In considering these results, it is to be noticed that there is a general unanimity of agreement except in the case of the albuminoid gelatin. In the proteolysis of this body, for some reason not explainable, the digestive products show no marked deviation from the composition of the mother-proteid, but in every other instance there is to be traced a distinct tendency toward diminution in the content of carbon, proportional to the extent of proteolysis. In the primary bodies, proto and heteroproteoses, the percentage of carbon is only slightly lowered; indeed, in some few cases, notably in elastin and casein, the primary products show a slight increase in their content of carbon, but in most instances there is a slight falling off in the percentage of this element. In the deuteroproteoses, however, the loss of carbon is very marked. The percentage loss, to be sure, varies with the different proteids, doubtless dependent in part upon the nature of the proteid itself, and also, I think, upon the strength of the proteolytic agent employed and the duration of the proteolysis. It is to be further noticed that peptones, whenever analyzed, show a still further loss of carbon and also a marked loss of sulphur. In nitrogen there is no constant difference.
On the assumption that these various products of proteolysis are formed by a series of hydrolytic changes, accompanied by cleavage of the molecule, we might at first glance look for a marked increase in the content of hydrogen. But when we consider the size of the proteid molecule, with the small proportion of hydrogen contained therein and the large amount of carbon, it is plain that hydrolytic cleavage might naturally leave its mark on the percentage of carbon, rather than on the percentage of hydrogen of the resultant products. In view of these facts, the above results show nothing inconsistent with the theory that pepsin-proteolysis, as a rule, is accompanied by a series of progressive hydrolytic cleavages in which the primary proteoses are the result of a slight hydration, these bodies by continued proteolysis being further hydrated with formation of secondary proteoses, which in turn undergo final hydration and cleavage into true peptones. In accord with this theory, true peptones always show a marked difference in composition from that of the mother-proteid, the most striking feature being the greatly diminished content of carbon, which may be taken as a measure, in part at least, of the extent of the hydrolytic change. And it is to be noticed that the crystallized phytovitellins are no exception to the general rule; the secondary vitelloses and peptones resulting from proteolysis bear essentially the same relationship to the mother-proteids that the albumoses from egg-albumin do. Moreover, the alcohol-soluble proteids, of which the zein of cornmeal is a good example, show the same general tendency, and it is an interesting fact that the proteoses, or more specifically the zeoses, formed from this peculiar proteid, are readily soluble in water and show the general proteose reactions. It may also be mentioned that these zeoses, as well as the elastoses, are very resistant to further hydrolysis by pepsin-acid, and yield only comparatively small amounts of true peptones.
In connection with this question of the composition of proteoses and peptones as formed by pepsin-proteolysis, it is interesting to note a recent observation recorded by Schützenberger.[138] This experimenter took 350 grammes of moist blood-fibrin, corresponding to 75.5 grammes of dry substance, and subjected it to proteolysis with 2.5 litres of a very strong pepsin-hydrochloric acid solution for five days. The resultant fluid was then freed from acid by treatment with silver oxide, after which the solution was evaporated to dryness on a water-bath and the residue dried in vacuo. This residue, termed by Schützenberger fibrin-peptone, was found on analysis to contain 49.18 per cent. of carbon, 7.09 per cent. of hydrogen, and 16.33 per cent. of nitrogen, thus agreeing very closely with true fibrin-peptone as analyzed by Kühne and myself. Further, Schützenberger showed that the fibrin in undergoing this transformation had taken on 3.97 per cent. of water. But to my mind, the most significant fact connected with this experiment is the positive evidence it affords, not only of hydration as a feature of peptonization by pepsin-acid, but that this greatly diminished content of carbon, so characteristic of peptones, and to a less extent of deuteroproteoses, is wholly independent of the methods of separation and purification ordinarily made use of. Thus, Schützenberger, in the above experiment, did not attempt any separation of individual bodies. Proteolysis was carried out under conditions favoring maximum conversion into peptone, and the resultant product, or products, was analyzed directly without recourse to any methods of precipitation or purification. To be sure, the substance analyzed could not have been peptone entirely free from proteose, but in any event it represented the terminal products of pepsin-proteolysis, and like true amphopeptone contained 3.5 per cent. less carbon than the original fibrin. Hence, we may conclude, without further argument, that peptonization in gastric digestion is the result of distinct hydrolytic action, in which the original proteid molecule is gradually broken down, or split apart, into a number of simpler molecules, the proteoses and peptones.
