- Email: [email protected]
Action of Rennin and Pepsin on β-Casein: Insoluble and Soluble Products
A C T I O N OF R E N N I N AND P E P S I N ON B - C A S E I N : AND S O L U B L E P R O D U C T S
INSOLUBLE
J. CERBULIS, J. H. CUSTER, ~N]) C. A. Z I T T L E Eastern Regional Research Laboratory, 1 Philadelphia 18, Pennsylvania
SUMMARY
B-Casein was hydrolyzed to a considerable extent by the enzymes pepsin and rennin at pH 6.4. Pepsin hydrolyzed B-casein more extensively than rennin when given sufficient time. Hydrolysis of B-casein was evidenced by an increase in solubility at pH 4.7, and an increase in solubility in 2% trichloroacetic acid (TCA). Solubility in 12% TCA was about the same before and after enzyme treatment; hence, it was not a suitable reagent for demonstrating hydrolysis. The insoluble portion (pH 4.7; 2% TCA) of ~-casein after enzyme treatment was heterogeneous, containing at least three components. The soluble fraction was very heterogeneous and contained ten or more components. The major products released by rennin and pepsin have similar electrophoretic behavior, but there were differences among the minor products. Reports on the action of the enzymes rennin and pepsin on B-casein have been diverse. Some have concluded that only a-casein is altered by rennin, whereas B-casein and ~,-casein are not attacked (3, 1), but are merely coprecipitated unchanged along with the altered a-casein in the presence of calcium salts (3). Others have reported that B-casein shows only a slow general proteolysis (6, 7 ) ; that the prolonged action of rennin on B-casein gives a product no longer preeipitable with calcium ion (9) ; and that B-casein is split into two components in 4 hr. by rennin (8). A recent report has shown that B-casein is solubilized considerably by pepsin in 5 rain. (10). The present investigation was undertaken to learn more about the action of the enzymes rennin and pepsin on B-casein, and to clarify the reports just cited. Since some of the conflict appeared to be due to the choice of conditions for precipitating the treated B-casein, precipitation at p H 4.7 as well as precipitation with two concentrations (2 and 12%) of trichloroacetic acid (TCA) were compared. The components of the insoluble and soluble fractions were investigated at the same time by eleetrophoresis and chromatography. EXPERIMENTAL PROCEDURE
Enzymes. Pepsin was a crystalline, Commercial product. Rennin was a highly purified product obtained through the courtesy of Dr. R. A. Sullivan, National D a i r y Research Laboratories, L. I., N. ¥ . This rennin had the same specific activity as crystalline rennin. B-Casein was prepared by the method of H i p p et al. (5), utilizing differential solubility in aqueous urea solutions. Preparation of dry fractions. B-Casein and all precipitates were dried by Received for publication June 24, 1960. Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. 1725
1726
J. CERBULIS, J. H. CUSTER, AND C. A. ZITTLE
washing with acetone and ether and removing the solvent in a stream of air. Solutions were concentrated and dried by a rapid, large-surface v a c u u m technique (4) at 30-35 ° C. I t was observed that all washings in all experiments, a f t e r evaporation of ether, left some yellowish, fat-like residue. These residues were not analyzed further. E)~zyme reaction. A 2% B-casein solution was p r e p a r e d by dissolving 5 g. of d r y protein in a m i x t u r e of 2.5 ml. of 1 N N a O H and 240 ml. of distilled water, and the solution was adjusted to p H 6.4. Five milligrams of enzyme was dissolved in 5 ml. of water and added to the casein solution at 30 ° C. The reaction was continued for a definite time at 30 ° C. The enzyme reaction was stopped either with T C A or heat, according to the type of experiment being run. (a) pH 4.7 precipitation. The enzyme reaction was stopped by placing the flask in a boiling w a t e r bath and stirring for 15 rain. The contents of the flask were quickly cooled to room temperature, adjusted to p H 4.7 with 0.1 N HC1, and centrifuged. Nitrogen and phosphorus were determined in the s u p e r n a t a n t solution. The precipitate was dried as described above. The p H 4.7-soluble portion was dried by a vacuum technique and was fraetionated with T C A as described for T C A precipitation. Three fractions were obtained: 2% T C A insoluble, 12% TCA-insoluble, and 12% TCA-soluble. (b) TCA precipitations. Twenty-five per cent T C A solution was added to a solution of the enzyme reaction products until a concentration of 2% was reached; the m i x t u r e was then centrifuged, and nitrogen and phosphorus were determined in the s u p e r n a t a n t solution. The s u p e r n a t a n t was extracted with ether six times to remove the T C A ; then the aqueous phase was adjusted to p H 7.0 with N a O H and evaporated to dryness by a r a p i d v a c u u m technique (2). The portion of the B-casein insoluble in 2% TCA was dissolved in dilute N a O H and then reprecipitated at p H 4.7 with HC1. The s u p e r n a t a n t of the p H 4.7 precipitation was dialyzed against water and dried by the r a p i d v a c u u m technique. Similarly, 50% TCA was added to another portion of the enzyme reaction products until a concentration of 12% was reached. Subsequent steps were the same as those for the 2% T C A precipitation. Analytical methods. The determination of nitrogen and phosphorus has been described previously (10).
