Part 5

Further, we are to remember that boiling dilute acid and superheated water tend to produce a cleavage along specific lines; viz., a cleavage into the anti- and hemi-groups of the molecule, and as representatives of these groups we may, in the hydration of every native proteid, look for two distinct rows of closely related substances.

In digestive proteolysis it will be our purpose to show that cleavage of much the same order occurs, not necessarily resulting, however, in the formation of identically the same products, but certainly accompanied with the production of bodies belonging to the hemi- and anti-groups, although they may be less sharply separated from each other than in the cleavage with dilute sulphuric acid.

The body originally described as hemialbumose, and identified as a product of every gastric digestion, is now known to be a mixture of closely related substances ordinarily spoken of as albumoses,[92] or generically as proteoses. These are primary products in the digestion of every form of proteid matter, intermediate between the mother-proteid and the peptone which results from the further action of the proteolytic enzymes. Associated with the hemialbumoses are corresponding antialbumoses, coming from the anti-half of the proteid molecule, and differing from their neighbors, the hemi-bodies, mainly in their behavior toward the ferment trypsin. Thus, we have the counterpart of the many bodies described by Meissner, although now arranged systematically and on the basis of structural and other differences not thought of in his day.

[92] Kühne und Chittenden: Ueber Albumosen, Zeitschr. f. Biol., Band 20, p. 11.

By the initial action of pepsin-acid, proteids are first transformed into acid-albumin or syntonin, then, by the further action of the ferment, this body is changed into the primary proteoses, proto and heteroproteose, of each of which there must be two varieties, a hemi and an anti. These may then undergo further transformation into what is known as a secondary proteose, viz., deuteroproteose, of which there must likewise be two varieties, corresponding to the hemi- and anti-groups respectively. By continued proteolytic action there results as the final product of gastric digestion peptones; approximately, an equal mixture of so-called hemipeptone and antipeptone, generally known as amphopeptone. Such a peptone exposed to the proteolytic action of trypsin should obviously break down in part into simple crystalline bodies, leaving a residue of true antipeptone. In truth, this is exactly what does happen when the peptone resulting from gastric digestion is warmed with an alkaline solution of trypsin. The so-called hemipeptone quickly responds to the action of the pancreatic ferment, and is converted into other products, while the so-called antipeptone resists its action completely, thus giving results in harmony with our general conception of the proteid molecule.

ALBUMIN MOLECULE.

(_Hemi-groups._ _Anti-groups._) ║ ╲ ╱ ║ ║ ║ ╲ ╱ ║ ║ ║ ╳ ║ ║ ║ ╱ ╲ ║ ║ ║ ╱ ╲ ║ ║ Protoalbumose Heteroalbumose Antialbumid (amphoalbumose) (amphoalbumose) ║ ║ | | ║ ║ ║ | | ║ ║ Deuteroalbumose Deuteroalbumose Deuteroalbumose (amphoalbumose) (amphoalbumose) (antialbumose) ║ | | ║ ║ ║ | | ║ ║ Amphopeptone Amphopeptone Antipeptone

On the basis of these facts, and others not yet mentioned, we may accept provisionally, at least, the above schematic view, suggested in part by Neumeister,[93] of the general line of proteolysis as it occurs in pepsin-digestion; a view which clearly expresses the significant relationship of the hemi- and anti-groups in the proteid molecule.

[93] Zur Kentniss der Albumosen, Zeitschr. f. Biol., Band 23, p. 391.

The dark and light lines in this scheme are intended to represent the relative share which the hemi- and anti-groups take in the formation of the individual bodies. Thus, we see that protoproteoses have their origin mainly in the hemi-groups of the molecule, although, as the fine line indicates, anti-groups are somewhat concerned in their construction. Heteroproteoses, on the other hand, come mainly from the anti-groups, but still some hemi-groups have a part in their structure. As previously stated, these two primary proteoses by further hydrolytic action may be transformed into secondary products; viz., into deuteroproteoses, but, as the above scheme indicates, the two deutero bodies will be more or less unlike in their inner nature. In one sense, they are both amphodeuteroproteoses, but they necessarily differ in the proportion of hemi- and anti-groups they contain. By the still further action of pepsin-acid, the deutero bodies may be changed, in part at least, into peptone, _i. e._, into amphopeptone, although, as Neumeister has pointed out, protoproteose tends to yield an amphopeptone in which the hemi-groups predominate, while the peptone coming from heteroproteose contains an excess of anti-groups. Moreover, in the gastric digestion of any simple proteid a certain number of anti-groups are split off in the form of antialbumid, a body which is only slowly digestible in pepsin-acid. By the very powerful proteolytic action of a strong gastric juice, however, antialbumid may be somewhat digested, and is then transformed into antideuteroalbumose, which in turn may be eventually changed into antipeptone.

