Enzymes

Front matter

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HX64141942 QP601 .C66 Enzymes, by Otto Coh

Preface

The following lectures were given under the Herter Foundation at the University and Belle vue Hospital Medical College in the City of New York in 1910.

They were delivered before an audience of physicians and medical students and the subject was treated from the biological point of view. Here let me take occasion to express my indebtedness to the members of the faculty of the College, as well as to many other colleagues in New York, for their kindness during a visit which I shall ever remember with pleasure.

I am also indebted to Dr. W. B. Cannon for assistance which has enabled me to deliver these lectures in a language foreign to me.

OTTO COHNHEIM.

Heidelberg, September, 1910.

COLL

NEW V

Contents

Preface Introduction

Chapter

I. Methods of Obtaining Enzymes II. The Purification of Enzymes

Introduction

The term ''ferments" was first used early in the nineteenth century in the time of Berzelius and Schwann. Subsequently, the ferments as chemical bodies became confused with the micro-organisms which cause fermentation. To avoid confusion, Kiihne gave the new name, " enzymes," to such digestive ferments as he knew — pepsin, trypsin, steapsin, and ptyalin. To-day we know that yeast, the "ferment" of the earlier authors, is a complete organism, containing enzymes like other cells and organisms, although we use the terms "ferments" and 1 ' enzymes ' ' promiscuously.

Some hydrolytis enzymes of the yeast and of the digestive glands have been known for a long time, but interest in the enzymes has grown in recent years, especially since the great discovery of zymase by Buchner. It is now the general opinion of physiologists that living organisms effect most chemical operations by means of enzymes — i.e., enzymes are the tools of the cell.

Earlier authors used for enzymes or ferments names ending in "in" like pepsin, trypsin, ptyalin. Recently most investigators have adopted names ending in " ase" which were suggested by French biologists, like maltase, lactase, protease; and they have tried to introduce a rational nomenclature, in which the name shows the action of the enzyme. The name of the chemical com-

vii vill INTRODUCTION

pound which is acted upon by the enzyme is taken, and is connected with the ending "ase." Thus sucrase is an enzyme which acts upon sucrose or cane sugar; protease acts upon proteins; and maltase upon maltose. I think it will be impossible to employ these designations very closely, because proteins can be dissociated by different enzymes, e.g., peptones by two enzymes with different qualities. Glucose is converted by zymase into carbon dioxide and alcohol, and by the enzyme of the Bacillus acidi lactici into lactic acid. Different qualities necessitate different names. I shall use the historical names, and employ the name that was given to the enzyme by its first discoverer, or that which has been generally adopted. Similarly the so-called rational nomenclature, as adopted by all chemists, employs really the old names. I shall therefore employ the terms pepsin, trypsin, crepsin, ptyalin, invertin, maltase, lactase, zymase, and steapsin. In the case of the enzyme that converts starch into dextrin and maltose, both names, diastase and maltase, are authorized because the existence of two enzymes in malt and in saliva has been suggested. The methods which enable us to obtain and to work with enzymes, have first to be dealt with, and later comes the discussion of the general properties of enzymes, after which the individual enzymes will be considered.

Chapter I

Chemistry cannot produce enzymes. They are found only as products of the living protoplasm of cells, and in all investigations on enzymes they must be separated from the cells and the organs. So far as ease of separation is concerned, a great difference exists between enzymes — the extracellular enzymes, that act outside of the cell, and the intracellular enzymes, or endo-enzymes, which in living organisms never leave the cell.

The enzymes of the first class are secreted by the glands, and in many cases these secretions can be obtained, and with them the enzymes. Human saliva is easily obtained; and following the methods of Pawlow1 and of Bayliss and Starling2 we can get pure saliva, pure gastric and pancreatic juice of the dog, cat, horse, goat, and other higher animals. The pure secretions of

1 J. P. Pawlow: "Arbeit der Verdauungsdnisen," 1898. — Nagel's "Handbuch," Bd. ii., 1906.

2 W. M. Bayliss and E. H. Starling: Journal of Physiology, 28 and 29

I the digestive glands of some invertebrates can also be obtained. One advantage of these methods is that the product is free from many undesired substances; the chief advantage is the certainty of having the correct solubility and the proper reaction. Where the actual secretions are obtainable, investigations should never be made with extracts.

