MICHIGAN STATE UNIVERSITY
fl

EAST LANSING, MICHIGAN

 

 

 

 

 

 

 

 

 

 

 

PHYSICAL AND CHEMICAL CHANGES PRODUCED BY
CHYMOTRYPTIC PROTEOLYSIS OF CASEINS

By

Rashid Ahmad Anwar

A THESIS

Submitted to the School of Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1957

 

 

 

 

ACKNOWLEDGMENTS

The author wishes to express his most sincere appreciation
to Dr. Hans A. Lillevik for his patience, deep interest, and
active guidance without which it would have been practically
impossible to complete this work.

Acknowledgment is also due to other members of the
chemistry department for help and advice from time to time,
and the.American Dairy Association for providing the funds in
support of this work.

In addition the author wishes to thank Miss Jane Rhae
Smith for technical assistance in the ultracentrifuge analysis.

Finally, he is grateful to all those who helped in the

completion of this manuscript, especially the one who helped
tracing the figures.

ii

 

VITA

Outline of Studies:
Major subject: Biochemistry
Minor subjects: Organic Chemistry, Chemical Engineering
Biographical Items:
'Born, October 15, 1930, Distt.Jullunder (India)

Undergraduate Studies: Panjab University Institute of
Chemistry, l9h8-1951.

Graduate Studies: Panjab University Institute of Chemistry,
1951-1952.

Michigan State University,
195h-1957.

Experiences: Lecturer Pharmaceutical Chemistry, Panjab
University Institute of Chemistry, Lahore
(Pakistan), l9S2-l95h;

Graduate Teaching Assistant, Michigan State
University, September, 1955-December 1956;

Special Graduate Research Assistant, Michigan
State University, January, l957-September, 1957.

Member of American Chemical Society, Society of the Sigma Xi,
and Pakistan Association for the Advancement of Science.

 

— “v—MV y.-:~_-' _— ~

 

 

 

PHYSICAL AND CHEMICAL CHANGES PROWCED BY
CHYMOTRYPTIC PROTEOLYSIS OF CASEINS .

By

Rashid Ahmad Anwar

AN ABSTRACT

Submitted to the School of Advanced Graduate Studies of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the
requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1957

Approved

 

 

 

 

 

 

ABSTRACT

A study was made of the action of chymotrypsin upon whole casein
and its purified alpha and beta fractions in more or less systematic
manner, since very little such work appears to have been reported with
this enzyme.

Proteolysis of 3 per cent caseins (whole, alpha or beta) with 0.010
or 0.0165 mg. crystalline chymotrypsin per m1. of digest at pH 7.5 was
studied by: electrophoresis; titration in aqueous, alcohol or acetone
media; conductivity change; and analysis for nitrogen and phosphorus
products made soluble in 10 per cent trichloroacetic acid (TCA).

By moving boundary electrophoretic analysis of isoelectric pre-
cipitable products in 0.1M veronal, pH 8.6, it was noticed that both
major components of whole casein gradually disappeared. Initially a
split in the alpha peak was observed but this was followed by increas—
ing development of both faster and slower peaks.

The same digestion mixture run at pH 5.6 by dilution with an equal
volume of l M acetate buffer produced a precipitate (at 30°C.) which,
after washing and reprecipitations, showed electrophoretically a single
component with a mobility of 5.3 Tiselius units. Repeating the experi-
ment upon pure preparations of alpha or beta casein produced the same
result. If a sample of the precipitate from alpha or beta casein was
mixed with a sample of the precipitate from whole casein the electro—

phoretic pattern of the mixture again showed a single peak of the same

 

 

II

 

 

 

mobility. This casein derivative from whole casein showed two peaks in
the ultracentrifuge with sedimentation coefficients of 7J6 (Svedbergs)
in peak 1, and 36.h.(Svedbergs) in peak 2, when extrapolated to zero
concentration. Its isoelectric point was found to be pH 6.1 and its

phosphorus and nitrogen were 0.3 and 15.1 per cent respectively.

The greater titration increaments of potassium hydroxide in alcohol,

shown by all casein chymotryptic digests (at 3000) at pH 7.5, compared
with those in either aqueous medium.or with hydrochloric acid in
acetone, indicate the liberation of acid groups additional to those
derived from peptide bond hydrolysis. This suggestion is further sub-
stantiated by the finding in these casein digests of phosphorus
products (mostly inorganic P) soluble in 10 per cent TCA.

The rate and extent of liberation of TCA soluble phosphorus was
greatest from digests of alpha casein and least from those of beta.
The inorganic portion of the total acid soluble phosphorus was greater
from all preparations. The organic phosphorus portion which was the
least of the total TCA soluble, was released more from whole casein
than.that from alpha casein.

One dimensional paper chromatography of TCA saluble products from
whole and alpha caseins showed 2 ninhydrin spots (peptides) with high
Rf values. The same two spots could be detected from early stages and
upwards to h hours of digestion. Beta casein TCA soluble products

showed mainly one spot with an Rf corresponding to the faster derived

from whole or alpha casein.

vi

 

 

 

 

 

 

 

The hydrolysates of TCA soluble peptides from whole alpha and beta

caseins showed in each case identically 12 amino acid spots by two

dimensional paper chromatography which were positively identified . In

addition to these residues TCA soluble peptides from whole and alpha

casein were found to contain tryptophan, showing that the fast moving

peptide(s) common to all 3 proteins did not contain tryptophan.

vii

 

 

 

TABLE OF CONTENTS

Page

I. INTRODUCTION............................................... 1
II. HISTORICAL................................................. 2
A. Casein and Its Fractions............................. 2

B. Enzyme Catalyzed Hydrolytic Reactions of Casein...... 5

1. Proteolytic Enaymes............................ 5

2. Effect of Phosphatases on Casein............... ll

3. Clotting of Casein............................. 12

C. Chymotrypsin......................................... 12

III. EXPERIMENTAL............................................... 16
A. Apparatus............................................ 16

B. Materials and Reagents............................... 18

C. Experimental Procedures.............................. 2h

D. Tables of Results. 38

E. Figures.............................................. h8

IV. DISCUSSION................................................. 75
V. SUMMERY.................................................... 91
BIBLIOGRAPHY 9h

APPENDIX1900000000000000000000000090.0000...09.000090000000000.-102

viii

 

 

 

LIST OF TABLES

TABLE

I Liberation of Acidic and Basic Groups During Proteolysis of
3% Casein Solution, using 0.0165 mg. of Chymotrypsin per
ml. of Digest, as Determined by Titration in Alcohol, Water
and.Acetone Media...........................................

II Liberation of Acid Soluble Phosphorus During Proteolysis of
3%m101-eCaseinWj-thCMOtIVpSinOOOOOOOO000.000.000.000.000

III Liberation of Acid Soluble Phosphorus During Proteolysis of
3%A1pha casein With CllymotrypSinOIOOOOIOO0.0.00.0...OI0....

IV Liberation of Acid Soluble Phosphorus During Proteolysis of
3% Beta Casein with Chymotrypsin............................

V Change in Conductivity During Proteolysis of 3% Whole,
Alpha, and Beta Caseins, with 0 .0165 mg. of Chymotrypsin
Per ml. 0f Digesto.00.00....0...0.0.0.000...OOOOOOOOOOOCOOOO

VI Non-Protein Nitrogen During Proteolysis of 3% Whole Casein
With ChymothSinOC.O...‘.....I....‘O0.000.000.0000....00...

VII Non-Protein Nitrogen During Proteolysis of 3% Alpha Casein
“Ii-til Chymotryij-nOUOCOCOOOOOOCOOOOOOCCCCOOOOOODOOOOOOOCOOOO.

VIII Non-Protein Nitrogen During Proteolysis of 3% Beta Casein
with CMGtryPSinoooooooo00000000000.000000900000000....0000

DI Electrophoretic Mobilities of a Casein Derivative Produced
by Chymotrypsin and Precipitable at pH 5 .6 at Different
Hydrogen Ion Concentrations.................................

X Approximate Sedimentation Coefficients of the asein
Derivative, Alpha Casein and Beta Casein at 21; In 0 .1 M
veronal Buffer pH 806......C....OOOOOOQOOOOOOOIIOOIOOCI.O

XI Some Physico-chemical Properties of the Casein Derivative
Produced by Chymotrypsin and Precipitable at pH 5 .6. . . . . . . . .

