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Proteolysis in Stored Eggs A. K. BALLSAND T. L. SWENSON, B u r e a u of Chemistry and Soils, Washington, D. C.

T

WO visible changes which eggs undergo during ware-

house storage-a weakening of the membranes around the yolk and a decrease in the proportion of thick white to thin white-indicate roughly the length of the storage period. These materials are largely protein and might therefore be susceptible to the action of the proteolytic ferments of the egg itself. The work reported here indicates that such is the case and identifies the cause of these changes as tryptic proteinase. Previous workers ( 3 ) have found in egg white a proteinase which resembles that of the pancreas in showing a n optimum activity in alkaline solutions. The present authors found that this ferment is actually identical with the proteinase of trypsin, as shown by the fact that it is also activated by the enterokinase of the intestinal mucosa. Tryptic proteinase is never found in whole egg white to any great extent and is sometimes not found at all. This is due to the fact that the ferment is apparently confined to the thick white, while the thin white contains a n inhibitory substance. B y mixing the two, an inactive or nearly inactive product results. There is, therefore, an error in the measurement of the activity of the thick white, since some thin white always adheres to it. This probably accounts for occasional irregular readings, and under the circumstances it mould be strange if anomalous results did not sometimes occur. Nevertheless, the crude thick white seems to be the best material to use for measuring proteolytic activity, for attempts to remove all the thin white by washing with water or buffer solutions, or t o remove the ferment from the egg white b y extraction processes, have usually led to complete inhibition of the enzyme. There are also great variations in the amount of active proteinase in individual eggs of the same lot, but the amount of total enzyme (i. e., the amount observed after activation with enterokinase) is reasonably constant. Since there is no way of measuring proteinase except by its enzymic action, it is impossible to say how much inhibited ferment may be present. The effective enzyme, however, clearly resides in the thick white alone. This may mean that the inhibitor occurs in both fractions, but in excess only in the thin white. The presence of a tryptic inhibitor in egg white has been noted by many investigators, some of whom have attributed this action to the proteins themselves (4, '7), others to the presence of an antitrypsin (1). PROTEOLYTIC ACTIVITYOF THICKAND THINEQQ WHITE The whites of three or five eggs were separated in the usual manner (5) by a l4mesh screen. Three-cc. portions of each fraction were diluted t o 10 cc. with water or with water containing enterokinase' and incubated for 30 minutes a t 30" C. Scrapings from the mucosa of 1 Enterokinase was prepared as follows: the first meter of the pig's small intestine were dried with acetone and ether i n t h e usual manner and powdered. Five grams of this powder were stirred with 350 cc. of 0.04 M ammonia for about 4 hours. The mixture was centrifuged, and t h e cloudy supernatant liquid neutralized with 1 M acetic acid. The kinase was then precipitated in the cold b y adding a n equal volume of acetone. The precipitate was filtered off with the aid of kieselguhr, washed with a little acetone, alcohol, and dry ether i n succession, and finally dried in t h e air. T h e dried residue was suspended in 200 cc. distilled water for 10 t o 15 minutes, then filtered. The filtrate was brought to p H 5 with 1 M acetic acid, centrifuged again to remove a small amount of coagulable protein, then neutralized again. The preparation contained no peptidase. It was kept in the ice box under toluene. Four-tentha cc. of the fresh kinase completely activated 1.0 cc. of the glycerol extract of pancreas used in these experiments. Kinase so prepared is rather pure b u t does not keep indefinitely.

Then 15 cc. of 6 per cent casein solution2 and 5 cc. of 1 M ammonia-ammonium chloride buffer of pH 8.4 were added. Ten cc. of the mixture were titrated at once by the alcoholic titration method of Willstatter and Waldschmidt-Leitz (11). After 20 minutes longer a t 30" C. another 10-cc. portion was titrated. The difference between the two titrations shows the quantity of carboxyl liberated by the enzyme in the aliquot used for the titration, corresponding to 1.0 cc. of egg white. The buffer used was made by mixing one volume of 1 M ammonium hydroxide with two volumes of 1 M ammonium chloride. The alcoholic titration for carboxyl was made as follows: A 10-cc. portion of the digestion mixture was pi etted into a flask containing about 20 cc. of 95 er cent alcohorand 1 cc. of 0.5 per cent alcoholic thymolphthagin. The mixture was then titrated with 0.1 N alcoholic potassium hydroxide to a distinct blue color. After this preliminary titration, 175 cc. of boiling 95 per cent alcohol were added, the contents of the titration flask were mixed, and the titration with alcoholic otassium hydroxide was continued in the hot mixture. By t&s means the precipitated casein remained finely divided and did not occlude titratable acids. The end point is a faint but true blue which requires a little practice to recognize. Daylight is preferable. The results, expressed in cubic centimeters of 0.1 N alkali, refer to the amount of proteinase and of casein in the 10-cc. portion used for the titration. The amount of kinase used for activation was just sufficient completely t o activate 1 cc. of an inactive glycerol extract of dried pancreas. This amount of protease after activation was diluted to 30 cc., and 10-cc. portions were titrated in alcohq.1; thus it was found that 0.33 cc. of the extract assayed as above after activation gave a titration difference of 1.5 cc. of 0.1 N alkali. In this article all subsequent mention of pancreatic extract refers t o this preparation. TABLEI. PROTEOLYTIC ACTIVITYOF THICKAND THIN EGG WHITEAGAINST CASEINAT PH 8.4 VOL.