In many cases, however, the secretions cannot be obtained. For instance, the small intestine secretes a litre, or more, of fluid, but the greatest part of this enteric juice is reabsorbed before running out of a fistula, and we can get but a few cubic centimetres from a large dog, or even from the large ruminants. From small animals, most invertebrates, and micro-organisms, it is naturally impossible to get secretions in quantities sufficient for any precise investigation. We are forced to make extracts from the organs or from the whole organism, and to examine the enzymes present in such extracts. The only fluid that dissolves enzymes is water; no enzyme is extractable by strong alcohol, ether, benzene, chloroform, strong glycerin, etc. Diluted glycerin was often used by the earlier investigators (Wittich). It has the advantage that the growth of bacteria is checked by a glycerin of seventy or eighty per cent, and that the changes which enzymes undergo in watery solutions are prevented or much retarded by glycerin. Thus a solution of pepsin or of trypsin in diluted glycerin can be preserved for many months or even years, and the quantity of the dissolved enzyme can be measured better than if the ferment were in a solid state. The enzyme, however, is not dissolved in the glycerin itself, but in the water diluting the glycerin; therefore diluted glycerin dissolves merely a small quantity of the enzyme, and the glycerin solutions of enzymes are weak solutions. If we want a solution comparable with the natural secreted juices, we must dissolve the organs in water, but we have the choice of pure water, or a salt solution, or a weak acid or alkaline solution.

On account of its low osmotic pressure, distilled water destroys the cells and thus makes ferments accessible, while weak salt solutions dissolve more proteins than does pure water. For enzymes, however, I have seen no difference between water and salt solutions. Acid solutions are good for enzymes secreted in association with acid, such as pepsin, some proteolytic enzymes of the blood corpuscles and of invertebrates, and also the enzymes of yeast. The other enzymes are not destroyed by very weak acids, but some cell proteins are precipitated, and many enzymes are absorbed in these proteins and precipitated with them. I shall discuss this in treating of the purification of enzymes. If there is no special reason for the use of acids, as in the case of pepsin, perhaps the best and simplest method is to extract tissues with water, and to avoid other solvents. It is only with tissues which become acid immediately after death, as in the case of the pancreas and the muscles, when oxygen is lacking, that it may be advantageous to use a weak solution of sodium carbonate similar in its reaction to that of most living tissues. A stronger alkaline reaction endangers most enzymes, and must be avoided. With water, or a very weak alkaline solution, it is very easy to extract all enzymes destined for secretion. I have obtained extracts of the stomach and of the pancreas containing more pepsin or trypsin than the natural juices. I have never seen peptone so rapidly split by the enteric juice as by a watery extract of the intestinal mucous membrane. The extraction is more nearly complete if the glands or the mucous membranes are finely minced and ground with sand. In this way, I have extracted from the intestinal mucous membrane all the erepsin in thirty minutes, using three portions of water. It is also very easy to extract diastase, maltase, and invertin from yeast, or the ptyalin from the salivary glands, in a short time.

More difficulty is experienced in extracting the endoenzymes from the organs. The submaxillary gland and the liver contain the same two enzymes, diastase or ptyalin, which split starch or glycogen into maltose, and maltase, which changes maltose into glucose.

There does not seem to be any great difference between the salivary gland and the liver in the quantity of the two enzymes contained in the glands. A great deal of glycogen is lost if the enzymes of the liver are not destroyed immediately after death; i.e., the liver yields much diastase and maltase. The enzymes cannot be extracted with equal ease from the salivary gland and liver.