Electrophoresis was carried type electrophoresis a p p a r a t u s length cell. All buffer solutions by NaC1. The buffering salts 5.6-acetate.
out with a 1% solution at 1 ° C. in a Tiselius for 3 hr. with a 11-ml. volume, standard, fullwere of ionic strength 0.1, with 80% contributed were: p H 8.6-veronal, p H 6.7-phosphate, p H
Paper electrophoretic analyses of proteins and peptides were p e r f o r m e d in a D u r r u m t y p e electrophoresis cell (Spinco Model R). 2 Electrophoresis was done at 5 ° for 16 hr. in a veronal buffer, p I I 8.6, ionic strength 0.075, on I t is n o t i m p l i e d the U S D A r e c o m m e n d s t he above c o m p a n y or i t s p r o d u c t to t h e p o s s i b l e exclusion of others i n the same business.
ACTION
OF
RENNIN
AND
PEPSIN
ON
fl-CASEIN
1727
W h a t m a n p a p e r No. 1. Most of the experiments were done with 175 v., which gave a current flow of about 4.5 ma. Usually, 0.01 ml. of a 6% solution of the substance u n d e r test was applied to the apex of the paper. P a p e r chromat o g r a p h y was carried out with n-butanol acetic acid water pyridine as solvent for 16 to 18 hr. by an ascending technique.
Sttdning was p e r f o r m e d with n i n h y d r i n and bromophenol blue (2). Soluble products. The hydrolysis of fl-casein resulting f r o m the action of rennin or pepsin, measured by the amount of soluble nitrogen and phosphorus with three precipitation conditions ( p H 4.7, 2% TCA, 12% T C A ) is shown in Table 1. Precipitation with 12% T C A gave a v e r y small soluble fraction, TABLE 1 ttydrolysis of fl-casein by rennin and pepsin at pK 6.4: soluble nitrogen and phosphorus obtained with three precipitation conditions Nitrogen (% of total N) Rennin
Precipitation conditions
Initial soluble
5 rain.
pH 4.7 2% TCA 12% TCA
0.80 0.20 0.20
3.46 0.92 0.27
pH 4.7 2% TCA 12% TCA
0.48 0.15 0.15
2.04 1.79 0.85
1 hr.
5 rain.
7.60 3.62 3.35 1.46 0.98 0.58 Phosphorus (% of total P) 2.39 3.53 1.51 0.77 0.69 0.77
Pepsin 1 hr.
5.5 hr.
17.49 15.81 2.37
35.00 ........ ........
2.70 1.05 0.77
........ ........ ........
f a r less t h a n the amounts obtained by other precipitants. Precipitation at p H 4.7 gave the largest soluble fraction, whereas those experiments wi~h 2% T C A gave somewhat less. U n d e r the conditions used, pepsin hydrolyzed fl-casein more extensively than did rennin, p a r t i c u l a r l y with longer periods of time. The results of p a p e r electrophoretic analysis of pepsin-treated /?-casein fractions are shown in F i g u r e 1. The rennin diagram for the p i t 4.7-soluble fraction is almost identical with the pepsin diagram shown (a), except t h a t in the Band I region, two v e r y close bands are obtained with rennin. The diagram for the 2% TCA-soluble fraction (not shown) indicates the same n u m b e r of components as t h a t for the p H 4.7-soluble fraction, but some were reduced in amount. The quantitative relationship between bands shown in F i g u r e 1 was obtained a f t e r 5 min. and 1 hr. of proteolysis. The p a t t e r n for the p H 4.7soluble fraction was identical, even when proteolysis was for 5.5 hr. I n the p H 4.7-insoluble fraction, however, when proteolysis exceeded I hr., the fl-easein band was no longer apparent. Bands I and I I I are the m a j o r components of the soluble fractions, with the other cross-hatched bands ( F i g u r e 1) intermediate in amount, and the remaining bands relatively minor in amount. Fractionation of the p H 4.7-soluble material with T C A revealed t h a t the bands corresponding to I, and I I I to VII, were found in the 2% T C A insoluble fraction ( F i g u r e l c ) . All bands in both insoluble fractions gave positive reactions with n i n h y d r i n and bromophenol blue reagents. A qualitative difference between the action of rennin and pepsin was noted in the 12% TCA-insoluble
1728
J. CERBULIS, J. It. CUSTER, AND C. A. ZITTLE
IT
I
I
I
H
Schematic representation of paper eleetrophoretie bands of fractions of fl-easein obtained after the action of pepsin for 5 or 60 min. Eleetrophoresis was at p t I 8.6 for 16 hr. (2). Protein bands were evident after staining with bromophenol blue. Cross-hatching is used to indicate the more strongly stained bands. Component I V is fl-easein. Components migrating to the left are negatively charged. FIo. 1. (a) p]X 4.7-soluble fraction; (b) p i t 4.7-insoluble fraction; (c) p i t 4.7-soluble, 2% TCA-insolub]e fraction; (d) p H 4.7-soluble, 1~% TCA-inso]uble fraction; (e) 12% TCA-insoluble, pH 4.7 soluble fraction; (f) 12% TCA-soluble fraction.