Page

38

39

LO

hl

I42

1:3

1:1;

145

I46

116

1:7

—’ ‘—'—-— ’F'ffit—im 4.11— I... 3.2:.

 

 

 

LIST OF FIGURES

FIGURE

1.

N

10.

Consumption of acid or base in alcohol, acetone and water
media, during digestion of 3% casein solutions with 0.0165
mg. of cmotr'y-psjn per ml. Of digestooonut-IIIIDOQOIOIbnoc

Liberation of phosphorus during proteolysis of 3% casein
solutions. Chymotrypsin concentration of 0.0165 mg. per
ml. ofdigest..-.I...I.....l.......l'..IIIUI'....III.I.I.IO

Liberation of phosphorus during proteolysis of 3% casein
solutions. Chymotrypsin concentration of 0.010 mg. per
ml. of digest....IOOCIIIIIOI‘30...OIOICIOODIIIOIQCOIOI'I...

Specific conductance changes during proteolysis of 3%
casein solutions. Chymotrypsin concentration of 0.0165
mg. perm-I of digest...‘I....'I...I.'...II......I....IIIQ.

Liberation of TCA soluble nitrogen and E280 absorbing
substances during proteolysis of 3% casein solutions.
Chymotrypsin concentration of 0.0165 mg. per ml. of

digeStOOIDIIIDCDOCIICODOOIOIOCOIIO.DIODIOCIIOIIDOIIOQI'UII'

Liberation of TCA soluble nitrogen and E280 absorbing sub-
stances, during proteolysis of 3% casein solutions.
Chymotrypsin concentration of 0.010 mg. per ml. of digest..

One dimensional paper chromatogram of TCA soluble peptides
from whole casein. Chymotrypsin concentration of 0.010
mg. per ml. ofdigest-OI.ClOIIIOOCIIIOOOIOIOOOOOIII.l.IIOII

One dimensional paper chromatogram of TCA soluble peptides
from whole, alpha and beta caseins.........................

One dimensional chromatogram of hydrolysates of TCA solu-
ble peptides. Chymotrypsin concentration of 0.010 mg. per
ml- of digest..-OI.IIIIO.III-00....DOOOOIIIUOIIO'OOOIOOIOII

One dimensional chromatogram of hydrolysate of TCA soluble
peptides. Chymotrypsin concentration of 0.0165 mg. per
ml. ofdigest-00000....CCDIOOOCIOIOIIDIDGO'IOQOICOII'OIIUOI

 

Page

h8

A9

50

51

52

53

56

5?

 

 

     
 

t 3’ I
. / ..

. / d
/ , ‘
. f ~ "I
o /' ‘
1‘ I. -
x x” " y
I I

 

 

LIST OF FIGURES - Continued

Figure Page

11, Two dimensional.chromatogram.of hydrolysate of TCA soluble
peptides from whole casein after 15 minutes digestion.. . . . . 58

12. Two dimensional chromatogram of hydrolysate of TCA soluble
peptides from whole casein.after h hours digestion......... 59

13. Two dimensional paper chromatogram of hydrolysate of TCA
soluble peptides from alpha casein......................... 60

1h. Two dimensional chromatogram of hydrolysate of TCA soluble
peptides from hem casein..............00.00.000.000.ICC... 6]-

15. Two dimensional chromatogram of known amino acids.......... 62

16. Two dimensional chromatogram of mixture of known amino
acids and hydrolysate of TCA soluble peptides.............. 63

17. Electrophoretic patterns, whole casein, alpha casein....... 6h
18. Electrophoretic patterns of whole casein during digestion.. 65

19. Electrophoretic patterns of alpha casein after different
digestion times............................................ 66

20. Electrophoretic patterns of beta casein during digestion... 67

21. Electrophoretic patterns of the casein derivative from
whole, alpha and beta caseins.............................. 68

22. Electrophoretic patterns of the mixture of the casein
derivative from whole and alpha casein and from whole and

bemOOOOCOOOOIOOOOIOOOOOOOOOOOOOOOIOOOOOO'O0.00.000.00.00.00 69

23. pH- Mobility curve of the casein.derivative from whole

“861110.00’0000000000000OOOOOOOOOOOOOCOOOCOO00......0.0...O 70

2h. Coneentration-Sedimentation coefficient curve of the
casein derivative from whole casein........................ 71

25. Ultracentrifuge patterns of the casein derivative from
“1019casejnOOCOCOCODCOUCCOOOOOCOOO0....OOIOOOOCOOOOOOOOOI. 72
26. Ultracentrifuge patterns of alpha caseina.................. 73

27. Ultracentrifuge patterns of beta casein.................... 7h

xi

  

-.' Ann's-m“ wh’hnrm -_,._‘__.__, ,

 

 

 

 

I . INTRODUCTION

Since the last quarter of the nineteenth century the biochemically
catalyzed reactions of casein with various enzyme preparations have
been the subject of many investigations. The principal aims of such
work have been of seeking information with regards to protein structure,
mechanism of reaction and biological significance of the protein and
its derivatives .

Very little of such information has been reported with catalytic
effect of crystalline chymotrypsin (an important proteolytic enzyme of
pancreatic juice) and no systematic examination appears to have been
done with this biocatalyst either on whole casein or its purified
fractions. .

The importance of casein in nutrition, the important role which
chymotrypsin plays in intestinal digestion and lack of information
concerning the action of chymotrypsin on casein and the nature of the
products resulting therefrom are strong enough basis to justify the
investigations carried out in this research.

In the. experiments to be described that follows, attempts have
been nade to study the action of chymotrypsin on whole casein and on
its purified alpha and beta fractions in more or less systematic manner
With regards to proteolysis, liberation of phosphorus and products

formed at different stages of digestion.

 

 

 

II . HISTORICAL

A. Casein and its Fractions

Casein, a phosphorus containing protein of milk, was one of the
first proteins to be isolated in relatively pure form. It is present
in the milk of all animals so far investigated and can be readily
precipitated by the addition of acid. Cow's milk, due to its avail-
ability, has been most completely investigated and same is true with
the principal protein product thereof; namely, bovine casein.
Therefore, the word casein generally refers to bovine casein unless
otherwise specified.

Mulder (66) in 1838 published a method for the separation of
casein from milk by acidification. Hannnarsten (32) prepared casein
from diluted skim milk by the addition of acetic acid. In general
practice, dilute hydrochloric acid is used for the preparation of acid
precipitated casein.

In 1956 Thugh (92) obtained a patent for the preparation of water
soluble casein. According to this procedure, casein was not exposed
to a higher hydrogen concentration than that of milk. He precipitated
casein by the addition of calcium chloride to skim milk and showed his
preparation to be highly mter soluble (30%) as compared to the acid
precipitated preparation (9-10%) . Waugh‘s water soluble casein was
recently shown to have close resemblance to acid precipitated casein by

Nielsen (67) from his studies on its osmotic pressure, molecular weight

and electrophoretic behavior.

 

 

 

 

 

Casein was considered to be a pure protein for a long time largely

due to the work of Harrmmrsten. The first evidence of its heterogeneity
can be attributed to the work of Osborn and Wakeman (72), who in 1918
isolated a small amount of alcohol soluble protein from isoelectric
casein. At that time this protein was merely considered to be the
contaminant. In 1925 Linderstrdm-Lang and Kodama (51) from their
solubility studies of casein in acid solutions, showed that it is a
mixture. Later in 1929 Linderstrdm-Lang (52) was able to obtain
fractions, differing greatly in phosphorus content and several other
properties, by treatment of casein with ethyl alcohol and hydrochloric
acid and precipitating the protein from the extracts with sodium
hydroxide. The fractionation studies of Cherbuliez and Meyer in 1933
(9) and Cherbuleiz and Schneider in 1932 (10) also give a strong support
to the heterogeneity of casein.

Groh at El. (28) in 1931:, reported on the separation of casein by
three different methods, namely fractionation by 1) urea, 2) phenol,
and 3) alcoholic ammonium hydroxide .