In 1863 my father1 obtained from the salivary gland a

1 J. Cohnheim: Virchow's Archiv, 28, 241 (1863).

solution splitting starch in one minute; and using the same method, he obtained from the liver a solution splitting starch in two hours. Forty years later, Borchardt 1 had no better results when isolating the ptyalin of the liver. Neither from yeast nor from the muscles can the glycolytic enzymes be extracted without complete disintegration of cells and cell structure. For fifty years biologists debated whether the fermentation of sugar by yeast was an enzymotic process, or whether only the living yeast could burn sugar. This was decided when E. and H. Buchner and Hahn 2 were able to isolate the zymase from the cell after thoroughly destroying the cell membranes. The chemical means for dissolving out substances like glycogen from the tissues, using strong sodium hydroxide or other strong alkalies, cannot be used, because they destroy the enzymes. The disintegration must be made by mechanical means. Buchner and many others have ground micro-organisms and tissues with sand and siliceous earth, and subjected the mass to considerable pressure in a hydraulic press. Macfadyen and Rowland have frozen yeast at a temperature of — 1800 C, and ground the solid ice without any addition. For muscles, glands, and other tissues, the best method is KosseFs.3 The tissues or the whole animal are frozen by solid carbon dioxide, or by a salt-and-ice freezing mix-

1 L. Borchardt: Pfluger's Archiv, ioo, 259 (1903).

2 E. Buchner, H. Buchner, and M. Hahn: "Die Zymasegarung," Munich, 1903.

3 Berichte d. d. chem. Ges., 7>2> (1900).

ture, and cut by rotating knives. In this manner a snow is obtained which has no visible microscopical structure; and this is mixed with siliceous earth and subjected to the high and slowly increasing pressure of a hydraulic press. Using this method, I have obtained 60 cc. of a clear liquid from 100 grammes of fresh muscles of the cat or ox, and 40 cc. of liquid from 100 grammes of liver, spleen, or kidney. This liquid contains a fair quantity of the intracellular enzymes, but only a small amount of the cell proteins. There is no certainty that we have in solution all the enzymes existing in the cells or organs. Recently Pringsheim and Zemplen,1 in the laboratory of E. Fischer, tried to extract the sucroclastic enzymes from some bacteria, and succeeded in many cases, but not in all, although all investigated bacteria contain the enzymes. The enzymes are retained by the proteins, while the other insoluble substances remain with the siliceous earth. We may now proceed to discuss the difficult and important question of adsorption, or attachment of enzymes to proteins and other substances. In many cases we can overcome the adsorption and render the enzymes soluble, if we change the reaction of the pulp before expressing it. Many enzymes resemble the nucleoproteins in solubility, and are precipitated by very weak acids. Those enzymes, especially, that convert disaccharides into monosaccharides, as lactase, maltase, and invertin, are fixed in, and retained by, the solids of the tissues. Another method of

1 "Studien iiber die Polysaccharide spaltenden Fermente in Pilzpresssaften"; Zeitschr. f. physiolog. Chemie, 62 (1909).

disintegrating cells or tissues is by drying them. Kiihne treated fresh pancreas with alcohol and ether in great excess; the well-dried powder contained all the enzymes of the gland. The dried pancreas powder was used also by Malloizel in his important investigations on the synthesis of fats induced by the enzyme steapsin. Buchner placed yeast in an excess of " acetone and thus obtained the "dauerhefe," i.e., a dry powder containing zymase. Wiechowski dissolved the fats and lipoids of the fresh liver in toluene, and dried the undissolved residue at 500 C. The enzymes can be preserved for a long time as dry powder, but they lose their solubility. The " dauerhefe" when added to a sugar solution ferments the sugar, but it is not possible to extract the zymase from this preparation. It is generally necessary to purify the enzymes and this is done by removing contaminating substances from the fresh extracts and then evaporating the pure solution to dryness. The pepsin and trypsin of commerce are dry powders prepared in this way.

