ENZYNIE-SUBSTRATE STABILIZATION WITH SURFACE- DENATURED PEPSIN-ALBUMIN

Teru Hayashi and George R. Ed{son Department of Zoology, Columbia University, New York, New York

Received July 3, 1950

INTRODUCTION

The earlier studies on the autolytic activity of pepsin-albumin films rolled into fibers (1,2) demonstrated that the phenomenon was an actual proteolytic activity, due to the presence of pepsin in the fiber (or film) itself. The pelbtic nature of the action was shown in the heat-sensitivity of the autodigestion phenomenon, the pi t dependence, and the formation of products soluble in trichloroacetic acid.

In the course of these earlier studies, it was recognized that there were two possible alternative roles of the pepsin molecules in the autodigestion process. These may be stated as follows:

a) The autodigestion of the rolled-up films was due to the action of pepsin molecules incorporated in the film itself. That is, the pepsin mole- cules responsible for the phenomenon had been actually "surface-dena- tured" in the sense that they had undergone certain physical changes at the air-water interface of the Langmuir trough, becoming part of the monomolecular film of pepsin and albumin molecules. These pepsin mole- cules, under certain rigid conditions of hydration and hydrogen-ion con- centration, had acted proteolytically on the albumin molecules.

b) The autodigestion was due to the action of pepsin molecules which had not actually undergone "surface-denaturation"; rather, undenatured, native pepsin molecules had become adsorbed on the film and become entrapped in the fibers. Thus, the autodigestion of the fibers was due tO the proteolytic activity of such native pepsin molecules present as con- taminants in the fiber.

The results of a number of previous experiments devised to test these alternatives adduced evidence in favor of the first possibility and against the second (2).

The present investigation was undertaken for the purpose of throwing additional light on the above question and also to begin the elucidation of the mechanism of the autodigestion. It may be stated that the results provide additional evidence in favor of the first possibility given above,

437

438 T E R U HAYASHI AND GEORGE R. EDISON

and indicate tha t the autodigestible pepsin-albumin film is a stabilized enzyme-subs t ra te complex.

M A T E R I A L S

The ovalbumin used was prepared from fresh hen's eggs according to the method of La Rosa (3). The albumin was thrice-recrystalized, then dialyzed overnight in several changes of distilled water, and finally frozen- dried. This lyophilized material was used in making up the experimental solutions, fresh, filtered solutions being used in each experiment.

The pepsin used was the crystalline Armour product, dialyzed over- night against distilled water refidered slightly acid with a drop of con- centra ted acetic acid, then frozen-dried. The frozen-dried pepsin was used in making fresh-filtered solutions for each experiment.

The ammonium sulfate and buffer solutions were all made of s tandard Merck products. Pyrex-d is t i l l ed water was used throughout the experiments.

M E T H O D S

The technique for making the pepsin-albumin film-fiber has been de- scribed in detail in a previous publication (2). Certain steps in the p ro- eedure are described below, since they bear on the present iovestigation.

1. Nine parts of a buffered albumin solution (5 mg./ml.) are mixed with 1 part of a buffered pepsin solution (5 mg./ml.). The pH of the resulting mixture is checked elec- trometrically ("mixture pH"). Either acetate buffer or phosphate-citrate buffer in the pH range 4.0-5.5 may be used without affecting the result; i.e., the fibers formed from this mixture are readily autodigestible. Both types of buffer are used in the present investigation.

2. Drops of the mixture of proteins are placed at the air-water interface of a Lung- muir trough in excess to form a monomolecular film: The trough is filled with 10 × di- luted McIlvaine's buffer (checked electrometrically) at pH 4.0 ("trough pH").

3. The film is compressed by the use of suitable barriers (Devaux effect) giving a fiber, in reality a rolled-up mouomolecular film of pepsin and albumin molecules. Auto- digestion takes place when such a fiber is treated with dilute HC1 at pH 1.5 ("digestion pH").

In the steps described above, it is to be noted tha t the "mixture p H " is tha t of the solution in which nat ive pepsin and albumin molecules are mixed prior to their application to the a i r -water interface and the for- mat ion of the monomolecular film. The opt imum " t rough p H " for the formation of autodigestible complex fibers had been found experimentally to be 4.0-4.2, while the opt imum "digestion p H " for digestion had been established sharply at 1.5 (2).

Observations of the autodigestion were made under the low-power mag- nification of the microscope to note only whether autodigestion occurred or not. In some cases, the t ime required for complete digestion was measured.