Hollander in 1939 (6)4) was the first person to demonstrate
Electrophoretically and beyond doubt the presence of at least three
distinct and more or less homogeneous components of casein,‘and
designated them as alpha, beta, and 33mm in the decreasing order of

their mobilities. At about the same time Cherbuliez and Jeanerat in
1939 (11), had successes in isolating a fourth component of casein
and named it delta casein, which appeared to be identical with the

Whey protein of Hammrsten.

 

 

 

 

Warner in 191;); (91) reported the isolation of each of the two

distinct fractions alpha and beta from whole casein, based on the
higher solubility of beta casein at pH 1.5 and 2° c. He, however,
pointed out that his fractions although distinct were not electro-
phoretically homogeneous at all pH's, particularly below their iso-
electric points.

In 1950 Hipp gt 2.2;. (38) separated the gamma fraction from whole
casein by taking advantage of its solubility in fifty per cent ethyl
alcohol. It was shown to be identical with the alcohol soluble protein
earlier described by Osborn and Wakeman (72). Based on the solubility
in 5 per cent ammonium sulfate at pH 6 and hOOC Cherbuliez and Baudet
(12) the same year isolated two subfractions from alpha casein, with
almost identical phosphorus, tyrosine and tryptophan content. The
soluble portion was designated as alpha-1 and insoluble as alpha~2
casein.

Hipp gt 3.1. in 1951 (1?) and 1952 (18) published several success-
ful methods for the fractionation of casein. Their urea method was
patented in 1955 and appears to be the most practical one at the present
time. Von Tavel and Signer (89) separated alpha and beta casein by
counter current distribution, using phenol-dwater-ethanol or phenol-
water-acetic acid as solvent system.

Waugh 3’3 9.3;. (93) in 1956 reported the presence of another ‘com-o
ponent in casein which they designated as kappa casein. On treatment
of once calcium precipitated casein described earlier (92) with 0.25M

calcium chloride at 37°C and pH 7 the alpha component was observed to

 

ll

 

 

 

 

 

 

dissociate and show a new component (named kappa) in the ultracentri-

fuge. Alpha and beta caseins rapidly precipitated by this treatment
but kappa casein tended to remain in the supernatant. They have
reported its phosphorus content to be less than 0.5%.

McMeekin and co-workers (63) at the American Chemical Society
Meeting of April, 1957, in Miami, Florida, reported the isolation of
fourth component from acid precipitated casein, which was other than
alpha, beta or gamma, and designated it as alpha-2 casein. The original
alpha casein minus the alpha-2 casein has been designated by them as
alpha-1 casein. Alpha-2 casein of McMeekin has phosphorus content of
0.1 to 0.15% and electrophoretic mobility of 5.0 Tiselius Units at pH
8.1; in 0.1M veronal buffer.

The properties of several casein fractions isolated thus far by
various workers are summarized by Nielsen (67) in the form of a table.
In conclusion, nothing can be said as to the total munber of
components present in casein. However, it may be pointed out that

for the purpose of investigations to be reported in the following
Pages, the alpha and beta components which comprise the major share

01' casein were isolated by the urea method of Hipp gt a}: (37) and

used .

3' Enzyme Catalyzed Hydrolytic Reactions of Casein

(1) Proteolytic Enzymes.

The PrOgress or research in the field of protein hydrolysis by
pmteOJ-Ytic enzymes has been slow in spite of the fact that the

proteins are the natural substrates for many proteolytic enzymes.

 

 

 

This is mainly due to the complex nature of proteins.

Enzyme catalyzed hydrolysis reactions upon casein have been under
investigation for a long time and the clotting of milk is perhaps the
oldest such enzymatic reaction known. Rimington and Kay (79) have
written a comprehensive historical smunmy of the work prior to 1926
concerning the action of pepsin and trypsin on casein.

Lubavin (56) in 1871 reported that a greyish deposit was gradually
formed when gastric Juice was allowed to act upon casein. It contained
phosphorus varying with the conditions of experiment. In 1891 this
greyish precipitate was given the name of paranuclein by Kossel (15)
and pseudonuclein by Hammarsten in 1893 (31) due to its physical
similarity with the insoluble nuclein produced from nucleoproteins by
the action of pepsin.

Salkowski and Hahn (1895) showed that in the presence of sufficient
pepsin the whole of the precipitate (paranuclein) goes into solution
(80). This observation was confirmed by Krehl and Matthes (I46) in the
same year and three years later by Alexander (3), but questioned by
Moraczewski (65).

Sebelien (83) also in 1895, working with crude pancreatic enzymes
(at that time called trypsin) showed that, unlike the action of pepsin,
no Paranuclein was formed but that the whole of the casein, except for
a negligible residue, went into solution. During tryptic proteolysis
0f casein Biffi in 1898 found that about 27% of the soluble phosphorus
°°u1d be precipitated by magnesia mixture (7). This observation was

later continued by Plimmer and Bayliss (1906) who reported the presence

 

 

 

 

__ __HH -——u---I- e.-

 

of, on the average, 35% of the phosphorus as phosphoric acid after
tryptic digestion (76).

According to Salkowski, 1899, pepsin first transforms casein in
such a way that no precipitate is obtained by the addition of acetic
acid and after that the separation of paranuclein begins gradually (80).

Plimmer and Bayliss (76) during 1906 also studied the rates of
separation of phosphorus from casein by trypsin, pepsin, papin and
alkali. From their studies they concluded that total trichloroacetic
acid (TCA) soluble phosphate was released in a way similar to that of
the acid soluble nitrOgen. Papain when allowed to react in neutral
media upon casein produced results similar to trypsin, whereas pepsin
was much slower and did not completely solubilize the protein phosphorus.

Rimington and Kay (1926) while studying the action of pepsin,
trypsin, bone and kidney phosphatases on casein made the following
observations: 1) No inorganic phosphorus was liberated by the action
of pepsin even after nine days. 2) Paranuclein containing a large
proportion of the original casein phosphorus was obtained. 3) Trypsin
brought about the complete hydrolysis of the organic to inorganic
Phosphorus in a slow process through an intermediate phosphopeptone
Stage. 1;) No hydrolysis was observed with bone phosphatase but there
was slight action with kidney phOSphatase.

In 1927 Posternak (77) digested casein with trypsin and from the
digest isolated a phosphopeptone, containing 5.9% phosphorus, 11.9%
nitrogen, and it was composed of glutamic and aspartic acids, serine
and isoleucine. He indicated that phOSphoric acid was bound to the

hydroxyl group of serine.

 

 

 

 

For the degradation of casein Levene and Hill (149) in 1933 used
trypsin and isolated a phosphodipeptide with the aim of finding out
where phosphate is attached and to what amino acid. They proposed the
structure of this dipeptide to be either phospho-seryl-glutamic acid or
glutamyl-serine-dphosphate. At about the same time Lipmann (55) iso-
lated phosphoserine from casein and thus proved that phosphoric acid
is attached to serine.

In 1935 the effect of various substances e.g. carbohydrates, heavy
metal salts, bile salts, etc., on the hydrolysis of casein by pancreatic
proteases was studied by Farber and Wynne (22). Theyfound that
carbohydrates and bile salts inhibited the enzymes whereas the heavy
metals had no effect. Damodaran and Ramachandran (16) in l9h1 digested
casein initially with pepsin to a paramclein containing 50 to 60% of
the total phosphorus and 20% nitrogen and then with trypsin until
constant amino nitrogen was obtained. Fromsuch a digest they were
able to isolate the barium salt of a phosphopeptone, containing h.314%
phosphorus and 6.146% nitrogen, which was composed of glutamic, iso-
leucine and serine residues.

Horwitt in 191414 (140), studied the first stage of casein hydrolysis
by chymotrypsin and crystalline trypsin. He observed that after the
addition of 1 mg. of chymotrypsin in 1 ml. of water to 10 ml. of 6 per
cent casein, pH 7.5 at 50°C, the solution became opaque in 1 minute and
Suggested that this could be used to determine the amount of chymotrypsin.
Similar reaction was observed with trypsin when used in greater amount.

Winnik (96) in 191114 studied the action of pepsin, trypsin, chymotrypsin,

 

 

 

 

papain and ficin on casein. After prolonged treatment the average

molecules were approximately pentapeptides in digests with chymotrypsin,
ficin or papin and heptapeptides in the pepsin and trypsin digests.