Between the endo-enzymes working within the protoplasm and the enzymes destined for secretion, are the enzymes of the mucous membranes of the stomach and of the intestine in which the chyme is absorbed. The chyme passes through the cells, but becomes no part of the protoplasm. It has been observed that the enzymes formed by the cells of the small intestine — the erepsin, the invertin, the maltase, and especially the lactase — are to be found in very large quantity in the extract of the mucous membrane, but in small quantity in the secretion or in the contents of the intestine. It seems to me that the simplest explanation of this fact is that the peptones and the disaccharides are converted to a small extent in the lumen of the intestine, and to a greater extent while they pass through the cells. For the stomach this explanation seems certain. Tobler,1 when investigating the digestion in the stomach, using a good quantitative method, found that twenty to thirty per cent of the proteins of fresh meat are absorbed in the stomach, particularly in the right side of the stomach in the antrum pylori. Bergmann 2 has discovered a protease or an erepsin which splits peptone into amino-acids, in the mucous membrane of the antrum pylori, but this protease is not present in the chyme ejected through the pylorus; at least I could find no traces of it.3 The function of this strong enzyme is to attack peptone passing through the cells of the absorbing membrane. These enzymes — the protease of the stomach and the erepsin and the lactase of the intestine — work within the cell, but they can be as easily extracted as those enzymes which are secreted.

Some authors have believed that they could investigate tissue enzymes without dissolving the tissues. Salkowski, Jacoby,5 Hedin and Rowland,6 and others have placed

1 L. Tobler: Zeitschr. f. physiolog. Chemie, 45 (1905).

2 P. Bergmann: Skandinav. Archiv f. Physiol., 18 (1906). aCohnheim: Miinchener med. Wochenschrift, 1907.

4 E. Salkowski: Zeitschr. f. klin. Medicin, 17 (Suppl.), 1891.

5 M. Jacoby: Zeitschr. f. physiolog Chemie, 30 (1910).

6 S. G. Hedin and S. Rowland: Zeitschr. f. physiolog. Chemie, 32 (1901). — O. Schumm, Hofmeister's Beitrage, 7 (1905).

finely minced liver in water saturated with chloroform or toluene, and allowed the enzymes of the dead cells to act on the substances surrounding them.

For some time I followed the same method in investigating the glycolytic enzymes of muscles. I placed the finely minced muscles in water containing glucose and the activator of the enzyme extracted from the pancreas. We can detect and estimate in this manner unstable enzymes which are destroyed whilst being extracted, but we lose the advantage of limiting the action to selected substances.

Many proteolytic or so-called autolytic enzymes of the liver, spleen, and kidney can be extracted without difficulty in the same manner as erepsin from the mucous membranes; and with the exception of this extract, we know only of the existence in these organs of some proteolytic enzymes. We can find, for instance, in the liver three classes of proteolytic enzymes of different characters and different biological importance. First, enzymes derived from the blood left in the vessels of the liver. These interesting enzymes, which Opie 1 has investigated so thoroughly, will be dealt with later. Secondly, there is erepsin, destined to act upon the peptones which have passed the intestinal wall unchanged and which circulate in the blood. Thirdly, there may be an enzyme which attacks the proteins of the living protoplasm, and thus in

1 E. L. Opie: Rockefeller Institute for Medical Research, 4 (1905); 6(1906); 8(1908).

metabolism draws upon the real body of the cells. Apparently there would be a great difference in metabolism whether the enzyme attacked absorbed protein or the protein of the cell itself. If we extract the enzyme, we can distinguish between erepsin and trypsin, because trypsin leaves untouched the true albumins and globulins of the protoplasm, while erepsin slowly attacks the histone of the nucleus. The strong proteolytic enzyme yielded by the so-called liver of molluscs nearly resembles erepsin. Vernon and Hedin 1 extracted and isolated the enzymes and demonstrated their resemblance to erepsin. The study of the enzymes within the tissue, and without extracting them, cannot be even approximately complete; we must always try to extract the enzymes.