From these statements it is evident that a given proteid exposed to pepsin-proteolysis may give rise to a large number of products; in fact, to a far larger number than is implied by the names in the above scheme. Thus, at first glance you would be inclined to say there can be only three deuteroalbumoses, for example; one, a pure antibody, the other two, amphoalbumoses, differing from each other simply in their content of hemi- and anti-groups. It must be remembered, however, that the inner constitution of these bodies, as implied by the relative proportion of the above groups, may vary to almost any extent. Thus, every variation in the number of anti-groups split off from the original albumin molecule to form antialbumid means just so much of a change in the relative proportion of hemi- and anti-groups entering into the structure of both primary and secondary albumoses. Hence, as you can see, digestive proteolysis, even in gastric digestion, is a somewhat complex process. We have to deal not only with a number of bodies superficially unlike, as the primary and secondary proteoses and peptones, but these bodies may show marked variations in structure dependent upon the exact conditions attending their formation.

Evidently, the complexities attending digestive proteolysis are connected primarily with the complex nature of the proteids themselves, while proteolysis, as a process, is made possible through the natural tendency of the proteids to undergo hydration and cleavage.

LECTURE II.

PROTEOLYSIS BY PEPSIN-HYDROCHLORIC ACID, WITH A CONSIDERATION OF THE GENERAL NATURE OF PROTEOSES AND PEPTONES.

PROTEOLYSIS BY PEPSIN-ACID.

Gastric digestion is essentially an acid digestion. As a proteolytic agent, pepsin can act only in the presence of acid, and we have every reason for believing that the enzyme and the acid form a compound, which in turn combines with the proteid undergoing digestion; or, what amounts to much the same thing, that the acid perhaps forms first a compound with the proteid, to which the pepsin can then unite to form a still more complex compound capable of undergoing hydration and cleavage. Pepsin-proteolysis, therefore, is strictly the proteolysis produced by pepsin-acid. In view of this fact, we may well give a moment’s thought to the nature and origin of this acid.

Without attempting any statement of the gradual development of our knowledge regarding the acid of the gastric juice, we may accept the now well-established fact that the acid is hydrochloric acid, and that it has its origin in the parietal, or so-called border-cells of the gastric glands. That the acid is derived from the decomposition of chlorides is practically self-evident, but Cahn[94] has added experimental proof which removes all shadow of doubt, through his study of the gastric secretion in animals deprived for many days of salt; the gastric juice in such cases being perfectly neutral in reaction, but normal as regards its content of pepsin.

[94] Die Magenverdauung im chlorhunger. Zeitschr. f. physiol. Chem., Band 10, p. 522.

The way in which the specific gland-cells manufacture free hydrochloric acid out of material contained in an alkaline medium is somewhat doubtful. There are, however, at the present day two theories worthy of special notice. The first is based upon observations made by Maly[95] many years ago, which tend to show that certain mineral salts present in the blood are capable of reacting upon each other with formation of hydrochloric acid. Thus, while the blood is an alkaline fluid, it really owes its alkalinity to the presence of two acid salts, viz., sodium bicarbonate (HNaCO_{3}) and disodium hydrogen phosphate (HNa_{2}PO_{4}). This latter compound, acted upon by the carbonic acid of the blood, is transformed into a dihydrogen sodium phosphate with simultaneous formation of acid sodium carbonate, as shown in the following equation:

Na_{2}HPO_{4} + CO_{2} + H_{2}O = NaH_{2}PO_{4} + HNaCO_{3}.

