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 : INSOLUBLE

AND S O L U B L E P R O D U C T S

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 copre-
cipitated 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 pre-
cipitation 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 differ-
ential 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 tech-
nique (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 out with a 1% solution at 1 ° C. in a Tiselius
type electrophoresis a p p a r a t u s for 3 hr. with a 11-ml. volume, standard, full-
length cell. All buffer solutions were of ionic strength 0.1, with 80% contributed
by NaC1. The buffering salts were: p H 8.6-veronal, p H 6.7-phosphate, p H
5.6-acetate.
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 chroma-
t 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 Pepsin
Precipitation Initial
conditions soluble 5 rain. 1 hr. 5 rain. 1 hr. 5.5 hr.
pH 4.7 0.80 3.46 7.60 3.62 17.49 35.00
2% TCA 0.20 0.92 3.35 1.46 15.81 ........
12% TCA 0.20 0.27 0.98 0.58 2.37 ........
Phosphorus (% of total P)
pH 4.7 0.48 2.04 2.39 3.53 2.70 ........
2% TCA 0.15 1.79 1.51 0.77 1.05 ........
12% TCA 0.15 0.85 0.69 0.77 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 4.7-
soluble 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) inter-
mediate 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 reac-
tions 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 H

Schematic representation of paper eleetrophoretie bands of fractions of fl-easein ob-

tained 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 ob-
tained 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 electro-
phoresis (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 Pepsin
Reaction
time with Altered Altered Altered Altered
enzymes pH ~ fl-caseln fl-cas,ein fl-casein fl-casein fl-casein I ~-easein I I
(%)
5 min. 8.6 92 8 72 28 ........
I hr. 8.6 74 26 12 88 ........
1 hr. 6.7 ........ 11 89
1 hr. 5.6 ........ 16 S4 62 ~6:
5.5 hr. 5,.6 ........ 0 100 36 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 elec-
t 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 .

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(6) K E ~ , W. Die Abspaltung von Peptiden aus reinen Casein Fractionen dutch Kristal-
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