1 Vernon: Journ. of Physiol., 30 and 33.

Chapter II

For many purposes we can use, without further treatment, the extracts of the tissues containing ferments. The investigations of zymase by Buchner1 and his collaborators were made with the press-juice of the yeast. Emil Fischer has studied the enzymes by hydrolyzing the disaccharides with the simple, unpurified extracts of yeasts and animal tissues. For the glycolytic enzymes in the tissues of the higher animals, no method is known to-day by which the enzymes may be purified without destroying them. Other enzymes withstand chemical treatment, and in many investigations it is necessary to remove proteins, nucleic acids, salts, carbohydrates, etc., accompanying the enzymes in the watery extract. Even the natural secreted juices are not pure solutions of enzymes. The saliva and the enteric juice contain sodium chloride and sodium carbonate; the pancreatic juice and the secretion of the liver of the cephalopods contain sodium carbonate and perhaps carbonates of organic bases; the gastric juice contains hydrochloric acid; and, in addition, all these juices contain proteins and nucleic acids, and perhaps also lecithins. The organism can secrete liquids

1 E. and H. Buchner: "Die Zymasegarung," Munich, 1903. — E. Buchner and his collaborators: Ber. d. deutsch. chem. Ges., 1896-1908.

II which do not contain proteins or nucleic acids, e.g., urine, bile, sweat; or liquids which contain these bodies in traces, e.g., the aqueous humor, the cerebrospinal fluid, and some liquids in invertebrates, for instance echinodermata. But these secretions are free from enzymes. The secretions containing enzymes are rich in proteins and nucleic acids. The quantity of these in the gastric and pancreatic juice varies proportionately with the enzymes, and their solubility is like that of the enzymes. Therefore many authors have thought them to be the enzymes; but they are mistaken, because we can separate the enzymes from the proteins accompanying them in extracts and in secretions. I shall discuss their chemical nature later on.

In order to purify substances chemically, we precipitate either the substance or the impurities. Now we do not know any reagent which will bring down enzymes in a specific manner, but the latter have the property of being adsorbed by finely divided solids, and especially by precipitates during their formation in a solution. We have to consider three possibilities, although practically it is not easy to tell which one we are dealing with in a special case.

i. Enzymes and other substances are similar to one another in solubility and precipitability. Thus all proteins occurring in animal or vegetable bodies are salted out by ammonium sulphate, and, so far as we know, so are all enzymes.

2. Enzymes are brought down by physical adsorption, by precipitates forming in an enzyme solution; for instance, by the precipitate of calcium phosphate formed on adding lime-water and phosphoric acid, or the precipitate of cholesterin formed on adding an alcoholic solution of cholesterin, to the aqueous solution of enzymes. Proteins, animal charcoal, and siliceous earth also bind enzymes by physical adsorption.

3. Enzymes are fixed and held by solid substances in chemical connection, and in this manner they are attached to many proteins, in addition to the mere physical adsorption, which occurs in the cases of animal charcoal and siliceous earth. Physical adsorption precipitates all enzymes, while the chemical connection is a specific one; but the connection is apparently a loose one which can be broken by hydrolysis. The completeness of hydrolytic dissociation depends on the amount of water, and we can thus separate enzymes and proteins, or enzymes and animal charcoal, by the addition of an excess of water; we can wash out the enzymes from the solids, though they are chemically connected with them.

