse of time and does not produce more than a certain amount of change. As we shall see later, this limitation is 2 GENERAL CHEMISTRY OF THE ENZYMES due, partly to the participation of the enzyme in the equilibrium of the reaction and partly to the chemical instability of these substances. One property which the enzymes exhibit and which is generally regarded as characteristic of them, is that of becom- ing inactive if their solutions are heated for a longer or shorter time at a high temperature about 100. This is not an absolute criterion, as inorganic catalysts and enzymes do not exhibit any fundamental difference in this respect. As soon as any con- stituent of an organ which accelerates a reaction is explained chemically or identified with a known compound, no reason remains for terming it an " enzyme"; whether the term enzyme is to be retained for such substances, or whether the name scientifically more accurate of catalyst is to be employed, is entirely a matter for the future. But the choice of a definition is of subordinate importance, as we are, in many cases, so far removed from any chemical explanation of the enzymes that this term will certainly persist for a long time. The distinction between the enzymes and the toxines is also, to some extent, arbitrary. Common to both classes of bodies are their origin in the living organism, their capacity of forming anti-bodies, and certain other properties, as also are their modes of action. On the other hand, the toxines are characterised with moderate sharpness by their poisonous action. We can, indeed, omit a treatment of this extensive subject all the more readily, as the physical chemistry of the toxines has undergone considerable development during recent years. Of far greater importance than deciding how the enzymes are to be limited is the definition of the physical and chemical prop- erties of typical representatives of these remarkable substances. The aim in view is, of course, the exact description of the enzyme by a chemical formula and by constants characteristic of the pure substance. For the general chemistry of the enzymes, clear views concerning the degree of purity and the composition of the various members are of the greatest importance. In the first place, the enzymes must be prepared and analysed, and here physico-chemical investigations also afford valuable aid. The first question to be decided is : Do the enzymes occur in a state of true solution, or must they be classed with colloidal substances? Or, speaking more strictly, which enzymes approach the one and which the other limiting case, and what can be INTKODUCTION 3 affirmed concerning their molecular magnitude and degree of dispersion? As criteria on these points serve diffusion, adsorption phenomena and also behaviour in the electric field. More recent measurements have shown that the influence of temperature on enzyme action can be defined more exactly than earlier data would have led one to suppose, and the " tem- perature of destruction " and " optimum temperature," which give little information, are now replaced by well-defined physico- chemical magnitudes. Undoubted and considerable success has followed the study, during the past few years, of activators or co-enzymes; men- tion need only be made here of the work of Harden and Young and of Buchner and Meisenheimer, which has led to the discovery of essential factors influencing alcoholic fermenta- tion. Also, with reference to inactivators remarkable regularities have been observed, those concerning the influence of configuration calling for special mention. Further, the action of poisons is now so far understood that, in enzyme investigations, we can make use of substances which prevent bacterial infection and yet have no harmful effect on the enzyme, thus avoiding the errors which have been so often caused in work of this kind by insufficient disinfection. Although the majority of the anti-bodies, so important physiologically, are classed among the toxines, yet such a large number of observations have been made on the anti-fer- ments that these must not remain unnoticed. When we have, in the first part of this book, obtained infor- mation concerning the chemical facts of enzymology, we must turn to the second part of the question: In what manner is a reaction induced or accelerated by an enzyme and how do enzymic reactions proceed? In the first place, we will consider the laws of chemical dynamics which come into play in enzymic reactions and, in particular, the results which have been obtained from a study of catalysts. Comparison of non-enzymic reactions with enzymic changes will show us that the same processes are being dealt with in the two cases and that the deviations from the classical examples of chemical dynamics, exhibited by many enzyme reactions, are readily explained by the simple assumption that enzyme and 4 GENERAL CHEMISTRY OF THE ENZYMES substrate unite to form more or less stable complexes, which are to be regarded as the " active " molecules and hence bring about the reaction. This assumption is adhered to all the more strongly, because it corresponds with our general conception of the role of catalysts. In the fourteen years which have passed since the synthesis of isomaltose by maltase was discovered (Croft Hill), the number of enzymic syntheses has become quite considerable. Not only do we now know enzyme reactions which proceed in both directions, but in several cases the synthetic action of the enzyme has been separated, and caused to take place apart, from the decomposing action. A knowledge of these syntheses is naturally of the utmost importance for the biochemistry of animals and plants. For chemistry in general these processes are the more import- ant, since, as is well known, the enzymes are extremely sensitive towards the steric configuration of the substrate and lead to the formation of asymmetric products. The enzymes hence place us in a position to effect asymmetric syntheses. CHAPTER I SPECIAL CHEMISTRY OF THE ENZYMES NOMENCLATURE THE already large and rapidly increasing number of enzyme actions necessitates a rational system of nomenclature. According to a proposal made by D u c 1 a u x (see Bourquelot, Les ferments solubles, Paris, 1896), the name of the enzyme is derived from that of the substance on which it acts: for example, lactase is the enzyme which decomposes lactose. Unfortunately, many departures have been made from this principle. In cases where the name derived from that of the substrate is not suf- ficiently definite, E. O. von Lippmann (Chem. Ber., 1903, 36, 331 ; see also B u c h n e r and Meisenheimer, Chem. Ber., 1905, 38, 621) proposes that both the name of the substance acted upon and that of the (principal) product formed from it should be indicated in the name of the enzyme; for example, amylo-maltase would be the enzyme which forms maltose from starch. But as this enzyme is not a maltase, but belongs rather to the class of amylases, it would be more convenient to term it malto-amylase. Names in general use, such as pepsin, zymase and erepsin, are retained. Although the employment of a rational method of naming enzymes is to be desired, yet, on the one hand, the right to give or alter a name must be left with the discoverer, and, on the other, the region of action and the individuality of enzymes, like pepsin are not yet so completely determined as to allow of the adoption of a perfectly suitable name. According to a suggestion by the author (H., 1911, 74, 13), the names of synthesising enzymes should be made to indicate the substances which they form and to terminate with the syllable "ese"; thus phosphatese would be the enzyme which syn- thesises organic esters of phosphoric acid, and nitrilese ( B-nitrilase according to Rosenthaler) that which forms nitriles. 5 GENERAL CHEMISTRY OF THE ENZYMES CLASSIFICATION Since very little is known concerning the nature of the enzymes, the classification of these bodies is based on the chemical reactions which they induce. And it is to be expected that the classifica- tion indicated by the chemical actions would also be brought out in the physical and chemical properties of the enzymes. An enumeration of all the enzymes described in the literature of the subject does not fall within the scope of this work; indeed, there are a very large number of such substances, the individuality of which has not been sufficiently well established. The following summary serves rather to indicate, in a general way, typical reactions in which enzymes play a part. Reac- tion. Substrate. Products. Enzyme. r Esters : Fatty acids +alcohols Esterases: Fats Higher fatty acids +glycerol Lipases Lower esters Chlorophyll +alcohol Lower fatty acids +alcohols Crystalline chlorophyll +phytol Butyrases Chlorophyl- luse Higher carbohydrates : Cellulose Cellulase Hemicellulose Cytase f Amylases and Starch, glycogeu Maltose (dextrins) j amylo-pec- [ tinases Inulin Fructose Inulinase Pectoses Pectin Pectase Glucosides including Polysaccharides : Hexoses and glucoside-residues a-Glucosides Glucose a-Glucosidase 1 /3-Glucosides Glucose +sugar, alcohol or phenol-residue (Maltase) 0-Glucosidase (Emulsin) 2 /3-Galactosides Galactose Lactase T3 >> Fructosides Fructose +sugar residues Invertase n Other glucosides Other sugars +phenols, etc. Rhamnase, Myrosin, etc. Phytin Inositol +phosphoric acid Phytase Hexosephosphate Hexose +phosphate Hexosephos- phatase Digallic acid Gallic acid Tannase (Tannin) Carbamide derivatives: R-CO-NH-R' R-COOH+R'-NH 2 Carbamases, proteinases Proteins Albumoses, peptones Pepsin, pap- ain Proteins, albumoses, peptones, peptides > Peptides, amino-acids / Trypsin, erep- ^ sin Arginine Urea +ornithine Arginase I Nucleic acids Nuclein bases +phosphoric acid Nuclease SPECIAL CHEMISTRY OF THE ENZYMES Reac- tion. Substrate. Products. Enzyme. Acid amides: 1 Urea Carbon dioxide +NH S Urease i . Amines : Desamidases 6 Amino-acids Hydroxy-acids +NHs Desamidase W Guanine Xanthine +NH, Guanase Adenine Hypoxanthine +NHj Adenase = . n Hydrogen peroxide Molecular oxygen +H*O Catalases |!l ' Hydroxvnitriles Aldehyde +HCN Nitrilases , * Benzaldehyde +HCN Mandelic acid nitrite d-Oxynitrilese = ?. , Na2HPO4 +carbohy- Ester of carbohydrate phos- 003 drate phoric acid Phosphatese ill? Peroxides f Reduction products of per- \ \ oxides +O Peroxydases Sgo >, ll|| Casein Paracasein ( +whey-albumen) Chymosin Fibrinogen Insoluble fibrin Fibrin ferment ?! S ' (thrombin) 5=^|S Pectins Pectinates Pectinase Glucose Lactic acid Zymase of lactic a acid bacteria o Glucose, fructose, man- Alcohol +CO Zymase (sum- g' nose, galactose total of the enzymes of al- 8 coholic f e r - mentation) Phenols Quinones Phenolases Aldehydes Acids Aldehydases f Alcohol Acetic acid Alcoholoxy d ase 3 of acetic acid bacteria Certain enzymes which exert actions other than those given above are mentioned hi the Appendix to Chapter I. SPHERE OF ACTION OF THE ENZYMES. THEIR PREPARATION AND PURIFICATION In this section are given such data as appear necessary for understanding the general behaviour of the enzymes. The dynamics of the enzymes suffers in considerable measure from the disadvantage that we know nothing of the composition of these bodies and hence can form beforehand no idea of the chemical processes taking place during their action. It is, there- 8 GENERAL CHEMISTRY OF THE ENZYMES fore, all the more necessary to investigate experimentally all the factors influencing enzymic reactions, in order to avoid the danger of missing a secure foundation for the theoretical treatment of the subject. Especially would the author point out that it is no t possible to pay too much attention to the preparation and purification of enzymes for use in physico-chemical measurements. Enzyme preparations are obtained either by subjecting the organs to pressure or by extracting them with suitable solvents. The consistency of the starting material, the admixtures which are always present, the age and especially the previous history of the preparation, influence not only the intensity, but also the mode of action of the enzyme to a greater extent than many investigators have supposed. A knowledge of the material is hence indispensable to a critical examination of the exper- imental results. Extracts or preparations of organs often exert several enzymic actions at the same time; thus, to choose an example from recent literatune, a preparation from croton seeds has been found by S c u r t i and Parrozzani (Gazzetta Chim. Ital., 1907, 37, i, 476) to hydrolyse, not only fats and esters of monobasic acids, but also cane sugar and proteins. From these results, it should be concluded, not that an enzyme exists possessing a general hydrolytic capacity, but that the preparation employed contains several enzymes. In order to study the separate components, the different actions have to be separated, and when a preparation has been obtained which exhibits only a single reaction, it is termed biologically pure. There still remains, however, the possibility that the various stages of the reaction are ccelerated by different constituents of the enzyme. It has, for example, been found to be probable that, in the hydrolysis of amygdalin, three enzymes take part, one of them effecting the resolution into glucose and the glucoside of mandelic acid nitrile, a second hydrolysing the latter compound to mandelomtrile and glucose, while the third decomposes the nitrile into benzal- dehyde and hydrocyanic acid. Even those enzymes which are biologically the purest are still very far removed from the state of chemical purity and we possess and on this stress must be laid no certain knowledge SPECIAL CHEMISTRY OF THE ENZYMES 9 that even an approximate isolation of any hydrolytic enzyme has yet been attained. This is explained by the instability of the enzymes, which, when subjected to protracted and energetic purifying processes, become inactive, so that their presence can no longer be detected; and also by the extremely small con- centrations in which the enzymes always seem to occur in nature, and by the large amounts of impurities especially of colloidal substances contained in the extracts. Of the oxydases, which are to some extent stable to heat, we have chemical knowledge of at least one member. We shall see later that the behaviour of enzymes towards ex- ternal influences, such as acids, alkalies, co-enzymes, etc., is often determined, wholly or partially, by the impurities present. On account of the importance which processes of purification have for enzymology, the methods employed are treated some- what in detail. As may be again mentioned, consideration of the whole of the literature on the different enzymes does not come within the limits of this work. In the first place, investigations will, of course, be omitted to which lasting value cannot be ascribed, and no attention will be paid to those dealing with purely phys- iological questions and with the distribution of the enzymes in the animal and vegetable kingdoms, since these are not directly connected with the general chemistry of the enzymes. ESTERASES The usual action of these enzymes consists in the hydrolysis of esters. The enzymes of this group prove to be more or less markedly specific, as will be shown more in detail in Chapter VIII. It must, however, be mentioned that the lipase of the stomach decomposes, not only true fats, but also the lipoids, lecithin, jecorin and protagon (P. Mayer, Biochem. Z., 1906, 1, 81; Schumoff-Simanowski and Sieber, H., 1906, 49, 50). Also pancreatic juice, according to A b d e r- h a 1 d e n and others, decomposes lecithin [but a negative result was obtained by Kalaboukoff and Terroine (Soc. Biol., 1909, 66, 176)]. A special class is formed by the L i p a s e s , which resolve more especially the natural fats, i.e., the glycerol esters of palmitic, stearic and oleic acids. 10 GENERAL CHEMISTRY OF THE ENZYMES Animal lipases play an important part in the stomach (gastric juice and mucous membrane l ), pancreas 2 and intestines 3 of the higher animals. Also serum contains lipases (Neuberg and collaborators) and, according to Pagenstecher (Biochem. Z., 1909, 18, 285), this is the case with all the organs, especially the liver and spleen, of the ox. These animal enzymes decompose both animal and vegetable fats and oils. A lipase has also been found in the albumen of hens' eggs. In general, it is relatively difficult to obtain active extracts from animal organs containing lipase and Connstein is of the opinion that it is best to employ the pancreatic juice of the crushed glands themselves. Aristides Kanitz, however, seems to have prepared active glycerol-extracts (H., 1905, 46, 482), and Lewkowitsch and M a c 1 e o d have worked with aqueous lipase solutions which attack neutral fat (Proc. Roy. Soc., 1903, 72, 31). The extraction of lipase-preparations from the pancreas has been investigated in detail by D i e t z and Pottevin (Bull. Soc. Chim., 1906, [iii], 35, 693; see also E. B a u r , Zeitschr. f. angew. Chem., 1909, 22, 97). . Rosen- h e i m (Journ. of PhysioL, 1910, 40) has recently made the interesting observation that the lypolytic enzyme can be separated from its activator or co-enzyme by mere filtration of the glycerol extract of pancreatic lipase. The substance remaining on the filter is sensitive to heat, whilst that in the nitrate is stable to heat. A mixture of these two exerts enzymic action, but each separately is inactive. There is a great amount of contradiction among the results obtained with the lipases ; it does, however, seem established that the lipases of the stomach and pancreas do not exhibit identical properties. The two animal lipases appear to be related in the same manner as the corresponding proteolytic enzymes, pepsin and trypsin; but they both differ essentially from the lipases of seeds. *F. Volhard, Zeitschr. klin. Med., 1901, 42, 414 and 43, 397; W. Stade, Hofm. Beitr., 1903, 3, 291; A. Zinsser, Hofm. Beitr., 1906, 7, 31; A. From me, Hofm. Beitr., 1905, 7, 51; A. Falloise, Arch. Internat. de Physiol., 1906, 3, 396, and 1907, 4, 405. 2 H. Engel, Hofm. Beitr., 1905, 7, 77; U m b e r and Br u g s c h , Arch. f. exp. Path., 1906, 55, 164. ' W. B o 1 d y r e f f , H., 1907, 50, 394. SPECIAL CHEMISTRY OF THE ENZYMES 11 Vegetable lipases are obtained from oily seeds, especially of Ricinus. The first work on these lipases was carried out by Reynolds Green (Proc. Roy. Soc., 1890, 48, 370) and S i g m u n d. Further contributions to the knowledge of them are due to Cojnnstein, Hoyer and Wartenberg (Chem. Ber., 1902, 35, 2988), Nicloux (Soc. BioL, 1904, 56, 840 and Proc. Roy. Soc., B., 1906, 77, 454), H. E. Armstrong (Proc. Roy. Soc., B., 1905, 76, 606) and others. A very good monograph has been written by C o n n s t e i n for the " Ergeb- nisse der Physiologic" (1904, 3). E. R o u g e (Centralbl. f. Bakt., 1907, II, 18, 403) gives a resume of the literature deal- ing more particularly with vegetable lipases. Ricinus-lipase is only active in relatively strongly acid solu- tion, and in the seeds, it is activated by the lactic acid present (Hoyer, H., 1906, 50, 414). According to Braun and Behrend (Chem. Ber., 1903, 36, 1142, 1900), the seeds of Abrus precatorius, which are nearly related to Ricinus seeds, decompose fats in neutral solution, but the effect is comparatively slight. Characteristic of the Ricinus-enzyme, as of most true lipases, is its insolubility in water; it is hence nec- essary to bring the pressed mass, remaining after the removal of the oil from the seeds, into intimate contact with the fat- emulsion. As to the other sources of plant lipases, mention may be made of the fungi, both higher and also lower, like P e n i c i 1 1 i u m (Gerard, C. R., 1897, 124, 370; Camus, Soc. BioL, 1897, 49, 192), Aspergillus niger (Camus, loc. cit.) and especially yeast (Delbriick). Lipases have also been detected in numerous bacteria; they cause the rancidity of butter and other natural fats (for the literature see Fuhrmann, Bakterien- enzyme, Jena, 1907). Butyrases. Against the extended application of the esters of the lower fatty acids and the monoglycerides to the study of the lipases, objection has often been raised. In particular, A r t h u s (Soc. BioL, 1902, 53, 381) and also D o y o n and M o r e 1 (C. R., 1902, 134, 1001 and 1254) have pointed out that H a n r i o t 's experiments with monobutyrin (Soc. BioL, 1896, 48, 925; C.R., 1896, 123, 753) which challenge criticism in many directions, give no information as to the presence and action of 12 GENERAL CHEMISTRY OF THE ENZYMES the true lipases. A distinction must therefore be drawn between the lipases and esterases (buty rases). The latter enzymes, which occur abundantly in many juices and organs (blood-serum, liver, kidneys) decompose not only the monovalent alkyl and the glyceryl esters of the lower fatty acids, but also amyl salicylate (H. Chanoz and H. Doyon, Soc. BioL, 1900, 52, 116, 717) and similar compounds. Schmiedeberg's histozyme is perhaps identical with these enzymes. D a k i n has effected asymmetric ester-decompositions by means of li ver-esterases ; these will be considered in Chapter VIII. Detailed studies on the same enzymes are due to K as tie, Loevenhart and E 1 v o v e , whose quantitative measure- ments will be referred to in the third chapter. They give the following method for the Preparation of li ver-esterases. The macerated liver (10 grms.) is extracted with water (100 c.c.) and the extract filtered through a linen cloth. Twenty c.c. of this extract are diluted with 72 c.c. of water and 8 c.c. of a 0-OlN-solution of hydrochloric acid added. When this mixture is heated to 40, a heavy precipitate of protein separates and, on filtering, a clear golden-yellow solution is obtained. The action of pancreas-lipase is participated in by a co- enzyme (R. Magnus, H., 1904, 42, 149), which is stable at a boiling temperature and the essential constituents of which are alkali salts of the bile acids (Magnus, H., 1906, 48, 376; see also Chapter V). Among the esterases must also be classed Chlorophyllase. This very interesting enzyme, which was discovered and described by Willstatter and S t o 1 1 (Lieb. Ann., 1911, 378, 18), accompanies chlorophyll and is wide-spread in its occurrence. The reaction which it produces is an alcoholysis. Chlorophyll contains three carboxyl groups, one of which is apparently free, and the others esterified with a methyl and a phytyl (from phytol) group respectively. Only the latter reacts with the alcohol, and then only under the influence of the enzyme, the phytoxyl group being replaced by ethoxyl. " In its action, chlorophyllase cannot be replaced by other esterases. On the other hand, for the enzyme found in the leaves, chlorophyll is a specific substrate. With phaeophytin SPECIAL CHEMISTRY OF THE ENZYMES 13 this enzyme does not react so well and with an ordinary wax no reaction takes place." ENZYMES OF THE HIGHER CARBOHYDRATES C y t a s e s . Whether true cellulose is decomposed enzym- ically is still uncertain. More is known, especially from the investigations of Mac Gil la wry, of H. T. Brown and Morris (Journ. Chem. Soc., 1890, 57, 497), of Reynolds Green (Annals of Bot., 1893, 7, 93) and, recently, of S c h e 1- lenberg (Flora, 1908, 98, 257), of the action of cytase, the substrates of which are the hemicelluloses and their reaction- products, mannose and galactose; also pentose-polysaccharides the pentosans are decomposed, but it is not yet known whether the hydrolysis is complete. Such enzymes occur in the intestines of herbivorous animals, in wood-destroying fungi (C z a p e k , Lotos, 1898, 46, 235; Schorstein, Centrabl. f. Bakt., 1902, II, 9, 446) and in bacteria. Also hydrocelluloses, which, so far as is known, are nearly allied to the hemicelluloses, are decomposed by cytases. To the same group of enzymes belong caroubinase, which dissolves the caroubin in the carob (C e r a t o n i a s i 1 i q u a) (E f f r o n t , C. R., 1897, 125, 116) and the enzyme described by Bourquelot and H e r i s- s e y (C. R., 1899, 129, 228, 391, 614; 1900, 130, 42, 340, 741) as s e m i n a s e , which occurs in lucerne, T r i g o n e 1 1 a and other plants. As well as in plants, cellulases or cytases are found in the animal body, especially in the intestines of graminivorous animals (H. T. B r o w n , Journ, Chem. Soc., 1892, 61, 352), in snails (Biedermann and M o r i t z , Pfltig. Arch., 1898, 73, 236) and in fishes (K n a u t h e) . Experiments by the author (Zeitschr. f. angew. Chem., 1912, 25, 46) indicate the occurrence in Merulius lacrimans (dry-rot fungus) of an enzyme which decomposes cellulose-dextrin. A m y 1 a s e s . These enzymes more accurately termed malto-amylases include all those which break down starch and glycogen forming maltose. Very little that is definite can be asserted with regard to the individuality of the amylases. After the view had been expressed by Brown and Mor- r i s and by Reynolds Green that at least two enzymes 14 GENERAL CHEMISTRY OF THE ENZYMES showing different biological relations are to be distinguished (diastase of translocation and diastase of secretion), Maquenne (C. R., 1906, 142, 124, 1059, 1387) carried out a series of notable investigations which explained the saccharification from a chemical point of view. Starch consists, indeed, of 80-85% of amylose and 15-20% of amylopectin. Amylase attacks dissolved amylose and also " soluble starch" very readily, but acts on amylopectin (starch-paste) very slowly. Amylopectinase, on the contrary, saccharifies amylopectin (starch-paste) with great ease. The diastases of the very varying organs of plants and animals must contain both of these diastatic enzymes. That the saccharifying and liquefying actions of the " diastases " are often parallel has been shown more especially by an investigation made by Chrzascz (Zeitschr. f. Spiritusind., 1908, 31, 52). Another recent noteworthy contribution on vegetable diastatic enzymes is due to Butkewitsch (Biochem. Z., 1908, 10, 314). Further investigations in this direction are, however, desirable, as well as a more detailed study of the diastatic decomposition of glycogen. Also the individuality of the true amylases, apart from amylo- pectin, is doubtful, if we consider the far-reaching decomposition necessary in order to pass from the highly-condensed starch through the dextrins to maltose. After Miss T e b b (Journ. of Physiol., 1894,15,421), Brown and Morris, Rohmann (Chem. Ber., 1894, 27, 3251), Hamburger (Pflug. Arch., 1895, 60, 543) and B e i j e r i n c k (Centralbl. f. Bakt., 1895, II, 1, 221) had detected, in the mixture of enzymes which saccharifies starch, an enzyme which effects the transformation starch-to-maltose and another which further breaks down the maltose to glucose, W i j s m a n (Rec. Trav. Chim. Pays-Bas, 1890, 9, 1), Pottevin and others assumed that the reactions starch-to-dextrin and dextrin-to-maltose are also effected by separate enzymes, 1 and recently Ascoli and Bonfanti (H., 1904, 43, 156) speak of several amylases. Occurrence. Enzymes which attack glycogen and starch are, as was discovered by Claude Bernard, widespread in the animal 1 L i n t n e r ' 8 statement that isomaltose is formed during the diastatic conversion of starch must, after subsequent work, more especially by Ling and Baker (Journ. Chem. Soc., 1895, 67, 702) and by Brown and Morris (ibid., 709), be regarded as disproved. SPECIAL CHEMISTRY OF THE ENZYMES 15 kingdom. Their occurrence in blood-serum which was detected by this investigator has been examined more closely by Bial, Pick, and A s c o 1 i and B o n f a n t i (H., 1904, 43, 156). According to N a s s e such an enzyme occurs in muscle-plasma, and this was confirmed by Halliburton (Journ. of Physiol., 1887, 8, 182). Carlson and Luckhardt (Amer. Journ. of Physiol., 1908, 23, 148) have found amylase in numerous other body-liquids. The fact that saliva dissolves starch has been known much longer, and this property was ascribed by L e u c h s in 1831 to an enzyme, ptyalin. Foster and von W i 1 1 i c h also found amylolytic enzymes in a great number of organs. In addition to the liver and pancreas, the muscles are especially rich in an enzyme which attacks glycogen; and Mendel and S a i k i (Amer. Journ. of Physiol., 1908, 21, 64), by experiments on the pig, found that this is the case in the embryo state, while other organs of different animals usually become richer in enzyme as development proceeds (P u g 1 i e s e and others). According to Roger (Soc. Biol., 1908, 64, 1137), an amylase occurs also in hens' eggs (white and yolk) and is partially soluble in ether. Of no less biological importance than animal amylases are those of plants. The starch-decomposing action of germinated barley was discovered by Kirchoff as long ago as 1814. An enzyme-preparation was made in 1833 by P a y e n and Persoz and was named " diastase"; this term is also much used at the present time, but, in the interests of as rational as possible a nomenclature, it should be replaced by the terms amylase, amylopectinase, etc. 1 The whole of the saccharifying enzyme-preparation together, that is, the mixture of amylase, dextrinase, etc., may meanwhile be called diastase. 2 In accordance with the function of the diastases of effecting the metabolism of the polysaccharides, these enzymes are widespread in all parts of plants, and are especially abundant in shoots and leaves, par- ticularly with the Leguminosse and grasses (Brown and Morris, 1 W i j s m a n ' s nomenclature is by no means an acceptable one. If two enzymes really take part in the formation of maltose, they should be distinguished as amylase and dextrinase. 2 It is most desirable that the use of the term "diastases" as a generic name for enzymes should be abolished from the French literature. For this use of the term there is, indeed, a historical explanation, but there is no justification for its continuance, especially as it often gives rise to mis- understanding. 16 GENERAL CHEMISTRY OF THE ENZYMES Journ. Chem. Soc., 1893, 63, 604). Amylases have further been detected in potatoes and the sugar-beet; also in germinating, starch-containing pollen-grains (Reynolds Green), in the bark of many plants (Butkewitsch, Biochem. Z., 1908, 10, 314), in the sap, in many higher and lower fungi, especially in several species of yeast here the diastase may be related to the glycogen-content and finally in bacteria. Special mention must be made of the so-called t a k a - d i- a s t a s e, the mixture of saccharifying enzymes from A s p e r - gillus o r y z a e , a fungus contained in koji-yeast. It sac- charifies starch and indeed, according to Stone and Wright, and T a k a m i n e , more energetically than does malt-diastase. Experiments with the view of preparing it in a pure state were made byWroblewski (Chem. Ber., 1898, 31, 1130). Preparation. Of the animal amylases, the ptyalin of saliva is the best suited for preparation. According to J. C o h n h e i m (Virch. Arch., 1865, 28, 241) the saliva is pre- cipitated with freshly-prepared calcium phosphate. From the precipitate the ptyalin is dissolved by means of water, and the aqueous solution precipitated with alcohol. Another method is given by K r a w k o w (J. Russ. Phys. Chem. Soc., 1887, 19, 387), who precipitates saliva-diastase by ammonium sulphate. Direct precipitation of the saliva with alcohol also yields a sac- charifying preparation. C o h n h e i m has found saliva-diastase to be free from protein, but he does not state on what absolute quantities of the preparation the tests were made. Von Wittich takes up pancreas-diastase in anhydrous glycerol. Larger and purer yields of amylase are obtained from vege- table material. From malt Lintner prepared diastase as fol- lows: one part of green malt (or air-dried malt) was extracted for 24 hours with 2-4: parts of 20 J o alcohol, the extract being precipitated with 2-5 times its volume of absolute alcohol and the precipitate washed with absolute alcohol and ether. Loew (Pflug. Arch., 1882, 27, 203; 1885, 36, 170) steeps ger- minated barley in a little water and then extracts with 4% alcohol. He precipitates the extract with lead acetate, suspends the precipitate in water, removes the lead from the solution by means of hydrogen sulphide, and finally precipitates the diastase with a mixture of alcohol and ether. SPECIAL CHEMISTRY OF THE ENZYMES 17 s b o r n e and Campbell (Journ. Amer. Chem. Soc., 1896, 18, 536) and also Wroblewski (H., 1897, 24, 73) salt out the diastase with ammonium sulphate. E f f r o n t (Enzymes and their Applications, London and New York, 1902, pp. 104 e t s e q.) proposes the extraction of malt with water and, in order to diminish the quantity of the extractive material possessing no diastatic action, he induces alcoholic fermentation in the infusion by yeast previously rendered very poor in nitrogen. E f f r o n t states that the fermentation destroys a large quantity of carbohydrates, removes considera- ble quantities of proteins and salts, and leaves the diastase absolutely untouched. Wroblewski (Chem. Ber., 1897, 30, 2289) gives the following method: Finely-ground malt is extracted, first with 70%, and then' twice with 45% alcohol. Sufficient strong alcohol is added to the last two extracts to bring the alcohol-content to 70%. The precipitate formed is washed with absolute alcohol and ether and dried in a vacuum. Like O s b o r n e and Campbell (loc. ei t.), Wrob- lewski (Chem. Ber., 1898, 31, 1130) effected further purifica- tion by salting out with ammonium sulphate. Wroblewski considered that, as a result of these exper- iments, he had shown with certainty that diastase is a protein substance nearly allied to the albumoses, whilst, according to T. B. O s b o r n e (Chem. Ber., 1898, 31, 254), diastase is a protein-like substance or "a compound of an albumin with a proteose." For his most active preparation he gives the fol- lowing composition (calculated for ash-free substance): C, 52-5; H, 6-72; S, 1 -90; N, 16-10%. The solution gives the character- istic reactions of the proteins. Wroblewski 's purest preparation had a nitrogen-content of 16-5%. If, however, the recent researches of S. Frankel and Hamburg (Hofm. Beitr., 1906, 8, 389) should be confirmed, diastase contains neither protein-groups nor reducing sugars. The non-enzymic substances were precipitated with lead acetate, the solution sterilised by filtration and further purified by fer- mentation with yeast rendered poor in nitrogen and subsequent filtration through a Pukall filter. After drying in a vacuum, the syrupy liquid yields a powder free from fermentable and reducing 18 GENERAL CHEMISTRY OF THE ENZYMES sugars and from protein. It represents a very active substance which does not give the biuret reaction or reduce F e h 1 i n g ' s solution but shows a faint M i 1 1 o n ' s reaction; it also gives M o 1 i s c h ' s reaction and the pentose reaction slightly. When dialysed into spring-water, the dissolved diastases are separated into two principal groups: the saccharifying diastases diffuse through the membrane, whilst the liquefying ones remain. A distinct advance seems to have been made by H. C. S h e r- m a n and M. D. Schlesinger (Journ. Amer. Chem. Soc., 1911, 33, 1195). They found that pancreas-diastase keeps well in 50% alcohol, and they purified such a solution by dialysis. A very active preparation (saccharifying power, 5000 on Lintner's scale at 40) contained 53-0% C, 6-6% H and 15-6% N. Inulinase. In addition to hemicelluloses, starch and glycogen, another carbohydrate, inulin, also occurs as a reserve material. The enzyme, inulase or inulinase, accompanying this, decomposes inulin into its simplest com- ponent, fructose. Starch is not attacked by inulinase. ReynoldsGreen (Annals of Bot., 1888, 1, 223) discovered this enzyme, which occurs in many of the Compositse, in the tubers of H e 1 i - anthus tuberosus (artichoke). Bourquelot (Bull. Soc. Mycol., 1893, 9, 230; 1894, 10, 49) detected inulinase in Aspergillus n i g e r and isolated it from the mycelium of this fungus. The enzyme appears to be widespread in the Eumycetes; Dean (Bot. Gaz., 1903, 35, 24) found it also in Penicillium glaucum. The best medium for its action is 0-001% hydrochloric acid. The enzymes which decompose trisaccharides, such as melicitase, etc., have not yet been sufficiently individu- alised. THE ENZYMES OF THE GLUCOSIDES AND DISACCHARIDES E. Fischer has stated that the disaccharides may be regarded as glucosides and can be classified with these according as they contain the glucose in the a- or (3-form. From the results of E. F. Armstrong (Journ. Chem. Soc., 1903, 83, 1305) and C. S. H u d s o n (Journ. Amer. Chem. Soc., 1909, 31, 1242), it appears that a-glucosides, which were originally characterised SPECIAL CHEMISTRY OF THE ENZYMES 19 by the fact that they are hydrolysed by a constituent of yeast- extract, generally yield a-glucose. p-Glucosides are hydrolysed by a component of almond extract, and from such a glucoside Hudson obtained ^-glucose. a-G lucosidase; maltase. As a-glucosidases will be designated those enzymes which hydrolyse a-glucosides specifically. They are therefore limited, on the one hand, by ^-glucosidase, which acts only on ^-glucosides, and, on the other, by lactase and invertase, which accelerate the hydrolysis of the ^-galactosides or fructosides. Occurrence. In both the animal and vegetable kingdoms, maltase almost always accompanies the diastases, from which it cannot often be separated. Thus, this enzyme has been found in blood and in serum (G 1 e y and B o u r q u e 1 o t , Soc. BioL, 1895, 47, 247; Ham- burger, Pfliig. Arch., 1895, 60, 543; Tebb, Journ. of Physiol., 1894, 15, 421 ; Fischer and N i e b e 1 , Sitzungsber. K. Akad. Berlin, 1896, 73) ; also in many tissues (Shore and Tebb, Journ. of Physiol., 1892, 13, 19), especially in the liver, intestines and pancreas. As Miss Tebb found, the maltase can be extracted from these organs in both the fresh and dried states by means of chloroform water; the oppo- site statement of Brown and Heron (Proc. Roy. Soc., 1880, 30, 393) is thus contradicted. The maltases occur in great abundance in the vegetable kingdom. Their occurrence in m a 1 1 and in y e a s t must -be especially mentioned. These two maltases do not appear to be absolutely identical (Fischer, H., 1894, 26, 74). The lactic acid yeasts and also kephir-grains always contain lactases in place of maltases. Saccharomyces Marxianus contains no maltase, but only invertase (E. C. Hansen; E.Fischer and P. Lindner, Chem. Ber., 1895, 28, 984). Excepting in this case, it can be said that invertase always accompanies maltases in yeast-extracts; according to Beijerinck and to E. Fischer and P. Lindner (Chem. Ber., 1895, 28, 984), invertase is lacking in Saccharomyces octosporus. In Saccharomyces apiculatus neither maltase nor invertase is found. Preparation. As starting material for the preparation of the yeast-enzymes, maltase and invertase, it is best to employ pure cultures. The yeast is used as fresh as possible and is well pressed and, according to E. Fischer (H., 1898, 26, 74), ground and well shaken two or three times with the ten-fold quantity of water. Removal of the mother-liquor is effected 20 GENERAL CHEMISTRY OF THE ENZYMES most suitably by a P u k a 1 1 flask-filter. The yeast is pumped as dry as possible and is then spread out in as thin a layer as possible on poro.us tiles and dried in the air at the ordinary tem- perature. Under these conditions it gradually shrivels up and assumes a dark-grey colour. After 1-2 days, it is powdered as finely as possible and left to dry in the air until it forms a loose powder. The final drying may also be carried out at 30-35. In this state the yeast can be kept for months without the maltase being destroyed. When the enzyme is to be used, the yeast is extracted with 10-15 times the quantity of water for 12-20 hours at 30-35, with occasional shaking, the liquid being then filtered through paper. Toluene serves as a suitable antiseptic. R 6 h m a n n (Chem. Ber., 1894, 27, 3251) heated the yeast for an hour at 105^110 before extraction, but this procedure, according to Croft Hill, is not to be recommended. The latter investigator gives the following method (Journ. Chem. Soc., 1898, 73, 636) : Good, pressed bottom-yeast is washed three times with distilled water by decantation, collected on a covered filter, spread out on a porous support and dried in a vacuum over sulphuric acid. The yeast dries in about two days and is then powdered and sieved through a cloth, a yellowish-white powder being obtained. This powder is then spread out on a double layer of fine tulle over the mouth of a glass vessel in an oven previously heated to 40. In successive quarters of an hour, the temperature is raised to 60, 70, 90 and 100, the last being maintained for 15 minutes, after which the prepara- tion is allowed to cool in a desiccator. The yeast is then weighed, ground in a mortar with 10 times its weight of 0-1% sodium hydroxide solution, filled into flasks with addition of toluene and left at the room-temperature for 3 days. The extract is now filtered, first through paper and afterwards through a Cham- berland filter. If 1 c.c. of this fresh extract is added to 20 c.c. of 2% maltose solution at 30, about 20% of the, sugar is hydrolysed in 40 minutes. Different yeasts appear to contain widely varying proportions of maltase, so that not every species of yeast is suitable for the preparation of maltase. Trehalase. Trehalose, a disaccharide composed of two molecules of glucose, is hydrolysed by an enzyme which was SPECIAL CHEMISTRY OF THE ENZYMES 21 found by Bourquelot in Aspergillus and other fungi, by Fischer (H., 1898, 26, 79) in the diastase of green malt and in yeasts of the Frohberg type, and by Kalanthar (H., 1898, 26, 97) in various other yeasts. Whether the enzyme, which acts best with a very slight concentration of hydrogen-ions, really differs from maltase is not yet established ; Bourquelot (Soc. Biol., 1895, 47, 515) regards it as a separate enzyme, but Fischer is not in agreement with this opinion. The ^-glucosidases hydrolyse (3-methylglucoside and also most of the natural glucosides, which are on this account placed in the ^-series. 1 It has been shown recently by Hudson and Paine (Journ. Amer. Chem. Soc., 1909, 31, 1242) that the hydrolysis of a typical natural glucoside, salicin, under the influence of emulsin, yields ^-glucose. It is better here not to apply the ordinary principles of nomenclature, but to name the glucosides according to the form of glucose to which they give rise and the enzymes so that they refer to the glucosides characterised in this way. Of special glucosido-glucoses hydrolysed by emulsin, mention may be made of: Isomaltose (E. Fischer, Chem. Ber., 1895, 28, 3024; compare also E. F. Armstrong, Proc. Roy. Soc., B, 1905, 76, 592). Gentiobiose (Bourquelot and H e r i s s e y, C.R., 1902, 135, 399). Cellose or Cellobiose (E. Fischer and G. Z e m p 1 e n, Lieb. Ann., 1909, 365, 1). C e 1 1 a s e . From the results of fractional filtration of extracts of Aspergillus niger by Holderer's method, G. B e r t r a n d and M. H o 1 d e r e r (C. R., 1909, 149, 1385 and 1910, 150, 230) assume the existence of an enzyme which differs from (3-glucosidase and acts specifically on cellose. An enzyme extracted from apricot seeds hydrolyses only cellose and not trehalose. x The opportunity must not be neglected of pointing out that E. Fischer, who introduced this method of reasoning, has issued a warning that it must not be regarded as absolutely safe (Lieb. Ann., 1909, 365, 1): "For it might be assumed that one and the same enzyme hydrolyses both the alcohol-glucosides and the glucosido-glucoses. But as long as no pure enzyme is obtained, complicated mixtures like emulsin or yeast-extract having to be used, no proof of this exists." 22 GENERAL CHEMISTRY OF THE ENZYMES Cellase occurs, together with other enzymes, in apricot kernels, almonds, barley, and the mycelium of Aspergillus. ^-Methylgalacto sides are also hydrolysed by the enzymes of the almond (E. Fischer, Chem. Ber., 1895, 28, 1429) and, since Fischer found that this enzyme likewise effects the hydrolysis of lactose, the latter is to be regarded as a p-galactoside. But we shall not go far wrong if we assume, with B o u r q u e 1 o t and H e r i s s e y (C. R., 1903, 137, 56) and with E. Fischer, that this latter action is not brought about by the same enzyme as hydrolyses (S-glucosides, but depends on the presence of an enzyme which decomposes (3- galactosides and is hence either identical with, or nearly related to, the lactase occurring in lactose-yeasts. The view that kephir- lactase and emulsin- lactase are different, has been advanced by H. E. and E. F. Armstrong and E. H o r t o n (Proc. Roy. Soc., B, 1908, 80, 321). They assume that the one enzyme is a galacto-lactase and the other a gluco-lactase, the first being taken up by the galactose-residue and the latter by the glucose-residue of milk-sugar. A preparation which is biologically purer is obtained, accord- ing to Pottevin (Ann. Inst. Pasteur, 1903, 17, 31), from Aspergillus niger, Aspergillu s-emulsin hydrolys- ing only ^-glucosides and not (3-galactosides or milk-sugar. Amygdalin is resolved by the enzymes of the almond into glucose, benzaldehyde and hydrocyanic acid, and mandelo- nitrile glucoside, formed by the action of yeast-enzymes on amygdalin, is also hydrolysed by emulsin into its simplest com- ponents (E. Fischer, Chem. Ber., 1895, 28, 1508). The glucosido-glucose contained in amygdalin is not identical with maltose, since, on the one hand, maltose is not liberated by the action of emulsin (C a 1 d w e 1 1 and Courtauld, Journ. Chem. Soc., 1907, 91, 666; R o s e n t h a 1 e r, Arch, der Pharm., 1908, 245, 684) and, on the other, maltose has no retarding action on the hydrolysis by emulsin (A u 1 d , Journ. Chem. Soc., 1908, 93, 1276). It is, therefore, best to indicate by "emulsin" the mix- ture of glucoside-resolving enzymes and to characterise the prepara- tion according to its origin, a distinction being drawn between Aspergillus -emulsin, almond -emulsin, etc. SPECIAL CHEMISTKY OF THE ENZYMES 23 For the enzyme-constituents, rational names are then chosen, the results of H. E. and E. F. Armstrong and H o r t o n , C a 1 d w e 1 1 and Courtauld, and Rosenthaler indicating at least four components of almond-emulsin, namely: (1) Amygdalase, characterised by the reaction: C 6 H 5 CH(CN) - O - CoHioO 4 O Amygdalin = C 6 H 5 CH(CN) Mandelonitrile glucoside (2) A (3-glucosidase, which acts on g-glucosides, among them mandelonitrile glucoside: C 6 H 5 CH(CN) O C 6 HiiO5+H 2 O Mandeloaitrile glucoside = C 6 H 5 CH(CN) - OH+C 6 Hi 2 6 . Mandelonitrile Glucose (3) A hydroxynitrilase: C 6 H 5 -CH(CN)-OH = C 6 H 5 -CHO + HCN. Mandelonitrile Benzaldehyde Hydrocyanic acid In addition to these three substances which take part in the decomposition of amygdalin, the existence in emulsin must be assumed of: (4) An enzyme which resolves milk-sugar, i.e., a lactase (which Armstrong terms gluco-lactase). According to N e u b e r g (Ergeb. der Physiol., 1904, 3, 446), the conjugated glycuronic acids are also decomposed by emulsin. The synthetic action of certain components of emulsin is treated more in detail in Chapter VII. Occurrence. 1. Phanerogams. As well as in almonds, emul- sin is found in the leaves of Prunus laurocerasus (where laurocerasin likewise occurs), in the seeds of many of the Rosacese, in manihot (Guignard) and in extracts of numerous plants, such asMonotropa, Polygala [Bourquelot, Journ. de Pharm. et Chim., 1904, (5), 30, 433], Malus communis, Hedera helix, etc. (H e r i s s e y , Thesis, " Recherches sur I'Emulsine," Paris, 1899). 2. Cryptogams. It was discovered simultaneously in P e n i c i 1 - Hum glaucum by Gerard (Soc. Biol., 1893, 45, 651) and in Aspergillus niger by Bourquelot, who also detected enzymes capable of attacking glucosides in many other fungi, especially 24 GENERAL CHEMISTRY OF THE ENZYMES in the Polyporus species found in wood. H 6 r i s s e y has found emulsin in many lichens and mosses. Bourquelot has recently observed hydrolysis of otherwise unknown glucosides by emulsin (Arch, der Pharm., 1907, 245, 172). Fermi and Montesano (Centralbl. f. Bakt., 1894, I, 15, 722), Gerard (Soc. Biol., 1896, 48, 44) and Twort (Proc. Roy. Soc., B, 1907, 79, 329) have detected emulsin in bacteria, 27 species out of 44 examined having the property of hydro- lysing glucosides. Worthy of note is the observation of Henry and A u 1 d (Proc. Roy. Soc., B, 1905, 76, 568) that many yeasts also exhibit "emulsin" action. Animal enzymes closely related to emulsin were found by G e* r a r d (Soc. Biol., 1896, 48, 44) in the kidneys of the horse and rabbit. In molluscs B i e r r y and G i a j a (Soc. Biol., 1906, 58, 1038) found enzymes capable of hydrolysing populin and phloridzin; extracts of cross-spiders also resolve amygdalin (Robert and W. Fischer). Decompositions of glucosides by animal extracts were also noted byGonnermann (Pfliig. Arch., 1904, 103, 225; 1906, 113, 168) and, more recently by K o b e r t ; according to the latter, extract of placenta hydrolyses amygdalin, arbutin, salicin and helicin. Preparation: Herissey (Thesis; compare Bour- quelot, Arch, der Pharm., 1907, 245, 172). One hundred grams of sweet almonds are steeped for about a minute in boiling water and, after draining, are carefully peeled. They are then ground as finely as possible in a mortar without water, the product obtained being macerated at room-temperature with 200 c.c. of a mixture of equal parts of distilled water and water saturated with chloroform. After about 24 hours, the mass is strained and pressed through a damp cloth. This pro- cedure yields 150-160 c.c. of liquid, to which 10 drops of glacial acetic acid are added to precipitate the casein. The clear nitrate (120-130 c.c.) is added to 500 c.c. of 95% alcohol, the precipitate thus formed being collected on a smooth filter and, after draining, treated with a mixture of equal volumes of alcohol and ether. After drying in a vacuum over sulphuric acid, horny, transparent plates are obtained and, when ground, these give an almost white powder. Invertase ( = a-Fructosidase) . Invertin or sucrase owes its name to its property of converting cane-sugar into invert- SPECIAL CHEMISTRY OF THE ENZYMES 25 sugar ( = glucose + fructose). The sphere of action of invertase extends to all synthetic a-methylfructosides; on the other hand,, ^-methylfructoside, a-glucosides and a-galactosides resist its action. Apart from these synthetic glucosides, gentianose a trisaccharide from Gentiana lute a is also attacked by invertase, which resolves it into fructose and g e n t i o - b i o s e. Further, melitriose (raffinose), a trisaccharide occurring in the sugar-beet, is decomposed by invertase, yielding fruc- tose and m e 1 i b i o s e . Occurrence. The distribution of invertase in the yeasts is as well known as important; in the majority of cases, it is accompanied by maltase and, in the lactose-yeasts, by lactase. Invertase occurs alone in only few yeasts, among them being Saccharomyces Marxi- anus (Fischer, H., 1898, 26, 75). S. apiculatus contains no invertase. Of other lower organisms which contain invertase, mention may be made of Fusarium, Streptococcus (Leuconostoc) mesenterioides, Aspergillus oryzae and M o n i 1 i a Candida. A long series of invertase-containing bacteria is also known, especially owing to the investigations of Fermi and M o n - t e s a n o (Centralbl. f. Bakt., 1895, 1, 482, 542). With the higher plants, invertase is found especially in the green leaves and young shoots (K a s 1 1 e and Clark, Amer. Chem. Journ., 1903, 30, 422), in ripe bananas, in mulberries, in resting and, still more abundantly, germinating pollen, and in wheat and barley embryos; in the crown leaves of Robinia viscosa and pseudacacia, Papaver rhoeas, Rosa species and Bougainvillea bracts. Also in fruits, such as dates, which, when unripe, contain the invertase as an insoluble endo-enzyme, this only becoming soluble when the fruit ripens (V i n s o n , Journ. Amer. Chem. Soc., 1908, 30, 1005). Invertase is found in human intestinal juice, even immediately after birth (Kriiger), but not in that of cattle (F i s c h e r and Niebel). Robertson (Edinburgh Med. J., 1894) found it in almost all organs. Preparation. If yeast-cells are to be extracted with water, it is first of all necessary to kill them, either by treatment for a short time with ether or for a longer time with alcohol (Osborne, H., 1899, 28, 399), etc., or by heating the dry yeast at 105 (S a 1 k o w s k i) or by plasmolysis (I s s a e w). After careful dehydration in a vacuum and subsequent heat- ing, yeast may yield about 12% of its invertase on extraction (Euler and Kullberg, H., 1911, 73, 94). 26 GENERAL CHEMISTRY OF THE ENZYMES Living yeast also gives up invertase to the surrounding liquid water or sugar solution but in relatively small quantities. Presumably it is more especially the old cells from which the invertase can be extracted directly. ' S u 1 1 i v a n and T o m p s o n left top-fermentation beer-yeast for a month at 15 so that it became completely liquid; [it was then pressed and the clear solution obtained precipitated with 47% alcohol. The precipitate, after deposition, was dissolved in water and sufficient alcohol added to bring its content in the liquid up to 28%; in this solution the invertase remained dissolved, whilst the majority of the protein substances separated. On raising the alcoholic content of the filtered liquid to 47%, a precipitate was again formed and this was washed with absolute alcohol and dried in a vacuum. The preparation thus obtained was very active, but still not quite pure; it contained about 5% of ash (magnesium and potassium phosphates), which the English investigators regarded as an admixture. Their further puri- fication experiments showed, as had already been indicated by the work of Osborne (H., 1899, 28, 399) and of Salkowski, that invertase is not a protein. Even the purest preparation contains, besides phos- phoric acid, a carbohydrate. Wroblewski regards this as an impurity, as also does shim a (H., 1902, 36, 42); the latter came to the conclusion that yeast-gum consists of a substance which contains d-mannose and a methyl-pentosan giving fucose on hydrolysis. H a f n e r , who carried out a thorough examination of pure invertin (H., 1904, 42, 1), regards it as by no means disproved that this peculiar carbohydrate always adhering to invertin is an integral constituent of the enzyme. A large part of the phosphorus of invertin preparations is combined organically. The specific activity of the enzyme is not con- nected with the presence of large nitrogenous groups like the albumoses or peptones; the absence of peptones is also supported by the failure of the biuret action. The nitrogen is probably present in the form of smaller groups, which have, however, not been investigated. Very active invertase solutions are also obtainable from pure cultures ofAspergillus niger. The best method for preparing invertase in as pure a form as possible consists in removing protein by lead acetate and kaolin, and in subsequently applying the following diffusion process (E u 1 e r and Kullberg, H., 1911, 73, 335): Bottom fermentation beer-yeast is subjected to autolysis for 3-10 days and then precipitated, as Hudson recommended, with excess of lead acetate; the whole mass is then ground with SPECIAL CHEMISTRY OF THE ENZYMES 27 kaolin and the liquid pumped off. The lead is precipitated by means of hydrogen sulphide and the nitrate ground several times with kaolin and a little charcoal and filtered. By means of a collodion dialysor, the enzyme solution is freed from the impuri- ties, which diffuse rapidly, and is finally precipitated with alcohol. Two kilos of pressed yeast, treated in this way, give about 8 grms. of a pure white powder, which is freed from further quantities of nitrogenous impurities by dialysis or diffusion. The preparations are protein-free, and the content of nitrogen varies between 0-3 and 2%. The molecular weight exceeds 25,000. The activity is d=0 = 10 minutes, i.e., 0-05 grm. of the preparation, dissolved in 25 c.c. of an 8% cane-sugar solu- tion, reduces the rotation of the cane-sugar to zero in 10 minutes at a temperature of 20. The sensitiveness of invertase to temperature (cf. Chapter V) is such that the activity of an invertase solution is diminished by one-half by heating for 30 minutes at 63 (H . E u 1 e r and af Ugglas). Its optimum temperature is 53-56. The influence of the acidity of the solution on the velocity of inver- sion has been investigated in detail by Sorensen and by Hudson (cf. Chapter IV). A poisonous action towards invertase is shown by mercury salts and potassium cyanide (and nearly all salts with an alkaline reaction); hydrocyanic acid and chloroform are less harmful, whilst thymol and toluene are without effect. OTHER ENZYMES WHICH HYDROLYSE GLUCOSIDES The number of different individuals in this group seems to be very large, but the sphere of action and specificity of the enzymes described are usually very indefinite. Closely related to g- glucosidase is: Gaultherase or betulase, the specific action of which consists in hydrolysing the glucoside of methyl salicylate (gaultherin) . Neither salicin nor amygdalin is attacked by this enzyme. Occurrence. Exclusively in plants. It was discovered by Schneegans (Arch, der Pharm., 1894, 232, 437) in the bark of 28 GENERAL CHEMISTRY OF THE ENZYMES Betula lenta. At the same time Bourquelot found it in several Polygala and Azalea species, and in Spiraea ulmaria, Monotropa hypopitys and Gaultheria procumbens (C. R., 1896, 123, 315; J. de Phann. et Chim., 1896, 3, 577). Preparation, according to Bourquelot (loc. cit.) : Monotropa plants are ground with sand and the glucoside removed by digesting for half an hour with 95% alcohol. The residue, which contains the enzyme, is quickly dried with alcohol and ether, after which the enzyme can be extracted with water. Cf. B e i j e r i n c k (Centrabl. f. Bakt., 1899, II, 5, 325). Schiitzenberger mentioned an enzyme which hydro- lyses populin and also phillyrin, a glucoside occurring in the bark of Phillyrea latifolia, but he did not investi- gate it further. Sigmund (Monatsh. f. Chemie, 1909, 30, 77) found an enzyme, which decomposes salicin but does not seem to be identical with emulsin, in certain species of S a 1 i x and P o p u 1 u s ; he also found one which hydrolyses arbutin in Calluna vulgaris and Vaccinium myrtillus. W . Sigmund (Monatsh. f. Chemie, 1910, 31, 657) dis- covered an enzyme, capable of hydrolysing sesculin, in the seed- coats of the horse-chestnut (Aesculus hippocastanum). It does not appear to be either an amygdalase or a lipase, but is not yet sufficiently defined. Sigmund proposes for it the names, salicase, arbutase and aesculase. According to T. Weevers (Rec. Trav. bot. J^Teerland., 1910, 8) an enzyme which hydrolyses salicin specifically occurs in Salix purpurea and Populus monilifera, and one that hydrolyses arbutin in Vaccinium vitis idaea and Pinus communis. B i e r r y and G i a j a (Soc. BioL, 1907, 62, 1117) found, in snails and Crustacea, an enzyme which is not identical with " emulsin " but which hydrolyses populin and phloridzin. It remains to be shown that these enzymes are not really general g-glucosidases. G e a s e is the name given by Bourquelot and H e r i s - s e y (C. R., 1905, 140, 870) to a specific enzyme from G e u m urbanum (Herb Bennett) and r i v a 1 e which liberates eugenol from a glucoside contained in these plants. SPECIAL CHEMISTRY OF THE ENZYMES 29 Elaterase, from Ecballium elaterium hydro- lyses elaterin (Berg). Rhamnase hydrolyses xanthorhamnin, yielding, according to G . and C h . Tanret (Bull. Soc. Chim., 1899, [iii], 21, 1065), rhamninose and rhamnetin. Rhamninose is regarded as a trisaccharide, which can be hydrolysed into 2 mols. of rhamnose (methylpentose) and 1 mol. of galactose. The enzyme occurs in Rhamnus infectoria. Besides these glucoside-enzymes, another series is known which hydrolyse glucosides of one or the other group in a specific manner. M y r o s i n decomposes sinigrin or potassium myronate into glucose, potassium hydrogen sulphate and allyl mustard oil (allyl isothiocyanate) according to the equation: CioH 18 OioNS 2 K = C 3 H 5 -CNS + C 6 Hi 2 6 + KHS0 4 Sinigrin Allyl mustard oil Glucose Also other sulphur-glucosides occurring in the Cruciferse are hydrolysed by myrosin; but, according to E. Fischer (Chem. Ber., 1894, 27, 3483), a- and ^-glucosides are not attacked. Occurrence. The distribution of myrosin has been shown by the investigations of Spatzier (Pringsheim's Jahrb. f. wiss. Bot., 1893, 25, 39) and, especially, of G u i g n a r d (C. R., 1890, 111, 249 and 920; also Journ. de Bot., 1894, 67 and 85). It is characteristic of the Cruciferse and certain allied families and is found also inManihot- species. It is localised in certain cells which are rich in proteins and are dispersed through the tissues. G u i g n a r d has isolated mechan- ically such cells and cell-layers, e.g., the pericycle of Cheiranthus. Roots contain the enzyme mainly in the cork, whilst, in the stem, it is met with especially in the pericycle. Leaves are often very rich in myrosin, which occurs in the young mesophyll. Poisons and antiseptics : The action of myrosin is prevented by tannin in a concentration of 1% or by salicylic acid in solutions stronger than 1-5%. Chloral in 1% concentration is less harmful, and borax quite harmless. Cf. Reynolds Green, " Soluble Ferments and Fermentation," 1899, p. 154. Erythrozyme is the name given to an enzyme (S c h u n c k , 1852) which" decomposes the ruberythrin or ruberythric acid of madder into alizarin, dihydroxyanthra- 30 GENERAL CHEMISTRY OF THE ENZYMES quinone and glucose. This hydrolysis is also effected, although more slowly, by emulsin, with a constituent of which, ery- throzyme is closely allied or identical. Indigo-enzymes. Breandat founa in the leaves of Isatis alpina, an enzyme which decomposes i n d i - can, the glucoside of indoxyl, into indoxyl and a sugar (indi- glucin). And according to Beijerinck (Malys Jahrb., 1900) an enzyme exists capable of hydrolysing the allied isatan (from Isatis tinctoria). L o t a s e from Lotus arabicus decomposes the glucoside lotusin into lotoflavin (1 : 3 : 3' : 5' tetrahydroxy- flavone), glucose and hydrocyanic acid. According to D u n - stan and Henry (Proc. Roy. Soc., 1900, 67, 224, and 1901, 68, 374), it is different from emulsin. Phaseolunatase, investigated by Dunstan, Henry and Auld (Proc. Roy. Soc., 5, 1907, 79, 315) seems to be identical with the linamarase of Jorissen and Hairs (Bull. Acad. Roy. Belgique, 1891, 21, 518) and possibly with maltase. As mentioned on p. 6, lactase decomposes milk-sugar into d-galactose and d-giucose and, according to E. Fischer, it hydrolyses p-galactosides generally. This action appears to be strictly specific, since neither ex- nor (3-glucosides are attacked. Maltase and lactase are found in various yeasts (E . Fischer, H., 1898, 26, 81) and fungi (B o u r q u e 1 o t and H e r i s - sey), and scarcely ever occur together. Eurotiopsis G a y o n i forms an exception to this rule, as, according to Laborde (Ann. Inst. Pasteur, 1897, 11, 1), it attacks both maltose and lactose. Occurrence. Its existence in lactose-yeasts was assumed by Beijerinck (Centralbl. f. Bakt., 1889, 6, 44) but was first definitely proved by E . F i s c h e r (Chem. Ber., 1894, 27, 3481). In the animal organism it does not occur very largely. It is found in both the freshly macerated placenta and in the dry powder. Human intestinal mucus, as well as that of the calf and dog, contain lactase, but only with the young organism. In the intestinal juice of adult man no lactase is found (Hamburger and H e k m a, J. de Physiol. et Pathol. gn., 1902, 4, 805). According to Plimmer (Journ. of SPECIAL CHEMISTRY OF THE ENZYMES 31 PhysioL, 1906, 35, 20) lactase always occurs in carnivora and omnivora, but with herbivora, with the exception of the rabbit, it is found only in the young animal. W e i n 1 a n d (Zeitschr. f . Biol., 1899, 38, 606) found this enzyme in the pancreas. Lactase must also occur in all those bacteria which decompose milk-sugar with formation of lactic acid and alcohol, the hexoses, which are formed as intermediate products, result- ing from the action of this enzyme ; Fuhrmann (Vorlesungen liber Bakterienenzyme, p. 96) mentions especially Bacillus acidi laevolactici and Bacterium coli. Preparation. (Fischer, Chem. Ber., 1894, 27, 2991 and 3481) . Air-dried yeast is carefully ground with powdered glass, the mass being then digested with 20 times its quantity of water for 20 hours at 30-35 and filtered through a P u k a 1 1 filter. The enzyme solution prepared in this way undoubtedly possessed the property of converting milk-sugar into hexoses, but its action was weaker than that of the aqueous extract of kephir-grains. M e 1 i b i a s e . Melibiose, a product of the hydrolysis of ramnose, can be further hydrolysed to d-galactose and d-glucose, so that it contains the same components as lactose. This hydrolysis may be effected by an enzyme occurring in certain bottom-yeasts, but not in top-fermentation yeasts. The enzyme is closely allied to maltase and is perhaps to be regarded as a maltase (Fischer, H., 1898, 26, 81). PHYTASE An enzyme which decomposes phytin or inositolhexaphos- phoric acid, C6Ho[OPO(OH)2]6, into inositol and phosphoric acid, was obtained by N. Suzuki, Yoshimura and Takaishi (Bull Coll. Agric., Tokyo, 1907, 7, 503) from rice- and wheat-bran. The preparation itself was free from phosphorus and hydrolysed neither amylose nor proteins. Pre- sumably the enzyme, like phytin, is widespread in the vegetable kingdom. According to M c C o 1 1 u m and Hart (Journ. of Biol. Chem., 1908, 4, 497), phytase is also contained in the liver and blood, but not in muscle- or kidney-extract. Quite recently, A. W. D o x and Ross Golden (Journ. of Biol. Chem., 1911, 10, 183) have detected phytase in lower fungi. 