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«Downloaded from on December 26, 2006 On the one hand, Kiibne (1) and Neumeister (2) concluded that the products of digestion arise by a ...»

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THE STAGES OF THE PEPTIC HYDROLYSIS OF EGG

ALBUMIN.

By JENNIE McFARLANE, VIOLET E. DUNBAR, HENRY BORSOOK, Am)

B.ARDOLPH WASTENEYS.

(From the Department of Biochemistry, University of Toronto, Toronto, Canada.)

(Accepted for publication, October 18, 1926.)

Numerous investigators have studied the stages in peptic digestion. The conclusions at which they have arrived may be grouped

under two main heads.

Downloaded from www.jgp.org on December 26, 2006 On the one hand, Kiibne (1) and Neumeister (2) concluded that the products of digestion arise by a serial degradation from the protein through acid albumin to proteose to peptone. On the other hand, Lawrow (3), Klug (4), Pick (5), Zunz (6), and Goldschmidt (7) found that the products of digestion appear more or less simultaneously.

The most comprehensive study of the stages of peptic digestion was made by Zunz. His general conclusion was that metaprotein, proteoses (primary and secondary), peptones, and simpler products, arise simultaneously and by inference directly from the protein molecule. The metaprotein and a large part of the primary proteose, he found to be converted into peptones. He also described large amounts of substances (51 per cent of the total nitrogen) simpler than peptones which appeared in the early hours of digestion and later decreased.

Klug concluded that the acid metaprotein formation in the early stages of digestion was due to the action of pepsin, while Maly and Boos (8) believed that it might be due to the action of acid alone.

A reexamination of the problem seemed desirable. The methods used were chiefly those described by Wasteneys and Borsook (9) for the fractional analysis of incomplete protein hydrolysates.

Metaprotein (acid albumin) was estimated by determination of the nitrogen content of the precipitate obtained on careful adjustment of the reaction to pH 6.0.

The proteose was precipitated by saturation of the solution with The Journal of General Physiology

438 STAGES OF PEPTIC HYDROLYSIS

sodium sulfate, at 33°C., and the peptone by precipitation with tannic acid under specially controlled conditions. The tannic acid filtrate was precipitated with alcohol and zinc sulfate. The nitrogen content of this precipitate was taken as subpeptone nitrogen, and the remainder as residual nitrogen, contained, it is assumed, in the simplest constituents of the hydrolysate.

No attempt was made to subdivide proteoses into primary and secondary classes except in one phase of the investigation which will be discussed later.

The results obtained are in accordance with those of Zunz and Pick that most of the products found in a peptic hydrolysate arise directly from the protein molecule. They differ in regard to the significance of acid metaprotein, and also in regard to the amount of, and variations in, the fraction simpler than peptone.

Downloaded from www.jgp.org on December 26, 2006 It was found that acid metaprotein is hydrolyzed more slowly than the native albumin from which it is derived; that the peptic hydrolysis of albumin can be effected at acidities less than pH 4.0, where no formation of metaprotein can be demonstrated; and that the hydrogen ion concentration for the minimum hydrolysis of undenatured albumin is distinctly different from that of acid metaprotein. For the former it is in the neighborhood of pH 6.0; for the latter at pH 4.0.

From these results it was concluded that acid metaprotein formation, while a product of the action on protein of acid alone, as is well known, is not a necessary stage in the peptic hydrolysis of albumin, and its influence, if any, on hydrolysis, is to retard it.

Two-thirds of the initial amount of protein is hydrolyzed in 4 hours. The reaction then becomes progressively slower, so that in 12 hours I0 per cent, and in 7 days 6 per cent of the protein remains still unhydrolyzed. This rate of hydrolysis, however, holds only under the conditions of this experiment, but the rapid initial decomposition is characteristic of peptic hydrolysis under any conditions.

The results indicate that the peptic hydrolysis of albumin progresses in two stages. The first stage, occurring in the first 12 hours, consists of the hydrolysis of practically 100 per cent of the protein, with the formation of products of which 85 per cent may be spoken of as primary, i.e. undergo no further hydrolysis. The products at MC~'AP.LAIWE, DUNBAR, BORSOOK, AND WASTENEYS this time consist of 55 per cent proteose, 17 per cent peptone, 12 per cent subpeptone, and 5 per cent residual nitrogen. A second stage, occurring later and progressing much more slowly, results in the hydrolysis of 15 per cent of the primary products into simpler fragments, which m a y be designated as secondary. T h e secondary hydrolysis occurs in b o t h the proteose and subproteose fractions.