MATERIAL

Single eggs 0 days old

A v . of 5 eggs 0 days old

Single eggs 6 months old b

OF ACTIVITYOF ENTIRE THIN WHITE^ E ~ QTHICK Without With WHITE WHITE kinase kinase Cc. Vol. % Cc. 0.1 N KOH

' I!: 23 25

69

25

76

.. .. ..

.. .. ..

25 23 25

40

....

-0.4

0

-0.3 -0.1 -0.3

-0.2 0.1 -0.1

.,.

...

64

26

...

...

Av. of 3 eggs 6 months .. 0 0 old b .. 0 0 a After 20 minutes a t 30' C. 1 I n ordinary commercial storage; temperature, 29' humidity, 8 5 % ; eggs of unknown origin.

ACTIVITYOF

THICKWHITE^ Without With kinase kinase Cc. 0.1 N

KOH

1.5

1.7

0

1.5 1.0

0.1

1.9 1.3 1.3 1 0

1.4

3.2

2.2 0.2

1.9 1.8

F. (-1.7'

C.);

The tryptic activity of the thick white varies in a remarkable way with the age of the egg. I n fresh eggs it is high, but it decreases with age until it practically vanishes. The variation, shown in Table 11, was observed with cold-storage eggs (temperature, -1.5" C . ) of unknown origin but of similar size, grade, and appearance. It was also found with eggs kept a t +4.5" C., which had been obtained a t the same time from a flock of hens of the same age and on the same diet. In this case the changes were more rapid but were analogous to those found in the storage eggs. The explanation of this behavior is not clear. It may be caused by a redistribution 6 grams of casein (Hammersten) were 2 Casein was prepared a s follows: placed in a dry mortar and 30 t o 40 cc. distilled water added in small portions, the caclein being rubbed to a smooth paste meanwhile. Then 6 cc. of 1 N sodium hydroxide were added, and the stirring was continued until the casein waB all dissolved. It was then diluted t o 100 cc. and kept under toluene. It is important not t o heat the casein.