fraction (Figure ld) ; Band I was the major one for pepsin and B a n d I I I was major for rennin. The 12% TCA-soluble fraction was small in amount and contained only one or two bands (Figure l f ) . These components gave a positive reaction with n i n h y d r i n only. fl-Casein and all of the insoluble and soluble fractions were studied by paper chromatography, fl-Casein and the enzyme-transformed fl-easein remained at the origin, with some forward streaking. All other fractions gave spots with greater movement. The results showed that Fractions d and e (See F i g u r e 1) were much more heterogeneous than expected from the eleetrophoretic results, with movement of 0 to 32 enl. in a 16-hr. run. F u r t h e r , the major bands (I and I I I ) of e could not be identical with the I and I I I bands of d, for the number and distribution of the components revealed by chromatography differed greatly
ACTION
01~ RENNIN
AND
PEPSIN
ON
fl-CASEIN
1729
for these two fractions. The 12% TCA-soluble fraction showed six to eight components by chromatography, more than observed by electrophoresis, with movement of 2 to 32 cm. in a 16-hr. run. Differences also were observed between the split products obtained with rennin and pepsin in both the pH 4.7-soluble, 12% TCA-insoluble, and the 12% TCA-soluble fractions. Insoluble products. The paper electrophoretic pattern of the fraction obtained by precipitation at pH 4.7 is shown in Figure lb. The 12 and 2% TCA precipitates gave, after reprecipitation at pH 4.7, electrophoretic patterns like Figure lb. The portion soluble at pH 4.7 obtained by reprecipitation of the 2% TCA-insoluble fraction was heterogeneous as shown by paper electrophoresis (Figure lc). It contained fl-casein (IV), enzyme-transformed fl-casein (V), and all the components made soluble by the two enzymes. Both moving boundary and paper electrophoretie studies at pH 8.6 showed that the insoluble fraction was not merely unaltered fl-casein, but that a new component differing in electrical charge resulted from the action of rennin and pepsin. This altered casein migrated much faster than fl-easein, and slower than a-casein. The concentration of this fraction increased with the reaction time, and in the 5.5 hr. treatment by pepsin all of the original fl-casein had been transformed to the new electrophoretic component, which contained 65% of the original fl-casein nitrogen (See Table 2). This new electrophoretic fraction was not TABLE 2 E l e c t r o p h o r e t i c c o m p o s i t i o n of the insoluble f r a c t i o n of fl-casein ( p H 4.7) a f t e r h y d r o l y s i s with rennin and pepsin Rennin
Reaction time with enzymes
pH ~
5 min. I hr. 1 hr. 1 hr. 5.5 hr.
8.6 8.6 6.7 5.6 5,.6
fl-caseln
Pepsin
Altered fl-cas,ein
fl-casein
Altered fl-casein
Altered Altered fl-casein I ~-easein I I
(%) 92 74 ........ ........ ........
8 26
72 12 11 16 0
28 88 89 S4 100
........ ........ 62 36
~6: 64
p H at w h i c h electrophoresis w a s p e r f o r m e d w i t h t h e m o v i n g b o u n d a r y technique.
homogeneous, for electrophoresis at pH 5.6 revealed two peaks. The ratio of the two peaks was not constant but with time the faster (II) increased at the expense of the slower, as shown in Table 2. DISCUSSION
The present results comparing the use of several precipitants indicate that the enzymes rennin and pepsin cause a considerable proteolysis of fl-casein. This was evident when pH 4.7 or 2% TCA precipitation was used after the action of these enzymes. W h e n 12% TCA was the precipitant, as was the case in the experiments of Nitschmann and collaborators (6, 7), the soluble fraction was negligible. On the basis of similar results, Nitschmann et al. (6, 7) had concluded that rennin did not hydrolyze fl-casein appreciably, a conclusion that the present results show to be erroneous.
