There are many theories extant to explain this peculiar method of chemical change, but few of them help us to any real understanding of the matter. These enzymes are typical catalytic or contact agents, and by their presence render possible marked changes in the character of the proteid or albuminoid matter with which they happen to be in contact. But the conditions under which the contact takes place exercise an important control over the activity of the ferment. Temperature, reaction, concentration of the fluid, presence or absence of various foreign substances, etc., all play a very important part in regulating and controlling the activity of these two proteolytic enzymes. In fact, as one looks over the large number of data which have gradually accumulated bearing upon this point, one is impressed with the great sensitiveness of these ferments toward even so-called indifferent substances. Their specific activity appears to hinge primarily upon the existence of a certain special environment, alterations of which may be attended with an utter loss of proteolytic power, or, in some less common cases, with a decided increase in the rate of digestive action. This constitutes one of the peculiar features of these proteolytic enzymes; powerful to produce great changes, they are nevertheless subject to the influence of their surroundings in a way which testifies to their utter lack of stability. Furthermore, as you well know, conditions favorable for the action of the one ferment are absolutely unfavorable for the activity of the other, and indeed may even lead to its destruction. Thus, while pepsin requires for its activity the presence of an acid, as 0.2 per cent. HCl, trypsin is completely destroyed in such a medium. Again, trypsin exhibits its peculiar proteolytic power in the presence of sodium carbonate, a salt which has an immediate destructive action upon pepsin. Hence, a medium which is favorable for the action of the one ferment may be directly antagonistic to the action of the other.
Another factor of great moment in determining the activity of these two enzymes is temperature. That which is most favorable for their action is 38° to 40° C., and any marked deviation from this temperature is attended by an immediate effect upon the proteolysis. Exposure to a low temperature simply retards proteolytic action, doubtless in the same manner that cold checks or retards other chemical changes. There is no destruction of the ferment, even on exposure to extreme cold, the enzyme being simply inactive for the time being. Exposure of either pepsin or trypsin to a high temperature, say 80° C., is quickly followed by a complete loss of proteolytic power, i.e., the ferment is destroyed. It is to be noticed, however, that the destructive action of heat is greatly modified by the attendant circumstances. Thus, fairly pure trypsin, dissolved in 0.3 per cent. sodium carbonate, is completely destroyed on exposure to a temperature of 50° C. for five to six minutes, while a neutral or slightly acid solution of the pure enzyme is destroyed in five minutes by exposure to a temperature of 45° C. On the other hand, the presence of inorganic salts and the products of digestion, such as albumoses, amphopeptone, and antipeptone, all tend to protect the trypsin somewhat from the destructive effects of high temperatures, so that in their presence the enzyme may be warmed to 60° C. before it shows any diminution in proteolytic power. Alkaline reaction, combined with the presence of salts and proteid, viz., just the conditions existent in the natural pancreatic secretion, constitute the best safeguard against the destructive action of heat, and under such conditions trypsin may be warmed to about 60° C. before it begins to suffer harm. But all this testifies in no uncertain way to the extreme sensitiveness of the ferment to changes in temperature; a sensitiveness which manifests itself not only in diminished or retarded proteolytic action, but terminates in destruction of the ferment when the temperature rises beyond a certain point.
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.
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.
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.
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.”
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 H2O + O2 = CO(OH)2 + H2O2.