Peptones, i. e., amphopeptones, are the final products of gastric digestion; but to how great an extent is actual peptonization carried on in pepsin-proteolysis? As we have seen, syntonin, primary proteoses, secondary proteoses, and peptones are all products of pepsin-digestion, and it might perhaps be assumed that ultimately all of a given proteid undergoing pepsin-proteolysis would be converted into amphopeptone. Examination, however, shows that such is not the case, at least in artificial digestive experiments. Peptones are truly formed, and many times in large amount, but never under any circumstances have I been able to effect a complete transformation of any proteid into true peptone by pepsin-proteolysis; there is always found a certain amount of proteoses more or less resistant to the further action of the ferment. Obviously, the nature and proportion of the individual products formed in any digestive experiment are dependent greatly upon the attendant conditions; but even with a large amount of active ferment, an abundance of free hydrochloric acid, a proper temperature, and a long-continued period of digestion, even five and six days, there is never found a complete conversion into peptone. Indeed, the largest yield of peptone I have ever obtained in an artificial digestion is sixty per cent., while the average of a large number of results under most favorable circumstances is somewhat less than fifty per cent.[139]
We understand that peptones are the products of the hydration and cleavage of previously formed proteoses. The primary proteoses pass into secondary proteoses and these into peptones, but for some reason this transformation after a time becomes a slow and gradual process. At first there is a marked and rapid progression; the proteid undergoing proteolysis is rapidly dissolved, and both proteoses and peptones may be detected in abundance. But if we continue to watch the changing relations of primary and secondary proteoses and peptones, we find that progression soon ceases to be rapid, and eventually travels onward at a snail’s pace. Thus, in one experiment with coagulated egg-albumin, there was found at the end of forty-eight hours’ digestion with pepsin-hydrochloric acid, only thirty-seven per cent. of peptones with fifty-eight per cent. of proteoses, and yet digestion had been sufficiently vigorous to allow of a complete solution of the proteid in two hours. At the end of seventy-two hours the amount of peptones had increased to about forty-two per cent., the proteoses having correspondingly diminished; but even at the end of seventeen days only fifty-four per cent. of peptones were to be found, thus affording striking evidence of the slow conversion of the first-formed products into peptones.
Naturally, the individual proteoses show marked differences in their rate of conversion into secondary or final products. Take as an illustration some results[140] obtained with caseoses formed in the digestion of the casein of milk. Thus, heterocaseose, a primary product, yielded only fifteen per cent. of peptone after ninety-four hours at 40° C. with a strong pepsin-acid solution. Protocaseose, however, containing some deuterocaseose, under like conditions, yielded thirty-two per cent. of peptone in one hundred and nineteen hours, while pure deuterocaseose gave sixty-six per cent. of peptone in one hundred and thirty-seven hours. Evidently, then, the first-formed soluble products of gastric digestion, i. e., the primary proteoses, are only slowly converted into peptone, since they must first pass through the intermediate stage of deuteroproteose, which is plainly not a rapid process. The deutero-body, on the other hand, once formed is more rapidly converted into peptone, but even this is in no sense a rapid process. Hence, in the artificial digestion of proteids with pepsin-hydrochloric acid, solubility of the proteids may be quite rapid, and even complete in a very short time, but the resultant products will be mainly proteoses and not peptones. The latter are truly formed and in considerable amount, but proteoses, either as primary or secondary bodies, are invariably present and usually in excess of the peptones.