They also reported the liberation of 1 to 3 per cent of the total
nitrogen as free amino acids, determined as carboxvl nitrogen. This
study was made on prolonged hydrolysis and with large quantities of

the enzymes as compared to the work to be described in this investi-
gation.

The effect of proteolytic enzymes on raw and heated casein was
investigated by Eldred and Rodney (21) in 19146. They first treated
casein with pepsin at pH 1.8 and then with trypsin and chymotrypsin at
pH 7.8 for three to four days. The digestibility of raw and heated
casein did not differ; whereas available lysine was found to be less
in the case of heated casein as determined by the specific enzyme
lysine decarboxylase. Reisen at. al. (78), using pepsin, whole
"pancrease" and erepsin, obserVed that longer heating of casein decreased
the rate of enzymatic liberation of amino acids.

A comparative study in 19147 of the liberation by pancreatin of
four amino acids from casein; namely, tyrosine, tryotophan, histidine,
511d arginine was made by Beck (5), and he observed that tyrosine was
liberated most rapidly.

Hoover and Kokes (39) the same year observed that the digestion
of casein by papain was characterized by a rapid production of peptides,
averaging four to six units, followed by the release of amino acids

Without much change in the average length of the peptides present.

 

 

   

10

They also pointed out that Winnik's experiments were concluded at a
point when the production of amino acids was Just becoming appreciable.
After six days of digestion with trypsin Sullivan 33; _a__l;. (86)

showed that 1:6 per cent of the total amino acids were liberated from
raw casein, whereas 35 per cent from Vitamin free and 26 per cent from
comercial dried casein became released. Benton and Elvehjem (18)
digested bovine casein and zein with pepsin at pH 2.0 followed by
pancrease and duodenal powder at pH 8.0. They noticed wide variations
in the liberation of amino acids from casein and zein when measured by
biological assay methods, whereas by chemical methods the rate and
actent of liberation were approximately the same.

Christensen (13) in 1951; reported his observations about the
action of the proteolytic enzymes plasmin, trypsin and chymotrypsin on
casein proteins. He studied the action of these enzymes on whole,
alpha, and beta caseins, with respect to viscosity changes and libera-
tion of acid soluble material as determined by E280 absorbancy measure-
ments. From the data he concluded that proteolytic hydrolysis of
casein does not follow a simple course but that several apparently
independent reactions occur and that the complexity of the reaction is
due to factors in addition to the presence of several proteins hydro-
lyzing at different rates.

Peterson at E}: (75) in 1951; separated the primary products formed
from beta casein by the action of trypsin. They were able to obtain
a. fraction free of phOSphorus and another fraction containing 3 P81;

cent PhOSphoms. They also reported the further fractionation 0f the

 

 

 

 

ll

digest into components, which were essentially electrophoretically
homogeneous.

The isolation of a pressor material, pepsitensin, produced by the
action of pepsin on casein was reported in 1955 by McGlory 9:2 §_._l_. (62).
It is apparently a polypeptide or a mixture of similar polypeptides
and a true product of enzymatic action rather than of autolysis.

PhosphOpeptones, obtained from alpha and beta casein by partial
hydrolysis with pepsin were isolated by Grove gt _a__l_. (29) in 1956.
They showed that a phosphOpeptone gel from alpha casein was insoluble
at pH 14.7 and contained essentially one component by electrophoresis,
whereas the phosphopeptone from beta casein was largely soluble at pH

14.7, insoluble at pH 3.5 and contained two components in equal amounts.

(2) Effect of Phosphatases on Casein.

An extensive investigation on the effect of phosphatase enzymes on
casein and its separated fractions was summrized in 1956 by Perlman
(73). From her results, she has been able to throw light on the nature
of phosphate bonds in alpha and beta caseins and has also explained
Why in certain cases phOSphatase has not released inorganic phosphorus
from whole casein.

Sundrarajan and Sarma (87) in 1956 reported the formation of
dePhOSPhOI'ylated casein by the action of ox spleen phosphoproteinphos-
phatase upon whole casein. They also studied the nature of acid soluble
nitrogenous products formed during enzymic dephosphorylation of casein

by one dimentionei paper chromatography. They concluded that during

 

 

 

 

12

the enzymic dephosphorylation of casein, the protein remained relatively
intact.

(3) Clotting of Casein.

The enzyme studied most for the clotting of casein is rennin.
Chymotrypsin also has milk cotting activity. The nature of the change
produced in casein when enzymically clotted is not yet fully understood.
Nitschmann and co-vworkers (l, 61, 61a, 69, 69a) have published a series
of papers in recent years, concerning the action of rennin on casein.
According to the knowledge at hand, clotting of casein with rennin is
considered to be a three step reaction; 1) Casein is changed to modi-
fied casein with the simultaneous liberation of non protein nitrogen,
2) Modified casein undergoes moderate thermal denaturation which takes
place above 15° C., and 3) Denatured casein crosslinks with calcium

ions and gives a clot.

C. Chymotrypsin

Chymotrypsin is one of the three major proteolytic enzymes of
pancreatic Juice, the others being trypsin and carboxypeptidase. Prior
to the individual separation of these enzymes, the combined activity
was believed to be due to a (single enzyme called trypsin. The term
trypsin is now applied to only one enzyme of this group.

Although the isolation and characterization of chymotrypsin was
first reported by Kunitz and Northrop (h8) in 1935, it was shown as
early as 1902 by Vernon (90) that the activity of pancreatic extract as

determined by the clotting of milk could be separated from proteolytic

 

 

 

13

activity as determined by direct methods detecting hydrolysis. He con-
cluded that there were two enzymes. He also showed that one of these
was more stable than the other and that the activation of the extract
was caused by the less stable one.

Trypsin and chymotrypsin do not exist as such in the pancreas but
as proenzymes, called trypsinogen and chymotrypsinogen. The activation
of chymotrypsinogen was first studied in 1935 by Kunitz and Northrop
(1;8). Jacobson (142) studied rather in some detail in 191.4? the activa-
tion of chymotrypsinogen. Since then a number of investigators have
worked in this field and have isolated several different chymotrypsins,
with practically the same activity and specificity. Their findings
indicate the complexity involved in the activation of this proenzyme.
According to Janddorf and Michel (1;3) in 1956 "Many of the postulated
intermediates may well be the result of proteolytic processes or changes
in the physical state of the proteins, and their importance in the min
pathway of activation is largely unknown at present." Some of the
different chymotrypsins are alpha, beta, gamna, delta and pi.

Sometimes, in enzymic studies, it becomes desirable to inhibit the
enzyme in such a way that the inhibiting agent does not effect the
substrate. Inhibition of chymotrypsin has been studied by a mnnber of
workers among which Ball and co-workers (141;) are the leading investi-
gators.

Ball and Jansen (1;) wrote a comprehensive review on the

stoichiometric inhibition of clwmotrypsin. Their own work was mainly
cOncerned with the inhibition of chymotrypsin in the absence of sub-

strate, with a view towards finding the active site.

 

 

 

 

In 19145 Sizer (81;) concluded from his work that sulfhydryl or

disulfide groups are not essential for chymotrypsin activity. Wood
and Ball (97) in 1955 using partially purified horseradish enzyme
showed that oxidation of tryptOphan residues reduces the enzynntic
activity of chymotrypsin.

Cohen gt 2.1. (114) on the basis of their work in 1955 suggested
that the final position of the dialkyl phosphoryl group introduced into
the chymotrypsin molecule by di-l-isopropyl fluorophosphate (DFP) was at
the hydroxyl group of a serine residue. 0n the other hand photoxidation
studies in 1953 by Weil gt. g1 . (91;) showed that chymotrypsin was com-
pletely inactivated and no longer reacted with or? when one histidine
and three tryptophan residues were destroyed.

In Gutfreund and Sturtevant (30) in 1956 presented the evidence that
both, serine hydroxyl and imidazole groups are important for proteolytic
enzyme activity. Also in the same year, Massey and Hartley (60)
supported the view that histidine is the active center of chymotrypsins.

In spite of all this work, no suitable procedure for the inhibition
0f Chymotrypsin in the presence of substrate appears to have been worked
out. Schwert 915.1. (81) while studying the amidase activity of trypsin
and Chymotrypsin in 19148 used saturated potassium carbonate to liberate
“Malia and assumed that this kind of enzyme activity stopped when the
reaction mixture came in contact with the reagent.