32 GENERAL CHEMISTRY OF THE ENZYMES HEXOSEPHOSPHATASE According to Harden and Young (Proc. Roy. Soc., B, 1910, 82, 327), this enzyme, which occurs in pressed yeast juice and also in yeast dried at room-temperature, separates the phosphoric acid from hexosephosphate. See later under " zymase." PECTASE It is undoubtedly most rational to employ the name p e c - t a s e s for those enzymes which convert pectoses into pectin and pectinic acids. The reaction, which yields also arabinose, consists certainly of a hydrolysis, but the details of the chemical changes occurring are unknown. The pectinic acids formed exist in the plants as calcium salts. The work of M a n g i n (C. R., 1888-1893) and of Devaux (Soc. phys. nat. de Bordeaux, 3) can only be mentioned here. The enzyme here termed pectase was obtained from malt-extract by B o u r - quelot and Herissey (C. R., 1898, 127, 191; 1899, 128, 1241), who called it pectinase; according to the general principle of naming the enzyme after the substrate, this should be altered to pectase. PECTINASE By the term pectinase should be indicated the enzyme which coagulates dissolved pectin-substances, e.g., in fruit-juices, in presence of lime, to gelatinous calcium salts of the feebly acid pectinic acids. Here also there is as yet no explanation of the chemical reaction taking place. A high concentration of acid retards or completely prevents coagulation, which, in the case of acid fruit-juices, proceeds only after neutralization with lime. The velocity of the reaction is conditioned by a certain equilib- rium between the enzyme and the concentrations of the acid and calcium salts. Without the action of the enzyme, the lime is unable to coagulate pectins to calcium pectates; soluble calcium salts can, indeed, induce pectins to coagulate, but, in this case another product is formed, namely a pectinate soluble in 0-2% hydrochloric acid. Calcium pectate, however, yields insoluble pectinic acid with 0-2% hydrochloric acid. It SPECIAL CHEMISTKY OF THE ENZYMES 33 should be noted that Bertrand and M a 1 1 e v r e , to whom is due a thorough investigation of this enzyme (C. R., 1894, 119, 1012; 1895, 120, 110, and 121, 726), named the latter pectase. Occurrence. It is found in nearly all plants, especially in young, quickly-growing organs, shoots, leaves, roots, and fruit. In great abundance and in an extremely active condition, the enzyme appears in the extract of clover or lucerne, and also in the leaves of the potato plant, rape, etc. CARBAMASES (P r ot einases) Three proteolytic enzymes or groups of enzymes are dis- tinguished: the pepsin of the gastric secretion, the trypsin of the pancreas and the erepsin of the intestinal mucus. It is very probable that these three substances are not individuals, but rather mixtures of enzymes, which yet act specifically on certain protein complexes. These three enzyme-groups can, however, be readily differentiated according to their origin. More difficult is the division of the vegetable proteinases, where classification is not possible according to either the localisation of the enzymes or the media in which they act. Hence, a dis- tinction between vegetable pepsins and vegetable trypsins can hardly be drawn. The only classification at present apparent depends, on the one hand, on a separation of the peptases from the true proteinases, and, on the other, on a limitation of the pepsinases, for which the absence of the lower hydrolytic products is characteristic. It is, however, doubtful whether trypsin itself carries the hydrolysis further than pepsin does, or whether it owes this property to an accompanying peptase. In any case, a strict division of the proteinases is at present difficult. Pepsin: Pepsinases The decomposition of proteins effected by pepsin extends, so far as is known, to all proteins; the products formed con- sist of albumoses and peptones, so that the hydrolysis is incomplete, lower polypeptides and amino- acids not, as a rule, appearing (Abder- h a 1 d e n and R o s t o c k i , H., 1905, 44, 265). 34 GENERAL CHEMISTRY OF THE ENZYMES Occurrence: In the gastric juice of all the vertebrates examined, with the exception of certain fishes. The pepsin of Brunner's glands is closely allied to gastric pepsin. In the gastric mucous membrane, pepsin occurs not in an active form but as a pro-enzyme (pepsinogen). It exists with new-born children and, with certain herbivorous animals, even in the foetal state, in the mucous membrane, but is not present at birth in those carnivora which have been examined, namely, the dog and the cat. Proteolytic enzymes, similar to pepsin and active in acid solution, have also been found in several invertebrates, but, with some animals at least, these enzymes are not identical with ordinary pepsin. Preparation : Very active and stable pepsin-solutions are obtained by extracting the gastric mucous membrane with glycerol; the pepsin, together with protein, may be precip- itated from the extract by means of alcohol. A considerable amount of pepsin can also be extracted from this membrane by acidified water. The best starting material is, however, pure gastric juice, which can be prepared by P a w 1 o w ' s well-known method from gastric fistulae. Attempts at the purification of pepsin have, up to the present, led to no final result, but Pawlow's gastric juice is the best material to work on for this purpose. Such gastric juice, on freezing, yields a solid product, which Mme. S c h u m o ff- Simanowski has called " granular pepsin " (Korniges Pepsin) . The preparation of the enzyme in a pure state has been worked at mainly by N e n c k i and S i e b e r (H., 1901, 32, 231; 33, 291) -and byPekelharing (H., 1902, 35, 8). According to Pekelharing's method, P a w 1 o w ' s gastric juice is dialysed for 20 hours at 0, the pepsin thus being deposited in transparent granules- at the bottom of the dialyser. The turbid liquid is centrifuged and the colourless residue pressed and dried; in Pekelharing's opinion, this represents pure pepsin. The percentage composition of the enzyme is found to be : C H N S Cl P Fe Ash Nencki and Sieber: 51-26 6-74 14-33 1-5 0-48 small variable 0-57 Pekelharing: 51-99 7-07 14-44 1-63 0-49 + 0-1 According to Nencki and Sieber, pepsin is com- bined with lecithin. When boiled with acids, pepsin yields an SPECIAL CHEMISTRY OF THE ENZYMES 35 albumose and a nucleo-proteid, which then yields purine-bases (Pekelharing found xanthine) and pentoses ( F r i e - denthal, Engelmann's Arch. f. PhysioL, 1900, 24, 181). They regarded the pepsin molecule as performing (by three different groups) the three functions of the gastric juice: protein digestion, clotting and plastein formation, a view with which Pekelharing agreed. The precipitate formed when gastric juice is coagulated by heat contains, according to Pekelharing, an acid pep- sinic acid which has the percentage composition: C, 50-79; H, 7-0; N, 14-44 and S,l-08 and gives M i 1 1 o n ' s and the biuret reactions. Formaldehyde does not act on " pepsin," and this fact caused Bliss and N o v y to doubt the protein character of pepsin. Further, as was shown by N e n c k i and S i e b e r and also by Pekelharing, it is possible to prepare pepsin solutions which vigorously digest proteins but do not show the protein reactions; indeed, this was shown to be the case as early as 1885 by Sundberg (H., 1885, 9, 319; cf . B r u c k e , Wiener Sitz.-Ber., 1861, 43, 601). The preparation of a protein-free pepsin solution, which has an energetic digestive action but does not produce clotting, was described by Schrumpf (Hofm. Beitr., 1905, 6, 396). The mucous membrane was separated from a fresh pig's stomach and was then ground with kieselguhr and pressed under a high pressure. The pressed juice was clarified by means of a Kitasato filter-candle and was found to remain clear on addition of uranyl acetate, ammonium sulphate, etc. H e r 1 i t z k a (Atti Real. Accad. LinceL, 1904, [v], 13, ii, 51) has deduced a proof for the opposite view that pepsin is a true protein from the fact that, in absence of hydrochloric acid, pepsin gradually loses its activity, peptones appearing at the same time. These may, however, result from admixtures, so that the investigation proves nothing concerning the chemical nature of pepsin. As regards the individuality of pepsin, it has already been indicated that the same molecule, by means of other side-chains, causes rennet-action and plastein-formation, and this hypothesis is not in discord with the facts at present known (cf . J a c o b y , Biochem. Z., 1906, 1, 53). On the other hand, justifiable 36 GENERAL CHEMISTRY OF THE ENZYMES objections have been raised against P a w 1 o w ' s assumption that the rennet-action is a reversal of the pepsin-action (Bang, H., 1904, 43, 358; Schmidt-Nielsen, H., 1906, 48, 92; especially Hammarsten, H., 1908, 56, 18). From its chemical characters, pepsin appears to be an acid, and this view is sustained by a number of different observations by various investigators. J a c o b y (Biochem. Z., 1907, 4, 471) found that pepsin, and also rennin, are soluble in alkali, and adsorption experiments described by M i c h a e 1 i s indi- cate marked adsorption by basic media. This view is also supported by the observation that pepsin migrates to the anode. Pepsin is inactive in an alkaline medium, but on acidification it recovers its activity more or less completely according to the nature and duration of the previous alkalinity ( T i c h o m i - row, H., 1908, 55, 107). Pepsin exerts its optimal activity at 40 and its stability is greater in acid than in neutral solutions and is further increased by the presence of salts. Trypsin: Tryptases The enzyme of the pancreas, which is active in alkaline or neutral solution, resolves the proteins into simple polypeptides, these being to some extent further broken down. It does not decompose all dipeptides, but presumably only those combina- tions occurring in the organism (cf. Chapter VIII). On the other hand, it can be stated that only those polypeptides are attacked which contain naturally-occurring, optically active amino-acids. Acid-amides, hippuric acid, etc., are not attacked (Gulewitsch, H., 1899, 27, 540; Sc.hwarzschild, Hofm. Beitr., 1903, 4, 155; Fischer and Bergell, Chem. Ber., 1903, 36, 2592 and 1904, 37, 3103). With nucleo- proteins the protein component is separated from the nucleic acid and decomposed further. From certain nucleo-proteins, the phosphoric acid is liberated ( B a y 1 i s s , Arch. Sci. Biol. St. Petersburg, 1904, 11, Suppl., 281). Not at all improbable is the assumption of Schaeffer % and T e r r o i n e (J. de Physiol. et Pathol. gen., 1910) that the trypsin of the pancreas is accompanied by an erepsin which SPECIAL CHEMISTRY OF THE ENZYMES 37 attacks directly .(without kinase) all substances split off by the gastric juice. Occurrence. Trypsin occurs in the pancreatic juice and in the tissues of the pancreatic glands as a pro-enzyme, which is trans- formed by specific activators or kinases into the active condition. Trypsins or tryptases have been found in all the vertebrata tested for them. H e d i n (Journ. of Physiol., 1903, 30, 155 and 195) discovered a tryptic enzyme in normal blood-serum ; it acts . in an alkaline medium and decomposes casein, gelatine, and coagulated serum- albumen, but globulins and coagulated egg-albumen are not attacked. Besides in the pancreas, animal tryptases are found in the urine, and in the spleen and other organs; also in hens' eggs. Enzymes which must be termed tryptases are contained in the leucocytes and, as Jochmann showed, not only in leucemic, but also in normal, leucocytes. This occurrence is limited, so far as is known to man, monkeys, and dogs (E r b e n , Munch. Med. Wochens., 1906 and 1907; Jochmann and Lockemann, Hofm. Beitr., 1908, 11, 449). The tryptase of the leucocytes is less sensitive to heat than that of the pancreas; its optimal temperature is 55. Tryptases have also been found in insects, protozoa, sponges, worms, and molluscs. Preparation. Active but impure solutions may be obtained by extracting finely-chopped pancreatic glands with chloroform- water. The best material is pancreatic juice obtained by P a w 1 o w ' s method and activated by intestinal secretion. The purification of trypsin has been recently attempted, especially by Mays (H., 1903, 38, 428 and 1906, 49, 124 and 188). He obtained a relatively very pure and active prepa- ration by salting-out the trypsin solution with sodium chloride and magnesium sulphate; but his investigations indicate little concerning the chemical nature of trypsin. It is not a nucleo- protein and does not give the biuret reaction (Mays, Schwarzschild). In aqueous solution, it is very labile and sensitive to heat, and, indeed, more so in alkaline than in neutral solution. Sub- strate and decomposition products exert a considerable protecting action (Bayliss, Vernon), so that the optimal tem- perature is given as 40. Trypsinogen is less sensitive to heat than trypsin. Acids denature it even in low concentrations. In contrast to pepsin, which dissolves in alkali, trypsin is 38 GENERAL CHEMISTRY OF THE ENZYMES soluble in acids. In the electric field, it behaves as an ampho- teric electrolyte, that is, it migrates either to the anode or to the cathode according to the reaction of the solution. It is affected but slightly by chloroform or thymol. Pollak (Hofm. Beitr., 1905, 6, 95) characterises glu- tinase as a separate enzyme, owing to its resistivity towards acids. It acts on glue, but not on certain other proteins. On the other hand, A s c o 1 i and N e p p i (H., 1908, 56, 135) have established the fact that the specificity of glutinase towards glue is only apparent and is due to the different proteins being influenced differently by activators and paralysers Er ep sin See Cohnheim (H., 1901, 33, 651; 1902, 35, 134; 1902, 36, 13; 1906, 49, 64). This enzyme decomposes albumoses, polypeptides, peptones and protamines completely into amino- acids. With the exception of casein, proteins are not attacked, but nucleic acids are decomposed (Nagayama, H., 1904, 41, 348). Occurrence: in the intestinal juice and the intestinal mucous membrane, the latter being the richer in the enzyme. It appears, especi- ally after V e r n o n ' s investigations (Journ. of Physiol., 1904, 32, 33) and those ofAbderhalden, to be the most widely distributed proteolytic enzyme; it has been detected in nearly all the animals examined for its presence. Preparation. According to Cohnheim, it can be prepared from the pressed juice of the intestinal mucous membrane; also extraction of the latter with glycerol or water yields very active solutions. From the aqueous solution, the enzyme is salted out with ammonium sulphate. Pressed yeast juice likewise contains much erepsin (Abderhalden). The optimal temperature is about 38 (in alkaline solution). PROTEOLYTIC ENZYMES OF PLANTS Proteinases is the name given to those enzymes which break down the true proteins. The decomposition appears to proceed as far as the albumoses and peptones. It is seldom that proteinases occur alone in the organs of plants where SPECIAL CHEMISTRY OF THE ENZYMES 39 proteins are decomposed; they are almost always accompanied by peptases, which correspond with the erepsins of the animal body. Vines (Annals of Bot., 1904, 18, 289; 1905, 19, 149, 171; 1906, 20, 113; 1908, 22, 103; 1909, 23, 1) to whom we owe a thorough investigation of the vegetable proteinases has, indeed, recently found both enzymes in the seeds of Cannabis s a t i v a . Occurrence. Proteinases occur especially in germinating and ungerminated seeds and more abundantly in oil-bearing than in starch- containing seeds particularly in Cannabis sativa, Sinapis, R i c i n u s and L i n u m ; further, in certain juicy fruits (F i c u s c a r i c a ) and leaves (Agave). They are generally found in insect- ivorous plants. Among those which have been thoroughly investigated are B r o m e 1 i n in acid banana-juice (Chittenden, Journ. of Physiol., 1894, 15, 249); according to C aid well (Bot. Gaz., 1905, 39, 407), this consists of two components, a pepsinase and a tryptase; and Papain or papayotin in the juice of the fruit, leaves and stem of the papaw tree (Carica papaya). These two enzymes are very closely allied if not absolutely identical. Animal proteins, such as fibrin, are also hydrolysed by bromelin (Chittenden). From the mixture of proteolytic enzymes in oily seeds, Vines isolated a fibrin-digesting proteinase. A similar action is exhibited by the papain of Carica which readily digests fibrin. After digestion of fibrin with Carica- sap and with an enzyme similar to papain and obtained from Bacillus fluorescens 1 i q u e f a c i e n s , Emmerling (Chem. Ber., 1902, 35, 695) found in addition to albumoses and pep- tones, also various amino-acids, such as leucine, tyrosine, etc. With "papayotin Merck," Kutscher and Lohmann (H., 1905, 46, 383) obtained analogous results. According to Abderhalden and Teruuchi, H., 1906, 49, 21), papain splits glycyl-1-tyrosine; this also indicates the simultaneous occurrence of proteinases and peptases, unless, indeed, papain is regarded as a tryptase with a wider sphere of action. Vegetable proteases accompany malt-diastase and taka- diastase. These proteinases are extractable by alcohol in a remarkable manner (Vines, Annals of Bot., 1910, 24, 213). The statements made concerning the acidity or alkalinity of the medium in which vegetable proteinases exhibit their maximal activity differ considerably (cf . Reynolds Green, Vines, Emmerling, Weis, and others) . This dis- 40 GENERAL CHEMISTRY OF THE ENZYMES agreement depends partly on qualitative and quantitative differences between the substrates employed and partly on varying composition of the enzyme preparations. The peptase present will always act best in neutral or faintly alkaline solu- tions and the proteinases, on the other hand, in acid ones. Proteinases occur abundantly in fungi and they can be extracted from the mycelia by water or glycerol. As extracellular enzymes, proteinases occur in insectivorous plants. In these they are liberated from zymogens in certain glands under the stimulative action of nitrogenous substances. With Nepenthes, the enzyme is distributed in the anti- septic, almost protein-free sap of the leaf -pitchers. This acid sap decomposes proteins not merely into albumoses, but also into amino-acids (Vines, Annals of Bot., 1897, 11, 563). But Abderhalden found that glycyl-1-tyrosine is not attacked. Hence uncertainty still prevails regarding the nature of the enzyme or enzymes of Nepenthes. The leaf-glands of D i o n a e a and D r o s e r a and the leaf -edge of P i n- g u i c u 1 a yield acid, mucilaginous secretions which attack proteins of cartilaginous and glutinous tissues. Extracellular proteinases also appear to be produced by bacteria, especially by those species which are capable of liquefying gelatine. Another group of fungus-proteinases consists of typical endo- enzymes; noteworthy among these is the Endot-ryptase of yeast (Hahn and Geret). So far as is known, its action extends to all proteins (gelatine, fibrin, casein, egg-albumin), which it resolves into amino-acids. It cannot be extracted from yeast by water, and is obtainable only byBuchner's method. Unlike papain, endotryptase is most active in acid solution. Its temperature-optimum is 40-45. ^-Proteases. Papayotin, Hahn and G e r e t ' s endotryptase and the enzymes which effect a partial decomposition of the protamines and complicated proteins in faintly acid solu- tion, are classed together by Takemura as ^-proteases. The action of pepsin on protamines would thus be attributable to the presence of a g-protease. Peptases are those enzymes which decompose albumoses, peptones and polypeptides into amino-acids. They generally accompany the proteinases and, as has been shown by Vines SPECIAL CHEMISTRY OF THE ENZYMES 41 (Annals of Bot., 1906, 20, 113), Abderhalden and S c h i t - tenhelm (H., 1906, 49, 26) and Euler (H., 1907, 61, 244), they occur abundantly in the seeds of lupins, rape, peas and maize. Abderhalden and his collaborators found it also in pressed yeast juice. According to certain investigators, among them W e i s (H., 1900, 31, 79) and Vines (Annals of Bot., 1903, 17, 237 and 1904, 18, 289), the most rapid hydrolysis takes place in faintly acid solution, whilst others, e.g., W i n d i s c h , assert that it occurs in a slightly alkaline medium. NUCLEASES The decomposition of the nucleo-proteins begins with their resolution into nucleic acids and protein-components, a reaction which is brought about by pepsin or trypsin. The further division of the nucleic acids is not, however, produced by the true proteinases (Sachs; Abderhalden and Schitten- helm, H., 1906, 47, 452), but by a special group of enzymes, the nucleases. These, therefore, resolve the nucleic acids into their constituents, purine or pyrimidine bases (adenine, guanine, cytosine, thymin), pentoses, and phosphoric acid. That the decomposition of nuclein, and the digestion of Buchner's pressed yeast juice are enzymic in character has been shown by Salkowski (H., 1889, 13, 506) and H a h n and G e r e t (Chem. Ber., 1898, 31, 2335) respectively, and more recent researches prove that a number of enzymes act on the nucleic acids. These enzymes may be divided into a number of groups (cf. B . B 1 o c h , Biochem. Centralbl., 1907, 5, 561). 1. Nuclease, which liberates the phosphoric acid from the nucleic acid molecule. 2. Hydrolytic enzymes, which split off ammonia from the aminopurines and replace the amino-group by hydroxyl. 3. Oxidising enzymes which oxidise hydroxypurines to uric acid. 4. Uric acid oxydases. According to Sachs (H., 1905, 46, 337), pure trypsin has no action at all on nucleic acids. Decomposition of nucleic 42 GENERAL CHEMISTRY OF THE ENZYMES acids takes place only in fresh pancreas extracts, which exert but slight proteolytic action. Occurrence. Very frequent in animal organs, e.g., in the spleen, liver (Jones), and in the pancreatic and thymus glands (Kutscher, H., 1901, 34, 114). It performs an important function in many organs of plants more particularly in germinating seeds in synthesizing and decomposing nucleo-proteins (Z a 1 e s k i , Bot. Ber., 1907, 25, 349). Worthy- of note also are the nucleases of higher (K i k- k o j i , H., 1907, 51, 201) and lower fungi (I w a n o f f , H., 1903, 39, 31), especially those of yeast. P. A. Levene and Medigreceanu (Journ. of Biol. Chem., 1911, 9, 389) have recently proposed the following classification and nomenclature. Nucleinases resolve the nucleic acid molecule into nucleotides. Nucleotidases decompose nucleotides into phos- phoric acid and a carbohydrate-base complex (nucleoside) . Nucleosidases hydrolyse nucleosides into ribose and purine bases. Arginase (Kossel and Dak in, H., 1904, 41, 321) This enzyme hydrolyses arginine specifically into urea and ornithine according to the following equation: NH NH 2 +NH 2 CH 2 CH 2 CH 2 CH COOH. I NH 2 Whilst trypsin hydrolysis may be indicated by the scheme: C CO-j-NH-C, the action of arginase is represented by Occurrence. In the liver, kidneys, thymus, and intestinal mucous membrane of the calf and also in the muscles of the dog and SPECIAL CHEMISTRY OF THE ENZYMES 43 the lymphatic glands of cattle. Shiga (H., 1904, 42, 502) found arginase also in yeast. It can be extracted from the organs by means of water. Ur ease This enzyme decomposes urea into carbon dioxide and ammonia. Occurrence. According to L e u b e (Virch. Arch., 1885, 100, 540), urease occurs inMicrococcus ureae, from which it passes readily into the surrounding liquid. In cystitic urine the enzyme occurs only when fungi capable of decomposing urea are present. Lea (Journ. of Physiol., 1885, 6, 136) has attempted to prepare urease in a pure state; by treatment of Micro- coccus ureae with alcohol, extraction of the precipitate with water and repeated reprecipitation with alcohol, a powder is obtained which yields a clear aqueous solution but contains protein. As later work has shown, urease is tenaciously retained by living protoplasm. Moll (Hofm. Beitr., 1902, 2, 344) also obtained the enzyme in a similar manner and from the same material; it readily undergoes decomposition. Schit- t e n h e 1 m obtained an enzyme-solution (free from purine- bodies) from the kidneys and K i k k o j i found urease in a pileate fungus. AMIDASES (DESAMIDASES) Decomposition of the proteins by trypsin and erepsin ceases when the amino-acids are reached, but the desamidases attack the latter, decomposing them into ammonia and hydroxy-acids. Such simple desamination, as was discovered byH. Prings- heim (Biochem. Z, 1908, 12, 15), takes place by virtue of an enzyme present in acetone-yeast. Similar action is exhibited by permanent preparations of Aspergillus niger (Shi- bat a, Hofm. Beitr., 1904, 5, 384). It has been shown by Pringsheim, Abderhalden and Schittenhelm that the amidases of yeast do not pass into the press-juice. Of biological importance is the detection of amidases in higher plants (Butkewitsch, H., 1909, 63, 103; cf. Kiesel, H., 1910, 65, 283). 44 GENERAL CHEMISTRY OF THE ENZYMES The so-called " alcoholic fermentation of the ammo-acids " discovered by F , E h r 1 i c h consists of a combination of two reactions, namely the desamination of the amino-acids to the corresponding hydroxy-acids and the subsequent loss of carbon dioxide from the latter; the total reaction can hence be formulated thus: Reactions similar in principle to those brought about by amidases are caused by the enzymes known as guanase and a d e n a s e . These convert guanine and adenine into the corresponding hydroxy-derivatives : N = C NH 2 N = C OH II II CH C NH, + H 2 = CHC NH, II II >CH || || >CH + NH 3 ; N C - W N C - W Adenine Hypoxanthine and NH CO NH 2 -C C NH X +H 2 = OH-C II II >CH || || CH+NH 3 N - C -- N^ Guanine Xanthine Concerning the individuality and mode of action of these enzymes of the purine-bases, various views have been expressed by the different investigators who have studied them (Schitten- helm, H., 1904, 42, 251; 1905, 43, 228; 45, 121, 152; 46, 354; Jones and his collaborators, H., 1904, 42, 35, 343; 1905, 44, 1; 45, 84; 1906, 48, 110; and also Burian, H., 1905, 43, 494) . Even if Schittenhelm's assumption that the two enzymes are identical is not correct, they are certainly very similar. Occurrence. Guanase and adenase have been found in the pancreas, spleen, lungs, and liver of the child, pig (not in the spleen), cattle, and also in numerous other organs. Preparation. (Schittenhelm, H., 1904, 42, 251). One or two spleens are extracted with about 2 litres of water for 12 hours and the enzyme then precipitated with SPECIAL CHEMISTRY OF THE ENZYMES 45 ammonium sulphate. The precipitate is dissolved in water and freed from ammonium sulphate by dialysis. Ni t r ilase According to Rosenthaler (Biochem. Z., 1909, 19, 186; 1910, 28, 408), the decomposition of amygdalin is effected by three enzymes an amygdalase, a ^-glucosidase and an hydroxynitrilase. The last of these exerts a catalytic influence on the reaction. OH C 6 H 5 -CH CH 2 -OH CH-OH HCH -CH 2 CH 3 CH 3 CH 3 | ~~OH-^| 4-1 ->- CH-OH CH-OH CH-OH CHO CHO CHO CH-OH CH-OH CH-OH CH-OH~OH ""C-OH^CO CH 2 -OH CH 2 -OH CH 2 -OH CH 2 -OH CH 2 CH 3 Glucose . Glyceraldehyde Methylglyoxal There is also uncertainty concerning the occurrence of methyl- glyoxal, which cannot be fermented by pressed yeast juice. Boysen-Jensen (Bot. Ber., 1908, 26, 666; also Dis- sertation, Copenhagen, 1910) supposes the intermediate product to be dihydroxyacetone, an isomeride of glyceraldehyde, but it cannot be said that this has been proved to be the case; this assumption is, however, rendered probable by the fact that dihydroxyacetone is fermented readily and glyceraldehyde only slowly. It is worthy of note that the transformation of glucose into alcohol + carbon dioxide can be effected by purely chemical means, the various reactions requiring, however, different catalysts: Glucose ^lactic acid (alkali as catalyst). Lactic acid >acetaldehyde+ formic acid (sulphuric acid as catalyst) . Acetaldehyde-f formic acid alcohol+ carbon dioxide (rhodium as catalyst). (Buchner, Meisenheimer and S c h a d e , Chem. Ber., 1906, 39, 4217; Schade, Zeitschr. f. physikal. Chem., 1906, 57, 1.) Further, the author's investigations (Arkiv for Kemi, 1911, 4, No. 8) show that, in ultra-violet light, lactic acid undergoes decomposition into alcohol and carbon dioxide. According to a new and interesting investigation by Franzen and Steppuhn (Chem. Ber., 1911, 44, 2915), formic acid is fermented as well as formed by living yeast and must hence be taken into account as an intermediate product. As substrate for fermentation, mannose, galactose, or fructose may be used instead of glucose. Glucose and fructose exhibit no difference in their velocity of decomposition, and in the case of mannose there is only a slight deviation. Galactose, on the SPECIAL CHEMISTRY OF THE ENZYMES 53 other hand, is fermented only by certain species of yeast, including bottom fermentation beer-yeasts, and by their pressed juices; the fermentation takes place far more slowly than that of glucose (E. Fischer and Thierfelder, Chem. Ber., 1894, 27,2031; E. F. Armstrong, Proc. Roy. Soc., B, 1905, 76, 600; SI at or, Journ. Chem. Soc., 1908, 93, 217). As, however, has been shown by S 1 a t o r and by Harden and Norris (Proc. Roy. Soc., 1910, 82, 645), the capacity of yeasts for fermenting galactose can be increased by culti- vating them in solutions containing this sugar. Mention must also be made of S 1 a t o r ' s view that the different hexoses are attacked by different enzymes: glucose and fructose by gluco-zymase, mannose by manno-zymase and galactose by galacto-zymase. The mechanism of alcoholic fermentation is considerably less simple than was formerly supposed, a number of enzymes and subsidiary substances taking part in the formation of alcohol and carbon dioxide. The first separation of zymase into a " zymase in a restricted sense " and a lactacidase must be given up, since the formation of lactic acid as an intermediate product has been shown to be improbable. And a special enzyme has now to be assumed for each of the changes indicated in the above scheme of reactions. Of great importance for the elucidation of the nature of fermentation is Harden and Young's discovery of the co-enzyme of zymase. By filtration through a film of gelatine under a pressure of 50 atmospheres, pressed yeast juice can be divided into a filtrate and a residue, which are separately inactive towards sugar but produce fermentation when again mixed (Harden and Young, Proceedings of the Physiol. Soc., Nov. 12, 1904, see Journ. of Physiol., 1904, 32, i; Proc. Roy. Soc., B, 1906, 77, 405). The substance in the dialysate resists boiling (thermostable) and undergoes hydrolytic decomposition and hence destruction by enzymes (lipases) of the yeast juice. On the other hand, the zymase itself which does not traverse the gelatine filter is destroyed when heated and is presumably a protein substance, being attacked by the proteinases or pro- teases of the yeast juice. From this attack it is protected by a thermostable substance a ntiprotease (Buchner and 54 GENERAL CHEMISTRY OF THE ENZYMES Haehn, Biochem. Z., 1910, 26, 171). This antiprotease is, like the co-enzyme, destroyed by lipase, but is more stable than the co-enzyme towards hydrolytic agents and towards heat. Both enzyme and co-enzyme are precipitated from the yeast juice by acetone, but the latter less readily than the former, so that a certain degree of separation can be attained by frac- tional precipitation ( B u c h n e r and Duchacek, Bio- chem. Z., 1909, 15, 221). Alcoholic fermentation by means of pressed yeast juice is facilitated by the addition of a phosphate. Harden and Young (Proc. Roy. Soc., B, 1906, 77, 405) have made the important discovery that, during the period of enhanced fer- mentation, the amount of carbon dioxide produced exceeds that which would have been formed in the absence of phosphate by a quantity exactly equivalent to the phosphate added C0 2 : R 2 / HP0 4 . These two investigators consider that the phosphate reacts with the hexose in the yeast juice in the following manner: (1) +2H 2 0+C6H 10 4 (P0 4 R 2 ) 2 . The result is an ester of hexosediphosphoric acid, the salts of which have been more closely investigated by Young (Biochem. Z., 1911, 32, 177). During the fermentation, the hexosediphosphate accumulates in the solution, but as soon as fermentation ceases, this ester undergoes hydrolysis in the yeast juice, thus. (2) C6Hi 4 (P0 4 R 2 ) 2 +2H 2 = C 6 Hi 2 06+2R 2 HP04. This hydrolysis is effected by a hexosephosphatase." These reactions will be considered further in Chapter VII. Occurrence. Zymases do not only occur in yeasts, but are extraordinarily widespread throughout the whole of the animal and vegetable kingdoms. There is now scarcely room for doubt that the combustion of sugar in the animal organism and also in higher plants begins with decompositions completely analogous to those brought about by yeast. Deviation from these occurs only in the final phase of the reaction, since in living animal organs and living plants alcohol is formed only when oxygen is lacking. On the other hand, the intramolecular respiration of sugar is absolutely identical with alcoholic fermentation. SPECIAL CHEMISTRY OF THE ENZYMES 55 This v.ew, as far as the higher plants are concerned, is due especially to E. Godlewski, Palladin and Kostytschew. Also the change known as glycolysis, occurring in the animal body, must be closely allied to fermentation. Animal glycolytic enzymes or zymases have been prepared especially from the blood, spleen, pancreatic tissues and muscle. Mme. N. Sieber (H., 1903, 39, 484; 1905, 44, 500) has obtained three glycolytic enzymes in the form of stable powders from blood-fibrin and spleen: (a) soluble in water, (b) soluble in neutral salt solutions, and (c) soluble in water or alcohol (peroxydase) . All these enzymes contain nitrogen and give the reactions of the proteins. Their ash contains iron, manganese and phos- phoric acid, but not copper. The second of them gave the fol- lowing mean composition: C, 52%; H, 7-5% and N, 15%. Tincture of guaiacum is turned blue directly by enzymes (a) and (b), but only in presence of hydrogen peroxide by (c). 11 6 h m a n n and S p i t z e r ' s reagent (a dilute alkaline solution of a-naphthol and paraphenylenediamine) is coloured by all three enzymes in absence of hydrogen peroxide. It is doubtful if these oxydase- or peroxydase-reactions are related in any way to the ability to bring about the combustion of sugar. In any case the essential constituents of the enzyme- complexes obtained by Mme. Sieber are fermentati^on- enzymes and not oxidation enzymes. Further the glycolytic enzyme found by Cohnheim (H., 1903, 39, 336; 1904, 42, 401) in muscle, from which he extracted it in an inactive state, is not oxydasic in character. It is a decided endo-enzyme like yeast-zymase and is obtained from the frozen, subdivided muscle by pressing or by extraction with an ice-cold, isotonic solution of sodium oxalate; the oxalic acid must then be precipitated with the calculated quantity of calcium chloride (H., 1906, 47, 253). Corresponding with this enzyme, there exists a co-enzyme or activator which, like that of yeast-zymase, is thermostable. Cohnheim pre- pared it from the pancreatic tissue of the cat. The objections raised by Glaus and E m b d e n (Hofm, Beitr., 1906, 6, 214 and 343) to the investigations of Cohn- heim have been refuted by the latter author. Reference 56 GENERAL CHEMISTRY OF THE ENZYMES must be made to Stoklasa's work (Chem. Ber., 1905,38, 664; Arch. f. Hygiene, 1904, 50, 165) which likewise indicates the existence of a zymase in the muscles and also in the milk. It is, however, remarkable that such a capable experimenter as A. Harden, working in conjunction with H . Maclean (Journ. of Physiol., 1911, 42, 64), was unable to detect alcoholic fermentation by animal tissues (liver, kidneys, pancreas, flesh, etc.) or by juices or powders prepared from them. That the intramolecular respiration of plants is to be regarded as a zymase fermentation has already been mentioned (Pal- ladin and Kostytschew, H., 1906, 48, 214; Kost- ytschew, Bot. Ber., 1908, 26, 167). Also by means of seedlings of Hordeum distichum, Pisum sativum and Lupinus luteus, Stoklasa, Ernest and C h o - c en sky (H., 1907, 50, 303; 1907, 51, 156) have clearly shown the action of the enzymes of intramolecular respiration. The arguments opposed to Stoklasa's earlier experiments can scarcely be advanced against the work just referred to. Pal lad in (Bot. Ber., 1905, 23, 240) holds the view that the carbon dioxide respired by plants arises in three ways: 1. By the enzymes combined with the protoplasm; Palladin calls this portion, nucleo-carbon dioxide and the corresponding enzymes, " carbonases." 2. By the protoplasm itself (apparently directly) under the influence of various irritants irritant-carbon dioxide. 3. By the action of oxydases. In another place (Zeitschr. f. physikal. Chem., 1909, 69, 187, Arrhenius- Festschrift), the author has indicated that he is unable to agree entirely with P a 1 1 a d i n ' s views, especially as regards car- bonase and the role of oxydases and reductases in the respiration process. The opportunity must, however, not be neglected to direct attention to the many remarkable observations com- municated by Palladin to the Berichte der deut. botan. Gesellschaft. Preparation of yeast-juice. One kilo of washed and well-pressed bottom-yeast is mixed with 1 kilo of fine sand and 300 grms. of kieselguhr and is ground, in 4-6 lots, in a large mortar until the mass becomes soft and doughy. In this way a large proportion of the yeast-cells are broken. The dough is then enveloped in a press-cloth and pressed in a hydraulic press, the pressure being raised to about 90 kilos SPECIAL CHEMISTRY OF THE ENZYMES 57 per sq.cm. and maintained at this value for an hour. About 400 c.c. of a clear, pale-brown, viscous juice, containing only a very small number of living cells, are thus obtained ( E d u a r d and Hans Buchner and M . H a h n , Die Zymase- garung, Munich, 1903, p. 58). From the yeast-juice the zymase can then be precipitated with alcohol or acetone, which, however, throws down a large amount of proteins, carbohydrates and salts at the same time. In order to obtain very active preparations, the yeast-juice must be poured into a large excess of acetone (10 volumes) ; . only in this way can the precipitate formed be obtained so free from water that the chemical changes occurring in it are reduced to a minimum (Buchner and Duchacek, Biochem. Z., 1908, 15, 221). The following simple method for obtaining zymase-prepara- tions has been recently discovered by von Lebedew (C. R., 1911, 152, 49; Bull. Soc. Chim., 1911, [iv], 9, 744): It consists in drying the yeast at a temperature of 25-30 and macerating with water for 2 hours at 35; the filtered liquid exhibits considerable fermentative activity. According to the author's experience, Munich yeast (from Schroder's factory) and many other yeasts are suitable for this purpose, but this is not the case with all yeasts. According to Rinckleben (Chem. Zeitung, 1911, 35, 1149), zymase can also be obtained by plasmolysing fresh yeast with glycerol. Preparation of Permanent Yeast. In addi- tion to those described above, another method is known by which the fermentative activity of yeast-cells can be separated from the true life functions. When yeast is introduced into alcohol or acetone (Albert, Chem. Ber., 1900, 33, 3775), the cells are killed without their fermenting power being destroyed. Well pressed yeast (500 grms.) is thoroughly disintegrated, placed on a hair-sieve and the whole dipped into a basin containing 3 litres of acetone. By alternately raising and lowering the sieve in the liquid, and by means of a small brush, the whole of the yeast-cells are passed through the sieve in 3 or 4 minutes. The yeast is then left in the acetone for 10 minutes, being fre- quently stirred meanwhile. Most of the liquid is next poured off and the yeast, after being pumped as dry as possible, is again 58 GENERAL CHEMISTRY OF THE ENZYMES treated with acetone, filtered and drained. The residue is well kneaded with 250 c.c. of ether, filtered, dried in the air, finely ground and then dried for 24 hours at 45. This preparation has been placed on the market under the name of " zymin" by A. Schroder, of Munich. The best antiseptics to use with yeast-juice are toluene and thymol. Lactic Acid B ac ter ia-zymase This is the enzyme by means of which lactic acid bacteria are enabled to decompose sugar into lactic acid. The enzymic nature of this transformation was demonstrated by Buchner and Meisenheimer (Chem. Ber., 1903, 36, 635; Lieb. Ann., 1906, 349, 125); they succeeded in obtaining an active permanent preparation of Bacillus Delbriickii which, however, only gave rise to small quantities of ^-lactic acid. Preparation. The organism was cultivated at 40-45 in wort prepared from malt and rye. The bacteria were subsequently separated by means of a centrifuge and dried on a porous tile. The mass was then intro- duced into 15-20 times its weight of acetone, with which it was ground for 10-15 minutes, the bacteria being pumped dry, washed with ether and dried in a vacuum. OXYDASES The action of oxydases is assumed in changes of very dif- ferent kinds: oxidations of purine bases, conversion of alcohols and aldehydes into acids, transformation of simple and substituted phenols, amines, amino-acids, and derivatives of these com- pounds into quinone derivatives. The following kinds are thus to be distinguished: 1. Purine-oxydases. 2. Alcoholases. 3. Aldehydases. 4. Phenolases. 5. Tyrosinase. SPECIAL CHEMISTRY OF THE ENZYMES 59 To these true oxydases must be added the peroxydases, which can hardly be separated sharply from the oxydases, and, especially with the latter, there remains still a good deal that is not clear, so that the division of this section can be considered only as a provisional one. For the recognition of oxydases use has been made of a number of reactions, some of which are referred to below; they are not, however, given by all oxydases, which are, to a great extent, specific in their action. Blue coloration of guaiacum tincture or of a-guaiaconic acid; violet coloration of tetramethylparaphenylenediamine; brown coloration of m- and p-phenylenediamine alone or in presence of hydrogen peroxide (A so); reddening of aniline acetate (C. R., 1896, 123, 315) and blue coloration of a-naphthol (B o u r q u e 1 o t , C. R., 1896, 123, 423; Soc. BioL, 1896, 46, 896); reddening of alo'in (S c h a e r , Arch, der Pharm., 1900, 38, 42; K a s 1 1 e); oxidation of phenolphthalin to phenolphthalein (K a s 1 1 e and S h e d d , Amer. Chem. Journ., 1901, 26, 526) ; oxida- tion of benzidine ; leucorosolic acid (K a s 1 1 e, Public Health and Marine Hospital Service of the U. S. Hygienic Lab. Bull., 1906, No. 26, 7-22), hydroquinone, pyrogallol, and guaiacol; oxidation of leuco-malachite green to malachite green (quantitative, spectro-photometric method of von Czyhlarz and von Fii r t h , Hofm. Beitr., 1907, 10, 358); oxidation of aldehydes, e.g., salicylic aldehyde (S c h m i e d e b'e r g , Arch. f. exp. Path., 1881, 14, 288, 379), formaldehyde (Pohl; Arch. f. exp. Path., 1896, 38, 65); oxidation of arsenious to arsenic acid; forma- tion of diaminophenacin from o-phenylenediamine, and of indophenol from p-phenylenediamine and a-naphthol (R 6 h m a n n and S p i t z e r , Chem. Ber., 1895, 28, 567). The separation of iodine from potassium iodide has also been regarded as an oxydase reaction. But this action, as has been pointed out by A s o and more recently by Wolff and d e S t o e c k 1 i n , C. R., 1908, 146, 1415), must be attributed to the nitrous acid occurring in plant juices. As regards the oxidising enzymes of the purine bases, the action of xanthine - oxydase is best under- stood, owing especially to the work of B u r i a n . This enzyme does not attack uric acid, which is so readily broken down by ordinary oxidising agents. B u r i a n supposes it to be quite a general oxidising enzyme for (cyclic?) amidines which is unable to attack the double linking between the (4) and (5) carbon atoms of the purine nucleus: 60 GENERAL CHEMISTRY OF THE ENZYMES NH CO NH CO NH CO CH C (4) NH\ CO C NHv CO C NH\ || || >CH | || >CH | || >CO N C (5) W NH C W NH C NEK Hypoxanthine Xanthine Uric acid An enzyme which oxidises uric acid to allantoin was obtained by Wiechowski and Wiener (Hofm. Beitr., 1907, 9, 232, 247, 295) from animal organs by treating the powdered organ (the cells being completely ruptured) with 05% soda solution. The nitrates obtained after grinding with the soda solution are inactive, but if the emulsions are subjected to dialysis into 0-05% soda solution for 5-6 days, the enzyme of dog's liver passes completely, and that of ox-kidneys partially, into the filtrate. Enzymic liquids were also obtained by centrifugating the emulsions. Repeated precipitation and nitration yielded a protein-free preparation, which exhibited the total enzymic activity of the starting material. Alcoholase : Oxydase of Acetic Bacteria. This enzyme oxidises ethyl alcohol to acetic acid and thus catalyses a reaction 'for which energetic oxidising agents are otherwise necessary. The enzyme has not yet been separated from the bacteria, but its existence can be proved by killing the bacteria with acetone (Buchner and M e i s e n - h e i m e r , Chem. Ber., 1903, 36, 637; B u c h n e r and Gaunt, Lieb. Ann., 1906, 349, 140). Preparation. Large quantities of acetic bacteria (best Bacterium aceti, which can always be obtained by leaving beer in a glass dish exposed to the air) are cultivated on beer-wort to which 4% of alcohol and 1% of acetic acid have been added. As culture-vessels, shallow glass basins, as wide as possible, are most suitable; these are covered with a layer of cotton wool in order to prevent the entry of germs from the air. In the course of 4 or 5 days after inocu- lation, the bacteria form a fairly thick coating on the surface of the nutrient solution. The clear liquid underneath is syphoned off and the residue centrifuged to remove most of the liquid; the brittle bacterial membranes are then washed superficially with water and dried on porous tile. On the following day the SPECIAL CHEMISTRY OF THE ENZYMES 61 mass is introduced into 10-20 times its weight of acetone, rubbed to a fine powder and left for 10 minutes, after which it is filtered, washed with ether and dried in a vacuum over sulphuric acid. This procedure yields a yellowish powder which, however, only oxidises very small quantities of ethyl alcohol. Aldehydases. Aldehydes, e.g., benzaldehyde and sal- icylic aldehyde, are oxidised by the extracts of many animal organs, but it is doubtful if this is an enzyme action. See the remarks of B a c h (Chem. Ber., 1904, 37, 3791) arid the results of Dony-Henault and van Duuren (Bull. Acad. roy. Belgique, 1907, 577). Of the vegetable oxydases mention must first be made of the laccase from the lac-tree, which was discovered by Y o s h i d a and studied in detail by Bertrand, who gives the follow- ing method for its preparation (Ann. de Chim. et de Phys., 1897, [vii], 12, 115). The thick sap of Rhus succedanea is mixed with 5-6 times its volume of alcohol, by which means the laccase is precipitated, whilst the phenolic derivatives which produce the blackening of the lac pass into the alcoholic solution. The solution is filtered through a cloth and the residue washed on the cloth several times with alcohol. It is then taken up in cold distilled water, only a small quantity of black substance remaining undissolved. The filtered solution is again precipitated with alcohol in large excess, the precipitate being collected on the filter and dried in a vacuum. Laccase is obtained as a white substance having a neutral reaction and readily soluble in water. Bertrand regards it as a protein, although his preparation contained only 0-44% of nitrogen. He assumes its composition to be as follows : Moisture (determined at 120) 7-40% Gum (arabans and galactans) 84-95 Laccase 2-50 Ash 5-17 The most essential constituent of the ash is manganese, which is present to the extent of 2-5%. Laccase is extremely sensitive to acids (cf. Chapter III) and is destroyed by short boiling of its solution. 62 GENERAL CHEMISTRY OF THE ENZYMES From other plants, such as Medicago sativa and Lolium perenne, Bertrand obtained preparations which he termed laccases, since, in neutral solution and in presence of manganese salts, they accelerate the oxidation of polyphenols. As the author has pointed out (H., 1909, 61, 1), they are quite different from R h u s - 1-acease. Preparation of Medieago-lacca.se. The fresh plants, at the commencement of the flowering stage, are chopped up and pressed. On standing, the juice obtained deposits dark flocks, from which it is separated by filtration. Alcohol is then added, the abundant precipitate thus formed being, to a large extent, taken up in water and again precipi- tated. This procedure is repeated thrice, the preparation ob- tained, after drying in a desiccator, being a white, dusty, highly hygroscopic powder, soluble in water with great readiness. According to E u 1 e r and B o 1 i n , it consists mainly of the calcium salts of aliphatic hydroxy-acids. The separation of the mixture, effected by fractional crystallisation of the correspond- ing barium salts, shows it to contain glycollic, citric, malic, and mesoxalic acids. The oxidising action of these salts is described in Chapter IV. Numerous attempts have been made to prepare laccases artificially. According to Bertrand, the oxidising agent of many plants is composed of manganese and a protein with a specific action. T r i 1 1 a t (C. R., 1904, 138, 94, 274) assumed that the laccase regarded as a specific enzyme can be replaced by any protein or, at any rate, by certain classes of proteins; he has, however, no good foundation for this view. Especially on the ground of his own experiments on R h u s - laccase, the author also regards as unsuccessful Dony-He*nault's attempts to attribute the action of laccase to the alkalinity of the preparations and thus to show that lac case-action is only an oxidation by means of manganese and hydroxyl-ions. The laccase preparations obtained by Bertrand's method are not alkaline, but are extremely active. Further efforts to prepare artificial oxydases and peroxydases have been made by M a r t i n a n d (C. R., 1909, 148, 182), Wo 1 f f (C. R., 1908, 147, 745), and d e S t o e c k 1 i n (C. R., 1908, 147, 1489). With other oxydases, so little has been done as regards puri- fication and isolation that is has not been decided to what class of bodies these substances belong SPECIAL CHEMISTRY OF THE ENZYMES 63 Slowtzoff (H., 1900, 31, 227) attempted to prepare the " lac- case" of potatoes in a pure state, his purest preparation containing 12- 8% NandO- 53% S. Sarthou (J. de Pharm. et Chim., 1900, [vi], 11, 482, 583; 1901, [vi], 13, 464) obtained quite different numbers for his schinoxydase, namely, 6-28% N, 0-2% S, and 1-34% ash, from which he concluded the enzyme to be a nucleoprotein. A s o and L o e w regard the oxydases as albumoses. But Rosenfeld's investigations (Disser- tation, St. Petersburg, 1906) on the oxydase of the radish (Raphanus s a t i v u s) appear to indicate that this enzyme does not belong to the proteins. According to R o s e n f e 1 d the oxydase would be a crystalline substance, containing C, N, S, P, and K but not Fe or Mg, S pn t z e r came to the conclusion that the oxydase of the liver is a nucleoprotein, but this enzyme, which effects the oxidation of salicylic aldehyde to salicylic acid, was further purified by J a c o b y (H., 1900, 30, 135). It is found to be soluble in water and non-diffusible, and to become inactive on heating, while it does not give the reactions characteristic of the proteins. According to Tschirch and Stevens (Arch, der Pharm., 1905, 43, 504), the oxydase of Japanese lac shows the pyrrole reaction. Bach and C h o d a t are of opinion that the oxydase consists of a peroxydase and an oxygenase, the former alone giving the pyrrole reaction (Bach, Chem. Ber., 1908, 41, 226). Theoenoxydaseof apples (L i n d e t , C. R., 1895, 120, 370), pears, plums, grapes, and the fungus Botrytis cinerea, parasitic to grapes, must be a tannin-oxydase ; by its action the flesh of the fruit is turned brown on exposure to the air. Oxydases of doubtful individuality have been detected in numerous plants, e.g., Arum maculatum, olives (" olease ")> barley and malt (" spermase "), coffee beans and yeast. Should B a 1 1 e 1 1 i ' s observation (C. R., 1904, 138, 651) on the oxidation of formic acid to carbonic acid by oxydases in presence of hydrogen peroxide be confirmed, an interesting organic oxidation will present itself. In this connection, mention may be made of Loevenhart's discovery (Chem. Ber., 1906, 39, 130) that formic acid is oxidised to carbon dioxide by hydrogen peroxide in presence of iron, copper, etc. A very remarkable oxidation, which is not yet understood, has been described by Z a 1 e s k i and R e i n h a r d (Bio- chem. Z., 1911, 33, 449); it consists in the oxidation of oxalic acid in 1% solution to carbonic acid. 64 GENERAL CHEMISTRY OF THE ENZYMES For information concerning other oxydases, see Battelli and Stern's resume in Ergeb. der Physiol., 1912, 12, 95. Ty rosinase (G.Bertrand, Bull. Soc. Chim., 1896, [iii], 15, 791.) From tyrosine and its derivatives this enzyme forms, by oxidation, melanins dark-coloured substances of unknown chemical com- position. Interesting data concerning the chemical aspect of the action of tyrosinase have recently been obtained by Abderhalden and Guggenheim (H., 1908, 54, 331). They were able to show that ozone and tyrosine alone do not yield melanins, but that these are synthesised by oxidation from tyrosine or polypeptides containing it, on the one hand, and from phenols or amino-acids on the other. Similar colorations are obtained if the oxidation is effected with potassium dichromate instead of tyrosinase, and it is hence probable that tyrosinase contains an oxidising agent, the action of which is exerted along with that of the amino-acids of the plant-juice. Tyrosinase attacks not only Z-tyrosine itself, but also dl- tyrosine, tyrosine anhydride (Bertrand and Rosen- blatt; Chodat), and a large number of polypeptides containing tyrosine. Suprarenin (adrenaline) is likewise oxidised, and all the cresols, resorcinol, m-toluidine, o-, m- and p-xylenols, thymol, carvacrol and naphthol; also, according to Ber- trand, phenol. The opinion expressed by Gonnermann that the specific action of tyrosinase consists of a hydrolysis, which is then followed by an oxidation (not specific), has been combated by Chodat and Bach. According to Chodat, Zahorski and F r e e d e - rickz (Arch. Sci. phys. nat., 1909, 27, 306), the specificity of tyrosinase is conditioned by the presence of an activator which is stable to heat. For the preparation of the enzyme, the disintegrated fungus is extracted with water and the extract precipitated with alcohol. Occurrence. In numerous fungi of the species Boletus, Russula, Lactarius, Coprinus, Paxillus, etc. Also SPECIAL CHEMISTRY OF THE ENZYMES 65 in Merulius lacrimans, beet-juice, dahlia bulbs, potato skins, and V i c i a f a b a . Tyrosinase is often accompanied by lac- cases. From sepia, C. Neuberg has extracted an enzyme which resembles tyrosinase and acts on adrenaline. PEROXYDASES Under this name are included those substances which activate peroxides. Their typical reaction is the transference of oxygen from hydrogen peroxide to guaiaconic acid or to polyphenols. Bach and Chodat (Chem. Ber., 1903, 36, 600) have given a method for the preparation of peroxydase from pump- kins and horse-radish roots. Bach and Tscherniak have recently (Chem. Ber., 1908, 41, 2345) obtained a peroxy- dase in the following manner: Thirty kilos of turnips were pounded up and pressed and the juice obtained (20 litres) mixed with 2 litres of 96% alcohol in order to coagulate the gummy matters. After nitration, the alcoholic juice was precipitated by 130 litres of strong alcohol, the precipitate being filtered off, washed with alcohol and freed from precipitant in a vacuum. The crude peroxydase thus obtained (52 grms.) was kneaded with 600 c.c. of water, only a small portion of the substance passing into solution. The undissolved residue was filtered off and washed with a little water, and to 600 c.c. of the filtrate, containing only about 7 grms. of dry matter, 40 grms. of powdered basic lead acetate were added; the precipitate was pumped off and the clear filtrate (600 c.c.) treated with powdered sodium carbonate (21 grms.) until no further turbidity was produced. The alkaline filtrate was dialysed through parchment into distilled water. After 13 days the dialysate (670 c.c.) was mixed with 4-5 litres of 99% alcohol, and the precipitate thus formed collected, after 24 hours, on a hardened filter, washed with absolute alcohol and freed from the latter in a vacuum. This preparation contained 7 87% of water, 81 66% of organic matter and 1-47% of ash; the percentage of nitrogen, calculated on ash-free material, was 3 44. The peroxydase prepared by E. de Stoecklinin C h o d a t ' s laboratory from Cochlearia armoracia 66 GENERAL CHEMISTRY OF THE ENZYMES contained 1 1 41% of water, 65 88% of organic matter and 22 71% of ash; the nitrogen-content was 3-43%. The activating power of this peroxydase was only one-tenth part of that of the above- mentioned preparation of Bach. Neither de Stoecklin nor C hod at (Schweiz. Woch. Chem. u. Pharm., 1905, 43) obtained protein reactions with their peroxydases. The author's experience (H., 1909, 61, 1) indicates that dialysis is the best known method of purifying peroxydase preparations. From horse-radish E u 1 e r and B o 1 i n ob- tained a preparation which increased continuously in activity when subjected to dialysis. The preparation showing the greatest activity per unit of weight contained 10-4% N and 2-5% of ash. Dialysis is rendered far more effective if the enzymic juice is previously treated with kaolin or other suitable adsorption agent in order to remove the proteins. D e 1 e a n o ( Biochem. Z., 1909, 19, 266) proposes the use of colloidal ferric hydroxide for this purpose. Bach (Chem. Ber., 1910, 43, 362) suggests the preliminary removal of the gummy matters by means of magnesium sulphate. During recent years it has been repeatedly pointed out that the action of peroxydases can be obtained partly by purely inorganic materials (see Wolff, C. R., 1908, 146, 781 and M a r t i n a n d , C. R., 1909, 148, 182), and partly by synthetic organic preparations (de Stoecklin, C. R., 1908, 147, 1489). As Moitessier, Lesser, von Fiirth and von C z y h 1 a r z , and Bertrand and Rogozinski (C. R., 1911, 152, 148) have shown, the well-known guaiacum-blue reaction of the blood depends not on an enzyme, but on the haemoglobin; it appears with undiminished intensity after boiling. Oxyhsemoglobin acts not only as a peroxydase but as an oxy- dase as well (de Stoecklin). Further, the oxidation phenomena in milk cannot depend on the presence of a peroxydase. Thermolabile peroxydases do, however, exist and these must for the present be classed as enzymes. SPECIAL CHEMISTRY OF THE ENZYMES 67 CATALASES ( O . L o e w , Rep. U. S. Dept. of Agric., 1901, No. 68.) After the ability to decompose hydrogen peroxide had been long regarded as a general property of the enzymes, O . L o e w demonstrated the existence of special catalases. The discoverer distinguished two catalases occurring in plants, viz., an a-catalase which is not extractable by water and was regarded as a nucleo- protein, and a g-catalase soluble in water which was regarded as an albumose. The chemical action of the catalases is analogous to that of the colloidal metals ( B r e d i g ), molecular (inert) oxygen being formed, together with water. Ethyl hydroperoxide is not decom- posed (Bach and Chodat, Chem. Ber., 1903, 36, 1757). Occurrence : Extremely widespread in the animal and vege- table kingdoms. In blood (S enter; Lesser, Zeitschr. f. Biol., 1906, 48, 1) ; in numerous organs (K a s 1 1 e and Loevenhart ; Liebermann; Battelli,C. R., 1904,138,923); in milk(Raud- nitz, Zeitschr. f. Biol., 1901, 42, 91; Reiss, Zeitschr. klin. Med., 1905, 56, 1; Faitelowitz, Dissertation, Heidelberg, 1904). Catalase is also found in virtually all plant-juices. Especially rich in this enzyme are many leaves, e.g., of clover, Rosa, Picea, which mainly contain the insoluble form of the enzyme; and certain seeds e.g., of the apple and peach, in which the enzyme occurs principally in the soluble form. Highly active catalases are obtained from fungi, e.g., Boletus scaber (E u 1 e r , Arkiv for Kemi, 1904, 1, 357), and from the lower fungi, yeasts, and bacteria. The preparation is usually carried out by precipitating the aqueous extracts with alcohol. S e n t e r gives the follow- ing method of obtaining the catalase of the blood: Defibrinated ox-blood is mixed with 10 times its volume of carbonated water and left over night. It is then centrifuged and filtered and the liquid precipitated with an equal volume of alcohol, the alcoholic solution of haemoglobin being poured off and the reddish-brown precipitate repeatedly washed with 50% alcohol. The precipitate is dried in a vacuum, ground to a fine powder, stirred with water and allowed to stand in ice for 2 or 3 days in order that the enzyme may be extracted completely. The solution is filtered through hardened filter-paper until it becomes quite 68 GENERAL CHEMISTRY OF THE ENZYMES clear; the faintly yellow liquid thus obtained vigorously decom- poses hydrogen peroxide with evolution of oxygen. For the preparation of highly purified catalase-products, pig- or ox-fat is the most suitable starting material. The fat is disintegrated by means of sand and extracted with water at about 30, the enzyme being then precipitated with alcohol and further purified by repeatedly dissolving in water and precip- itating with alcohol (Euler, Arkiv for Kemi, 1904, 1, 357; Bach, Chem. Ber., 1905, 38, 1878). In his first experiments ( 1 o c . c i t . ) the author obtained a preparation which still showed faint protein reactions and contained 14-5% N and 1-2% S but no phosphorus. A prepa- ration made from defibrinated blood and similarly purified gave 14-1% N. A continuation (not yet published) of this investi- gation has yielded enzyme-preparations which are certainly of a higher degree of purity. The nitrogen-content diminishes as the purification proceeds, the final product, which exhibits con- siderable activity, containing 6 2% N and exhibiting M i 1 1 o n 's and M o 1 i s c h ' s reactions. Fat-catalase appears therefore to be not a protein, but mention must be made of the opposite results obtained by Bach (loc. cit.). The question of the protein character of catalase hence requires further investigation. Reducin.g Enzymes (Reductases; Reducase) A substance termed " philothion " was described by d e Rey-Pailhade and was regarded by him and also by Pozzi-Escot as a reducing enzyme. With this view, however, the author is unable to agree, since the reactions described by de Rey-Pailhade and Pozzi-Escot are not enzymic in character. The existence of reducing enzymes has, indeed, not yet been demonstrated with absolute certainty. In most cases in which reduction has been observed, no attempt has been made to show that it is really due to an enzyme-action, i. e., a catalytic reaction, and is not merely a stoichiometric reduction by a readily oxidisable substance. A substance which, at 70, accelerates the reduction of meth- ylene blue by formaldehyde, has been found to exist in milk (Schardinger). That this is a catalytic action has been SPECIAL CHEMISTRY OF THE ENZYMES 69 rendered probable by the investigations of S. Oppenheimer (Arb. a. d. Inst. f. exp. Therapie in Frankfurt, 1908, 4) and of Trommsdorff (Centralbl. f. Bakt., 1909, 49, 291). Schardinger's reaction is also hastened by colloidal platinum or iridium ( B r e d i g and S o m m e r , Zeitschr. f. physikal. Chem., 1910, 70, 34). Bach has recently (Biochem. Z., 1911, 31, 443) endeavoured to ascertain if Schardinger's enzyme, for which he proposes the name perhydridase, is related to the reduc- ing enzymes of the liver and other tissues. He is of the opinion that the " reducase " of the liver is a mixture of enzymes and contains that of Schardinger. Further, the same reaction underlies the action of the systems : palladium methylene blue hypophosphate water; palladium methylene blue aldehyde water; milk-enzyme methylene blue aldehyde water, this reaction consisting in decomposition of the water by the oxidis- able substance with the help of a catalyst which forms a labile, strongly reducing compound with the hydrogen of the water. In the researches of Abelous and his collaborators on horse-kidneys, bacterial action was not excluded. Indeed, as Abelous himself stated and the author has confirmed, no reduction takes place if the extract is filtered through a Chamber- land candle (cf . the work of Heffter, Arch. f. exp. Path., Schmiedeberg- Festschrift, 1908, 29). In the roots of plants and in seedlings, strongly reducing substances occur but these are not enzymic metabolic products (O. Schreiner and M. Sullivan). The same holds for reduction by micro-organisms. K a s 1 1 e and E 1 v o v e (Amer. Chem. Journ., 1904, 31, 606) have also given an interesting study on the reducing actions of plant-juices. APPENDIX In the above short account, mention has only been made of those enzymes, of the individuality and mode of action of which something definite is known. A large number of enzymes, which have received special names, have not been considered, as they do not differ essentially from the better-known repre- sentatives of the same groups. But certain other enzymes, which have found no place in the 70 GENERAL CHEMISTRY OF THE ENZYMES main enzyme groups, may be briefly referred to here, since their further study appears to be not without interest. 