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571

When tryptic proteinase is added to thick white, thus increasing the amount of ferment present, the effect of the enzyme on the white itself becomes more marked. -4rapid increase in the carboxyl titration, accompanied by a disTABLE11. CHANGES IN TRYPTIC ACTIVITYOF THICKWHITE appearance of the thick white is observed. WITH TIME The characteristic protein of the thick white is mucin, as (Technic a s previously described; each determination on material from McSally (6) has shown. A sample of the isolated mucin three eggs) STORAQE ACTIVITYOF THICKWHITE was found to behave in a manner entirely analogous to TEMP. AQE OF THICK Without With the original thick white. The mixture of mucin and buffer OF EQGS EGGS WHITE kinase kinase (pH 9.1) showed an increase in a free carboxyl after incubaC. Days Cc. 0.1 N K O H tion with tryptic proteinase (for 20 minutes a t 30" C. as 1.9 1.5 0 0 0.2 1 previously described). During the course of the digestion the 0. +4.5 2 0.9 5 68 0.2 0.4 .. protein, which is insoluble in the buffer solution, gradually 0.3 10 55 1.o dissolved. 0.8 50 0.9 16 0.3 0.5 28 52 Since the pH of egg white is quite suitable for tryptic acMonths tivity, the same effect may be produced in the whole egg by 3 50 0.4 0.5 injecting a small quantity of trypsin solution through a hole 4 48 0.7 0.1 -1.6 5 39 1.0 1.3 in the shell into the thick white with a hypodermic needle. 6 50 2.2 1.9 Fresh eggs handled in this way acquired in a few hours (at 7 39 1.4 3.2 room temperature) the appearance of eggs held in cold storage for many months. There was a large proportion of thin IXHIBITIOE; O F PROTEOLYSIS BY T H r N WHITE white; the yolks had the characteristic flattened appearance While the thick white has a decided proteolytic action, the and had become extremely fragile. Within 2 days the inthin white in comparable concentrations is inhibitory to jected eggs had lost practically all the thick white, and it was tryptic proteolysis. Active pancreas extract and activg thick almost impossible to break the shell without breaking the yolk white behaved alike toward thin white in this respect. S o a t the same time. This effect is evidently due to proteolysis. inhibitory action of crystalline egg albumin was observed with Eggs injected with malt or aspergillus amylase remained unconcentrations comparable to those of thin white. The altered. Papain, on the other hand, produced the same amount of free carboxyl produced from casein under the condi- change as trypsin, but to a lesser degree. The lon7er efficiency tions already described decreased with the addition of the of papain is doubtless due to the fact that the pH of egg white inhibitory thin white. is not near the optimum for papain activity. Apparently little or no extra enterokinase exists in the fresh eggs, for TABLE111. INHIBITORY EFFECTOF THIN WHITE ON CASEIX inactive pancreatic extracts produced almost no effect. HYDROLYSIS However, when the thick whites taken from the eggs in[Quantities refer t o the portion of the digestion mixture used for jected with inactive pancreas extract were mixed with a titration (10.0cc.)] CARBOXYL enterokinase, rapid solution of the thick white occurred. ENZYME INHIBITOR INCREABE Since ordinary trypsin and pancreatic extract contain Cc. 0.1 N K O H 0 . 8 6 cc. pancreas extract other enzymes besides the proteinase, the action of purified 1.6 0.86 cc. enterokinase None pancreas proteinase was also investigated. The purified Same 0 . 2 5 cc. thin white 0.7 Same 0.50 cc. thin white 0.5 preparation, however, acted in the same manner as crude Same 2 . 0 cc. thin white 0.3 None 3 . 0 cc. thin white 0.0 trypsin which confirms the view that the proteinase, rather than an auxiliary ferment, is responsible for the phenomenon. 3 . 0 cc. thick white None 0.7 3.0 cc. thick white The purification of pancreas extract consisted in the reNone 1.3 0.2 cc. enterokinase Same 1 . 0 cc. thin white -0.2 moval of carboxypolypeptidase. iln extract of dried and Same 3.0 cc. thin white -0.3 powdered pancreas gland was made with ten times its weight 1 . 0 cc. thick white None 0.6 of 75 per cent glycerol and separated from the undissolved Same 2 .O mg. crystalline egg albumin 0.8 tissue by filtration through paper. Fifty cc. of the original Same 3.3 mg. crystalline egg extract were diluted to 100 cc. with water and adjusted by albumin 0.6 Same 10.0 mg. crystalline egg one or two drops of 1 N sodium hydroxide and 1 N acetic acid 0.7 albumin until neutral to litmus. To the neutral solution eight successive 10-cc. portions of BF,HAVroR OF PRoTEoL*lC alumina C, (S)3 were added (10 cc. = 0.5 gram alumina), FERMENTS

of both the inhibitory substance and the kinase of the eggs. It indicates definitely that fermentative processes are a t work in the stored material.

O

I

1

1

Thick white alone when stored at 30" C. under toluene undergoes autolysis. The digestion is shown by an increase in the titration and by the formation of thin white which readily flows through the wire screen. TABLEIv. DIGESTION

+ +

+

THICKWHITE

(WASHED WITH

WATER) AND

(Quantities apply to the 10-cc. portions used for titration) SUBSTRATE INCREASE I N CARBOXYL^ Cc. 0.1 N K O H 0.10 g . mucin 0 2 .0 - C F t h i r k whqtn .. 0

ENZYME None None 0.33 cc. oancreas extract 0 . 3 3 cc. pancreas extract kinase 0.66 cc. purified pancreas proteinase 0.33 cc. pancreas extract kinase 0.33 cc. pancreas extract 0.66 cc. purified pancreas proteinase 0.60cc. purified pancreas proteinase 0.33 cc. pancreas extract kinase a I n 20 minutes a t 30° C.