In this connection the question naturally arises how far we are to trust these results in their bearing on the natural process of digestion as it occurs in the living stomach. Obviously, the conditions are quite different in the two cases. In artificial digestions, we have especially the influence of an ever-increasing percentage of soluble products on the activity of the ferment, a condition of things generally considered as more or less inhibitory to enzyme action. We have attempted to measure the real value of this influence by experiments[141] conducted in parchment dialyzing tubes, in which the conditions are made favorable for the removal of at least some of the products of digestion as fast as they are formed. In these experiments, the dialyzer tubes containing the proteid and pepsin-acid were immersed in a large volume of 0.2 per cent. hydrochloric acid (about three litres), which was gradually changed from time to time, the whole mixture being kept at 40° C. during the entire period of the experiment. The extent of peptonization was then ascertained by analysis of both the contents of the dialyzer tubes and of the surrounding acid, the results being compared with those obtained from control experiments carried on in flasks. Without considering the results in detail, it may be mentioned that the slow and incomplete peptonization so characteristic of artificial gastric digestion is not materially modified by this closer approach to the natural process. The several digestions carried on in the dialyzer tubes were certainly accompanied by a fairly rapid withdrawal of the diffusible products of digestion, yet no noticeable increase in the amount of peptone formed was observed. The results certainly favor the view that the conversion of the primary products of gastric digestion into true peptone is a slow and gradual process, even under the most favorable circumstances, and that this lack of complete peptonization is not due to accumulation of the products of digestion, but is rather an inherent quality of pepsin-proteolysis under all circumstances.
In these dialyzer experiments it was observed that not only did peptones diffuse, but also the proteoses. In fact, it was found that six to eight per cent. of the proteoses formed passed through the parchment walls of the dialyzer tubes into the surrounding acid in the nine hours’ digestion. This led to a study of the diffusibility of proteoses in general, from which we were led to conclude that these bodies possess this power to a greater degree than had hitherto been supposed. As might be expected, it was also found that the attendant conditions modify materially the rate of diffusibility; the two factors especially prominent being temperature and the volume of the surrounding fluid. Thus, 1.9 grammes of protoalbumose dissolved in 200 c.c. of water and suspended in 4.5 litres of water heated to 38° C., diffused through the parchment tube to the-extent of 5.09 per cent., while at 10° C. diffusion amounted to only 2.57 per cent. Under somewhat similar conditions, pure peptone diffused to the extent of eleven per cent. in six hours at 38° C. Somewhat singular, however, was the result obtained with deuteroalbumose; this proteose showing a diffusibility considerably less than that of the proto-body. But as Kühne[142] has independently obtained essentially the same results, this apparent anomaly cannot depend upon any errors of work.
It is of course to be understood that diffusion experiments made with dead parchment membranes cannot necessarily be expected to throw much light upon the rate of absorption of these bodies through the living membranes of the stomach and intestine, where, as Waymouth Reid[143] has well said, we have to deal with an absorptive force dependent, no doubt, upon protoplasmic activity, and comparable, in part at least, to the excretive force of a gland-cell. Furthermore, in considering absorption as it occurs in the living stomach, we must necessarily give due weight to the selective power of the epithelial cells, a power which may be far more potent even than we suppose. Hence, without attempting at this point to draw any broad deductions from our experiments we may simply lay stress upon the facts themselves, viz., that the primary products of pepsin-proteolysis are diffusible, and, like true peptones, are capable of passing through animal and vegetable membranes, although to a less extent. We may further emphasize the fact that experiments of this character on diffusibility can, at the most, only indicate general tendencies, since every variation in the attendant conditions will exercise some influence upon the final result.
With reference to the bearing digestive experiments made in dialyzer tubes have upon the natural process as carried on in the living stomach, we must necessarily grant that the conditions approximate only in the crudest way to those existent in the alimentary tract. At the same time, if complete peptonization is characteristic of pepsin-proteolysis in the stomach, and failure to obtain such results in an artificial digestion is due to lack of withdrawal of the diffusible products formed, then certainly the experiments carried on in dialyzer tubes, with abundant opportunity for diffusion, and with a large excess of free hydrochloric acid, should show some indications of increased peptone-formation. But none were obtained.