The chemical connection, the physical adsorption, and the precipitability of enzymes in the presence or absence of proteins, are available for the purification and separation of enzymes chiefly by three methods :

1. We can precipitate both enzymes and proteins with alcohol (Vernon1), with ammonium sulphate (Kiihne 2

1 H. M. Vernon: Journ. of Physiology, 30 (1903).

2W. Kiihne: Naturhist. Med. Verein, Heidelberg, 3 (1886).

and others), with uranyl acetate (Jacoby 1 — the aldehydase of the liver; Roselle2 — trypsin), or with phosphoric acid and lime-water (Brucke and others — gastric juice and saliva). We allow the precipitates to stand for some time, until the proteins become insoluble, and then dissolve out the enzyme with water, or in the case of pepsin, with hydrochloric acid. Using the phosphoric acid and lime-water method, Brucke isolated pepsin, though with great loss, as the first enzyme to be obtained at all free from proteins; my father employed the same method for ptyalin, which was not destroyed by this treatment. So with ammonium sulphate the erepsin of the intestinal mucous membrane was separated from a great mass of the proteins and other impurities. The ammonium sulphate and the other crystalloids were subsequently removed by dialysis, leaving a solution rich in ferments but poor in all other substances.

2. We can split the proteins wholly into amino-acids or peptones, and then precipitate the enzyme with ammonium sulphate. By this method Kuhne obtained nearly pure trypsin. This method is used for pepsin and for the enzyme oxidizing aldehydes in the liver, but cannot be applied to most enzymes which are destroyed by strong proteolytic ferments. Even trypsin itself, purified in this manner, loses a great deal of its power.

3. We can precipitate many proteins, nucleic acids, and other substances, by very dilute acetic acid, while the

1 M. Jacoby: Zeitschr. f. physiol. Chemie, 3 (1900).

2 Roselle: Diss. Strassburg, 1901.

enzyme remains in solution. In such manner Kossel and Dakin i freed liver arginase from most proteins. Essentially the same principle applies when fresh tissue-extracts are allowed to stand for some time; coagulation being then due to the weak acid reaction resulting in muscles and other tissues shortly after death. If we dialyze the extract at this time in running water, we remove both the proteins which become insoluble and the salts and other crystalloid substances which pass through the parchment. After the dialysis, or after standing, we filter off the precipitate. As a rule it is useless to filter under pressure through a Chamberland or similar filter.

Nevertheless, the advantage of Buchner's method consists not only in the complete disintegration of tissues and structures, but likewise in the filtration of the press-juice through siliceous earth, which retains the nucleoproteins, the globulins, and all proteins coagulating spontaneously. Almost the only proteins found in the press-juice of the muscles and the liver are albumin and the haemoglobin of the blood. These juices are in most cases very rich in enzymes.

Using one of these methods it is easy to obtain proteolytic enzymes and steapsins, but many sucroclastic enzymes are retained by the proteins, and cannot be dissolved out again by water. Sometimes it is possible to dissolve them by very weak alkali, but the necessary conditions have not been sufficiently studied.

1 A. Kossel and H. D. Dakin: Zeitschr. f. physiol. Chemie, 41, 42 (1904).

If we do not succeed in bringing the enzymes into solution, we must add the solid precipitate to the solution containing carbohydrates or fats or proteins, which are to be acted upon by the enzyme. We do not know whether the enzyme works under such conditions in a solid state and converts carbohydrates and other substances only by contact, or whether the enzyme passes into solution. We shall see that we must assume a chemical connection and combination between enzyme and substance. Perhaps the chemical compound thus newly formed is more soluble than the free enzyme. This would be a good explanation of the action, for instance, by which the mucous membrane of the small intestine converts milk sugar, although it is impossible to find lactase in the extracts, or the ability of dried pancreas powder to dissolve protein, which is lacking in extracts made from this powder. We must always remember, however, that the difference can be produced also by an improper reaction of the extract. It is an important fact, however, that we can use the dry powder for many purposes.