OF

1 The process of preparing this adsorbent ie substantially as follow: 700 cc. of water containing 2 2 grams of ammonium sulfate and 1.43 per cent ammonia (by weight) are maintained a t 58' C. While the solution is being vigorously stirred, a solution of 76.7 grams ammonia aluni in 150 cc. water, also kept a t a temperature of 58' C., is added all a t onre. (Footnote continued on next page.)

__. 0.30 g. casein n m _. o.

+ kinase + kinase

ISOLATED MUCIN REMARKR

0.2

Mucin not dissolved in 72 hr. (control) Substrate not dissolved in 72 hr. (control) Control Control Control Mucin dissolved in 30 min.

1.1

Mucin dissolved in leas than 18 hr.

I__"

0.30g. casein 3.0 cc. thick white 3 . 0 CC. thick white 7.0 cc. thick white 7.0 cc. thick white 0 . 1 0 g. mucin

OF

0

1.5 1.4

0.3 2.0 1.0

.,......... ...........

INDUSTRIAL AND ENGINEERING CHEMISTRY

572

Vol. 26, No. 5

TABLE V. FERMENT ACTIONON THICKWHITE (NOT WASHED total volume to 5 cc. After an incubation of one hour a t 30” WITH WATER)UNDER TOLUENE C., the digestion mixture was washed with a little water INCREASE IN CARBOXYL into a titrating flask containing 75 cc. of 95 per cent alcohol and 1 cc. of 0.5 per cent alcoholic phenolphthalein. Titration TITRATION(0.1N KOH SUBTHICKWHITE PER 10 Q. SUB ST RAT^ was made in the cold with 0.1 N alcoholic potassium hydroxide. STRATE After E N Z Y MMIXTURE) ~ As a control a similar mixture of the enzyme, peptide solution, ENZYME A N D OTHER (THICK At 18 hr. After After After and water was titrated as soon as prepared, without incubation. ADDITIONS WHITE) s t a r t a t 30’ C. 24 hr. 48 hr. 72 hr. The amount of carboxypolypeptidase was roughly proportional cc. Volume % cc. cc. cc. to the increased titration value. It was exactly proportional E G G S APPROX. 8 MONTHS OLD (NOV.) t o the monomolecular constant of the observed reaction velocity. 10 mg. commercial trypsin (activity = With powerful enzyme preparations a precipitate of 1-tyrosine 1 cc. of pancreas exsometimes forms. It is best to dissolve this at the end of the tract) 50 100 60 . . . . . . . . . digestion by the addition of a small measured quantity of stsnd10 mz. commercial napal’n (equivalent- to ard sodium hydroxide, the amount of which must then be added 2.5 units, 10) 50 100 60 ... ... ... t o the alkali required for the subsequent alcoholic titration. 10 mg. commercial Tables I V to VI show the alteration in thick white caused by takadiastase 50 100 80 ... ... ... 10 mg. commercial tryptic proteinase. malt amylase 50 100 94 . ., . . . . ..

None 2.0 cc. purified pancreas proteinase 2.0cc. kinase 2.0 cc. enterokinase

+

50

100

94

...

50 50

100 100

70 88

... ...

8 MONTHS

OLD (JAN.)

EGGS APPROX.

...

... ... ...

...

...

At s t a r t After 72 hr.

1.0 cc. purified pan-

creas proteinase 1.0 cc. kinase 1.0 cc. water None

+

50 50 50

100 100

25 50

100

66

2.0 0.4

...

2.2 0.3 1.7

1.4

0.7 1.7

CONCLUSION I n view of the foregoing experiments it is reasonable to conclude that the disappearance of thick white from eggs in storage is due to a slow proteolysis catalyzed by tryptic proteinase. Either the egg mucin is broken down considerably, or it is hydrolyzed just sufficiently to form products of a less hydrophilic type, thereby liberating water which was pre-

OF FERMENT SOLUTION INTO EGGS TABLEVI. EFFECTOF INJECTION

[Volume per cent of thick white, 18 hours (at room temperature) after injection] OF 10 EGGS) FRESH EGQS(.\V. OF 5 ) FRESH EGGS(Av. OF 5) FRESH EGGS(Av. OF 12) commeGia1 comme+ial 0 25 cc. 0.66 cc. trypsin papain 2.5 mg. inactive purified No (titer as in (titer as in commercial NO pancreas NO 0.25cc. N? pancreas injection Table 11’) Table IV) takadiastase injection extract injection enterokinase injection proteinase 46 37 36 53 58“ 60b 68 62 67 40 54 cc.,of 100% thick white (collected from these eggg) and kept a t 30’ C. as a control to the following test marked ( 6 ) contained after 36 hours 47 cc(877) thick white. b 80 cc. of 100% thick white (collected from these injected eggs) 1.0cc. enterokinase solution contained, after 18 hours a t 30’ c.,only 37 cc. (73%) thick white.