Gergely gt g1. (25) in 1955 used di-isoprOpylfluorophosphate to
stop the chymotryptic digestion of myosin. Li 33 9.3.. (50) a year later

Stated but without any evidence that one drOp of glacial acetic acid

 

 

15

served to stop the reaction of this enzyme upon hypophyseal growth

harmone, a polypeptide.

In 1956, Harris (33) demonstrated that chymotrypsin is irreversibly

denatured with 8 M urea. However, he expressed the view that in the

presence of substrate, the enzyme is stabilized to a considerable

extent against this urea inactivation.

‘MOst of the detailed physicochemical measurements have been

carried out with alpha chymotrypsin and some of them are as reported

below.

Nitrogen 15.5% (71)
Isoelectric point 8.1-8.3 (h?)
Sedimentation constant 320w" 2.5 S (79)
Molecular weight 27,000 (9h)
Crystalline form Rhombohedrons (71)

pH optimum for casein
digestion 7-9 (71)

pH optimum for coagulation 6.5-7.0 (57)

 

16

III . EXPERDIENTAI.

A. Apparatus

Tmerature Control - A constant temperature bath with a 1/14 inch
plate glass front window and constructed in the Kedzie Chemical Labora-
tory was used for controlling the temperature. It was provided with a
reservoir bottle to maintain autonatically a constant level of water.
The R. B. Instrument Company thermoregulator with Fisher Serfass

electronic relay controlled the temperature at 30 1: 0.0300.

pH Meter -- A Beckman Model H2, glass electrode, line operated pH

meter was used for hydrogen ion activity measurements.
Timer -- A Meylan stop watch was used to time the reaction periods.

Glassware -- All pipette and volumetric glassware used were of

Kimble glass brand.

Spectrophotometers -- Absorbance measurements at 280 xgl were wade
uSing the Beckman Model DU spectrophotometer. The Beckman Model B

Spectrophotometer was used in the determination of total and inorganic
PhOSphorus.

Centrifuges -- The International Model 2 centrifuge was used in
Preparation of acid precipitated casein. This was equipped with a
basket attachment. For the determination of inorganic phosphorus an

International clinical centrifuge with a size 213 rotor for 15 ml.

 

 

17

centrifuge tubes was used. The Servall refrigerated centrifuge with
size SS-l rotor for 50 ml. stainless steel tubes was used in the
preparation and purification of protein precipitated at pH 5.6 after

action. of chvmtrypsin on whole, alpha and beta caseins.

Dialysis -- All the dialyses were made in Visking cellophane tub-

ing, on an external rotating liquid dialyzer constructed by Djang,
Lillevik and Ball (19).

Electroghoretic Analyses -- Were made with the Tiselius electro-
phoresis apparatus Model 138 (Perkin Elmer Corp.) . For conductivity
measurements, the Model RC-IB conductivity bridge (Industrial Instruments

Inc.) equipped with a cell (Perkin Elmer) of 0.14893 constant was used.

Freeze DIES, ~- Was carried out with the Virtis Freeze Dryer
(Virtus Co.) .

Digestion Rack - Was one manufactured by the American Instrument

Co., and was used in the digestion of samples for nitrogen and total

PhOSphorus analysis .

Semi-micro Kjeldahl Amatus -- Fifty ml. digestion flasks were

used for the digestion of total phosphorus and nitrogen samples. The

distillation apparatus employed was one modified and used in the Kedzie
Chemical Laboratory .

9W -- The Chromatocab Model B, (Research Equipment

Corp.) was used for descending runs and ascending chromatograms were

 

18

developed in the chromatography cabinet manufactured by University
Apparatus Co. The chromatograms were dried in the (Research Equipment

Corp.) oven constructed for this purpose.

Electromagetic Stirrer - An electromagnetic stirrer (Labline Inc.)

was used in the alcohol, acetone and water media titration work.

Analytical Ultracentrifuge -- The Spinco Model E (Specialized

Instruments Corp.) was utilized for studying the sedimentation behavior

of proteins .

B. Materials and Reagents

Chemicals - All inorganic and organic chemicals used were either

C. P. or reagent grade unless otherwise specified.

Mme Source -- Crystalline chymotrypsin (salt free from ethanol)
supplied by the Nutritional Biochemicals Corp. , Cleveland, Ohio, in

one gram quantities was used.

Estrates - Acid precipitated casein was prepared from cow's
fresh raw skim milk by the procedure described by Dunn (20). It con-
tained 15.9 per cent moisture and 15.29 per cent nitrogen on moisture
free basis, and was stored at -20°C until used. Electrophoretic behavior
01' the preparation in 0.1M veronal buffer of pH 8.6 is seen from
(Figure 17) and appears similar to the one shown by Hipp _e_t_ 2.3;. (37).
Alpha and beta caseins were very kindly supplied by H. C. Nielsen
who Prepared these according to a modification described in his

doctoral dissertation (67) of the urea procedure of Hipp 23 El. (37)-

 

 

 

19

Alpha casein contained 2.6 per cent moisture whereas beta had 3.8 per
cent moisture. Their electrOphoretic patterns were also similar to

those obtained by Hipp gt 2.1. (37) under the same conditions.

Casein Stock Solutions - Six grams of air dried whole, alpha or
beta casein was weighed into a 125 ml. erlenmeyer flask. Sixty to
seventy ml. of glass distilled water was added in small portions until
a smooth paste. (Distilled water mentioned hereafter refers to glass
distilled water.) To this was then gradually added 16 ml. of 0.2N
SOdium hydroxide in the case of whole and beta caseins and 20 m1. of
0.2N sodium hydroxide was used in the case of alpha casein. A mechanical
shaker was used to disperse the proteins. After diapersion the solu-
tion was heated in a boiling water bath for 15 minutes, cooled to room
temperature and its pH was adjusted to 7.5 by the dropwise addition of
0.2M sodium hydroxide. The pH 7.5 solution was then quantitatively
transferred to a 100 ml. volumetric flask, made to volume, filtered
and supplied with a crystal of thymol as preservative. It was always
Stored in cold room at 5°C and used within two weeks of its preparation.

Time was produced a 6 per cent (w/v), pH 7.5 stock solution for use as
substrate in the enzymatic studies.

Wen Stock Solution -- Five mg. of crystalline chymotrypsin

was weighed into a 50 m1. volumetric flask and dissolved to volume with
distilled water.

MPer Cent (wig) Trichloroacetic Acid, -- Twenty grams of tri-

Chloroacetic acid was dissolved in water and volume made to 100 m1.

 

 

 

 

 

20

For ten per cent trichloroacetic acid, the above solution was diluted

with equal volume of distilled water.

Fisk-Subbarow Phosphorus Analypis Reagents, -- These reagents were
prepared as described by Hawk, Deer and Summerson in the thirteenth

edition of their text Practical Physiological Chemistry (31;) .

_R_e_§.gents for Separation and Analypis of Inorganic Phosphorus -

§_ij_e_1\_1_ sodium hydroxide—one hundred grams of sodium hydroxide was
weighed out on an analytical balance, transferred quantitatively to a
500 m1. volumetric flask, and diluted to the mark.

0.5N sodium hydroxide—twenty grams of sodium hydroxide was weighed
on an analytical balance and transferred quantitatively to a 1.0 liter
volumetric flask, and diluted to the mark.

10 Per cent (w/v) calcium chloride reagent-«ten grams of calcium
chloride was dissolved in an ammonium chloride buffer pH 9.0 (prepared
as below), diluted to 100 ml., and saturated with ammonium hydroxide.
The reagent was good for one week, when stored in Pyrex bottle and

filtered just before use.

Wash reagent was a one to five dilution of the above ten per cent

calcium chloride with distilled water.

Ammonium Chloride buffer pH 9.0-was prepared by dissolving 26.7
gms. of ammonium chloride in water and adding concentrated ammonium

hydroxide gradually until it reached pH 9.0.

 

21

Bromoflml Blue Indicator for use in inorganic phosphorus analy-
sis was prepared by dissolving 0.0h gm. bromothymol blue in 100 ml. of

95 per cent ethanol.

10 N Sulfuric Acid--Accurately measured 280 ml. of concentrated
sulfuric acid was diluted with water transferred quantitatively to one
liter volumetric flask and made to volume by the addition of distilled

water .