No methods are available for following subsequent changes in the subproteose fraction. In any case they affect the main picture of peptic hydrolysis v e r y little, as only a v e r y small fraction, about 6 per cent at most, of the total N is involved.

–  –  –

Metaprotein Formation and Its Relation to Hydrolysis.

Downloaded from www.jgp.org on December 26, 2006 Egg albumin (Merck) and pepsin (Merck) have been employed throughout.





The metaprotein was prepared by allowing a solution of albumin at pH 1.6 to stand at room temperature for several days, thymol being employed as antiseptic.

The rate of formation of metaprotein is shown in Table I. After several weeks it attains a value of about 80 per cent of the total N, leaving approximately 15 per cent of the nitrogen still in the form of albumin soluble at neutrality.

A solution of metaprotein, on the other hand, prepared by solution at pH 1.6 of the precipitate obtained at pH 6.0 remains unchanged after standing for days at room temperature.

To compare the relative rates of hydrolysis by pepsin of metaprotein, and of albumin which has not been denatured by acid, two simultaneous hydrolyses were followed. The first hydrolysis was of an albumin solution which had been standing at room temperature at pH 1.6 for several days. The second was of an albumin solution of equal nitrogen content, to which the enzyme was added simultaneously with an amount of acid necessary to bring the pH to 1.6. This experiment was performed with 2.0, 1.5, 1.0, 0.5, and 0.25 per cent stock pepsin solutions, lasting 45 minutes. Considering the lowest enzyme concentration as 1 unit the others were, respectively, 10, 8, 6, 4, and 2 units. The results are recorded in Fig. 1.

It is clear from Fig. 1 that in mixtures of undenatured albumin and acid rectaprotein, the velocity of peptic hydrolysis is greater where the concentration of acid metaprotein is less. Fig. 1 also shows the surprising result, that, at least in these experiments, strict proportionality between velocity of hydrolysis and concentration of pepsin obtains in those solutions where the conversion of undenatured protein to metaprotein has attained approximate equilibrium.

The slower rate of hydrolysis of acid metaprotein, whether the solution consists almost entirely of metaprotein, as in the previous experiment, or of a mixture of

440 S T A G E S OF P E P T I C HYDROLYSIS

metaprotein and undenatured albumin, is shown in an experiment the result of which is indicated in Fig. 2. In this experiment the relative rates of hydrolysis were compared in three solutions of identical nitrogen and enzyme content, one containing undenatured albumin, a second consisting of equal parts of undenatured albumin and acid metaprotein, and a third containing only metaprotein. As the curves show, peptic hydrolysis is most rapid in the undenatured albumin solution, and least rapid in the metaprotein solution, while the relative rate of hydrolysis of the mixture is intermediate.

Since acid metaprotein was hydrolyzed more slowly than undenatured albumin, it seemed probable that the formation of metaprotein indeed was not, as Maly, and Boos (8) maintained, a necessary stage in peptic hydrolysis. To test this, experiments were performed to ascertain the possibility of obtaining peptic hydrolysis of albumin at hydrogen ion concentrations where no metaprotein is formed.

TABLE I.

–  –  –

It was necessary first to define the limit of acidity beyond which metaprotein formation does not occur. Portions of a neutral solution of 3.2 per cent albumin were adjusted to various acidities with hydrochloric acid. These were set away in a water bath at 37.7°C. for 45 minutes. They were then assayed for metaprotein by precipitation at pH 6.0. The results are given in Table II. They show that no metaprotein formation occurs at acidities less than pH 4.0.

As a result of this experiment a number of hydrolyses were carried out at acidities slightly less than pH 4.0 with several concentrations of pepsin. Typical results are given in Table III.

–  –  –

changes occurring in the protein substrate, it is possible to detect the progress of hydrolysis at acidities less than pH 4.0. Michaelis (10) found that peptic hydrolysis of albumin ceases at pH 4.0.

