Part 7

I have introduced these dry chemical facts, none of which are especially new, because I deem them of considerable importance and because they are not very generally known. In fact, there seems to be a tendency on the part of some who are more or less familiar with the advances made in our knowledge of the products of pepsin-proteolysis to question the existence of these different bodies, or to show at least a spirit of indifference toward these recent facts which have been gradually accumulated, and I may say accumulated at the expense of considerable labor. The time is past for calling the products of gastric digestion peptones; it is time for a full recognition of the fact that pepsin-proteolysis is synonymous with the production of a row of bodies, chemically and physiologically distinct from each other, each endowed with individuality enough to admit of certain detection, and all bearing a certain specific and harmonious relationship to their neighbors, the other members of the series.

Further, it is not enough to admit the formation of a single intermediate body, midway between syntonin and peptone. The so-called propeptone of the past is simply a mixture of proteoses, of ever changing composition, varying with each change in the proportion of the component proteoses. Each of these proteoses can be detected, under suitable conditions, in the products of every artificial digestion as well as in the stomach-contents, and no better measure of the proteolytic power of the natural stomach-secretion can be devised than a study of the character of the individual bodies present in the stomach-contents after a suitable test meal. The proper tests and separations can be made with a small amount of the filtered fluid, and much light thrown upon the digestive power of the secretion by even a rough estimate of the proportion of primary and secondary proteoses and peptones formed in a given time, after the ingestion of a certain amount of proteid food.

In pepsin-proteolysis we have to deal, in my opinion, with a series of progressive hydrolytic changes in which peptones are the final products of the transformation. Commencing with the formation of acid-albumin or syntonin, hydrolysis and cleavage proceed hand in hand, under the guiding influence of the proteolytic enzyme, and each onward step in the process is marked by the appearance of a new body corresponding to the extent of the hydrolysis; each body, perhaps, being represented by a row or series of isomers, all externally alike, but different in their inner structure, according to the proportion of hemi- and anti-groups contained in the molecule. As opposed to this theory, we have the older views of Maly,[120] Herth,[121] Henninger[122] and others, based upon observations which tend to show that peptones do not differ in chemical composition from the proteids which yield them. As a matter of fact, the products then analyzed were not peptones at all; they were merely the _primary_ products of pepsin-proteolysis, _i. e._, what we now term primary proteoses, and it is time we stopped using such data to enforce the theory that peptones are polymers of the proteids from which they are derived.

[120] Ueber die chemische Zusammensetzung und physiologische Bedeutung der Peptone. Pflüger’s Archiv f. Physiol., Band 9, p. 585.

[121] Ueber die chemische Natur des Peptones und sein Verhältniss zum Eiweiss. Zeitschr. f. physiol. chem., Band 1, p. 277.

[122] De la Nature et du rôle physiologique des peptones. Paris, 1878.

In 1886, the writer, in conjunction with Professor Kühne, commenced a study of the various cleavage products[123] formed by the action of pepsin-hydrochloric acid from the better characterized and purer proteids, this being a continuation of our earlier work on the proteoses and peptones formed from blood-fibrin, serum-albumin, etc. This work I have continued in my laboratory up to the present time, with many co-workers, and as a result we have to-day a series of observations gradually accumulated during these last seven years, some the results of work carried on this last year, which speak in no uncertain way of the character of both the primary and secondary products of pepsin-proteolysis. Furthermore, in attempting to settle this question once for all, I have selected for study examples from the various classes of both animal and vegetable proteids; and as representatives of the latter have had carried out two lengthy series of experiments on the crystallized proteids which occur so abundantly in some seeds, on the assumption that these crystalline bodies would furnish a certain guarantee of purity which might naturally be lacking in the amorphous proteids of animal origin. Some of these results are now placed together in the following tables, a study of which reveals some very interesting facts:

[123] Globulin and Globuloses. Studies in Physiol. Chem. Yale University, vol. ii., p. 1.