With these methods we can easily remove proteins and other harmless, indifferent substances. The chief difficulties which complicate the purification, extraction, and investigation of enzymes, are the presence of other enzymes, and the anchorage of the enzymes in the cells. The avoidance of these difficulties is the chief advantage of the secreted natural juices as compared with the extracts.

i. By no method can one enzyme be separated from another without great loss. The gastric juice contains only the well-known proteolytic enzyme, pepsin, which dissolves natural proteins and converts them into peptones; but the process never goes beyond the stage of yielding biuret-giving substances. The mucous membrane of the stomach, and therefore its extracts, contains, besides pepsin destined for secretion, an erepsin acting intracellularly and splitting peptones into amino-acids. The pepsin and the erepsin have the same solubilities, and if we free the proteolytic enzyme from impurities, we concentrate both enzymes, getting an artificial juice with properties other than those of the natural one. The contradictions of authors on the question of trypsin and trypsinogen is believed by Bayliss * to be susceptible of the same explanation.

2. If we microscopically study the pancreas or a salivary gland at rest, we see the cell crowded with small, refractive granules forming a mass that leaves only a very narrow clear space next to the basement membrane. In the salivary gland the cell can be wholly studded with the granular mass. During glandular activity the granules become much fewer in number, and retract to an inner narrow margin; in the salivary gland they may even disappear. There can be no doubt that during the act of secretion, the granules are discharged to form part of the secretion. They become the solid matter of the juices; therefore they must be soluble in water. If we extract

1 W. M. Bayliss: Archiv. des Sciences Biol, de St. Petersbourg, n (i Suppl.), 1901.

the pancreas or the parotid gland while studded with these granules, they, and with them the enzymes, are dissolved immediately. As has been pointed out by Pekelharing,1 we find in the extracts of the gastric mucous membrane the most characteristic chemical compound of the gastric juice.

There is something else to be noticed in dealing with the intracellular enzymes that never leave the cell. In the state of activity, both these enzymes and the substances hydrolyzed or oxidized by them must be in solution, for " corpora non agunt nisi soluta." To avoid the action of enzymes at rest, the two bodies must be separated from each other; they must be fixed outside the real fluid protoplasm. We know another substance which, in this respect, is analogous to the endo-enzymes; glycogen, which is soluble in water, but which may be stored in insoluble form within the cell. Ehrlich 2 found that glycogen is fixed in the liver cell by a "Tragersubstanz," a holding-substance, and that one of the color reactions attributed to glycogen is due to this substance.

Glycogen and this Tragersubstanz are rather firmly bound together. We know with what difficulty the glycogen is completely dissolved out of the organs, and with what difficulty it is freed from the last traces of impurities. It seems to me that we must also assume such a holding-substance for the intracellular enzymes which restrains the action of the enzyme even after the almost

1 Pekelharing: Zeitschr. f. physiol. Chemie, 22 (1896); 38 (1902).

2 P. Ehrlich: in a paper in French's Zeitschr. f. klin. Med., 6 (1883).

complete disintegration of the tissues. I have cut frozen muscles using Kossel's * rotating knives, and I have studied the glycolytic enzyme of this snow; I have also compared the snow simply dissolved and floating in water, with the press-juice I obtained by mixing the snow with siliceous earth and pressing it. The glycolytic power of the press- juice was much greater, I think, because the siliceous earth had retained with the other proteins the Tragersubstanz of the enzyme.

1 Kossel: Zeitschr. f. physiol. Chemie, 33 (1901).

NEW VG

Chapter III

All enzymes are colloids, like the native proteins of the seralbumin type. Like these, they cannot be dialyzed through parchment, and are irrevocably changed on heating in aqueous solution to 6o° to 700 C. The albumin is coagulated by heat, and this coagulum cannot be dissolved without conversion into peptones; the enzymes, however, disappear entirely on heating. It is possible that they are coagulated and become insoluble; it is also possible that they are chemically changed and decomposed. The only sign of the presence of enzymes is their action, and we see that the hydrolyzing or oxidizing power of a solution is lost by heating, and cannot be restored. Pfeffer was able to show that the enzymes do not volatilize with the vapor of water and thus do not leave the solution.