Part 3

Similarly, pepsin dissolved in 0.2 per cent. hydrochloric acid feels the destructive effect of heat when a temperature of 60° C. is reached. In a neutral solution, on the other hand, destruction of the ferment may be complete at 55° C. Here, too, peptone retards very noticeably the destructive action of heat, especially in an acid solution of pepsin, so that under such circumstances the ferment may not be affected until the temperature reaches 70° C. I have tried many experiments along this line, not only with pepsin and trypsin, but also with many other ferments. We may briefly summarize, however, all that is necessary for us to consider here in the statement that the pure isolated ferments are far more sensitive to the destructive action of heat than when they are present in their natural secretions. This, as stated, is due not only to the reaction of the respective fluids but also to the protective or inhibitory action of the inorganic salts and various proteids naturally present. We may thus say with Biernacki[36] that the purer the ferment the less resistant it is to the effects of heat.

[36] Das Verhalten der Verdauungsenzyme bei Temperaturerhöhungen. Zeitschr. f. Biol., Band 28, p. 49.

It is thus plain that these enzymes, capable though they are of accomplishing great tasks, are nevertheless exceedingly unstable and prone to lose their proteolytic power under the slightest provocation. When, however, they are surrounded by their natural environment, the acid or alkali of the respective secretion, together with salts and proteids, they then appear more stable; their natural lability becomes for the time being transformed into semi-stability, and the temperature, for example, at which they lose their peculiar power, is raised ten degrees or more. I have also found the same to be true of the vegetable proteolytic ferments, and also of the amylolytic ferment of saliva.

The above facts furnish us, I think, a good illustration of how dependent these proteolytic enzymes are upon the proper conditions of temperature, to say nothing of other conditions, for the full exercise of their peculiar power. Toward acids, alkalies, metallic salts, and many other compounds they are even more sensitive than toward heat, and much might be said regarding the effects, inhibitory or otherwise, produced by a large number of common drugs or medicinal agents on these two ferments. Any lengthy discussion of this matter, however, would be foreign to our subject, and I will only call your attention in passing to one or two points which have a special bearing upon the general nature of the enzymes. Take, for example, the influence of such substances as urethan, paraldehyde, and thallin sulphate on the proteolytic action of pepsin-hydrochloric acid[37] and we find that small quantities, 0.1 to 0.3 per cent. tend to increase the rate of proteolysis, while larger amounts, say one per cent., decidedly check proteolysis. Similarly, among inorganic compounds, arsenious oxide, arsenic oxide, boracic acid, and potassium bromide[38] in small amounts increase the proteolytic power of pepsin in hydrochloric acid solution, while larger quantities check the action of the ferment in proportion to the amounts added. Again, with the enzyme trypsin, similar results with such salts as potassium cyanide, sodium tetraborate, potassium bromide and iodide[39] may be quoted as showing not only the sensitiveness of the ferment toward foreign substances, but likewise its peculiar behavior, viz., stimulation in the presence of small amounts and inhibition in the presence of larger quantities.

[37] Chittenden and Stewart: Studies in Physiol. Chem., Yale University. Vol. 3, p. 64.

[38] Chittenden and Allen, _Ibid._, vol. 1, p. 76.

[39] Chittenden and Cummins, _Ibid._, vol. 1, p. 112.

Furthermore, we have found that even gases, as carbonic acid and hydrogen sulphide, exert a marked retarding influence on the proteid-digesting power of trypsin. Moreover, while it is generally stated that proteolytic and other enzymes are practically indifferent to the presence of chloroform, thymol, and other like substances that quickly interfere with the processes of the so-called organized ferments, pepsin and trypsin certainly do show a certain degree of sensitiveness to chloroform, and indeed even to a current of air passed through their solutions. Thus, very recently, Bertels[40] and Dubs[41], working under Salkowski’s direction, have called attention to the peculiar behavior of pepsin to chloroform; their results showing, first, that small amounts of this agent tend to increase the proteolytic power of the enzyme, while larger amounts decrease its digestive power. Another interesting point brought out especially by Dub’s experiments is the fact that an impure solution of the ferment, viz., an acid extract, for example, of the mucous membrane of the stomach containing more or less albuminous matter, is far less sensitive to chloroform than an acid solution of the purified ferment, thus showing again the protective influence of proteids and other extraneous matters; the latter guarding the enzyme to a certain extent from both the stimulating and inhibitory action of various agents.