COMPOSITION OF PROTEOLYTIC PRODUCTS FORMED BY PEPSIN-HYDROCHLORIC ACID.

_Proteolysis of Blood-fibrin._

===================================================== | Mother | Proto- | Hetero- | Deutero- | Ampho- |Proteid.|fibrinose.|fibrinose.|fibrinose.|peptone. | | [124] | [124] | [124] | [125] --+--------+----------+----------+----------+-------- C | 52.68 | 51.50 | 50.74 | 50.47 | 48.75 H | 6.83 | 6.80 | 6.72 | 6.81 | 7.21 N | 16.91 | 17.13 | 17.14 | 17.20 | 16.26 S | 1.10 | 0.94 | 1.16 | 0.87 | 0.77 O | 22.48 | 23.63 | 24.24 | 24.65 | 27.01 --+--------+----------+----------+----------+--------

[124] Kühne and Chittenden: Zeitschr. f. Biol., Band 20, p. 40.

[125] Kühne and Chittenden: Studies in Physiol. Chem., Yale Univer., vol. ii., p. 40.

_Proteolysis of Paraglobulin._[126]

============================================ | Mother | Proto- | Hetero- | Deutero- |Proteid.|globulose.|globulose.|globulose. --+--------+----------+----------+---------- C | 52.71 | 51.57 | 52.10 | 51.52 H | 7.01 | 6.98 | 6.98 | 6.95 N | 15.85 | 16.09 | 16.08 | 15.94 S | 1.11 }| | | O | 23.24 }| 25.36 | 24.84 | 25.59 --+--------+----------+----------+----------

[126] Kühne and Chittenden: Studies in Physiol. Chem., Yale Univer., vol. ii, p. 12.

_Proteolysis of Coagulated Egg-albumin._

================================================== | Mother | Proto- | Hetero- |Deutero- | Hemi- |Proteid.|albumose.|albumose.|albumose.|peptone. | | [127] | [127] | [127] | [128] --+--------+---------+---------+---------+-------- C | 52.33 | 51.44 | 52.06 | 51.19 | 49.38 H | 6.98 | 7.10 | 6.95 | 6.94 | 6.81 N | 15.84 | 16.18 | 15.55 | 15.77 | 15.07 S | 1.81 | 2.00 | 1.63 | 2.02 | 1.10 O | 23.04 | 23.28 | 23.81 | 24.08 | 27.64 --+--------+---------+---------+---------+--------

[127] Chittenden and Bolton: _Ibid._, vol. ii, p. 153.

[128] Kühne and Chittenden: Zeitschr. f. Biol., Band 19, p. 201.

_Proteolysis of Casein from Milk._

=================================================== | Mother | Proto- |Hetero- |α Deutero-|β Deutero- |Proteid.|caseose.|caseose.| caseose. | caseose. | | [129] | [130] | [129] | [129] --+--------+--------+--------+----------+---------- C | 53.30 | 54.58 | 53.88 | 52.10 | 47.72 H | 7.07 | 7.10 | 7.27 | 6.93 | 6.73 N | 15.91 | 15.80 | 15.67 | 15.51 | 15.97 S | 0.82 }| | | | O | 22.03 }| 22.52 | 23.18 | 25.46 | 29.58 --+--------+--------+--------+----------+----------

[129] Chittenden: Studies in Physiol. Chem., Yale Univer., vol. iii., p. 80.

[130] Chittenden and Painter: _Ibid._, vol. ii., p. 195.

_Proteolysis of Myosin from Muscle._[131]

==================================================== |Mother Proteid.|Protomyosinose.|Deuteromyosinose. --+---------------+---------------+----------------- C | 52.82 | 52.43 | 50.97 H | 7.11 | 7.17 | 7.42 N | 16.77 | 16.92 | 17.00 S | 1.27 | 1.32 | 1.22 O | 21.90 | 22.16 | 23.39 --+---------------+---------------+-----------------

[131] Kühne and Chittenden: _Ibid._, vol. iii, p. 147.