1730
J. CERBULIS, J. H. CUSTER, AND C. A. ZITTLE
P a p e r e l e c t r o p h o r e t i c a n a l y s i s of t h e soluble f r a c t i o n s s h o w e d t h a t t h e v a r i a . t i o n i n t h e a m o u n t f o u n d w i t h t h e d i f f e r e n t p r e c i p i t a n t s was n o t d u e m e r e l y to d i f f e r e n c e in d e g r e e of s o l u b i l i t y of t h e e n z y m e - t r a n s f o r m e d fl-casein. T h e r e a r e m a n y m o r e c o m p o n e n t s p r e s e n t in t h e p i t 4.7 soluble f r a c t i o n t h a n in t h e 12% T C A - s o l u b l e f r a c t i o n . F r o m this s t a n d p o i n t , the p o r t i o n s o l u b l e a t p H 4.7 is t h e t r u e r m e a s u r e of h y d r o l y s i s . T h e a m o u n t of p r o t e o l y s i s l e a d i n g to a n i n c r e a s e in t h e soluble f r a c t i o n i n c r e a s e d w i t h t h e t i m e of a c t i o n of t h e enzymes. T h e a c t i o n of p e p s i n was m u c h m o r e e x t e n s i v e t h a n t h a t of r e n n i n , p a r t i c u l a r l y w i t h l o n g e r p e r i o d s of time. S i n c e t h e p a p e r e l e c t r o p h o r e t i c p a t t e r n s of t h e soluble f r a c t i o n were e s s e n t i a l l y t h e s a m e o v e r a p e r i o d of 5.5 hr., a g e n e r a l p r o t e o l y s i s p r o b a b l y t a k e s p l a c e f r o m t h e b e g i n n i n g of t h e r e a c t i o n . D u r i n g t h i s same p e r i o d , f r e e e l e c t r o p h o r e s i s a t p H 8.6 shows t h a t t h e fl-casein is t r a n s f o r m e d to a n e w elect r o p h o r e t i c c o m p o n e n t of g r e a t e r m o b i l i t y . E l e c t r o p h o r e s i s a t p i t 5.6 shows t h a t this f r a c t i o n is h e t e r o g e n e o u s a n d c h a n g e s in c o m p o s i t i o n in a r e g u l a r w a y w i t h time. P a p e r e l e c t r o p h o r e t i c p a t t e r n s a n d p a p e r c h r o m a t o g r a p h y dis. closed t h a t t h e soluble f r a c t i o n s w e r e v e r y complex, c o n t a i n i n g e i g h t to t e n o r m o r e c o m p o n e n t s . Some of these c o m p o n e n t s w e r e p r e s e n t o n l y i n t r a c e s , a n d it was i m p o s s i b l e to give t h e e x a c t n u m b e r of p e p t i d e s . REFERENCES (1) ALAIS, C., MOCQUOT, G., ~ITSCtt~AI'~Iq, ~t., AND ZAHLER, P. Das Lab und seine Wirkung auf das Casein der Milch. VII. Ober die Abspaltung von Nicht-Protein-Stickstoff (NPN) aus Casein durch Lab und ihre Beziehung zur Primfirreaktion der Labgerinnung der Milch. Helv. Chim. Acta, 36: 1955. 1953. (2) C~ULIS, J., C u s ~ , J. H., AN~) ZITTLE, C. A. Action of Rennin and Pepsin on a-Casein: Paracasein and Soluble Products. Arch. Biochem. Biophys., 84: 417. 1959. (3) CH~ULn~Z, E., ANn BAVDF.!r, P. Rccherches sur la Cas~ine. VI. Sur la Transformation de la Cas6ine en Paracas6ine. IIelv. Chim. Acta, 33: 1673. 1950. (4) C~IQ, L. C., GP~E~ORV,J. D., AN]) HAUSMANN, W. Versatile Laboratory Concentration Device. Anal. Chem., 22: 1462. 1950. (5) HIPP, ~ . J., G•ovzs, M. L., CUSTER, J. H., AND MCM~.~KIN, T. L. Separation of a-, fl-, and "y-Casein. J. Dairy Sci., 35: 272. 1952. (6) K E ~ , W. Die Abspaltung von Peptiden aus reinen Casein Fractionen dutch Kristallisiertes Lab. Dissertation, Bern. 1954. (7) NITSC~AN~, tI., AN]) K E L P , W. Das Lab und seine Wirkung auf das Casein der Milch. IX. ~3ber die Abspaltung yon Nicht-Protein-Stickstoff (NPN) aus Isoliertem a- und fl-Casein durch Lab. Holy. Chim. Acta, 38: 942. 1955. (8) TSUGO, T., AND "YAMAUCHI, K. The Break-Up of Casein by Rennin and Its Relation to Milk Coagulation. 2, Pt. 2, 588. 14th Intern. Dairy Congr., Rome. 1956. (9) WAUG~, D. F. The Interactions of as-, fl, and a-Caseins in Micella Formations. Discussions, Faraday Soc., No. 25: 186. 1958. (10) ZI~rL~, C. A., AND C~ULIS, J. Clotting of Casein with Pepsin: Amount and Nature of the Soluble Products. J. Dairy Sci., 41:241. 1958.