[40] Ueber den Einfluss des Chloroforms auf die Pepsinverdauung, Virchow’s Archiv, Band 130, p. 497.

[41] Der Einfluss des Chloroforms auf die künstliche Pepsinverdauung, Ibid., Band 134, p. 519.

Another point to be emphasized just here is that any chemical substance, such as a metallic salt, having a specific action upon proteid matter, will almost invariably interfere more or less with the proteolytic action of these enzymes, both through a direct action upon the soluble ferment itself, and also through an indirect action in modifying or inhibiting the digestibility of the proteid exposed to proteolysis. All of these facts emphasize more or less the proteid-like nature of the enzymes, or at least the carriers of the ferments. It is further very suggestive that the destruction of these enzymes by heat happens to occur at approximately those temperatures which are generally recognized as the coagulation points of ordinary proteids. Moreover, the apparent lack of stability so characteristic of these ferments, their inherent proneness to alteration, their marked susceptibility to every change in environment, all point to large complex molecules, such as we have in proteids and are familiar with in living protoplasm.

Whatever the exact nature of these proteolytic enzymes, they are certainly endowed with the power of transforming relatively large amounts of proteid matter into soluble products, even though they themselves are present in very small quantity. They are derived, as we have seen, from living protoplasmic cells, and we might perhaps, with v. Nägeli[42] and Mayer,[43] consider them as retaining a portion of that molecular motion so characteristic of living protoplasm, by which the equilibrium of the dead food-proteid may be disturbed and thus changes started which result in what we call proteolysis. However this may be, we must look to some phase of catalytic or contact action as the true explanation of this power of proteolysis. At first glance, any explanation or theory involving the use of catalysis seems exceedingly vague and indefinite, and yet many illustrations can be given of chemical reactions where the dominating agent evidently acts in this manner. “We call a force catalytic,” says the philosopher of Heilbron, “when it holds no communicable proportion to the assumed results of its action. An avalanche is hurled into the valley.... A puff of wind or the fluttering of a bird’s wing is the catalytic force which has given the signal for, and which is the cause of the wide-spread disaster.”[44] In the older theories of catalytic action, the catalytic agent was supposed to remain passive, but not so in the more modern conception of catalysis. The ferment by its presence makes possible certain changes and combinations which could not occur in its absence, at least under the existing circumstances, although all the other conditions might be favorable. The proteids, for example, have a natural tendency to undergo hydration; thus, simple boiling with dilute acid or exposure to the action of superheated water alone,[45] will produce many if not all of the products formed in natural digestive proteolysis. To be sure, they are not formed as readily as in artificial or natural digestion, and there may be some minor points of difference, but still proteolysis can be imitated in this manner. The proteolytic enzymes simply help on this natural tendency of proteid bodies to undergo hydration, and by their presence and action enable it to occur at lower temperatures than it otherwise could, and at the same time render it more rapid and complete. This is not accomplished, however, by simple contact. The enzymes, we may assume, combine in some manner with the proteid undergoing digestion, starting thereby a train of reactions in which the proteid and the water present are the main actors, they being, however, perfectly passive in the absence of the inciting agent, the enzyme. As expressed by Ganger, “the ferment phenomena resemble those in which there is apparently a periodic synthesis and dissociation of the catalyzing agent, which acts in a similar manner to the agent which explodes a train of gunpowder.”

[42] Theorie der Gährung. München, 1879.

[43] Die Lehre von den chem. Fermenten. Heidelberg, 1882.

[44] Quoted from Gamgee’s Physiological Chemistry of the Animal Body, vol. 2, p. 7.

[45] Chittenden and Meara. A study of the primary products resulting from the action of superheated water on coagulated egg-albumin. Journal of Physiol., vol. 15, p. 501.