_Proteolysis of Elastin._[132]

==+=============================================== |Mother Proteid.|Protoelastose.|Deuteroelastose. --+---------------+--------------+---------------- C | 54.24 | 54.52 | 53.11 H | 7.27 | 7.01 | 7.08 N | 16.70 | 16.96 | 16.85 S}| | | O}| 21.79 | 21.51 | 22.96 --+---------------+--------------+----------------

[132] Chittenden and Hart: Studies in Physiol. Chem., Yale Univer., vol. iii, p. 37.

_Proteolysis of Gelatin._[133]

==+=============================================== |Mother Proteid.|Protogelatose.|Deuterogelatose. --+---------------+--------------+---------------- C | 49.38 | 49.98 | 49.23 H | 6.81 | 6.78 | 6.84 N | 17.97 | 17.86 | 17.40 S | 0.71 | 0.52 | 0.51 O | 25.13 | 24.86 | 26.02 --+---------------+--------------+----------------

[133] Chittenden and Solley: Journal of Physiol., vol. xii, p. 33.

_Proteolysis of Phytovitellin_[134] _(Crystallized) from Squash Seed._

==+===============+===============+================= |Mother Proteid.|Protovitellose.|Deuterovitellose. --+---------------+---------------+----------------- C | 51.60 | 51.52 | 49.27 H | 6.97 | 6.98 | 6.70 N | 18.80 | 18.67 | 18.78 S | 1.01} | | O | 21.62} | 22.83 | 25.25 --+---------------+---------------+-----------------

[134] Chittenden and Hartwell: _Ibid._, vol. xi, p. 441.

_Proteolysis of Phytovitellin_[135] _(Crystallized) from Hemp Seed._

============================================================= |Mother Proteid.|Protovitellose.|Deuterovitellose.|Peptone. --+---------------+---------------+-----------------+-------- C | 51.63 | 51.55 | 49.78 | 49.40 H | 6.90 | 6.73 | 6.73 | 6.77 N | 18.78 | 18.90 | 17.97 | 18.40 S | 0.90 | 1.09 | 1.08 | 0.49 O | 21.79 | 21.73 | 24.44 | 24.94 --+---------------+---------------+-----------------+--------

[135] Chittenden and Mendel: _Ibid._, vol. xvii, p. 48.

_Proteolysis of Glutenin_[136] _from Wheat._

==+========================================= | Mother | Proto- | Hetero- | Deutero- |Proteid.|glutenose.|glutenose.|glutenose. --+--------+----------+----------+---------- C | 52.34 | 51.42 | 51.82 | 49.85 H | 6.83 | 6.70 | 6.79 | 6.69 N | 17.49 | 17.56 | 17.43 | 17.57 S | 1.08 | 1.34 | 1.59 | 0.80 O | 22.26 | 22.98 | 22.37 | 25.09 --+--------+----------+----------+----------

[136] Formerly called gluten-casein, and the products gluten-caseoses. Chittenden and E. E. Smith: Journal of Physiol., vol. xi, p. 420.

_Proteolysis of Zein._[137]

============================================ |Mother Proteid.|Protozeose.|Deuterozeose. --+---------------+-----------+------------- C | 55.23 | 53.29 | 51.31 H | 7.26 | 6.87 | 6.88 N | 16.13 | 16.10 | 16.27 S | 0.60 | 1.54 | 1.08 O | 20.78 | 22.20 | 24.46 --+---------------+-----------+-------------

[137] Chittenden and Williams: Not heretofore published.

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.

[138] Recherches sur la constitution chimique des peptones. Comptes Rendus, vol. 115, p. 208.

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]

[139] Chittenden and Hartwell: The Relative Formation of Proteoses and Peptones in Gastric Digestion. Journal of Physiol., vol. xii, p. 12.

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.

[140] Chittenden and Hartwell, loc. cit., p. 22.