INSOLUBLE
J. CERBULIS, J. H. CUSTER, ~N]) C. A. Z I T T L E Eastern Regional Research Laboratory, 1 Philadelphia 18, Pennsylvania
SUMMARY
B-Casein was hydrolyzed to a considerable extent by the enzymes pepsin and rennin at pH 6.4. Pepsin hydrolyzed B-casein more extensively than rennin when given sufficient time. Hydrolysis of B-casein was evidenced by an increase in solubility at pH 4.7, and an increase in solubility in 2% trichloroacetic acid (TCA). Solubility in 12% TCA was about the same before and after enzyme treatment; hence, it was not a suitable reagent for demonstrating hydrolysis. The insoluble portion (pH 4.7; 2% TCA) of ~-casein after enzyme treatment was heterogeneous, containing at least three components. The soluble fraction was very heterogeneous and contained ten or more components. The major products released by rennin and pepsin have similar electrophoretic behavior, but there were differences among the minor products. Reports on the action of the enzymes rennin and pepsin on B-casein have been diverse. Some have concluded that only a-casein is altered by rennin, whereas B-casein and ~,-casein are not attacked (3, 1), but are merely coprecipitated unchanged along with the altered a-casein in the presence of calcium salts (3). Others have reported that B-casein shows only a slow general proteolysis (6, 7 ) ; that the prolonged action of rennin on B-casein gives a product no longer preeipitable with calcium ion (9) ; and that B-casein is split into two components in 4 hr. by rennin (8). A recent report has shown that B-casein is solubilized considerably by pepsin in 5 rain. (10). The present investigation was undertaken to learn more about the action of the enzymes rennin and pepsin on B-casein, and to clarify the reports just cited. Since some of the conflict appeared to be due to the choice of conditions for precipitating the treated B-casein, precipitation at p H 4.7 as well as precipitation with two concentrations (2 and 12%) of trichloroacetic acid (TCA) were compared. The components of the insoluble and soluble fractions were investigated at the same time by eleetrophoresis and chromatography. EXPERIMENTAL PROCEDURE
Enzymes. Pepsin was a crystalline, Commercial product. Rennin was a highly purified product obtained through the courtesy of Dr. R. A. Sullivan, National D a i r y Research Laboratories, L. I., N. ¥ . This rennin had the same specific activity as crystalline rennin. B-Casein was prepared by the method of H i p p et al. (5), utilizing differential solubility in aqueous urea solutions. Preparation of dry fractions. B-Casein and all precipitates were dried by Received for publication June 24, 1960. Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. 1725
1726
J. CERBULIS, J. H. CUSTER, AND C. A. ZITTLE
washing with acetone and ether and removing the solvent in a stream of air. Solutions were concentrated and dried by a rapid, large-surface v a c u u m technique (4) at 30-35 ° C. I t was observed that all washings in all experiments, a f t e r evaporation of ether, left some yellowish, fat-like residue. These residues were not analyzed further. E)~zyme reaction. A 2% B-casein solution was p r e p a r e d by dissolving 5 g. of d r y protein in a m i x t u r e of 2.5 ml. of 1 N N a O H and 240 ml. of distilled water, and the solution was adjusted to p H 6.4. Five milligrams of enzyme was dissolved in 5 ml. of water and added to the casein solution at 30 ° C. The reaction was continued for a definite time at 30 ° C. The enzyme reaction was stopped either with T C A or heat, according to the type of experiment being run. (a) pH 4.7 precipitation. The enzyme reaction was stopped by placing the flask in a boiling w a t e r bath and stirring for 15 rain. The contents of the flask were quickly cooled to room temperature, adjusted to p H 4.7 with 0.1 N HC1, and centrifuged. Nitrogen and phosphorus were determined in the s u p e r n a t a n t solution. The precipitate was dried as described above. The p H 4.7-soluble portion was dried by a vacuum technique and was fraetionated with T C A as described for T C A precipitation. Three fractions were obtained: 2% T C A insoluble, 12% TCA-insoluble, and 12% TCA-soluble. (b) TCA precipitations. Twenty-five per cent T C A solution was added to a solution of the enzyme reaction products until a concentration of 2% was reached; the m i x t u r e was then centrifuged, and nitrogen and phosphorus were determined in the s u p e r n a t a n t solution. The s u p e r n a t a n t was extracted with ether six times to remove the T C A ; then the aqueous phase was adjusted to p H 7.0 with N a O H and evaporated to dryness by a r a p i d v a c u u m technique (2). The portion of the B-casein insoluble in 2% TCA was dissolved in dilute N a O H and then reprecipitated at p H 4.7 with HC1. The s u p e r n a t a n t of the p H 4.7 precipitation was dialyzed against water and dried by the r a p i d v a c u u m technique. Similarly, 50% TCA was added to another portion of the enzyme reaction products until a concentration of 12% was reached. Subsequent steps were the same as those for the 2% T C A precipitation. Analytical methods. The determination of nitrogen and phosphorus has been described previously (10).