The reason for the difference between our findings and those of Michaelis lies in the probability that the latter worker was dealing

–  –  –

with a solution consisting largely of metaprotein. The hydrogen ion concentration for the threshold of minimum hydrolysis of acid metaprotein is at pH 4.0; for egg albumin, which has not been denatured, it is in the neighborhood of pH 6.0. The optimum pH is approximately the same for both, near pH 1.6. This is shown in Fig. 3.

In this experiment 3.2 per cent solutions of albumin and of acid

442 STAGES OF PEPTIC IIYDROLYSIS

metaprotein were hydrolyzed for 1 hour at 37.7°C. with 0.2 per cent pepsin at the acidities indicated.

In the peptic hydrolysis of coagulated egg albumin observations were made, which, in contradistinction to the findings with uncoagulated albumin, do not conform to the view that acid metaprotein is not a necessary stage in the peptic hydrolysis of albumin. A 3.2

–  –  –

per cent solution of albumin, acidified with dilute acetic acid, was coagulated by boiling. The coagulate was filtered and washed thoroughly with distilled water. It was then suspended in dilute HC1, and the reaction of the suspension adjusted to pH 1.6. Pepsin was added to a concentration of 0.2 per cent, and the suspension was incubated at 37.7°C. for 3 hours. At the end of this time all of the coagulum had disappeared. Part of the digest was neutralized;

McFARLANE~ DUNBAR~ BORSOOK, AND WASTENEYS 443 another part was treated with trichloroacetic acid. In the neutralized solution a heavy precipitate appeared. The total nitrogen of the solution was 42 rag. The nitrogen in the filtrate from trichloroacetic acid precipitation was 30.6 rag. indicating that 11.4 mg. of protein nitrogen had not been hydrolyzed. The filtrate of the neu

–  –  –

tralized solution contained 31.9 mg. of nitrogen. The precipitate thrown out on neutralization contained, therefore, 10.1 rag. The nitrogen precipitated by trichloroacetic acid was 11.4 rag., by neutralization 10.1 rag. In view of the difference of the two methods the correspondence is sufficiently close to indicate that the same amount of material was precipitated by neutralization and by triSTAGES OF PEPTIC IIYDROLYSIS chloroacetic acid. Evidently there is a material produced in the early stages of the hydrolysis of coagulated egg albumin which is soluble in dilute acid, insoluble at the neutral point, and precipitated by trichloroacetic acid. This agrees with the definition of metaprotein. We are led to the conclusion, for the time being, that though metaprotein is not a stage in the peptic hydrolysis of soluble unde

–  –  –

natured albumin, it is probably the first stage in the hydrolysis of coagulated egg albumin.

The phenomena associated with heat coagulation of albumin have been fully discussed by Wu and Wu (11). This experiment, however, discloses an interesting anomaly. It is ordinarily supposed that in the coagulation of egg white by heat, albumin passes through the

MCFARLANE, DUNBAR, BORSOOK, AND WASTENEYS

stage of acid metaprotein. On suspending the coagulum in acid so that the reaction of the suspension is pH 1.6, contrary to the behaviour of tmcoagulated albumin, no metaprotein is formed. Immediately on adding pepsin, however, metaprotein appears. Either the change from metaprotein to coagulated egg white is a reversible phenomenon with pepsin as a necessary adjuvant for reversion, or else we are dealing with different kinds of metaprotein.

The Stages in the Peptic Hydrolysis of Albumin.

The method of analysis was that described by Wasteneys and Borsook (9) for the fractional analysis of incomplete protein hydrolysates. Two types of experiments were carried out. One was a preliminary survey, in which the pH of the digest was allowed to Downloaded from www.jgp.org on December 26, 2006 rise with the progress of the hydrolysis. In the other, the pH was kept approximately constant at 1.6 throughout the course of the experiment by the occasional addition of acid. The findings in both types of hydrolyses were essentially the same, but the latter experiment was more thoroughly controlled, and the results there obtained are taken as the basis for the present discussion.

6 litres of a 5 per cent solution of egg albumin (Merck) were adjusted to pH 1.6 and sufficient solution of scale pepsin (Merck), also adjusted to pH 1.6, was added to make the concentration of enzyme

0.2 per cent.



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