: To precipitate the proteins, t i t ra t ion with a saturated, filtered solution of ammonium sulfate was employed, A measure of the precipitate thus

STABLE PEPSIN--ALBUMIN COMPLEX 439

formed was obtained by determining the optical density of a well-stirred sample of the precipitate suspension in a Coleman Junior spectropho- tometer and also in a Klett-Summerson photoelectric colorimeter using monochromatic light of 420 mg. Instead of blanks in the determinations, the instruments were set for zero optical density (100% transmission) in the clear protein solution containing the same reagents but before the formation of any precipitate (before the addition of the last increments of ammonium sulfate beginning the onset of precipitation).

EXPERIMENTS AND RESULTS

From the foregoing descriptions, it may be assumed that, in the auto- digestible fibers, both pepsin and albumin molecules are present. Since autodigestion involves a proteolytie action, some sort of association be- tween the enzyme and substrate molecules would appear to be necessary. The question then may be asked whether this association is established at the time of mixture of the two proteins or at the time of application to the surface of the trough. The question was tested initially by making films and fibers incorporating both pepsin and albumin molecules but in such a way that mole-to-mole association of the enzyme and substrate molecules was prevented. This was done in two ways.

In the first method, paraffined threads were placed transversely across the surface of the trough dividing the trough surface into sectors in such a way that any protein film formed in any sector was effectively contained therein. Albumin and pepsin solutions were placed on the surface in alter- nate sectors to form r.espective films separated by the threads. With certain variations in procedure, the threads were then removed and the film com- pressed to form a pepsin-albumin fiber which was then tested for auto- digestion. The procedural variations, after spreading the films and waiting 20 see., were (a) remove threads and compress immediately; (b) remove threads, wait 5 rain., then compress; (c) remove threads, wait 20 rain., then compress; and (d) remove threads, compress immediately to 50% of the original area, wait 5 min., then compress.

In the second method, no separating threads were used. Instead, albu- min and pepsin solutions were placed dropwise separately on the trough at varying distances apart, the films thus formed being compressed into fibers and tested for autodigestion.

In the foregoing experiments, i t is necessary t ha t each film and fiber tested be made on a clean t rough containing fresh buffer solution. Anomalous results will be obtained if the same t rough is used over and over for making the films and fibers. This is because in applying the drops of protein solution to the surface some of the protein molecules go into solution in the bulk of the trough. Such molecules in solution will make their way to the surface and become incorporated into films spread subsequently at the a i r -water interface.

440 T E R U t tAYASFI I AND G E O R G E R. E D I S O N

From the results (see Table I) it was apparent tha t a necessary condi- tion for making autodigestible fibers was the mixing of the enzyme and substrate prior to spreading on the t rough; tha t is, an interaction between the pepsin and albumin occurred in the mixture. Attent ion was therefore directed to the effect of the mixture pH on autodigestion.

Protein mixtures of 9 parts of albumin to 1 part of pepsin in McIlvaine 's buffer with final protein concentrations of 5 mg./ml. , and final mixture p I I ' s of 4.4, 5.0, 6.0, 6.6, and 7.2 were made. Fibers were formed from each mixture, and tested in the usual fashion. I t was found that, at pH 7.2, the fibers were not autodigestible, whereas all the other mixtures formed auto- digestible fibers.

The results of the foregoing experiments are summarized in Table I. Since there seemed to be a definite difference in the behavior of pepsin.

and albumin molecules when mixed at pH 7.2 as contrasted to tha t when mixed at the lower pH's, some clue as to the nature of this behavior was

TABLE I

Observations on A utodigestion

Conditions for the formation of autodigestible (-t-)fibers as contrasted to non-auto- digestible ( - ) fibers. Solutions for Expts. 1 and 2: pepsin 2 mg./ml.; albumin 10 mg./ml. For Expt. 3: final protein concentration of mixture 5 mg./ml.

Conditions

Auto- Expt. pH digestion

Alb.

5.3~ 5.3~ 5.3~ 5.3~

5.3~

5.3~ 5.3~ 5.3~ 5.3~ 5.3~

4.4 4.4 4.4 4.4 4.4

Pep. Mixt . Trough

5.3~ 4.0 5.3~ / 4.0 5.3~ 4.0 5.3~ 4.0

5.3~ 5.35 4.0

Fiber formation

Dig.

1.5 Alb. and pep. films 1.5 separated on trough 1.5 by threads, threads 1.5 removed before

compression. 1.5 Control Standard fibers, mixing pepsin and

albumin prior to spreading.