1. An isomerising enzyme, which converts man- nose into glucose, is thought by Gat in (Soc. BioL, 1908, 65, 903), to exist in the seeds of Borassus flabelli- f o r m i s . 2. According to J. Parnas (Biochem. Z., 1910, 28, 274) the liver contains a soluble enzyme which is able to accelerate Cannizzaro's aldehyde transformation, i.e., it converts aldehydes anaerobically into the corresponding alcohols and acids. Par- nas suggests the name aldehydemutase, whilst Battelli and Stern (Biochem. Z., 1910, 29, 130) apply the term aldehydase to a similar enzyme studied by them. 3. Kotake (H., 1908, 57, 378) refers to an enzyme of ox-liver which demethylates caffeine, giving xan- thine, hypoxanthine, ^-methylxanthine, etc. 4. In the kidneys and liver, Gottlieb and Stangas- singer (H., 1907, 52, 1; 1908, 55, 295, 322) found substances which convert creatine into creatinine and are apparently enzymic in character. CHAPTER II PHYSICAL PROPERTIES OF THE ENZYMES ALTHOUGH the courses followed by most enzymic reactions can be represented by formulae which hold for catalyses in homo- geneous systems, and although also dynamics as yet affords little means of taking the state of solution or the colloidal condition of the enzymes into account, yet, in experiments with enzymic liquids, adsorption phenomena always make themselves more or less strongly felt and have, indeed, a determining influence on the general chemical behaviour of the enzymes. Wherever a liquid is bounded by a vaporous space, there is formed at the surface a layer possessing properties different from those of the body of the liquid this layer is termed the surface-layer. The latter has a tendency to diminish, and it is to this that the well-known capillary phenomena are due; on the interior of the liquid a pressure is exerted, termed the internal pressure. The thickness of this surface-layer, which differs from the remainder of the liquid, is very small. The pressure with which the surface-layer presses on the internal liquid is also very small, unless the relation between the surface of a substance and its volume " the specific sur- face " is very large, i.e., the distribution of the substance is considerable. The latter is especially the case with the so- called colloidal solutions. 1 We shall begin with a short theoretical consideration of surface phenomena, employing the method of representation given by Maxwell. The wire EF (Fig. 1) is to be regarded as capable of moving freely along the rectangular wire A BCD. Within the frame EBCF is a layer of liquid which, in consequence of the surface- tension, tends to diminish and so draw the side EF upwards. 71 72 GENERAL CHEMISTRY OF THE ENZYMES The weight is so chosen that it exactly compensates the pull of the liquid layer on the wire; increase of the load G would then result in rupture of the layer and decrease, in rise of EF. The pull of the layer is caused by its surface-tension, and, since both surfaces of the layer are active, cor- responds with double the surface- tension. If now the movable wire is displaced, by means of the weight G, from its F IG i highest position BC to the position EF, an amount of work has been done against the surface-tension which is proportional to G (made equal to the surface-tension) and to the magnitude of the sur- face produced. 1 This work evidently represents the surface- energy and is given by : Surface-energy = Surface-tension X surface. The surface-tension may also be expressed by the energy acting on unit-surface or by the force acting on unit-length, and is usually given in dynes per cm. Owing to the small absolute values possessed by the known surface-tensions of liquids and aqueous solutions about 100 dvnes the surface-energy of a liquid attains a considerable cm. magnitude only when the surface is large, and hence becomes comparable with the other forms of energy of a substance only when the ratio of surface to volume the " specific surface " (Wo. Ostwald ) exceeds a certain value, namely, about 10,000. The physiologically most important and most interesting phenomena in this region are met with not in pure liquids, but partly in solutions and partly in " heterogeneous systems," in which a substance occurs in a very fine state of division and which Wilh. Ostwald has named disperse systems. Solutions. It can be stated generally that the con- 1 The surface-tension of liquids is independent of the expanse of the surface. PHYSICAL PROPERTIES OF THE ENZYMES 73 centration at the surface of a solution is different from that prevailing inside the solution. This can be demonstrated experi- mentally and also follows from thermodynamical considerations. The derivation given below follows that given by M i 1 1 n e r [Phil. Mag., 1907, (6), 13, 96] and was also deduced by H . Freundlich in his" Kapillarchemie." Suppose n molecules to be dissolved in a certain volume v. The concentration has then not the uniform value --, but is greater (or smaller) in the surface-layer (or interior). If the excess per unit of surface w is indicated by a, the excess of concentration in the whole surface will be aw and the concentration in the interior of the liquid, c = - . v Consideration of a thermodynamic cycle leads to the differential equation: dr dp where T is the surface-tension and p the osmotic pressure. This expresses the fact that the surface-tension changes with the volume and, therefore, also with the concentration, if the osmotic pressure changes with the magnitude of the surface, and this can only happen if the concentration of the solution depends on the magnitude of the surface and is therefore different from the concentration in the latter. If equation (I) is developed as a function of c, it gives rise to dv dc dv v dc (II) dt dr dc c dv dc and -r~ = T'7~- 'T" (HI) aw dc aw v dc The excess of concentration per unit of surface is hence given by C?T 4 and K2HPO4, G e r b e r explains as due to the precipitation of lime by these salts, lime being less necessary with the vegetable than with the animal enzymes. By small quantities of acid sulphates, such as KHSO4, both animal and vegetable rennets are accelerated. Concerning the action of activators in general, it may be said that: As far as the " kinase " of tryptase is concerned, this can be regarded, on the one hand, as a catalyst of the reaction tryp- sinogen trypsin and, on the other, as functioning like the activat- ing acids. It is highly desirable that a more extended series of exper- iments should be made to decide if the conversion of pro-enzyme into enzyme is a reversible process. There still remains the possibility of a chemical reaction occurring between zymogen and kinase and in order to obtain information on this question, the manner in which the activation varies with the time would have to be studied more closely. That the process is a relatively rapid one has been shown by P a w 1 o w and his collaborators. In one way or another, the zymogen is often activated " spontaneously/' In most cases the substrate or some foreign substance succeeding it yields the activator as the result of a slow reaction of some kind. In an interesting investigation H o y e r (loc. cit.) observed the appearance of lactic acid as such an activating substance. If the activator is a normal product of the substrate under the given conditions, the well-known case of auto-catalysis presents itself. A typical example of such a reaction is the spontaneous decomposition of ethyl acetate in aqueous solution; in this instance the liberated acetic acid is the catalyst which accelerates the subsequent hydrolysis in proportion to the concentration of its hydrogen ions. Meanwhile there are no grounds for making an essential distinction between the activation of the zymogen with initiation of the reaction and the action of acids, alkalis and many salts in accelerating the reaction. The action of the latter substances often termed co-enzymes rests undoubtedly on the reversible formation of compounds of these activators, partly with the substrate and partly with the 112 GENERAL CHEMISTRY OF THE ENZYMES enzyme. In most cases it is a salt-formation which takes place (E u 1 e r , Hofm. Beitr., 1905, 7, 1); this occurs instantaneously, so that the " incubation periods " of the accelerating alkalis, e.g., with catalase, or those of the inhibiting acids are without influence of course, only so long as disturbing secondary reactions, decomposing chemically the enzyme or substrate, are avoided. In many cases the minimal concentration determining the optimum of acid- or alkali-action must correspond exactly with the quantity of acid or alkali necessary for the neutralisation of the solution (cf. Cole, Journ. of PhysioL, 1903, 30, 202). The sensitiveness of the enzymes towards acids and alkalis is, indeed, very great but is quite conceivable if the very small concentrations in which the enzymes themselves are present in solution are considered. More or less complete analogies are found with many well-known catalytic processes. Thus, hydro- quinone in a solution containing 0-001 normal-manganese sul- phate undergoes oxidation very slowly; but if sufficient alkali is added to combine with the majority of the sulphuric acid and thus to liberate manganese hydroxide, the oxidation is enormously accelerated. The manganese sulphate corresponds with the enzyme in its inactive state, the alkali with the activator. In hydrolytic changes, e.g., the enzymic inversion of cane-sugar, the chemical reaction presumably consists in the activating acid liberating the enzyme which in neutral solution is present as a salt and so bringing it into the active condition (cf. E u 1 e r and B. af U g g 1 a s , H., 1910, 65, 124). As well as to salt-formation, an important part in the activa- tion of enzymes must be attributed to the formation of com- plex compounds. It is in this way that the specific properties of phosphoric acid and of calcium and manganese are exerted, as described above. With manganese the capacity to form com- plex compounds with hydroxylic bodies has been long known, and with calcium, physico-chemical investigations have rendered necessary the assumption that it also yields such complexes. The reactivity of phosphoric acid with polyhydric alcohols has often been studied qualitatively, and quantitative experiments would be of biological interest. In the description of pepsin action on p. 95, it was mentioned that hydrochloric acid not only acts on the enzyme but also accelerates the digestion by forming salts with the proteins. ACTIVATORS, PARALYSORS AND POISONS 113 Similarly, it is doubtless the alkali salts of peptones and peptides which constitute the active molecules of tryptic diges- tion. This complete alteration of the reactivity of a substance by salt-formation has many chemical analogies: nitrous acid diazotises, whilst nitrites do not ; in alkaline solution polyphenols are oxidised by the oxygen of the air with great readiness, but in acid solution only with difficulty. The velocity of decomposi- tion of hydrogen peroxide is influenced by the acids or alkalis present in a similar manner, no matter whether the reaction is brought about by " catalases " or by inorganic oxides (colloidal platinum, ferric hydroxide). A similar argument can be applied also in other cases, e.g., that of invertase-action, where it may be assumed that the compound (cane sugar-mineral acid), which represents the active molecule in the process of inversion, is resolved catalytically by the enzyme into glucose, fructose and free acid. Being amphoteric electrolytes and hence capable of forming salts with either 'acids or alkalis, the proteins, peptones and amino-acids often exert indirectly an accelerating, though seldom a powerful, action; as the author has already emphasised (Ergeb. der Physiol.; 1907, 6), they regulate' the concentration of the free acid or base. 1 Finally, those cases must be considered where the activator represents the common solvent, that is, the con- necting link between enzyme and substrate, where the two alone cannot form a homogeneous system. The activation of the Upases by the bile acids is possibly to be explained in this way. The purely chemical (not enzymic) splitting of fats by naphthalene-stearosulphonic acid has thus been inter- preted by T w i t c h e 1 . The causes underlying the actions of neutral salts are also very varied. These actions are partly due to simple chemical transposi- tions between the electrolytes present and the consequent altera- tion of the acidity or alkalinity. So that sodium sulphate, salicylate and phosphate hinder peptic digestion (P a w 1 o w, Danilewski), the strong hydrochloric acid being replaced 1 In many instances the addition of proteins to an enzyme solution miti- gates the harmful action of tryptic ferments on the enzyme. Beneficial effects of another type are also observed, e.g., with diastase, ptyalin, etc. 114 GENEKAL CHEMISTRY OF THE ENZYMES by the weaker, less active acid of the salt. With higher con- centrations of the salts, the influence of dissociation comes into play, sodium chloride diminishing the electrolytic dissociation of the protein hydrochloride. Further those influences come into action which produce the " neutral salt action " in non- enzymic hydrolyses and which vary widely in different reactions. The actions of small quantities of neutral salts, such as NaCl, KC1, etc., would indeed seldom be observable if the salt were added to enzyme solutions previously free from electrolytes. Many investigations indicate that the small quantities, of salts occurring in the organs with the enzymes are essential for the activity of the latter; if these small amounts of salts are removed by dialysis, the enzyme action, for example, of amylase, ceases. Very large quantities of salt have a coagulating action and precipitate enzymes more or less directly from enzyme solutions, which usually contain colloidal substances. Yet even quite considerable concentrations of salts are often without harmful effect; thus the activity of ptyalin solutions is not weakened by large proportions of magnesium and ammonium sulphates 1 (Patten and Stiles, Amer. Journ. of Physiol., 1906, 17, 26). In addition to the reversible influence on the time- course of enzyme reactions, acids, alkalis and salts exert also an influence on the irreversible changes of the enzyme- substance. These two actions are essentially different. The latter of the two has been studied in the case of invertin by Fernbach (Recherches sur la sucrase, Thesis, Paris, 1890). This investigator found that dissolved invertin, exposed to the oxygen of the air, gradually undergoes oxidation and becomes permanently inactive; this oxidation occurs far more rapidly in alkaline than in acid solution. (Cf . also Hudson and Paine, Journ. Amer. Chem. Soc., 1910, 32, 774). 1 It is regarded as unnecessary to mention the many cases in which smaller proportions of salts have no marked effect, ACTIVATORS, PARALYSORS AND POISONS 115 PROTECTIVE AGENTS Lastly, the influence of neutral salts on enzyme reactions makes itself felt in an indirect way, a beneficial effect being pro- duced by the destruction, alteration or removal of substances which may be classed together as inhibiting agents. These causes play a part in the interesting biological phenomena accompanying fertilisation described some years ago by Jacques Loeb. The favourable influence of certain other substances on enzyme solutions is also often due to the counteraction of the inhibiting action of the paralysors. In this way we may regard the acid salts often termed "buffers " and the proteins too as protective agents. The latter may, for instance, unite with part of the alkali in the case of tryptic digestion and thus protect the enzyme, especially at relatively high temperatures, from destruction (Vernon, Journ. ofPhysiol., 1904, 31, 346). Also soda, calcium carbonate, etc., which are able to protect yeast from the poisonous effect of various substances, do so in virtue of their neutralising action (Henneberg, Centralbl. f. Bakt., 1908, II, 20, 225). INHIBITING AGENTS (PARALYSORS) The term " poison," which has recently been largely used for all substances which delay or prevent catalytic reactions, is not justifiable in the light of recent knowledge. The expression poisoning must therefore be reserved for the disturbance of the life functions by paralysors. Concerning the chemistry of the action of paralysors we are almost completely in the dark, but these bodies are of con- siderable interest in enzymology since they are indispensable as sterilising agents in all protracted experiments. The influence which paralysors, such as chloroform etc., may at times exert on enzymic decompositions is shown, for example, by E. F i s c h e r's observations on the hydrolysis of glucosides by yeast-enzymes (Chem. Ber., 1895, 28, 1436). Just as with the action of activators, that of poisons and other inhibiting substances is dependent on the concentration of the enzyme and the purity of the solution. It is found that the 116 GENERAL CHEMISTRY OF THE ENZYMES injurious action of poisons on enzyme solutions increases as the concentration of the enzymes diminishes; this behaviour seems to indicate that an addition of the poison to the enzyme takes place. Inorganic Salts NaF. According to A r t h u s and H u b e r (C. R., 1892, 115, 839) this salt is without influence on the soluble enzymes, but kills bacteria. Its effect on lipase is greater than that of any other antiseptic (Loevenhart, Journ. of Biol. Chem., 1907, 2, 391 ; K a s 1 1 e and Loevenhart, Amer. Chem. Journ., 1900, 24, 491). Chymosin is injured by it, but not stronger solutions of trypsin (Kaufmann, H., 1903, 39, 434). The action of erepsin on dipeptides is partly retarded, partly accelerated by sodium fluoride (Abderhalden, Caemmerer and Pinkussohn, H., 1909,59, 293). According toVandevelde it has no effect on pepsin and trypsin. B u c h n e r states that ammonium fluoride annuls the action of zymase. The influence of fluorides on tjirombin has been thoroughly investigated by B o r d e t and G e n g o u (Ann. Inst. Pasteur, 1904, 18, 98) and that on diastases by Ef front . HgCl2i even in 00005N-solution has a harmful effect on catalase. In 0-001% solution, it is poisonous to amylase and still more so to urease. It injures ptyalin or trypsin in 0-005% solution, but its action on erepsin is much less marked (E u 1 e r , Arkiv for Kemi, 1907, 2, No. 39). Invertin is also weakened by it, but to a relatively small extent (D u c 1 a u x) . Hg(CN)2: exerts a decided inhibiting action, although less than that of HgCl2, on catalase (F a i t e 1 o w i t z). B(OH)s. D u c 1 a u x found that this retards the action of chymosin, but the more recent results of A g u 1 h o n (C. R., 1909, 148, 1340) indicate an accelerating effect. According to the latter author, the enzymes which hydrolyse carbohydrates, glucosides and proteins act, without alteration, in cold saturated boric acid solution; catalase is somewhat retarded. As2Os. As Buchner has shown by an extended series of experiments, arsenic is i n j u r i o u s to cell-free fermentation, but proteins and sugar act as protecting agents against this ACTIVATORS, PARALYSORS AND POISONS 117 poison. Amylase (K j e 1 d a h 1) and pepsin (Asher) are harmfully affected by arsenic salts. EbS: has an injurious influence on catalase but is without action on pepsin, trypsin, diastase and emulsin (Fermi and P e r n o s s i , Zeitschr. f. Hygiene, 1894, 18, 83). Os (ozone). Whilst hydrogen peroxide accelerates many enzyme actions and, so far as is known, has a slow destructive action only on catalase, ozone has a harmful influence, as has been shown by S i g m u n d for most of the more important enzymes, by K a s 1 1 e especially for lipase (Journ. Chem. Soc., Abs., 1906, i, 615) and by Buchner and H o f m a n n for zymase (Biochem. Z., 1907, 4, 215). H202: according to Vandevelde (Hofm. Beitr., 1904, 5, 558), most enzymes are beneficially affected, only catalase being retarded. Of salts which have a more specific injurious action, mention may be made of the following: Alkali sulphates retard peptic' digestion, as was found by Grutzner (Pfleiderer, Pfliig. Arch., 1897, 66, 605) (with trypsin there is either slight retardation or no effect at all) . CaCl2 has an especially marked weakening action on invertin (D u c 1 a u x) . Iron salts are injurious to pepsin (Asher). Potassium permanganate strongly inhibits lipase (K a s 1 1 e and Loevenhart). Nitrates and chlorates are stated to be intense catalase- poisons. For certain other substances which hinder the action of blood-catalase, S e n t e r (Zeitschr. f. physikal. Chem., 1905, 51, 673) gives the following concentrations as necessary to diminish the velocity of reaction to one-half its original value: Paralysor Grm.-mol. per litre I 2 in KI 1/50,000 Hydroxylamine hydrochloride 1/80,000 KNO 3 1/40,000 KC1O 3 1/40,000 For the catalase from frog's muscle, C. G. Santesson (Skand. Arch. f. Physiol., 1909, 23, 99) has recently obtained similar results. 118 GENERAL CHEMISTRY OF THE ENZYMES Inorganic colloids (gold, platinum, silver, arsenic, copper, mercury, bismuth) do not accelerate pepsin, but in high concen- trations rather inhibit it (Pinkussohn, Biochem. Z., 1908, 8, 387). Organic Poisons and Inhibiting Agents Chloroform. The original statement made by M ii n t z , that chloroform injures only the micro-organisms but not the enzymes, has had to be considerably modified in recent times. Chloroform injures maltase (according to L i n t n e r and Krober), amylase, ptyalin, yeast-glucase, pepsin, rennin and urease, but has no, or but slight action on trypsin, erepsin, invertin (Fischer), Aspergillus-maltase (H e r i s s e y) or zymase. For the literature on this subject see K a u f m a n n (H., 1903, 39, 434). Chloral completely destroys oxydase (from L e p i o t a americana) (Kastle and Loevenhart, Chem. Zentralbl., 1906, 77, i, 1554) but injures myrosin only slightly. Formaldehyde. In 40% concentration, this does not destroy Lepiota-oxydase (Kastle and Loevenhart, 1 o c . c i t .) but it has an injurious effect on chymosin and amy- lase. In 1% solution, it is without action on erepsin (E u 1 e r , loc. cit.). Zymase is harmfully influenced by formaldehyde but pepsin only by concentrated solutions. G 1 y c e r o 1 has an appreciable inhibiting influence on rennet-action (R e i c h e 1 and S p i r o , Hofm. Beitr., 1905, 7, 485). Toluene. This hydrocarbon, which E. Fischer introduced for the sterilisation of enzyme solutions, is harmless in the great majority of cases, but urease is weakened by it. Phenol: exerts a deleterious action on pepsin, amylase and catalase. Cresols: harmless for liver-butyrase (Kastle, Chem. Zentralbl., 1906, 77, i, 1555). Thymol: injurious to oxydases (Kastle and Loeven- hart) and saliva-diastase (Schlesinger, Pugliese) and markedly so to zymase (B u c h n e r) and chymosin (Freudenreich). It does not weaken the action of the more ACTIVATORS, PARALYbORS AND POISONS 119 concentrated trypsin solutions (Kaufmann, H., 1903, 39, 434) or that of yeast-maltase (E . Fischer). Maltose retards peptic digestion (Sailer and F a r r) . Salicylic acid: stated to have a weakening effect on pepsin and trypsin and also on lipase. Autolytic action and that of xanthine-oxydase (B u r i a n , H., 1905, 43, 494) are, however, accelerated. Hydrocyanic acid. Whilst this acid has proved itself an extremely powerful poison towards catalase, its deleterious action on other enzymes is considerably weaker and sometimes very faint. Zymase is, indeed, completely inactivated, but this action is reversible (B u c h n e r) . Also, according to F u 1 d and S p i r o , chymosin is not injured and the same is the case with pepsin. The decomposition of polypeptides by erepsin is accelerated by small, but inhibited by larger, quantities of potassium cyanide (Abderhalden, Caemmerer and Pinkussohn, H., 1909, 59, 293). In 1% solution, hydro- cyanic acid weakens but does not destroy the proteolytic enzyme of yeast-juice (G e r e t and H a h n , Chem. Ber., 1898, 31, 202). The sensitiveness of catalase towards hydrocyanic acid is shown by Senter's results which, with those obtained with certain other organic poisons are given below. 1 The concen- trations required to diminish the velocity of reaction to one-half are: Paralysor. Grm.-mol. per litre. HCN 1/1000000 Phenylhydrazine 1/20000 Aniline 1/400 A 1 k a 1 oi d s. The older literature on this subject is given by N a s s e (Pflug. Arch., 1875, 11, 159) and in the work of 1 Faitelowit2 has arranged various paralysors, according to the concentrations in which they affect milk-catalase, in the following order: i n HCN H 2 C 2 O 4 KCN HNO 3 KCNS Ba(NO 3 ) 2 HgCl 2 HC1 H 2 S CHsCOOH Hg(CN) 2 120 GENERAL CHEMISTRY OF THE ENZYMES Chittenden, to whom a very thorough investigation is due. In general, alkaloids retard enzyme action, but not very strongly (Chittenden; Gockel); cf. also L a q u e u r (Arch. f. exp. Path., 1906, 55, 240) who studies especially the effect of quinine on enzymes. According to the latter author, the autolytic enzyme and blood-oxydase are the most strongly inhibited by quinine. A s h e r observed a deleterious action of quinine preparations on peptic digestion. Oxydases appear to be especially sensitive to alkaloids (R o s e n f e 1 d) . Hor- denine sulphate retards peptic and tryptic digestion (Camus), but not the action of maltase, invertase or lipase. Of practical importance for the technique of enzymology are the investigations dealing with the action of alcohol on enzymes : Small quantities of alcohol have an accelerating influence on lipase (G i z e 1 1 , Zentralbl. f. Physiol., 1905, 19, 769, 851) but in other cases a more or less complete inhibiting action, e.g., on trypsin (Gizelt, loc. cit.), rennet (R e i c h e 1 and S p i r o , Hofm. Beitr., 1905, 7, 485) and diastase. Only tyro- sinase is able to act in 50% methyl-ethyl alcohol. Inhibition by alcohol is almost always reversible. Even very large quantities of alcohol are withstood for a time by all enzymes, as the ordinary precipitation methods show. If the alcohol is removed after precipitation of the enzyme, the latter resumes its activity (Schondorff and V i c t o r o w , Pflug. Arch., 1907, 116, 495). Like the activators, inhibiting agents exert their action by combining partly with the substrate, partly with the enzyme and partly with the activator. Especially often where the paralysors are acids or bases must these actions play a part. Also, salts of the heavy metals alter the state of solution of colloidal substrates such as proteins, starch, etc. The behaviour exhibited by antiseptic and narcotic media, such as toluene, thymol and chloroform, towards living c e 1 1 s is determined principally by the behaviour and the altera- tions of the lipoidal plasma-skin. The plasma undergoes change only after the penetration of the plasma-skin by the narcotic. The enzymic actions of living cells are influenced in various ways. By toluene or chloroform, fermentation, for example, is interrupted, although zymase itself as is shown by experi- ACTIVATORS, PARALYSORS AND POISONS 121 ments with press yeast-juice is not injured by chloroform. On the other hand, yeast-cells invert cane-sugar equally quickly in absence or presence of chloroform or toluene. H. Euler and Beth af Ugglas (H., 1911, 70, 279) have arrived at the result, that the activity of those enzymes which are combined with the plasma in the living cell, e.g., zymase, is annulled by narcotics, whilst the enzymes occurring free in the cells, as, for example, invertase, are not influenced by these substances. Euler and K u 1 1 b e r g have investigated the connection between the sensitiveness to poisons, extractability, and relative quantities of the enzymes of a beer-yeast called " H " from a Stockholm brewery (H., 1911, 73, 85), the results being given in the following table : Zymase. Monilia- invertase. Maltase. Beer-yeast invertase. Living yeast Relative velocity of reaction in8% sugar solution Extractability . . . Action of poisons: Chloroform Thymol 1 inhibited completely ditto 1 (to 2) inhibited completely ditto 1 (to 2) inhibited completely considerable 170 slight scarcely any weakening no weaken- Toluene Weakening on drying ditto 20 : 1 ditto 25 : 1 weakening considerable weakening ing ditto 2 : 1 Dried yeast Extraetability . . . Action of poisons : Chloroform. . . Toluene very slight weakened . weakened very slight weakened very in- complete weakened about 20% not weak- ened not weak- ened Concerning the chemical compounds presumably formed in the poisoning of the free enzymes we have no data, since we do not even know the nature of the reacting enzymic substance. These chemical combinations often appear to be only loose ones; the ability of the enzyme to regain wholly or partially its original activity after removal of the paralysor, is especially marked with " poisoning " by hydrocyanic acid. 122 GENERAL CHEMISTRY OF THE ENZYMES If the paralysor is present in only very small quantities, it may be destroyed by any oxidising agent present or by other enzymes without external action. To this must be attributed the spontaneous re-activation which is known as " recovery." Analogies to known chemical processes are here also not lacking. A comparison has often been drawn between the actions exerted by paralysors on catalase and on B r e d i g ; s so-called " colloidal metals " which are, indeed oxides and other oxidis- ing catalysts. But even S e n t e r ' s thorough experiments, referred to above, are not able to explain to what chemical changes the active oxygen, the action of which is here in question, is subjected by the paralysors. Besides, the " poisonings '" with catalase and colloidal platinum follow by no means parallel courses. The principal result of the interesting contributions of H 6 b e r (Pfliig. Arch., 1900, 82, 631) and of Loevenhart and K a s 1 1 e (Amer. Chem. Journ., 1903, 29, 397) to' this sub- ject may be summed up in the sentence: " That the effect of any particular substance on the catalyser can be explained, in the majority of cases at least, upon purely chemical grounds." In many respects the anti-bodies correspond with the inhibit- ing substances here considered and the limits of the term anti- bodies are determined by characteristics somewhat similar to those which define the limits of the enzymes in the wider field of the catalysts. A number of substances which retard enzymic reactions in the normal organism should also be termed inhibiting substances in contradistinction to the anti- enzymes, which the organism forms as protective substances after the introduction of foreign enzymes. A whole group of inhibition phenomena are to be attributed to the adsorption of enzymes. To such phenomena belongs especially the inhibition of trypsin, rennet and invertase by charcoal, studied by S. G. H e d i n and by A. Eriksson. H e d i n showed that the retardations caused by white of egg and serum-albumin are analogous to those produced by charcoal, that is, they are due to adsorption phenomena. To this group belong therefore all the non-specific inhibiting effects of serum which were formerly ascribed to anti-bodies. H e d i n has collected the literature on this subject in the ninth yearly volume of the Ergebnisse der Physiologic (1910) (cf. H., 1911, 72, 313). ACTIVATORS, PARALYSORS AND POISONS 123 The data are, however, too incomplete to permit of a critical classification of these inhibiting substances, so that a resume of all substances referred to in the literature as anti-enzymes will be given in a later chapter. Reference must finally be made to those inhibiting effects observed when heated enzyme solutions are added to the active enzymes. Such retardation has been observed: With tryptase by Pollak (Hofm. Beitr., 1904, 6, 95) in the digestion of gelatine. With peptase by Schwarz (Hofm. Beitr., 1905, 6, 524), according to whom the inhibitory substance resists the action of heat and exists ready-formed in fresh solutions of the enzyme. With peptase, rennet and taka-diastase by Cramer and Beam (Proceedings of the Physiol. Soc., June 2, 1906, see Journ. of Physiol., 1906, 34, xxxvi; Biochem. J., 1907, 2, 174), who heated the enzyme solutions to 50-60; at 100 the inhibit- ing substance is destroyed. With invertase by A. Eriksson (H., 1911, 72, 330), who assumes the existence of an inhibiting agent, resistant to heat, in invertase solutions. Here belong also the results obtained by P o r t e r (Biochem. Z., 1910, 25, 301) with peptase, tryptase, rennet, lipase, saliva- amylase, amygdalase and taka-diastase. In contact with collo- dion membranes these enzymes, with the exception of taka- diastase, lose their activity, all of them except saliva-amylase then exhibiting a retarding action on the corresponding enzymes. CHAPTER IV CHEMICAL DYNAMICS OF ENZYME REACTIONS THE relations required by chemical dynamics for the simplest cases of catalytic reactions are found to be fulfilled by enzymic processes to very varying extents. In some cases the time-law for unimolecular reactions and the proportionality between veloc- ity of reaction and amount of catalyst are closely followed. But in the majority of instances the experimental data are in agreement with the doctrine of reaction only within a limited region of concentration, and not a few reactions will be described for which no simple theoretical representation has yet been found possible. The arrangement of the various enzymic processes according to the mathematical expressions which they follow is apparently the simplest method to adopt. But this can only be carried out at the expense of brevity and clearness since, as already mentioned, the kinetics of the action of one and the same enzyme may be altered completely by change of the external conditions. Separate treatment will therefore be given for each of the more important enzymes, beginning with the hydrolysing enzymes and passing on to the fermentation enzymes, oxydases and catalases. The opportunity must not be neglected of emphasising the necessity of a critical examination of the numerical data of the quantitative results given below. Even in the most favourable cases, where a chemically individual substrate has been employed, the solutions dealt with not only contained a chemically unknown catalyst in unknown concentration, but were also contaminated with the foreign constituents of the enzyme preparation and, in- deed, with substances which, sometimes even in minimal amounts, might exert a deciding influence on the course of the reaction to be observed. In short, one is in the doubtful position of making 124 CHEMICAL DYNAMICS OF ENZYME REACTIONS 125 quantitative observations on a system insufficiently investigated qualitatively. It might then be asked: Are quantitative results obtained with enzyme solutions of any value at all and is any detailed treatment of them advisable? If the observations made on a preparation are not very comprehensive and if the experimental conditions are varied only inconsiderably, the value of a physico- chemical investigation of an enzyme is, indeed, small. But our knowledge of enzymes has been widely extended by a number of studies of the kinetics of reaction, which, especially when considered as a whole, have elucidated the general relations of enzyme action and have furnished valuable aid in the elabora- tion of enzymological methods. Before the experimental results are examined in detail, it will be well to consider the theoretical principles to be applied in judging these results. THEORETICAL PRINCIPLES OF ENZYMIC DYNAMICS A knowledge of the law of mass action may be assumed. This states that tlie active mass of a substance is proportional to its osmotic pressure and hence (within rather narrower limits) to its concentration. If a single type of molecule A with a con- centration C A is changed by a chemical reaction into new sub- stances, without the concentration of any other kind of mole- cule present being appreciably altered, then for given external conditions of temperature, pressure and medium the quantity of substance dC A changed in every interval of time dt per unit of volume will be given by the equation: (la) where k f is a constant, termed the velocity constant (also reaction constant) of the process. This constant k r retains and on this emphasis must be laid its value, undiminished, no matter how far the reaction has pro- ceeded or what initial concentrations may be chosen. On the other hand, the velocity v, that is, the amount of substance changed per unit of time and in unit volume, is 126 GENERAL CHEMISTRY OF THE ENZYMES dependent on (proportional to) the concentration C A . It assumes the same numerical value as k', if C A = 1, i.e., if the substance transformed amounts to one grm.-mol. per litre and is in some way maintained at this concentration. This simplest equation allows, therefore, of the representa- tion of a chemical process which proceeds (practically) completely in one direction. Integration of (la) gives the constant for so-called u n i m o- lecular reactions: A/=.Zn or fc = .l og -- . . . (16) t ax t & a x' if we indicate decimal logarithms by log and hence make /b = 0-4343 A:'. 1 An example of an enzyme reaction in which molecules of only one kind undergo change, is the decomposition of hydrogen 1 If a reaction proceeds so that equimolecular quantities of t w o sub- stances combine to form the product of the reaction, i.e., according to the scheme, A +->2 = fci [ester] /^[acid] [alcohol] or, if we start from a pure ester solution of the concentration a and indicate the amount changed in time t by x: = v = vi V2 = ki(a x)k2X 2 ..... (21) The ratio between the two velocity constants is, as was shown byvan't Hoff (compare Chapter VI), the constant of chemical equilibrium, so that ki__ [ester] &2 [acid] [alcohol]' When the constant of the " reverse reaction " is not vanish- ingly small, the time course of the total reaction v, as can be easily seen, is changed if the product of the reaction x is previously added to the system, and, in general, the progress of a reversible reaction must be retarded by addition of the products of the reaction. When ki and k% have been experimentally determined, it is easy to calculate how v changes with increasing additions of x. CHEMICAL DYNAMICS OF ENZYME REACTIONS 139 For dilute solutions and inorganic catalysts these relations have been completely worked out. 1 With enzyme reactions another circumstance, which has to be considered, complicates matters to some extent. We have already discussed the assumption, now generally accepted, that the enzymes form compounds with the substrate and with the products of reaction. In no case do we know in what meas- ure an enzyme enters into such combination, but we assume that these compounds exert considerable influence on the time course, so that the velocity of an enzymichydrolysis is altered by addition of the products of the reaction to the system, not only according to Eq. (21), but also by combination (inactiva- tion) of the catalyst. This influence has not only been observed qualitatively, but has, in many cases, been measured. Of the experiments showing the retarding influence of the hydrolytic products, that quoted by W . K ii h n e (Lehrbuch der physiol. Chem., 1866, p. 39) is one of the earliest: If a digestion solution, filtered from excess of undigested fibrin, is placed in a dialyser, most of the peptones diffuse into water, whilst the pepsin remains behind. When the pepsin solution is restored to its original volume by evaporation and to its initial acidity, it dissolves almost exactly as much fibrin as was previously dissolved. It is consequently the peptones which hinder the digestion. We may also recall the investigation, already mentioned, of Tammann (H., 1891, 16, 271) which showed in a con- vincing manner that the products of hydrolysis generally influence the completeness of enzymic reactions; but, as he says, " not only by removal and destruction of the decomposition products can an enzyme reaction be rendered complete, as the same end is attained by repeated additions of enzyme." Of Tammann's numerous experiments, the following may be mentioned : To different solutions, each containing 0-51 grm. of amygdalin, were added equal amounts of emulsin and varying quantities of saturated benzaldehyde solution, all the solutions being then 1 In a modification of this equation devised for reversible enzyme reactions, B. Moore (1906) makes an attempt to take account of the activity of the enzyme. 140 GENERAL CHEMISTRY OF THE ENZYMES made up to 25 c.c. When the final state was reached (at 20 ), the following quantities of amygdalin were decomposed: Volume of aldehyde solution. Percentage of amygdalin decomposed. c.c. 20-3 1 c.c. 18-8 5 c.c. 14-7 10 c.c. 11-31 Precipitation Solution saturated with benzaldehyde. 5-7J of emulsin. Hydrocyanic acid has a still more marked action : 25 c.c. of solution at 30 contained 0-5 grm. emulsin and 0-001 grm.-mol. of amygdalin: Hydrocyanic acid added. Amygdalin decomposed. 0-0000 grm.-mol. 23-7 0-0001 " 18-7 0-0002 " 16-4 ,0-0003 " 12-1 The effect of the third product of the decomposition, glucose, is much less marked, as is shown by the following table, due to Auld : Minutes. Grm. of Glucose added. Amygdalin hydrolysed. 30 0-0 13-5% 30 0-2 13-3 30 0-75 11-8 30 1-0 11-6 In the further investigation of this influence it should be remembered that emulsin consists of several specifically-acting enzymes. That the diastatic hydrolysis of starch gives an end point which is influenced by the sugar formed, is pointed out by M o r i t z and Glendinning (Journ. Chem. Soc., 1892, 61, 689). Henri has made numerous experiments on the effect of added glucose and fructose on the inversion of cane-sugar, the establishment of an effect of this kind being of importance for the development of his formula. As has been often pointed out, it is greatly to be regretted that his experimental numbers are considerably distorted owing to the mutarotation of glucose, so that quantitatively they are almost valueless. It does appear, CHEMICAL DYNAMICS OF ENZYME REACTIONS 141 however, that invert-sugar lessens the velocity of inversion. The following table is compiled from Henri's numbers (1 o c . c i t . , p. 202) : 2 c.c. of enzyme solution +50 c.c. 0-2-normal cane-sugar solution. t Without addition. X +0-3-normal invert-sugar. X a a 99 0-138 0-072 215 0-301 0-160 299 0-407 0-224 459 0-594 0-340 586 0-700 0-417 1202 0-927 0-672 The 2-normal cane-sugar solution which is also 3-normal with respect to invert-sugar was therefore hydrolysed only half as rapidly as that without invert-sugar. This retarding influence of invert-sugar seems to be due exclusively to the fructose, as is shown by the following values of cc given by H e n r i . Time. 0-2 N-cane-sugar. 2 N-cane-sugar. + 0-2 N-glucose. 0-2 N-cane-sugar + 0-2 N-fructose. 0-2 N-cane-sugar. + 0-2 N-invert- sugar. 75 0-142 0-144 , 0-123 0-119 184 0-385 0-362 0-317 0-306 275 0-564 0-532 0-457 0-446 445 0-798 0-746 0-672 0-648 605 0-906 0-878 0-799 0-794 As will be seen, the retardation of a reaction by the hydro- lytic products is decidedly specific. That this is the case was also brought out very clearly in a table given by E. F. A r m - strong (Proc. Roy. Soc., 1904, 73, 516) to show the effect of the hexoses in delaying the hydrolysis of sugars. Still more striking examples of the specificity of such retardations will doubtless be obtained from the investigation of the polypeptides which has recently been taken in hand. 142 GENERAL CHEMISTRY OF THE ENZYMES K ii h n e 's experiment on the influence of the decomposi- tion products on the peptic digestion of fibrin has already been quoted. B a y 1 i s s has made experiments on the retardation of the tryptic digestion of casein by albumoses, peptones and amino-acids (Arch. Sci. Biol. St. Petersburg, 1904, 11, Supple- ment, pp. 261 et seq.), his results showing that the amino-acids glycine and leucine were examined are the most active in this respect. In investigating the hydrolysis of dipeptides (glycylglycine) by erepsin E u 1 e r also made experiments on the influence of the amino-acids (glycine) (H., 1907, 61, 213). In this case it was found that addition of glycine has only a subordinate effect. 0- 10 N-glycylglycine; 0-04 N-NaOH. 0-05 N-glycylglycine; 0-10 N-glycine; 0-04N-NaOH. Minutes. 1000(a-a;). 1000&. Minutes. 1000(o -x). lOOOfc. 955 480 8 852 6-25 10 414 6-40 16 766 6-00 18 372 6-20 25 678 5-95 27 329 6-08 0-20 N-glycylglycine; 0-05N-NaOH. 0-10 N-glycylglycine; 0-2 N-glycine; O-OSN-NaOH. 1860 _ 900 _ 6 1692 6-9 6 829 5-9 12 1545 6-7 12 767 5-8 20 1376 6-55 30 1210 6-2 It is here, of course, essential that the relation between the NaOH and the acid present (dipeptide+amino-acid) is not appreciably changed by the addition of glycine. Special mention must further be made of the work of Abderhalden and Gigon (H., 1907, 53, 251) on the hydrolysis of glycyl-Z-tyrosine. In this case the tyrosine in the solution produces a considerable retardation. Similar effects are produced by the previous addition of active amino-acids, especially of those occurring in nature : d-alanine, Z-serine, Meucine, d-glutaminic acid, Z-phenylalanine, d-tryptophane and Z-diamino- trihydroxydodecanic acid. CHEMICAL DYNAMICS OF ENZYME REACTIONS 143 The order of magnitude of the retardations is shown by the following results : 0-1 grm. glycyl-Z-tyrosine + 1 c.c. pressed yeast-juice. 0-1 grm. glycyW-tyrosine+1 c c. pressed yeast- juice + 0-05 grm. d-glutaminic acid. Rotation corrected Minutes. Rotation. Minutes. Rotation. for that of the glutaminic acid. +0-70 +0-70 +0-55 9 +0-51 7 +0-55 +0-47 23 +0-20 19 +0-54 +0-46 34 +0-00 29 +0-54 +0-46 49 +0-53 +0-45 79 +0-53 +0-45 105 +0-47 4-0-39 On the other hand, glycine, Z-alanine and d-leucine exert no retarding action, whilst with the racemic compounds, such as dZ-alanine, the effects are small. From the specific influences which the hydrolytic products thus show towards the digestion of polypeptides, it must again be concluded that the hydrolysing enzyme enters into direct union with these protein decomposi- tion products. At about the same time C h o d a t (Arch. Sci. phys. nat., 1907, 26, 112) made similar measurements with the anhydrides of Z-tyrosine and glycyl-Z-tyrosine. In extremely high dilutions, the amino-acids have an accelerating action. Abderhalden and G i g o n also point out the difference between digestion in vitro, where the decomposi- tion products of the proteins have a considerable retarding influence, and digestion in the intestinal canal, where these inhibit- ing products are rapidly removed by resorption. In other words: In the organism we have (in certain periods) so-called stationary states, in which the substance, acted on by the enzyme and then removed from the sphere of action of the latter by diffusion or some other method, is continually replaced by an equivalent quantity of fresh starting material. Such cases of stationary chemical processes, for example, with the unimolec- ular reaction A +B+C, can be represented by the following scheme. Entry of A : *> C o x grm.-mols. !|2 per unit of time. Field of reaction: conversion of x II grm.-mols. per unit time. ~ Exit of B and C: x grm.-mols. of each per unit time. 144 GENERAL CHEMISTRY OF THE ENZYMES For the given conditions, we have the simple relation : dx -j- = const. at For such a stationary condition to obtain, it is by no means necessary that a system should be limited by solid walls. H. Goldschmidt (Zeitschr. f. physikal. Chem., 1899, 31, 235) has studied a chemical process in which the above equation is realised. If an excess of an ester, slightly soluble in water, is shaken with a dilute hydrochloric acid solution, in which the ester (in so far as it is dissolved) is hydrolysed with a certain velocity, the concentration of the dissolved ester is kept constant merely by the shaking, since the amounts destroyed by hydrolysis are continually removed by the diffusion of the solution. If the velocity of reaction is not very great, the ester disappearing from the aqueous phase owing to the reaction, can be carried away .completely by diffusion, so that the concentration of the ester in the aqueous solution is maintained at a constant value, namely, that corresponding with saturation. The usual equation for a unimolecular reaction, = (a-x}k becomes dx _ , since a x = a = const. A condition for such a reaction is, therefore, that the phase in which the reaction occurs, is always saturated with reference to the substrate; this condition may be fulfilled by vigorous shaking of the heterogeneous system, or by employing the body to be dissolved in an extremely finely divided state, so that its surface of contact with the solution is very great. Excessively laige surfaces of this kind occur particularly in so-called colloidal solutions. CHEMICAL DYNAMICS OF ENZYME REACTIONS 145 In this connection, it must be again pointed out that the acceleration or retardation suffered by an enzyme reaction is by no means always to be attributed to combination of the enzyme with the substrate. In many, perhaps even in most, cases it is a question of the alteration of the concentration of the activators (co-enzyme, etc.), either by the reaction products and the substrate acting in a reversible manner on these substances or by the latter being changed or destroyed by secondary reactions. The opportunity has already been taken of pointing out that the deviation from the theoretical course of the reaction which is termed S c h ii t z 's rule might, according to the data as yet to hand concerning peptic digestion, very well be caused by the gradual fixing of the hydrochloric acid by the albumoses and peptones formed, the acid being withdrawn from both the sub- strate and the pepsin. It is to be hoped that digestion exper- iments carried out in vitro and with a constant concentration of hydrochloric acid, may decide this not unimportant question. According to theory, all reactions, even if they apparently proceed to completion, are finally arrested at a position of equilib- rium. Chemical reactions often go so far in one direction that, under ordinary conditions, the equilibrium cannot be detected analytically; only in very concentrated solutions is the equilib- rium apparent. The manner in which the equilibrium of a rever- sible reaction A^2B depends on the velocities of the two opposed processes A 2B and A< 2B, and the way in which natural equilib- ria and the final states of enzymic reactions are connected, will be described in Chapter VI. This short resume of the theory of enzymic dynamics may now be brought to an end. It will be seen later how the exper- imental data agree with the requirements of theory. 146 GENERAL CHEMISTRY OF THE ENZYMES H. EXPERIMENTAL DATA ON THE COURSE OF ENZYME REACTIONS ESTERASES AND LIPASES With esterases, that is, with enzymes which hydrolyse lower esters but not neutral fats, K a s 1 1 e and Loevenhart (Amer.Chem. Journ., 1900, 24, 491) and Kastle, Johnston and E 1 v o v e (ibid, 1904, 31, 521) have carried out a number of experiments; they used turbid aqueous extracts of pig's liver and pancreas filtered through cloth. Tubes containing 4 c.c. water, 0-1 c.c. toluene, and 0-26 c.c. ethyl butyrate were heated for 5 minutes at 40. One c.c. of a 10% extract was then added and after a definite time the solu- tion titrated with N/20-KOH solution: Minutes. X k. ID* K.E 5 (6-53) 1354 (0-45) 10 (8-66) 907 (0-40) 15 8-53 597 0-26 20 9-54 500 0-24 25 10-67 500 0-24 30 (9-41) 329 (0-16) 60 17-32 316 0-28 120 25-35 244 0-32 180 28-36 184 0-28 The constant k, calculated from the equation of a reaction of the first order, diminishes regularly and very considerably. Also interpolation shows that the errors of observation, pre- sumably owing to the difficulty of pipetting small volumes of the ester exactly, were very large; the values given in brackets fall quite away from the curve. For the other numbers, the values of K.E calculated from formula (18) a In a x EXPERIMENTAL DATA OF ENZYME REACTIONS 147 show satisfactory agreement. This formula was deduced (pp. 133-135) on the assumption that the concentration of the free enzyme is inversely proportional to that of the products of the reaction. The same formula is obtained on the supposition highly probable in this case at least that the activity and not the concentration of the enzyme is inversely proportional to the reaction products, chief among these being the acid. As was found by K a s 1 1 e and Loevenhart, the lipase of the pancreas is very sensitive to acids. It would doubtless be of value to ascertain if the formula for unimolec- ular reactions does not hold with good approximation if a cer- tain quantity of strong acid were previously added in order to depress the action of the products of the reaction. With a moderate degree of accuracy the_ above figures and those given below agree with the formula x/\/t = k. The measurements made by S t a d e on an emulsion of egg- yolk and neutralised gastric juice (Hofm. Beitr., 1902, 3, 291) have been calculated according to the above formula by Arrhenius and found to agree closely with it, as is seen from the following table : Hours. x (observed). x (calculated). 2 0-204 0-186 K# = 10 4 0-256 0-257 6 0-298 0-308 8 0-353 0-348 10 0-376 0-383 25 0-495 0-552 29 0-515 0-582 31 0-554 0-596 35 0-609 0-620 75 0-775 0-784 Further, Engel (Hofm. Beitr., 1905, 7, 77), in a careful investigation with emulsion of egg-yolk and pancreatin on the lines of Volhard's and S t a d e ' s experiments, arrived at the result that S c h u t z 's rule holds for the enzymic saponifica- tion of fats ; that is, for a constant period of digestion, the amounts of digestion are in the ratio of the square roots of the quantities of enzyme, and for the same quantity of enzyme the products 148 GENERAL CHEMISTRY OF THE ENZYMES of digestion are proportional to the square roots of the times of digestion. Hence, for one and the same juice, the equation must hold. This is found to be the case. Pancreatin. 4 hours. 9 hours. 25 hours. x (obs.) x (calc.) VEi x (obs.) x (calc.) X V~Ei x (obs.) x (calc.) X VTsi 0-04grm. 0-09 " 0-16 " 17-6 20-9 35-2 16-8 24-5 31-6 4-4 3-5 4-4 18-4 36-3 48-4 24-5 35-0 44-6 3-1 4-0 4-0 35-0 58-2 72-1 38-3 53-0 65-0 3-5 15s 3-8L1 3.6(| The values of x (calc.) have been obtained by means of A r r- h e n i u s 's formula, the constant K being taken as 1. Although the experimental errors are considerable, it is clear that the numbers follow S c h ii t z ' s rule. The experimental data obtained by Z e 1 1 n e r (Monatsh. f . Chemie, 1905, 26, 727) with lipase from fly agaric (A m a n i t a m u s c a r i a) have been subjected to calculation by K a n i t z , who found that the results vary, as in two series of experiments T* the quotient y was constant and in another series the expression x vl- E u 1 e r (Hofm. Beitr., 1905, 7, 1) hydrolysed ethylbutyrate in aqueous solution with esterases (from the fatty tissues of the pig) and found the law for unimolecular reactions to hold. Minutes. t Vol. of baryta solution used in the titration (C.C.). x Vol. of baryta solution not used. a x Unimolecular reaction constant. fc.10* 2-70 2 0-3 2-40 256 6 0-75 1-95 235 9 1-05 1-65 237 16 1-65 1-05 250 From his observations on the lipase of Lactarius sanguifluus, Rouge (Centralbl. f. Bakt., 1907, II, EXPERIMENTAL DATA OF ENZYME REACTIONS 149 18, 403, 587) also drew the conclusion that, in dilute solutions, the action of the enzyme is directly proportional to its amount. Experiments with the true lipases all relate to systems with limiting surfaces perceptible macroscopically (suspensions), since most of the lipases are insoluble in water. Glycerine extracts showing lipolytic activity can be obtained from the pancreatic glands of the pig (A. K a n i t z , H., 1905, 46, 482). This author followed the course of hydrolysis of olive and castor oils. Into each of a series of test-tubes were placed 10 c.c. of olive oil, 3-9 c.c. 0-lN-sodium hydroxide, 0-25 c.c.N-calcium chloride, and 1 c.c. of lipase extract, the contents of the tubes being titrated after t minutes. The numbers of c.c. of 0-lN-sodium hydroxide used are given under x. X X t X t vT 0-0 _ 70 9-2 0-131 1-10 140 12-3 0-088 1-04 288 19-0 0-065 1-12 405 23-1 0-057 1-15 1455 34-2 0-023 0-90 The time-law of the vegetable lipases was first investigated by Connstein, Hoyer and Wartenberg (Chem. Ber., 1903, 35, 3988), who established the essential fact that considerable quantities of free acid are necessary for the lipases to exhibit their action. If, as suggested by S i g m u n d (Monatsh. f. Chemie, 1890, 11, 272), powdered castor-oil seeds are ground with water and left for 24 hours at about 40, small quantities of acid are detectable by titration. Later, however, the quan- tity of acid formed rises suddenly. This " jump " occurs after 2-3 days at 35-40 or after 4-6 days at 15-20. For instance, 5 grms. of castor-oil seeds were pounded with 5 grms. of 1% chloral hydrate solution and the mass kept at 16. Immediately. After 2 days. Per cent of ricinoleic acid found 1 3 4 days. 6 days. 8 days. 52 59 59 This is a case of autocatalysis, in which one of the products of the reaction exercises an accelerating action. As to the most 150 GENERAL CHEMISTRY OF THE ENZYMES favourable concentration of acid, information is given by the following results, obtained with sulphuric acid: Normality of the acid 0-02 0-05 0-10 0-12 0-2 0-5 Percentage hydrolysed in 18 hours ... 