[95] Untersuchungen über die Quelle der Magensaftsäure. Annalen d. Chem. u. Pharm., Band 173, p. 227.

This acid sodium phosphate dissolved in a fluid containing sodium chloride, gives rise to free hydrochloric acid by a very simple reaction:

NaH_{2}PO_{4} + NaCl = Na_{2}HPO_{4} + HCl.

It is also to be noted that the disodium hydrogen phosphate, may, likewise, give rise to hydrochloric acid through its action on calcium chloride, as indicated by the following equation:

2Na_{2}HPO_{4} + 3CaCl_{2} = Ca_{3}(PO_{4})_{2} + 4NaCl + 2HCl.

It is thus evident that hydrochloric acid may originate in the inter-reaction of these several salts which are known to be present in the blood; but obviously, the above reactions cannot take place in the blood itself, and we must look to the selective power of the epithelial cells of the gastric glands, as suggested by Gamgee,[96] for the withdrawal of the needed salts from the blood. Once present in the acid-forming cells, and perhaps aided by the inherent qualities of the protoplasm, the necessary chemical reactions may be assumed to take place, after which the newly formed acid may pass from the gland-cells into the secretion of the gland.

[96] Physiological Chemistry of the Animal Body, vol. 2, p. 113.

A later theory regarding the formation of the acid of the gastric juice emanates from Liebermann.[97] This investigator claims the existence in the mucous membrane of the stomach of an acid-reacting, nuclein-like body, which is apparently a combination of the phosphorized substance lecithin with a proteid. To this compound body Liebermann gives the name of lecithalbumin. It is apparently located in the nuclei of the gastric cells, is strongly acid in reaction, and, according to Liebermann, is an important agent in the production of the free hydrochloric acid of the gastric juice, although its action is somewhat indirect. According to this theory, the free acid is formed in the mucous membrane of the stomach from sodium chloride, through the dissociating action of the carbonic acid coming from normal oxidation. The thus-formed acid then diffuses in both directions, viz., through the lumen of the gland into the stomach-cavity, and in part in the opposite direction into the veins and lymphatics. It is the assumed function of the lecithalbumin to react with the alkaline sodium carbonate, produced simultaneously with the hydrochloric acid. This naturally gives rise to the liberation of carbonic acid and to the formation of a non-diffusible sodium-lecithalbumin compound, which is retained for the time being in the body of the cell. When the circulation of the blood, accelerated by the digestive process, returns to its ordinary pace, this latter compound is slowly decomposed by the carbonic acid with formation of the readily diffusible sodium carbonate, which passes into the blood-current. The rate of this latter reaction is impeded, or, perhaps regulated, by the swelling up of the lecithalbumin-containing cells, thus rendering the imbibition of the carbonic acid a slow process. The rate of production of the hydrochloric acid by this hypothetical process depends primarily upon the blood supply, and the oxidative changes by which carbonic acid is formed.

[97] Studien über chemische Processe in der Magenschleimhaut. Pflüger’s Archiv f. Physiol., Band 50, p. 25. Neue Untersuchungen über das Lecithalbumin. Liebermann, Ibid., Band 54, p. 573.

There is much that might be said for and against this theory,[98] but we cannot stop to discuss it here. Like the previous theory, it implies the production of hydrochloric acid from a chloride or chlorides, through chemical processes taking place in the stomach-mucosa, and presumably in the large border-cells of the peptic glands. This hydrochloric acid, as you know, in the act of secretion, reacts upon the pepsinogen with which it may come in contact, transforming it into pepsin. It also has the power of combining with all forms of proteid matter, not excepting the products of proteolytic action, to form acid compounds in which the so-combined acid, although equal quantitatively to the original amount of free acid, is less active in many ways. Thus, it does not possess in the same degree a destructive action on the amylolytic ferments;[99] it does not play the same part in aiding the proteolytic action of pepsin, and its antiseptic power is far from equal to that of a like amount of free acid.[100]

[98] See discussion by Plósz and Liebermann in Jahresbericht für Thierchemie, Band 22, p. 260.

[99] Chittenden and H. E. Smith: Studies in Physiol. Chem., Yale Univer., vol. i., p. 18.