Electrophoresis was carried type electrophoresis a p p a r a t u s length cell. All buffer solutions by NaC1. The buffering salts 5.6-acetate.
out with a 1% solution at 1 ° C. in a Tiselius for 3 hr. with a 11-ml. volume, standard, fullwere of ionic strength 0.1, with 80% contributed were: p H 8.6-veronal, p H 6.7-phosphate, p H
Paper electrophoretic analyses of proteins and peptides were p e r f o r m e d in a D u r r u m t y p e electrophoresis cell (Spinco Model R). 2 Electrophoresis was done at 5 ° for 16 hr. in a veronal buffer, p I I 8.6, ionic strength 0.075, on I t is n o t i m p l i e d the U S D A r e c o m m e n d s t he above c o m p a n y or i t s p r o d u c t to t h e p o s s i b l e exclusion of others i n the same business.
ACTION
OF
RENNIN
AND
PEPSIN
ON
fl-CASEIN
1727
W h a t m a n p a p e r No. 1. Most of the experiments were done with 175 v., which gave a current flow of about 4.5 ma. Usually, 0.01 ml. of a 6% solution of the substance u n d e r test was applied to the apex of the paper. P a p e r chromat o g r a p h y was carried out with n-butanol acetic acid water pyridine as solvent for 16 to 18 hr. by an ascending technique.
Sttdning was p e r f o r m e d with n i n h y d r i n and bromophenol blue (2). Soluble products. The hydrolysis of fl-casein resulting f r o m the action of rennin or pepsin, measured by the amount of soluble nitrogen and phosphorus with three precipitation conditions ( p H 4.7, 2% TCA, 12% T C A ) is shown in Table 1. Precipitation with 12% T C A gave a v e r y small soluble fraction, TABLE 1 ttydrolysis of fl-casein by rennin and pepsin at pK 6.4: soluble nitrogen and phosphorus obtained with three precipitation conditions Nitrogen (% of total N) Rennin
Precipitation conditions
Initial soluble
5 rain.
pH 4.7 2% TCA 12% TCA
0.80 0.20 0.20
3.46 0.92 0.27
pH 4.7 2% TCA 12% TCA
0.48 0.15 0.15
2.04 1.79 0.85
1 hr.
5 rain.
7.60 3.62 3.35 1.46 0.98 0.58 Phosphorus (% of total P) 2.39 3.53 1.51 0.77 0.69 0.77
Pepsin 1 hr.
5.5 hr.
17.49 15.81 2.37
35.00 ........ ........
2.70 1.05 0.77
........ ........ ........
f a r less t h a n the amounts obtained by other precipitants. Precipitation at p H 4.7 gave the largest soluble fraction, whereas those experiments wi~h 2% T C A gave somewhat less. U n d e r the conditions used, pepsin hydrolyzed fl-casein more extensively than did rennin, p a r t i c u l a r l y with longer periods of time. The results of p a p e r electrophoretic analysis of pepsin-treated /?-casein fractions are shown in F i g u r e 1. The rennin diagram for the p i t 4.7-soluble fraction is almost identical with the pepsin diagram shown (a), except t h a t in the Band I region, two v e r y close bands are obtained with rennin. The diagram for the 2% TCA-soluble fraction (not shown) indicates the same n u m b e r of components as t h a t for the p H 4.7-soluble fraction, but some were reduced in amount. The quantitative relationship between bands shown in F i g u r e 1 was obtained a f t e r 5 min. and 1 hr. of proteolysis. The p a t t e r n for the p H 4.7soluble fraction was identical, even when proteolysis was for 5.5 hr. I n the p H 4.7-insoluble fraction, however, when proteolysis exceeded I hr., the fl-easein band was no longer apparent. Bands I and I I I are the m a j o r components of the soluble fractions, with the other cross-hatched bands ( F i g u r e 1) intermediate in amount, and the remaining bands relatively minor in amount. Fractionation of the p H 4.7-soluble material with T C A revealed t h a t the bands corresponding to I, and I I I to VII, were found in the 2% T C A insoluble fraction ( F i g u r e l c ) . All bands in both insoluble fractions gave positive reactions with n i n h y d r i n and bromophenol blue reagents. A qualitative difference between the action of rennin and pepsin was noted in the 12% TCA-insoluble
1728
J. CERBULIS, J. It. CUSTER, AND C. A. ZITTLE
IT
I
I
I
H
Schematic representation of paper eleetrophoretie bands of fractions of fl-easein obtained after the action of pepsin for 5 or 60 min. Eleetrophoresis was at p t I 8.6 for 16 hr. (2). Protein bands were evident after staining with bromophenol blue. Cross-hatching is used to indicate the more strongly stained bands. Component I V is fl-easein. Components migrating to the left are negatively charged. FIo. 1. (a) p]X 4.7-soluble fraction; (b) p i t 4.7-insoluble fraction; (c) p i t 4.7-soluble, 2% TCA-insolub]e fraction; (d) p H 4.7-soluble, 1~% TCA-inso]uble fraction; (e) 12% TCA-insoluble, pH 4.7 soluble fraction; (f) 12% TCA-soluble fraction.