25 80 86 84 86 13 In addition to the seed-lipases, only pepsin acts in such strongly acid solutions . According to the authors named above, the idea that the acid transforms a pro-enzyme (zymogen) present in the seeds into an active enzyme, is untenable, since when the seeds are treated for a long time with acid and the latter then removed, they exhibit no alteration; in fact they show as little fat-splitting action in neutral solution as before treatment, and they also become active in presence of acid. In their examination of the influence of various organic acids on Ricinus-lipase, H. E. Armstrong and O r m e r o d (Proc. Roy. Soc., 1906, 78, 378) obtained the following numbers: Concentration of the acid. 0-01 N. 0-02 N. 0-10 N. 0-50 N. k. 10*. Acetic acid 5-45 14-9 14-6 13-6 1-8 Succinic acid 2-80 14-6 15-4 12-2 6 Citric acid 7-25 15-3 14-7 1-1 82 Tartaric acid 6-95 15-4 14-2 97 Here also there occurs a rather flat maximum. No relation is evident between the strength of the acid the dissociation constants are given in the last column and the extent to which the reaction is accelerated or the enzyme activated. The nature of the added acid seems, indeed, to have but slight influence, and this suggests the idea that the action consists of a liberation of the true activator, itself presumably a weak acid. In order to ascertain the influence of the quantity of the enzyme on the velocity of hydrolysis, quantities of 0-5 grm. of the Ricinus-seeds were mixed well with 5, 10, 15, 20, 25, and 50 grms. respectively of castor oil and with similar amounts of 2% acetic acid. Assuming that the active mass of the lipase is proportional to the total quantity, and calculating the results obtained after a certain time by means of the formula previously deduced (p. 136) : 1000 EXPERIMENTAL DATA OF ENZYME REACTIONS 151 the following numbers are obtained, these agreeing satisfactorily with the observed values excepting in the case of the largest proportions of enzyme: Action of 0-5 grm. castor-oil seeds on After 1 day, After 2 days, grms. of oil. ' Solution. x (obs.) x (calc.) x (obs.) x (calc.) 50 50 49 49 49 59 25 25 60 65 74 74 20 20 71 69 80 78 15 15 77 75 87 84 10 10 81 83 86 91 5 5 89 94 92 98 K = 186 K = 300 The time-course of the reaction is shown by a number of the experiments carried out by these authors, among them Nos. 26, 28, 38, and 46. The last two were carried out by pounding 5 grms. of castor-oil seeds with 6 5 grms. of castor-oil and either 4 grms. of 1 N-sulphuric acid (No. 46) or 4 grms. of acetic acid (No. 38). The results were as follows: EXPERIMENT 46. EXPERIMENT 38. 0-10 N-sulphuric acid. 0-10 N-acetic acid. 0-40 N-acetic acid. t (mins.) x (obs.) x (calc.) t (hours) * (obs.) x (calc.) t (hours) x (obs.) x (calc.) 15 12 20 1 52 48-6 1 65 63-9 30 20 27 2 65 62-8 2 86 78-8 45 30 32 3 70 71-5 3 84 86-5 00 33 36 4 72 77-6 4 84 91-2 90 41 43 24 80 99-5 24 91 99-9 150 54 53 *E= 180 K.i = 380 210 59 59 330 68 69 1620 81 97 With the longer times, the deviations of the observed numbers from the calculated ones are considerable and on this account Arrhenius supposes equilibrium to be set up in these cases. N i c 1 o u x (Soc. Biol., 1902, 54, 840) has also made a thorough investigation of Ricinus-lipase or, as he terms it, the lipolytic action of the cytoplasm of Ricinus-seeds. N i c 1 o u x like- 152 GENERAL CHEMISTRY OF THE ENZYMES wise found his preparation to be insoluble in water; he emulsified the cytoplasm in the oil mostly cottonseed oil to be hydro- lysed and added dilute acetic acid. The following numbers were obtained at 18: t (minutes) ....... 30 45 60 90 127 150 210 450 Percent, hydrolysed 23-6 33-1 40-4 54-8 67-0 73-2 85-5 94-4 K E = lOOOZn -- x 1-10 1-58 1-89 2-73 3-45 3-89 5-12 4-31 1000 -x 100 100 k= - log - 0-388 0-387 0-375 0-382 0-392 0-381 0-399 0.278 t 100-x It is evident that the observed numbers do not follow S c h u t z 's rule; on the contrary, the course of the hydrolysis agrees moderately well with the formula for unimolecular reactions. A thorough series of experiments with Ricinus-lipase has recently been carried out by Jalander (Biochem. Z., 1911, 36, 435). For short times (up to 60 minutes) be found proportionality with enzyme-concentrations of 1-4 per 1000. For more protracted action this proportionality disappears, S c h ii t z ' s rule, x:VE = const, then holding approximately. Bodenstein and Dietz (Zeitschr. f. Elektrochem., 1906, 12,605; Dietz, H., 1907, 52, 279) have also studied a heterogeneous system. Pancreatic lipase, in the shape of shavings of the tissue of the pancreatic glands of the pig, was emulsified with amyl alcohol containing water and butyric acid or amyl butyrate in solution. One would expect the velocity equation, ~TT = ^1 * ^ acid " C alcohol K>2 ' C ester ' C water at to be fulfilled, all the concentrations relating to the enzyme phase. The concentrations of water and alcohol are approx- imately constant, since only very small quantities of these dis- appear during the process. On the other hand, the substances must be divided between enzyme and liquid according to Nernst's law of distribution, i.e., C en zyme = a. C so i u tion. The proportionality factors, like the constant concentrations of alcohol and water, become included in the constants, so that -- = ki Cacld &2 Cester = &1 (a x) k 2 X. EXPERIMENTAL DATA OF ENZYME REACTIONS 153 If but little water is dissolved in the amyl alcohol, the reverse reaction is very slight and the process goes on according to the simple equation:. dx , as is shown by the following table : a = 10 normal 4% H 2 0. 2% H 2 O. t (hours). Titre of 5 c.c. k t (hours). Titre of 5 c.c. k 0-00 13-55 0-00 13-55 - 2-00 12-75 0-013 2-50 13-15 0-0064 5-53 11-90 0-010 9-57 11-80 0-0062 10-33 10-35 0-012 14-35 10-95 0-0064 15-17 9-15 0-012 24-45 9-45 0-0063 25-20 7-05 0-012 31-88 8-45 0-0063 32-58 5-85 0-012 47-89 6-55 0-0065 48-83 4-25 0-012 55-27 5-90 0-0064 55-92 3-90 0-011 76-67 4-60 0-0060 77-73 3-00 0-010 100-42 3-25 0-0062 308-00 1-50 308-00 1-10 On passing to higher contents of water, a final condition attainable from either side is set up and the velocities of the two opposed reactions become measureable. Such a case is presented by the following tables. Formation of ester. Hydrolysis of ester. t (hours). Titre of 5 c.c. ki t (hours). Titre of 5 c.c. h 0-00 13-40 0-00 0-00 1-98 12-60 0-014 3-63 0-75 0-0075 4-00 11-82 0-014 7-60 1-45 0-0077 6-98 10-80 0-014 16-77 2-35 0-0064 11-55 9-10 0-016 24-05 2-95 0-0070 14-98 8-40 0-016 89-30 4-20 25-10 6-55 0-016 96-95 4-25 Mean 0-015 Mean 0-0072 154 GENERAL CHEMISTRY OF THE ENZYMES Both constants are approximately doubled if the quantity of enzyme is doubled: Quantity of enzyme. ^ Constants^ 1 0-015 0-0072 2 0-028 0-014 A . E . Taylor, who hydrolysed triacetin, the acetic ester of glycerine, with powdered Ricinus-seeds, found the course of the reaction to be as with unimolecular reactions. He gives the following results for experiments in which 0-5, 1, and 2% solu- tions of triacetin were employed. The constants k refer to 18. t (hours). 4 8 16 24 28 32 40 48 0-5% x (obs.) 0-096 0-162 0-287 0-418 0-489 0-477 0-623 0-652 k 109 96 92 98 104 88 106 96 1-0 x (obs.) 0-083 0-174 0-338 0-418 0-488 0-542 0-609 0-655 k 94 104 112 98 104 106 102 96 2-0 x (obs.) 0-098 0-174 0-323 0-431 0-502 0-485 0-595 0-636 k 112 104 106 102 108 90 98 91 How it happens that in all these experiments the quantity of fat hydrolysed can be calculated by means of the very simple law holding for homogeneous" systems, is not very easy to under- stand. With the hydrolysis of fats and of triacetin this is all the more remarkable, since this hydrolysis takes place in three stages, which doubtless occur with different velocities. New experiments with esterase from pig's liver have been made by G. Peirce (Journ. Amer. Chem. Soc., 1910, 32, 1517). The principal results are as follows: (1) In a solution of given volume and given acidity, the time required for the hydrolysis of a definite quantity of ethyl butyrate is inversely proportional to the concentration of the enzyme. Under similar conditions of acidity, each particle of enzyme hydrolyses the same absolute amount of ester per instant, no matter what the concentration of the enzyme. (2) With a given concentration of enzyme, the time taken to hydrolyse a given amount of ethyl butyrate is dependent on the acid-concentration but not on the ester-concentration, pro- EXPEKIMENTAL DATA OF ENZYME REACTIONS 155 vided this is above N/200. In other words, for each concentra- tion of acid a given amount of enzyme hydrolyses very nearly the same amount of ethyl butyrate over a wide range of ester-con- centration. (3) This phenomenon can be brought into conformity with the law of mass action by assuming that the enzyme and the ester form an intermediate compound, which, in concentrations of the ester above N/200 contains most of the enzyme. AMYLASE The chemical process of the hydrolysis of starch presumably takes place in several phases, in which the dextrins formed as intermediate products are broken down; amylase may con- sequently be composed of several enzymes. In experiments with diastase (amylase), the difficulties attending the varying constitution of the enzyme are supplemented by the complication introduced by a non-individual substrate. As has been shown, more especially by Maquenne's inves- tigations, starch consists in reality of two components, namely, 80-85% of amylose and 15-20% of amylopectin. A m y 1 o s e forms no paste and in solution is coloured an intense blue by iodine; only in solution is it attacked by malt-diastase. There exist a series of condensation products increasingly soluble in water, amylose being the last of these; the lower members more especially are largely contained in " soluble starch." In the saccharification of pure amylose maltose alone is formed. Amylo- pectin is a gelatinous substance which is insoluble in water and alkalis and gives dextrin as well as maltose (?) on hydrolysis. It is not yet certain, but is probable that this is brought about by a special amylopectinase and not by. the amylase. In any case, in quantitative experiments on amylase the purest possible amylose or, at any rate, " soluble starch " should be employed. For the technical determination of diastase, potato starch is treated according to L i n t n e r 's method (see Appendix). As regards the hydrolysis curves, the experiments made by different authors do not agree especially well. The earliest of these experiments, carried out by H . Brown and G 1 e n d i n - n i n g (Journ. Chem. Soc., 1902, 81, 388) with malt-extract do 156 GENERAL CHEMISTRY OF THE ENZYMES not correspond with the simple logarithmic curve. are expressed better by the formula 1 , a+x The results as is shown by the following numbers. 3% solution of starch with 0-25 c.c. malt-extract per 100 c.c.: Minutes. X fc.10 5 fctf.10 5 Temperature, 51-52. 10 0-1084 498 '472 20 0-2250 553 497 40 0-4350 620 506 60 0-6150 690 518 80 0-7385 728 514 100 0-8150 732 495 120 0-8800 762 497 140 0-9220 791 497 160 0-9500 813 492 3% solution of starch with 1 c.c. malt-extract per 100 c.c. 10 0-081 366 352 Temperature, 21 20 0-163 386 357 40 0-308 399 345 60 0-440 419 341 70 0-506 437 345 Henri's experiments (Lois generates etc.), on the other hand, follow the logarithmic law; whether the method employed (determination of the change of reducing power) is free from objection depends on the purity of the amylase used. Also with saliva-diastase, A . E . Taylor obtained the following values: Substrate 0-25% t (minutes.) 30 45 60 75 90 120 150 180 fc(X10 6 ) 490 465 455 470 465 455 460 455 Substrate 0-5% k (X10 6 ) 430 420 390 415 405 395 430 410 Substrate 0-75% k (X10 6 ) 390 370 385 390 380 370 365 370 As Taylor emphasised, the values of k vary considerably with the concentration of the substrate. In the course of a valuable investigation on pancreas-amylase, Kendall and Sherman (Journ. Amer. Chem. Soc., 1910, EXPERIMENTAL DATA OF ENZYME REACTIONS 157 32, 1087) found: (1) that the initial speed of conversion for a constant amount of enzyme was the same for different concentra- tions of starch; (2) that the speed of the reaction diminished the more rapidly, the smaller the initial concentration of the starch. For the formula deduced by these investigators to express the course of the reaction, their original paper must be consulted. As regards the influence of the concentration of the amylase, Henri found, with a vegetable preparation, proportionality between the concentration of the enzyme and the quantity of starch hydrolysed per unit of time. Taylor arrived at the same result with saliva-diastase. Brown and Glendinning (Journ. Chem. Soc., 1902, 81, 381), however, found the velocity of reaction to be propor- tional to the square-root of the concentration of the saliva-diastase, and Klempin (Biochem. Z., 1908, 10, 206), from experiments with oat-diastase, concluded that S c h ii t z 's rule, E^/i= K, holds for this enzyme. P a w 1 o w 's experiments (Arbeit der Verdauungsdrlisen, Wiesbaden, 1898), which were carried out with M e 1 1 's capillary tubes and are of interest in themselves, hardly give any informa- tion concerning the course of the saccharification of starch, since they dealt with the liquefaction of starch-paste and since also the velocity of diffusion exerts a determining influence on the velocity of the process. A large number of experiments were made by Mdlle. P h i 1 o c h e (Journ. de Chim. physique, 1908, 6, 213, 355) with " Merck " diastase and taka-diastase, starch and glycogen being used as substrates. The constants of the formula for unimolecular reactions di- minish rapidly as the reaction proceeds. The following numbers were obtained with 1% starch solution, the concentration of the diastase being 1 : 20,000. Minutes. X 1 a a t ax' 21 0-06 123 51 0-11 100 113 0-15 62 224 0-26 58 390 0-36 50 158 GENERAL CHEMISTRY OF THE ENZYMES The influence of the concentration of the starch on the amount of maltose formed in 60 minutes is shown by the following table: Concentration of starch (%). Quantity of maltose (60 mins.). 1 0-24 1-5 0-30 2 0-338 2-5 0-397 3 0-397 Activators and Inhibitors. The following substances favour the hydrolysis of starch: vanadium and aluminium salts, ammo- nium and calcium phosphates, asparagine, ammo-acids, proteins and picric acid (Ef front, Enzymes and their Applications, p. 117; Soc: Biol., 1904, 57, 234; Allg. Brauer- und Hopfenzeitung, 1905, 45). The accelerations are sometimes very considerable; thus an addition of 0-05 grm. of asparagine to 100 c.c. of a starch solution containing amylase increases the velocity sevenfold. Carbon dioxide also has an accelerating action, especially when under increased pressure (D e t m e r , Mtiller-Thurgau). The optimum activity of vegetable diastases occurs with a slight excess of hydrogen-ions (small quantities of organic acids); hydroxyl- ions, even in very small concentration, retard the hydrolysis, but the inactivation is annulled immediately the alkali is neutralised. The optimum temperature (measured by K j e 1 d a h 1 by the reducing power of the hydrolytic products) is 63. INVERTASE On the hydrolysis of cane-sugar by invertase numerous quantitative investigations have been carried out. Of these, besides the earlier work of K j e 1 d a h 1 (Medd. fra. Carlsberg Lab., 1881), mention must first be made of the researches of T a m m a n n (Zeitschr. f. physikal. Chem., 1889, 3, 25) and of those executed almost simultaneously by O ' S u 1 1 i v a n and T o m p s o n (Journ. Chem. Soc., 1890, 57, 834). These investiga- tors showed first that the reaction is a catalytic one and O ' S u 1 - 1 i v a n and T o m p s o n found it to be unimolecular. This result was contested later by D u c 1 a u x (Traite de Micro- biologie, Vol. 2, 129), while Henri (Zeitschr. f. physikal. Chem., 1902, 39, 194) also arrived at a formula differing from the unimolecular one, namely, the expression (compare p. 129), 1 a+x EXPERIMENTAL DATA OF ENZYME REACTIONS 159 which corresponded moderately well with his experimental data. Still later Bodenstein deduced the complicated formula mentioned on p. 129, and finally Henri (Theses, p. 92) arrived at the following equation: a 1 . 1 i a J +- f log -_-, which he obtained by integration of the differential Eq. (12) of p. 131. As has been already stated, it has recently been found that the experimental data of Henri and his collaborators are considerably influenced by the mutarotation of glucose and therefore give no definite information as to the time-course of the hydrolysis of cane-sugar; further, in these experiments the concentration of the hydrogen-ions was not defined. Hud- son (Journ. Amer. Chem. Soc., 1908, 30, 1160, 1564) has per- formed a valuable service not only in demonstrating the reliabil- ity of O ' S u 1 1 i v a n and Tompson's data, but also in continuing and considerably extending the investigations of these workers. O ' S u 1 1 i v a n and T o m p s o n had rightly recognised that glucose formed from cane-sugar by inversion appears firstly in a mutarotating condition and that therefore the optical activity of a solution which has been inverted by enzyme affords no measure of the progress of the reaction. To the test-portions removed after definite times from their solutions they hence added a little alkali, which annuls the mutarotation almost instantaneously. Their results show that experiments carried out in this way correspond with the unimolecular formula, but that, if the pre- caution mentioned is not taken, the constant increases con- siderably. Their numbers are as follows : Rotation. 1 . a k =ln . x ax Minutes. Without alkali. With alkali. Without alkali. With alkali. 74-5 69-4 37 57-9 37-6 0-0021 0-0046 152 0-6 -20-4 0-0037 0-0074 268 -19-1 -24-8 0-0041 0-0058 00 -27-8 -27-8 160 GENERAL CHEMISTRY OF THE ENZYMES The constancy of the figures in the final column leaves some- thing to be desired and Hudson's numbers may well be given here: Rotation. 10*-^. log -^-. t a-x Minutes. Without alkali. With alkali. ^Without alkali. With alkali. 24-50 24-50 30 16-85 14-27 396 558 60 10-95 7-90 399 530 90 4-75 3-00 464 539 110 1-95 0-80 482 534 130 -0-55 -1-49 511 559 150 -2-20 -2-40 522 533 00 -7-47 -7-47 As is seen from this and other tables given by Hudson, the values of k are constant. According to Sorensen (Biochem. Z., 1909, 21, 131) and Michaelis and Davidsohn (Biochem. Z., 1911, 35, 386), however, this constancy of the reaction-coefficient is observed only with a certain concentration of the hydrogen-ions. With different H'-concentrations, Sorensen obtained the following results: Temperature 52 H'=0-2.10- 6 H' = 0-1.10- 3 H-=0-2.10~ 3 t k t k / k 2 2 2 17 91 17 127 17 53-6 32 103 32 127 32 39-3 47 111 47 132 47 26-1 62 127 62 135 62 18-2 92 147 92 149 92 15-3 122 230 122 126 122 11-2 While, therefore, with H'-concentration of 0-2.10 6 , the coefficient k increases, with H* =0- 1.10~ 3 it remains constant and with H'=0-2.10~ 3 it diminishes. Michaelis and Davidsohn explain this behaviour on the assumption that EXPERIMENTAL DATA OF ENZYME REACTIONS 161 the constancy of the values of k is brought about by the com- pensating effect of the destruction of the enzyme on the increasing values of the coefficient. Considering the high temperature, 52, chosen for Sorensen's experiments, this view is most prob- ably accurate, and at lower temperatures, and higher concentra- tions of hydrogen-ions (H' = 10~ 3 ) M i c h a e 1 i s and David- s o h n found increasing values for k. That other experimenters have been unable to obtain agreement with the law of mass action, cannot therefore, be due solely to non- removal of the mutarotation. Thus, H. E. Armstrong and Glover, in comparing the actions of invertase on cane-sugar and on raffinose (Proc. Roy. Soc., 1908, 80, 312), rendered the sugar solutions alkaline before reading them in the polarimeter, but still obtained no greater constancy, as is shown by the figures in the left-hand part of the following table: 34-2 grms. cane-sugar +4 c.c. invertase-extract per 1000 c.c. 59-4 grms. raffinose +4 c.c. invertase-extract per 1000 c.c. Minutes. Percentage hydrolysed. &.10 5 . Percentage hydrolysed. fc.10 5 . 0-0 0-0 5 8-3 753 1-8 157 15 25-9 868 5-9 176 25 39-5 865 13-4 249 40 62-4 1062 20-1 243 60 78-2 1102 29-3 251 95 01-1 1106 41-6 246 140 93-7 859 53-4 237 200 95-1 656 66-6 238 260 96-0 537 77-9 252 oo 100-0 These tables show that invertase hydrolyses cane-sugar into fructose and glucose about four times as rapidly as this enzyme decomposes raffinose into fructose and melibiose. Our knowledge of the other conditions governing the action of invertase is due to the work ofO'Sullivan and T o m p - son and of H u d s o n, confirmation of which has been supplied by Taylor (Journ. of Biol. Chem., 1909, 5, 405). The pro- portionality found by the first-named authors to exist between the velocity of inversion and the concentration of the enzyme is completely confirmed by H u d s o n 's results. 162 GENERAL CHEMISTRY OF THE ENZYMES O ' S u 1 1 i v a n and T o m p s o n give the following table (1 o c . c i t . , p. 848) : Temp. Invertase prepara- N-H 2 SO4. Time readings. Reading in 2 d.m. Minutes taken to reach zero rotation. tion, grms. tube. Beginning. End. A (obs.). B (calc.). 15-5 0-15 0-00187 11 40 4 41 - 2-0 283-0 291 15-5 0-45 0-0031 3 00 4 40 - 1-8 94-8 96-3 15-5 1-50 0-0050 11 56 12 26 + 1-0 30-7 29-1 56-5 0-0345 0-00025 11 00 12 43 + 16-5 157-6 157-1 56-5 0-0722 0-000375 11 22 12 15 + 13-5 74-8 75-1 The third column gives the acidity of the solution, the con- centrations of acid used having been found by preliminary experiments to be the most favourable to the velocity of reaction. In the last column but one, marked A, are given the times elapsing before the rotation falls to and in the last column, B, the times which would be necessary for this to occur on the assumption of proportionality between concentration of enzyme and velocity of reaction. Also with change of the concentration of sugar, the require- ments of theory seem to be satisfied within very wide limits by O' S u 1 1 i v a n and Tompson's results, that is, in equal times one and the same quantity of enzyme hydrolyses equal proportions of the sugar, no matter what the concentration of the latter may be. On the other hand, A . J . Brown (Journ. Chem. Soc., 1902, 81, 373) gives numbers indicating that a given quantity of enzyme inverts the same absolute amount of sugar in a definite time: Grms. of cane-sugar per 100 c.c. Grms. of cane-sugar inverted in 60 minutes. Percentage of cane-sugar inverted in 60 minutes. 4-89 1-230 25-2 9-85 1-355 13-8 19-91 1-355 6-8 29-96 1-235 4-1 40-02 1-076 2-7 But in dilute sugar solutions containing relatively large amounts of enzyme, the action of a given quantity of enzyme is, according to Brown, proportional to the concentration of the sugar. EXPERIMENTAL DATA OF ENZYME REACTIONS 163 Grms. of cane-sugar per 100 c.c. Grms. of cane-sugar inverted in 60 minutes. ; lO'fc^log^-. t ax (2-0) (0-308) (132) 1-0 0-249 219 0-5 0-129 239 0-25 0-060 228 Hudson collects his results in the following table : Influence of the Concentration of Inver- tase on the Velocity of Inversion at 30 Percentage of cane-sugar inverted with the Concentration of the Minutes. Product. following amounts of cane-sugar per litre. invertase, E. t E.t. A c c grms. 90-9^: litre o_o grms. litre litre 2-00 15 30 73-2 45-3 11-2 2-00 30 60 93-0 74-2 22-0 1-50 20 30 73-2 44-8 11-2 1-50 40 60 92-8 74-5 22-7 1-00 30 30 72-9 45-3 11-5 1-00 60 60 93-0 74-7 22-3 0-50 60 30 72-9 45-2 11-4 0-50 120 60 92-7 74-5 22-6 0-25 120 30 73-1 45-2 10-9 0-25 240 60 92-7 74-7 21-9 The results are therefore as follows : 1. Proportionality exists between the amount of sugar inverted per unit of time and concentration of the enzyme. 2. The concentration functions are, in addition, dependent on the relative quantities of substrate and enzyme. So long as the enzyme is not present in large excess the relative amount of hydrolysis diminishes as the amount of substrate is increased. With his erroneous method, Henri observed a relation indicated by the following table. The number of milligrams of sugar inverted after the first minute in a c-normal cane-sugar solution is denoted by n: 0-58 0-01 1-41 0-025 2-40 0-05 2-96 4-65 0-10 0-25 5-04 0-50 4-45 2-82 1-00 1-50 1-15 2-00 Apart from the fact that Hudson' s investigation has led again to a method which is free from objection, the observation 164 GENERAL CHEMISTRY OF THE ENZYMES that cane-sugar gives rise first of all to a-glucose possesses on little interest. (Its bearing on the enzymic synthesis of disac- charides will be considered later.) From the work referred to we may take the following extract: From cane-sugar, not only glucose but also fructose is formed in a labile (characterised by high rotatory power) modification. But the disappearance of the mutarotation measured at 30 in the case of fructose (constant of reaction k = 186) proceeds about 11 times as fast as with glucose (k = 0-0167). Dissolved invertase-preparations have no influence on these velocity con- stants. Hence, in a cane-sugar solution undergoing enzymic hydrolysis, the difference between the apparent and the actual degree of hydrolysis depends almost entirely on the alteration of the rotation of the glucose. If by means of a very active invertase, a cane-sugar solution can be inverted almost instan- taneously, all further alteration in the rotation of the solution must be attributed almost wholly to the mutarotation of the glu- cose and the velocity of this change must be nearly coincident with the fall of rotation occurring with pure glucose. This has now been actually confirmed. Use was made of an invertase solution so active that 72% of the cane-sugar was inverted within half a minute. The final rotation was determined after addition of a little sodium hydroxide solution: Minutes. Rotation without alkali. 10' . a -log t ax Minutes. 103 , a log t ax 33-50 3 11-88 99 4 7-32 99 5 4-77 93 9 - 0-35 72 10 - 1-35 69 13 - 3-57 63 Commencement 16 - 4-90 57 3 32-3 19 - 6-03 54 6 33-4 23 - 7-15 50 10 33-6 29 - 7-92 44 16 28-7 30 - 8-22 45 17 . 30-7 00 -10-22 31 '7 EXPERIMENTAL DATA OF ENZYME REACTIONS 165 In an equally concentrated solution of invertase the velocity constant of the disappearance of the mutarotation of glucose was found to be /b.!0 3 = 29-9, which is in good agreement with the value, 31-7, given above. Hudson's excellent investigations have been treated at length, because the results obtained with invertase are of impor- tance to the consideration of the enzymic decomposition of other disaccharides and of the glucosides, where the mutarotation of the hexoses makes itself felt to a greater or less extent as a source of error. Worthy of notice is the influence of the products of the reac- tion on the course of inversion. As is shown by the experiments ofE.F. Armstrong (Proc. Roy. Soc., B, 1904, 73, 500), Barendrecht (Zeitschr. f. physikal. Chem., 1904, 49, 456) and also Henri (loc. cit.), fructose retards inversion to a much greater extent than glucose does. According to Baren- drecht, galactose also exercises a retarding influence, which is, however, less than that of fructose. The marked acceleration of enzymic inversion by acids has already been mentioned. It was found by O'Sullivan and T o m p s o n that invertase is very sensitive to small amounts of acid, and this observation has been confirmed by Hudson (loc. cit.). According to the work of the latter, invertase shows its optimum activity in about 0006 normal hydrochloric acid. Sorensen, who made a very thorough inves- tigation of the influence of acidity on enzyme actions (Biochem. Z., 1909, 21, 131) found the optimum concentration of the hydrogen-ions for invertase to be W~ 4 ' 4 10 ~ 4 ' 6 . Very weak acids, like carbonic acid, produce corresponding accelerations. The slightest excess of OH-ions brings the reaction. to a standstill. The enzyme is almost entirely uninfluenced by antiseptics such as toluene and chloroform. The temperature-optimum is given as 50-60. The inactivation constant, & t !0 3 , of invertase from a yeast of Frohberg type was found to have the value 4 at 60 and with a H'-concentration of 10 ~ 6 in aqueous solution (a f U g g 1 a s , H., 1910, 65, 124). The velocity of inversion by living yeast has been studied byEuler andS.Kullberg (H., 1911, 71, 24). In this case the system is macro-heterogeneous, so that the velocity of diffusion should exert an influence on the course of the process. 166 GENERAL CHEMISTRY OF THE ENZYMES Here also the inversion corresponds with the formula for unimoleeular reactions. Temperature, 20. Inversion mixture. Time (mins.). Rotation. A-x &.1O>. la 0-25grm. fresh distillery yeast +20 c.c. 20% cane-sugar solu- tion +5 c.c. H2O + 1 c.c. chlo- i reform; the reaction was stopped by 5 c.c. 0-4N-NaOH solution 17 25 34 00 7-15 6-25 5-80 5-25 -2-29 9-44 8-54 8-09 7-54 26 27 29 7-15 9-44 16 Same as above, but without ad- dition of chloroform 17 25 6-20 5-84 8-49 8-13 27 26 36 5-30 7-59 26 00 -2-29 It will be seen that chloroform exerts no influence on inversion by living yeast. Between 20 and 30 , the effect of the tem- perature is the same as with inversion by dissolved enzyme. As is well known, invertase attacks the trisaccharide, raffinose, decomposing the cane-sugar group present. This hydrolysis proceeds more slowly than that of free cane-sugar; it has been investigated by H. E. Armstrong and Glover (Proc. Roy. Soc., 1908, 80, 317). MALTASE As with emulsin, so also with maltase, E.F.Armstrong (Proc. Roy. Soc., 1904, 73, 508) found the reaction-constants of the first order to diminish considerably. Maltose, 5%. Hours. X k. 10* Hours. X k.W* 1 7.3 329 1 4-7 209 2 13-9 325 3 11-7 180 4 24-4 304 5 17-8 170 7-25 31-7 229 23 23-9 52 23 35-2 82 28 25-0 45 47 31-4 35 Maltose, 10% EXPERIMENTAL DATA OF ENZYME REACTIONS 167 In striking contrast to these are the numbers of Henri and Mdlle. P h i 1 o c h e (Soc. Biol., 1904, 57, 171), of H e n r i and of Terroine (Archivio di Fisologia, 1904, 2, 1), who found that the constants k for a unimolecular reaction at first rise, whilst the constants kn for a definite initial concentration remain comparatively constant. Terroine (loc. cit., p. 4). Maltose, 4%. P h i 1 o c h e (loc. cit., p. 6). Maltose, 4%. Minutes. fc.lO*. 2k H .W 5 . Minutes. X a, k. 105. 50 88 167 63 0-176 134 112 86 156 120 0-312 135 175 103 170 181 0-441 139 230 127 202 241 0-588 163 349 119 176 363 0-753 167 470 134 184 480 0-824 157 588 122 166 600 0-859 142 780 113 148 750 0-869 117 903 106 148 930 0-900 107 The constants vary, however, with the dilution of the mal- tase, so that Henri first employed formula (3), p. 129, for the calculation (m = 3, w=l), but as the agreement with the exper- imental data was not satisfactory, he proposed the introduction of new constants into this formula. Further, H e r z o g (Zeitschr. f. allg. Physiol., 1904, 4, 177) obtained different results, which he calculated according to formula (le), p. 129, with variable coefficients. Maltose, 3-67%. =1-12. Maltose, 3-217%. e=2. Minutes. X a k *i(l-e)10. Minutes. X a t &i(l-e)io. 30 0-128 195 27 20 0-066 149 16 60 0-215 175 23 40 0-138 161 19 120 0-328 144 22 80 0-193 116 15 180 0-415 127 22 120 0-248 103 14 240 0-471 115 20 180 0-312 90 15 370 0-567 76 21 240 0-358 80 15 590 0-631 73 20 620 0-687 81 22 168 GENERAL CHEMISTRY OF THE ENZYMES It is very probable that the action of maltase, like invertase- action, depends on the concentration of the H'-ions present. It is most desirable that a new investigation should be made in which this influence is considered; more simple laws of reaction would then probably be found to hold. According to Croft Hill and to Lintner and K r 6 b e r (Chem. Ber., 1895, 28, 1050) the optimum temperature is 40. A s p e r - g i 1 1 u s - maltase is stated to be only slightly sensitive towards chloro- form (H 6 r i s s e y , Soc. Biol, 1896, 48, 915). In working with yeast- maltase Fischer recommends the use of toluene. LACTASE Quantitative measurements have been made by E . F . A r m - strong (Proc. Roy. Soc., 1904, 73, 506). In the various series of experiments, the reaction-constants of the first order diminish considerably, but not regularly. As examples, the following tables may be given- Two GRMS. MILK-SUGAR PER 100 c.c. I. II. 100 c.c. enzyme-extract. 40 c.c. enzyme-extract. Hours. X fc.lO< Hours. X .10* 1 22-1 1085 0-33 3-2 423 2 31-2 812 0-66 6-4 430 3 38-9 713 1 9-6 438 4 45-8 665 1-5 13-2 410 5 51-5 629 2 16-4 389 6 56-6 664 3 20-8 338 10 69-0 509 5 25-2 252 17 84-2 471 23 47-6 122 23 92-4 461 100 89-6 82 The constants are evidently dependent on the concentration ratio, enzyme : substrate. With a relatively large amount of enzyme, the constant diminishes continuously, but if less enzyme is present, equal amounts of sugar are at first hydrolysed in equal intervals of time. The following table shows the influence of the concentration of lactase on the velocity of hydrolysis of a 5% milk-sugar solu- tion; the quantities of sugar hydrolysed are given in percentages: EXPERIMENTAL DATA OF ENZYME REACTIONS 169 C.c. Lactase. 1 -5 hour. 20 hours. 25 hours. 45 hours. 68 hours. 1-0 0-15 2-2 2-6 3-9 4-8 2-5 0-4 5-8 6-8 10-2 12-6 10. 1-6 23-3 38-6 48-5 20. 3-2 45-8 54-5 1 The quantities hydrolysed are approximately proportional to the enzyme-concentrations, so long as these are not too high. As is shown by the next table , very small amounts of enzyme are able to hydrolyse only small amounts of sugar; their activity then ceases, indicating that the products of hydrolysis, glucose and galactose, combine with the enzyme and so withdraw it from the reaction with the substrate. 5% Milk-sugar solution; amounts hydrolysed in percentages. c.c. Lactase. 24 hours. 144 hours. 0-66 2-3 2-3 1-0 3-2 3-5 2-0 6-3 7-4 5-0 15-4 34-0 If the quantity of milk-sugar is varied, it is found that, with large proportions of enzyme, the amount hydrolysed in unit time is proportional to the concentration of the sugar, so that the values of k are equal. PERCENTAGES OF MILK-SUGAR HYDROLYSED Milk-sugar per 100 c.c. Hydrolysed after 3 hours. k. 10*. 1-0 grm. 0-5 " 0-185 0-098 296 298 0-2 " 0-0416 337 . For comparatively large amounts of substrate the percentage of sugar hydrolysed is inversely proportional to its concentration, so that in unit time equal absolute amounts of sugar are decom- posed, no matter what the concentration. 170 GENERAL CHEMISTRY OF THE ENZYMES According to H. E.Armstrong, E. F. Armstrong and H o r t o n , emulsin contains a gluco-lactase. In a series of experiments carried out by Armstrong (Proc. Roy. Soc., 1904, 73, 507) with milk-sugar and emulsin, the values of k fell rapidly. 2 grms. lactose 2 grms. lactose \ per 100 c.c. -2 grm. emulsin /*" 0-4 grm. emulsin ^^ Minutes. X &.10*. X vr Minutes. X k. 10*. X ~VT 0-5 3-2 282 4.5 1-0 4-8 214 4-8 1-0 4-9 218 4-9 2-0 6-4 143 4-5 2-0 7-5 169 5-3 3-0 7-6 114 4-4 4-5 9-0 91 4-2 4-5 9-4 95 4-4 6-0 10-0 91 4-1 6-0 10-6 81 4-3 23-0 19-7 41 4-1 23-0 30-5 69 2-0 29-0 22-0 37 4-1 29-0 35-0 64 2-0 48-0 29-0 31 4-2 48-0 47-8 59 2-2 53-0 30-7 30 4-2 53-0 50-0 57 2-2 144-0 62-2 29 5-2 144-0 84-0 55 7-0 264-0 77-5 24 4-8 No simple relation between the time and amount of reaction is at first evident from these figures, and the tables given in this paper indicate none between the velocity of reaction and the concentration of enzyme. The diminution of the constant may be explained by the retarding influence of the products formed by the reaction. Later investigations (Proc. Roy. Soc., 1908, 80, 326) show good agreement with a unimolecular reaction, if relatively large quantities of enzyme (almond-extract) are employed. 5% Milk-sugar solution Hours. 40 c.c. enzyme-solution. 60 c.c. enzyme-solution. X fc.KM. X &.10*. 2 15-2 358 18-5 444 3 18-5 296 22-7 373 5 21-1 251 36-5 394 7 33-5 253 44-9 380 9 42-2 264 52-5 360 10 44-0 252 53-5 332 24 73-4 240 76-0 258 EXPERIMENTAL DATA OF ENZYME REACTIONS 171 As is seen from the third and fifth columns, there is here also no proportionality between concentration of enzyme and velocity. Only relatively small amounts of enzyme have hydro- lysing actions proportional to their concentrations, as is shown by the following figures: Amount of enzyme 10 20 40 60 c.c. Velocity constant, /c.10 4 107 212 279 385 Like invertase and maltase, lactase exhibits its optimal activity in faintly acid solution: according to B i e r r y and S a 1 a z a r (C. R., 1904, 139, 381), with 0-002-0-004% of HC1, which corresponds with a concentration of hydrogen-ions of about 10~ 3 . Lactic acid is stated by Bokorny (Maly's Jahrb., 1903, 33) to exert a specific accelerating influence. ENZYMES OF EMULSIN 1. (i-G lucosidase The first quantitative investigation of emulsin was made byTammann (H., 1891, 16, 298 et seq.), who examined the action of this enzyme on amygdalin, salicin, arbutin and coniferin. As the following comparison of the calculated and observed values of (ax) shows, at 25 the process appears to be unimolecular : DECOMPOSITION OF SALICIN BY EMULSIN t (hours). a x ft found. calc. 1 87 88 0-061 3 68 67 0-057 5 42 52 0-075 8 35 35 0-058 12 24 21 0-052 26 9 3 0-040 Important progress is marked by the work of Hudson and Paine (Journ. Amer. Chem. Soc., 1909, 31, 1242) on the decomposition of salicin. These authors paid attention to the facts that the hydrolysis of salicin yields ^-glucose and that the reaction is extremely sensitive towards hydrogen and hydroxyl- 172 GENERAL CHEMISTRY OF THE ENZYMES Ions. The following numbers show that, under suitable exper- imental conditions, satisfactory constancy of the velocity constant k of the first order is obtained. Minutes. Specific rotation (alkaline solution). IWk. -62-0 Temperature, 30 10 OT: * O 360 20 -48-7 330 Concentration of 30 -41-6 353 salicin, 35 -39-5 339 5% 85 -15-8 374 145 + 2-9 350 00 +32-2 ~~~ The influence of acids and bases is indicated by the following table: Concentration of NaOH. Activity of the emulsin. 1 Concentration of HC1. Activity of the emulsin. 0-005 0-00027 222 0-0009 138 0-0005 225 0-0005 195 0-0018 242 0-00009 222 0-005 255 0-009 206 The optimal activity is hence shown with 0-005 grm.-mols. of HC1 per litre. A u 1 d ' s experiments (Journ. Chem. Soc., 1908, 93, 1251) on the hydrolysis of salicin by an enzyme (phaseolunatase) present in Phaseolus lunatus also seem to indicate constancy of the values of k (Table III). Hours. X k Hydrolysis of salicin by phaseolunatase at 39-5 0-5 1 15-8 30-9 141 145 2-0 3-0 59-0 6*7 156 147 H e r z o g (K. Akad. v. Wetensch., Amsterdam, Sitzungsber., 1903, and Zeitschr. f. allg. PhysioL, 1904, 4, 163) has likewise made experiments on the decomposition of salicin by emulsin: EXPERIMENTAL DATA OF ENZYME REACTIONS 173 Minutes. X a fc.10 5 . 10< . o - (X k H=~ lOS ^C Temp. 25. 24 0-174 346 15 54 0-354 351 16 Salicin solution, 86 0-450 302 ' 14 0-07 N. 210 0-691 243 13 270 0-775 239 14 s=0-6 371 0-847 . 219 14 2. Amygdalase and Hydroxynitrilase The investigations of Armstrong, on the one hand, and Rosenthaler, on the other, indicate that the name amygdalase should be given to that enzyme which hydrolyses amygdalin into mandelonitrile glucoside and glucose. A g- glucosidase present in " emulsin " then decomposes the mandelo- nitrile glucoside further into glucose and mandelonitrile, and the latter product is finally broken down into benzaldehyde and hydrocyanic acid by the hydroxynitrilase. So that three enzymes take part in the hydrolysis of amygdalin. It can, therefore, hardly be expected that the formation of the final products should correspond with a simple reaction-formula. The first investigation of the system amygdalin-emulsin was made by T a m m a n n ; certain of his experiments on the retardation of the reaction by the products formed have already been referred to in the preceding section (p. 139). Auld (Journ. Chem. Soc., 1908, 93, 1251) has recently made a very thorough investigation of the hydrolysis of amygdalin. He followed the reaction by titrating the liberated hydrocyanic acid with iodine and found increasing values for the unimolec- ular constant: 300 c.c. 2% amygdalin solution+15 c.c. 2% emulsin solution. Minutes. X fc.10 5 . 10 6-1 255 80 45-2 295 Temperature 40 100 54-9 309 150 75-3 347 1360 93-3 (x indicates the quantities of amygdalin decomposed, in percentages.) 174 GENERAL CHEMISTRY OF THE ENZYMES H . E . and E. F. Armstrong and H o r t o n (Proc. Roy. Soc., 1908, 80, 330) give their results in the form of the following curves (Fig. 4) : The curve representing the glucose formed is not coincident with that showing the hydrocyanic acid liberated, which would be understandable if these two substances were set free in two different reactions effected by two different enzymes. These authors have therefore done right in not calculating the reaction- HYDROLYSIS OF AMYGDALIN BY EMU LSI NAT 25 10 12 14 Time in Hours FiG. 4. constants for the complex of reactions comprised in the hydro- lysis of amygdalin. Similar results are given by A u 1 d (loc. cit., p. 1268) for the temperature 41. Quantity of enzyme 2 3 4 6 12 25 50 c.c. Percent, hydrolysis in 10 mins. 3-7 6-05 8-7 12-5 17-2 21-6 24-1 If dilute amygdalin solutions are employed, the constants are, as they should be theoretically, independent of the concen- tration of amygdalin (A u 1 d , loc. cit., p. 1270). Hydrocyanic acid and glucose produce retarding effects. EXPERIMENTAL DATA OF ENZYME REACTIONS 175 Mention must finally be made of Henri and L a 1 o u ' s polarimetric experiments (Soc. Biol., 1903, 55, 868) on the simultaneous decomposition of amygdalin and salicin. t minutes. 2% salicin. 2-5%. amygdalin. 2% salicin +2-5% amygdalin 4% salicin. 1-25% amygdalin. 46 0-67 0-97 1-05 1-08 0-90 130 1-58 2-38 3-63 2-25 1-57 268 2-32 3-15 4-22 3-45 1-56 GO 3-15 3-17 6-32 6-30 1-59 It will be seen that the hydrolysis of the mixture takeb place much more slowly than that of the two constituents separately. This fact also indicates combination of enzyme and substrate. The enzyme is moderately resistant to chloroform and toluene and its optimum temperature is given as 45. PROTEOLYTIC ENZYMES The first accurate experiments on the time-course of the decomposition of protein by pepsin are due toE. Schiitz (H., 1885, 9, 577). They were carried out with solutions of globulin-free egg-albumin (about 1 grm. per 10 c.c.), to which were added 5 c.c. of 5% hydrochloric acid and a pepsin solution of definite strength; the solutions were then diluted to 100 c.c. and kept at 37-5 for 16 hours. The albumin was then removed from the solutions and the amounts of peptone formed determined by means of the optical rotations. In this way S c h ii t z found the velocity of digestion to be proportional to the square-root of the concentration of the pepsin. The results of the first of the three series of experiments are given here : Quantity of Rotation of the peptone in minutes. pepsin. Observed (mean). Calculated. 1 7-3 7-4 2 9-75 10-4 3 12-8 12-7 4 14-8 14-7 5 16-5 16-4 6 18-45 18-9 176 . GENERAL CHEMISTRY OF THE ENZYMES Mention must also be made of the experiments ofBorissow with trypsin and of Samojloff with pepsin, these exper- imenters also arriving at the relation x = K^/Et. (Dissertation, St. Petersburg, 1901; Arch, des Sci. BioL, 1893, 2, 699; see P a w 1 o w , Arbeit der Verdauungsdriisen) ; there was here no intention of obtaining a representation of the chemical dynamics of enzyme action and, owing to the experimental methods employed in these investigations, no conclusions concerning this can be drawn. The same may be said of Walther's researches (Arch, des Sci. BioL, 1899, 7, 15). Very extensive numerical data on the digestion of protein by pepsin were given in 1895 by J. S j 6 q v i s t (Skand. Arch, f. PhysioL, 1895, 5, 317), who followed the course of the digestion by measuring the electrical conductivity. Every 100 c.c. of solution, 0-05N with reference to hydrochloric acid, contained 2-23 grins, of albumin (almost freed from salts by dialysis) and also 2-5, 5, 10 or 20 c.c. of pepsin solution. The con- ductivity of these solutions fell during the experiment from the initial value [i = 188-4 (old units) to a final value of about 83-4. The amount of albumin acted on, x, was taken as proportional to the fall A in the conductivity. The following tables contain the observed values of A at 37, together with the corresponding 2-5 c.c. of pepsin solution per 100 c.c. Hours. Conductivity, M Change of conductivity, A X *4 k - H'S / Vt 188-4 _ 0-5 1 . 2 177-3 11-1 10-5 2-97 7-45 4 171-1 17-3 16-41 3-78 8-21 6 167-4 21-0 19-93 3-81 8-13 8 164-5 (23-9) 22-68 3-77 8-02 9 163-1 25-3 24-00 3-82 7-90 12 159-9 28-5 27-04 3-70 7-70 16 156-4 (32-0) 30-36 3-62 7-59 20 152-9 35-5 33-68 3-70 7-53 32 146-2 42-2 40-04 3-40 7-08 48 139-8 (48-6) 45-06 3-20 6-50 64 135-0 (53-4) 50-78 3-13 6-34 96 127-9 60-5 57-41 2-80 5-87 00 3-49 EXPERIMENTAL DATA OF ENZYME REACTIONS 177 proportional values of x, the calculated values of the constant K A of the A r r h e n i u s formula (18) or (20) and, finally, the values of the constant of S c h ii t z 's formula x = K s -\/t. 5 c.c. of pepsin solution per 100 c.c. Hours. Conductivity, M Change of conductivity, A X KA X KS = -7=- Vt 188.4 _ _ 0-5 1 178-2 10-2 9-68 4-93 9-8 2 172-8 15-6 14-80 6-09 10-5 4 164-7 23-7 22-49 7-49 11-2 6 159-5 28-9 27-42 7-70 11-2 8 155-5 (32-9) 31-22 7-62 11-0 9 153-5 (34-9) 33-11 7-88 11-0 12 149-4 39-0 36-58 7-47 10-6 16 145-2 (43-2) 40-15 8-25 10-0 20 141-0 47-4 44-98 7-39 10-0 32 133-1 (55-3) 52-47 6-85 9-3 48 126-2 (62-2) 59-02 6-30 8-5 64 121-4 (67-0) 63-58 5-85 7-9 96 114-4 74-0 70-21 5-21 7-3 00 6-84 10 c.c. of pepsin solution per 100 c.c. Hours. Conductivity, M Change of conductivity, A X KA X KS= , Vt 188-4 0-5 179-2 9-2 8-73 8-04 12-35 1 174-2 14-2 13-47 10-34 13-47 2 165-9 22-5 21-35 13-40 15-10 4 154-8 33-6 31-88 16-30 15-94 6 148-0 40-4 38-34 16-65 16-01 8 143-2 (45-2) 42-88 16-42 15-31 9 140-8 47-6 45-14 16-51 15-05 12 136-1 52-3 49-62 14-09 14-32 16 130-9 (57-5) 54-56 15-23 13-64 20 125-7 62-7 59-50 15-44 13-30 32 119-4 69-0 65-46 12-90 11-60 48 113-1 75-3 71-45 11-25 10-31 64 109-1 (79-3) 75-25 10-08 9-41 96 101-8 86-6 82-17 9-60 8-39 00 13-30 178 GENERAL CHEMISTRY OF THE ENZYMES 20 c.c. of pepsin solution per 100 c.c. Hours. Conductivity, M Change of conductivity, A X KA X KS = 7= Vt 188-4 '0.5 176-0 12-4 11-77 15-0 16-64 1 167-8 20-6 19-55 22-2 19-55 2 157-9 30-3 28-75 25-9 20-33 4 144-5 43-9 41-66 30-6 20-83 6 137-2 51-2 48-58 29-7 19-83 8 133-0 (55-4) 52-57 25-1 18-54 9 130-1 58-3 55-52 28-0 18-44 12 125-8 62-6 59-40 25-6 17-15 16 121-6 (66-8) 63-34 25-1 15-81 20 117-3 71-7 68-03 22-5 15-21 32 109-8 78-6 74-58 19-5 13-18 48 102-3 86-1 81-70 18-3 11-79 64 97-4 (91-0) 86-34 14-4 10-49 96 91-2 97-2 92-24 18-0 9-41 oo 22-7 S j 6 q v i s t 's observations hence indicate that the relation x = K s \/t holds moderately well during the first half of the reaction. Calculation of the constants for the various concentrations of enzyme E shows that these are approximately in the propor- tions, A/0^2 : VoT : VtTo^ : Vo-025. K S .^ 7-6 10-4 14-4 18-2 VE 0-500 0-707 1-00 1-42 Quotient K S : VE 15-2 14-7 14-4 12-8 So that, for small and equal values of t, S c h ii t z 's rule holds, i.e., the quantity of substance transformed is inversely proportional to the square-root of the concentration of the enzyme. The proportionality between the amount of albumin hydrolysed and the square-root of Et (enzyme-concentration X time) is much more general, as is shown by the following table in which Arrhenius (Immunochemistry, p. 67) has collected to- gether the diminutions of conductivity given by S j 6 q v i s t. Et= 0-05 0-1 0-2 0-4 0-8 1-6 3-2 4-8 6-4 9-6 # = 0-025 11-1 17-3 23-9 32-0 42-2 53-4 0-05 10-2 15-6 23-7 32-9 43-2 55-3 67-0 74-0 0-01 9-2 14-2 22-5 33-6 45-2 57-5 69-0 75-3 79-3 86-6 0-02 12-4 20-6 30-3 43-7 55-4 66-8 73-6 78-6 86-1 Mean. . . . 10-2 Calculated 11 14-9 15-6 22-7 22 32-2 31-1 43-8 44 55-4 67-6 74-3 79-0 EXPERIMENTAL DATA OF ENZYME REACTIONS 179 Further investigation of peptic digestion is due to Julius Schutz (H., 1900, 30, 1), who coagulated the undigested protein remaining after 15 hours and determined the nitrogen in the filtered liquids by K j e 1 d a h 1 ' s method. At a tem- perature of 38, he obtained the following quantities of hydrolysed protein ( bs.) the values calculated from Schiitz's rule being given in the final column. Quantity of pepsin. 10 4 .Zobs. 10 4 .Z ca ic. 1 212 213 4 471 426 9 652 639 16 799 852 25 935 1065 36 1031 1278 In the same year E . Schutz and H u p p e r t (Pfliig. Arch., 1900, 80, 470) gave further data concerning peptic diges- tion. The decomposition products termed secondary albumoses of the protein were determined polarimetrically. " The quan- tities of secondary albumoses formed are proportional to the square-roots of the times." Further, the quantities of digested protein, the sum-totals of the intermediate products and the amounts of secondary albumoses, are in the same ratios as the amounts of protein employed, namely, 1:2:3:4. E . Schutz and H u p p e r t also investigated the influence of hydrochloric acid. Under certain conditions, secondary albumoses are formed in proportion to the quantity of protein, to the square-root of the time, and to the concentrations of pepsin and acid. The conditions for this rule to hold are a moderately rapid reaction and a concentration of acid not exceeding 2%. If we denote the amount of secondary albumoses by S, that of albumin by A, the time by t, the concentration of hydrochloric acid by s, and the quantity of pepsin by P, the velocity with which the secondary albumoses are formed is expressed by It must be stated that objections have been raised by Sjoqvist (loc. cit.) to the methods used in these experi- ments. 180 GENERAL CHEMISTRY OF THE ENZYMES Gross (Berl. klin. Wochens., 1908, 45, 643) expressed the view that S c h (i t z 's rule does not hold for peptic diges- tion, but that the amount of digestion is proportional directly to the quantity of enzyme and inversely to the time of digestion. His experiments were carried out as follows: Increasing amounts of pepsin were added to constant quantities of an acid solution of casein, observation being then made in each case of the time when the whole of the casein was digested, i.e., when no turbidity was produced by addition of saturated sodium acetate solution. The observations were made at intervals of 10-20 seconds, the temperature being 40. The following series of results may be quoted : Casein solution, 50 c.c. Casein solution, 50 c.c. Gastric juice, c.c. Digestion complete in minutes. Griibler pepsin, (0-1%) c.c. Digestion complete in minutes. 1-0 52-7 1-0 64-0 2-0 25-0 2-0 31-7 4-0 12-2 4-0 16-7 8-0 6-25 In order to test Gross's results, Kurt Meyer (Berl. klin. Wochens., 1908, 45, 1485) made a number of experiments by F u 1 d ' s edestin method. 1 The quantity of pepsin in any tube was four times, and that of protein twice, that in the pre- ceding tube. M e y e r collects his results in two tables, of which one is given here: 1% edestin solu- 1% pepsin solution (G r u b 1 e r) in 0-03 HC1. tion in 0-03 HC1. c.c. 0-0025 c.c. 0-01 c.c. 0-04 c.c. 0-16 c.c. 0-1 4. _ 0-2 + 0-4 -f. _ 0-8 -f 1-6 3-2 1 The method was so modified that the amounts of digestion could be obtained in one series of experiments; thus, series with increasing quantities of edestin and similar series with increasing quantities of pepsin were carried out. EXPERIMENTAL DATA OF ENZYME REACTIONS 181 Incipient turbidity is indicated by +. No exact idea can be formed of the magnitude of the exper r imental error is these investigations. K . Meyer himself, however, draws the conclusion that S c h ii t z ' s rule is valid for peptic digestion. 0-05 c.c. gastric juice. Quantity of casein, c.c. Time of digestion in minutes. Quantity of casein, c.c. Time of digestion in minutes. 5 6-3 10 13-3 6 7-5 12 14-3 7 8-7 14 17-0 8 10-0 16 21-2 For the sake of completeness it may be mentioned that S p r i g g s (Journ. of PhysioL, 1902, 35, 465) followed the course of pepsin-action by measurements of the viscosity with s t w a 1 d ' s viscosimeter. Against these experiments the objection may, however, be raised that the relation of viscosity to the degree of protein hydrolysis is not suffi- ciently known, so that no safe conclusions can be drawn from the results of these measurements. W e i s (Medd. fra Carlsberg Lab., 1903, 5, 127) has made a very thorough investigation of the action of vegetable proteases. He found that peptic action proceeds relatively rapidly, whilst the tryptic decomposition of the albumoses is more gradual; the two actions can, to some extent, be separated. From the results obtained, which are extremely difficult to deal with in detail, it is to be concluded that, for the proteolysis of vegetable protein at any rate within a certain region of concentration S c h u t z ' s rule appears to hold. According to the results given on p. 176, etc., the exponent of the enzyme -concentration increases, with increasing dilution of the enzyme, from 0-5 (Schiitz 's rule) to 1. In the separate series of experiments, the amounts of sub- stance transformed are proportional to the square-roots of the times. From the table on p. 183 of the above paper we extract the following numbers, the values of x:\/T being given in addi- tion. 182 GENERAL CHEMISTRY OF THE ENZYMES Amount of change. Amount of change. Hours. Mgrms. N. Ratio^_ Hours. Mgrms. N. Ratio. / t X x : -\t t X x : Vt 1 5-34 5-34 I 5-80 5-80 2 8-42 5-96 2 8-54 6-04 3 9-82 5-67 3 (12-00) 5-50 4 11-92 5-96 4 11-34 5-67 5 12-98 5-81 5 12-94 5-79 6 13-70 5-59 6 13-32 5-44 9 17-22 5-74 9 14-20 4-73 The author has also calculated the results of the experiments in which W e i s varied the concentration a of the substrate (pro- tein). The third column gives the constants k for unimolecular reactions; the fourth, the product ka and the fifth, the constant K S of the formula x^/a = K s . AFTER 5 HOURS l Protein Amount of N trans- concentra- tion. formed as percentage of total N. 7 1 1 k= -log t ax k.a.lQG K s a X 1% 36-2 0-00065 65 36-2 2 25-9 0-00043 86 36-6 3 20-3 0-00033 100 35-2 4 16-0 0-00025 100 32-0 5 13-2 0-00020 102 29-5 AFTER 2 HOURS Protein Amount of N trans- concentra- tion. formed as percentage of total N. , 1 , a k= log t ax fc.a.lO" K s a X 1% 22-0 0-00090 90 22-0 2 17-0 0-00067 134 24-0 3 13-1 0-00051 153 22-7 4 9-1 0-00035 140 18-2 5 7-9 0-00030 150 17-7 1 The numbers in the two tables were obtained in two separate series of experiments md are hence not comparable. EXPERIMENTAL DATA OF ENZYME REACTIONS 183 It will be seen that the amount of protein changed diminishes with increasing values of a. Arrhenius (Immunochemistry, p. 85) explains this as follows: When, for example, 10% of the protein is digested, the absolute amount of the products of the reaction is doubled if the initial concentration a is doubled. But the velocity of reaction is inversely proportional to the absolute quantity of the products and, therefore, also to the initial con- centration. According to what was stated on p. 133, the amount of change, expressed as a percentage of the total amount of pro- tein, is hence approximately inversely proportional to the square- root of a, as is shown by the fifth columns of the two tables given above. The work of W e i s on vegetable proteinases deals also with the trypsins, that is, with those proteolytic enzymes which act in alkaline solution. From the results of experiments carried out virtually by M e 1 1 ' s method, H.M.Vernon (Journ. of Physiol., 1901, 26, 421) has drawn the conclusion that the digestion of fibrin by trypsin follows S c h ii t z ' s rule if the times of digestion are corrected for the destruction of the trypsin in the soda solution. Amount of enzyme- extract E in c.c. Time of digestion t in minutes. Corrected time of digestion in minutes. txVE. 2 11-8 11-14 16-2 1 17-7 16-25 16-3 0-5 26-8 23-62 16-7 0-25 36-1 30-57 15-3 0-125 80-4 57-32 20-3 0-0625 176-0 93-57 23-4 As has long been known qualitatively, the concentration of the acid present in peptic digestions exerts a marked influence on the time-course of the reaction. This influence is expressed quantitatively in Schiitz's formula mentioned on p. 179, according to which with certain definite conditions of the enzyme- and substrate-concentrations the velocity of reaction is pro- portional to the square-root of the concentration of the hydro- chloric acid. 184 GENERAL CHEMISTRY OF THE ENZYMES The optimal concentration of the hydrogen-ions has been the subject of a recent and thorough investigation bySorensen (Biochem. Z., 1909, 21, 288). In this work the concentration of the H-ions was determined electrometrically, the progress of the reaction being measured by the amounts of protein pre- ciptable after different times by stannous chloride or tannic acid. The results are given in the following table, the " exponents of the hydrogen-ions," PH, being given in the first column; by this Sorensen understands the logarithm to base 10 of the recip- rocal of the normality-factor of the solution as regards hydrogen- ions. The concentration of these ions is given also in the ord- inary form (column 2). It will be seen that the acidity-optimum increases with the duration of the peptic action. The influence of the hydrochloric acid must be explained, as already mentioned, by the protein hydrochloride being more readily acted on than the free protein. Further the enzyme itself may be in the form of a salt, this pepsin hydrochloride showing increased activity; this possibility has recently been emphasised by J. L o e b (Biochem. Z., 1909, 19, 534) but we have no definite indications on this question. As the author has pointed out (Ergeb. der Physiol., 1907, 6), the " pepsin-hydro- chloric acid " the existence of which has been so often assumed- can mean nothing but a pepsin salt of hydrochloric acid. The older investigations ofBorissow on tryptic digestion have already been mentioned. L . P o 1 1 a k (Hofm. Beitr., 1904, 6, 95) also employed M e 1 1 ' s method to examine a tryptic enzyme, g 1 u t i n a s e , which he isolated, and which acts on gelatine. He arrived at the result that the action of this enzyme does not correspond exactly with S c h ii t z ' s rule, which, however, it approaches far more closely than does the mixture of enzymes of an ordinary pancreas infusion or Griibler's trypsin. V. Henri and Larguier des Bancels have inves- tigated the action of trypsin on gelatine (C. R., 1902, 136, 1581). By regarding, as S j 6 q v i s t did, the change of conductivity as proportional to the progress of the reaction, they obtained confirmation of the formula a-x EXPERIMENTAL DATA OF ENZYME REACTIONS 185 JS 3 00 "# I > ^2 05 * 00 (N C^> GO (N - t^ Tt< * iO iO l> 00 CO CO (N (N (M C5 (N CO (M O O O rH rH (N ^ 186 GENERAL CHEMISTRY OF THE ENZYMES Since, however, the final value of the process is not observed (or, at any rate, not given) and the observations are only spread over the very short period of about an hour, the real decomposi- tion of protein cannot have proceeded very far and these exper- iments tell little about the course of tryptic digestion. As has been calculated by Arrhenius, the conductivity is approx- imately proportional to the square-root of the time of digestion. / (minutes) 10 20 30 40 50 Conductivity (mean) 27-3 44-0 53-0 58-7 65-7 7-37\A 29-3 41-5 50-0 58-7 68-8 Of wider scope are the experiments of B a y 1 i s s (Arch. Sci. Biol. St. Petersburg, 1904, 11, Supplement), in which also the conductivity method was employed. The substrate used was partly caseinate in faintly ammoniacal solution and partly gelatine. The experimental results are given mostly in the graphic form. It is found that the constants for unimolecular reactions diminish considerably with lapse of time, this being attributed to the retard- ing effect of the products of the reaction. The numbers obtained by B a y 1 i s s are indeed in good agreement with the formula, aln- x = KEt. a x With concentrations of casein up to about 4%, the velocity of digestion is proportional to the concentration of the substrate; in 4-8% solutions, the velocity is independent of the concentration of the substrate, whilst with more than 8%, inverse proportionality sets in. As regards the relation between the velocity and the concentration of the enzyme, approximate proportionality exists during the first quarter of the reaction; but even in the second quarter, the velocity of reaction is considerably less than would correspond with the concentration of the trypsin (loc. cit., p. 26). Using V o 1 h a r d ' s method for estimating pepsin and trypsin which will be referred to in the Appendix W. L 6 h - lein (Hofm. Beitr., 1905, 7, 120) arrived at the result that tryptic digestion does not follow S c h u t z ' s rule. . F a u - b e 1' s results (Hofm. Beitr., 1907, 10, 35) point to the same conclusion. The measurements which have as yet been made do not indicate, with the certainty that might be desired, the con- EXPERIMENTAL DATA OF ENZYME REACTIONS 187 ditions for simple proportionality between velocity of reaction and the concentration of the enzyme. But far more often than in experiments with pepsin have the conditions been such that the relations : Amount of digested protein = const. and Amount of digested protein = const. \/T, have been found to hold only over a very limited range. But, as with pepsin (cf. p. 179), it appears to be quite general that the same quantity a of protein is digested if the amonut of enzyme is made to vary inversely with the time, i.e. x = const. f(E.t) ....... (22) This rule evidently holds only for those cases in which the proportionality between x and Et is direct. This occurs with undisturbed unimolecular catalytic reactions, so long as the products of the reaction x are small in amount compared with the substrate a. Further, according to the measurements of Bayliss, Hedin and others, this rule is obeyed, in the case of trypsin, with small quantities of enzyme and also with conditions so chosen that x = const. \/Et. This rule is contained in the widely-used formula (18), as is shown by the derivation of the latter. Formula (22) evidently means that the progress of the diges- tion depends only on the quantity of protein already hydrolysed, no matter whether this has been obtained by the action of much enzyme for a short time or by that of less enzyme for a longer time. This relation has been confirmed by H e d i n (Journ. of Physiol., 1905, 32, 468; ibid, 1906, 34, 370; H., 1908, 57, 471). One of the tables from the last of these papers is given below. The amount of casein digested was measured by the quantity of nitrogen not precipitated by tannic acid. The values given in the various columns under E.t indicate the numbers of tenths of a c.c. of normal acid required to neutralise the ammonia obtained by K j e 1 - 188 GENERAL CHEMISTRY OF THE ENZYMES d a h 1 ' s method from equal volumes of the filtrate from the tannin precipitate : Et... 1 .. 