Isobutanol Benzene mixture was prepared by mixing equal volumes of

isobutanol and thiophene free benzene.

10 Per cent Ammonium Molybdate-chcurately weighed 10 gms. of
ammonium molybdate was dissolved in distilled water and volume made to

100 ml,

2.2 Per cent (v/v) Sulfuric acid in Absolute Ethanol-~Thirty-two
ml. of concentrated sulfuric acid was dissolved in 968 m1. absolute
ethanol.

Stannous Chloride stock solution--Ten grams of starmous chloride
(Dihydrated) was dissolved in 25 m1. concentrated hydrochloric acid
and kept in a refrigerator.

Stannous Chloride working solution-~Stock solution of stannous
Chloride was diluted 200 times with 1N sulfuric acid. This was always

freshly Prepared immediately before use.

3.31 Sulfuric Acid was prepared by diluting 10 N sulfuric acid ten

times with distilled water.

 

22

Reagegts for Nitrogen Determinations
’7

Digestion Mixture--Five hundred m1. of concentrated sulfuric acid

was added to 500 m1. of distilled water containing 100 gms. of sodium

sulfate and two gms. of copper sulfate.

2 Per cent (w/v) Boric acid was prepared by dissolving 20 gms. of

boric acid in distilled water and making the volume to one liter.

Boric Acid-«Indicator solution--Two m1. of freshly prepared 0.02
per cent methyl red and one m1. of 0.02 per cent methylene blue was
added to 100 ml. of two per cent boric acid solution. It was always

prepared just before use.

6 N Sodium Hydroxide-~Accurate1y weighed 2).;0 gms. of sodium
hydroxide was quantitatively transferred to one lite-:- volumetric flask,

dissolved in distilled water and volume made to the mark.

Veronal buffer pH 8.6 ionic strength--0.1 was prepared by dis-
solving 21.197 gms. of veronal (5.5 diethyl barbituri-c acid U.S.P.)

and 0.1 mole of sodium hydroxide in distilled water and making the

volume to one liter.

0.05 N Alcoholic Potassium Hydroxide-3.75 gms. potassium hydroxide

 

was dissolved in 62.5 ml. distilled water and diluted to one liter
With 95 per cent ethanol. The reagent was standardized against 0.106? N

hydrochloric acid with phenolphthalein as indicator.

Emlphthalein Indicator-«The indicator solution for the

 

 

23

“1115tatter and Waldschmidt-Leitz (1921) titration was prepared by
diluting Six m1. of 0.5 per cent thymolphthalein in 95 per cent ethanol

to 100 ml. with absolute alcohol.

0.05 N Alcoholic Hydrochloric Acid--O.2 m1. of concentrated hydro-

 

chloric acid was diluted to one liter with 90 per cent ethanol and

finally standardized. against 0.05 N alcoholic potassium hydroxide using

phenolphthalein as indicator.

Naphthyl Red Indicator-«0.1 gm. of Naphthyl red (h-benzene-azo-
naphthylamine-l) was dissolved in 96 per cent alcohol and volume made

to 100 m1.

1 M Acetate Buffer-Mas prepared by the gradual addition of one

 

normal sodium hydroxide to one mole of acetic acid (57.1; ml. of glacial
acetic acid) until the required pH 5.6 was attained. A buffer of pH

14.6 was also similarly prepared. Approximate amounts of sodium hydroxide
required in each case were precalculated using Henderson-Hasselbach

equation as described by Gortner (26) .

Other Buffers-«All other buffers used in electrophoretic determin-

ations were of ionic strength 0.1 and necessary amount of monobasic
acid required for 0.1M sodium hydroxide was calculated using Henderson-
Hasselbach equation. For phosphate buffers both the Lewis ionic
strength equation (58) and the Henderson-Hasselbach equation ( 26) were
solved Simlltaneously to get the necessary amounts of acid and alkali.

In every case the pH was checked and adjusted on the pH meter.

 

 

 

2h

Solve t S Stems for Pa er Chromato a h

 

Butanol : acetic acid : water (h:l:5) was prepared according to
Slotta (1951) (85). For the preparation of water saturated phenol, 39
ml. of distilled water was added to 100 gm. of phenol and made 0.1 per
cent with respect to alpha benzoin oxime as recommended by Consden
gal. (15).

2,6-Lutidine : collidine : water (1:1:1) was made according to

Dent (l7), and was added 1-2 per cent diethylamine.

Emdrin solution for the detection of spot was prepared by dis-

solving 0.1 gm. of ninhydrin in 100 ml. of ethanol containing 5 per cent

v/v collidine .

C. Experimental Procedures

mac Digestion -- A suitable volume of 6 per cent casein
solution (alpha, beta or whole) was pipetted into one arm (50 ml.
Capacity) of a bifurcated test tube. Into the other arm was added an
equal volume of suitable diluted chymotrypsin solution (0.01 per cent
w/v stock solution diluted one to three or one to five was used). When
the total volume of the digestion mixture was expected to be more than
140 "11., two 125 ml. Erlenmeyer flasks, one for the substrate and the
other for the enzyme solution, were used. The digestion vessel(s)
Has/were placed into a constant temperature water bath held at 300C.
Before mixing, the solutions were allowed to stand for 20 minutes in
the bath to bring them to temperature. The digestion was started by

lelng the two solutions thoroughly. The time of initial contact of

 

 

 

 

 

 

25

the W0 SOlutions was taken as zero digestion time and was noted by
starting the stop watch. Appropriate aliquots of digestion mixture
were removed at specified times and proteolysis arrested for the type

of analysis to be described.

Alcoholic potassium hydroxide titration for total acidity change-

The enzyme concentration used was 0.0165 mg./ml. of digest (stock solu-
tion diluted one to three). One ml. aliquots were removed from the
digestion mixture at intervals and were immediately titrated in alcohol
according to the method of Willstatter and Waldschmidt-Leitz (95).
This method is a modification of Foreman‘s (Zha) original alcoholic
sodium hydroxide titration. The aliquots removed were directly
pipetted into three ml. of absolute alcohol-indicator mixture contained
in 25 x 100 mm. test tubes. Each sample was then titrated against 0.05
N alcoholic potassium hydroxide solution to a distinct blue color;

Six ml. of absolute alcohol was added and the sample again titrated to
the appearance of permanent blue color. A five ml. burette calibrated
to 0.02 ml. was used. The sample was kept well stirred during the
Process of titration with the aid of electromagnetic stirrer which

also aids in keeping minimum time for minimum carbon dioxide inter-
ference. The initial titer obtained from the aliquot taken immediately
after mixing (zero time) was subtracted from subsequent titers to get
the increment in ml. (A ml.) of standard alcoholic potassium hydroxide
required for titration of the acid groups produced during digestion,
per "11- 0f the digest. The results obtained are reported as PM of

POtassium hYdroxide required to neutralize the acid groups produced

 

 

26

(mi-311% digestion, per ml. of the digest in Table I and shown in

Figure 1. .

Ageous potassium hydroxide titration-- For aqueous titrations,

one ml. aliquots were removed from the same digestion mixture at
specified intervals of time and added directly to 9 ml. of distilled
water containing thymolphthalein indicator, already placed in 25 x 100
mm. test tubes. Each aliquot thus removed was immediately titrated
against the same standard alcoholic potassium hydroxide, using the same
burette as mentioned above, to the appearance of blue color. The solu-
tion was kept well stirred during titration with the help of an
electromagnetic stirrer. The initial titer obtained from the aliquot
taken immediately after mixing was subtracted from the subsequent titers
as mentioned in alcoholic potassium hydroxide titration. The results
were also treated and expressed in the same manner as in alcoholic

potassium hydroxide titration. See Table I and Figure 1.