Variation (a) Variation (b) Variation (c) Variation (d)

5.3~ 5.N 5.3~ 5.3~ 5.3~

4.4 4.4 4.4 4.4 4.4

4.0 1.5 4.0 1.5 4.0 1.5

14.0 1.5 5.35 4.O 1.5

4.4 4.0 1.5 5.0 4.O 1.5 6.0 4:0 I1.5 6.6 4.O 1.5 7.2 4.0 1.5

Alb. and pep. films Placed 30 era. apart placed dropwise sep- Placed 10 era. apart arately on trough. Placed 2 cm. apart Film then compressed. Placed 0 cm. apart Control. Standard fiber.

Standard fiber with the Standard fiber with the Standard fiber with the Standard fiber with the Standard fiber with the

mixture pH varied. mixture pH varied. mixture pit varied. mixture pH varied. mixture pH varied.

m

2-

÷

÷ +

+ + + +

STABLE PEPSIN--ALBUMIN COMPLEX 441

sought by utilizing the differential susceptibility of the pepsin and albu- min molecules to precipitation by saturated ammonium sulfate.

Figure 1 shows the curves obtained for pepsin and for albumin when titrated separately with saturated ammonium sulfate at pH 4.4. It is to be noted that these two molecular species have a clear-cut difference in their susceptibility to precipitation by ammonium sulfate, the pepsin responding to less reagent. The curves are smooth, indicating a homo- geneity in the response of each protein to the precipitating action.

1.2,

.9 "2 o E O

O

e -

r~

"6 ._o

pH =4A

.4

I R 5mg/ml

Y P. 2.5 mg/rr

0 0 1.6

A.5mg/ml

I A.2.Smg/ml

g/ml

1.2

ml. added saturated (NH4)zS04

FIG. 1. Precipitation curves for various concentrations of pepsin (P) solutions, and albumin (.4) solutions, showing differential susceptibility to (NH4)2S04. Phosphate- citrate buffer st pH 4.4.

When the pepsin and the albumin are mixed in a 1:1 ratio and then titrated, however, this difference in susceptibility is not reflected, as shown in Fig. 2. Instead~ the two proteins are precipitated out together uniformly in a manner characteristic of the pepsin. I t may be concluded that, an in- teraction occurs between these molecules such that the pepsin and the albumin precipitate out together.

That the pepsin and the albumin may be precipitated independently out of the mixture can be shown readily by varying the mixture pH. Mixtures in a 1 : 1 proportion of the two proteins were made at final pH's of 4.4, 5.0, 6.0, 6.6, and 7.2. The lower mixture pH's gave precipitation curves similar to that observed in Fig. 2, but at the highest pH of

442 T E R U H A Y A S H I A N D G E O R G E R. E D I S O N

O

0 0

t--

E 3

C} , m

o

.9 pH= 4.4

A:P= I

.6

3

5

0 .4 .8 1.2 1.6 ml. added saturated (NH4)zS04

FzG. 2. Precipitation curves for various concentrations of albumin-pepsin mixture. AlP = 1, in phosphate-citrate buffer at pH 4.4.

7.2 the pepsin and the a lbumin precipi ta ted out separately. Figure 3 shows the curve for a 1 : 1 mixture of pepsin and a lbumin at p H 7.2, along with the curves for pepsin alone and a lbumin alone at the same final pH. The shape of the curves shows clearly the separat ion of the pepsin and albumin.

The results give evidence for the belief tha t the pepsin and a lbumin in m i x t u r e b e t w e e n p H 4.4-6.6 form an enzyme-subs t r a t e complex. This

30 pH= 7.2 A'P= I

" I

X

G

a I0

°

0 2 4 6 8 ml. added saturated (NH4)2S04

FzG. 3. Precipitation curves for pepsin (P), albumin (A) and albumin-pepsin (A/P -- !) mixture at pH 7.2. Protein concentration = 2.5 mg./mi, in phosphate-citrate buffer.

STABLE PEPSIN--ALBUMIN COMPLEX 4:43

0

E 0 ~o ¢/)

E3 N 0

o

.8 pH=4.4

P

.6

.4

2.

0 ._~_= / 0 0.4 0.8 I:?_ 1.6

ml. added saturated (NH4)zS04

FIG. 4. Precipitation curves for pepsin (P), albumin (A) and mixture solutions at various albumin-pepsin ratios. Protein concentration = 2.5 mg./ml., buffered in phos- phate-citrate at pit 4.4.

belief was further substantiated when mixtures with varying proportions of albumin and pepsin were titrated with saturated ammonium sulfate. As Fig. 4 shows, at the higher albumin to pepsin ratios, the albumin-pepsin complex precipitated out initially, to be followed by the subsequent pre- cipitation of the excess albumin.