1 5 8 2-0 10-35 2 12 5 95 5-0 20-15 7-5 24-50 10 [26 95 15-0 31-0 20-0 34-05 2.. . . 3.... 4 . . 5 .. 5 6 85 70 10 10-75 10 85 10-75 13 13 13 -25 35 15 20-40 20-30 19-90 24-65 24-35 23-80 L *^ 27 27 26 8 95 31-7 31-05 30-75 33-75 33-55 As regards the investigation of the kinetics of the digestive enzymes, the difficulties which beset all enzymic problems are here supplemented by others due to the complicated nature of the proteins. Observations made by any of the physico-chemical methods, whether optical or electrical, represent the effect of a large number of decompositions proceeding simultaneously, and it is, as the author has repeatedly emphasised, surprising that simple direct relations are so often found. The processes of decomposition would hence become decidedly more apparent if the dipeptides, which F i s c h e r 's work has rendered accessible, were employed as substrates. The first experiments in this direction were made by E u 1 e r (H., 1907, 51, 213) on glycylglycine, not however with the trypsin of the pancreas, but with the proteolytic enzyme of the walls of the intestines, the so-called erepsin. The course of the reaction was followed by the change in conductivity of an alkaline solution of glycylglycine. The final conductivity, obtainable by complete hydrolysis of the glycyl- glycine, was determined beforehand with glycine solutions of corresponding concentration. In the possibility of a certain knowledge of this final value seems to lie one of the principal advantages of using dipeptides as substrates in tryptic and ereptic decompositions. It was also found that the diminution of the conductivity is proportional to the diminution of the concentra- tion of the dipeptide. 1 The first result obtained was that the velocity of hydrolysis of glycylglycine depends greatly on the alkalinity of the erepsin solution. 2 1 It is to be noted that the existence of such proportionality, which forms the basis of further calculations, was not shown in previous investigations where tryptic or peptic digestion of protein was followed by means of the conductivity. 2 The alkalinity is expressed as the normality of the total soda added. The actual alkalinity, that is, the concentration of the free base, is very much smaller, since the greater part of the soda is used up in neutralising the glycylglycine. EXPERIMENTAL DATA OF ENZYME REACTIONS 189 N/10-glycylglycine. 5 grms. powdered erepsin per 100 c.c. Concentration of alkali 0-04 0-05 0-075 Reaction constant X 1000 <0-05 7-0 6-2 2-6 0-10 0-2 Different preparations of the enzyme showed varying sensitive- ness towards alkali. It was next ascertained that the decomposition of glycylglycine is a reaction of the first order and that, under favourable con- ditions, the corresponding velocity coefficients k remain constant until the reaction is half completed. 1 N/10-glycylglycine. 5 grms. powdered erepsin per 100 c.c. 0-04 N-NaOH. b. 0-05N-NaOH. Minutes. 1000 (a x). 1000/fc. Minutes. 1000(o -x). lOOOfc. 920 935 7 819 7-20 10 806 6-46 15 721 7-08 17 739 6-00 22 649 6-88 25 654 6-18 30 579 6-70 30 622 5-90 In most cases a considerable fall in the velocity occurs even after about half an hour. This depends principally on destruction of the erepsin, the rapidity of the destruction increasing with the amount of free alkali in the solution. Within certain limits the velocity of reaction is almost independ- ent of the concentration of the dipeptide. This independency holds, however, only for certain relations between the concen- trations of the enzyme and substrate. With small amounts of enzyme, the velocity of reaction rises with increase of the con- centration of the glycylglycine. 0-1 N-glycylglycine; 0-04 N-NaOH. 1000/c=0-35 . 2 N-glycylglycine; . 05 N-NaOH. 1000/0 = 0-55 Since, as has already been mentioned, the concentration of free alkali has here also a considerable influence on the velocity of reaction, the influence of the concentration of the substrate is very difficult to explain. The author concludes, from the results 1 As the behaviour of erepsin, like that of other enzymes, depends on the absolute activity of the solution, it is advisable, in order to make differene results comparable, to establish a standard action. A normal preparation might be taken as one which decomposes glycylglycine or an optically active dipeptide, in 5% solution, to the extent of one-half in 1 hour. 190 GENERAL CHEMISTRY OF THE ENZYMES of these experiments, that only the alkali salt of the d i p e p t i d e undergoes hydrolysis (Arkiv for Kemi, 1907, 2, No. 39). As regards the effect of the concentration of enzyme, it may be said that, in most of the conditions of concentration examined, the velocities of reaction were proportional to the enzyme-con- centrations ; the Schiitz-Borissow law did not hold in any instance. The following numbers serve as examples: Concentration of erepsin 5 4 3 2 1000/c: 6-5 5-4 4-3 2-8 With low concentrations of the enzyme, especially with feeble preparations of pancreatin, k increases far more rapidly than the concentration of the enzyme. With relatively high concentrations of enzyme, deviations from proportionality in the sense of S c h ii t z's rule do, indeed, occur; but the experimental errors are so great in these reactions, which take place in a few minutes, that conclusions cannot be drawn from the results. If optically active polypeptides are employed as substrates, the enzymic hydrolysis can often be followed polarimetrically. Such measurements have been carried out byAbderhalden and his collaborators. The following results, obtained with d-alanyl-d-alanine, indicate the time-law of this reaction (Abderhalden and Koelker, H., 1907, 51, 294; Abderhalden and M i c h a e 1 i s , H., 1907, 52, 326) : 0-45 grm. dipeptide+6 c.c. pressed juice. 0-45 grm. dipeptide +4 c.c. pressed juice +2 c.c. physiological salt solution. Min. Rotation X 10"z k.W*. Min. Rotation. X 10"z &.10*. t t -1-21 -1-31 3 -0-96 0:39 130 453 3 -I'll 0-18 60 192 7 -0-74 0-59 84 390 7 -0-98 0-37 53 183 11 -0-51 0-84 71 382 10 -0-81 0-54 54 202 18 -0-20 1-15 64 380 16 -0-56 0-79 49 214 20 -0-16 1-19 59 373 25 -0-21 1-14 46 268 27 +0-01 1-36 50 451 30 -0-09 1-26 42 294 34 +0-05 1-40 41 430 35 +0-02 1-37 359 40 +0-07 1-42 35 43 +0-07 1-42 55 +0-10 1-45 54 +0-08 1-43 65 +0-10 1-45 EXPERIMENTAL DATA OF ENZYME REACTIONS 191 0-45 grm. dipeptide+3 c.c. pressed juice +3 c.c. physiological salt solution. Minutes. Rotation. X IWx t fc.10 4 o-o -1-31 5-0 -1-16 0-19 380 125 6-5 -1-09 0-26 400 132 7-5 -1-05 0-30 400 134 16-0 -0-76 0-59 369 142 22-0 -0-54 0-81 368 161 28-0 -0-32 1-03 368 192 30-0 -0-25 1-10 314 209 38-0 -0-09 1-28 332 232 45-0 +0-01 1-36 265 60-0 +0-07 1-42 ~ 0-6 grm.d-Alanyl-d-alaninein 7 -6 c.c. pressed juice +0-4 c.c. physiological salt solution. 0-6 grm. d-Alanyl-d-alanine +5-7 c.c. pressed juice +3 -3 c.c. physiological salt solution. Min. Rotation X X ~t &.10* Min. Rotation. X X T fc.10* 5 12 19 26 30 -1-30 -1-08 -0-85 -0-59 -0-23 -0-09 22 45 71 107 121 44 38 38 40 40 148 140 162 241 289 7 13 26 37 50 -1-23 -1-09 -0-91 -0-46 -0-11 21 39 84 119 140 30 30 32 32 28 60 92 144 216 40 30 It will be seen that the theoretical formula, k = -log - , t a x leads to reaction-coefficients which show a continuous rise, the amount of this increasing with diminution of the amount of enzyme for a constant amount of substrate. On the other hand, with relatively small amounts of enzyme, x the value of the expression is strikingly constant, and this appears to indicate that the change proceeds independently of the amount of substrate still to be decomposed. But it is more probable that here, as is always the case, the velocity of reaction, dx : dt, is proportional to the quantity of substrate present, and that the reaction, as it proceeds, is subjected to some secondary acceleration. 192 GENERAL CHEMISTRY OF THE ENZYMES The velocity of reaction is approximately proportional to the amount of enzyme: 40 : 7-6 = 5-4, 30 : 5-7 = 5-4. This proportionality is much less sharp with d-alanylglycine, with which Abderhalden and K o e 1 k e r (H., 1908, 55, 416) have experimented; the two following tables appear on p. 422 of this paper : 2 c.c. Dipeptide solution =0-001 normal. -5 c c. pressed yeast juice. 4 c.c. water. 2 c.c. Dipeptide solution =0-001 normal. 2-0 c.c. pressed yeast juice. 2-5 c.c. water. Minutes. Rotation. i *.10*. Minutes. Rotation. ft. 10'. 12 +0-85(extrap.) 0-80 22 12 +0-85 (extrap.) 0-70 70 31 0-71 25 31 0-54 64 62 0-63 22 62 0-38 56 86 0-53 24 86 0-28 56 118 0-49 23 118 0-19 55 190 0-36 20 Here, therefore, the velocity of reaction increases considerably more slowly than the concentration of the enzyme, but there is no indication of the validity of S c h ii t z 's law. With reference to the effect of the concentration of the sub- strate, Abderhalden andKoelker (H., 1908, 54, 363) have made experiments with active material, the following table giving some of their results : (rfr-mol.) ( 2 BB-mol.) (sfo-mol.) 3-0 c.c. d-Alanyl-rf- alanine solution, 1 -0 c.c. pressed yeast juice. 2-0 c.c. water. 4-0 c.c. d-Alanyl-d- alanine solution, 1 -0 c.c. pressed yeast juice. 1 -0 c.c. water. 5-0 c.c. d-Alanyl-d- alanine solution, 1 -0 c.c. pressed yeast juice. Minutes. Angle. Angle. Angle. 4 -1-36 -1-83 -2-15 12 -1-31 -1-75 -2-10 32 -1-17 -1-58 -1-97 61 -0-95 -1-31 -1-73 92 -0-67 -0-97 -1-43 128 -0-42 -0-69 -1-16 167 -0-18 -0-38 -0-86 190 -0-08 -0-23 -0-69 238 +0-03 +0-01 -0-35 308 +0-05 +0-13 +0-02 357 +0-05 +0-12 +0-15 377 +0-11 +0-18 +0-29 EXPERIMENTAL DATA OF ENZYME REACTIONS 193 The dipeptide employed in these experiments was, as these investigators state, not free from the racemic compound, so that the course of the reaction and the calculations become more complicated. It can, however, be seen from the above numbers that the amounts of dipeptide hydrolysed in a certain time are, as a first approximation, independent of the initial concentration of the dipeptide. The velocity constants calculated according to the unimolecular formula would, therefore, diminish considerably with increasing concentration of the dipeptide instead of being independent of this concentration, as theoretically they should be. Within a certain region of concentration or with a certain ratio between the concentrations of substrate and enzyme, this relation is found in the case of most enzymes. It has already been shown, by experiments made by the author, how greatly the degree and character of the reaction change with the alkalinity of the solution, and confirmation of this result is afforded by the investigations of Abderhalden and K o e 1 k e r (loc. cit., p. 378). To obtain definite results in any study of the kinetics of trypsin and erepsin, careful attention rrust be paid to the alkali-content of the solutions. If, as the above measurements indicate, it is the alkali salts of the dipeptides or proteins which undergo hydrolysis and thus function as the " active molecules," the concentration of the substrate is not merely that of the dipeptide or protein but depends also on that of the alkali added; hence no general simple relations for the velocity of reaction will be found if the concentration of the dipeptide or protein alone is varied. It is of more value to ascertain how the concentration of the alkali must be altered at the same time for the theoretical requirement independence of the reaction constant on the concentration of the substrate to be fulfilled. Abderhalden and Koelker (H., 1908, 55, 416) have examined also the course of the reaction in the case of tri- and tetra-peptides. Study of such higher polypeptides not only serves to characterise the polypeptides, with which the velocity of hydrolysis is a characteristic constant, but they are also of interest in indicating the course of the reaction when, as happens with the true proteins, several hydrolyses take place at the same time. As examples are given the results of two series of exper- iments on d-alanylgly cylglycine ; since the separate observations 194 GENERAL CHEMISTRY OF THE ENZYMES are subject to considerable errors, the results given here have been interpolated graphically. 3-32 c.c. Peptide solution =0-002 mol. 4-98 c.c. Peptide solution =0-003 mol. 1 -0 c.c. pressed yeast juice. 2 18 c.c. H2O. 1-0 c.c. pressed yeast juice. 0-52 c.c. HzO. Minutes. Angle (interp.) k. 10*. Minutes. Angle (interp.) k.10*. 1-70 2-55 20 1-15 85 20 1-80 76 40 0-70 96 40 1-20 82 60 0-41 103 60 0-80 83 80 0-20 116 80 0-55 83 120 0-26 82 It will be seen that the constants calculated from the unimo- lecular formula and given in the third column, do not increase very greatly with the time. A dynamic investigation of the vegetable ereptases discovered by V i n e s (Annals of Bot., 1910, 24, 213), would be of considerable interest. At about the same time as the author, A . E . Taylor car- ried out experiments on alkaline proteolysis, using a chemically definite substrate in order to render the results more definite. To this end he prepared protamine sulphate from the salmon by K o s s e 1 ' s method and hydrolysed it with G r u b 1 e r ' s pancreatin. Unfortunately the results of these researches are given only very briefly (Berkeley: On Fermentation, 1907, p. 152). The numbers do, however, show that the law x = n\/t does not hold for trypsin. The results of each series of exper- iments can be calculated by the formula for unimolecular reac- tions: but the constants vary if at different times the concentra- tion of the substrate is altered. Further, under constant external conditions, the velocity of protamine digestion is simply and directly proportional to the concentration of the trypsin. These results were obtained with the optimal concentration of hydroxyl- ions. Protamine, 0-100 grm. in 50 c.c. Temperature 40 C Quantity of trypsin, grm. Mean time of digestion, t Quantity of trypsin X 0-008 0-006 0-004 0-003 0-002 0-001 0-0005 37 50 76-5 103 156 313 657 296 300 306 309 312 313 328 EXPERIMENTAL DATA OF ENZYME REACTIONS 195 Here also, then, the relation x = K S \/E does not hold. In connection with the above investigations on the action of proteolytic enzymes in vitro, reference may be made to the relations deduced by Arrhenius (H., 1909, 63, 323) from the results of the numerous experiments made by E . S . London on digestion in the stomach of the dog. For the experimental details reference must be made to London's original papers (H., 1905, 45, 381; 46, 209; 1906, 47, 368; 49, 324; 50, 125; 1907, 61, 241, 468; 52, 482; 53, 148, 240, 246, 326, 334, 356, 403, 429; 54, 80; 1908, 55, 447; 56, 378-416, 512-553; 57, 113, 529; 1909, 60, 191-283; 61, 69; 62, 443-464; 1910, 65, 189-218; 68, 346-380). The follow- ing cases are to be distinguished: 1. Digestion of different quantities of meat introduced by the mouth. The fact, estab- lished by L o n d o n , that the amount digested in a given time is not proportional to the quantity of nutriment taken, is explained as follows: " The first 100 grms. of meat lie close to the stomach-wall in a layer of uniform thickness. Within this lies another layer of 100 grms. of meat, this being rather thicker, as its surface dimin- ishes approximately as the cross-section of a cone as the apex is approached. Further layers of 100 grms. follow, the thickness continually increasing. Into these layers the gastric juice diffuses from the mucous membrane. The quantity of gastric juice in each layer diminishes considerably and the diminution increases in extent as the layers lie farther away from the mucous membrane. This is conditioned partly by their increasing thickness and partly by the laws of diffusion, which require such increasing diminution even for layers of equal thickness. Hence it comes about that the innermost layers are not perceptibly digested and the amount digested in a given time increases, as the amount of food taken is augmented, only to a maximum." As far as digestion is concerned, every layer is independent on every other and the total quantity digested is the sum of those digested in the various layers. On the simple assumption that the digestion in the outermost layer is always the same, the jmmber of layers which may lie inside has been calculated by Arrhenius from London's results by means of an 196 GENERAL CHEMISTRY OF THE ENZYMES empirical formula. The latter gives the quantities of food digested in a certain time in the various layers and corresponds very well with the experimental figures. With the aid of this formula Arrhenius has prepared a table showing the time-course of the digestion which he represents also graphically (Fig. 5). 1 icoo DIGESTION OF DIFFERENT QUANTITIES OF MEAT, 100-1000 GRAMS. \ 400 200 \\ \ 10 11 1 From C h i g i n and Lobassow's results (Dissertation, St. Petersburg, 1896), H e r z o g (Zeitschr. f. allg. Physiol., 1904, 4, 163) has attempted to calculate the time-course of the secretion of gastric juice. Determinations were made of the quantities of pepsin (measured by the number of c.c. of gastric juice and its specific digestive power) secreted in a blind sac in the fundus part of the stomach, when food was introduced into the stomach. According to Herzog's calculations, the secretion of pepsin follows the formula for unimolecular reactions. With introduction of solid food the values of k obtained are moderately constant. But in what degree the measurement and calculation of the quantities of pepsin by the Russian workers are free from objection, cannot well be judged. C h i g i n and Lobassow's results have recently been referred to in a paper by Arrhenius (H., 1909, 63, 323). EXPERIMENTAL DATA OF ENZYME REACTIONS 197 From the curve for 1000 grms. the quantities remaining undigested after a certain number of hours have been calculated, the results being as follows: Time in hours: 1 2 3. 4 5 6 7 8 9101111-5 Amount undigested: 1000 875 750 627 507 390 276 180 100 51 24 7 3 Difference per hour: 125 125 123 120 117 114 96 80 49 27 17 It will therefore be seen that the quan- tity digested is at first very nearly pro- portional to the time, but gradually di- minishes later. This apparent contradiction ofSchiitz's rule depends on the fact that in vivo the products of digestion are continually removed, whilst in vitro they remain in the solution and hence retard the reaction. The above table leads to a very important rule. It will be seen that for practically complete digestion of 1000 grms. of meat 11-5 hours are necessary. The time t required to digest another quantity M is calculated by subtracting from 11-5 hours the time taken to digest (1000-M) grms. of meat when the initial quantity is 1000 grms. In this way the times b s . given below for varying values of M have been obtained. M W kale. (kbs. kale.) 1000 11-50 10-81 +0-69 900 10-70 10-26 -fO-44 800 9-90 9-67 +0-23 700 9-09 9-05 +0-04 600 8-27 8-38 -0-11 500 7-44 7-65 -0-21 400 6-60 6-84 -0-24 300 5-73 5-92 -0-19 200 4-74 4-84 -0-10 100 3-45 3-42 +0-03 50 2-40 2-42 -0-02 25 1-55 1-71 -0-16 Besides the values of obs ., those of ca i c . are also given, these being calculated from the formula 198 GENERAL CHEMISTRY OF THE ENZYMES Hence, the time required for the virtually complete digestion of flesh-food introduced per o s is very nearly proportional to the square-root of the quantity of food. This square-root law is repeated in numerous series of experi- ments and also with different nutriment. It holds not only for ordinary meat but, as was shown byArrhenius, for the digestion of gliadin (London, H., 1908, 56, 394), of the albumin of hens' eggs (London, H., 1908, 56, 405) and of bread (London, H., 1906, 49, 359), and is therefore of the greatest importance for calculating the digestion periods of solid foods. 2. Digestion of meat introduced directly into the stomach by a fistula. If the nutriment is taken per o s , a reflex secretion of gastric juice occurs, so that the digestion is greatly accelerated. When, however, the introduction of food takes place through a fistula, the secretion of gastric juice produced by the stimulating action of the meat on the stomach-wall is at first gradual and increases with the time; indeed, as the following calculation shows, the quantity of gastric juice in the stomach is about proportional to the time elapsing since the introduction of the meat. Thus, if the digestion is proportional to the quantity of gastric juice present and the latter to the time, the amount digested per unit of time, , is proportional to the time t and to the quantity at of meat (M x) still present (M being the amount initially added), or, mathematically: dt from which follows: London and Pewsner's results, given below, have been calculated according to this formula. EXPERIMENTAL DATA OF ENZYME REACTIONS 199 A. Feeding of dog per fistulam. Eyes and nose covered. t (hours) Undigested quantity (Mx) (M-x) C aic. , Difference. 100 100 2 84 84-7 -0-7 4 56 51-5 +4-5 6 20 22-5 -2-5 8 7 7-0 9 3-5 -3-5 B. Feeding per fistulam. Eyes and nose uncovered. t (hours). Undigested quantity (Mx). (M-a) ca i c . Difference. 100 100 2 84 83-7 +0-3 4 53 49-1 +3-9 6 18 20-1 -2-1 8 5 5-8 -0-8 9 2-7 -2-7 In the experiment A, the constant had the value k = 0-0180 and in B, k = 0-0193. 3. If we now ask how the total course of the digestion is to be represented (i.e., not only as regards the time necessary for local digestion), it may in general be stated that, in so far as small quantities of food (the layer which, according to the above assumption, is in immediate contact with the stomach-wall) are concerned, digestion follows the law holding for unimolecular reactions. This is shown by the following examples : Digestion of boiled protein, 200 grma. Digestion of meat, 200 grms. t (100 -z) (100-aOcalc. Diff. t (100-3) (lOO-z)calc. Diff. 100 100 100 100 1 70 65 +5 1 60 56 +4 2 32 42 -10 2 31 32 -1 3 28 27 + 1 3 15 18 -3 4 18 18 4 5 10 -5 5 15 12 +3 5 6 6 6 4 7 -3 (7 5 -5) 200 GENERAL CHEMISTRY OF THE ENZYMES By (a x) is denoted the quantity of undigested protein still in the stomach at the time t. The calculated figures are obtained by means of the formula: 7 1 100 fc=-7-log t & 100-ar The same formula holds for the digestion of dissolved carbohydrates (amylodextrin) . 4. The resorplioii of a sugar solution in the intestines also follows the unimolecular law. If a solution of glucose is introduced into the intestine, the latter gives up water to it if the solution is concentrated, but takes up water from a dilute solution. The quantity of water yielded to the solution reaches a maximum, amounting to 900 c.c., corresponding with the capacity of the body to give up water From London's results, Arrhenius has calculated the concentration of the solution when no water is taken up o give out by the intestine, this being 10 5% of glucose. As was to be expected, it is found that the amount of water taken from the intestine is proportional to the excess of the concentra- tion over 10-5%. The law followed is hence expressed by the equation: where W is the amount of water given up by the intestine and (C 10-5) represents the excess of the concentration over 10 5%. RENNET (CHYMOSIN) By the action of rennet, casein is converted into paracasein (compare p. 45); whether at the same time some component, possibly an albumose, is split from the casein molecule is a dis- puted question. On account of this supposed partial hydrolysis, and of the occurrence of pepsin and chymosin together, the latter is classed with the true proteolytic enzymes. It must here be borne in mind that the clotting of milk by rennet consists of two processes (Hammarsten) : chymosin only accelerates the conversion of casein into paracasein, a change which proceeds without calcium salts. These salts are necessary only for the precipitation of the curd (para- casein) for which, indeed, no rennet is required. It would therefore be expected that the time which clotting takes to begin is made up of two periods: (1), that necessary for EXPERIMENTAL DATA OF ENZYME REACTIONS 201 the casein to be transformed, almost completely, into paracasein time of conversion, and (2), that necessary for the formation of a visible clot time of separation. Such an assumption was advanced by Fuld (Hofm. Beitr., 1902, 2, 169). According to this author, the time of separation amounts to several days or to a few minutes or even less, in dependence on the temperature of the paracasein solution, and is so inappreciable in comparison with the long period of conversion of the experiments referred to above, that it may be unhesitatingly neglected. On the other hand, R e i c h e 1 and S p i r o , in their most recent investiga- tion, regard such an assumption as that of F u 1 d as unjustified, and express the view that, with reference to its time-course, the whole clotting process must be considered as a single process (Hofm. Beitr., 1906, 8, 15). According to Bang (Skand. Arch. f. Physiol., 1911, 25, 105), the clotting of milk by rennet is an extremely complicated process, in which the calcium salts present are distributed between organic acids, lactalbumin and lactoglobulin, whilst, on the other hand, the casein is distributed among the basic constituents of the solution. The clotting process obeys the law, [E].t = const., or The product of the enzyme-concentra- tion [#] a n d time of clotting t is constant. This relation was first observed by Segelcke and S t o r c h (Ugeskrift for Landmaend, 1870) and was subsequently fully confirmed by H a n s e n and S o x h 1 e t (Milchzeitung, 1877). The most complete investigations of the action of rennet are due to F u 1 d and to R e i c h e 1 and S p i r o . At 40, E . F u 1 d (Hofm. Beitr., 1902, 2, 172) obtained the following numbers, which proved the exact and general validity of the time-law even with small amounts of rennet: Quantity of rennet. Time of clotting, t, in seconds. Product. 0-4 6 24 0-4 6-6 26 0-2 13 26 0-1 25 25 0-8 6 48 0-4 17 44 0-2 22 44 0-1 45 45 202 GENERAL CHEMISTRY OF THE ENZYMES According to C. G e r b e r (Soc. BioL, 1907, 63, 575), who has recently made a thorough study of the clotting process, it is essen- tial, when working with the rennet of commercial pepsin, to employ temperatures between 25 and 30 so that the enzyme shall be under normal conditions. Within these limits of tem- perature, G e r b e r finds that the law of Segelcke arid S t o r c h holds closely for parachymosin. Drops of parachymosin #....1 2 3 46 7 8 9 10 Time of clotting, t 29-66' 14-75' 10-30' 7-60' 5-50' 4-50' 3-66' 3-16' 2-92' E.t 29-6 29-5 31 30-3 33 31-5 29-3 28-4 29-2 M a d s e n has also investigated the duration of milk-clotting, the method used being similar to that employed with pepsin action (see Arrhenius, Immunochemistry, p. 72). He adds, for instance, varying quantities of rennet to equal amounts of milk in test-tubes, places the mixtures in a water-bath at a definite temperature, takes them out after time t, cools them quickly and deter- mines the smallest quantity of rennet L which has produced clotting. COAGULATION OF MILK BY RENNET SOLUTIONS OF DIFFERENT CONCENTRATIONS AT 36-55 Minutes. L. Lt. Minutes. L. Lt. 4 0-08 0-32 35 0-007 0-25 6 0-05 0-30 50 0-005 0-25 9 0-033 0-30 70 0-004 0-28 11 0-024 0-26 80 0-0032 0-26 12 0-019 0-23 100 0-0028 0-28 14 0-0175 0-25 120 0-0025 0-30 20 0-013 0-26 180 0-00185 0-33 25 0-010 0-25 240 0-0017 0-41 30 0-007 0-21 Bang (Pflug. Arch., 1900, 79, 425) found that the relation, [E] t = const., did not hold for parachymosin. By a series of careful experiments R e i c h e 1 and S p i r o (Hofm. Beitr., 1905, 7, 485) showed that the relation between quantity of rennet L and period of clotting t can be expressed generally by the formula: L n .t = const. EXPERIMENTAL DATA OF ENZYME REACTIONS 203 The exponent n of L changes with the nature and amount of the other substances in the solution, and in a liquid of the com- position of milk it assumes exactly the value 1, so that the law holds in its simplest form, L.t = const. In the following experiments the solutions were prepared by dilution with whey, the clotting action of which was negligible in comparison with that of the enzyme added. Milk. c.c. of 35% rennet diluted with whey. Whey. Time of clotting t in seconds. Whey I. Whey II. Whey III. 0-5 0-5 4-0 22 22 33 1-0 0-5 3-5 17 22-5 27 1-5 0-5 3-0 18 27 28 2-0 0-5 2-5 17 25-5 26-5 2-5 0-5 2-0 18 24-5 25-5 3-0 0-5 1-5 16 26 26 3-5 0-5 1-0 16 26 24 4-0 0-5 0-5 15 25 26 4-5 0-5 ~ 16 24 27 As will be seen from these results, the constant Lt or, since L is the same in all cases, the time of clotting, is independent of the concentration of milk (casein) in the solution from 20% up to 90 %. Replacement of the whey by 0-9% sodium chloride solution resulted in constancy of the time of clotting for more dilute as well as for more concentrated solutions. More extended investigations then showed that the difference between the times of clotting for dilute and concentrated milks was approximately proportional to the difference between the dilutions. Hence, if V denotes the volume of the diluted milk, M that of the undiluted milk, and t and t' the times of clotting in the two media, then: M V-M const. 204 GENERAL CHEMISTRY OF THE ENZYMES This is shown by an extract from Table VII of R e i c h e 1 and S p i r o ' s paper. Rennet. Milk. Whey. Time. -<'> (T^T) 1-0 0-2 8-8 110 1-60 1-0 0-6 8-4 50 1-81 1-0 1-0 8-0 39 1-93 1-0 2-0 7-0 28-6 1-75 1-0 4-0 5-0 24 1-60 1-0 8-0 1-0 22 1-60 The influence of the Ca-ions is also expressed by a simple and important relation, Time t for amount of rennet. Constant, Ca" Xt for amount of Milk, CaClj, / _ rennet. c.c. u /oo 1-0 0-5 0-25 1-0 0-5 0-25 8-0 95 48 24 57-0 28-8 14-4 8-0 0-05 88-6 45-6 23 57-6 29-6 15-0 8-0 0-1 79 41-6 22 55-3 29-1 15-4 8-0 0-2 66-4 36 19 53-1 28-8 15-2 8-0 0-5 48 26-4 14 52-8 29-0 15-4 8-0 1-0 30 18-2 10-6 48-0 29-1 17-0 8-0 2-0 17 11 6-8 44-2 28-6 17-7 8-0 5-0 10 7-4 5-4 56-0 41-4 30-2 8-0 10-0 13 9-2 6-2 137-8 97-5 65-7 8-0 20-0 22 15 8-6 453-2 309-0 177-2 The values of the constant, which show little change up to about 0-02% of calcium, are calculated on the assumption that the Ca-ions in the milk amount to 0-6%o; about 15% of the total calcium of the milk must then be in an ionised condition. Further, as was shown in a subsequent paper (Hofm. Beitr., 1906, 8, 15) the rennet-action a is, in general (not merely at the clotting point), directly proportional to the quantity of enzyme L and to the time t, that is = L.t. const. EXPERIMENTAL DATA OF ENZYME REACTIONS 205 The more recent assertion of H.Kottlitz (Arch, intern, de Physiol., 1907, 5, 140) that S c h ii t z ' s rule holds also for rennetic action in no way diminishes the importance of R e i c h e 1 and S p i r o ' s results. Several attempts have been made to estimate the value of the time-laws determined, on the one hand, for the action of rennet and, on the other, for that of pepsin, as criteria for the disputed identity of chymosin and pepsin. The discussion of this question would not be in place in a chapter dealing with the chemical dynamics of the enzymes; but it may at least be asserted that, especially since H a m - marsten's comprehensive investigation (H., 1908, 56, 18), it is no longer possible to regard the clotting of milk simply as a peptic action. The influence of concentration, and also that of temperature, are so divergent in the cases of clotting and diges- tion, that it must at the least be assumed that two different enzymic groups are united in one molecule. This assumption, so long as the preparation of the enzymes in a pure state is not achieved, can be neither refuted nor proved, and is indeed, under present circumstances, of subordinate importance. FIBRIN-FERMENT E . F u 1 d (Hofm. Beitr., 1902, 2, 514) mixed the plasma of bird-blood with the enzyme solution (obtained by extracting muscle with 0-8% sodium chloride solution). The velocity of clotting increased more slowly than the concentration of the enzyme, the results agreeing approximately with S c h ii t z ' s rule but more accurately with the expression : ^ \ 0-585 aj ' where vi and v% are the velocities of clotting and E\ and E% the concentrations of the enzyme. For protracted clotting periods, that is, for low enzyme-concentrations, this relation fails, the duration of clotting then showing disproportionate increase. It has been shown, especially by M a r t i n (Journ. of Physiol., 1905, 32, 207), that the rule obeyed by chymosin and numerous other enzymes that the same amount of action occurs for equal 206 GENERAL CHEMISTRY OF THE ENZYMES values of the product E.t, no matter what the values of E and t holds also for the fibrin-ferment. ZYMASE Although the " zymase " of pressed yeast-juice is accompanied by an extremely large quantity of various other substances and is further removed from a state of purity than is the case with any other enzyme, yet in recent years, especially by the work of Harden and Young, important information has been obtained concerning the chemistry of alcoholic fermentation. If the final products, alcohol and carbon dioxide, of pressed yeast-juice containing sugar are studied quantitatively, as has been done by the author (H., 1905, 44, 53), the following results are obtained: The expression log == k gives moderately constant numbers during the first half of the reaction, but subsequently the value of k exhibits considerable diminution. To this effect, two principal causes contribute, firstly, the separation of protein substances which carry down part of the fermentation enzymes, and secondly, alteration of one of the activators of the zymase; this activator consisting of organic compounds of phosphoric acid is attacked by the lipase of the yeast-juice, the latter gradually becoming impoverished in the " co-enzyme " which is of s\ich great importance to alcoholic fermentation. The quantity of carbon dioxide evolved per unit of time was determined partly by the loss in weight of the solution and partly volumetrically. 4 grms. glucose in 20 c.c. of yeast-juice. Minutes. x (grms. CO 2 ). (ax). fc.10 4 . 1-800 80 0-078 722 2-4 315 0-299 501 2-51 379 0-360 440 2-56 505 0-460 340 2-54 1024 0-779 021 2-41 1180 0-810 0-990 (2-20) 1544 0-899 0-901 (1-95) 2119 0-955 0-845 (1-55) EXPERIMENTAL DATA OF ENZYME REACTIONS 207 For about 18 hours, the velocity of reaction is here moderately constant. This is, however, a favourable case and under other conditions, as is shown by the following table, the pressed juice remains unchanged only for 6-8 hours. The influence of the concentration of the substrate is also shown by the following results. 20 c.c. juice +8 grms. of glucose in 20 c.c. of solution. Minutes. : x(grms.) (o-*). k. 10*. Minutes. x(grms.) (a-x). fc.105. 3-909 3-909 3-900 3-900 161 0-078 3-822 5-44 160 0-0815 3-8185 5-38 260 0-120 3-780 5-24 257 0-1195 3-7805 5-24 358 0-161 3-739 5-11 355 0-159 3-741 5-05 404 0-181 3-719 5-10 404 0-180 3-720 5-10 5-21 5-19 20 c.c. juice +2 grms. of glucose in 20 c.c. of solution. Minutes. x(grms.) (a-x). fc.10*. Minutes. x(grms.) (a-x). k.lW. 0-977 0-977 0-968 0-962 167 0-0855 0-8825 2-40 153 0-081 0-881 2-50 240 0-123 0-845 2-46 260 0-123 0-839 2-27 332 0-152 0-816 2-29 331 0-182 0-780 2-75 2-38 2-51 In addition to these series of experiments, three others were carried out, also with concentrations of sugar in the ratio 4:1. The constants obtained were as follows: i fci . 10 5 :*,. 10 5 No. 4. 5-2 : 24 4 = 1 : 4 7 11 5. 2-5 : 12 = 1 : 4 8 11 7. 15-0 : 75 = 1 : 5 " 6. 20-0 : 97 1 : 4 85 It will be seen that the velocities are not, as they should be according to theory, independent of the concentration of the substrate; also they are not inversely proportional to the initial 208 GENERAL CHEMISTRY OF THE ENZYMES concentration a, so that ka is not a constant magnitude but, in the region of concentration investigated, increases as a decreases. In each of the four series of experiments, ai : a2 = 4 : 1. ftiai.10 6 ' & 2 a 2 .10 6 No. 4. 208 244 " 5. 100 120 " 7. 600 750 " 6. 800 990 Whilst, in general, the velocity of reaction increases either proportionally to, or slower than, the concentration of enzyme, the velocity of fermentation increases more rapidly than the concentration of the pressed juice but always slower than its square. If the exponents n are calculated according to the equation In k\ In k% 1 7 > In ci ln 2 it is found that, for a constant sugar-content, n increases with diminution of k, that is, with diminution of the concentration of the zymase: ki . 10 6 n 100 1-29 86 1-33 35 1-52 12 1-67 The numbers appear to indicate that, with very high fer- mentative activity, proportionality between the concentration of the pressed juice and the velocity of fermentation is attained. Finally, if pressed juice containing sugar is diluted, that is, the volume increased while the absolute amounts of the juice and sugar remain constant, the following relations are found: f Concentration"32 : 52 = 1:1-63 f 20: 30: 50 = 1:1-5 :2-5 ) [Velocity 192:282=1:1-47 () { 120 : 188 : 315 = 1: 1-57: 2-63 The mean of these two experiments indicates, within the limits of concentration employed, approximate proportionality between concentration and velocity. EXPERIMENTAL DATA OF ENZYME REACTIONS 209 Prior to these experiments with pressed yeast-juice, A b e r s o n Rec. Trav. Chim. Pays-Bas, 1903, 22, 78) had studied the course of the alcoholic fermentation of glucose by living yeast-cells. It must however be emphasised that Aberson's observa- tions were made polarimetrically, so that he measured the amount of sugar disappearing during the reaction. But new measurements made by E u 1 e r and Johansson (H., 1912, 76, 347) with living yeast show that the diminution in rotation of a fermenting sugar solution is by no means proportional to the evolution of carbon dioxide. This is indicated by the following numbers: CO 2 evolved. Change in rotation. Concen-i ^ Dif- No. Temp. tration of Sugar. yeast in 25 c.c. solution Time. Min. Grms. Per cent. A Degrees. Per cent. B fer- ence. B-A (1 30 10% 0-5 125 0-0936 7-7 5-37-4-67=0-70 13 1 5-4 12 30 10 0-5 181 0-1492 12-2 5-32-4-46 = 0-86 16 2 4-0 3 30 10 1 42 0-0644 5-3 5-33-4-80=0-53 10-0 4-7 4 30 10 1 63 0-1160 9-5 5-33-4-48 = 0-85 16-0 6-5 5 30 10 1 232 0-4918 40-3 5-33-2-84 = 2-49 47-0 6-7 6 30 10 1 406 0-7256 59-4 5-33-1-85 = 3-48 65-9 6-5 The cause of this difference has not been fully elucidated. Owing to this circumstance, Aberson's experiments give information concerning only the first phase of alcoholic fermentation, namely, the transformation of the sugar. The results of his numerous experiments do not correspond with the expected law, but the values given in the last column of the fol- lowing table which is taken from that given on p. 97 (1 o c . c i t . ) are far more nearly constant. Minutes. Rotation of the glucose solution. 1 . a fc= log t ax 1 a+x KH log t ax 34-1 Temperature, 29-3. 31 33-0 45-9 90-0 Volume, 600 c.c. 91 30-9 47-0 90-0 Amount of yeast : 125 29-7 48-0 90-0 mins. : 0-288grm. 213 26-7 50-6 90-0 dry yeast per 50 c.c. 306 23-5 51-2 91-0 514 mins. ': 0-294 393 20-9 54-1 90-0 grm. dry yeast per 514 17-7 55-4 90-3 50 c.c. 210 GENERAL CHEMISTRY OF THE ENZYMES In the fermentation of similar sugar solutions with varying quantities of yeast, A b e r s o n obtained proportionality between the velocity of reaction and the amount of yeast (1 o c . c i t ., p. 84): Grms. yeast k 60 271 20 93-4 Grms. yeast k 25 165 15 104 SI at or (Journ. Chem. Soc., 1906, 89, 128), who measured volumetrically the carbon dioxide evolved, also found propor- tionality between the velocity of fermentation and the quantity of yeast added. He showed likewise that the initial velocity of fermentation is independent except with very dilute solu- tions of the sugar-content. The fermentation experiments of H e r z o g (H., 1902, 37, 149) and G r i g o r i e w (H., 1904, 42, 299) also refer to hetero- geneous systems. The course of fermentation with permanent yeast (which, however, contained glycogen) is shown by the following table from H e r z o g ' s paper. Concentration, 1-136 grms. glucose (a = l) and 1-2 grms. zymase (perma- nent yeast) in 100 c.c. Temperature 24-5. t a x 10 6 a 10 6 a+x . '*8 t ax . '*S t ax 120 0-961 144 141 240 0-931 129 133 1200 0-687 137 117 1440 0-627 140 118 1740 0-560 145 117 2690 0-403 146 111 3000 0-365 146 108 4140 0-271 137 99 The exponent n of the enzyme-concentration which shows the increase of the velocity of the reaction, is greater in the exper- iments with permanent yeast than in those with the pressed juice. The following table indicates the behaviour of living yeast, acetone-permanent yeast and yeast-juice from a dynamical point of view. EXPERIMENTAL DATA OF ENZYME REACTIONS 211 Reaction constant. Living yeast. Acetone permanent yeast, Zymase solution (pressed juice). k = Influence of the amount of yeast or concentration of zymase log ki log kz 1 a+x 1 l a 1 1 a+x 1 a . Jog t a x 1 k . a i n - creases with in- crease of a log or log t a-x t a-x 2 (Herzog) log t a-x (loc. cit., p. 62) 1-67-1-29 log ci - log c 2 Influence of the concentration of sugar, a k.a increases with diminution of a , within the region' of concentration examined. As has been already stated, B u c h n e r and M e i s e n - h e i m e r arrived at the conclusion that zymase using the word in its wider sense is a mixture of at least two enzymes, namely, zymase in a restricted sense which decomposes fermentable hexoses into lactic acid, and lactacidase, which breaks down the lactic acid into alcohol and carbon dioxide. There has been a considerable amount of discussion concerning the constituent chemical processes which take part in the transformation of sugar into alcohol and carbonic acid. Since grave objections have been advanced to the intermediate formation of lactic acid (compare the critical review byBuchner and M e i s e n - h e i m e r in Landw. Jahrbiicher, 1909, 38, Erganz.-Band 5, 265), nothing final can be said as regards the number 'and nature of the participating enzymes. That a " co-enzyme " plays an important part in fermenta- tion must now be considered an established fact. The same is the case with the observation that addition of phosphates to pressed yeast-juice containing glucose not only accelerates the fermentation but also increases the total fermenting power or the amounts of alcohol and carbon dioxide which can be formed from a given quantity of sugar by the pressed juice (Harden and Young, Proc. Roy. Soc., 1905, 77, 405; 1906, 78, 369; 1908, 80, 299). These investigators have studied quantitatively the accelera- tion of fermentation by phosphates. Of their results, which are 212 GENERAL CHEMISTRY OF THE ENZYMES expressed in the form of tables and curves,the following may be cited (Proc. Roy. Soc., 1908, 80, 307): Time after addition, in minutes. Carbon dioxide evolved in preceding 5 minutes, with n c.c. of 0-3 molar potassium phosphate solution added. n = c.c. n = 10 c.c. n = 15 c.c. 5 4-0 11-1 7-7 10 3-2 16-0 9-7 15 4-2 20-2 12-1 20 3-6 22-4 16-1 25 4-3 17-4 18-4 30 3-6 6-6 19-4 35 4-3 4-6 20-4 40 3-2 4-7 16-7 45 4-5 12-7 50 4-2 6-0 55 ~ 4-1 4-0 It is evident that a marked optimum of phosphate-con- centration exists, the velocity of fermentation diminishing if this is exceeded. The results of another series of experiments are shown graph- ically in the curves given below (Fig. 6). The concentrations of the solutions were as follows : Curve A : 25 c.c. pressed juice + 5 c.c. phosphate +15 c.c. bicarbonate " B : 25 c.c. +10 c.c. +10 c.c. " C : 25 c.c. +15 c.c. + 5 c.c. " D : 25 c.c. +20 c.c. + c.c. " These results suggest that the phosphate is capable of form- ing two or more different unstable associations with the fermenting complex. One of these, formed with low concentrations of the phosphate, has the composition most favourable for the decom- position of sugar, whilst the others, formed with high concen- trations of phosphate, contain more of the latter, probably associated in such a way with the fermenting complex as to ren- der the latter partially or wholly incapable of effecting the decom- position of the sugar molecule." The influence of arsenates and arsenites on the evolution of carbon dioxide produced by yeast-juice has also been studied EXPERIMENTAL DATA OF ENZYME REACTIONS 213 in detail by H a r d e n and Young (Proc. Roy. Soc., B, 1911, 83, 451). " A striking feature of the effect of the addition of a phos- phate to yeast-juice is that the marked acceleration only con- tinues until an amount of carbon dioxide has been evolved which is chemically equivalent to the phosphate added. Moreover, at the close of this period of enhanced fermentation, the added phosphate is no longer present in a form precipitable by magnesium citrate mixture, but has become converted into a hexosephosphate. 5 10 15 20 25 30 50 60 70 .Time in Minutes FIG. 6. Neither of these phenomena occurs when an arsenate is substituted for the phosphate. The enhanced rate of fermentation continues long after an equivalent of carbon dioxide has been evolved and no organic combination of arsenic is formed. The sharp contrast between the actions of arsenate and phos- phate is clearly shown, when the effects of equivalent amounts of phosphate and arsenate on the same sample of yeast-juice are directly compared, as is done in the following experiments." 214 GENERAL CHEMISTRY OF THE ENZYMES 20 c.c. Yeast-juice +5 c.c. 0-3 mol. phosphate. +5 c.c. 0-3 mol. arsenate. +0-75 c.c. 0-3 mol. arsenate Time. Total Rate per 5 minutes. Total. Rate per 5 minutes. Total. Rate per 5 minutes. 5 7-1 7-1 11 4 11-4 21-7 21-7 10 19-8 12-7 26-5 15-1 46 24-3 15 36 1 16-3 43 16-5 71 25 20 43-8 7-7 59 16 95-3 24-3 25 45-7 1-9 75 16-3 119-8 24-5 30 47-5 1-8 " Formation of the Phosphoric Ester Contrary to the opinion of I wan of f (H., 1907, 50, 281; Centralbl. f. Bakt., 1909, II, 24, 1), Harden and Young (Centralbl. f. Bakt., 1911, II, 26, 178) regard the formation of the ester as due, not to a special enzyme, but to the zymase. According to E u 1 e r and K u 1 1 b e r g ' s and E u 1 e r and O h 1 s e n ' s experiments with the extract of dried yeast (H., 1911, 74, 15, 1912, 76, 468), the enzymic formation of the phosphoric ester can, however, be separated from the other fermentative processes, and the author (H., 1911, 74, 13) hence suggests the name phosphatese for this synthesising enzyme. If glucose is fermented by pressed yeast-juice in presence of phosphates, the latter very soon become combined organically. This also occurs with extract of Munich yeast, no matter whether this be dried at 40 or, as von Lebedew suggests, at 25-35; in both cases, as was described by Harden and Young, fermentation and formation of phosphate proceed together. But if another race of yeast with a lower fermenting power is dried at 40 in a vacuum and then extracted in the usual manner, no formation of phosphoric ester occurs with glucose (or mannose or fructose) (Table a, below) . If, however, the glucose is partially fermented beforehand in absence of living yeast, organic combina- tion of the phosphate takes place (Table 6) (E u 1 e r and O h 1 - sen, Biochem. Z., 1911, 36, 313). EXPERIMENTAL DATA OF ENZYME REACTIONS 215 TABLE a. 25 c.c. yeast-extract. 20 c.c. glucose solution (20%). 10 c.c. 5% Na 2 HP0 4 . Minutes. Grms. of Mg 2 P 2 Or from 10 c.c. of the mixture. 150 269 0-0399 0-0394 0-0394 TABLE 6. 25 c.c. yeast-extract. 20 c.c. fermenting glucose solution (2Q%). 10 c.c. 5% Na 2 HP04. Minutes. Grms. of Mg 2 P 2 O7 from 10 c.c. of the mixture. 0-0414 74 0-0134 264 0-0000 Like other enzymes, the phosphatese may be precipitated from the aqueous yeast-extract by means of alcohol, a considerable proportion of its activity remaining after this treatment. It is remarkable that the synthetic enzymic action of the extract is greatly increased by heating it at 40 for half an hour. CATALASES The decomposition of hydrogen peroxide effected by the action of unknown constituents termed, after O . L o e w , catalases of the animal and vegetable body, is so easily followed quanti- tatively, that this process has formed the subject of a large number of investigations and is to-day one of the best- known enzymic changes. Especially from the fatty tissues of animals can solutions be prepared which, beyond their ability to decompose hydrogen peroxide, exhibit few enzymic activities; particularly small are the amounts of organic matter present in these liquids, which must therefore be comparatively pure. Also blood-serum, the organs of plants, etc., by precipitation with alcohol and suitable treatment of the precipitates, yield prepara- tions which, per unit of weight, rapidly decompose hydrogen peroxide (O . L o e w , Rep. U. S. Dep. of Agric., 1901, No. 68; S enter, Zeitschr. f. physikal. Chem., 1903, 44, 257 and Proc. Roy. Soc., 1904, 74, 201; E. J. Lesser, Zeitschr. f. Biol., 1906, 48, 1; L. van Italie, Soc. Biol., 1906, 0, 150; K a s 1 1 e and Loevenhart, Amer. Chem. Journ., 1903, 29,563; L.von Liebermann, Pfliig. Arch., 1903, 104, 176 and Chem. Ber., 1905, 38, 1524). Catalase follows the theoretically simplest formulae and relations with an approximation shown with few other enzymes, 216 GENERAL CHEMISTRY OF THE ENZYMES possibly because the simple nature of the chemical process con- ditions especially simple relations, or possibly owing to the exclusion of any considerable complication by the relatively high degree of purity of the enzyme. The most important general conclusions arrived at are as follows : The enzymic decomposition of hydrogen peroxide in neutral or acid solution is a reaction of the first order. In dilute solutions of the peroxide, decomposition follows the law of mass action exactly. Numerous physico-chemical measure- ments of the action of catalases have been carried out, the fol- lowing series of numbers being given by G. Senter and by H. Euler : Blood-catalase. Senter, loc. cit., p. 282. jJs-molar H2O2-solution. Fungus-catalase. Euler, Hofm. Beitr., 1905, 7, 1. 2 Vmolar H 2 O 2 -solution. Minutes. a x fc.10*. T=0. Minutes. a x. k.lW. r = i5. 46-1 8-0 5 37-1 190 6 6-9 107 10 29-8 192 12 5-8 116 20 19-6 190 19 5-0 107 30 12-3 193 55 2-5 100 50 5 194 I s s a e w (H., 1904, 42, 102) also obtained very constant values of k with dilute solutions of hydrogen peroxide; with more concentrated solutions (0-01 N and above) the constants diminish. Catalases are extremely sensitive preparations of different origins to varying degrees towards higher concentrations of hydrogen peroxide. Thus, blood-cat alase is appreciably destroyed (perhaps oxidised) in about 0-01 N-H2O2, this .action being naturally more rapid at higher than at low temperatures, the values of k being greatly diminished even at 30. It is hence advisable to employ low temperatures in working with catalase. According to Waentig and Steche (H., 1911, 72, 226), this injurious action is observable even at in about N/80- hydrogen peroxide solution. The reaction constants are, as they should be theoretically, nearly independent of the concentration of the peroxide, equal percentage amounts being decomposed per EXPEKIMENTAL DATA OF ENZYME REACTIONS 217 unit of time by equal quantities of enzyme. Senter (loc. c i t . , p. 283) gives the following resume: CH 2 0, /c.10 4 CH 2 o 2 fc.10 3 Cn 2 o 2 fc.10 3 290* 120 i N 175 Io^ N 192 1 N 122 AT 188 1 225 1100 N 460 (The two values of k in each of these pairs are comparable, one with the other, but not with those of the other pairs.) The following results, given by Senter, indicate the influence of the concentration of the enzyme: Concentrations, E, in the proportions 3 6 8 24 fc.10 4 28 58 72 230 k : C 9-3 9-7 9-0 9-6 Within the limits of experimental error the velocities of reaction in very dilute (^iir-niolar) solutions of hydrogen peroxide are therefore proportional to the concentrations of the enzyme. With stronger peroxide solutions, Senter found deviation from this rule. While fc.10 4 is about 100 in weak catalase solutions, its value in presence of the threefold quantity of enzyme is approx- imately 360. That, under these conditions, the velocity of reac- tion increases more rapidly than the concentration of the enzyme, is confirmed by experiments made by Bach, who investigated the dynamics of the catalases with a preparation from beef-fat (Chem. Ber., 1905, 38, 1878). With N/210-peroxide solutions, I s s a e w observed deviation from proportionality in the opposite direction, that is, a slower increase of the velocity than of the enzyme-concentration. But the yeast-catalase he employed must, like all yeast-enzymes, have been very impure. As was shown originally by L o e w , catalases are extremely sensitive to acids. The action of acid has also been the subject of numerous quantitative investigations, which show that the enzyme is not permanently changed by acids, neutralisation of these being accompanied by return of the catalytic activity. 218 GENEKAL CHEMISTRY OF THE ENZYMES 100 c.c. of catalase solution were poisoned, mixed with 100 c.c. of H2O2 solution and 25 c.c. then titrated; concentration of H2(>2 in the mixture 0-005 -normal. snVu norm. HC1. Tffs0o norm. HC1. Without addition. Minutes CH 2 2 /c.10 4 Minutes CH 2 2 /c.10 4 Minutes Cn 2 o 2 fc.10 4 27-9 27-9 27-9 70i 25-3 6 15* 25-6 26 5| 19-8 278 136 23-0 6 35| 22-4 27 15 10-5 275 195 20-9 6 66^ 18-7 26 25i 5-8 258 1305 3-0 6 185 10-1 24 Yuggg-norm. HOI. Incubation period, 2 hours; then a small excess of NaOH added, and afterwards the H 2 O 2 . Without addition. Minutes. CH O! fc.10 4 Minutes. CH 2 O 2 fc.10 4 28-7 28-7 5| 15* 23-8 18-5 121 113 5 25-2 16-9 120 121 26i 14-9 86 31* 11-9 127 41* 11-7 70 53 6-5 120 As is shown by the latter table, the catalase is not changed by the dilute hydrochloric acid solution, even after two hours. The incubation period, that is, the time during which the enzyme remains in contact with the acid, has been repeatedly shown to be without influence on the subsequent activity of the enzyme. The catalytic power of catalases is also reduced by quite small amounts of baryta. Temperature, 10 TTO norm. Ba(OH) 2 . ah norm. Ba(OH) 2 . Without Catalase from fatty tissue Incubation period, Incubation period, addition (E u 1 e r , Hofm. Beitr., 15 minutes. 1905,7,12) fc.!0 3 = 40 40 minutes. A;.10 3 =40 of baryta. /c.10 3 =60 This sensitiveness towards alkali varies considerably with catalases of different origins. A very slight increase of the alkalinity, however, appears to Increase the velocity of the decomposition to some extent. Thus, the author found (1 o c . c i t . ) that the velocity of reaction of catalase from Boletus scaber is doubled by suspending pure magnesium hydroxide in the solution. EXPERIMENTAL DATA OF ENZYME REACTIONS 219 As is brought out most clearly by'Sorensen's results '(Biochem. Z., 1909, 21, 131), the optimal activity of catalase is always exhibited in almost neutral solution. The actions of acids and bases on this enzyme depend, in all probability, on the formation of salts by these electrolytes with the catalase. Bach (Chem. Ber., 1905, 38, 1878) has also investigated catalase quantitatively, his results in general agreeing with those required by theory. OXYDASES Substances of unknown constitution and composition, formed in the animal and vegetable kingdoms and capable of initiating oxidation changes, have been designated oxydases, generally without any definite proof of their mode of action; their sensitive- ness to heat was, however, established and they were then classed with the enzymes. That these substances effected catalytic acceleration of oxidation processes was seldom, and could indeed only with difficulty be, proved, especially when isolated con- stituents of an organ or juice were not examined. On the other hand, many other enzymes are not catalysts in the strictest sense of the word, so that no limit could easily be drawn. The wide distribution of oxydases in vegetable and animal organisms ren- ders it probable that these substances perform an important function in all life-processes; but what this function is, what reactions the oxydases bring about in the living animal or plant, still remains unknown. The members of this class of bodies which have as yet been obtained exhibit a somewhat limited sphere of action. Aldehydases Medwedew has made a very complete study of the oxidising agent of the liver, with reference to its action on salicylic aldehyde (Pfliig. Arch., 1896, 65, 249; 1899, 74, 193; 1900, 81, 540 and 1904, 103, 403). As regards the final state or equilibrium, the following results were obtained. Case 1 : relatively high concentration of salicylic aldehyde in neutral-acid solution. The concentration of the oxidation product (salicylic acid) is inversely proportional to the square-root of the amount of substance to be oxidised and approx- 220 GENERAL CHEMISTRY OF THE ENZYMES imately proportional to the square of the concentration of the aldehydase. Case 2: relatively high concentration of salicylic aldehyde in neutral-alkaline solution. One and the same quantity of the oxydase gives at the end of the reaction, that is, on complete exhaustion of the oxidising power, one and the same amount of acid, no matter what the concentration of the aldehyde. In relation to the velocity it was found : (a) If to the quantity of oxydase m an excess of aldehyde a is added, the velocity of oxidation is proportional to the square-root of the concentration of aldehyde. In Medwedew's opinion, liver-oxydase is inactivated by the oxidation. But this must, in reality, only be a question of the consumption of an oxidising agent obtained from the liver. (b) If the concentration of aldehyde is less than that which the oxydase present is able to oxidise, the velocity of oxidation is proportional to the square of the aldehyde-concentration, so that dx : dt = k(ax) 2 , where x is the concentration of the alde- hyde changed up to time t and a the initial concentration. How far these relations are quantitatively reproducible and hence are independent of the non-controllable composition of the liver-extract, and how far this interpretation of the numbers obtained is fitting, remain undecided. Doubts have, however, been expressed on these questions (compare Bach's remarks, Chem. Ber., 1905, 38, 3791) ; but D o n y - H e n a u 1 1 and van Duuren's experiments have led to different results (Bull. Acad. roy. Belgique, 1907, 577). Slowtzpff's observation (H., 1900, 31, 227) that " potato- laccase " oxidises paraphenylenediamine solution with a velocity proportional to the square-root of the quantity of this " laccase," also appears to the author to be insufficiently proved. What Slowtzoff investigated must have been a very impure mixture of a peroxydase and a substance Allied toMedicago- laccase. The author obtained the latter component from potatoes by precipitation with alcohol. The nitrogen-content of the first precipitate amounted to 2 84% ; after solution of the prepa- ration, treatment with animal charcoal and further precipitation with alcohol, the proportion of nitrogen present fell to 1-6%, whilst the ability to accelerate the oxidation of hydroquinone solutions in presence of manganese salts remained undiminished. EXPERIMENTAL DATA OF ENZYME REACTIONS 221 As regards the " laccases," the investigations of E u 1 e r and B o 1 i n (H., 1908, 57, 80; 1909, 61, 1 and 72) show that the oxydase of Rhus vernicifera and Rhus succe- d a n e a differs considerably from those of Medicago s a t i v a , etc., which, according to Bertrand, are also, termed laccases. As Bertrand himself showed, the Rhus- preparations are rich in manganese, whilst this is not the case with laccases of the Medicago -type. The former alone turn guaiaconic acid solutions blue directly and redden solutions of guaiacol. This effect of Rhus -laccase and also the power to transfer molecular oxygen to phenols (hydroquinone, pyrogallol) are destroyed by heating the solution to 100 for a short time, whilst Medicago -laccase is unchanged by heating. In the case of Rhus -laccase, Bertrand (Bull. Soc. Chim., 1897, [iii], 17, 619) established approximate proportionality between the manganese-content and the catalytic activity. The extraordinary sensitiveness of this preparation to acid is shown by the following measurements (Ann. Inst. Pasteur, 1907, 21, 673). The oxidation of guaiacol to tetraguaiacoquinone was measured. To a 2% guaiacol solution were added a little (0 1 grm. per litre) laccase (from Rhus succedanea) and sufficient sulphuric acid to give the mixture the acidity shown in the table. The numbers represent the intensities of the red colour measured in a colorimeter produced after 5 hours by the tetraguaiacoquinone formed. Normality of the acid. Serie3 1. Series 2. 1 100 100 100 73.4 48-8 20-3 100 90-9 75-2 60-4 60-4 60-4 500,000 1 400,000 1 200,000 1 100,000 1 50,000 222 GENERAL CHEMISTRY OF THE ENZYMES Oxydases of the M e d i c a g o -type, in presence of neutral manganese salts, accelerate the transference of molecular oxygen to the poly phenols. A representation of the course followed by this action is given by the following experiments : 50 c.c. of a solution, 0-2-normal as regards hydroquinone and 0-001 -equivalent normal as regards manganese acetate, and containing a weighed quantity of Medicago -oxydase, were shaken in a glass vessel from which the air had been replaced by pure oxygen. The oxidation of the hydroquinone to quinone or quinhydrone, produced by the manganese and oxydase, was measured by the diminution in the volume of oxygen in the vessel. 