We can find many illustrations among chemical phenomena where one body, even though present in small quantity, acts as a go-between and makes possible an almost indefinite exchange of matter and energy. Take as an illustration, the part played by water in determining the explosion of oxygen and carbon-monoxide gas. Some years ago, Dixon[46] called attention to the fact that a mixture of these two gases when perfectly dry would not explode even by contact with red hot platinum wire. The presence, however, of a small amount of aqueous vapor would at once cause an explosion to occur. In confirmation of this observation, Traube[47] has reported that a flame of carbon-monoxide gas introduced into a perfectly dry atmosphere is at once extinguished. In a moist atmosphere, on the other hand, the flame will continue to burn indefinitely, that is as long as the CO gas is supplied. In these cases, the water, which is so necessary for the appearance of the reaction, and which furnishes a striking illustration of the action of a contact or catalytic substance is not purely passive. To be sure, only a minimal amount is necessary for the combustion of an indefinite amount of carbonic oxide, but the water enters into the reaction itself. It is to be noticed that carbonic oxide and water alone, even at high temperatures, will not react, but in the presence of oxygen the water is decomposed with formation of hydrogen-peroxide, thus:

CO + 2 H_{2}O + O_{2} = CO(OH)_{2} + H_{2}O_{2}.

[46] Chemical News, vol. 46, p. 151.

[47] Bericht. d. Deutsch. chem. Gesellsch., Band 18, p. 1890.

The hydrogen-peroxide thus formed combines with carbonic oxide to form carbonic acid, which in turn is decomposed into the anhydride CO_{2}, with regeneration of water, the latter being available for further action of the same order:

H_{2}O_{2} + CO = CO(OH)_{2}

2 CO(OH)_{2} = 2 CO_{2} + 2 H_{2}O.

Indeed, as can be readily seen from the equations, this may be kept up indefinitely, a small amount of water, _i.e._, the go-between, the catalytic agent, sufficing to accomplish the transformation of almost any amount of carbon-monoxide. This, I think, furnishes an excellent illustration of the way in which catalytic agents, such as the proteolytic enzymes, may be supposed to act. It is truly contact action, but the agent is not purely passive; the enzyme combines with the substance undergoing proteolysis, and the resultant compound thus formed is enabled now to combine with water and undergo hydrolysis, something which could not be accomplished by the proteid and water alone, that is at body temperature. This new and more complex compound is naturally less stable and soon undergoes dissociation or cleavage with a splitting off of the original enzyme for one product, which is thus available for further action of the same order; while, as other products, we find the hydrated and otherwise altered substances coming from the proteid, and whose formation is the ultimate object of the whole process.

The parallelism between this hypothetical action of the proteolytic enzymes and the known reactions in the above combustion of carbonic oxide is certainly very close, and leaves little doubt that this explanation of enzyme action is, in a general way at least, correct. Thus the carbonic oxide, CO, brought in contact with pure, dry oxygen gas (apparently all that is necessary for its direct oxidation into carbonic acid, CO_{2}), undergoes no change; the burning CO gas is at once extinguished. Evidently, something more is necessary in order to start the process of oxidation. So, too, in proteolysis; the process, as we shall see later on, is essentially one of hydration, but bring the proteid and the water, or acid-water, together and although all the conditions are apparently favorable for hydration there is, as you know, little or no change. But introduce the catalytic agent and immediately the reaction commences. In the case of the burning CO gas in contact with oxygen, the water acting as contact agent makes oxidation possible, enabling the main actors in the transformation to react upon each other. But, as we have seen, the contact agent is something more than a mere looker-on, it becomes for the time being an integral part of the molecule, undergoing change, combining with it and thus making possible the subsequent alterations characteristic of the specific transformation, in which, however, the regeneration of the contact agent is a prominent feature. So, too, with the proteolytic enzymes, pepsin and trypsin, they are the go-betweens, making possible the union of the proteids with water by combining with the proteid molecule and thus paving the way for both hydration and cleavage. In the cleavage of the complex molecule, we have the regeneration of the ferment as a prominent feature, and in proteolysis we understand that the regenerated ferment may act not only upon more of the original proteid, but likewise upon the primary products of its action, thus giving rise eventually to a row of more or less closely related cleavage products. Finally, we can conceive that the enzyme may gradually be affected by the process, that its regeneration may become less complete, and thus digestive power be eventually diminished.