EGGSSTORED 6 MONTHSAYER RAG^ 2.5 mz. 2.5 me.

(I

+

After each addition the alumina was thrown down in the centrifuge. The resulting solution measured 171 cc. Of this solution 0.66 cc. gave an increase in carboxyl by the usual casein method amounting to 1.4 cc. 0.1 N potassium hydroxide, after activation with 0.66 cc. enterokinase. Without added kinase the carboxyl increase was 0.80 cc. 0.1 N potassium hydroxide. With chloroacetyl-1-tryosine, by the method to be described, 0.66 cc. enzyme gave an increase in carboxyl of 0.20 cc. 0.1 N potassium hydroxide which showed that six-sevenths of the original carboxypolypeptidase had been removed. This purification is a somewhat abbreviated form of the method of Waldschmidt-Leitz and Purr (9). The newly discovered protaminase of pancreas (8) is not removed by this process, but, since the protaminase is not activated by enterokinase, the phenomenon of egg proteolysis cannot be ascribed to that ferment. The action of carboxypolypeptidase, one of the constituent enzymes of trypsin, was measured as directed by WaldSchmidt-Leitz and Purr (9) on chloroacetyl-1-tryosiiie. The technic (essentially that of the authors cited) was as follows: An aqueous solution of chloroacetyl-1-tyrosine was made to contain 1.08 grams of the peptide and 60 cc. of 0.2 M disodium phosphate (Na2HP04)in 100 cc. It was then adjusted to a pH of approximately 7.2 by a few drops of 1 N sodium hydroxide. An appropriate quantity of the enzyme was mixed with 3.0 cc. of this solution, and water was added to bring the The temperature rises t o about 61’ C. and is maintained between 58’ a n d 61° C. for 15 minutes. The precipitate is then poured into 5 liters of water, allowed t o settle, and washed b y decantation until the supernatant liquid remains turbid: i t is then washed twice more. I n order to decompose basic ammonium sulfate, 80 cc. of 20 per cent ammonia are added t o the wash water used for one washing-namely, the fourth. A total of about twenty decantations are required. The gelatinous precipitate is then stored under water for about a year a t room temperature, when i t IS ready for use.

viously bound to the mucin. The present data do not permit a choice between the two alternatives which thus present themselves. The presence in eggs of an inhibitor of tryptic activity may perhaps make the latter alternative somewhat more probable. The writers hope that further experiments on the enzymic breakdown of egg mucin and a study of the characteristics of the thin egg white may answer this question. I n either case the proteolytic system of the egg is a dominant factor in producing the physical changes which take place in eggs during storage. ACKNOWLEDGMENT

The authors are indebted to E. McNally of the Bureau of Animal Industry for furnishing the sample of isolated mucin. LITERATURE CITED Delezenne, C., and Pozerski, E., Compt. rend. SOC. biol., 55, 935 (1903). Grassmann, W., “Neue Methoden und Ergebnisse der Enzymforschung,” Hirschmaldsche Buchhandlung, Munich, 1928. Halpern, M., Z. physiol. Chem., 39,377-89 (1903); Koga, T., Biochem. Z., 141, 430 (1923). Hedin, S. G . , 2. phusiol. Chem., 52, 412 (1907). Holst, W.F., and Almquist, H. J., Hilgardia, 6, 52 (1931). blcNally, E., Proc.SOC.Ecptl. Biol. X e d . , 30, 264 (1933). Vernon, H. M., J. Physiol.,31, 497 (1904). Waldschmidt-Leita, E., and Kofranyi, E., 2. physiol. Chem.. 222, 148 (1933). Waldschmidt-Leitz, E., and Purr, A , Ber., 62, 2217 (1929). Willstatter, R., and Grassmann, W.,Z . physiol. Chern., 138, 204 (1924). Willstatter, R., and Waldschmidt-Leita, E , Ber., 54, 2988 (1921); WilljtBtter, R . , Waldschmidt-Leits, E., Dunaiturria, S., and Kunstner, G., Z. physiol. Chern., 161, 209 (1926). RECEIVEDFebruary 15, 1934. Contribution 204 of the Food Research Division, Bureau of Chemistry and Soils.