0-2 grm. oxydase per 50 c.c. 1 grm. oxydase per 50 c.c. Minutes. Oxygen absorbed (c.c.). Minutes. Oxygen absorbed (c.c.). 5 1-8 5 1-0 10 2-3 10 1-7 15 3-0 15 2-2 20 4-1 20 2-5 30 5-9 30 3-1 It has been shown that Medicago -oxydase is a mixture of calcium salts of organic mono- and poly-basic hydroxy-acids, among which are glycollic, citric, malic and mesoxalic acids. The catalytic action of these pure salts corresponds closely with that of Medicago -oxydase, as is shown by the following numbers obtained under the conditions described above. 0-2 grm. calcium oxalate per 50 c.c. 1 grm. calcium glycollate ] 0-05 grm. calcium malate ^ per 50 c.c. 0-05 grm. calcium mesoxalate J Minutes. Oxygen absorbed (c.c.). Minutes. Oxygen absorbed (c.c.) 5 1-8 5 2-3 10 2-9 10 3-4 20 4-5 15 4-1 30 5-7 20 4-8 30 5-9 As is well known, the oxidation of polyphenols, either alone or, more markedly, in presence of manganese salts, is considerably EXPERIMENTAL DATA OF ENZYME REACTIONS 223 increased by small quantities of alkali, so that especial care must be taken as regards the neutrality of the oxydase and of the calcium salts. In the above investigation they were absolutely neutral. Since the salts of all the aliphatic hydroxy-acids appear to be more or less active in this direction, oxydases of this group must be of very frequent occurrence in the vegetable kingdom. The indophenol reaction, which was first employed by Ehrlich in 1885 and consists in the formation of indophenol from a-naphthol and ^-phenylenediamine, has been used recently by V e r n o n (Journ. of PhysioL, 1911, 42, 402) as the basis of a quantitative method. With the help of his method, this author has investigated quantitatively numerous oxidation phenomena induced by tissues. According to the concentration of the substrate (naphthol and diamine), the amount of indophenol formed was found to be proportional either to the square of the quantity of enzyme, or to this quantity itself or to its square root. PEROXYDASES Presumably still more widespread and consequently of more general action are those substances of the animal and vegetable body which activate 'peroxides, including hydrogen peroxide, i.e., transfer the peroxide-oxygen to other substances; these are termed peroxydases. The changes which the peroxydases themselves undergo, during this transference of oxygen from the peroxides to sub- stances like a-guaiaconic acid, are unknown, and it has often been doubted whether the peroxydases should really be regarded as catalysts and as enzymes. Our conception of the latter, in particular, is so indefinite, that at the present time, when so little is known concerning the exact chemical nature of the peroxydases, discussion of this question is of little value. Owing more especially to the work of J. Wolff and E. d e S t o e c k 1 i n (C. R., 1911, 153, 139), substances such as potas- sium ferrithiocyanate, K 3 Fe(CNS) 6 , are however known which are not of organic origin and yet behave like the peroxydases. 1 1 It appears to be established that, in most biological oxidations which are regarded as enzymic, the true oxidising agent is peroxydic in character. This view was advanced almost simultaneously by C h o d a t and Bach 224 GENERAL CHEMISTRY OF THE ENZYMES We shall proceed at once to a consideration of the numerical relations due to C h o d a t and Bach and their pupils characterising the action of these enzymes. In the experiments which will first be described (Chem. Ber., 1904, 37, 1342), definite quantities of peroxydase (from horseradish), hydrogen peroxide and pyrogallol were mixed in aqueous solution, . the purpurogallin formed in 24 hours being collected on a tared filter, washed with 100 c.c. of water, and dried at 110 until of constant weight. Pyrogallol is not appre- ciably attacked by either peroxydase or hydrogen peroxide alone. Three series of experiments were carried out : A. With variation only of the amount of peroxydase. B. With variation only of the amount of hydrogen peroxide. C. With variation only of the amount of pyrogallol. Temperature, 15-17. Volume of the mixture, 35 c.c. A HYDROGEN PEROXIDE, 0-10 GRM.; PYROGALLOL, 1 GRM. Amount of peroxydase, in grm. : 0-01 0-02 0-03 0-04 0-05 0-06 0-07 0-08 0-09 0-10 Purpurogallin formed, in grm.: 0-021 0-042 0-066 0-083 0-102 0-123 0-145 0-166 0-167 0-162 B PEROXYDASE, 0-10 GRM.; PYROGALLOL, 1 GRM. Hvdrogen peroxide, in grm.: 0-01 0-02 0-03 0-04 0-05 0-06 0-07 0-08 0-09 0-10 Purpurogallin, in grm.: 0-205 0-42 0-60 0-78 0-99 0-121 0-142 0-168 0-168 0-165 Experiments A show very clearly that with a constant amount (in excess) of hydrogen peroxide, the yields of purpurogallin are exactly proportional to the quantities of peroxydase employed; whilst, with varying amounts of hydrogen peroxide and a constant quantity (in excess) of peroxydase, the amount of change is, as experiments B show, proportional to the former. An excess of peroxydase or of hydrogen peroxide is without influence on the oxidising capacity of the system peroxydase- hydrogen peroxide. From these results Bach and C h o d a t (Chem. Ber., 1902, 35, 1275, 2487, 3943; 1903, 36, 600, 606, 1756) and by K as tie and Loevenhart (Amer. Chem. Journ., 1901, 26, 539). The application of these noteworthy theories is often hindered by the un- certainty of their chemical foundations, so that the theoretical results need not be repeated here. EXPERIMENTAL DATA OF ENZYME REACTIONS 225 drew the conclusion that peroxydase and hydrogen peroxide take part in the reaction always in constant relations. They arrived thus at the hypothesis which had been previously advanced by K a s 1 1 e and Loevenhart (Amer. Chem. Journ., 1901, 26, 593), namely, that the peroxydase forms with the hydrogen peroxide a definite compound exhibiting more energetic oxidising properties than the peroxide alone. The result obtained by the two first-named investigators that excess of hydrogen peroxide does not affect the change indicates that the peroxydase is not used up during the course of the oxidation. c PEROXYDASE, 0-10 GRM.; HYDROGEN PEROXIDE, 0-10 GRM. Pyrogallol, in grm. 1-5 2 3 4 Purpurogallin, in grm. 0-205 0-203 0-208 0-202 From the results of experiments C it is seen that the concentra- tion of the pyrogallol is without influence on the magnitude of the change. Since the formation of purpurogallin and the procedure described above are not suitable for the determinination of the time-law of peroxydase-action, Bach and C h o d a t (Chem. Ber., 1904, 37, 2434) chose for this purpose the oxidation of hydrogen iodide. Five c.c. of a solution, g^^-normal with respect to acetic acid and hydrogen peroxide, were added to 45 c.c. of a solution containing ^^-equivalent of potassium iodide and varying quantities of peroxydase. The iodine separated after a certain time was estimated by titration with thiosulphate. 1-25 -EQUIVALENT PEROXYDASE * luU.uUU Minutes 1 2 4 6 8 10 12 20 c.c. thiosulphate. 1-3 2-4 4-4 6-1 7-1 7-9 8-1 8-4 PARALLEL EXPERIMENT WITHOUT PEROXYDASE Minutes 2 4 6 8 10 12 20 c.c. thiosulphate 0-25 0-45 0-6 0-8 0-9 1-1 1-6 2-5.10~ 5 -EQUIVALENT PEROXYDASE Minutes 1 2 4 6 8 10 20 c.c. thiosulphate 2-4 4-7 8-4 10-2 11-7 11-8 12-3 * The peroxydase-preparation was the same as irf the above experiments. It activated exactly its own weight of hydrogen peroxide, or, according to Bach and C h o d a t ' s mode of expression, it had the activating power 1. 226 GENERAL CHEMISTRY OF THE ENZYMES 5 . 10~~ 5 -EQUIVALENT PEROXYDASE Minutes '. . . 1 2 46 8 10 20 c.c. thiosulphate 4-9 8-9 14-0 14-7 15-0 13-1 15-4 10.10~ 5 -EQUIVALENT PEROXYDASE Minutes 1 2 4 6 8 10 20 c.c. thiosulphate 10-1 15-7 17-0 17 1 17-2 17-2 17-4 15.10~ 5 -EQUIVALENT PEROXYDASE Minutes 1246 8 10 20 c.c. thiosulphate 15-0 18-6 19-1 19-1 19-2 19-2 19-4 It will be seen first of all that, in the oxidation of hydriodic acid just as in that of pyrogallol, the peroxydase ultimately loses its activity, since after some time the procedure of the reaction becomes exactly that exhibited when no peroxydase is added; the rate at which the activity of the peroxydase diminishes increases with the concentration of the peroxide. Hence the results obtained with different concentrations of peroxydase are comparable only for those phases of the reaction where the enzyme still exerts almost its full activity This is still the case at the end of the first minute, after which time the magnitudes of the change are exactly within the limits of experimental error- proportional to the amounts of peroxydase: Peroxydase-concentration X 10 5 2-5 5 10 15 Amount of change after 1 minute (c.c.) 2-4 4-9 10-1 15-0 Comparison of the final states reached in the system hydriodic acid-peroxydase-hydrogen peroxide shows that the amount of the product of the reaction (iodine) is not, as with the oxidation of pyrogallol, directly proportional to the quantity of peroxydase, but increases more slowly than the latter. It /y is to be noted that the activating power of the peroxydase, - a (where x denotes the amount of hydrogen peroxide activated by a weight a of peroxydase), is considerably greater in the oxidation of hydriodic acid by hydrogen peroxide than in the oxidation of pyrogallol. These experiments with hydriodic acid were extended by Bach (Chem. Ber., 1904, 37, 3785), who determined the EXPERIMENTAL DATA OF ENZYME REACTIONS 227 separation of iodine occurring in 10 minutes at 22 in differently concentrated mixtures of HI, H202 and horseradish-peroxydase ; his results are collected in the following table : Increase of the change measured in c.c. of 0-01 N-thiosulphate. Peroxy- dase. I. 12-5HI. II. 25HI. ill. 37-5HI. IV. 50HI. V. 75 HI. VI. 100HI. A 1-25 3-1 4-4 5-0 5-4 5-3 5-4 B 2-5 5-1 7-1 7-9 8-5 8-6 8-5 C 5-0 7-0 9-5 11-3 13-2 13-6 13-6 D 10-0 9-3 12-9 15-7 17*9 21-4 25-1 E 15-0 10-9 15-9 18-5 21-6 26-0 29-3 F 21-0 10-7 17-6 21-4 25-5 29-1 33-6 G 25-0 10-8 17-8 24-0 28-4 32-6 37-1 Control 0-7 1-4 2-0 2-7 4-1 5-4 The increase in the amount of action in a given time rises both with the concentration of the peroxydase and with that of the hydriodic acid and, for each concentration of enzyme and acid, attains a certain limiting value and then remains constant; it must therefore be concluded that peroxydase, hydriodic acid and hydrogen peroxide react together in definite proportions. It is readily seen from the preceding table that the pro- duct of the concentrations is (within certain limits) constant; for instance, DXl = CXlI, etc. Finally, comparison of the concentrations of hydriodic acid which correspond with different increments in the change shows that these increments are almost exactly proportional to the square-roots of the concentrations of the acid; this is evident from the following summary: I. II. III. IV. V. VI. Series D 9-2 12-9 15-7 17-9 21-4 25-1 Calculated 9-2 13-0 15-9 18-6 22-4 25-9 Series E 10-9 15-9 18-5 21-6 26-1 29 3 Calculated 10-9 15-3 18-8 21-8 26-5 30-7 Bach did not, however, obtain such regularity with another preparation, this giving a different relation, which had not pre- viously been observed. 228 GENERAL CHEMISTRY OF THE ENZYMES Increase of the change measured in c.c. 0-01 N-thiosulphate. Peroxy- dase. 12-5HI. II. 25HI. ill. 37-5HI. IV. 50HI. v. 75HI. VI. loom. A 1-25 1-2 2-4 3-3 4-2 4-2 4-1 B 2-50 2-2 4-2 6-1 8-1 8-3 8-2 C 5-0 3-6 6-0 9-4 .12-1 15-2 15-8 D 10-0 4-4 8-3 12-2 14-6 20-7 25-9 E 15-0 5-0 9-6 13-8 18-5 27-4 36-6 F 20-0 5-0 10-1 15-1 20-1 30-2' 41-0 G 25-0 5-1 10-2 15-6 20-4 30-0 40-8 Control 0-7 1-6 2-2 2-9 4-1 5-6 The results of series F and G show that, after the p e r - oxydase-maximum is reached, the increase in the amount of t r a n s f o r m a t i o n i s e x a c 1 1 y proportional to the concentration of the hydriodic acid. For the complete utilisation of peroxydase, a definite acidity (concentration of the hydrogen-ions) of the liquid is necessary, the nature of the anion of the acid being without influence. The darkening of many plant-juices owing to enzymic oxida- tion was found by G r a f e and Weevers to be conditioned by the presence of catechol and this discovery has recently been confirmed by Miss Wh el dale (Proc. Roy. Soc., B, 1911, 84, 122). The study of the oxidation of catechol by plant-juices from a kinetic standpoint would be of great interest. TYROSINASE In conjunction with an investigation on melanotic pigments and the enzyme formation of melanins, 1 O. von Ftirth and E. Jerusalem (Hofm. Beitr., 1907, 10, 131) studied the mode of action of tyrosinase. The very complicated relations found by these authors led Bach (Chem. Ber., 1908, 41, 216, 221), who regarded tyrosinase as a mixture of an oxygenase and a peroxydase, to establish the conditions of action of this enzyme. The amounts of melanin formed were determined as follows: The juice was diluted tenfold with distilled water and 10 c.c. of this solution 1 A step towards the elucidation of the chemistry of tyrosinase action has been made in a valuable investigation by Abderhalden and Guggenheim (H., 1907, 54, 331). EXPERIMENTAL DATA OF ENZYME REACTIONS 229 mixed with 10 c.c. of tyrosine solution (containing 0-05% of tyrosine and 0-04% of sodium carbonate) and 30 c.c. of water; after 24 hours, the solution was acidified with 1 c.c. of 10% sulphuric acid and titrated with 0-002-normal permanganate until the latter was decolorised. The tyrosinase was extracted from Russula delica. 1. Dependence of melanin-formation on the concentration of tyrosinase. Into each of a series of eight beakers were placed 10 c.c. of the tyrosine solution and a certain quantity of the enzyme solution, the volume being then made up to 50 c.c. with water. Permanganate solution used, in c.c. I. II. .III. IV. V. VI. VII. VIII. Volume of enzyme solution (c.c.) 0-5 1-0 1-5 2-0 5-0 10-0 15-0 20-0 A After 24 hours 10-8 14-2 17-3 19-8 25-8 30-4 33-6 35-8 B. After 48 hours 13-2 16-0 17-8 20-4 25-6 31-2 34-4 35-4 These results show that : (1) the amount of the product of the reaction increases with that of the enzyme, although more slowly than the latter, and (2) the rapidity with which the reaction comes to a standstill increases with the concentration of the enzyme. In these respects, tyrosinase behaves similarly to horse- radish-peroxydase (see p. 227). 2. Velocity of reaction and concentration of enzyme: Three 750 c.c. Erlenmeyer flasks were each charged with 100 c.c. of tyrosine solution, a certain volume of enzyme solution, and water up to a volume of 500 c.c. Quantity Time in 1 of enzyme solution per 500 c.c. . lours : 1 10 c.c. c.c. 0-0 20 c.c. c.c. 1-4 30 c.c. c.c. 2-8 2 0-0 3-9 5-7 3 1-6 5-8 8-8 4 2-7 7-8 11-1 6 5-5 11-1 16-1 * ' 9 9-4 16-3 20-8 14 15-9 19-0 22-3 24 16-0 19-9 2-8 Volume of permanganate solution required. 230 GENERAL CHEMISTRY OF THE ENZYMES A reaction constant can scarcely be calculated with any degree of certainty, since the position of the end-value is doubtful. But if the times corresponding with equal amounts of change are compared, inverse proportionality between the amount of enzyme and the time of reaction is clearly evident: the product of the quantity of enzyme and the time is hence constant. That the initial and final stages of the reaction must be disregarded is explained by the slower initiation of the reaction with low than with higher concentrations of enzyme, whilst these higher con- centrations lead to more rapid inactivation of the enzyme. 3. Velocity of reaction and concentration of substrate: Three flasks, containing 25, 50 or 75 c.c. of the tyrosine and 30 c.c. of enzyme solution diluted to 500 c.c. Volume of permanganate solution required. Volume of tyrosine solution, in c.c After 1 hour 25 1-0 3-1 4-8 7-0 8-4 50 1-9 6-3 9-4 12-4 12-6 75 3-0 9-2 13-9 14-7 16-2 1 ' 3 hours " 5 " " 8 " 11 24 " With a constant concentration of enzyme, the quantity of melanin formed per unit of time is apart from the final stages of the reaction proportional to the amount of tyrosine present. Tyrosinase hence corresponds well with the law of mass action. OXIDATION OF XANTHINE In conclusion, it may be pointed out that B u r i a n (H., 1905, 43, 497) has investigated dynamically the oxidation of xanthine to uric acid. This reaction is, however, not one of pure oxidation, so that mention of this paper must suffice. CHAPTER V INFLUENCE OF TEMPERATURE AND RADIATION ON ENZYMIC REACTIONS TEMPERATURE influences chemical systems in two ways: It is, first of all, one of the factors which determine the posi- tion of equilibrium between the substances taking part in a rever- sible reaction. The degree to which the equilibrium changes with the temperature in any case is closely related to the heat- change of the reaction. If the equilibrium constant of a reaction is indicated, as before, by K, while U denotes the total heat- change determined calorimetrically, T the absolute temperature and 72 the gas-constant, van't Hoff's fundamental thermo- chemical law states that : U dT ' R.T 2 ' If, therefore, the heat-change accompanying any reaction is small and this is the case with most enzymic processes t h e equilibrium is only slightly dependent on the temperature. A much greater alteration with temperature is shown by the velocity with which a system proceeds towards its equilibrium or final position. In most cases, a rise of temperature of 10 doubles or trebles this velocity a phenomenon to which van't Hoff first directed attention. With non-enzymic reac- tions, indeed, still higher temperature-coefficients are observed. From the abundant experimental data, the following figures, referring to reactions of biological interest, may be quoted: Author. Reaction. Jemp^ *?10 ^ Price, Svenska Vet. Akad. Forh, 1899 . . Ethyl acetate +H 2 O 28-50 2 4 17,390 E u 1 e r , Chem. Ber., 1905, 38, 2551 ..... Formaldehyde +NaOH 50-85 3 6 24,900 S p o h r , Z. physikal. Chem., 1888, 2, 194 . Inversion of cane-sugar 25-55 3-6 25,600 231 232 GENERAL CHEMISTRY OF THE ENZYMES k The values of the quotient, ^ 10 , hold only for a certain hi interval of temperature, the increase of the velocity of reaction per degree diminishing with rise of temperature. On the other hand, the constant [L retains its value over a very wide range of temperature. This constant is given by the formula derived theoretically byArrhenius and found to be generally valid : /* (T,-TA k 2 = k l eR\ T lTz ) t (23); it represents therefore an exact expression for the dependence of the Velocity of reaction on the temperature. In this equation, ki and 2 denote the reaction-constants at the absolute tem- peratures TI and T2, while R is the constant of the gas-laws and e the base of the natural system of logarithms. An influence as great as on the velocity of chemical reactions is exerted by the temperature on the vapour pressure of liquids and on the equilibria of certain dissociations. In the latter case this is explainable on the assumption that organic reactions are also effected by ions. The constant [L gives the heat of transformation accompanying the conversion of the participating molecules from the " normal " into the " active " state, and hence corresponds with the sum of the heats of dissociation of the substances taking part. If, for instance, the inversion of cane- sugar is considered, [i is the sum of the heat of dissociation of water, t/idi ss . and that t/2diss.> f cane-sugar (the latter taken as a base). The heat of dissociation of water is 13,450 Cals.; the value of E/2diss. is still unknown, but it is known that with such extremely weak electrolytes as cane-sugar must be, the heats of dissociation are, in general, approximately of the same magnitude as that of water. On this assumption for t/2diss.> ^ would be 26,900, while calculation of the experimental results according to A r r h e n i u s 's formula leads to the value 25,600 (E u 1 e r , Zeitschr. f. physikal. Chem., 1904, 47, 353). The influence of temperature on the compound, substrate-catalyst (i.e., the hydrochloride, sulphate, etc., of the cane-sugar) is here apparently neglected. But, since the dissociation of strong acids and of salts changes comparatively slightly with the temperature, this influence is determined principally by the heats of dissociation of the cane-sugar and the water. INFLUENCE OF TEMPERATURE AND RADIATION 233 On the velocity of enzyme reactions, temperature has a twofold influence : that just referred to and, in addition, an action on the activity of the catalysing enzyme, which becomes more rapidly destroyed or permanently inactivated as the tem- perature rises. The processes resulting from these two actions were first treated, theoretically and experimentally, by Tammann (Zeitschr. f. physikal. Chem., 1895, 18, 426). The simplest assumption is that the enzyme is inactivated in aqueous solution by a unimolecular reaction, independently of whatever else is in the solution. So that, if E is the initial concentration of the enzyme and y the concentration at time t, then: hence E-y = E.e~ k E, . . , .... (24) where e is the base of the natural system of logarithms, and If further, a denotes as usual the original concentration of the substrate, which undergoes decomposition according to an equation of the first order, it follows that v = ~ = k(a-x)(E-y), ...... (26) that is, the velocity with which the reaction proceeds at time t must be equal to the product of the amounts of enzyme and sub- strate still present. Substitution in the last equation of the value of E y from Eq. (24) gives, as Tammann showed, the integral equation: (27) 234 GENERAL CHEMISTRY OF THE ENZYMES From Eq. (27), T a m m a n n calculated the "false equi- librium," that is, the final state of the enzymic reaction, and compared the result with that determined experimentally with the system emulsin-salicin. The following tables are taken from those given by Tammann. To solutions of salicin, previously heated, were added varying quan- tities of emulsin, 100 c.c. of each of the mixtures containing 3 -007 grms. of salicin and the amount of enzyme shown in the table. PERCENTAGE OF SALICIN HYDROLYSED Emulsin (grin.). 72 hours. 104 hours. 148 hours. 0-250 63-4 65-1 65-4 } 0-125 48-3 50-2 50-4 \ Temp., 65 0-0312 16-4 17-0 16-8 J Emulsin (grm.). 0-250 0-125 0-0156 45 hours. (101-2) 97-5 59-3 86 hours. 99-2 97-5 65-7 166 hours. (100-2) 67-6 Temp., 45 c Emulsin (grm.). 45 hours. 93 hours. 334 hours. 0-250 96-5 98-0 100.2 ) 0-125 96-5 97-5 99-6 [ Temp., 26 0-0156 85-8 92-1 98-0 J As will be seen, the higher the temperature, the sooner the reaction comes to a standstill, i.e., the more rapidly the enzyme is inactivated. It must particularly be pointed out that a distinction is to be made between the permanent non-reversible inac- tivation which every enzyme undergoes, especially at higher tem- peratures, and the inactivation due to the products of reaction and disappearing when these are removed. In the latter case, where the catalysing enzyme is held by the reaction-products, the reaction retards itself by consuming its own catalyst. Such catalytic retardation has been treated by O s t w a 1 d (Lehrbuch, II, 2, 271). The differential equation for this case, INFLUENCE OF TEMPERATURE AND RADIATION 235 gives, on integration, ki(Ak 2 k As O s t w a 1 d pointed out, k 2 x at the beginning of the reaction must not be greater than fci, as otherwise the reaction does not take place. The reaction therefore leads here to a " false equilibrium " or end-state. The influence of rise of temperature on an enzymic reaction is hence two-fold: 1, Acceleration of the reaction and, 2, inactivation of the enzyme. So that both k and ks increase as the temperature rises, and when the influence of temperature on an enzymic reaction is to be defined, the temperature-coefficient should be give n a s was done by Tammann b oth for k and for k E- The temperature-function of the two magnitudes is best expressed by Arrhenius's formula. Owing to the dependence of the stability of the catalyst on the temperature, the temperature-curves of enzymic reactions differ in appearance from those of most other chemical processes. Thus, they show an optimum, the temperature-coefficient at a certain temperature being zero, owing to the increased velocity of decomposition of the substrate being exactly compensated by the increased rate of destruction of the enzyme; further rise of temperature is then accompanied by decrease of the velocity of reaction. It was assumed on p. 233 that the decomposition of enzymes obeys the formula for unimolecular reactions. 1 This is best ascertained by keeping the enzyme for a certain time in aqueous solution at the temperature to be investigated, then mixing it where possible at a lower temperature with the substrate and calculating the constant k E from the initial velocity. The values of k E obtained at different temperatures then give the constant y. of the temperature-formula of Arrhenius given on p. 232, 1 Whether all enzymes decompose according to this law is still question- able. According to S e n t e r it is not the case with catalase. 236 GENERAL CHEMISTRY OF THE ENZYMES Spontaneous decomposition of: ji Emulsin in 0-5% solution 45,000 T a m m a n n Rennet in 2% solution l 90,000 ] Trypsin in 2% solution 62,034 V M a d s e n and W a 1 b u m * Pepsin in 2% solution 2 75,600 J Yeast-invertase 72,000 E u 1 e r and a f U g g 1 a s Dry emulsin 26,300 T a m m a n n For yeast-invertase, E u 1 e r and K u 1 1 b e r g (H., 1911, 71, 134) have shown that (JL is independent of the impurities arising from the yeast and hence represents a well-defined constant. Mention must also be made of the measurements made by N i c 1 o u x with lipase (Soc. Biol., 1904, 56, 701, 839, 868), the value obtained for [L being about 26,000. S e n t e r (Zeitschr. f. physikal. Chem., 1903, 44, 257) states that the destruction of blood-catalase at 55 is about 6-7 times as rapid as at 45; [L is about 50,000. Special emphasis must be laid on the high values of \L com- pared with those of the corresponding constants for 'other reac- tions, such as those given on p. 231. The result obtained with dry emulsin shows the slight sensitiveness to heat of dry enzymes relatively to that shown in the dissolved state. Similar observa- tions of a qualitative character have often been made with other enzymes. The numerical values obtained under these conditions for [L are, as is easily understood, dependent in a high degree on 1 Merely by shaking for a few minutes, even at room-temperature, enzyme solutions are rapidly inactivated. This action was observed almost simul- taneously by Signe and Sigval Schmidt-Nielsen with rennet, Abderhalden and Guggenheim with tyrosinase, and S h a k 1 e e and M e 1 1 z e r with pepsin. As was formerly assumed by the author, inactivation by shaking or denaturation and skin-formation are due partly to a surface-action and partly to the influence of atmospheric oxygen; with many colloidal solutions, shaking in the air produces flocculation (S. and S. Schmidt-Nielsen, H., 1910, 68, 317). "According to Shaklee (Zentralbl. f. Physiol., 1909, 23, 4), at 37 pepsin loses its activity according to the formula for bimolecular reactions at a x where a is the original amount of pepsin and x the amount changed (destroyed) in time t. After 12 days, 86% of the enzyme is destroyed. 3 Calculated by Arrhenius (Immunochemistry, p. 98). INFLUENCE OF TEMPERATURE AND RADIATION 237 the previous treatment and especially on the moisture-content of the enzyme-preparations, and they have a real significance only if the compositions of the preparations are suitably denned. The velocity of decomposition of dissolved enzymes and its temperature-coefficient are also largely dependent on the other substances present in the solution. Small proportions of acids or bases often influence the stability enormously; thus, for example, bases occasion a very considerable acceleration of the destruction of rennet and of trypsin (Arrhenius, Immunochemistry, p. 88). A very complete study of the influence of acids and alkalies on the destruction of invertase has been made by Hudson and Paine (Journ. Amer. Chem. Soc., 1910, 32, 985), from whose paper the following figure (p. 238) is taken. An extract from Table 2 of Hudson and P a i n e ' s paper shows that at 0-45, the temperature-coefficient of the destruction by acid or alkali does not differ from that of ordi- nary reactions. This is very remarkable, for, at the optimal sta- bility (H'= about 10~ 5 ), the temperature-coefficient of enzyme- destruction is, at any rate between 55 and 65, extremely high. It recalls the temperature-coefficients of the denaturation of pro- teins recently measured by Martin, and it is to be supposed that with the enzymes it is a case of similar denaturation, i.e., of a change of their state of solution. In this connection, an ultramicroscopical investigation would be of interest. In many cases, salts produced a marked increase in the stability of enzymes; thus, according to V e r n o n (Journ. of Physiol.,* 1901, 27, 174), the optimum of pancreas-diastase in a starch solution containing 2% of sodium chloride is at 50, whilst in pure aqueous starch solution the optimum temperature is 35. Apart from this, it is known that many neutral substances, particularly proteins l and other colloids (Journ. of Physiol., 1904, 31, 346), but more especially the specific substrates and reaction- products, increase the stability of the enzymes to a marked extent. This latter fact was pointed out by O ' Sullivan and T o m p - son (loc. cit.). Biernacki (Zeitschr. f. BioL, 1891, 28, 49) and Vernon (Journ. of Physiol., 1901,27, 269; 1902, 28, 375, 448; 1904, 31, 346) made the same observation in the 1 Non-specifically hydrolysable proteins often act as "buffers." 238 GENERAL CHEMISTRY OF THE -ENZYMES case of trypsin, 1 and W o h 1 and G 1 i m m (Biochem. Z., 1910, 27, 365) in that of amylase. In other instances, for example, according to the author's measurements, with invertase, the protective action of the substrate is slight. The stability of this enzyme is, however, considerably increased by the presence .10 .08 .06 .04 .02 Concentration of Acid-< .02 .04 .06 .08 -> Concentration of Alkali .10 FIG. 7. of fructose (Hudson Soc., 1911, 32, 988). and Paine, Journ. Amer. Chem. 1 In this connection mention must be made of the observations of W . Cramer and Beam (Proceedings of the Physiol. Soc., June 2, 1906; see Journ. of Physiol., 1906, 34, XXXVI), according to whom active pepsin is retarded by the addition of pepsin solutions inactivated at 60, whereas preparations inactivated at 100 produce little or no retardation. INFLUENCE OF TEMPERATURE AND RADIATION 239 In dilute solution diastase keeps better than in more concen- trated ones (E f f r o n t , Enzymes and their Applications, 1902, p. 56). The t emper atur e- coef f i c i ent s of the enzy- mic reactions themselves, that is, the alterations of the velocity-constants k with the temperature are of the same order of magnitude as those of other chemical processes. An attempt should always be made to measure the temperature- coefficient in a region of temperature where the destruction of the enzyme comes into consideration as little as possible. In most of the previous measurements, the distance from the op- timum is so slight that the constants [i are influenced by the destruction of the enzyme and are consequently too low. Fur- ther, as the following examples show, the errors of observation are generally very large, even in the work of reliable experimenters. A u 1 d (Journ. Chem. Soc., 1908, 93, 1275) : measurements on amygdalin-emulsin. kv, :/Ci5 = 2-37 /c 3 o :fc 20 = l-81 35 : & 25 =2-14 fc 40 :/c 30 =l-68 45 &50 K a s 1 1 e and Loevenhart (Amer. Chem. Journ., ] 900> 24, 501) left tubes containing 4 c.c. of water, 0-1 c.c. of toluene and 1 c.c. of a 10% liver- or pancreas-extract for 5 minutes in baths at 40, 30, 20, 10, and - 10, so that they assumed these temperatures. Ethyl butyrate (0-26 c.c.) was then added and the solutions titrated after 30 minutes. Percentage hydrolysed. By liver-extract. By pancreas-extract. 40 ll-29(?) 2-82 30 5-96 3-16 20 5-27 2-51 10 3-89 1-88 2-26 1-25 -10 0-70 240 GENERAL CHEMISTRY OF THE ENZYMES The value obtained with liver-extract at 40 must be due to an error of experiment. H a n r i o t (C. R., 1897, 124, 778) obtained similar results with his esterases from serum and pancreas. Reference must finally be made to a series of experiments made by C h o d a t (Arch. Sci. phys. nat., 1907, 23, 13) on the action of tyrosinase on tyrosine. The second row below gives the times in which the solution had attained a certain intensity of colour. Temperature 10 20 30 40 45 50 Time (minutes) 180 100 60 40 30 20 10 Very unreliable quantitatively are the temperatures given by Tammann (loc. cit.) for invertase and cane-sugar, Lindner and Krober (Chem. Ber., 1895, 28, 1053) for maltase, H a n r i o t and Camus (C. R., 1897, 124, 235) for serum-esterase and monobutyrin, M i q u e 1 (see H e r - z o g , 1 o c . c i t .) for urease, and by experiments made accord- ing to M e 1 1 ' s method. Consequently reference will only be made to the calculation of these results by H e r z o g (Zeitschr. f. allg. Physiol., 1904, 4, 189). In the following table (p. 241) are collected the data as yet obtained concerning the temperature-coefficients of enzyme- reactions. 1 In other cases the value of [JL changes considerably with temperature; this is shown, for instance, byMuller-Thur- gau's results with amylase (Landw. Jahrb., 18.85, 795). An extended investigation of the temperature coefficients of alcoholic fermentation by living yeast has been made by S 1 a t o r , with the following results : t vt+io : v t 5 5-6 10 3-8 15 2-8 20 2-25 25 1-95 30 1-60 1 Complicated biological processes, such as the assimilation of carbon by green leaves, are not considered here. INFLUENCE OF TEMPERATURE AND RADIATION 241 Author. Substrate and enzyme. Temp.- interval. kt+io k t (A K a s 1 1 e and Loevenhart (1 o c . c i t .) Ethyl butyrate, esterase 20-30 1-3 4,650 Tammann (loc. cit.) K j e 1 d a h 1 (Medd. fra Carlsberg Lab 1881 335) Cane-sugar, invertase Cane-sugar, invertase 20-30 30-40 1-4 1-5 6,000 7;800 O ' S u 1 1 i v a n and T o m p s o n (loc cit) Cane-sugar, invertase 40-50 1-4 6,800 S e n t e r (Zeitschr. f. physikal. Chem., 1903, 44, 257) Chodat (loc cit ) HzOa, catalase 0-10 20-30 1-5 1-5 6,200 7 200 E u 1 e r and af Ugglas (loc. cit) 0-20 2-0 11,000 Vernon (Journ. of Physiol., 1901, 27, 190) Starch, amylase 20-30 2-0 12,300 Vernon ( ibid. ) Vernon (ibid. 1903, 30, 364). . . Tammann (loc. cit.) Vernon (Journ. of Physiol., 1903, 30, 364) Taylor (Journ. of Biol. Chem., 1906, 2 87) Milk, rennet Witte's peptone, trypsin Salicin, emulsin Witte's peptone, erepsin Triacetin, lipase 20-30 15-25 15-25 15-25 18-28 2-1 2-3 2-4 2-6 2-6 13,400 14,300 15,000 16,400 16 700 F u 1 d (Hofm. Beitr., 1908, 2, 169) B a y 1 i s s (Arch. Sci. Biol., St. Petersburg, 1904, 11, 261, Sup- plement) Milk, rennet Casein, trypsin 30-40 20-7-30-7 3-2 5-3 22,000 37 500 Heterogeneous systems A b e r s o n (Rec. Trav. Chim. Pays-Bas, 1903, 22, 100) Sugar, living yeast Sugar, permanent yeast 18-28 15-25 2-7 2-8 15,600 18,000 Herzog (H., 1902,37, 160).. ... In consequence of the foregoing data it must again be emphasised that the influence of temperature on enzymic reac- tions is exactly denned only by determination of the inactivation constant k E at a given temperature and measurement of the initial velocities of the reaction itself at temperatures at least 20 lower than the above. The following very instructive numbers, obtained by G e r - ber (Soc. Biol., 1903, 63, 375), show how the temperature- coefficient of an enzymic reaction may depend on the quantity of enzyme present: 242 GENERAL CHEMISTRY OF THE ENZYMES Concen- Time of coagulation of milk at different temperatures. rennet. 25 30 33 36 39 42 45 0-005 0-010 30' 20" 14 45 29' 00" 11 30 ( no coag. \ in 360' r oo" 1 no coag. in 360' (no coag. in 360' No No 0-015 9 40 7 25 4 40 5' 35" ( coag. coag. 0-020 7 30 5 00 2 30 3 15 5' 30" 0-025 6 15 3 30 2 05 2 20 2 40 in 0-030 4 40 2 50 1 40 1 30 1 40 360' in 0-040 3 40 2 20 1 30 1 00 50 0-050 3 00 1 50 1 10 55 40 2' 05" 360' 0-075 2 20 1 20 50 40 30 1 00 0-100 1 40 1 00 40 30 25 35 0' 45" Very striking is the result of a comparison between the values of [L for the acceleration of the inversion of cane-sugar by an acid and by invertase respectively; the data on pp. 231 and 241 give: Cane-sugar-hydrochloric acid. Cane-sugar-invertase. H = 25,600 [1 = 11,000 Although enzymic temperature-coefficients had been deter- mined by very reliable experimenters (Kjeldahl, T a m - m a n n , O ' Sullivan and Tompson), new investiga- tions on this point were to be desired. On this account, the author and Miss B. af Ugglas have made fresh determina- tions of these coefficients under various experimental conditions (H., 1910, 65, 124); at the optimum concentration of hydrogen- ions, it was found that /CSQ : 20 = 1-9 -2-1. This is in agree- ment with the result obtained by V i s s e r (Zeitschr. f. physikal. Chem., 1905, 52, 257), namely 2, for the ratio k 2 o : kio. The first conclusion that could be drawn is that the con- stant [L includes the heat-change occurring during the forma- tion of the compound between the cane-sugar and the acid or invertase. It is also possible that the small rise of temperature taking place during enzymic inversion may be due to rise of tem- perature not only irreversibly destroying the invertase but also reversibly inactivating it; apart from the destroyed portion, the invertase resumes its original activity at the lower temperature. Hence, rise of temperature renders the substrates, cane-sugar INFLUENCE OF TEMPERATURE AND RADIATION 243 and water, more active and the catalysing enzyme less active. Other reactions, for instance, the hydrolysis of esters, also exhibit smaller temperature-coefficients for enzymic than for acid catal- ysis. For non-enzymic reactions, S 1 a t o r (Zeitschr. f . physikal. Chem., 1903, 45, 547) found temperature-coefficients varying with the catalyst. Very ill-defined is the so-called " optimum temperature," the position of which depends entirely on the period or phase of the reaction considered. Indeed, even at the optimal tem- perature the enzyme undergoes partial destruction during the reaction, so that if comparison is made of the times taken for the reaction to proceed to the extent of one-half, the optimum is apparently lower than if only the first one-fifth of the reaction is considered. The real initial velocity will, in general, show no optimum if the time of observation is made short enough. For practical purposes it may be of interest to know the temperature at which the reaction proceeds most rapidly, and it would then be best to consider the times in which, say, 90-95% of the sub- strate is decomposed. In any case, in giving the optimum tem- perature, it must be stated for which stage of the reaction it holds. Still more indefinite are most of the data on "maximum temperatures" (temperatures of destruction). Measure- ments of these temperatures are of value only when the duration of the experiment and the magnitude of the weakening are deter- mined. It is therefore advisable to give that temperature at which the enzyme is weakened, for example, to the extent of one- half in 30 minutes; still better is it to measure the inactivation constant ks of the dissolved enzyme at a given temperature. The presence and concentration of other substances in the solution may influence both the optimal and the maximal tem- peratures very considerably; marked alterations of these tem- peratures are produced especially by acids and bases, so that in measurements of the stability it is necessary to define the con- centration of the H'- or OH'-ions. Non-electrolytes so long as they do not constitute the substrate or a product of the reaction appear to have little influence, the stability-constant being, therefore, readily reproducible (cf. the measurements of B. a f U g g 1 a s and Kullberg, loc. cit.). The very great differences existing, according toR.Huerre 244 GENERAL CHEMISTRY OF THE ENZYMES (C. R., 1909, 148, 300), between the maltase of white maize and that of yellow maize, may be due to the action of a foreign substance of some kind; but it can, by no means, be denied that the two sorts of maize may contain different maltases. For most enzymes the optimal temperature is stated to be between 37 and 53, and the maximal temperature between 60 and 75. Many oxydases resist surprisingly high degrees of tem- perature. That known as Medicago -laccase is especially stable to heat, as is to be expected from its composition (see p. 62). Other oxydases are destroyed only at 80-90 (K a s 1 1 e , Chem. ZentralbL, 1906, 77, i, 1554). Also peroxydases, at any rate in the natural juices, are only slightly injured by heating. Thus, a preliminary experiment with the juice from pressed horseradish showed that heating for two hours at 60 diminished the activity of the peroxydase on guaiaconic acid only in the proportion of 7 : 5. For ^ the very low value, 4000, was obtained. Apparently still more resistant is myrosin, which, according to Guignard's experiments (C. R., 1890, 111, 249, 920; Bull. Soc. Bot. de France, 1894, [3], 418), is not destroyed even at 81, although a knowledge of the duration of the heating is to be desired. Peculiar behaviour towards high temperatures has been observed byDelezenne, Mouton and P o z e r s k i (Soc. Biol., 1906, 60, 68, 390) in the case of papain. At tem- peratures up to 40, papain exerts no digestive action on egg- albumin or blood-serum. Digestion only occurs, and then very rapidly, on further heating of the solution. These results were completely confirmed byJonescu (Biochem. Z., 1906, 2, 177), who studied the differences between ordinary and " heat- digestion," while Gerber (Soc. Biol., 1909, 66, 227) has also remarked the notable resistance of papain to high temperatures. Towards low temperatures, enzymes appear to be highly resistant; thus, Miss White showed that the enzymes of cereals are not destroyed by exposure to the temperature of liquid air for two days (Proc. Roy. Soc., ., 1909, 81, 440). To sum up, the sensitiveness of enzymes to heat is, indeed, very great, but is not so marked as with the toxines, for which INFLUENCE OF TEMPERATURE AND RADIATION 245 M a d s e n found the temperature-constants of spontaneous decomposition in solution to be as high as [L = 198,500. Enzymic reactions appear to have rather lower temperature- coefficients than the corresponding non-enzymic catalyses. INFLUENCE OF RADIATION The manifold action exerted by light on the processes occurring in living cells and tissues has naturally given rise to the impres- sion that enzymes are sensitive to rays of various wave-lengths. The success of the modern photo-therapeutic methods of F i n s e n and others, on the one hand, and the knowledge that toxines undergo very rapid destruction in the light, on the other, endow this subject with considerable practical and scientific interest. 1. Light-rays It may be mentioned firstly that, in general, enzymes do not appear to exhibit so high a degree of sensitiveness to light as do the toxines. This observation was, indeed, made some years ago by O . E m m e r 1 i n g (Chem. Ber., 1901, 34, 3811). Hertel subjected a number of enzymes and toxines to the influence of light-rays and observed, among other results, that trypsin and also diastase and rennet are weakened by rays having the wave-length 280 ^. His investigations also showed that the destruction of enzymes requires a longer exposure than does that of the toxines, the enzymes being therefore decidedly more photo-stable substances than the toxines (Biol. Zentralbl., 1907, 27, 510). Just as in the study of the thermo-lability of the enzymes, so also in investigating the action of light, a distinction must be drawn between the destruction of the enzyme by light and the alteration of the enzymic action under the influence of the radiation. In so far as the results already obtained indicate, the former influence predominates, the observed retardations of enzyme-action being therefore due principally to a partial annihilation of the enzyme molecule. In his paper cited above, E m m e r 1 i n g states that, in absence of air, invertase, lactase, emulsin, amylase, trypsin 1 1 According to Fermi and P e r n o s s i (Zeitschr. f . Hygiene, 1894, 18, 83), pepsin and trypsin are weakened in sunlight. 246 GENERAL CHEMISTRY OF THE ENZYMES and pepsin are injured but slightly by daylight; yeast maltase and rennet are rather more sensitive to these conditions. The activity of the last-named enzyme, in 1% aqueous solution, was reduced to one-half by diffused sunlight, and to one-third by direct sunlight, in the course of five days. D o w n e s and Blunt found this action of light to be of slight extent and the same result was obtained by F. W e i s (Medd. fra Carlsberg Lab., 1903, 5, 135) with the proteolytic enzyme of malt and by Schmidt-Nielsen (Medd. fra Finsens med. Lysinstitut, 1903) with chymosin. On the other hand, F. G . Kohl (Beitr. z. Bot. Zentralbl., 1908, 23, 64) states that invertase is considerably affected even by diffused daylight. The disagreement between this statement and the earlier observations is explained by the results of J a m a d a and Jodlbauer (Biochem. Z., 1908, 8, 61), who showed that the rays of sunlight which pass through glass are alone capable of injuring invertase, but to a marked extent only in presence of oxygen. With reference to the stability of catalase under the action of light, a comprehensive investigation was carried out by Lockemann, Thies and W i c h e r n (H., 1909, 58, 390). The inhibiting action of light on blood-catalase, both when the blood-solution is kept and during the reaction with hydrogen peroxide, is greatest with white and least with red light, blue occupying an intermediate position. 1 Also in this case, according to Z e 1 1 e r and Jodlbauer (Biochem. Z., 1908, 8, 84), appreciable injury is produced by visible rays only when oxygen is present; a similar result was arrived at by Z e 1 1 e r and Jodlbauer, and almost simultaneously by Bach (Chem. Ber., 1908, 41, 225), with peroxydase. The results of a large number of investigations are in agree- ment in indicating that the inhibiting influence of ultra- violet rays is much greater than that of the visible rays. The action of these rays was investigated firstly and very com- 1 In absence of catalase, additions of sodium chloride retard the decom- position of hydrogen peroxide by light. For the sensitiveness to light of solutions of the peroxide either containing or free from catalase, W o . O s t w a 1 d (Biochem. Z., 1908, 10, 1) found the influences of different kinds of light to be in the following order of diminishing magnitude: white, violet, yellow, dark. INFLUENCE OF TEMPERATURE AND RADIATION 247 pletely by Reynolds Green (Trans. Roy. Soc., 1897, 188, 167), who showed that violet and ultra-violet rays destroy diastase, but that the action of this enzyme_is enhanced by visible rays owing to activation of the zymogen. As was mentioned above, visible rays have only a slight retarding action on chymosin, catalase and peroxydase, but these enzymes are rapidly and per- manently inactivated by ultra-violet radiation (Schmidt- Nielsen, Zeller and Jodlbauer). Signe and Sigval Schmidt-Nielsen made a detailed, kinetic study of the destruction of rennet by ultra-violet light (H., 1908, 58, 235), whilst shortly beforehand Georges Dreyer and O 1 a v H a n s s e n (C. R., 1907, 145, 564) showed that the destruc- tion of enzymes by radiation follows the law for unimolecular reactions. Of Schmidt-Nielsen's experiments, which were made in the Finsen Institute with a mercury-vapour lamp, the following may be described : A 1% solution of dry, commercial rennet powder was exposed to the radiation for a definite period and the time required for the coagulation of cow's milk subsequently measured; this time was assumed to be a direct measure of the amount of unaltered enzyme in the solution. Temperature, C. Exposure to the light, minutes. Time of clotting, minutes. 1000/t. 24-2 8-5 24-2 1-0 23-5 442 24-2 1-5 39-25 443 24-2 2-0 71-0 461 7-7 12-75 1-0 19-5 405 12-80 1-5 34-5 434 12-85 2-0 59-0 442 13-90 2-0 56-0 431 12-95 2-0 54-5 425 As was, indeed, to be expected, these numbers show that the destruction of rennet by light is undoubtedly a unimolecular reaction. But what deserves special attention is the extraor- dinarily small temperature-coefficient of this reaction. That such 248 GENERAL CHEMISTRY OF THE ENZYMES processes possess low temperature-coefficients has been repeatedly observed and seems to be a general rule. It may be assumed in the above case that the temperature of the chymosin-mole- cules, after exposure to the light, is considerably higher than that of the surrounding solution and is not very different in the two series of experiments; the temperature of the solution had therefore little influence on the thermal condition of the chymosin. In Schmidt-Nielsen's experiments, the reaction constant diminished with increase of the concentration. This is not surprising, since the destruction by heat of enzymes proceeds more slowly in their concentrated than in their dilute solutions. Hence, as their concentration increases, enzymes become more stable to both heat and light. Of the total effect of the radia- tion of the mercury lamp, 96% is due to rays with the wave- lengths 220-250 \L[L and only about 0-3% to the visible rays. Very interesting experiments have been made byvonTap- p e i n e r and his co-workers on the action of sunlight on dias- tases and invertase in presence of fluorescent substances (sensi- tisers). Very small quantities of eosin, Magdala red or quinoline red are sufficient to cause sunlight which of itself is with action to exert a marked inhibiting effect. In diffuse daylight the fermentative power of yeast is destroyed by fluorescent bodies. With living yeast, only certain fluorescent substances are active; but with permanent acetone-yeast and, still more, with yeast-juice, all the fluorescent bodies examined, such as eosin, methylene blue, fluorescein, dichloranthracene- disulphonic acid, etc., induce considerable diminution of the fermentative power (von Tappeiner, Biochem. Z., 1908, 8, 47). Also on catalase all the fluorescent substances investigated have a sensitising action, whilst with peroxydase this is the case only with eosin and Bengal red; in both these instances, the action only occurs when the ultra-violet rays are, as far as possible, lacking ( J a m a d a and Jodlbauer, Biochem. Z., 1908, 8, 61 ; Z e 1 1 e r and Jodlbauer, ibid., 84; Karamit- s a s , Dissertation, Munich, 1907). Thus the biological action of light is of two kinds (Jodl- bauer and von Tappeiner, Deut. Arch. f. klin. Med., 1906, 85, 386): One requiring the presence of oxygen and accelerated by fluorescent substances, and the other produced INFLUENCE OF TEMPERATURE AND RADIATION 249 only by ultra-violet rays without any part being played by oxygen or fluorescent substances. The results mentioned above show that light exerts actions of two kinds on enzymes : (1) A destroying action, corresponding with denaturation by heat. (2) An activating effect, due to conversion of the " zymogen " into the active enzyme. 2. Other Forms of Radiation By Rontgen rays, enzymes are not weakened. This was shown by P. F. R i c h t e r and Gerhartz (Berl. klin. Wochens., 1908, 45, 646) to be the case with chymosin, yeast, pepsin, pancreatin and papain, while Lockemann, Thies and W i c h e r n obtained the same result with blood-catalase. Radium rays and radium emanation, how- ever, do appear to exert an action on enzymes, although, accord- ing to W i 1 c o c k (Journ. of Physiol., 1907, 34, 207), tyrosinase is not affected by radium rays, while Schmidt-Nielsen found that even a very active preparation of radium has a very slight effect on chymosin; Henri and Mayer (C. R., 1904, 138, 521) state that invertase, emulsin and trypsin are injured. Against these negative assertions are arrayed a number of other positive results. B e r g e 1 1 and B i c k e 1 (Verhandl. d. Kongr. f. inn. Med., Wiesbaden, 1906) first showed that peptic digestion is favoured by the emanation. N e u b e r g (Verhandl. d. deutsch. path. Ges., 1904) and Wohlgemuth (ibid) observed accelera- tion of the autolytic processes by radium radiation, while Loewenthal and E d e 1 s t e i n (Biochem. Z., 1908, 14, 484) found these processes to be facilitated by the emanation. Also Loewenthal and Wohlgemuth (Biochem. Z., 1909, 21, 476) have recently proved that radium emanation is capable of accelerating the action of the diastatic enzyme of the blood, liver, saliva or pancreas. " This favourable action is not always observable immediately; very often retardation occurs during the first 24 hours, this being gradually neutralised and then replaced, if the experiment is sufficiently prolonged, by an acceleration. In other cases, the emanation produced only 250 GENERAL CHEMISTRY OF THE ENZYMES inhibition, which was not compensated when the duration of the experiment was extended." Acceleration of enzyme-action by the emanation has been established with pepsin and trypsin. Of importance from a therapeutic standpoint isGudzent's discovery that the enzyme of purine-metabolism is activated by radium emanation. Action ofmesothorium. The mesothorium bromide discovered by 0. H a h n emits rays of three kinds: a-, @- and y-rays. Of these, the @- and y- rays pass without alteration through a mica plate, if this is not too thick; they are also able to traverse a thin sheet of glass, but are then weakened to some extent. B i c k e 1 and M i n a m i (Berl. klin. Wochens., 1911, 48, 1413) found that exposure of carcinoma, sarcoma and liver to the radiation of mesothorium bromide the action of emanation or a-rays being excluded has no influence on the autolytic enzymes. These authors regard their results as of fundamental importance. If it is a fact that the P- and y-rays of mesothorium are identical in every respect with the @- and y-rays of radium, it must be concluded that the activa- tion of the autolytic enzymes observed as a result of the action of radium is s o 1 e 1 y an effect of the a-rays or emanation. The same is probably the case with other enzymes. According to Minami (Berl. klin. Wochens., 1911, 48, 1798) the - and y-rays of mesothorium exert a very slight influence on the digestive enzymes, amylase, pepsin and trypsin. The biological action of thorium emanation has been studied by B i c k e 1 (Berl. klin. Wochens., 1911, 48, 2107) ; like that of radium emana- tion, it consists sometimes of a retardation and sometimes of an activation of enzymic action, and is more intense than that of g- and y-rays. In general, it may be stated that the healing action found by experience to be exerted by radium emanation depends on the activation of enzymes. The promotion of plant-growth by the emanation (F a 1 1 a and S c h w a r z , Berl. klin. Wochens., 1911, 48) is also to be attributed to enzyme-activation. CHAPTER VI CHEMICAL STATICS IN ENZYME REACTIONS THE position of equilibrium of a chemical system is deter- mined, as is well known, by the law of mass action. If 1 mol. of acetic acid reacts with 1 mol. of alcohol, so that 1 mol. of ester and 1 mol. of water are formed, then, according to the law of mass action- [ester] [acid] [alcohol] if the concentrations of the substances in dilute aqueous solution are indicated by [ ] and K denotes the equilibrium constant. According tovan't Hoff, chemical equilibrium of the above reaction is due to the equality of the velocity v\ of ester- formation and of the velocity V2 of ester-decomposition, so that, v\ = &i[acid] [alcohol] = V2 = fetester], and therefore [ester] _T^_&I [acid] [alcohol] ~ ~ k 2 ' The position of equilibrium is independent of the rapidity with which it is reached and also as exact experiments show independent of the presence and concentration of a catalyst, in so far as this does not combine to an appreciable extent with the components of the system. As was mentioned in Chapter IV (p. 128), a number of investigators have arrived at the conclusion that enzymic reactions are effected by means of a compound of the enzyme and the substrate. With the non-enzymic hydrolyses which have been as yet investigated and in which, according to the author's theory, the 251 252 GENERAL CHEMISTRY OF THE ENZYMES increase of active molecules is due to the formation of a salt of the substrate with the catalysing acid, the concentration of the compound substrate-catalyst is so small that no great alteration occurs in the concentration of the substrate or catalyst, either during the course of the reaction or when equilibrium has been attained. Enzymic decompositions usually differ from those effected by inorganic catalysts in that their velocity is determined not only by the absolute concentration of the catalyst (enzyme) but also, and to a far greater extent, by the concentration-r a t i o between enzyme and substrate. If the substrate is in excess, the velocity of reaction is approximately proportional to the concentration of the enzyme, whilst if excess of the latter is present, the velocity will be very nearly proportional to the concentration of the substance; in every case, the velocity of reaction appears to be proportional to the concentration of the " intermediate product." On the other hand, quantitative study of enzyme-reactions has shown that the products of reaction are also fixed by the enzyme (Henri, Bodenstein, and others). The question now to be considered is the relations in the case of enzymic reactions. A distinction must here be made between true equilibria and end-states. A. Equilibria The assumption of the existence of the molecules enzyme- substrate and enzyme-reaction product which bring about the reaction presumes that the mutual action between these mole- cules proceeds far more rapidly than those between the free substrate molecules and their decomposition products. It is, hence, principally the concentrations of the molecules of the enzyme-substrate and enzyme-reaction product which condi- tion the end-state. The simple assumption may first be made that, in miitMime, equal numbers of enzyme-substrate and enzyme-reaction pro- duct molecules take part in the reaction. The velocity constant ki of the decomposition of the substrate is CHEMICAL STATICS IN ENZYME REACTIONS 253 proportional to the concentration of the molecules of the enzyme- substrate compound; or if _ [enzyme-substrate] [enzyme] [substrate] ' then ki=Ki[enzyme] [substrate]. In a similar manner the velocity constant of the forma- tion of the substrate is expressed by 2 = .^[enzyme] [reaction product] 2 , if, as is often the case, 2 mols. of reaction product are formed from 1 mol. of substrate. According tovan't Hoff, the equilibrium is then given by the quotients KI [substrate] _ _ ~ ^[reaction product] 2 ' As will be at once seen, the numerical value of this " enzymic " end-state coincides with that of the natural stable equilibrium as reached with an inorganic catalyst, only if K\=K%, hence only in the case where the combinations enzyme-substrate and enzyme- reaction product are exactly equal. No convincing cause has, however, yet been suggested for such an assumption; on the contrary, the results obtained by Henri with invertase bear the interpretation that this enzyme is combined equally by cane-sugar, glucose and fructose. If the simple assumption, that equal numbers of the two " active " molecules react per unit of time, is abandoned and it is assumed that n% of enzyme-substrate and m% of enzyme- reaction product molecules react in equal times, then _g - Jil _ KI ^[substrate] &2 J2?w [reaction product] 2 ' and K is identical with the constant of stable equilibrium if Hence it will in general be expected that enzymes lead to an end-state different from that given by inorganic catalysts and 254 GENERAL CHEMISTRY OF THE ENZYMES the first question to be decided is the position of the natural equilibrium. The natural equilibrium can be determined directly in many cases, for instance, in the system fatty acid-alcohol- ester-water. In order to accelerate the attainment of equilibrium, a strong mineral acid may be employed, since the position of equilibrium is not altered by such an ideal catalyst. In certain other cases, an indication of the position of equilibrium may be obtained from the heat-change of the reaction (van't Hoff, Sitzungsber. K. Akad. Berlin, 1909, 42, 1065). As was shown above, the heat-effects of enzymic processes are mostly very small. For such changes, however, the equi- librium is of a simple nature. " Optical antipodes which form no racemic compound present the ideal example, and it has been shown both theoretically and experimentally that, in the solid state, the two antipodes are in equilibrium, whilst in the vaporous, fused and dissolved conditions, they form an inactive mixture of equal amounts. The relation between the equilib- rium constant K, i.e., the quotient of the concentrations of the two antipodes, and the work of transformation E may be expressed thermodynamically (van't Hoff, Svenska Vet. Akad. Handl., 1886) by Hence in this case # = and K l. What applies strictly to optical antipodes also holds approx- imately with reactions of small heat-effect and the equilibrium is not far removed from that corresponding with thermo-neutrality. How far true enzymic equilibrium may differ from the natural equilibrium cannot be stated exactly. It will depend on the proportion of the components of the equilibrium which combines with the enzyme or if it is assumed that separate enzymes accelerate the reaction in the two directions two enzymes to form complex compounds. Since the concentration of the enzyme is usually very low, the concentrations of the mole- cules of enzyme-substrate and enzyme-reaction product must also be low, and the enzymic and natural equilibria will then differ but little. In other words, if the corresponding enzyme or mixture of enzymes is added to & system in equilibrium, the CHEMICAL STATICS IN ENZYME REACTIONS 255 latter undergoes only slight change. This is required by thermo- dynamics, which also shows that in the case where the catalyst and the reacting substances do not (practically) unite, no change in the equilibrium should be produced. Otherwise the equilib- rium could be altered by alternate removal and introduction of the catalyst and perpetual motion thus attained. B. End-states and Stationary States Starting with a system not in chemical equilibrium, the natural equilibrium is not necessarily arrived at by addition of an enzyme-preparation capable of acting on the system. An end-state differing from the equilibrium will be attained. 