fraction (Figure ld) ; Band I was the major one for pepsin and B a n d I I I was major for rennin. The 12% TCA-soluble fraction was small in amount and contained only one or two bands (Figure l f ) . These components gave a positive reaction with n i n h y d r i n only. fl-Casein and all of the insoluble and soluble fractions were studied by paper chromatography, fl-Casein and the enzyme-transformed fl-easein remained at the origin, with some forward streaking. All other fractions gave spots with greater movement. The results showed that Fractions d and e (See F i g u r e 1) were much more heterogeneous than expected from the eleetrophoretic results, with movement of 0 to 32 enl. in a 16-hr. run. F u r t h e r , the major bands (I and I I I ) of e could not be identical with the I and I I I bands of d, for the number and distribution of the components revealed by chromatography differed greatly
ACTION
01~ RENNIN
AND
PEPSIN
ON
fl-CASEIN
1729
for these two fractions. The 12% TCA-soluble fraction showed six to eight components by chromatography, more than observed by electrophoresis, with movement of 2 to 32 cm. in a 16-hr. run. Differences also were observed between the split products obtained with rennin and pepsin in both the pH 4.7-soluble, 12% TCA-insoluble, and the 12% TCA-soluble fractions. Insoluble products. The paper electrophoretic pattern of the fraction obtained by precipitation at pH 4.7 is shown in Figure lb. The 12 and 2% TCA precipitates gave, after reprecipitation at pH 4.7, electrophoretic patterns like Figure lb. The portion soluble at pH 4.7 obtained by reprecipitation of the 2% TCA-insoluble fraction was heterogeneous as shown by paper electrophoresis (Figure lc). It contained fl-casein (IV), enzyme-transformed fl-casein (V), and all the components made soluble by the two enzymes. Both moving boundary and paper electrophoretie studies at pH 8.6 showed that the insoluble fraction was not merely unaltered fl-casein, but that a new component differing in electrical charge resulted from the action of rennin and pepsin. This altered casein migrated much faster than fl-easein, and slower than a-casein. The concentration of this fraction increased with the reaction time, and in the 5.5 hr. treatment by pepsin all of the original fl-casein had been transformed to the new electrophoretic component, which contained 65% of the original fl-casein nitrogen (See Table 2). This new electrophoretic fraction was not TABLE 2 E l e c t r o p h o r e t i c c o m p o s i t i o n of the insoluble f r a c t i o n of fl-casein ( p H 4.7) a f t e r h y d r o l y s i s with rennin and pepsin Rennin
Reaction time with enzymes
pH ~
5 min. I hr. 1 hr. 1 hr. 5.5 hr.
8.6 8.6 6.7 5.6 5,.6
fl-caseln
Pepsin
Altered fl-cas,ein
fl-casein
Altered fl-casein
Altered Altered fl-casein I ~-easein I I
(%) 92 74 ........ ........ ........
8 26
72 12 11 16 0
28 88 89 S4 100
........ ........ 62 36
~6: 64
p H at w h i c h electrophoresis w a s p e r f o r m e d w i t h t h e m o v i n g b o u n d a r y technique.
homogeneous, for electrophoresis at pH 5.6 revealed two peaks. The ratio of the two peaks was not constant but with time the faster (II) increased at the expense of the slower, as shown in Table 2. DISCUSSION
The present results comparing the use of several precipitants indicate that the enzymes rennin and pepsin cause a considerable proteolysis of fl-casein. This was evident when pH 4.7 or 2% TCA precipitation was used after the action of these enzymes. W h e n 12% TCA was the precipitant, as was the case in the experiments of Nitschmann and collaborators (6, 7), the soluble fraction was negligible. On the basis of similar results, Nitschmann et al. (6, 7) had concluded that rennin did not hydrolyze fl-casein appreciably, a conclusion that the present results show to be erroneous.