Much more might be said in support of the above hypothesis. On the other hand, some objections might be raised against it, but I know of no more reasonable explanation of enzyme action than that here presented, or one which so well accords with all of the known facts concerning the conditions which modify proteolytic action.[48] Thus, the influence of heat, of the products of proteolysis, of acids, alkalies, and various organic and inorganic salts on the action of these digestive enzymes is such as lends favor to the above view rather than opposes it.

[48] Compare L. de Jager, Erklärungsversuch über die Wirkungsart der ungeformten Fermente, Virchow’s Archiv, Band 121, p. 182. Also Chandelon: Bulletin de l’Academie Royale de Méd. de Belgique, 1887, 1, p. 289.

THE GENERAL NATURE OF PROTEIDS.

Proteids are confessedly among the most complex bodies the physiologist has to deal with, while at the same time they are perhaps the most important, not only in view of their wide-spread distribution through animal and vegetable tissues, but because of the prominent part they take in the nutrition of the body. The more our knowledge is broadened concerning these varied substances, the more we are impressed with their complexity, and at the same time with the necessity for a more accurate study of both their composition and constitution. Concerning the latter, full fruition of our hopes is probably in the distant future, but every step of advance in this direction adds greatly to our resources in the interpretation of the varied and complex changes characteristic of proteid metabolism. Every study of proteid decomposition adds something to our store of knowledge, and gives perhaps an added fact available for broadening our deductions.[49] Moreover, the composition and general reactions of the proteids may be investigated with full confidence of obtaining many useful results, which must necessarily be an aid in interpreting the changes accompanying digestive proteolysis.

[49] See Drechsel: Der Abbau der Eiweisstoffe. Du Bois Reymond’s Archiv f. Physiol., 1891, p. 248.

Take, for example, the single question of peptonization by gastric digestion. What is the nature of the process? Is the proteid transformed into a soluble and diffusible peptone as a result of hydration and cleavage, or is it a transformation which results from a simple depolymerization of the proteid molecule, _i.e._, are we to consider albumin and peptone as isomeric bodies? These questions, on which physiologists seem loath to agree, can certainly be answered definitely; not, however, by arguments but by careful experimentation, in which the composition of the proteid undergoing digestion must be a necessary preliminary factor, and the composition of the resultant product, or products, a secondary factor of equal importance. Further, the question needs to be answered not with reference to one proteid merely, but with reference to every proteid capable of digestion by either gastric or pancreatic juice. When these questions have been fully answered in this manner, we shall have positive data to deal with, and our conclusions will rest upon a foundation of fact not easily set aside. This is one of the problems upon which I have been at work for some years, and although progress may in one sense be slow, yet it is sure and gives results of no uncertain character.

First, then, let us consider briefly the nature of the proteids whose proteolysis we may be interested in; remembering, however, that in so doing we can merely touch upon the points essential for our purpose. Allow me to say in parenthesis that there is being published in Moscow a work on proteids alone of five volumes, 900 pages each, which it is supposed will constitute an exhaustive treatise of the subject.[50]

[50] Die Einheit der Proteinstoffe. Historische u. experimentelle Untersuchungen, by L. Morokhowetz.

If we attempt to classify all of the proteid bodies hitherto discovered and studied we are at once confronted with a problem of no small proportions. So varied are they in their reactions, solubilities, and behavior toward general reagents, so inclined to merge into each other by almost insensible gradations that it becomes an extremely difficult matter to make an arrangement that will satisfy all the requirements of the case. I have to suggest, however, the following classification, which is merely a modification of several existing ones, based primarily upon chemical composition, and solubility in the more common menstruums.

Proteids may first be divided into three main groups as follows:

I. _Simple Proteids._--Composed of carbon, hydrogen, nitrogen, sulphur, and oxygen, and yielding by decomposition aromatic bodies such as tyrosin, phenol, indol, etc.

II. _Compound Proteids._--Composed of a simple proteid united to some non-proteid body.

III. _Albuminoids._--A class of nitrogenous bodies related to and derived from proteids, but differing especially from the latter by great resistance to the ordinary solvents of true proteids.