DISCUSSION

The first question to be considered in the light of the experimental results is whether an enzyme protein retains its specific activity after it has been subieeted to the physical changes concomitant to the forma- tion of a monomolecular film. The published information pertaining to this question does not give an unqualified answer because of technical considerations.

Thus, the question has been approached by using films deposited on slides. Using this technique various workers (4-7) have studied the enzymes catalase, saccharase, urease, pepsin (milk-clotting), and trypsin. In all of these with the exception of trypsin (7) positive activity of greater or less degree was reported. However, the point that could not be resolved was: I s the activity due to the spread protein film, or is it due to (a) adsorbed, unspread protein; (b) "incompletely spread" protein; or (c) adsorbed and

4 4 4 TERU HAYASt t I AND GEORGE R. EDISON

subsequently desorbed protein? Because of this consideration, Rothen, in his review (7), points out that the belief in enzymatic activity of spread fihns is not substantiated.

The possibilities (a), (b), and (c) have been considered in some detail earlier (2). In addition, it may be pointed out that whether a protein molecule is Completely spread or not, the fact that protein molecules in clear solution are transformed into a tangible, insoluble fiber is indicative of marked physical change in the protein molecules. Whether these mole- cules can be more completely spread is not the question; the point is that they are in a physically different state from the same molecules in solution.

The albumin-pepsin system of the present investigation differs from the work of other investigators in that the enzyme and substrate are both incorporated in the film. The results show that the pepsin and albumin molecules must be associated in a specific manner prior to (and probably during) the physical changes taking place at the air-water interface in order that the autodigestive capacity be maintained. The enzyme and substrate molecules, when mixed and then formed into a film, form auto- digestible fibers. The molecules, if placed on the same interface separately but incorporated into the same film and fiber, do not form autodigestible fibers. The physical conditions for the adsorption of native pepsin mole- cules is the same in the two instances, yet with completely different results. The conclusion can be drawn that autodigestion is due to surface-spread pepsin molecules. This conclusion is supported by the work of Kaplan (8) who finds that surface-denatured catalase also retains its activity.

Parenthetically, it may be added here that autodigestible fibers may be formed on a trough filled with saturated ammonium sulfate. 1 In view of the work of Seastone (9) showing a minimal amount of protein going into solution in the underlying bulk of the trough under these conditions, it is difficult to see how adsorbed native pepsin can be responsible for the autodigestion.

As a tentative hypothesis, we may say that the establishment of inter- molecular linkages between the pepsin and albumin molecules while in solution mixture is a necessary part of the formation of autodigestible fibers. The results given in Table I indicate then that such linkages are formed when the two proteins are mixed at pH 4.4, 5.0, 6.0, and 6.6. How- ever, when mixed ~t pH 7.2, the necessary interaction of the pepsin and albumin does not occur, resulting in fibers that do not undergo autodi- gestion. I t is probable that the pepsin-albumin complex formed in mixture is similar in nature to the pepsin-edestin complex demonstrated by North- rop (10). However, Northrop's work did not involve the subsequent sur- face-denaturation of the protein complex.

Data obtained in the precipitation of the proteins with saturated

1 Unpublished experiments.

STABLE PEPSIN--ALBUMIN COMPLEX 4 4 5

ammonium sulfate support the hypothesis. The results show that at the same mixture pH range of 4,4-6.6 for the formation of autodigestible fibers, pepsin and albumin precipitate out simultaneously, whereas inde- pendently they have completely different solubilities to the precipitating action of ammonium sulfate. At mixture pll 7.2, where no autodigestible fibers are formed, the two proteins show a lack of interaction by precip- itating out separately. The notion, therefore, that within the pH range of 4.4-6.6, there is an intermolecular association of the pepsin and the albu- min in mixture seems to be substantiated.

The autodigestible fibers formed from the proper mixtures would then be the enzyme-substrate complex in the sense of Michaelis and Menten (II), but in a stabilized form. When this complex is placed in a medium of the proper hydrogen-ion concentration, it dissociates with the formation of digestion products of proteolytie activity.

The above scheme brings out the fact that the autodigestion of com- plex fibers is not an enzymatic activity of the pepsin as it is ordinarily considered. We may write the reaction in the standard form

E + S ~ ES, [11

ES -~ E ~- P, [21

where E and S represent enzyme and substrate, respectively, ES the com- plex, and P the products. In the mixture of pepsin and albumin at the pH range considered, reaction Eli predominates, while in the autodigestion of the complex fibers, reaction r2~ occurs. Since the ordinary concept of enzyme action involves both EI~ and r2~, the autodigestion phenomenon cannot be considered as an enzymatic activity of the pepsin, but only the degradation of the enzyme-substrate complex. Thus, the autodigestible fiber becomes, according to this-scheme, a stabilized complex.