1730
J. CERBULIS, J. H. CUSTER, AND C. A. ZITTLE
P a p e r e l e c t r o p h o r e t i c a n a l y s i s of t h e soluble f r a c t i o n s s h o w e d t h a t t h e v a r i a . t i o n i n t h e a m o u n t f o u n d w i t h t h e d i f f e r e n t p r e c i p i t a n t s was n o t d u e m e r e l y to d i f f e r e n c e in d e g r e e of s o l u b i l i t y of t h e e n z y m e - t r a n s f o r m e d fl-casein. T h e r e a r e m a n y m o r e c o m p o n e n t s p r e s e n t in t h e p i t 4.7 soluble f r a c t i o n t h a n in t h e 12% T C A - s o l u b l e f r a c t i o n . F r o m this s t a n d p o i n t , the p o r t i o n s o l u b l e a t p H 4.7 is t h e t r u e r m e a s u r e of h y d r o l y s i s . T h e a m o u n t of p r o t e o l y s i s l e a d i n g to a n i n c r e a s e in t h e soluble f r a c t i o n i n c r e a s e d w i t h t h e t i m e of a c t i o n of t h e enzymes. T h e a c t i o n of p e p s i n was m u c h m o r e e x t e n s i v e t h a n t h a t of r e n n i n , p a r t i c u l a r l y w i t h l o n g e r p e r i o d s of time. S i n c e t h e p a p e r e l e c t r o p h o r e t i c p a t t e r n s of t h e soluble f r a c t i o n were e s s e n t i a l l y t h e s a m e o v e r a p e r i o d of 5.5 hr., a g e n e r a l p r o t e o l y s i s p r o b a b l y t a k e s p l a c e f r o m t h e b e g i n n i n g of t h e r e a c t i o n . D u r i n g t h i s same p e r i o d , f r e e e l e c t r o p h o r e s i s a t p H 8.6 shows t h a t t h e fl-casein is t r a n s f o r m e d to a n e w elect r o p h o r e t i c c o m p o n e n t of g r e a t e r m o b i l i t y . E l e c t r o p h o r e s i s a t p i t 5.6 shows t h a t this f r a c t i o n is h e t e r o g e n e o u s a n d c h a n g e s in c o m p o s i t i o n in a r e g u l a r w a y w i t h time. P a p e r e l e c t r o p h o r e t i c p a t t e r n s a n d p a p e r c h r o m a t o g r a p h y dis. closed t h a t t h e soluble f r a c t i o n s w e r e v e r y complex, c o n t a i n i n g e i g h t to t e n o r m o r e c o m p o n e n t s . Some of these c o m p o n e n t s w e r e p r e s e n t o n l y i n t r a c e s , a n d it was i m p o s s i b l e to give t h e e x a c t n u m b e r of p e p t i d e s . REFERENCES (1) ALAIS, C., MOCQUOT, G., ~ITSCtt~AI'~Iq, ~t., AND ZAHLER, P. Das Lab und seine Wirkung auf das Casein der Milch. VII. Ober die Abspaltung von Nicht-Protein-Stickstoff (NPN) aus Casein durch Lab und ihre Beziehung zur Primfirreaktion der Labgerinnung der Milch. Helv. Chim. Acta, 36: 1955. 1953. (2) C~ULIS, J., C u s ~ , J. H., AN~) ZITTLE, C. A. Action of Rennin and Pepsin on a-Casein: Paracasein and Soluble Products. Arch. Biochem. Biophys., 84: 417. 1959. (3) CH~ULn~Z, E., ANn BAVDF.!r, P. Rccherches sur la Cas~ine. VI. Sur la Transformation de la Cas6ine en Paracas6ine. IIelv. Chim. Acta, 33: 1673. 1950. (4) C~IQ, L. C., GP~E~ORV,J. D., AN]) HAUSMANN, W. Versatile Laboratory Concentration Device. Anal. Chem., 22: 1462. 1950. (5) HIPP, ~ . J., G•ovzs, M. L., CUSTER, J. H., AND MCM~.~KIN, T. L. Separation of a-, fl-, and "y-Casein. J. Dairy Sci., 35: 272. 1952. (6) K E ~ , W. Die Abspaltung von Peptiden aus reinen Casein Fractionen dutch Kristallisiertes Lab. Dissertation, Bern. 1954. (7) NITSC~AN~, tI., AN]) K E L P , W. Das Lab und seine Wirkung auf das Casein der Milch. IX. ~3ber die Abspaltung yon Nicht-Protein-Stickstoff (NPN) aus Isoliertem a- und fl-Casein durch Lab. Holy. Chim. Acta, 38: 942. 1955. (8) TSUGO, T., AND "YAMAUCHI, K. The Break-Up of Casein by Rennin and Its Relation to Milk Coagulation. 2, Pt. 2, 588. 14th Intern. Dairy Congr., Rome. 1956. (9) WAUG~, D. F. The Interactions of as-, fl, and a-Caseins in Micella Formations. Discussions, Faraday Soc., No. 25: 186. 1958. (10) ZI~rL~, C. A., AND C~ULIS, J. Clotting of Casein with Pepsin: Amount and Nature of the Soluble Products. J. Dairy Sci., 41:241. 1958.