1 . If two enzymes exist which catalyse the reaction in opposite directions. The final state then depends on the relative quan- tities of these two enzymes. 2. If two enzymes are present, one catalysing the formation of a compound A of the components by the non-reversible reaction Enzyme 1 B+C -* A, and the other using up this compound A according to another r.on-reversible reaction. Enzyme 2 A - D+F. This case may evidently lead to widely varying stationary states, depending on the relative concentrations of the two enzymes 1 and 2. T a m m a n n (H., 1892, 16, 271) gave an account of the experimental material obtained before 1892 and also of his own investigations. From the results of the latter he deduced the important and undoubtedly correct consequence, that the end- states of enzymic reactions do not coincide with the positions of stable equilibrium of the reactions. Concerning the end-states attained under the influence of lipases, two more recent papers have been published: 256 GENEBAL CHEMISTRY OF THE ENZYMES Bodenstein and D i e t z (Zeitschr. f. Elektrochem., 1906, 12, 605) have compared the equilibrium formed between amyl butyrate, water, amyl alcohol and butyric acid with the end-state attained by this system under the influence of lipase. The measurements of the velocity-constants, k\ and 2, with which the formation and resolution of the ester proceed, have already been referred to on p. 152. The mean values obtained were: ki -0-015 & 2 = As should theoretically be the case, the quotient of these two velocity constants was found to be equal to the equilibrium constant determined directly. 1 Hence The end-state determined in these two ways showed, however, considerable and regular deviations from the natural stable equi- librium. Thus, while the natural equilibrium constant had the value 1-96, the enzyme experiments gave the following results: Initial concentration of the reacting substances. & 0-05 0-45 0-10 0-74 0-20 1-12 The fact that these end-states were reached from both direc- tions proved that they were not dependent on retardations of the reaction. Unfortunately, these data cannot serve for proving the above relations quantitatively, since the system examined was hetero- geneous (macro-heterogeneous) . A. E. Taylor (Journ. of Biol. Chem., 1906, 2, 87) also worked with Ricinus-lipase in the form of a moderately finely- divided suspension. The substrate employed was triacetin, the triglyceride of acetic acid. The natural equilibrium was investigated with 0-5, 1-0 and 2-0% solutions of the triacetin; 1 Since water and amyl alcohol were present in excess, the constant K simplifies to [amyl butyrate] [butyric acid] CHEMICAL STATICS IN ENZYME REACTIONS 257 mixtures of equal volumes of these solutions and of normal sulphuric acid, left for several months, gave the values: Initial concentration of the ester. Composition of the equilibrated liquid. 0-5% 12% ester, 88% hydrolysed 1 18 " 82 2 22 " 78 For the enzymic end-state, the following numbers were obtained : Initial concentration of the eater. Composition at the end-state. 0-5% 14% ester, 86% hydrolysed 1 21 " 79 " 2 30 " 70 From these numbers, Taylor drew the conclusion that the enzyme does not displace the equilibrium; but the dif- ferences between these two series of numbers are so large and so regular that, in the author's opinion, they do not indicate identity of the natural and enzymic equilibria. Whether such a dif- ference exists generally and how it depends on the concentrations of enzyme and substrate are questions of great interest, and exper- iments in this direction should give valuable results. Scarcely any other quantitative determinations of enzymic end-states have been made which are comparable with the natural equilibria. From Croft Hill's results on the equilibrium between maltose and glucose, Pomeranz (Wien. Sitzungsber., II B, 1902, 111, 554) has, indeed, calculated the equilibrium con- stants, which are in good agreement. From a qualitative point of view however, this end-state is by no means clear; maltose is re-formed either not at all or only in inappreciable amounts, being replaced by dextrins and isomaltose. A paper communicated byvan't Hoff, shortly before his death, to the Berlin Academy of Sciences (Sitzungsber. K. Akad. Berlin, 1910, 48, 963), treats of the equilibrium of glucosides in presence of emulsin. Measurements were made first with the natural glucoside salicin, in presence of solid salicin and solid saligenin. It was found that the formation of solid salicin from the solid products of hydrolysis is accompanied by an expansion in volume of 9-47 c.c. per grm.-molecule. The result was that the hydrolysis 258 GENERAL CHEMISTRY OF THE ENZYMES of salicin proceeds to practical completion; the equilibrium was, however, not measurable. [Also V i s s e r (Zeitschr. f. physikal. Chem., 1905, 52, 257) had previously obtained only indications of a synthesis of salicin.] With arbutin and aescillin, the hydrolyses were also virtually complete. On the other hand, the system glycerol-glucose-water-glyc- erolglucoside gives a measurable condition of equilibrium. Experiments were made with the molecular proportion 1 : 4 between glucose and glycerol and with increasing amounts of water. If the number of mols. of the latter is expressed by b and the fraction of the glucose changed by a, then glucosideX water a (6+ a) glucose X glycerol ( 1 a) (4 a) = k. Formation of glucose still occurred with 6 = 15 and a = 0-38, the value for k being 2-6. With molecular proportions of glycerol and glucose, as much as 70% may undergo change. T a m m a n n has investigated experimentally the dependence of the end-state on the quantities of the enzyme and of the react- ing compounds. In the action of emulsin on arbutin and on coniferin, it was found that the amount of substance hydrolysed at the end-state increases to a maximum as the quantity of enzyme increases. This indicates that, with increasing concen- tration of the enzyme, the number of the active molecules, enzyme-product of reaction, increases more than that of the molecules enzyme-substrate. When constant amounts of emulsin and different amounts of amygdalin are dissolved in 25 c.c. of water, the following amounts of amygdalin are decomposed at 40: Original quantity of amygdalin. Amounts hydrolysed Absolute amounts. Percentage amounts. 0-51 grm. 1-02 grms. 2-04 " 0-11 grm. 0-15 " 0-24 " 20 15 12 CHEMICAL STATICS IN ENZYME REACTIONS 259 Similar relations are found for the end-states of the system emulsin- arbutin. The solution contained 0625 grm. of emulsin and the follow- ing amounts of arbutin at 35: Original quantity of arbutin. Hydrolysed after 48 hours. 72 hours. 0-576 grm. 4-000 grms. 52-3% 44-0 52-3% 44-0 With constant amounts of enzyme, relatively more amygdalin and arbutin are hydrolysed in dilute than in concentrated solutions; the same probably holds for the hydrolysis of coniferin by emulsin. The question now arises : Within what limits is the e n d - state of an enzymic chemical system variable? That these limits must be quite wide is shown at once by the above facts. They depend not only on the concentration, but also on the previous history of the enzyme. Of the equilibria of biological importance with which considerable variations have been observed, that between starch and sugar in the living plant deserves special mention. The synthesis, but not the hydrolysis of starch is largely influenced by even trifling variations of temperature or by narcosis. These phenomena may be explained in two ways: (1) The existence may be assumed of two different enzymes, one responsible for the synthesis and the other for the hydrolysis; this is, of course, only conceivable on the supposition that different quantities of the two enzymes combine preferably with the starch or sugar, so that both starch and sugar participate in two equilibria: and [enzyme a][starch] =& m [enzyme a starch] [enzyme b] [starch] =/c w [enzyme b starch]. If corresponding equilibria hold for the compounds of the two enzymes with the sugar, it is obvious that there arise two enzymic end- states, which may assume widely different values. Their relations, one to the other, depend only on the total concentration of the starch. None of the facts are contradictory to this hypothesis, for which, however, no experimental proof is forthcoming. In such a case, indeed, the enzymes are far removed from ideal catalysts. (2) Or it remains to be tried as the author has emphasised in another place (Pflanzenchemie, II and III, p. 237) whether the assumption of a single catalyst or a single 260 GENERAL CHEMISTRY OF THE ENZYMES equilibrium constant does not meet the case. The very varying ways in which the opposed processes of building-up and breaking down react towards external influences, would then be attributed to the different constitutions of the media in which condensation and hydrolysis proceed. The catalyst and the reacting substances are then regarded as distributed between the aqueous cell-sap and the protoplasmic hydrosol. In the former, owing to the great excess of water present, the hydrolysis may proceed far; but in the protoplasts, which are rich in lipoids and proteins, such a relatively small proportion of water dissolves that, with a given attainable magnitude of the sugar-concentration, the opposite reversionary changes predominate. CHAPTER VI I ENZYMIC SYNTHESES THE suggestion expressed byvan't Hoff in 1898, that enzymes are able to effect or accelerate chemical syntheses (Zeitschr. f. anorg. Chem., 1898, 18, 1), has since then been confirmed by the results of numerous investigations. It was in the above year that Croft Hill (Journ. Chem. Soc., 1898, 73, 634) observed a synthetic action in the case of y east-malt ase. Croft Hill found that when yeast-maltase is allowed to act for a month at 30 on a 40% solution of glucose, the reducing and rotatory powers of the solution are so altered as to indicate formation of maltose. Shortly afterwards, however, E m m e r 1 i n g (Chem. Ber., 1901, 34, 600, 2207) showed that the effect observed by C r o f t Hill depends on the formation, not of maltose, but of isomaltose and dextrinous products. Isomaltose is not again hydrolysed by maltase. Similar behaviour was noted byE. Fischer and E. F. Armstrong (Chem. Ber., 1902, 35, 3144) with kephir-lactase, which from galactose and glucose synthesises not lactose but isolactose, a carbohydrate not attacked by the lactase. Finally, E.F. Armstrong (Proc. Roy. Soc., B, 1904, 73, 516) made a number of interesting observations which extended the discovery of E m m e r 1 i n g referred to above: the behaviour of emulsin is the opposite of that of maltase, as it hydrolyses isomaltose but synthesises glucose to maltose. These results led Armstrong to the generalisation that " Enzymes build up just those molecules which they are unable to break down." This is a problem of fundamental theoretical importance, If Armstrong's view is correct, it must be assumed thai those enzymes which give rise to a chemical equilibrium froir both directions represent mixtures of a synthesising and a hydro- lysing enzyme. Against the admissibility of this hypothesis 261 262 GENERAL CHEMISTRY OF THE ENZYMES which is only weakly supported by experiment, no fundamental objection can be advanced, and we may again consider the facts favouring such a two-enzyme theory. It has been emphasised, especially by Bayliss, that the experimental foundation for this view is indeed rather weak, inasmuch as Croft Hill made use of ordinary brewers' yeast, which has been shown by H e n r y and A u 1 d (Proc. Roy. Soc., B, 1905, 76, 568) to contain " emulsin " and hence a ^-glucosidase. This emulsin may have occasioned the formation of isomaltose. The forma- tion of isomaltose and isolactose admits, however, of another possible interpretation, which has been given by E . F . Arm- strong (Proc. Roy. Soc., B, 1905, 76> 513). It has been known since O ' S u 1 1 i v a n and Tompson's work, and has been confirmed by H u d s o n , that the hydrolysis of cane-sugar yields a d-glucose distinguished by its high rotatory power; this sugar, to which T a n r e t to whom we owe a very complete investigation of this sugar gave the name a-glucose, passes gradually into e-glucose. This s-glucose appears to con- sist of a- and ^-glucoses in equilibrium. In aqueous solution there is little a-glucose and a relatively large proportion of ^-glucose. On adding to such a solution, an enzyme the synthetic action of which is to be studied, an excess of the ^-modification is available and it is to be expected that the synthetic biose will contain the glucose-residue mainly in a form corresponding with ^-glucose. The biose corresponding with the oc-hexose should also be formed in smaller amount at the same time, but this has not been observed. Nothing, however, is yet known as to what constitutes the dif- ferences between these modifications. This view is capable of experimental proof in various ways. Firstly, it might be expected that the bioses, such as iso- maltose, synthesised from glucose solutions, should yield ^-glucose directly, but no such result appears to have been obtained; this should also be the case with those bioses and glucosides which undergo the same enzymic hydrolyses. Further, it would be expected that maltose could be synthesised from a-glucose i.e., from a freshly-prepared solution of glucose and a very active enzyme capable of effecting the synthesis, before the change from a- to ^-modification is complete. 1 1 With reference to certain statements in the literature, it must be pointed out that originally, the names a-glucose and a-glucoside were not connected. ENZYMIC SYNTHESES 263 Apart from the facts mentioned above with reference to the synthesis of maltose and lactose, numerous other statements have been made relating to enzymic syntheses which are regarded as pure reversions. Ethyl butyrate is formed from butyric acid and ethyl alcohol by the action of pancreas-lipase (K a s 1 1 e and L o e v e n - hart, Amer. Chem. Journ., 1900, 24, 491). Glyceryl butyrate (H a n r i o t , C. R., 1901, 132, 212) and amyl butyrate (Bodenstein and D i e t z , Zeitschr. f . Elektrochem., 1906, 12, 605) are also formed from their com- ponents. An extensive series of experiments on the formation of ester from methyl alcohol and oleic acid by pancreas-lipase has been carried out by Pottevin (Bull. Soc. Chim., 1906, 35, 693; Ann. Inst. Pasteur, 1906, 20, 901). These show, among other results, that the equilibrium between glycerol and oleic acid is independent of the quantity of enzyme added. Quantity of Percentage of ester formed. pancreatin employed. 1 day. 2 days. 20 days. 1 8 56 84 2 12 66 82 5 21 66 84 10 43 74 85 The following results, relating to the synthesis of a true fat, indicate the influence of the amount of water present. 40 grms. of oleic acid + 3 grms. powdered pancreas, experiment, 20 days. Temperature, 33. Duration of Amounts of Percentage esterified. Glycerol. Water. 130 grms. grms. 3 120 10 77 110 20 64 100 30 51 64 66 20 28 102 5 8 122 264 GENERAL CHEMISTRY OF THE ENZYMES Syntheses of true fats from various higher fatty acids (of (ocoanut oil, etc.) and glycerol are described by Welter cZeitschr. f. angew. Chem., 1911, 24, 385). Glyceryltriacetate is formed from its components by Ricinus- lipase (Taylor, Journ. of Biol. Chem., 1906, 2, 87). Cf. p. 154. Amygdalin is formed from mandelonitrile glucoside and glucose (E m m e r 1 i n g , Chem. Ber., 1901, 34, 3810). Benzaldehydecyanohydrin results from benzaldehyde and hydrocyanic acid (Rosenthaler, Biochem. Z., 1908, 14, 238; 1909, 19, 186). Triacetylglucose is given by acetic acid and glucose under the influence of pancreatin (A c r e e and H i n k i n s , Amer. Chem. Journ., 1902, 28, 370). Glycogen is formed in pressed yeast-juice from sugar (C r e m e r , Chem. Ber., 1899, 32, 2062). A condensation, the nature of which is not clearly understood, is produced in invert-sugar solutions by the revertase of Mucor mucedo, etc. (P a ri t a n e 1 1 i , Atti Real. Accad. Lincei, 1907, [v], 16, ii, 419; Bot. Ber., 1908, 26a, 494). Also no definite conclusions can be drawn concerning the action of the yeast-revertase investigated by Kohl (Beitr. z. bot. Zen- tralbl., 1908, 23, i, 64). Reference must also be made here to the studies of Maquenne (Bull. Soc. Chim., 1906, [iii], 35; lecture) and of Wolff and Fern bach (C. R., 1903, 137, 718) on the re-formation of amylose from its decomposition products; men- tion should also be made ojf the equilibrium attained under the action of malt-diastase (M o r i t z and Glendinning, Journ. Chem. Soc., 1892, 61, 689). Possibly the reversion of starch in plasmolysed vegetable cells, observed by Overton (Vierteljahrsschr. d. naturf. Ges. in Zurich, 1899, 44, 88), also represents such an enzyme action. Kendall and Sherman (Journ. Amer. Chem. Soc., 1910, 32, 1087) found that a state of equilibrium is also set up in the decomposition of starch by amylase (pancreas-amylase) . In 1 per cent starch solution, equilibrium is attained independently of the amounts of salt and alkali present when the amount of maltose is about 85% of the initial weight of the starch. The formation of hippuric acid from benzoic acid (benzyl ENZYMIC SYNTHESES 265 alcohol) and glycine by the action of kidney-extract has been observed by Abelous and Ribaut (Soc. Biol., 1900, 52, 543), but confirmation of this result is desirable. As regards the synthesis of protein substances, mention must first be made of the experiments on plastein-formation, which must undoubtedly be regarded as syntheses. D a n i 1 e w s k i established the fact that, in concentrated solutions of Witte's peptone, rennet produces characteristic protein precipitates. This phenomenon, " plastein-formation," which also occurs under the influence of pepsin preparations, was further investigated in Danilewski's laboratory and has since been examined more especially by Russian workers, for instance, S aw j alow (Centralbl. f. Physiol., 1902, 16, 625) and Okuneff (Dissertation, St. Petersburg, 1895). Kurajeff (Hofm. Beitr., 1901, 1, 121; 1903, 4, 476) found papain to possess a similar coagulating property. L a w r o w and S a 1 a s k i n (H., 1902, 36, 277) showed that the precipita- tion of concentrated Witte's peptone solutions by gastric juice occurs with albumoses of all types. Our knowledge of plastein- formation has recently (H., 1907, 51, 1) been considerably extended by L a w r o w , according to whom, not only the albumoses but also substances of the amino-acid type can be coagulated best in faintly alkaline solution. The coagulums exhibit the reactions of proteins but contain less nitrogen than these. Plastein-formation is favoured by increasing the concen- trations of the reacting solutions and occurs especially under conditions which retard the hydrolysis of proteins. The precipitation of plasteins may possibly consist of a salting- out process. Everything seems to indicate that Danilewski's reac- tion is really a synthesising action 1 of the pepsin or rennet, although true reversibility of the peptic action, i.e.-, re-forma- tion of the starting material has not been proved. Rosen- f e 1 d (Hofm. Beitr., 1906, 9, 215) has shown that the hydrolytic products of casein-plastein differ, at any rate quantitatively, from those of casein. This is proved very clearly by a more recent investigation of Henriques and G j a 1 d b a k (H., 1 Cf . also R. O. Herzog (H., 1903, 39, 305) and A . Number g (Hofm. Beitr., 1903, 4, 543). 266 GENERAL CHEMISTRY OF THE ENZYMES 1911, 71, 485), who followed the reaction by means of S 6 r e n - s e n 's method of titration with formaldehyde. An interesting pepsin-synthesis from the hydrolytic products of casein has been communicated by Robertson (Journ. of Biol. Chem., 1907, 3, 95) who obtained a body which con- tained 3-17% P2Os and which he termed u paranuclein." Taylor succeeded in effecting an enzymic synthesis with trypsin. He hydrolysed protamine sulphate from R o c c u s 1 i n a t u s completely into its components and treated the mix- ture of amino-acids with trypsin obtained from the liver of the mollusc Schizothaerus Nuttalii (Journ. of Biol. Chem., 1907,3,87). The same author has recently described further experiments both with glycerine liver-extract of Schizothaerus Nut- talii and with pancreatin (G r ii b 1 e r ' s) , the substrate being obtained as before from salmin sulphate. After a lapse of 4 months, the solutions, which proved to be still germ-free, were diluted with 4 parts of water, acidified with sulphuric acid and mixed with 3 parts of absolute alcohol; the control solutions showed no change, whilst the two containing enzyme gave thick white precipitates. The product was purified by means of its picrate, after which it was found to have a composition closely agreeing with that of salmin. Indications of a synthetic action of trypsin on the hydro- lytic products of casein were obtained by B a y 1 i s s (Arch. Sci. Biol. St. Petersburg, 1904, 11, Supplement, 261), while the number of cases in which retardation of the hydrolysis by the reaction products has- been observed show that a state of rever- sible equilibrium is assumed. The experiments of Beitzke and N e u b e r g (Verh. d. deutsch. path. Ges., 1905, 160; Virch. Arch., 1906, 183, 169) are of special interest, both in themselves and in relation to the representation of enzymic end-states given on p. 253 et seq. It was found that subcutaneous injection of emulsin (in rabbits) leads to the formation of anti-emulsin, which is able to synthesise glucose to maltose or to a disaccharide similar to maltose. Should further experiments show that our enzyme-prepara- tions contain in general a resolving and a synthesising con- stituent, the prevailing views concerning the formation and com- bination of anti-enzymes may require modification, the deter- ENZYMIC SYNTHESES 267 mining factor in the equilibrium between enzyme and anti- enzyme being ascribed to the chemical substrate and the products it yields. Anti-enzymes Like the toxines, many enzymes are able to cause production, in the living organism, of anti-bodies which retard the actions of the enzymes. Anti-enzyme action was first observed by Hildebrandt in 1893. From their actions, anti-enzymes appear, like enzymes, to be organic catalysts. They seem to correspond with the enzymes in physical properties and also in chemical lability, although the observations in this direction are few and not very definite. They are, as far as is known, approximately as unstable towards high temperatures as the enzymes themselves. Among the most stable anti-enzymes are the anti-lipase of Ricinus which, according to Bertarelli (Centralbl. f. Bakt., 1905, I, 40, 231), is not weakened at 70 and slowly loses its activity only at 80. The degree of purity of the enzyme appears to be without influence on the formation of anti-body, and a co-enzyme is apparently unnecessary to this reaction. This behaviour corresponds closely with that of the antitoxines, which are generally injured at 70. Like enzymes, antitoxines are more stable in the dry state than in solution. Judging from the results of physico-chemical investigations, the relations between enzymes and anti-enzymes are essentially identical with those between toxines and anti-toxines. For further information on this interesting problem, the monographs of Arrhenius and of M i c h a e 1 i s should be consulted. The most important facts concerning anti-enzymes are here brought together, because the problem of the reversibility of enzymic reactions is bound up with the action of anti-enzymes. By the term anti-enzymes, in its stricter meaning, is to be understood those specifically-acting secretions produced in the organism, in presence of enzymes, by immunisation. But normal serum also contains substances which, for instance, annul tryptic action more or less completely. It is not probable, from the facts yet known, that these differ essentially from the anti-bodies 268 GENERAL CHEMISTRY OF THE ENZYMES produced by immunisation, and they will therefore be discussed with the anti-enzymes. On the other hand, the thermo-stable and inorganic substances which prevent enzyme action to a greater or less extent will be termed, after Neuberg's suggestion, inhibiting agents. Anti-steapsin was prepared by A. Schlitze (Deut. med. Wochens., 1904, 30, 308), who, after injecting steapsin, obtained in two cases rabbit-serum showing strong anti-lipolytic action. His results have more recently been confirmed by B e i t z k e and N e u b e r g (Virch. Arch., 1906, 183, 169) and by B e r t a r e 1 1 i . The latter author (Centralbl. f. Bakt., 1905, I, 40, 231) was not able to obtain the anti-enzymes corresponding with the lipases from ox-liver and ox-blood serum, but by injection of various vegetable lipases (Ricinus- and nut-lipase) he separated from dog-serum anti-lipases with specific actions. Thus the anti-ricinus-lipase influenced neither the serum-lipase, nor the liver-lipase, nor the nut-lipase. Anti-emulsin, the first known anti-enzyme, was discovered by Hildebrandt (Virch. Arch., 1893, 131, 12) who, like B e i t z k e and N e u b e r g (Virch. Arch., 1906, 183, 169), immunised rabbits and precipitated the anti-body from the anti-ferment-serum with the globulin fraction. The synthetic action of this preparation is discussed later. Immunisation with diastase may perhaps have been observed by Kussmaul (Arch. f. klin. Med., 14), but his results *are uncertain. A s c o 1 i ' s work (H., 1904, 43, 156) also led to no definite result. Subcutaneous injection of diamalt a commercial enzyme solution prepared from green malt leads to the production in rabbit-serum of substances which retard the saccharifying action of diastase, while the serum originally showed an inverting action (B r a u n and S c h ii t z e , Med. Klin., 1907, No. 9, quoted in Biochem. Zentralbl., 1907, 6, 389). S a i k i prepared an anti-inulinase by injection (Journ. of Biol. Chem., 1997, 3, 395). An anti-invertase of slight activity was obtained by Schtitze and Berg ell (Zeitschr. klin. Med., 1907, 61, 366). S c h ii t z e (Zeitschr. f. Hygiene, 1904, 48, 457) also prepared an anti-lactase by injecting ' kephir-lactase under the skin ENZYMIC SYNTHESES 269 of rabbits or into the breast-muscles of the dog; the anti-body appears in the serum, which in the normal state did not contain it. Anti-pepsin. Sachs ^(Fortschr. d. Med., 1902, 20, 425) immunised geese against pepsin, the serum exhibiting sufficient anti-peptic action to annul 20 times its amount of pepsin. The anti-pepsin which was discovered by Weinland (Zeitschr. f. Biol., 1903, 44, 45) and is regarded as a normal secretion of the gastric mucous membrane, corresponds with the normal anti-trypsin of blood-serum. It retards peptic digestion in vitro and doubtless prevents auto-digestion of the mucous membranes. The same author has detected anti-enzymes of pepsin and trypsin in the pressed juice of Ascaris, and, according to R.O. Herzog (H., 1909, 60, 306), the action of rennet preparations is also retarded by Ascaris juice. Unlike these anti-enzymes, an agent which inhibits peptic action and was found by B 1 u m in gastric juice is stable to heat. While B e r g e 1 1 and S c h ti t z e tried in vain to obtain an anti-pancreatin (Zeitschr. f. Hygiene, 1905, 50, 305), J o c h - m a n n and Kantorowicz, in a recent preliminary com- munication (Munch, med. Wochens., 1908, 55, 728), refer to an anti-enzyme to pancreas-trypsin which must be identical with the anti-body of the leucocyte-enzyme. The same inves- tigators state that blood contains at least two anti-pepsins, one of which inhibits the digestion of serum-albumin, being destroyed at 80-85, while the other prevents the digestion of solidified hens' egg-albumin, being thermo-stable. Attempts at immunisation against papain have as yet been unsuccessful (B e r g e 1 1 and Schiitze, loc. cit.; von Stenitzer, Biochem. Z., 1908, 9, 382). Anti-tryptic Paralysors The experiments of H a h n (Berl. klin. Wochens., 1897, 34, 499) and those made almost simultaneously by P u g 1 i e s e and Coggi (Boll. Sci. Med., 1897, 8) first established the fact that normal serum retards tryptic digestion. Fermi had, however, previously observed that trypsin disappears soon after injection. Further work on this subject has been done by A c h a 1 m e (Ann. Inst. Pasteur, 1901, 15, 737), Camus and 270 GENERAL CHEMISTRY OF THE ENZYMES G 1 e y (Soc. Biol., 1897, 47, 425), Charrin and Levaditi, S i m n i t z k i and Glaessner (Hofm. Beitr., 1903, 4, 79). This anti-tryptic action is bound up with the serum- albumin (Landsteiner, Centralbl. f. Bakt., 1900, I, 27, 357; Cathcart, Journ. of Physiol., 1904, 31, 497; H e d i n , Journ. of Physiol., 1905, 32, 390). With a number of diseases, such as diabetes, tuberculosis, etc., increased anti-tryptic activity of the serum is observed (B r i e g e r and T r e b i n g , Berl. klin. Wochens., 1908, 45, 1041). According to A. D 6 b 1 i n (Zeitschr. f . Immunitatsforsch. u. exp. Therap., 1910, 4, 229), the anti-trypsin of serum is stable to heat, which weakens the anti-tryptic action of urine only slightly. The inhibiting body is not a lipoid but is colloidal in character. Delezenne (Soc. BioL, 1903, 55, 112) found that the retarding action of normal serum is not a direct action on the proteolytic enzyme, but is to be attributed to neutralisation of the corresponding kinase. He describes the following experiments: After preliminary determination of the amount of serum just necessary to annul the digestive action of a mixture of pancreatic and intestinal juices, three tubes were each filled with equal quantities of the substance to be digested, the pancreatic-intestinal mixture and the corresponding quantity of serum. After the lapse of some hours, when it was found that no digestion had occurred in any of the tubes, to one (A) was added an excess of pancreatic juice and to another (B) an excess of gastric juice, while C served as control; digestion took place only in B. From these results Delezenne inferred that, in the digestion with the pancreatic-intestinal juice only the intestinal juice (the kinase) was neutralised, and that the serum has no action on the pancreatic enzyme. A s c o 1 i and B e z z o 1 a (Centralbl. f. Bakt., 1903, I, 33, 783) arrived at somewhat similar conclusions. Against this view objections have, however, been advanced. According to Delezenne, the anti-trypsin would be an anti- kinase, and. the existence of an anti-trypsinogen might also be expected. B a y 1 i s s and Starling (Journ. of Physiol., 1904, 32, 129) were unable to detect such a body in blood-serum after subcutaneous injection. It was also shown that normal rabbit-serum possesses, in addition to its anti-tryptic properties, the ability to neutralise enterokinase, this power being enhanced ENZYMIC SYNTHESES 271 by injection of enterokinase. On the other hand, " anti-kinase " produced in serum does not increase its anti-tryptic properties. These observations have been confirmed and extended by Zunz (Bull. Acad. Roy. Med. de Belgique, 1905, [4], 19). Normal blood-serum contains not only anti-trypsin, but also a proteolytic enzyme, serum-protease, the action of which is retarded by the anti-trypsin. Serum-protease can be separated from anti-trypsin by salting-out, the former passing into the globulin fraction and the latter into the albumin fraction. That the amount of anti-trypsin in serum can be increased considerably by injection of trypsin solutions, has been shown by Achalme (Ann. Inst. Pasteur, 1901, 15, 737) and by W e i n 1 a n d (Zeitschr. f. Biol., 1902, 44, 1, 45). As regards the sensitiveness of the " anti-proteolase " to heat, Vandevelde's experiments (Biochem. Z., 1909, 18, 142) appear to indicate that weakening takes place at 55. According to F e r m i (Centralbl. f. Bakt., 1909, I, 50, 225), anti-tryptic action is exhibited by various organic tissues and by certain protein substances, such as yolk of egg and milk; casein alone also has an anti-tryptic action. The clinical significance of the tryptases and anti-tryptases, which cannot be treated in detail here, will be found discussed in a comprehensive paper by von Bergmann and Kurt Meyer (Berl. klin. Wochens., 1908, 45, 1673). Anti-urease. As was discovered by L . Moll (Hofm. Beitr., 1902, 2, 344), normal (rabbit-) serum and normal albumin- free urine always exert a retarding action on urease. This action is markedly increased by injection of small doses of a urease- preparation from Micrococcus ureae Pasteuri. Moll does not regard the anti-bodies of normal and of immu- nised serum as identical, since the latter loses the excess of its inhibiting power and hence becomes normal in this respect if heated for an hour at 65 (but not at 56), whilst the retarding capacity of normal serum is not altered by heating either for an hour at 65 or for six hours at 56. Anti-bodies of clotting enzymes. The important discovery of Morgenroth, that subcutaneous injection of rennet produces an anti-rennet in the serum and milk of the immunised animal (Centralbl. f. Bakt., 1899, 26, 349; 1900, 27, 721), directed attention to these anti-enzymes. 272 GENERAL CHEMISTRY OF THE ENZYMES As regards anti-rennet itself, Morgenroth and more especially M a d s e n and W a 1 b u m , and Bashford have inves- tigated its action quantitatively, whilst F u 1 d and S p i r o (H., 1900, 31, 132) have made a comprehensive study of the rennetic and anti-rennetic action of the blood. Arrhenius has pointed out the analogy existing between the behaviour of the clotting enzymes and that of certain precipitins towards the anti-bodies. It is hence unnecessary to describe the equilibrium phenomena between rennet or the fibrin-ferment and the anti- bodies. Emphasis must be laid on the fact discovered by H a m - m a r s t e n and R 6 d e n as early as 1887 that the normal serum of various animals contains a substance which inhibits the action of rennet. This substance is, however, not identical with that produced by active immunisation (Bashford, Journ. of Pathol., 1902, 8,52). According to Fuld and Spiro, the " anti-rennet " contained in horse-blood serum is a pseudo- globulin which acts by fixing a portion of the calcium ions and so retarding coagulation; these authors separate the chymosin and anti-chymosin of normal blood by precipitation with ammo- nium sulphate. For the relations between anti-pepsin and anti-rennet, see J a c o b y (Biochem. Z., 1907, 4, 471). Anti-fibrin-ferment. Bordet and G e n g o u (Ann. Inst. Pasteur, 1901, 15, 129) obtained this anti-body in the following manner: They injected guinea-pigs with normal rabbit-serum, by which means the guinea-pig-serum acquires the property of retarding the coagulation of rabbit-blood, i.e., the anti-fibrin-ferment is formed. This acts, as these authors showed, in a somewhat markedly specific manner on the sera of different animals. This anti-body is not affected by heating to 55. The composition of the anti-fibrin-ferment is not definitely known. Anti-laccase was thought to have been obtained by G e s s a r d (Soc. Biol., 1903, 55, 227) in rabbit-serum by injec- tion of laccase; the shortness of his communication renders criticism of the work impossible. The anti-catalase of Battelli and Stern (Soc. Biol., 1905, 58, 235, 758) behaves like ferric sulphate and should be ENZYMIC SYNTHESES 273 classed with the inhibiting agents. A true anti-enzyme of catalase does not appear to exist (D e W a e 1 e and Vandevelde, Biochem. Z., 1908, 9, 264). Although anti-enzymes corresponding with vegetable enzymes have often been produced in the animal body (M a g n u s and Friedenthal), no appreciable formation of anti-enzymes in plants has yet been observed. The most remarkable property of the anti-enzymes, in the narrow meaning of the word, is the rigid specificity of their action; this property they possess in common with the anti- toxines. Whether this is a peculiarity of the anti-bodies formed as protective agents in the organism, or whether closer investiga- tion will show that the anti-enzymes act specifically only in the same sense as do the enzymes, cannot at present be decided. It can, however, be asserted from the experimental material at present available, that the specificity of the enzymes does not differ fundamentally from that of other catalysts. CHAPTER VIII SPECIFICITY OF ENZYME ACTION THE question of the acceleration of one and the same reaction by different enzyme-preparations does not lend itself to a critical examination, owing to the impossibility of judging the physiological purity of the preparations. If therefore it is stated, for example, that emulsin hydrolyses fats, this can scarcely have any other meaning than that the preparations considered con- tain lipases, unless indeed it is shown that the actions on glucosides and fats always exhibit parallel courses. Meanwhile, the decid- ing factors in such cases are physiological in nature, whilst the chemist is concerned more nearly with the other problem, namely, that of determining what different reactions are always initiated by one and the same enzyme-preparation and enzyme. 1 The investigations of recent years have rendered it probable that in many enzyme-preparations in which formerly the presence of only one enzyme was assumed, a number of different enzymes exist with more restricted spheres of action. It has, indeed, been mentioned that emulsin contains at least five enzymes, and that diastase is presumably composed of a number of enzymes which effect the degradation of starch in stages. The term specificity is applied to cases where the action of an enzyme is exerted only on separate representatives of a larger class of bodies. Some of these cases are understood in so far as the course of the chemical change can be followed. Thus, the well-known fact that only sugars with six or nine carbon atoms, and not, for instance, the pentoses, are fermentable, is much less remark- (Zeitschr. f. physikal. Chem., 1903, 45, 513) has studied the interesting case in which different, inorganic catalysts (stannic chloride, iodine chloride, etc.) cause or accelerate catalytically different reactions of the substrate (chlorine and benzene). 274 SPECIFICITY OF ENZYME ACTION 275 able now that a representation of the intermediate products of the reaction has been attained. The specificity of the oxydases, to which attention has been repeatedly drawn, would most readily, arise as a consequence of purely chemical facts. In the cases where the specific nature of the oxydases is most pronounced, namely, with the phenolases, it may be asserted that the reactivity of the simple and substituted mono-, di- and tri-phenols is dependent on the constitution in the same way when " oxydases," as when non-enzymic manganese com- pounds, form the oxidising catalysts. To choose the simplest example: of hydroquinone, pyro- catechol and resorcinol, the first is oxidised rapidly and the second considerably more slowly, whilst the last is extremely resistant to oxidation (cf. BertrandBull. Soc. Chim., 1896, [iii], 15, 791). Another case is that of the Upases. From the results of K a s 1 1 e and Loevenhart's measurements, it is known that the ester-resolving action of pancreas-extract is by no means exerted on all esters. Apart from the fact that the true fats are only very slightly hydrolysed by this extract, enormous differences are observed between the velocities of hydrolysis of such closely-allied chemical individuals as ethyl acetate and ethyl butyrate. A further list of similar differences has been given by H. E. Armstrong and O r m e r o d . But with catalytic decompositions, completely analogous behaviour is shown. For example, according to R. Lowenherz, the constants of hydrolysis (with hydrochloric acid as catalyst) of ethyl formate and methyl benzoate are in the ratio of 1-1 : 0-0003, and still larger differences can easily be found. In this connection, it is to be noted that in enzyme reactions very small velocities do not show, since the extended duration of the action results in the enzyme becoming inactive. Also the results ofH. BierryandGiaja's experiments (C. R., 1908, 147, 268) on the action of maltases and lactases of various origins appear to depend on differences of degree, and not of kind, in the activities; the resolution of lactose, lactobionic acid and lactosazone is effected by an active preparation, whilst another preparation which resolves only lactose must be generally weaker. 276 GENERAL CHEMISTRY OF THE ENZYMES Fischer andAbderhalden (H., 1905, 46, 52; 1907, 50, 264) have collected a large mass of data concerning the power possessed by pancreatic juice of decomposing polypeptides- The behaviour of these substances towards P a w 1 o w ' s pan- creatic juice is shown in the following table: Hydroly sable. Non-hydrolysable. *Alanylglycine 1 Glycylalanine *Alanylalanine Glycylglycine *Alanylleucine A Alanylleucine B *Leucylisoserine Leucylalanine Glycyl-Z-tyrosine Leucylglycine *Alanylglycylglycine Aminobutyrylglycine *Leucylglycylglycine Aminobutyrylaminobutyric acid A * Glycylleucylalanine Aminobutyrylaminobutyric acid B * Alanylleucylglycine Aminoisovalerylglycine Dialanylcystine Glycylphenylalanine Dileucylcystine Leucylproline Tetraglycylglycine Diglycylglycine Triglycylglycine ester (Cur- Triglycylglycine t i u s ' s biuret base) Dileucylglycylglycine The hydrolysis of these substances by acids would, no doubt, likewise reveal considerable differences between the velocities. But, as K a s 1 1 e and Loevenhart's experiments with esters show, the order is not always the same for hydrolysability by enzymes and by other catalysts. The lack of parallelism between the two cases may be due to enzymes and acids being combined to different extents by different esters. The enzymes exhibit the strictest specificity towards optical antipodes. After E . Fischer had shown how new optically active products are obtained by purely chemical syntheses (Chem. Ber., 1894, 27, 3230), the fundamental difference assumed by Pasteur between natural and artificial syntheses fell to the ground. Four years later Fischer arrived at the conclusion that the specificity of enzymes towards optical antipodes is con- ditioned by the stereochemical structure of the enzymes (H., * 1898,26,60). 1 Peptides marked * are the racemic compounds. SPECIFICITY OF ENZYME ACTION 277 He himself, partly in conjunction with his collaborators, obtained in the hydrolysis of the methylglucosides the most striking examples of the influence of configuration on the attack- ability of a substrate. Both a- and @-methyl-d-glucosides are acted on by enzymes, but a- and $-methyl-/-glucosides remain unchanged. While, however, a-methyl-d-glucoside is hydrolysed only by yeast- enzymes, emulsin attacks (3-methyl-d-glucoside alone. In general, it appears that a-glucosides are decomposed by maltase and ^-glucosides by almond-emulsin. OCHs CH 3 HO-C-H r. H-C K-C OH CH 2 OH a-methylglucoside. H-C H-C-OH I CH 2 OH /3-methylglucoside. Further cases in which enzymes hydrolyse stereoisomeric compounds with very unequal velocities are given by the inves- tigations of Fischer and of Abderhalden on poly- peptides. Some of their results are as follows: Hydrolysed. d-Alanyl-d-alanine d-Alanyl-Z-leucine Z-Leucyl-Meucine /-Leucyl-c?-glutamic acid Not hydrolysed. d-Alanyl-Z-alanine Z-Alanyl-rf-alanine Z-Leucylglycine Z-Leucyl-d-leucine d-Leucyl-Weucine Since the view was advanced that enzymes act as optically active catalysts, numerous cases of enzymic, asymmetric syntheses and decompositions have been observed. Asymmetric Syntheses. If a symmetrical mole- cule gives rise to an asymmetric one, the dextro- and laevo- 278 GENERAL CHEMISTRY OF THE ENZYMES modifications are formed in equal quantities, so that an inactive, racemic preparation is obtained. It is, however, otherwise if a molecule which is already asymmetric is employed for further syntheses. If in one of the two optical antipodes, I and II, say, in I, one of the substituents 6 is replaced by another radicle r, two new forms, i and @i, may arise. Since in these two forms the new group r is at different distances from the remaining constituents of the molecule, it is evident that the molecules i and gi will be formed with unequal velocities. Just as two diastereomeric, 1 asymmetric products i and @i are formed from configuration I, so also II, which is the mirror- image of I, yields two corresponding diastereomerides, a 2 and @ 2 , these being mirror-images to ai and gi. The forms I and II thus give the two pairs of optical isomerides, ai+a2 and Pi + fe, in different amounts. As an example of an asymmetric synthesis analogous to those occurring in the living organism, the following case, which was investigated by Marckwald, may be taken : Methylethylmalonic acid was converted into an acid salt: C0 2 H C0 2 M CO 2 M CH 3 -C-C 2 H 5 a CH 3 -C-C 2 H 5 & C 2 H 5 -C-CH 3 C0 2 H C0 2 H C0 2 H The two forms of the acid salt, a and g, . as optical antipodes' possess similar properties, except as regards the sense of their rotation. This is the case, however, only if M itself is optically inactive. But if M itself be an optically active radicle, as, for instance, if the acid brucine salt of the acid were formed, the 1 Stereoisomeric compounds related as an object to its image in a mirror, are termed optical or enantiomorphous isomerides. On the other hand, stereoisomeric compounds which are not mirror imageSj one of the other, are named diastereoisomerides or dias- tereomerides. SPECIFICITY OF ENZYME ACTION 279 two forms a and @ will no longer be enantiomorphs but diastereo- merides and hence will exhibit different physico-chemical be- haviour. If the mixture of a- and $-forms is heated so as to remove the free carboxyl groups, the dextro- and laevo-salts must be formed in unequal amounts. The free valeric acids obtained by removal of the brucine residue, form an optically active mixture. According to Fischer's expression, from one active molecule (brucine), " another is born." A series of interesting syntheses was also carried out by McKenzie (Journ. Chem. Soc., 1904, 85, 1249) who, by reduction of Z-menthyl benzoylformate with aluminium-amalgam, obtained a mixture of Z-menthyl d-mandelate with a slight excess of Z-menthyl Z-mandelate. Shortly afterwards, by the reduction of Z-menthyl pyruvate, he succeeded in preparing laevo-lactic acid (Journ. Chem. Soc., 1905, 87, 1373). With the help of Grignard's reaction, other asymmetric syntheses, such as that of laevo-atrolactinic acid from menthyl benzoylformate and magnesium menthyl iodide, were effected (Journ. Chem. Soc., 1906, 89, 365). Mc- Kenzie and Wren prepared the optically active tartaric acids by oxidation of d- and /-bornyl and menthyl fumarates (Journ. Chem. Soc., 1907, 91, 1215). Of the investigations in this direction those of D a k i n deserve special mention. After W. Marckwald and A. McKenzie had succeeded in showing that the velocities of esterification of two opposed optically active acids by one and the same optically active alcohol were not equal (Chem. Ber., 1899, 32, 2130; 1901, 34, 469), Da kin found (Journ. of Physiol., 1903, 30, 253) that, when partially hydrolysed by lipase, inactive menthyl mandelate yields a strongly dextro- rotatory mandelic acid, while the remaining ester is correspond- ingly laevo-rotatory; the dextro-component of the ester is hence hydrolysed more rapidly than the laevo-component. 1 Further experiments by this investigator have led to a number of inter- esting conclusions (Journ. of Physiol., 1905, 32, 199). It must be remembered that two optical antipodes, in combining with one and the same asymmetric substance, do so with unequal velocities and that, on the other hand, the products of such 1 The hydrolysis of racemic esters has found practical application also in the preparation of optically active amino-acids (Warburg). 280 GENERAL CHEMISTRY OF THE ENZYMES reactions decompose at different rates. This was the case with D a k i n ' s asymmetric ester-hydrolysis by lipase; the latter must therefore be an optically active substance which enters into combination with the ester it hydrolyses. Experiment showed further that, in the fractional hydrolysis of a series of structurally allied racemic esters, the components which are the more rapidly attacked always possess similar configurations but not necessarily rotations of the same sign. Asymmetric hydrolysis by means of lipase is also effected if an asymmetric carbon atom is present, not in the acid- but in the alcohol-residue of the ester. D a k i n therefore drew the conclusion that combination between enzyme and ester takes place, not exclusively at the acid-group but probably with the molecule of the ester as a whole. Finally, a number of earlier (S c h u 1 z e and Bosshard, H., 1886, 10, 134) or isolated observations, and also the more recent ones of A. McKenzie and A . Harden (Journ. Chem. Soc., 1903, 83, 424) show that the specificity of the action of micro-organisms on optical antipodes is not complete. The enantiomorph less preferred as nutriment is also consumed by micro-organisms, although considerably more slowly and imper- fectly, and even in cell-free (active) enzyme solutions, in certain cases at least, neither of the two forms seems to remain unat- tacked. Here also, quantitative measurements of the relative attackability of the antipodes promise valuable results. While D a k i n ' s experiments dealt entirely with asymmetric hydrolyses, Rosenthaler has recently described (Biochem. Z., 1908, 14, 238) a true asymmetric synthesis, namely, the forma- tion of d-benzaldehydecyanohydrin from benzaldehyde and hydrocyanic acid under the influence of emulsin. Of his exper- iments the following may be described : To 5 grms. of emulsin, macerated with 20 c.c. of water, was added 0-675 grm. of hydrocyanic acid; after an hour, 20 grms. of benzaldehyde were slowly added, the liquid being kept thoroughly shaken meanwhile. The liquid was then agitated in a shaking machine for an hour, after which the nitrile was isolated and hydrolysed, and the mandelic acid extracted from the aqueous solution by means of ether. The residue from the ether, after crystallisation from benzene, showed a specific rotation of []/>= -153-78, which is in good agreement with the value SPECIFICITY OF EJNZYME ACTIOJN for mandelic acid. The cyanohydrin formed was free from the laevo- form, as was shown by hydrolysis to mandelic acid. As Rosenthaler found, in the emulsin there is a sub- stance which conditions the asymmetry of the synthesis and another constituent which accelerates the addition of hydro- cyanic acid to aldehyde or ketone. The latter of these substances proves to be a compound of magnesium, calcium or potassium. The explanation of this phenomenon is probably to be sought in Franzen's recent investigation (Chem. Ber., 1909, 42, 3293), which showed that aldehydes and ketones, with calcium, barium, strontium or magnesium cyanide, lead to the formation of the salt of the corresponding nitrile. This reaction proceeds as follows: /0-Ca-Ov 2C 6 H 5 CCH - C 6 H 5 . X H \CN CW Of great interest is the fact that Rosenthaler (Biochem. Z., 1910, 26, 1 and 28, 408) has succeeded in preparing from emulsin, besides the nitrile-synthesising enzyme (a-emulsin) which we shall term n i t r i 1 e s e , an enzyme which exerts solely a hydrolytic action and was named by him B-emulsin. By protracted heating at 40-45 the B-emulsin is inactivated completely and the hydrolytic enzyme partially, whilst the nitrilese remains active. Suitable treatment with acid and subsequent neutralisation with alkali also destroys the amygdalin-resolving action of emulsin, while the synthetic action (of the nitrilese) is to some extent retained. The nitrates obtained after precipitating with copper sul- phate, saturating with magnesium sulphate or half saturating with ammonium sulphate, contain no nitrilese but still hydrolyse amygdalin. In the decomposition of amygdalin, three enzymes must hence take part (cf. p. 23): an amygdalase, a g-glucosidase and a n i t r i 1 a s e which resolves the mandelonitrile; the two first are hydrolysing enzymes, whilst the last has a purely decom- posing action. Rosenthaler has attempted to separate his -emulsin further into these three constituents in the following manner: 282 GENERAL CHEMISTEY OF THE ENZYMES 1. The filtrates obtained after precipitation with copper sul- phate and half saturating with ammonium sulphate contained all the three enzymes. 2. The filtrate obtained after saturating with magnesium sulphate contained hydrolysing enzyme, but no nitrilase. No other means could be discovered of separating the hydror lysing enzyme from that which decomposes the nitrile. No satisfactory theoretical treatment of the co-existence and co-operation of a synthetic and a decomposing enzyme has, in the author's opinion, yet been advanced. Mention must, however, not be omitted of the theory developed by F a j a n s (Dissertation, Heidelberg, 1910; Zeitschr. f. physikal. Chem., 1910, 73, 25). An interesting case, closely related to the above, has been described by B r e d i g and F a j a n s (Chem. Ber., 1908, 41, 752). The two optically active camphocarboxylic acids, which readily decompose into camphor and carbon dioxide when heated : CioHi 5 CO 2 H = CioHi G 0+C0 2 , do so with different velocities when they are dissolved either in pure nicotine or in a solvent containing nicotine. The follow- ing results were obtained: Velocity of liberation of carbon dioxide in nicotine at 70. Per 1 grm. dextro-acid. fed Dissolved in 3 c.c. nicotine . 0-00493 " 5 0-00493 " 10 " . 0-00479 Mean.. . 0-00488 Per 1 grm. laevo-acid. Dissolved in 5 c.c. nicotine . 0-00436 " 5 " . 0-00444 10 " . 0-00421 Mean.. . 0-00434 Hence in nicotine as solvent, the d-acid decomposes about 13% more rapidly than the Z-acid. Salt-formation evidently takes place between the active acid and the active base, the diastereomeric bodies thus formed differing in their chemical behaviour. In the case studied by B r e d i g , the carbon dioxide liberated is evolved, so that the nicotine previously combined with the camphocarboxylic acids becomes free after the decomposition and can form salt with fresh quantities of acid. SPECIFICITY OF ENZYME ACTION 283 The difference between the experiments of Marckwald and those of B r e d i g consists in the employment by the latter of a weaker base. The reaction studied by B r e d i g hence assumes the character of a catalytic process. Nevertheless, both the results obtained by Marckwald and those of B r e d i g support the view, first expressed by Fischer, that the enzymes are optically active catalysts. Their mode of action may apparently be expressed as follows: By combination with the racemic substrate, the optically active enzymes give rise to diastereomeric substances, which decompose with different velocities and hence result in the forma- tion of optically active material. E . Fischer made an interesting experiment to ascertain if two oppositely active acids, d- and Z-camphoric acids, hydro- lyse cane-sugar with different velocities, but the result was neg- ative. The probability of the assumption that catalysing acids, like enzymes, combine with the substrate, suggests the extension of this experiment and the making of others in which a substrate consisting of two enantiomorphs shall be decomposed by an optically active catalyst. The author has been occupied with such experiments for several years. It is evident that all facts are of value which furnish further knoweldge of the union between enzyme and substrate. By the representation of the key fitting the lock, that is, by the hypothesis that the enzymes are optically active catalysts, the enzymes are brought into close relation with other catalysts. The development of this hypothesis is undoubtedly one of the most important aims of the chemistry of the enzymes. CONCLUSION WHAT then can be given as the results of the investigation of the enzymes? As regards the chemical nature of the enzymes, the result of our survey is but negative, inasmuch as the analyses and chem- ical reactions of various enzyme preparations furnish no evidence in support of the statement often found in the literature 1 that enzymes are protein substances; further there is nothing to indicate that all enzymes belong to a single class of substances. 1 See, for instance, V e r n o n , Ergeb. der Physiol., 1910, 9, 227. 284 GENERAL CHEMISTRY OF THE ENZYMES On the other hand, no fact is known which definitely disproves the protein character of any of the hydrolytic enzymes, since the results indicating the failure, of enzyme solutions to give protein reactions do not give the concentrations of the solutions and have not been sufficiently controlled by means of similarly dilute solu- tions of undoubted proteins. All that has yet been stated concerning the chemical con- stitution is mere supposition. Better than from the purely chemical investigations, we could, from the physico-chemical measurements of thermo-sensibility, i.e., from the inactivation constants, attempt to derive certain relations with the proteins, the denaturation of these by heat bearing a close resemblance to that of the enzymes. But perhaps the saponins exhibit still more marked analogies to those remarkable colloidal poisons, the physico-chemical behaviour of which is still insufficiently investigated. The view that, for the development of their activity towards the substrate, certain enzymes require the presence of other substances co-enzyme, acid, etc. which are classed together as activators, has resulted in a thorough qualitative investigation of the chemistry of enzyme-action, and the consideration of these activators is of the utmost importance to the chemico- dynamic study of the enzymes. The many deviations of the best-known enzymes, e.g., invertase, from the simple relations required by the law of mass action, had led to a formal treatment of enzymic reactions, but the results of this correspond, by no means, with the amount of labour expended. Only in the most recent times has the nec- essary revision of the earlier experiments been commenced; never- theless, it cannot be regarded as premature to assert that the reactions induced by enzymes the enzymic hydrolyses being here especially referred to follow the laws which hold generally for catalytic reactions in solution and are deducible theoretically from the law of mass-action. Correspondence of the time-law with that for unimolecular reactions, and proportionality between concentration of the enzyme and velocity of reaction were fre- quently observed. Where these relations are not obeyed, the disturbing influence exerted by the products of the reaction either is known with certainty or may be assumed with a high degree of probability. In individual cases, the ultimate cause SPECIFICITY OF ENZYME ACTION 285 of this disturbance is still uncertain; sometimes it must be the enzymes themselves, but in many instances the activators, which are combined, the latter case appearing to be the more common. In any case, we have seen that a simple explanation is forthcoming for S c h u t z ' s rule; relations similar to that of S c h ii t z can also occur with inorganic catalysts, one of the best-known, apparent peculiarities of enzymic reactions thus falling to the ground. For, that the numerous " laws " such as 7 1 a+x etc., possess neither real significance nor validity may be regarded as an established and pleasing fact. The enzymes are, therefore, catalysts. Do they, like the inorganic catalysts of the best-known reactions, for instance, hydrolysis of esters, leave the equilibrium of the reaction unchanged? Perhaps, under some circumstances and more fre- quently than now appears to be the case, they do so, but it can be stated with certainty that this does not always happen, and the conception of a catalyst must hence be made more compre- hensive than experience of non-enzymic reactions requiies. 1 But this leads to no disagreement with the principles of chemical dynamics or with the fundamental laws of thermodynamics, it being only necessary to assume that a considerable proportion of the enzyme can combine with the substrate or with the pro- ducts of the reaction. The equilibrium must evidently depend on the concentration of these new molecules, enzyme-substrate or enzyme-reaction product, and any circumstance altering this concentration alters also the equilibrium or the stationary con- dition of the reaction. The configuration of the substrate, the spatial arrangement of its atoms, is, as was seen in the preceding chapter, of deter- mining importance for the occurrence of an enzymic reaction. Emil Fischer has given us the theory for these facts, the underlying assumption being that an enzyme is an optically active catalyst. This forms, with the two components of a racemic mixture, " active molecules " which are not enantiomorphous but diastereomeric products, differing in their chemical properties 1 Cf. Taylor, Journ. of Biol. Chem., 1910, 8, 503. 286 GENERAL CHEMISTRY OF THE ENZYMES and hence leading to the decomposition of the components with unequal velocities. Thus, what formerly appeared to char- acterise the catalysts of living matter presents itself, in the light of this theory, as a consequence of our views of the configuration of molecules. But what is the nature of the combination between the enzyme and the substance succumbing to chemical attack, and how does the living organism maintain the equilibrium between enzyme and substrate so important to its existence? Here we meet the great riddle of the formation of enzymes and anti-enzymes in the organism which opens out a region of investigation of immeasurable breadth. APPENDIX PRACTICAL METHODS IN what follows, a short description is given of those methods of investigating enzyme-preparations and of following enzymic decompositions which have been or might be generally applied either in medicinal practice or in industrial work. Scientific investigation of enzymic reactions has often been effected with the acid of physico-chemical methods, but a detailed account of these would occupy too much space here, so that reference must be made to the special literature of the subject. Particular mention may be made of : O s t w a 1 d and Luther : Manual of Physico-chemical Measurements. W. A . R o t h : Exercises in Physical Chemistry, London, 1909. Hamburger : Osmotischer Druck und lonenlehre in den medizinischen Wissenschaften, Wiesbaden, 1902-1904. Also shorter references by: H. Friedenthal, L. Michaelis, etc., in Abder- halden's Handbuch der biochemischen Arbeitsmethoden, Berlin, 1910-1912. A . K a n i t z , in Oppenheimer's Handbuch der Biochemie des Menschen und der Tiere, Band I, 1908. As regards the methods of obtaining enzymes, these have been given in the first chapter of this book, where also the preparation of the separate enzymes in a pure state has been described. It is only necessary here to call attention to the fact that enzymes which exert their actions within the walls of a cell are often either not at all or only very incompletely extractable, so 287 288 GENERAL CHEMISTRY OF THE ENZYMES long as the cell- wall is living and uninjured; the dead cell-wall allows of much readier passage to the enzyme, which can thus be obtained in several ways. 1. The enzymic material is dried as rapidly, and at as low a temperature, as possible. By this treatment, the cell-walls are rendered, in st)me cases, more permeable and in others, more easily ruptured; these effects are enhanced if the dehydrated cells are heated at about 50-70 [cf. E. Fischer's method of preparing invertase (Chem. Ber., 1894, 27, 2985); and Wiechowski and Wiener's method (Hofm. Beitr., 1907, 9, 232) for preparing from the kidneys the enzyme which oxidises uric acid]. 