A puzzling situation arises when the substrate to enzyme ratios are examined. As reported previously (2), there is an apparent complete diges- tion of the fibers at a substrate to enzyme ratio as high as 18: 1. Yet, in the precipitation curves of Fig. 4 there is a sizable excess of uncombined substrate even at ratios as low as 3 : 1. It may be that the association of the enzyme and substrate molecules while in mixture is maintained but radi- cally altered at the time of transformation from molecules in solution to molecules in a monomolecular film. On the other hand, the paradox may be only apparent, to be resolved by improved methods of analysis. Work is planned along these possibilities.

The nature of the bonds involved in the formation of the complex is as yet a matter of speculation. The dependence on the hydrogen-ion con- centration for the establishment of the complex indicates that the complex binding is brought about by the opposite signs of electrical charges on the

446 TERU HAYASHI .%ND GEORGE R. :EDISON

respective molecules. From the data of Tiselius (12) pepsin would have a net negative charge, whereas albumin, which has an isoelectric point of about 4.8 (13), would have a net positive charge at pH's below 4;8 and a net negative charge above this. This, of course, would fit in with Michaelis' proposal (14) for the mechanism of pepsin action. However, according to this, pepsin and albumin molecules would exhibit no attraction for each other at pH 5.0, 6.0, and 6.6; yet the data of the present investigations indicate that a complex forms in these pH ranges. Thus, if salt linkages are involved, some explanation, such as localization of charges on the molecular surface, would have to be invoked to explain complex formation at these higher pH's.

SUmmARY

1. Pepsin and albumin molecules will form autodigestibie fibers from a compressed monomolecular film if they are mixed in solution prior to application to an air-water interface.

2. When pepsin and albumin molecules in solution are placed separ- ately on an air-water interface, the fibers formed are not autodigestible.

3. Autodigestible fi[)ers are formed from pepsin-albumin mixtures in the pH range of 4.4-6.6. When the proteins are mixed at pH 7.2, the fibers formed are not autodigestible.

4. Curves obtained by precipitating these proteins with ammonium sulfate show that, although pepsin and albumin have distinctly different sensitivities to the action of this reagent, they precipitate out together when in mixture.

5. The two proteins precipitate out together in the Same pi t range (4.4-6.6) as found for autodigestible fibers. At pH 7.2 they come down out of solution separately, according to their different solubilities.

6. Mixtures containing albumin in excess show at ptI 4.4 two separate precipitates.

7. A tentative scheme is proposed to explain the data. According to this scheme, pepsin and albumin mixed at pH 4.4-6.6 combine to form an enzyme-substrate complex, which is stabilized when spread at an air- water interface and compressed to a fiber. The autodigestiou of this fiber at pH 1.5 is taken to be the degradation of this stabilized complex.

REFERENCES

1. MAZlA, D., ttAYASHI, T., AtCD YUDOW~TCH, I~., Cold Spring Harbor Symposia Quant. Biol. 12, 122 (1947).

2. MAZIA, D., AND HAYASHI, T. (1950), in press. 3. LA ROSA, W., Chemist-Analyst 12, 2 (1927). 4. LANOtCZUIR, I., .~¢D SCHAEFER, V. J., Chem. Revs. 24, 181 (1939). 5. HARKINS, W. D., FOURT, L., AND FOURT, P. C., J . Biol. Chem. 132, 111 (1940). 6. SOBOTKA, It., AND BLOCH, E., 6 r. Phys. Chem. 45, 9 (1941).

STABLE PEPSIN--ALBUMIN COMPLEX 447

7. ROTHEN, A., Advances in Protein Chem. 3, 123 (1947). 8. KAPLAN, J. G., Federation Proc. 9, 69 (1950). 9. SEASTON~, C. V., J. Gen. Physiol. 21, 621 (1938).

10. NORTHROP, J. H., J. Gen. Physiol. 17, 165 (1933). 11. ~/[ICttAELIS, L., AND ~ENTEN, ~V[. L., Biochem. Z. 49, 333 (1913). 12. TISELIUS, A., HENSCI-IEN, G. E., AND SVENSSON, H., Biochem. J. 32, 1814 (1938). 13. MO~ER, L. S., J. Phys. Chem. 42, 71 (1938). 14. MICHAELIS, L., Die W~sserstoffionen-Konzentr~tion. Julius Springer, Berlin, 1914.