Linderstr m-Lan 's Acetone titratiOn with drochloric acid (53)--
In principle the procedure followed was the same with minor modifi-
cations as discussed in detail by Jacobson (I42) . The concentration of
Chymotrypsin was 0.0165 mg./ml. of digestion mixture. Two ml. aliquots
°f diSest were removed at intervals and quantitatively transferred to
25 x 100 mm. test tubes, containing a pro-determined quantity of
aqueous 0.1067 N hydrochloric acid. This amount (0.3-0.6 ml.) was such
that the mixture of acid, protein solution and acetone together with

Indicators in the beginning of the experiment showed a suitable

 

 

2 7

redeyellow color. Each aliquot was then immediately titrated with
small increments of 0.05 N hydrochloric acid to the appearance of a
permanent red color, during which time eight ml. of acetone was gradually
supplied. Not all of the acetone was added to the sample tube before
starting the titration but was added in portions to avoid protein
precipitation. A burette of one ml. capacity and calibrated to read
to 0.01 ml. was used. During titration the solution was well stirred
with the aid of an electromagnetic stirrer. The initial titer obtained
from the aliquot taken immediately after mixing (zero time) was sub-
tracted from the subsequent titers. Thus the increment ml. (A ml.) of
standard alcoholic hydrochloric acid required to titrate the basic
groups produced during digestion was obtained from 2 ml. of the digest.
The results obtained are reported in terms OfJJM of hydrochloric acid
required to neutralize the basic groups produced during digestion per

ml. of the digest in Table I and shown in Figure l.

gonductivity changes -- The resistance changes during proteolysis
of digests containing 0.0165 mg. of chymotrypsin per ml. of the digest
were recorded at desired time intervals. The specific conductance was
calculated from the ohm3 measured and cell constant value of 0.h893 and

is reported in Table V.

Inhibition Studies of Chymotrypsin Activity on Caseins - To study
the electr0phoretic changes produced during digestion, it was desirable
to inhibit the chymotrypsin activity at desired tims intervals and yet

produce Practically no effect on the protein or products under

 

 

28

investigation. A number of possibilities were tested. The exact pro-
cedul‘e was as follows. To three ml. of six per cent casein solution
we added an equal volume of diluted 0.1 per cent (w/v) chymotrypsin
solution (stock solution diluted one to five). Immediately after mix-
ing, a three ml. aliquot of the solution (pH 7.5) was removed and
accorded the inhibition treatment under consideration. (These treat-
ments are listed in the next paragraph.) The solution was then adjusted
to a protein concentration of about 1.5 per cent by the addition of
the appropriate buffer (3 ml.), dialyzed against this buffer to equi-
librium, and electrophoretically analyzed (electrophoresis procedure
to be described). When the electrophoretic patterns of the inhibitor
treated casein-enzyme mixture and the normal casein (no enzyme) solu-
tion were similar, it was regarded as having produced inhibition of
chymotrypsin activity. When digests inhibited by hydrogen ion concen-
trations of pH 6 and less formed a precipitate during dialysis against
buffers of same pH, no electrophoretic analysis was made. Since the
appearance of such a precipitate was not evident for enzyme free casein
solutions under similar conditions, it was taken to be a sufficient

Proof that chymotrypsin was not inhibited.

Inhibition Treatments of an aliquot of the digestion mixture were
node by:

 

1. Addition of three drops of glacial acetic acid to one ml. of
digest, dialysis against 0.1M veronal buffer pH 8.6.

2' Digestion mixture plus three drops of glacial acetic acid and

 

 

29

heated to boiling for 30 seconds. It was dialyzed against
veronal buffer pH 8.6.
3. Digestion mixture was heated to boiling for 30 seconds on

direct flame and then supplied with three drops of glacial

acetic acid. Dialysis was done against veronal buffer pH 8.6.

J:’

. Digestion mixture was precipitated with trichloroacetic acid,
filtered, precipitate redissolved in veronal buffer pH 8.6 and

dialyzed against veronal buffer.

\J‘L

. Digestion mixture was brought to pH 5.6 by the addition of 1 M

acetate buffer pH 5.6 and dialyzed against the same buffer.

0\

. Digestion mixture was brought to pH 12 by the addition of

phosphate buffer and dialyzed against the same buffer.

7. Three ml. of digestion mixture was added directly to 3 ml. of
distilled water containing enough urea so that the total
concentration of urea after the addition of digestion mixture
was 8 M. The mixture was heated to bring all the urea in solu-
tion and allowed to stand for a few hours. It was then
dialyzed against veronal buffer pH 8.6. (Harris 1956).

8. The digestion mixture was added to l M acetate buffer pH h.6

to produce isoelectric precipitation. The precipitate formed

was washed, reprecipitated, and finally dissolved and dialyzed
in.veronal buffer pH 8.6.

Isoelectric precipitation, washing five times with two reprecipi-

tations was found to be the only satisfactory way of getting rid Or and

Pmducing inhibition of chymotrypsin. Thus a study of proteolytic

 

 

30

changes by electrophorectic analyses of the digests was accomplished by
taking 3 ml. of aliquots (which were removed from the digestion mixture)
at desired intervals of time and then the enzyme was removed by iso-
electric precipitation procedure with washing, etc., as described above.
The precipitate from each aliquot rendered practically free from enzyme,
was dissolved, dialyzed and electrophoretically analyzed in six ml. of

0.1 M veronal buffer pH 8.6, ionic strength 0.1.

Electrophoresis -- The procedure described in the instruction
manual for the Perkin-Elmer electrophoresis instrument was used.
Usually, one to one and a half per cent protein solution was equilibrated

by dialysis, against 300 ml. of the selected buffer (mostly O.lM

veronal of pH 8.6), at 5°C.

Eggstion Products Soluble in 10 per cent LwZv) Trichloroacetic
{.2213 - Ten to 15 ml. aliquots were removed from the digestion mixture
at selected time intervals and pipetted directly into an equal volume
01‘ 20 per cent (w/v) of trichloroacetic acid. These samples were
Shaken intermittantly during a 30 minute period and then filtered
through Whatmann No. 2 filter paper. The filtrates were analyzed for
t0tal acid soluble phosphorus, inorganic phosphorus, products absorbing
at 280 mu, non-protein nitrogen, peptides and amino acids hydrolysable
therefrom by paper chromatography, which is described as follows:

1. ibsorbang at 280 191 - The filtrate from the zero digestion
time sample was set at 100 per cent transmission or (absorbancy) in the

DU Beckman spectrophotometer at 280 ran as a blank. The subsequent time

‘s 4-
‘ 1
l; ‘l
.l‘_ ~
I - w I -_
. A . . .
. ‘ .1 I m

 

31

samples were compared against the blank and the changes found in
absorbancy units are reported in Tables VI, VII and VIII and shown in
Figures 5 and 6.

2. Total Phosphorus (T.C .A. Soluble) -- The procedure followed was
essentially the same as described in the text by Hawk and co-authors
(3b.). Five ml. of the above protein free filtrate was pipetted into a
50 ml. micro Kjeldahl digestion flask and 2.5 ml. of 5N sulfuric acid
(along with two glass beads to prevent bumping) was added. The flask
was heated, on the micro-Kjeldahl digestion rack until the evaporation
was complete and the mixture turned brown or black, with no further
change. The sample was cooled slightly, treated with one dr0p of 30
per cent hydrogen peroxide (Baker's) and heated again. The addition
of Ivdrogen peroxide and heating was repeated until the contents of the
flask were colorless. About 3 to )4 ml. of distilled water was then
added to the cooled flask and heated momentarily to boiling. The flask
was cooled again and its contents were rinsed into a 25 ml. volumetric
flask. The total phosphorus present was determined by the Fisk and
Subborow (23) method. A blank and phosphorus standard solutions were
run in the same marmer. The results reported in ug./.ml (or per cent)
are given in Tables II, III and IV and shown in Figures 2 and 3.

3. We phosphorus .-- The method devised and found applicable
to proteolysates for the analysis of inorganic phosphorus is the result
of Parts of procedures described by Norberg (70) and the Berenblum
and Chain method (8) as modified by Martin and Doty (59). None of these

methods alone could give recoverable results upon analysis of known

 

 

32

amounts of phosphate added to proteolytic digests. One ml. of protein-
free trichloroacetic acid filtrate was pipetted into an 11 ml. glass
stoppered conical centrifuge tube. The filtrate was neutralized by
initial dropwise addition of S N sodium hydroxide and finally O.SN
sodium hydroxide to the green color of bromo thymol blue indicator

( PH (:3. 7.0) .