2. The finely-divided material, e.g., yeast, after being freed mechanically from water, is introduced into absolute alcohol or anhydrous acetone. Here also, the dehydration of the mate- rial by the organic solvent should be as rapid as possible. 3. The cell-walls may be destroyed by autolysis (see O ' Sul- livan and Tompson's method, p. 26). 4. The cells, while still living, are ruptured mechanically. The method so successfully employed by Buchner (see p. 56) is well known. Rowland (Journ. of PhysioL, 1901, 27, 53) gave a somewhat different method, in which a mixture of the cells with sand is made to assume a vigorous rotatory motion; the action resembles that of a sand-blast. Bacteria, soft organs, etc., can be hardened by cooling in liquid air and are then readily broken up. The analytical methods employed may now be mentioned. L i p a s e s . Pancreas-lipase is very suitably tested by means of an aqueous emulsion of egg-yolk, as in the work of V o 1 h a r d and others. The quantity of fat hydrolysed in time t is measured and the total amount hydrolysable then cal- culated. To this end, the egg-yolk emulsion containing the lipase is extracted with ether: (I) An aliquot part (50 c.c.) of the ethereal extract is titrated after addition of 50 c.c. of alcohol and then hydrolysed with 10 c.c. of normal sodium hydroxide solution, the salts of the fatty acids being decomposed after 24 hours by means of 10 c.c. of normal sulphuric acid. (II) The fatty acids APPENDIX 289 obtained by hydrolysis are estimated by titration and the per- centage x of fatty acid split off by the enzyme calculated by the formula, I : I+II = z : 100. In shaking the fat-emulsion with ether, more rapid separation is effected if 2-10 c.c. of alcohol are added to the ether. In this connection see Stade, Hofm. Beitr., 1902, 3, 291, and E n g e 1 , Hofm. Beitr., 1905, 7, 78. Esterases of lower esters. Ethyl butyrate is best employed as substrate. The course of the reaction is fol- lowed by direct titration or by observation of the change of the electrical conductivity. Vegetable lipases. Ricinus seeds are skinned, freed from oil by pressing and treating the pressed cake with ether, and finely ground. The seed-juice formed is separated from the inactive constituents of the seed in a centrifuge. This juice is allowed to stand for 24 hours, during which time the enzymic emulsion, in which the acid (lactic) necessary for activation is formed, collects at the surface and can be removed. One hun- dred grms. of oil and 0-2 grm. of manganous sulphate are stirred up with this emulsion (5-10 grms.) and the mixture left. Here also the lipolysis can be followed by titration. A m y 1 a s e s . For the estimation of the diastatic power of malt for brewery purposes, L i n t n e r (Zeitschr. f. prakt. Chem., 1886, 34, 386) gave the following method, which, in prac- tised hands, gives good results. Separate volumes; of 0-1, 0-2, 0-3, .... 1-0 c.c. of malt extract [25 grms. of the ground malt +500 c.c. of water, allowed to stand at 21 (70 F.) for 3 hours and then filtered bright] are added to a series of 10 test-tubes, each containing 10 c.c. of 2% soluble starch solution, the contents of each tube being well mixed. After exactly 1 hour's rest at 21, 5 c.c. of Fehling's solution are mixed with the liquid in each tube and the tubes then immersed in a boiling water-bath for exactly 10 minutes, after which the precipitate is allowed to settle. If the Fehling's solution in the tube containing 0-1 c.c. of malt extract is just completely reduced, the diastatic power of the malt is taken as 100; if that in the one containing 0-2 c.c. of the extract, the diastatic power is 50, and so on. A more exact result may be 290 GENERAL CHEMISTRY OF THE ENZYMES obtained, if necessary, by taking 0-1, 0-15, 0-2, 0-25, etc., c.c. of malt extract for the series of tubes. The cold-water malt extract itself contains a small amount of sugars which reduce Fehling's solution; the extent of this reduction may be determined by direct experiment, but for all ordinary purposes it is sufficiently accurate to deduct 1*5 from the value obtained for the diastatic power in the manner described above. The following modification of the above method has been recently devised by Ling and is widely used : 3 (or 1 0, 2 0, or 4-0, according to the expected diastatic power) c.c. of the malt extract (prepared as already described) are added to 100 c.c. of 2% soluble starch solution in a 200-c.c. flask, the mixture being kept at 21 (70 F.) for 1 hour. At the end of this time, 20 c.c. of N/10-sodium hydroxide solution are added and the liquid made up to 200 c.c. with water. After mixing, this solu- tion is introduced into a burette and gradually run into 5 c.c. of Fehling's solution diluted with a little water and kept boiling; this is continued until the solution just loses its blue colour or fails to give a brown coloration with a drop of ferrous thiocyanate solution on a white tile. If, say, 25 c.c. of the liquid (100 c.c. of which corresponds with 1 grm. of soluble starch and 1-5 c.c. of malt extract) are required tc reduce 5 c.c. of Fehling's solution, the diastatic power of the malt will be 1000 =26-7. 25X1-5 This method gives excellent results, in exact agreement with those given by L i n t n e r 's method. With preparations of diastase, L i n t n e r dissolves 2-0 5 grm. (according to the activity) in 50 c.c. of water and adds 0-1, 0-2, . . .1-0 c.c. of this solution to a series of 10 test-tubes, each charged with 10 c.c. of 2% starch solution. The subsequent procedure is exactly similar to that employed in the case of malts. A soluble starch which can be readily prepared with constant properties, is obtained by allowing potato starch to remain under 7 5% hydrochloric acid solution for 7 days at the ordinary temperature, then removing the acid completely by washing with cold water, and drying the starch in the air. This method APPENDIX 291 yields a product dissolving readily in hot water to a clear solution (cf. G.C.Jones, Journ. Inst. of Brewing, 1908, 14, 13). The determination of the velocity of hydrolysis by oxidation of the liquid with F e h 1 i n g ' s solution (cf . Wroblewski, H., 1898, 24, 173) is, indeed, the most reliable of the methods yet developed, although it is still capable of improvement. A gravimetric method, which seems to give good results, has recently been proposed by Sherman, Kendall and Clark (Journ. Amer. Chem. Soc., 1910, 32, 1073), who have also compared the older methods. A number of other methods are based on the colorations produced by iodine in starch and dextrin solutions. Of the earlier methods, those of D e t m a r (H., 1882, 7, 1) and Roberts (Proc. Roy. Soc., 1881, 32, 145) may be referred to, whilst the following method, given by Wohlgemuth (Biochem. Z., 1908, 9, 1), deserves special mention. To each of a series of test-tubes containing different quanti- ties of the enzyme solution to be tested are added 5 c.c. of 1% starch solution, each tube being placed immediately in a wire basket standing in ice-water, so as to prevent the slightest enzyme action. When all the tubes are ready, the wire basket contain- ing them is transferred to a water-bath at 40; by this means, the enzyme action in each tube begins at the same moment. After 30 or 60 minutes, the basket is placed again for a short time in ice-water, so that all the actions are interrupted at the same instant. The strength of the enzyme solution is then deter- mined as follows: All the tubes are filled with water up to within about the thickness of the finger from the top, and to each is added a drop of decinormal iodine solution, the liquid being then mixed. Different colorations dark blue, bluish-violet, reddish-yellow and yellow are thus obtained. The tubes showing a yellow or reddish-yellow colour contain disregarding further degradation of the starch to maltose or isomaltose and dextrose only achro- odextrin or erythrodextrin, the bluish-violet ones contain a mixture of erythrodextrin and starch, whilst those with a dark-blue colour contain mainly unaltered starch. As the lower limit of the activity (limes) Wohlgemuth takes the first tube in which the blue colour cannot be detected, i.e., the one showing 292 GENERAL CHEMISTRY OF THE ENZYMES a violet colour. In the preceding tube, the whole of the starch is broken down to the dextrin stage at least; from this, calcula- tion is made of the number of c.c. of 1% starch solution degraded to dextrin by 1-0 c.c. of enzyme in the time employed for the experiment. Suppose that the tube which has become just colourless contained 0*02 c.c. of saliva; this amount was then able to trans- form 5 c.c. of 1% starch solution into dextrin in 30 minutes, so that 1 c.c of saliva corresponds with 250 c.c. of 1% starch solution. To indicate the diastatic power of 1 c.c. of the enzyme solution, Wohlgemuth contracts the word diastase to D and suggests that the temperature and duration be given for each experiment. The result of the above determination would then be stated thus: D 3Q , = 250. To calculate the diastatic power from an experiment in which tube No. 5 was taken as the lower limit, while tube No. 4, containing 0-0125 c.c. of saliva, was coloured red (see the scale of colour given, 1 o c . c i t .) , we must proceed as follows: In 30 minutes, 0-0125 c.c! degrades 5 c.c. 1% starch solution. In 30 minutes, 1-0000 c.c. degrades 400 c.c. 1% starch solution, Other colorimetric iodine methods are given by J u n g k and by Johnson (Journ. Amer. Chem. Soc., 1908, 30, 798). A criticism of these methods will be found in Sherman, Kendall and C 1 a r k 's paper (1 o c. cit.). G 1 i n s k i and Walther (Pawlow, Arb. d. Ver- dauungsdrlisen, Wiesbaden, 1898) have applied M e 1 1 ' s method to the estimation of diastase. Narrow glass tubes, open at both ends, are filled with starch-paste and immersed in the enzyme solution, the length of the dissolved cylinder of starch being meas- ured after a certain lapse of time. The velocity of this reaction is evidently influenced considerably by the diffusion of the enzyme to the surface of the starch and by the rate at which the hydrolytic products are removed from this surface; movement of the liquid or of the tubes in the liquid has, therefore, considerable effect. On the other hand, this method of procedure measures the liquefac- tion of the starch-paste which is not a direct measure of the sac- charifying action of the amylase. The method is therefore applicable only in special cases. APPENDIX 293 According to E d . M u 1 1 e r (Zentralbl. f. inn. Med., 1908), the use of plates of starch-paste presents certain advantages. Enzymes of the Disaccharides and G 1 u- c o s i d e s . In these cases, the change of the optical rotation affords a simple and accurate method of following the reaction. As was pointed out on p. 159, it is essential to destroy the muta- rotation of glucose; this is best effected by the addition of soda immediately before reading the rotation. Another method consists in determining the reducing power of the solution. This is carried out with Fehling's solution in one of a number of ways, of which that of Bertrand is one of the most accurate and convenient. Bertrand (Bull. Soc. Chim., 1906, 35, 1285) boils the sugar solution to be tested with Fehling's solution of definite com- position for three minutes, the time being reckoned from the instant when the first bubbles form. The precipitated cuprous oxide is filtered on an asbestos filter and washed with hot water. The cuprous oxide remaining in the E r 1 e n m e y-e r boiling flask and also that collected on the filter are dissolved in a solution of ferric sulphate in sulphuric acid, the following reaction occurring: Cu 2 +Fe 2 (S0 4 ) 3 +H 2 S0 4 = 2CuS0 4 +H 2 +2FeS0 4 . The ferrous salt is titrated with permanganate solution, standardised by means of ammonium oxalate. Bertrand has prepared tables for the most important reducing sugars, so that the amount of sugar can readily be obtained from that of the cuprous oxide formed. The solutions employed have the following compositions: F~e hling's solution. Iron solution. Copper sulphate 40 grms. Ferric sulphate 50 grms. Rochelle salt 200 Sulphuric acid 200 Sodium hydroxide 150 " Water to 1 litre Water to 1 litre Permanganate solution 5 grms. potassium permanganate per litre. The iron solution should not reduce the permanganate. If this does occur, the permanganate solution is gradually added to the iron solution until the latter assumes a slight pink colour; it is then ready for use. 294 GENERAL CHEMISTRY OF THE ENZYMES When a sugar solution is to be titrated, 20 c.c. of it are intro- duced into an Erlenmeyer flask of 125-150 c.c. capacity. The amount of sugar in this volume of the solution is preferably 0-010-0-090 grm. and should not exceed 0-100 grm. To the sugar solution are added 20 c.c. of the copper sulphate solution and 20 c.c. of the alkaline tartrate solution which are best kept separate the liquid being then boiled for just 3 minutes after the first appearance of bubbles. For separating the cuprous oxide, use is made of a Gooch crucible packed with asbestos and fitted as usual to a pump- flask. The precipitate is washed with hot water and as little as possible of it collected on the filter. When the washing is complete, the main quantity of cuprous oxide in the Erlenmeyer flask is dissolved in a known volume of the ferric sulphate solution; the oxide changes from bright red to blue-black and finally yields a clear, pale-green solution. This is then poured through the filter to dissolve the remaining cuprous oxide, more ferric sulphate solution being added if necessary. When all the oxide is dissolved, the Erlenmeyer flask and the filter are washed with water and the combined liquids titrated in the pump-flask with the permanganate solution. The change in colour from green to pink is extremely sharp. The equation given above shows that 2 atoms of copper correspond with 2 mols. of ferrous sulphate and hence with 2 atoms of iron to be oxidised by the permanganate. The iron-titre of the permanganate has thus only to be multiplied by the ratio, 63-6 : 55-9-1-1377, in order to obtain the amount of copper, the corresponding quantity of the sugar being then given by the tables. The permanganate solution is standardised as follows: a weighed quantity of about 0-25 grm. of ammonium oxalate is dissolved in a beaker in 50-100 c.c. of water and 1-2 c.c. of pure sulphuric acid. The liquid is heated to 60-80 and the perman- ganate solution run in from a burette until a pink colour is obtained. One molecule of ammonium oxalate, (NH4) 20264 -HH^O (mol. wt., 142-1) corresponds with 2 Fe and hence with 2 Cu. rO > vx O Multiplication of the weight of oxalate by ~ 142 -1 ' *' 6 '' ^ APPENDIX 295 0-8951, gives the quantity of copper corresponding with the volume of permanganate solution required to produce the pink coloration. One litre of the permanganate solution will cor- respond with about 10 grms. of copper. The tables to be used with this method are given at the end of this section (pp. 306-311). I. B a n g (Biochem. Z., 1906, 2, 271) has described a new method for the estimation of reducing sugars which may be applied to the study of enzymic processes. It depends on the fact that, in presence of potassium thiocyanate, cuprous oxide separates as white, insoluble copper thiocyanate. The description of the method is readily accessible and will not be given in detail here. E m u 1 s i n . In discussing the possible methods for measur- ing the decomposition of amygdalin, A u 1 d points out that the estimation of the sugar liberated is affected by a number of considerations. Here also, the influence of mutarotation must be removed. He employs, therefore, in the investigation referred to on p. 173, D u n s t a n and Henry's titrimetric method (Proc. Roy. Soc., 1903, 72, 287) of estimating the free hydro- cyanic acid by means of standard iodine solution. The reaction proceeds according to the equation: HCN+I 2 = CNI+HI. Excess of sodium bicarbonate is employed to combine with the hydriodic acid formed. Proteolytic Enzymes On account of the great importance of measurements of digestive action to pure enzymology and also to practical medicine, the number of communications dealing with methods employed in these measurements is very large. 1 . An optical method was employed byE.Schiitz (compare p. 175). He removed the undigested protein from the peptic albumin solutions and estimated the quantity of peptone formed from the optical rotation of the residual liquid. S c h ii t z and H u p p e r t also used this polarimetric method (see p. 179) whilst Abderhalden and K o e 1 k e r 296 GENERAL CHEMISTRY OF THE ENZYMES have recently studied the action of tryptic enzymes on optically active polypeptides by direct measurement of the change of rotation of the solution. Ib. Obermayer and Pick (Hofm. Beitr., 1905, 7, 331) attempted to apply the alteration of the refractive index to the study of enzymic reactions. But this magnitude changes only in the case of tryptic digestion. Griitzner (Pfliig. Arch., 1874, 8, 452; 1905, 106, 463) has devised a colorimetric method. Fibrin, which has been softened by immersion in 0-1% hydrochloric acid containing carmine, is distributed as uniformly as possible into test-tubes of equal diameters, each containing 15 c.c. of 0-1% HC1, the pepsin solution to be tested being then added. The fibrin stained with carmine is dissolved and thus reddens the liquid; the inten- sity of the red colour indicates approximately the extent of the digestion. The digested liquid is compared with a number of tubes containing carmine solutions of definite dilutions, which are so chosen that the pepsin-content is proportional to the num- bers of the colour scale. This method has been modified by Roaf (Bio-Chemical Journ., 1908, 3, 188). 2. Measurement of the electrical conductivity was first employed for the study of peptic digestion by Sjoqvist (cf. p. 176). This method was subsequently largely used and was applied in investigations on tryptic digestion by Henri and by B a y 1 i s s (Journ. of PhysioL, 1907, 36, 221) and on the hydrolysis of dipeptides by erepsin (E u 1 e r, see p. 188); in the last experiments, a similar quantity of alkali solution was added to each solution in order to increase the variation of the conductivity. 3. Valid objections have been raised against the suggestion made by S p r i g g s (cf. p. 181) to determine the progress of proteolysis by measuring the viscosity of .the protein solutions, and this method cannot be recommended. 4. A purely chemical method of general applicability and great accuracy was proposed several years ago by Sorensen (Biochem. Z., 1908, 7, 45). By this method, the content of protein or its decomposition products in a solution is determined from the number of free carboxyl-groups. The latter can be estimated by titration if APPENDIX 297 ' the free amino-groups of the protein are first combined. This is readily effected by addition of excess of formaldehyde, which unites with the amino-groups, giving methylene-compounds. The increase of carboxyl-groups represents the extent of pro- teolysis, which can hence be expressed by the number of c.c. of N/5-barium hydroxide solution employed in the titration. On the assumption that each carboxyl-group formed during proteolysis corresponds with one amino-group, the amount of proteolysis can also be stated as milligrams of nitrogen, this being obtained by multiplying the number of c.c. of the N/5- barytaby 2-8. The titration is best carried out in presence of thymolphthalein as indicator, the solutions used being as follows: (a) 0-5 grm. of thymolphthalein (G r ii b 1 e r ' s) dissolved in 1 litre of 93% alcohol. (6) 50 c.c. of commercial formaldehyde solution are mixed with 25 c.c. of absolute alcohol and 5 c.c. of the thymolphthalein solution, N/5-baryta being then added until a faint green or blue colour results; this solution should be prepared fresh for each series of experiments. As a control solution, 20 c.c. of boiled water are used. To this are added 15 c.c. of the formaldehyde solution (b) and about 5 c.c. of the baryta solution, the liquid being then titrated back with N/5-HC1 until it assumes a faint blue opalescence. An addition is then made of two drops of baryta, which should change the colour to a distinct blue, and finally of two further drops, which should produce a vivid blue colour. It is this last colour which is obtained in titrating the protein solution, 20 c.c. of which is mixed with 15 c.c. of the formalde- hyde solution (6) and a slight excess of baryta solution; it is then titrated back with HC1 until the colour is fainter than that of the control solution, the baryta solution being finally added in drops until the deep blue of the control is obtained. For the description of the titration with phenolphthalein, the original paper must be consulted. The methods of V o 1 h a r d (Munch. Med. Wochens., 1903, No. 49) and L 6 h 1 e i n (Hofm. Beitr., 1905, 7, 120) are based on methods given by T h o m a s and Weber, and by M e u - nier (1901). In both, casein is employed; Thomas and Weber dissolve 100 grms. of casein in 1900 c.c. of water with 298 GENERAL CHEMISTRY OF THE ENZYMES the aid of 3-2 grms. of sodium hydroxide ( = 80 c.c. normal NaOH) or 5-04 grms. of hydrochloric acid ( = 138 c.c. normal HC1). The alkaline and acid solutions serve for the estimation of trypsin and pepsin respectively. After the digestion, the liquid is acidified, if necessary, with sulphuric acid and salted out with 20% sodium sulphate solution. After filtration, the precipitate is washed on the filter with hot water until the last trace of sulphuric acid is removed; the filter and precipitate are dried and weighed and the weight of undigested protein compared with that obtained in a blank experiment without pepsin or trypsin. The amount of protein dissolved gives a measure of the digestive power of the gastric juice examined. In Meunier's method, the gastric juice (14 c.c.) is mixed with pure hydrochloric acid (0-4 c.c.) and casein (1 grm.). After the casein has settled, 2 c.c. of the clear liquid are removed and the content of free hydrochloric acid estimated. The remainder of the liquid with the undissolved casein is kept for 24 hours in a water-bath at 40, the hydrochloric acid being again estimated in 2 c.c. of the filtrate. Since hydrochloric acid com- bines with protein during peptic digestion, the diminution in the amount of the free acid expresses the extent of the action. V o 1 h a r d employs a modification of the gravimetric method given by Thomas and Weber. As we have seen, the latter method is based on the observation that pure, unal- tered casein, dissolved in the hydrochloric acid of the digest, is completely precipitated by sodium sulphate. Hence, if different quantities of the enzyme are allowed to act on similar amounts of casein solution for equal intervals of time at 40, the precipitate produced by addition of sodium sulphate will be the smaller, the less the proportion of casein remaining undigested, i.e., the larger the proportion peptonised by the enzyme; the larger the residue, the smaller the quantity of enzyme. Thomas and Weber collect the precipitate on a tared, pleated filter-paper, wash with distilled water and dry and weigh. The difference in weight between the residues from experiments in which pepsin has, and has not been employed, serves as a measure of the peptic action. V o 1 h a r d avoids the inconvenience of this weighing by titration of the filtrate. He proceeds on the assumption that peptonisation of the casein solution is accompanied by increase APPENDIX 299 of the acidity of the filtrate, the peptone hydrochlorides being non-precipitable by sodium sulphate and hence passing through the filter. His experiments showed that, when equal quantities of the same acid casein solution without pepsin were used, the acidity of the filtrate was always constant and much smaller than corresponded with the true acidity of the original solution. This depends on the fact that, under similar experimental con- ditions, the casein precipitate always contains the same amount of hydrochloric acid, only the free acid passing into the filtrate. It is therefore justifiable to refer the excess of acidity over this constant value to the peptone hydrochlorides in the filtrate, and hence to regard the increase of acidity as a measure of the extent of digestion. The undigested residues are in inverse proportion to the acidities of the filtrates. As example may be given the following results of V o 1 h a r d taken from L 6 h 1 e i n ' s paper (1 o c . c i t .) : One hundred c.c. of casein solution, previously heated with 150 c.c. of water, were digested with O'l, 0-4, or 0-9 c.c. of gastric juice (acidity 59 : 87) for one hour. Each solution was then made up to 300 c.c. in a graduated cylinder and precipitated with 100 c.c. of 20% sodium sulphate solution. Titration of 200 c.c. of the filtrate from the solution which contained no gastric juice in presence of phenolphthalein, gave the acidity as 19-15. Two hundred c.c. from the other experiments gave Increase of acidity. 1. 0-lc.c. gastric juice, 22-25-19- 15 -acid of the juice (0-043)=3'06 2. 0-4c.c. ,25-5-19-15- (0-17) =6-18 3. 0-9c.c. ,28-5^19-15- " (0-387)=8-96 The casein precipitates were collected on weighed filters, washed, completely dried, washed again and finally dried until constant in weight. Weight of precipitate from original solution, A =4- 104 grms. 1 3-607 " 2 3-053 " 3 2-585 " The amount digested is hence, b v 1 (0-1 c.c.) AI =0-497 grm. ' 2 (0-4 c.c.) A -2 = 1-051 grms. " 3 (0-9 c.c.) A -3 = 1-519 '' 300 GENERAL CHEMISTRY OF THE ENZYMES The proportionality between the degree of acidity of the nitrate and the digested amount determined by weighing, is shown by the quotients: Of the investigations in which this method has been widely used, that ofS.Kuttner (H., 1907, 52, 63) may be mentioned. In the practical application of Volhard's method, the formulation of the results proposed by V o 1 h a r d himself is to be recommended. The pepsin-unit is taken to be that quan- tity of enzyme which renders the nitrate from the whole of the casein used more acid by 1 c.c. of decinormal acid. The digestion which would be produced by 1 c.c. of gastric juice in 1 hour is found from the experimental numbers by dividing the increase of acidity by the product of the time, t, and number of c.c. of juice employed, /. This number is to be multiplied by 2 or by 4, according as 200 or 100 c.c. of the nitrate are titrated. The values thus obtained for the increase of acidity follow S c h ii t z ' s rule, the pepsin-unit being given by the formula, _v*_ '"f-f Example: Suppose the acidity of 200 c.c. of the original solution, after precipitation and nitration, is 18*0, i.e., 36-0 per 400 c.c., and that of the juice, 20 c.c. per 100 c.c. of juice. Then, if in the digestion of 100 c.c. of casein solution made up to 300 c.c. with 3 c.c. of gastric juice for 3 hours, 200 c.c. of the nitrate obtained after adding 100 c.c. of sodium sulphate solution show an acidity of 32 -7( =65-4 per 400 c.c.), the calculation is as follows: less 36-0, for the original solution, and less 0-6, for the gastric juice v=28-8 OQ C o Xo = 3 2, and x = 10 24 pepsin-units. Two simple methods, apparently well suited to the clinical estimation of pepsin, are due to J a c o b y and F u 1 d . According to Jacoby's method (Biochem. Z., 1906, 1, APPENDIX 301 58), 0-5 grm. of ricin is dissolved in 5% sodium chloride solution and filtered. An opalescent solution is obtained which becomes turbid on addition of decinormal hydrochloric acid. Equal volumes of this solution are mixed with diminishing quantities of differently diluted gastric juice and then made up with dis- tilled water or boiled gastric juice to a constant volume. After 3 hours in a thermostat, the liquids are examined to ascertain the smallest quantity of gastric juice able to clear the solution, i.e., to digest the protein present completely. If, after dilution of the juice a hundredfold, 1 c.c. is just sufficient for this purpose, the number of pepsin-units in the original gastric juice is taken as 100 (normal gastric juice con- tains 100-200 pepsin -units). F u 1 d ' s method (F u 1 d and L e v i s o n , Biochem. Z., 1907, 6, 473; see also Zeitschr. klin. Med., 1907, 64, 376) is as follows: A clear boiled solution (1 : 1000) of crystalline edestin in N/300-hydrochloric acid is prepared, the edestin being thus converted into the so-called edestan. The gastric juice to be examined is now diluted in the pro- portion 1 : 20 and a series of dry test-tubes charged with diminish- ing amounts of this diluted juice by means of a 1 c.c. pipette reading to 0-01 c.c. These tubes should have a diameter of not more than about 1 c.m., so that mixing may be avoided on subsequent addition of ammonia. The selected amount, say 2 c.c., of edestin solution is then rapidly added to each tube and after a lapse of 30 minutes ammonia solution is poured carefully into each tube, starting with the one containing most pepsin. The tubes are then observed in incident light against a black background, the one containing the smallest amount of pepsin and showing no ring being noted. The number of c.c. of pepsin solution or gastric juice con- tained in this tube is divided by the product of its dilution and the number of c.c. of edestin solution digested. If, therefore, 0-25 c.c. of the 1 : 20 concentration of the gastric juice is suf- ficient to prevent the formation of the ring in 2 c.c. of edestin solution, the number required is 0-25:20X2 = 1:160. The gastric juice is then termed a 1 : 160 pepsin or is said to contain 160 pepsin-units. The methods of J a c o b y and F u 1 d have been repeatedly tested and found to be of general utility. 302 GENERAL CHEMISTRY OF THE ENZYMES Witte (Berl. klin. Wochens.j 1907, 44, 1338) suggests in Jacoby's method a slight but not unimportant modification : before use, the gastric juice is exactly neutralised; the results are thus rendered more exact. Also Reicher (Wien. klin. Wochens., 1907, 20, 1508) gives these methods the preference over all the older quantitative methods. He, too, found the influence of acidity to be very considerable and agreed with W i 1 1 e ' s proposal to neutralise the gastric juice. S o 1 m s (Zeitschr. klin. Med., 1907, 64, 159) likewise obtained favourable results with Jacoby's method. E i n h o r n (Berl. klin. Wochens., 1908, 45, 1567) suggests the simplification of Jacoby's method by the use of an appa- ratus of special construction. This apparatus, which is a glass vacuum-vessel, contains water at 50-60 and a stand holding graduated test-tubes charged with the digestion mixture. The time of a test may thus be shortened to 30 minutes. Of the researches in which F u 1 d and L e v i s o n ' s method has been successfully employed, those of Wo 1 f f and von Tomaszewski (Berl. klin. Wochens., 1908), 45, 1051) deserve special mention. In M e 1 1 ' s method, hens'-egg albumin is drawn up into a glass tube 1-2 m.m. in diameter and coagulated in the tube at 95, lengths of about 2 c.m. of the tube, cut sharply off, being then immersed in the peptic liquid. The length of the digested cylinder of the albumin is measured after 10 hours; it should not exceed about 6 m.m. The amount of pepsin is proportional to the square of this length. According toNierenstein and S c h i f f (Archiv. f. Verd. Krank., 1902, 8, 559), the gastric juices to be compared should be brought to equal degrees of acidity. These methods, which are open to the objections indicated on p. 292, resemble that of F e r m i founded on the solution of layers of solidified gelatine. H a 1 1 o r i (Arch, internat. de Pharm. et de Therap., 1908, 18, 255), points out that, in general, gelatine is digested much more rapidly than coagulated albumin and that two different enzymes may be in question. Such a statement is, however, hypothetical and the criticism of Fermi's method based thereon insufficiently supported. APPENDIX 303 Estimation of Trypsin and Erepsin Most of the methods given above for the estimation of pepsin may be used, with suitable modification, to follow tryptic diges- tion. This is the case with the optical methods, the conductivity method (cf. Henri, and B a y 1 i s s , p. 186), etc. The application of V o 1 h a r d' s method (cf . p. 297) to the estimation of trypsin is described by L 6 h 1 e i n (1 o c . c i t .) ; the only difference is that the hydrochloric acid is added to the casein after the digestion, whilst in the investigation of pepsin it is added before digestion. A similar method was employed by R. Goldschmidt (Deut. med. Wochens., 1909, 35, 522). J a c o b y ' s ricin method also serves for the estimation of trypsin (Biochem. Z., 1908, 10, 229). Two c.c. of a solution of 1 grm. of Merck's ricin in 100 c.c. of 1-5% sodium chloride solution are placed in each of a series of tubes together with 0,0-1, 0-2,0-3, 0-5, 0-7, 1-0 c.c. of a 1% solution of Griibler's trypsin. Water is added to bring the volume to 3 c.c. in each tube, to which 0-5 c.c. of 1% soda solution is then added. The tube without trypsin remains persistently turbid whilst the others gradually clear, that with 0-1 c.c. of trypsin becoming quite bright after 6 hours in an incubator. C h y m o s i n . The activity of a solution of this enzyme is estimated by determining in what dilution it just coagulates a certain quantity of milk in 30 minutes at40(K.Glaessner, Hofm. Beitr., 1901, 1, 1, 24; Hammarsten, H., 1896, 22, 103). Since the milk used for the estimation of rennet varies very considerably as regards its chymosin-content, Blum and Fuld (Berl. klin. Wochens., 1905, 42, 107; Biochem. Z., 1907, 4, 62) propose to replace the milk by a preparation of milk- powder, which is prepared commercially and is of constant composition. Three grms. of the milk-powder are mixed with 9 times the weight of water in the following manner : the weighed (or measured, after pressing down and smoothing in a measure) quantity of the powder is added in small portions to, and stirred 304 GENERAL CHEMISTRY OF THE ENZYMES with, sufficient distilled water to form a semi-solid paste, to which the remainder of the water is then added. On stirring, almost the whole of the powder goes into solution without heating, and this solution, which can be prepared in a couple of minutes, can be used immediately if the sediment is rejected. It will, on the other hand, keep for 3 days in an ice-chest. An addition of calcium salt which was recommended by these authors in their first communication, is unnecessary. Twenty tubes are now charged with the following amounts: (1) of the undiluted gastric juice, 0-10, 0-15, 0-21, 0-32, 0-46, 0-68, 1-0 c.c.; (2) of the dilution 1 : 10, 0-10, 0-15, 0-21, 0-32, 0-46, 0-68 c.c.; (3) of the dilution 1 : 100, 0-10, 0-15, 0-21, 0-32, 0-46, 0-68 c.c. The last tube, contained 1-5 c.c. of the boiled gastric juice, serves as a control. The solutions are then made up to 10 c.c. with the milk solu- tion, so that they contain the gastric juice in dilutions varying from 1 : 10 to 1 : 10,000 (a preliminary test being thus unneces- sary); the tubes are then placed in a large water-bath at 17-5. At the end of 2 hours, a drop of 20% calcium chloride solution is added to each of the tubes, which are then transferred to a water-bath at 40 for 5 minutes. The ratio, gastric juice : milk solution in the clotted liquid containing the least amount of the juice gives directly the rennetic value of the gastric juice and also its enzyme-content in general. A more accurate es- timation can afterwards be made, either immediately or, if an ice- chest is available, on the following day. Z y m a s e . For technical purposes and, indeed, whenever great accuracy is not desired, the fermenting power of pressed yeast-juice or permanent yeast is determined by placing in a small Erlenmeyer flask (100 c.c.) furnished with a M e i s s 1 valve, 20 c.c. of the pressed juice, 8 grms. of cane-sugar (or 2 grms. of permanent yeast, 10 grms. of water and 4 grms. of cane-sugar), and a little toluene, the loss in weight being determined after 1, 2, 3 or 4 days at 22. The evolution of carbon dioxide amounts to about 1-2 grms. (E. and H. Buchner and M. H a h n , Die Zymasegarung, 1903, p. 80). APPENDIX 305 For more accurate estimations, the carbon dioxide is expelled from the solution by a gentle stream of air, or the evolution of gas is allowed to take place under diminished pressure, the amount of carbon dioxide liberated being then determined volumetrically. An excellent volumetric method has been devised by S 1 a t o r (Journ. Chem. Soc., 1906, 89, 128). Oxydase and peroxydase. The numerous reac- tions which have been employed for the detection and estimation of the oxydases have been referred to on p. 59. Since, as was previously mentioned, no general method for the quantitative determination of the oxydases exists, the methods given for the study of special oxydases will not be described here. Reference may be made to the brief outlines given on pp. 220-223. C a t a 1 a s e. Since the determination of the catalase- content of the blood and other liquids of the body is now one of the more common tests of physiological chemistry, the most important methods may be shortly mentioned. With aqueous solutions of purified catalases, the undecom- posed hydrogen peroxide is usually determined by titration with potassium permanganate. The most suitable concentrations of the peroxide are N/20 N/50; the solutions are acidified with sulphuric acid and titrated with centinormal permanganate. In many cases this method is, as B r e d i g found in his researches on colloidal metals, preferable to measurement of the volume of oxygen evolved. Where the fluid of an organ is investigated directly, volume- and pressure-methods may possess decided advantages. A volume-method was employed by L. von Liebermann (Pflug. Arch., 1904, 104, 176) and more recently also by S a n - tesson, while W. L 6 b (Biochem. Z., 1908, 13, 339) has described an arrangement, which apparently allows of rapid and accurate measurement of catalase-content. In the same com- munication, Lob describes a pressure-method, which also serves well in certain cases. 306 GENERAL CHEMISTRY OF THE ENZYMES TABLES FOR THE ESTIMATION OF SUGARS BY BERTRAND'S METHOD Glucose 4-07X50-l c.c. Fourth crystallisation: [a]/> 1-960 grm.X2d.m. +52.' Sugar in m grins. Cu in mgrms. Sugar in mgrms. Cu in mgrms. Sugar in mgrms. Cu in mgrms. 10 20-4 41 79-3 71 131-4 11 22-4 42 si -i 72 133-1 12 24-3 43 82-9 73 134-7 13 26-3 44 84-7 74 136-3 14 28-3 45 86-4 75 137-9 15 30-2 46 88-2 76 139-6 16 32-2 47 90-0 77 141-2 17 34-2 48 91-8 78 142-8 18 36-2 49 93-6 79 144-5 19 38-1 50 95-4 80 146-1 20 40-1 51 97-1 81 147-7 21 42-0 52 98-9 82 149-3 22 43-9 53 100-6 83 150-9 23 45-8 54 102-3 84 152-5 24 47-7 55 104-1 85 154-0 25 49-6 56 105-8 86 155-6 26 51-5 57 107-6 87 157-2 27 53-4 58 109-3 88 158-8 28 55-3 59 111-1 89 160-4 29 57-2 60 112-8 90 162-0 30 59-1 61 114-5 91 163-6 31 60-9 62 116-2 92 165-2 32 62-8 63 117-9 93 166-7 33 64-6 64 119-6 94 168-3 34 66-5 65 121-3 95 169-9 35 68-3 66 123-0 96 171-5 36 70-1 67 124-7 97 173-1 37 72-0 68 126-4 98 174-6 38 73-8 69 128-1 99 176-2 39 75-7 70 129-8 100 177-8' 40 77-5 APPENDIX 307 Invert-sugar A 0-5% solution was prepared by hydrolysing 4-750 grms. of cane-sugar with 50 c.c. of 2% hydrochloric acid. The solution was heated for 10-15 minutes, cooled, neutralised, and diluted to a litre. Sugar in mgrms. Cu in mgrms. Sugar in mgrms. Cu in mgrms. Sugar in mgrms. Cu in mgrms. 10 20-6 41 79-5 71 130-8 11 22-6 42 81-2 72 132-4 12 24-6 43 83-0 73 134-0 13 26-5 44 84-8 74 135-6 14 28-5 45 86-5 75 137-2 15 30-5 46 88-3 76 138-9 16 32-5 47 90-1 77 140-5 17 34-5 48 91-9 78 142-1 18 36-4 49 93-6 79 143-7 19 38-4 50 95-4 80 145-3 20 40-4 51 97-1 81 146-9 21 42-3 52 98-8 82 148-5 22 44-2 53 100-6 83 150-0 23 46-1 54 102-3 84 151-6 24 48-0 55 104-0 85 153-2 25 49-8 56 105-7 86 154-8 26 51-7 57 107-4 87 156-4 27 53-6 58 109-2 88 157-9 28 55-5 59 110-9 89 159-5 29 .57-4 60 112-6 90 161-1 30 59-3 61 114-3 91 162-6 31 81-1 62 115-9 92 164-2 32 63-0 63 117-6 93 165-7 33 64-8 64 119-2 94 167-3 34 66-7 65 120-9 95 168-8 35 68-5 66 122-6 96 170-3 36 70-3 67 124-2 97 171-9 37 72-2 68 125-9 98 173-4 38 74-0 69 127-5 99 175-0 39 75-9 70 129-2 100 176-5 40 77-7 308 GENERAL CHEMISTRY OF THE ENZYMES Galactose 16-03X25c.c. Fifth crystallisation: [a]z> = 2 5 grms. X 2 d.m. Sugar in mgrms. Cu in mgrms. Sugar in mgrms. Cu in mgrms. Sugar in mgrms. Cu in mgrms. 10 19-3 41 75-6 71 126-6 11 21-2 42 77-4 72 128-3 12 23-0 43 79-1 73 130-0 13 24-9 44 80-8 74 131-5 14 26-7 45 82-5 75 133-1 15 28-6 46 84-3 76 134-8 16 30-5 47 86-0 77 136-4 17 32-3 48 87-7 78 138-0 18 34-2 49 89-5 79 139-7 19 36-0 50 91-2 80 141-3 20 37-9 51 92-9 81 142-9 21 39-7 52 94-6 82 144-6 22 41-6 53 96-3 83 146-2 23 43-4 54 98-0 84 147-8 24 . 45-2 55 99-7 85 149-4 25 47-0 56 101-5 86 151-1 26 48-9 57 103-2 87 152-7 27 50-7 58 104-9 88 154-3 28 52-5 59 106-6 89 156-0 29 54-4 60 108-3 90 157-6 30 56-2 61 110-0 91 159-2 31 58-0 62 111-6 92 160-8 32 59-7 63 113-3 93 162-4 33 61-5 64 115-0 94 164-0 34 63-3 65 116-6 95 165-6 35 65-0 66 118-3 96 167-2 36 66-8 ' 67 120-0 97 168-8 37 68-6 68 121-7 98 170-4 38 70-4 69 123-3 99 172-0 39 72-1 70 125-0 100 173-6 40 73-9 APPENDIX 309 Maltose Third crystallisation: [ Mannose 3-48X25c.c. 1-25 grm.X 5d.m = +13-92(=21), Sugar in mgrms. Cu in mgrms. Sugar in mgrms. Cu in mgrms. 10 20-7 60 113-3 20 40-5 70 130-2 30 59-5 80 146-9 40 78-0 90 163-3 50 95-9 100 179-4 INDEX OF AUTHORS Abderhalden, 9, 33, 38, 39, 40, 41, 43, 64, 103, 107, 116, 119, 142, 143, 190, 192, 193, 195, 196, 228, 236, 276, 277, 287, 293 Abelous, 69, 265 Aberson, 209, 210, 241 Achalme, 269, 271 Acree, 264 Agulhon, 116 Albert, 57 Armstrong, E. F., 18, 21, 22, 23, 53, 141, 165, 166, 168, 170, 173, 174, 261, 262 Armstrong, H. E., 11, 22, 23, 97, 128, 150, 161, 166, 170, 174, 275 Arrhenius, 75, 76, 131, 133, 134, 135, 136, 147, 151, 178, 183, 200, 202, 232, 236, 237, 267, 272 Arthus, 11, 116 Ascoli, 14, 15, 38, 268, 270 Asher, 117, 120 Aso, 59 Auld, 22, 24, 30, 101, 140, 172, 173, 174, 239, 262, 295 Bach, 61, 63, 64, 65, 66, 67, 68, 69, 108, 128, 217, 219, 220, 224, 225, 226, 228, 246 Baker, 14 Bang, 36, 45, 105, 109, 201, 202, 295 Barendrecht, 165 Barker, 74 Bashford, 272 Battelli, 63, 64, 67, 70, 272 Baur, 10 Bayliss, 36, 37, 84, 91, 103, 104, 106, 128, 142, 186, 241, 262, 266, 270, 296, 303 Beam, 123, 238 Bechhold, 84 Behrend, 11 Beijerinck, 14, 19, 28, 30 Beitzke, 266, 268 Benjamin, 48 Berg, 29, 95 Bergell, 36, 249, 268, 269. Bergmann, von, 271 Berkeley, 194 Bernard, Claude, 14 Bertarelli, 267, 268 Bertrand, 21, 33, 61, 62, 64, 66, 105, 107, 108, 221, 275, 293 Bezzola, 270 Bial, 15 Bickel, 249, 250 Biedermann, 13 Biernacki, 237 Bierry, 24, 28, 83, 87, 91, 100, 109, 171, 275 Blake, 85 Bliss, 35 Bloch, 41 Blum, 269, 303 Blunt, 246 Bodenstein, 128, 129, 131, 152, 159, 252, 256, 263 Bokorny, 171 Boldyreff, 10, 93 Bolin, 62, 66, 79, 83, 221 Bonfanti, 14, 15 Bordet, 116, 272 Borissow, 176 Bosshard, 280 Bourquelot, 5, 13, 18, 19, 21, 22, 23, 24, 28, 30, 32, 59, 98 Boysen-Jensen, 52 Braun, 11, 268 Breandat, 30 Bredig, 67, 69, 122, 282, 283, 305 Brieger, 270 Brown, A., 128, 162 Brown, H. T., 13 ; 14, 15, 19, 128, 155, 157 Briicke, 35 Brugsch, 10 Bruno, 92 Bruschi, 48 BuchnerE., 3, 5, 41, 51, 52, 53, 57, 58, 60, 81, 83, 84, 94, 105, 116, 117, 118, 119, 211, 288, 304 314 INDEX OF AUTHORS Buchner, H., 57, 304 Buckmaster, 91 Buglia, 93 Burian, 44, 59, 119, 230 Butkewitsch, 14, 16, 43, 100 Caemmerer, 107, 116, 119 Caldwell, 22, 23, 39 Campbell, 17 Camus, 11, 120, 240, 269 Carlson, 15 Cathcart, 270 Chanoz, 12 Charrin, 270 Chigin, 196 Chittenden, 39, 98, 109, 120 Chocensky, 56 Chodat, 48, 63, 64, 65, 66, 67, 143, 224, 225, 240, 241 Chodschajew, 83 Chrzascz, 14 Clark. 25, 291, 292 Claus, 55 Coehn, 85 Coggi, 269 Cohnheim, J., 16 Cohnheim, O., 38, 55, 91, 92 Cole, 97, 98, 110, 112 Connstein, 10, 11, 149 Courtauld, 22, 23 Cramer, 123, 238 Cremer, 264 Czapek, 13 Czyhlarz, von, 59, 66 Dakin, 12, 42, 279, 280 Dam, van, 105 Danilewski, 92, 113, 265 Davidsohn, 160, 161 Dean, 18 Delbruck, 11 Deleano, 66, 84 Delezenne, 91, 93, 106, 244, 270 Detmer, 158, 291 Devaux, 32 Dietz, 10, 152, 256, 263 Dietze, 102 Doblin, 270 Donath, 92, 93 Donnan, 74 Dony-Henault, 61, 62, 220 Downes, 246 Dox, 31 Doyon, 11, 12 Dreyer, 247 Drjewezki, von, 101 Duchacek, 54, 57 Duclaux, 5, 116, 117, 158 Dunstan, 30, 295 Durham, 108 Duuren, van, 61, 220 Edelstein, 249 Edmunds, 46 Effront, 13, 17, 97, 109, 110, 116, 158, 239 Ehrenreich, 82 Ehrlich, 44, 223 Einhorn, 302 Elvove, 12, 69, 146 Embden, 55 Emmerling, 39, 245, 261, 264 Engel, 10, 147, 289 Engler, 137 Erben, 37 Eriksson, 122, 123 Ernest, 56 Euler, 13, 25, 26, 27, 41, 62, 66, 67. 68, 79, 83, 88, 100, 102, 104, 105, 107, 112, 113, 116, 118, 121, 137, 142, 148, 165, 188, 193, 206, 209, 214, 216, 218, 221, 231, 232, 236, 241, 259, 283, 296 Faitelowitz, 67, 106, 116, 119 Fa jans, 282 Falloise, 10 Falta, 250 Farr, 119 Faubel, 186 Fermi, 24, 25, 83, 117, 245, 269, 271, Fernbach, 83, 108, 114, 264 Finsen, 245 Fischer, E., 18, 19, 21, 22, 25, 29 30, 31, 36, 53, 79, 115, 118, 119, 168, 261, 276, 277, 279, 283, 285, 288 Fischer, W., 24, 283 Foa, 98 Ford, 93, 97 Foster, 15 Frankel, 17 Franzen, 52, 281 Freederickz, 64 Freudenreich, 118 Freundlich, 73, 76, 88 Freidenthal, 35, 273, 287 Fromme, 10 Furth, von, 59, 66, 93, 228 Fuhrmann, 11, 31 Fuld, 48, 49, 119, 201, 205, 241, 272, 300, 301, 302, 303 Gatin, 70 Gaunt, 60 Gengou, 116, 272 Gerard, 11, 23, 24 INDEX OF AUTHOKS 315 Gerber, 48, 49, 104, 109, 111, 202, 241, 244 Geret, 40, 41, 119 Gerhartz, 249 Gessard, 272 Gewin, 47 Giaja, 24, 28, 275 Gigon, 108, 142, 143 Gizelt, 120 Gjaldbak, 265 Glaessner, 270, 303 Glendinning, 128, 140, 155, 157, 264 Gley, 19, 270 Glimm, 238 Glinski, 292 Glover, 161, 166, 170 Gockel, 120 Godlewski, 55 Golden, 31 Goldschmidt, H., 144 Goldschmidt, R , 303 Gonnermann, 24, 64 Gottlieb, 70 Grafe, 228 Green, Reynolds, 11, 13, 16, 18, 39, 49, 96, 247 Gries, 95 Grignard, 279 Grigoriew, 210 Gross, 180 Grass, 82 Griitzner, 96, 117, 296 Gudzent, 250 Guggenheim, 64, 228, 236 Guignard, 23, 29, 244 Gulewitsch, 36 Guthrie, 93 Hagglund, 75 Haehn, 54 Hafner, 26 Hahn, 40, 41, 57, 95, 119, 250, 269, 304 Hairs, 30 Hall, 92 Halliburton, 15 Hamburg, 17 Hamburger, 14, 19, 30, 91, 287 Hammarsten, 36, 45, 46, 47, 48, 49, 50, 83, 97, 104, 200, 205, 272, 303 Handovsky, 85, 87 Hanriot, 11, 128, 240, 263 Hansen, 19, 201 Hanssen, 247 Harden, 3, 32, 53, 54, 56, 94, 109, 206, 211, 213, 214, 280 Hardy, 89 Hart ,'31 Hattori, 302 Hedin, 37, 77, 78, 81, 82, 101, 122, 128, 187 Heffter, 69 Hekma, 30, 91 Henneberg, 115 Henri, 87, 91, 128, 129, 130, 132, 140, 141, 156, 158, 159, 163, 165, 167, 175, 184, 249, 252, 253, 296, 303 Henriques, 265 Henry, 24, 30, 262, 295 Herissey, 13, 21, 22, 23, 24, 28, 30, 32, 118, 168 Herlitzka, 35 Heron, 19 Hertel, 245 Herzog, 79, 137, 167, 172, 196, 210, 240, 241, 265, 269 Higuchi, 49 Hildebrandt, 101, 267, 268 Hill, Croft, 4, 20, 83, 168, 257, 261, 262 Hinkins, 264 Hober, 88, 122 Hoff, van't, 138, 231, 251, 253, 254, 257, 261 Hoffmann, 96, 117 Hofmeister, 87, 89 Holderer, 21, 82 Homfray, Miss, 75 Hongardy, 91 Horton, 22, 23, 174 Hoyer, 11, 94, 97, 108, 111, 149 Huber, 116 Hubert, 83 Hudson, 18, 19, 21, 26, 27, 90, 98, 99, 100, 114, 127, 159, 160, 161, 163, 165, 171, 237, 238, 262 Huerre, 243 Huiskamp, 50 Huppert, 179, 293 Issaew, 25, 216, 217 Italic, van, 215 Iwanoff, 42, 214 Izar, 109 Jacobson, 105 Jacoby, 35, 36, 83, 84, 272, 300, 301, 302, 303 Jalander, 152 Jamada, 246, 248 Jastrowitz, 136 Jerusalem, 228 Jochmann, 37, 269 Jodlbauer, 246, 247, 248 Johansson, 209 Johnston, 146 Jones, 42, 44 Jones, G. C., 291 Jonescu. 244 316 INDEX OF AUTHORS Jorissen, 30 Johnson, 292 Jungk, 292 Kalaboukoff, 9, 93 Kalanthar, 21 Kanitz, 10, 102, 107, 148, 149, 287 Kantorpwicz, 269 Karamitsas, 248 Kasarnowski, 79 Kastle, 12, 25, 59, 67, 69, 116, 117, 118, 122, 128, 146, 147, 215, 224, 225, 239, 241, 244, 263, 275, 276 Kaufmann, 116, 118, 119 Kayser, 108 Kendall, 156, 264, 291, 292 Kiesel, 43 Kikkoji, 42, 43, 101 Kirchoff, 15 Kjeldahl, 117, 158, 241, 242 Klatte, 81, 84 Klempin, 157 Knauthe, 13 Kobert, 24 Koelker, 103, 190, 192, 193, 295 Kottlitz, 205 Kohl, 246, 264 Kossel, 42 Kostytschew, 55, 56 Kotake, 70 Krafft, 79 Krawkow, 16 Krober, 118, 168, 240 Kriiger, 25, 110 Kudo, 102, 110 Kiihne, 139, 142 Kuttner, 300 Kullberg, 25, 26, 79, 104, 121, 165, 214, 236, 243 Kurajeff, 265 Kussmaul, 268 Kutscher, 39, 42 Laborde, 30 Lalou, 175 Landsteiner, 270 Langley, 94, 98 Lanzenberg, 108 Laqueur, 93, 120 Larguier des Bancels, 184 Larin, 95 Lawrow, 265 Lea, 43 Lebedew, von, 57, 214 Lesser, 66, 67, 215 Leube, 43 Leuchs, 15 Levaditi, 270 Levene, 42 Levison, 301, 302 Levites, 110 Lewkowitsch, 10 Liebermann, von, 67, 215, 305 Lindberger, 102 Lindet, 63 Lindner, 19, 240 Ling, 14, 290 Lintner, 16, 97, 100, 110, 118, 168, 288, 290 Lippmann, von, 5 Lobassow, 196 Lockemann, 37, 110, 246, 249 Loeb, Jacques, 107, 115, 184 Loeb, L., 50 Lob, W., 305 Lohlein, 186, 297, 299, 303 Lorcher, 104 Loevenhart, 12, 63, 67, 92, 93, 116, 117, 118, 122, 128, 146, 147, 215, 224, 225, 239, 241, 263, 275, 276 Loew, 16, 67, 107, 215, 217 Loewenherz, 275 Loewenthal, 249 Lohmann, 39 London, 195, 198, 200 Luckhardt, 15 Lundeqvist, 137 Luther, 137, 287 McCollum, 31 MacGillawry, 13 McKenzie, 279, 280 Maclean, 56 Macleod, 10 Madsen, 203, 245, 272 Magnus, 12, 92, 93, 273 Malfitano, 106 Malleyre, 33, 107 Mangin, 32 Maquenne, 14, 155, 264 Marchand, 108 Marckwald, 278, 279, 283 Martin, 205, 237 Martinand, 62, 66 Martini, 101 Maxwell, 71 Mayer, 9, 249 Mays, 37 Medigreceanu, 42 Medwedew, 128, 219 Meisenheimer, 3, 5, 51, 52, 58, 60, 94, 211 Meltzer, 236 Mendel, 15 Mett, 222 Meunier, 297, 298 Meyer, Kurt, 180, 271 Michaelis, 36, 77, 78, 82, 84, 85, 86, 87, 160, 161, 190, 267, 287 Millner, 73 INDEX OF AUTHORS 317 Minami, 250 Miquel, 240 Moitessier, 66 Moll, 43, 271 Montesano, 24, 25 Moore, 139 Moraczewski, von, 95 Morawitz, 50, 91, 107 Morel 11 Morgenroth, 271, 272 Moritz, 13, 140, 264 Morris, 13, 14, 15 Mouton, 244 Miiller, Ed., 293 Muller-Thurgau, 158, 240 Muntz, 118 Nagayama, 38 Nasse, 15, 119 Neilson, 110 Nencki, 34, 35, 47, 92 Neppi, 38 Neuberg, 10, 23, 65, 249, 266, 268 Nicloux, 11, 151, 236 Niebel 19, 25 Nierenstein, 302 Norris, 53 Novy, 35 Niirnberg, 265 Obermayer, 296 Ohlsen, 214 Okuneff, 265 Oppenheimer, C., 287 Oppenheimer, S., 69 Ormerod, 97, 150, 275 Osaka, 137 Osborne, 17, 25, 26, 109 Oshima, 26 Ostwald, Wilh., 72, 129, 234, 287 Ostwald, Wo., 72, 246, 287 O'Sullivan, 26, 98, 158, 159, 161, 162, 165, 237, 241, 242, 262, 288 Overton, 264 Pagenstecher, 10 Paine, 21, 100, 114, 171, 237, 238 Palladin, 55, 56 Pantanelli, 264 Parnas, 70 Parrozzani, 8 Pasteur, 276 Patten, 114 Pauli, 85, 87, 88, 89 Pawlow, 34, 36, 37, 47, 91, 92, 106, 111, 113, 157, 176, 276, 292 Payen, 15 Peirce, 154 Pekelharing, 34, 35, 47, 49, 50, 86 Pernossi, 83, 117, 245 Persoz, 15 Pewsner, 198 Pfleiderer, 94, 96, 117 Philoche, 157, 167 Pick, 15, 296 Pinkussohn, 107, 116, 118, 119 Plimmer, 30 Pohl, 59 Pollak, 38, 102, 123, 184 Pomeranz, 257 Porter, 123 Posternak, 88 Pottevin, 10, 14, 22, 107, 263 Pozerski, 244 Pozzi-Escot, 68 Preti, 110 Price, 231 Pringsheim, 43 Pugliese, 15, 118, 269 Quincke, 80 Rachford, 92, 93 Rapp, 105 Raudnitz, 67 Reichel, 94, 118, 120, 201 Reicher, 302 Reinhard, 63 Reiss, 67 Resenscheck, 84 Rey-Pailharde, de, 68 Ribaut, 265 Richter, 249 Rinckleben, 57 Ringer, 86 Roaf, 296 Roberts, 291 Robertson, 25, 104, 266 Roden, 272 Rohmann, 20, 59 Roger, 15, 109 Rogozinski, 66 Rona, 78, 84, 97 Rosenberg, 108 Rosenblatt, 64 Rosenfeld, 63, 120, 265 Rosenheim, 10, 92, 93 Rosenthaler, 5, 22, 23, 45, 173, 264 280, 281 Rostocki, 33 Roth, 287 Rouge, 11, 48, 148 Rowland, 101, 288 Rozenband, 105 Sachs, 101, 269 Saiki, 15, 268 Sailer, 119 Salaskin, 265 318 INDEX OF AUTHORS Salazar, 100, 171 Salkowski, 25, 26, 41 Samojloff, 176 Santesson, 117, 305 Sarthou, 63 Sawitsch, 47 Saw j alow, 265 Schade, 52 Schaeffer, 36, 83, 87, 110 Schaer, 59 Schapirow, 92 Schardinger, 68, 69 Schellenberg, 13 Schiff, 302 Schilow, 137 Schittenhelm, 41, 43, 44 Schlesinger, 18, 118 Schmidt, Alex., 49 Schmidt, C. L. A., 104 Schmidt, G. C., 75, 76, 78 Schmidt-Nielsen, Signe, 36, 47, 236, 247 Schmidt-Nielsen, Sigval, 236, 247, 248, 249 Schmiedeberg, 12, 59 Schneegans, 27 Schondorff, 120 Schorstein, 13 Schreiner, 69 Schrumpf, 35 Schiitz, E., 132, 133, 175, 179, 295 Schiitz, J., 93, 110, 179, 285 Schiitze, 268, 269 Schutzenberger, 28 Schulze, 280 Schumoff-Siemanowski, 9 Schumoff-Siemanowski, Mme., 34 Schunck, 29 Schwarz, 123, 250 Schwarzschild, 36, 37 Schwiening, 101 Scurti, 8 Segelke, 201 Senter, 67, 105, 106, 117, 119, 215, 216, 217, 236, 241 Shaklee, 236 Shaw-Mackenzie, 93 Shedd, 59 Sherman, 18, 156, 264, 291, 292 Shibata, 43 Shiga, 43 Shigeji, 49 Shore, 19 Sieber, 9, 34, 35, 47 Sieber, Mme., 55 Siedentopf, 80 Sigmund, 11, 28, 117, 149 Simnitzki, 270 Sjoqvist, 95, 96, 176, 178, 296 Slator, 51, 53, 210, 240, 243, 274, 305 Slowtzoff, 63, 220 Sorensen, 27, 90, 96, 98, 99, 100, 160, 161, 165, 184, 219, 266, 296 Solms, 302 Sommer, 69 Souder, 93 Soxhlet, 201 Spatzier, 29 Spiro, 48, 94, 118, 119, 120, 201, 203, 272 Spitzer, 59, 63 Spohr, 231 Spriggs, 181, 296 Stade, 10, 147 Stangassinger, 70 Starling, 91, 106, 270 Steche, 216 Stenitzer, von, 269 Steppuhn, 52 Stern, 64, 70, 272 Stevens, 63 Stiles, 114 Stoecklin, de, 59, 62, 65, 66, 223 Stoklasa, 56 Stoll, 12 Stone, 16 Storch, 201 Strada, 83 Sullivan, 69 Sundberg, 35 Suzuki, 31 Takaishi, 31 Takamine, 16 Takemura, 40 Tammann, 139, 158, 171, 173, 233, 240, 241, 242, 255, 258 Tanret, Ch., 29, 262 Tanret, G., 29 Tappeiner, von, 248 Taylor, 47, 104, 154, 156, 157, 161, 194, 241, 256, 257, 264, 266, 285 Tebb, Miss, 14, 19 Terroine, 9, 36, 93, 167 Terry, 110 Teruuchi, 39 Thierfelder, 53 Thies, 110, 246, 249 Thomas, 297, 298 Tichomirow, 36 Titoff, 75 Tomaszewski, von, 302 Tompson, 98, 158, 159, 161, 162, 165, 237, 241, 242, 262, 288 Trebing, 270 Trillat, 62 Trommsdorff, 69 Tscherniak, 65 Tschirch, 63 INDEX OF AUTHORS 319 Twitchel, 113 Twort, 24 Ugglas, af, 27, 100, 112, 121, 165, 241, 242, 243 Umber, 10 Vandevelde, 83, 109, 116, 117, 271, 273 Vernon, 37, 38, 46, 103, 115, 183, 223, 237, 241, 283 Victorow, 120 Vines, 39, 40, 41, 100, 194 Vinson, 25 Visser, 242, 258 Volhard, 10, 83, 97, 147, 288, 297, 298, 299, 300, 303 Vulquin, 101 Waele, de, 273 Waentig, 216 Walbum, 272 Walker, 92 Walther, 176, 292 Warburg, 279 Wartenberg, 11, 149 W T eber, 297, 298 Weevers, 28, 228 Weinland, 31, 269, 271 Weis, 39, 41, 100, 181, 246 Welter, 264 Wheldale, Miss, 228 White, Miss, 244 Whitney, 85 Wichern, 110, 246, 249 Wiechowski, 60, 288 Wiener, 60, 288 Wijsman, 14, 15 Wilcock, 249 Wilhelmy, 127 Willstatter, 12, 108 Windisch, 41 Witte, 302 Wittich, von, 15, 16 Wohl, 51, 238 Wohlgemuth, 93, 94, 110, 249,291,292 Wolff, J., 59, 66, 109, 223, 264 Wolff, W., 302 Wren, 279 Wright, 16 Wroblewski, 16, 17, 26, 96, 291 Yoshida, 61 Yoshimoto, 101 Yoshimura, 31 Young, 3, 32, 53, 54, 94, 109, 206, 211, 213, 214 Zahorski, 64 Zaleski, 42, 63 Zeller, 247, 248 Zellner, 148, 246 Zemplen, 21 Zinsser, 10 Zsigmondy, 80 Zunz, 91, 106, 107, 271 INDEX OF SUBJECTS Acids; activation by, 94 Activators, 90 et seq. Adenase, 7, 44 Adenine, 7,^44 Adsorption, 75 et seq. Adsorption media; acid or basic, 84 ; neutral, 81 Aesculase, 28 Alcoholic fermentation, 51 et seq. Alcoholoxydase of acetic acid bac- teria, 7, 60 Aldehydases, 7, 70, 219 Alkaloids, 119 Amicrons, 80 Amidases, 43 Amygdalase, 23, 173 Amygdalin, 22, 23, 171-175 Amylase, 6, 13, 155 ; 'activation by acids and salts, 97, 158, 289 ; dynamics, 155 ; occurrence, 14 ; preparation, 16 Amylopectinase, 6, 14, 155 Anti-enzymes, 267 Arbutase, 28 Arginase, 42 Arginine, 42 Arsenic, 109, 116 Asymmetric syntheses, 277 Autocatalysis, 111, 128 Autolytic enzymes, 101 Bases; activation by, 94 Betulase, 27 Blood; clotting of, 49, 50, 205 Borax, 29 Boric acid, 116 Bromelin, 39 " Buffers," 106, 115 Butyrases, 6, 11 Carbamases, 6, 33 Carbonases, 56 Caroubinase, 13 Casein, 45, 200 Catalase, 7, 67, 87, 116, 117, 305 ; activation by acids and alkalis, 105 ; dynamics, 215 ; occurrence, 67 ; preparation, 67 Catalysis, 127 Cellase, 21 Cellulase, 13 Charcoal as adsorbent, 81 Choral, 29, 118 Chloroform, 118 Chlorophyllase, 6, 12 Chymosin, 45, 303 ; activation by acids, 104 ; dynamics, 200 ; occurrence, 45 ; preparation, 46 Classification of enzymes, 6 Coagulating enzymes, 45 et seq. Co-enzymes, 12, 53, 90 Colloids, 78 ; inorganic, 118 Coupled reactions, 137 Creatine, 70 Cresols, 118 Cynarase, 48 Cytase, 6, 13 Desamidases, 7, 43 Diastase, 6, 13, 97, 118, 120, 289 et seq. ; preparation, 16, 17 Digestion; dynamics of, 195 Dihydroxyacetone, 52 Dynamics; chemical, of reactions, 124 'Elaterase, 29 Electric transference of enzymes, 85 Emulsin, 6, 22, 87, 261, 266, 295 ; activation by acids, 101 ; dynamics, 171 ; occurrence, 23 ; preparation, 24 321 322 INDEX OF SUBJECTS Endotryptase of yeast, 40 Enterokinase, 91 Enzymic equilibria, 252 Erepsin, 6, 38, 116, 118, 303 activation by alkali, 102 dynamics, 188 occurrence, 38 preparation, 38 Erythrozyme, 29 Esterases, 6, 9 et seq., 289 dynamics, 146 preparation, 10, 12 Fermentation; mechanism of, 51 Fermentation enzymes, 50 Fibrin ferment : see thrombin Fibrinogen, 49 Formaldehyde, 118 Galacto-lactase, 22 Galacto-zymase, 53 Gastric juice, 34, 97 Gaultherase, 27 Gease, 28 Gels, 89 Gluco-lactase, 22, 23, 170 a-Glucosidase, 6, 19 /3-Glucosidase, 6, 19, 21, 171 Glucp-zymase, 53 Glutinase, 38, 102, 184 Glyceraldehyde, 52 Glycerol, 118 Glycolytic enzymes, 55 Guanase, 7, 44 Guanine, 7, 44 Hexosephosphatase, 32, 54 Hexosephosphatese, 104, 214 Histozyme, 12 Hydrocyanic acid, 119 Hydrogen peroxide, 67, 117 Hydrogen sulphide, 117 Hypoxan thine, 44, 60 Indigo enzyme, 30 Inhibiting agents, 115 Internal pressure, 71 Intestinal juice, 101 Inulinase, 6, 98 occurrence, 18 Invertase, 6, 24, 87, 117, 284 activation by acids, 98 destruction by acids and alkalis, 237 dynamics, 158 occurrence, 25 preparation, 25 Isomaltose, 14 Isomerising enzyme, 70 Kaolin as adsorbent, 84 Kinases, 90, 91 Laccases, 61, 62, 105 ; artificial, 62 Lactacidase, 53 Lactase, 6, 30 ; activation by acids, 100 ; dynamics, 168 ; occurrence, 30 ; preparation, 31 Lactic acid bacteria; zymase of, 7, 58 Light; influence of, on enzymes, 245 Linamarase, 30 Lipases, 6, 9, 10, 116, 117, 118, 119, 263, 264, 275, 288 ; dynamics, 146 ; occurrence, 10-11 Lotase, 30 Maltase, 6, 19-20 ; dynamics, 166 ; occurrence, 19 ; preparation, 19 Mandelonitrile glucoside, 23 Manno-zymase, 53 Maximum temperatures, 243 Melibiase, 31 Mercuric chloride, 116 Mercuric cyanide, 116 Mesothorium; influence of, on en- zymes, 250 Methylglyoxal, 52 Microns, 80 Myrosin, 6, 29, 118 ; occurrence, 29 Neutral salts; activation by, 106 Nitrilase, 45 Nitrilese, 5 Nomenclature of enzymes, 5 Nuclease, 6, 41 Nucleinases, 42 Nucleosidases, 42 Nucleotidases, 42 Oenoxydase, 63 Olease* 63 Optimum temperatures, 243 Ornithine, 42 Oxydase of acetic bacteria, 60 Oxydases, 58, 118, 219, 305 ; detection, 59 Oxynitrilase, 45 Ozone, 117 Pancreatic juice, 107, 276 Papain or papayotin, 39, 40, 101 Paracasein, 45, 200 Parachymosin, 45, 105 INDEX OF SUBJECTS 323 Paralysors, 115 Pectase, 6, 32 Pectinase, 7, 32, 100 Pepsin, 6, 33, 46, 47, 83, 116, 117, 118 175 et seq., 265, 295 et seq. ; activation by acids, 94 st seq. ; dynamics, 175 ; occurrence, 34 ; preparation, 34 Pepsinogen, 34 Peptases, 40 Permanent yeast, 57 Peroxydases, 7, 65, 105, 223, 305 ; dynamics, 223 ; preparation, 65 Phaseolunatase, 30, 172 Phenol, 118 Phenolases, 7 Philothion, 68 Phosphates, 109 Phosphatese, 5, 7, 104 Phytase, 6, 31 Plastein formation, 265 Poisons, 115 et seq. Potassium cyanide, 119 Preparation of enzymes, 7 et seq. Press yeast juice, 53, 54, 56 ; dynamics, 206 ; preparation, 56 Prochymosin, 46 0-Proteases, 40 Protective agents, 115 Proteinases, 6, 33, 38, 295 Proteolytic enzymes of plants, 38, 181 et seq. Ptyalin, 15, 97 ; preparation, 16 Purification of enzymes, 7 et seq. Radiation; influence of, on enzymes, 245 Radium; influence of, on enzymes, 249 Reductases or reducing enzymes, 68 Rennet: see chymosin Reversible reactions, 137 Revertase, 264 Rhamnase, 6, 29 Rontgen rays; influence of, on en- zymes, 249 Salicase, 28 Salicylic acid, 29, 119 Salts, activation by, 106 ; inhibition by, 116 Schinoxydase, 63 Schiitz's rule, 132 Seminase, 13 Sodium chloride, 109 Sodium fluoride, 116 Specificity of enzyme actions, 274 Spermase, 63 Statics; chemical, in enzyme reac- tions, 251 Steapsin: see lipase Stimulin, 92 Submicrons, 80 Sucrase: see invertase Surface energy, 72 Surface tension, 72 Syntheses by enzymes, 261 Taka-diastase, 16 Tannase, 6 Tannin, 29 Temperature; influence of, on en- zyme reactions, 231 Temperature coefficients of enzyme reactions, 239 Thrombin, 7, 49, 107, 116, 205 Thymol, 58, 118 Toluene, 58, 118 Transference; electric, 85 Trehalase, 20 Trypsin, 6, 36, 43, 84, 86, 102, 116, 117, 118, 119, 123, 183, 184, 186, 194, 266, 303 ; dynamics, 184 ; occurrence, 37 ; preparation, 37 Tyrosinase, 64, 120, 228 Urea, 43 Urease, 7, 43 , Uric acid, 60 ; enzyme oxidising, 41, 60 Xanthine, 44, 60 Yeast; permanent, 57 Zymase, 7, 54 et seq., 94, 105, 109, 116, 118, 120, 304 ; co-enzyme, 211 ; dynamics, 206 ; occurrence, 54 ; preparation, 56, 57' Zymin, 58 Zymogen, 40, 49 ; activation of, 96 SHORT-TITLE CATALOGUE OF THE PUBLICATIONS OF JOHN WILEY & SONS NEW YORK LONDON: CHAPMAN & HALL, LIMITED ARRANGED UNDER SUBJECTS Descriptive circulars sent on application. 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