[100] Compare F. O. Cohn: Ueber die Einwirkung des künstlichen Magensaftes auf Essigsäure-und Milchsäuregährung. Zeitschr. f. physiol. Chem., Band 14, p. 74.

With relatively large amounts of proteid, we may have half or even quarter saturated proteid molecules, in which the weakness of the combined acid is far more pronounced than in the case of the fully saturated molecule. Such a condition of things must obviously exist in the early stages of gastric digestion. With an excess of proteid matter in the stomach, some time must elapse before the secretion of hydrochloric acid will be sufficient to furnish acid for all of the proteid matter present, yet pepsin-proteolysis does not wait the appearance of free acid. Indeed, the proteid matter may not have combined with more than half its complement of hydrochloric acid before digestive proteolysis is well under way. I have made many analyses of the stomach-contents after test meals, and under other conditions, where no free acid could be detected by the tropaeolin test, or better, by Günzburg’s reagent (phloroglucin-vanillin), although phenolphthalein as well as litmus showed strong acid reaction, and yet not only could acid-albumin be detected in the filtered fluid, but likewise proteoses and peptones. In other words, pepsin-proteolysis can proceed in the absence of free hydrochloric acid, although not at the same pace. Hence, proteoses and even peptones may make their appearance in the stomach-contents at a very early period of digestion, _i. e._, the final products of proteolysis may be found in a mixture containing even a large proportion of wholly unaltered proteid, and obviously at an early stage in the process. Expressed in other language, a portion of the first formed acid-albumin or syntonin may be carried forward by the digestive process to the secondary proteose and peptone stage, before the larger portion of the ingested proteid food has even combined with sufficient acid to insure the complete formation of acid-albumin. This introduces another factor, to be referred to later on, viz., the relative combining power of different forms of proteid matter, especially the proteoses and peptones, as contrasted with native proteids.

In proof of the statement that pepsin-proteolysis can proceed in the absence of free hydrochloric acid, provided combined acid be present, allow me to cite one or two experiments bearing on this point. A perfectly neutral solution of egg-albumen, containing 0.8169 gramme of ash-free albumin per 10 c.c. of fluid, was employed as the proteid material. In order to completely saturate the proteid contained in 20 c.c. of this neutral albumen solution, 50 c.c. of 0.2 per cent. HCl were required. Two mixtures were then prepared as follows:

_A._ Twenty c.c. of the neutral albumen solution + 50 c.c. 0.2 per cent. HCl + 30 c.c. of a weak aqueous solution of pepsin, perfectly neutral to litmus. This mixture gave only the faintest tinge of a reaction for free acid when tested by Günzburg’s reagent.

_B._ Twenty c.c. of the neutral albumen solution + 25 c.c. 0.2 per cent. HCl + 30 c.c. of the neutral pepsin solution. In this mixture, the proteid matter was obviously only half saturated with acid.

The two solutions were placed in a bath at 40° C., where they were allowed to remain for forty-four hours, a little thymol being added to guard against any possible putrefactive changes. At the end of this time the amount of undigested albumin was accurately determined. The 20 c.c. of original albumen solution contained 1.6338 grammes of dry coagulable albumin. At the end of the forty-four hours, _A_ contained only 0.5430 gramme of unaltered albumin, or acid-albumin, while _B_ contained 1.2225 grammes. That is to say, in the mixture _A_, where the acid existed wholly in the form of combined acid, but with the albumin completely saturated, 1.0908 grammes of the proteid were converted into soluble albumoses and peptones. In _B_, on the other hand, where the albumin was only half saturated with acid, 0.4113 gramme of the proteid was converted into soluble products. This difference in action is made more striking by the statement that where the proteid was only half saturated with acid, 25.1 per cent. of the albumin was digested; while with a complete saturation of the proteid, 66.7 per cent. of the albumin was digested.

To give emphasis to this matter, a second experiment may be quoted as follows: The proteid used was the same neutral solution of egg-albumen containing 0.8169 gramme of albumin per 10 c.c. Two mixtures were prepared as follows:

_A._ Ten c.c. of the neutral albumen solution + 21.7 c.c. 0.2 per cent. HCl, the amount needed to completely saturate the proteid, + 40 c.c. of a weak solution of pepsin, perfectly neutral.