The individual members of these three groups may be arranged as follows on the basis of solubility, coagulability, etc.:

I. SIMPLE PROTEIDS.--_A._ _Soluble in water._--_a._ Coagulable by heat, and by long contact with alcohol. Albumins: serum-albumin, egg-albumin, lacto-albumin, myo-albumin, vegetable albumins. _b._ Non-coagulable by heat and by long contact with alcohol. Proteoses:[51] protoproteoses, deuteroproteoses. Peptones:[51] amphopeptones, antipeptones, hemipeptones.

[51] Used in the generic sense; the proteoses including albumoses, globuloses, myosinoses, elastoses, etc., and the peptones the peptone-products formed in the digestion of any or all proteids.

_B._ _Insoluble in water, but soluble in salt solutions._--_a._ More or less coagulable by heat. Globulins. 1. Soluble in dilute and saturated NaCl solutions. Vitellins. 2. Soluble in dilute NaCl solutions, but precipitated by saturation with NaCl. Myosins, paraglobulin[52] or serum-globulin, fibrinogen, myo-globulin, paramyosinogen, cell-globulins. _b._ Non-coagulable by heat, soluble in dilute NaCl solution and precipitated by saturation with NaCl. Heteroproteoses.

[52] Not completely insoluble in saturated NaCl solution.

_C._ _Insoluble in water and salt solutions, soluble in dilute alcohol_--Zein, gliadins.

_D._ _Insoluble in water, salt solutions and alcohol; soluble in dilute acids or alkalies._--_a._ Coagulable by heat when suspended in a neutral fluid. Acid-albumins, alkali-albumins or albuminates. _b._ Non-coagulable by heat when suspended in a neutral fluid. Antialbumids, dysproteoses, glutenins.

_E._ _Insoluble in water, salt solutions, alcohol, dilute acids and alkalies; soluble in strong acids, alkalies, and in pepsin-hydrochloric acid and alkaline solutions of trypsin._--Coagulated proteids, fibrin.[53]

[53] In blood-fibrin we have a good illustration of the fact that these divisions are not absolutely exact, since this form of proteid matter, for example, is somewhat soluble in dilute acids and in salt solutions, although requiring a long time for marked solution.

II. COMPOUND PROTEIDS.--_A._ _Compounds of a proteid (globulin) with an iron-containing pigment, soluble in water and coagulable by heat and alcohol._ Hæmoglobin, oxyhæmoglobin, methæmoglobin, etc.

_B._ _Compounds of proteids with members of the carbohydrate group. Insoluble in water; soluble in very weak alkalies._--_a._ True mucins. _b._ Mucoids or mucinoids.

_C._ _Compounds of proteids with nucleic acid. Phosphorized bodies yielding by decomposition metaphosphoric acid. Insoluble in water and in pepsin-hydrochloric acid, but more or less soluble in alkalies._--Nucleins.

_D._ _Compounds of proteids with nucleins. Very soluble in dilute alkalies._--Nucleoalbumins, as casein of milk, and nucleoalbumins of cell-protoplasm and cell-nuclei, etc.

III. ALBUMINOIDS.--_A._ _Soluble in boiling water with formation of gelatin and yielding by decomposition leucin and glycocoll._--Collagen (gelatin).

_B._ _Insoluble in boiling water, and yielding by decomposition much leucin and some tyrosin, together with glycocoll and lysatin. Slowly hydrated by boiling dilute acids and by treatment with pepsin-hydrochloric acid._--Elastin.

_C._ _Insoluble in water, dilute acids and alkalies, also in gastric and pancreatic juice. Yield leucin and tyrosin by decomposition._--Keratin, neurokeratin.

We may now advantageously consider the composition of a few of the more prominent representatives of the individual groups, taking for illustration those bodies which have been most thoroughly studied, and which we may have occasion to refer to in our discussion of proteolysis. I have not included in the table any of the alteration-products of the proteids formed by the action of pepsin-acid, trypsin, or boiling dilute acids, confining myself here simply to those bodies which occur ready-formed in nature.