_B._ Ten c.c. of the albumen solution + 10.9 c.c. 0.2 per cent. HCl + 40 c.c. of the pepsin solution, making a mixture half saturated with acid.

These two solutions were warmed at 40° C. for seventeen hours. The extent of digestive action was then determined, when it was found that in _A_ only 0.1638 gramme of the proteid was undigested, while in _B_, 0.6088 gramme remained unaltered. In other words, where the proteid was completely saturated with acid, but with an utter lack of free acid, 79.9 per cent. of the albumin was converted into albumoses and peptone, while in the mixture half saturated with acid only 25.4 per cent. was digested.

These two experiments thus give striking proof that free acid is not absolutely essential for pepsin-proteolysis. Digestion is, to be sure, more rapid and complete when free hydrochloric acid is present, but proteolysis is still possible, and even vigorous, when there is a marked deficiency of free acid. Further, as we have seen, proteolysis may proceed to a certain extent even though the amount of acid available is not sufficient to combine with more than half the proteid matter present.

These facts at once raise the question whether the products of proteolysis may not have a stronger affinity for acid than the native proteids; an affinity so strong that they may be able to withdraw acid from the acid-albumin first formed. One of our conceptions regarding pepsin-proteolysis is that acid is necessary for every step in the proteolytic process. A primary albumose, for example, cannot be further changed by pepsin, unless there is acid present for it to combine with. This being true, it is clear, in view of the fact that even peptones may appear in a digestive mixture containing an amount of acid insufficient to combine even with the albumin present, that the products of proteolysis must withdraw acid from the acid-albumin first formed. In regard to the first point, my own experiments certainly tend to show that the products of gastric digestion do combine with larger amounts of hydrochloric acid than undigested proteids; and further, that of the several products of proteolysis, the secondary proteoses combine with a larger percentage of acid than the primary proteoses, while true peptones combine with still larger amounts. In other words, the simpler and more soluble the proteid, the larger the amount of acid it is capable of combining with; a statement which accords with results obtained by other workers[101] in this direction. Further, another factor of considerable importance in connection with the natural digestive process is that a dissolved proteid, such as protoalbumose for example, will combine more readily with free acid than an insoluble proteid; from which Gillespie[102] is led to infer that in pepsin-proteolysis where there is no free acid present, only acid-albumin, proteoses may be formed to a limited extent at the expense of some of the acid of the acid-albumin, a portion of the latter being perhaps reconverted into albumin. The ability of the proteoses, however, to withdraw acid from its combination with a native proteid is perhaps best indicated by Kossler’s[103] experiments, which show that a solution of acid-albumin containing only enough hydrochloric acid to hold the albumin dissolved, on being warmed at 40° C. for some hours with addition of a neutral solution of pepsin, may undergo partial conversion into albumose or peptone.

[101] See especially Gillespie: Gastric Digestion of Proteids. Journal of Anat. and Physiol., vol. 27, p. 207.

[102] Loc. cit.

[103] Beiträge zur Methodik der quantitativen Salzsäurebestimmung im Mageninhalt. Zeitschr. f. physiol. Chem., Band 17, p. 93.

In spite of these facts, there is some evidence that while proteoses and peptones have the power of combining with more acid than a like weight of native proteid, the latter, leaving out all action of the pepsin, has a stronger affinity for the acid; in fact, the firmness or strength of the union appears to diminish as the products become simpler.[104] Hence, a peptone separated from a digestive mixture, will part with its combined acid somewhat more readily than acid-albumin for example, although on this point there is not complete unanimity of opinion.[105] In digestive proteolysis, however, where the pepsin is accompanied by a minimal amount of hydrochloric acid, insufficient perhaps to even half saturate the proteid present, the formation of proteoses and peptones must be accompanied by a withdrawal of acid from its combination with the native proteid.

[104] Compare Blum: Ueber die Salzsäurebindung bei künstlicher Verdauung. Zeitschr. f. klin. Medicin, Band 21, p. 558.