To the neutralized aliquot was added one ml. of precipitating
agent, which consisted of 1 ml. of 10 per cent (w/v) calcium chloride
in 0.5M ammonium chloride buffer of pH 9.0, saturated with calcium
hydroxide. After the mixture stood for 30 minutes, the precipitate
which formed was centrifuged and washed with 5 ml. of a one to five
dilution of the precipitating reagent. The washed precipitate was
redissolved in 3 ml. of 10 per cent trichloroacetic acid, and treated
with 5 ml. of 1:1 isobutanol-benzene mixture, 0.5 ml. of MN sulfuric
acid and 0.5 ml. of 10 per cent ammonium molybdate. The mixture was
W311 shaken in the glass stoppered centrifuge tube for 15 seconds.

After separation of the two layers which occurred after mild
centrifugation, 3 ml. of the upper phase was transferred with the aid
0f Pipette into a new 15 ml. glass centrifuge tube. To this was then
added 2 ml. of'3.2 per cent sulfuric acid in absolute ethanol and 0.5
ml. of diluted stannous chloride solution. Immediate mixing produced
8. blue color whose intensity was measured at 625 119.1 in the Beckman
Model B Spectrophotometer. A blank and a phOSphOI'uS standard solutions
were run in the same manner. The results obtained in terms of ug./ml.
digest and Per cent of total protein phosphorus are given in Tables

II: III and 1V and shown in Figures 2 and 3.

g

-—~—_.—w-m——-

 

       

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33

h. Non-Protein Nitrogen -- Five ml. of trichloroacetic acid filtrate
was pipetted into 50 ml. digestion flask and two ml. of digestion mixture
added. The sample was digested for several hours on micro-Kjeldahl
digestion rack to a pale blue-green color. The flask was placed on
distillation apparatus, and 10 to 12 ml. of 6N sodium hydroxide was
used to liberate the ammonia. The ammonia was distilled into 10 ml.
of a boric acid-indicator mixture and titrated against 0.0m hydrochloric
acid. A reagent blank was also run along with the unknown samples.

The results are given in Tables VI, VII and VIII and shown in Figures
5 and 6. .

5. Paper Chromatography -- An attempt was made to characterize the
trichloroacetic acid soluble split products produced during digestion
by paper chromatography. Eight to 10 ml. of each of the filtrates,
taken at different time intervals, was extracted six times with ether
to remove T.C.A. The aqueous layer, after final extraction, was
separated and evaporated to dryness under vacuum in a desiccator. The
residue was dissolved in about 2 ml. of distilled water. About 103111.
of this concentrated solution was applied as a spot onto a sheet of
What-man No. 1 paper (18%" x 221;") and the chromatograms were develOped
by the ascending technique. Three different solvent systems were tried.
3111381101 3 acetic acid : water (h:l:5) and phenol saturated with water
gave almost identical results, whereas no movement of the material
could be detected using the lutidine-collidine solvent system. Two
Spots very close to each other and with high Rf values were detected in

case of whole and alpha caseins, whereas beta casein gave a Single SPOJc

W

 

3h

corresponding to the faster of the two derived from either whole or:
alpha casein. From the chromatographic analysis it appeared that the
split products were the same throughout the digestion (up to four hours),
only increasing in amount. Traced patterns of the chromatograms are
shown in Figures 7 to 16.

The remainder of the concentrated filtrate solution was again
evaporated to dryness under reduced pressure. Five ml. of 6N hydro-
chloric acid was added to the residue and it was hydrolyzed in sealed
glass tubes at 110°C for )40 to h8 hours. During hydrolysis black humin
was formed in the samples only from whole and alpha caseins, but no
humin was observed in the samples from beta casein. ‘

After hydrolysis, hydrochloric acid was repeatedly evaporated in
w and the residue was taken up in about 2 to 3 ml. of water. One
directional ascending chromatograms were run using water-saturated-
phenol as the solvent system. At least eight spots were detected with
ninhydrin reagent and were similar from all the samples and from all
three caseins (whole, alpha and beta). To completely identify the

amino acids, two dimensional chromatograms were run. Butanol : acetic
acid : water solvent system was used on a descending run and water
saturated phenol for ascending (other right angular direction) run.
For the detection of the spots, the chromatograms were sprayed with
0.1 per cent ninhydrin in a mixture of 5 per cent collidine and 95 per
Cent ethanol and heated in a chromatography oven at 10000 for a few
Minutes. ‘ ‘

 

 

35

At least 12 spots could be detected with the aid of two dimensional
chromatograms and these spots were identified as specific known amino
acids by running standard amino acids along with the unknown solution.
The results are shown in Figures 11 to 16.

In addition to the 12 amino aCids found, the presence of tryptOphan
was indicated by the formation of humin in the case of whole and alpha

caseins (see Lillevik and Sandstrom (SM).

Preparation of A Casein Derivative Precipitable at pH 5.6 with
CMotgypsin -- This precipitate was first observed while trying to
inhibit chymotrypsin below pH 6.0 as suggested from the data of Northrop
(71). Appropriate amount of 6 per cent casein solution was mixed with
equal volume of diluted chymotrypsin solution (stock solution diluted
one to five). One molar acetate buffer pH 5.6 equal to the combined
volume of casein and chymotrypsin solutions was then added and the
mixture allowed to stand at 30°C. A white precipitate separated out
after three hours. The appearance of the precipitate was much earlier
in the case of pure beta casein and quite slower in the case of pure
alpha casein. In every case it was observed that the digestion mixture
when allowed to stand overnight gave cleaner precipitate.

The precipitate thus formed was separated in the refrigerated
Centrifuge operated at 8 to 10 thousand R.P.M. and 0°C. This precipitate
was washed 5 times with water containing a small amount of pH 5.6
acetate buffer, then redissolved with aid of 0.2N sodium hydroxide and

r epr eCiPitated with the same buffer twice more. Finally the precipitate

 

 

 

 

“WV——

 

36

was lyophyllized and stored in the deep freezer for further studies.
The same precipitate was also obtained, as indicated by electrophoretic
analysis, by digesting at pH 7.5 for 35 mimtes and then adding an

equal volume of pH 5.6 acetate buffer.

Studies on the Casein Derivative Precipitatable at pH 5. --

1. Electrophoretic studiesrrBetween a 1.0 to 1.5 per cent (w/v)
solution of the casein derivative (or pH 5.6 precipitate) was electro-
phoretically analyzed in 0.1M veronal pH 8.6 as previously described and
this precipitate from whole casein showed essentially a single peak.
Preparations from pure alpha and beta caseins under similar conditions,
gave similar results. For further proof of similarity in the precipi-
tates from all three protein preparations, the precipitate derived from
whole casein was mixed with that from alpha. The mixture was electro-
phoretically analyzed and found to show again a single peak. Similar
treatment accorded to the precipitate mixture from whole and beta
caseins gave same results.

2. Isoelectric pH of the casein derivative. This was determined
by running the electrophoretic analysis at different hydrogen ion con-
centrations, both below and above the isoelectric point. The results
are shown in Figure 23 and given in Table IX.

3. Nitrogen and Phoghorus Content. An accurately weighed 30 mg.
quantity of pH 5.6 precipitate from whole casein was dissolved in three
ml. 01' concentrated hydrochloric acid. One ml. of this solution was
digeSted and analyzed for nitrogen content, as described under non-

Protein nitrogen analysis, and 1 ml. was digested for phosphorus

 

37

determination as described under total phosphorus procedure. The
results are given in Table XI. .

h. Analygis in the Ultracentrifuge. The sedimentation behavior of
the casein derivative when dissolved in 0.1M veronal buffer of pH 8.6
was studied using the Spinco analytical ultracentrifuge run at 2h°C
and 59780 R.P.M. For comparison, pure alpha and beta preparations were
similarly studied for their sedimentation behaviors under identical

conditions. The results are given in Table X and shown in Figures 25
to 27.

 

 

 

38

TABLEI

LIBERATION 0F ACIDIC AND BASIC GROUPS DURING PROTEOLYSIS 0F 3%
CASEIN SOLUTIONS, USING 0.0165 MG. 0F CHYMOTRYPSIN PER ML.
0F DIGEST, AS DETERMINED BY TI'I'RATION IN ALCOHOL,

WATER AND ACETONE MEDIA

 

Alcohol Hedimn Water Medium Acetone Medium
Digestion 13ml. of 011M 4ml. of A3114 21ml. of ANN
Time 0.0L. N KOH 0.011 N KOH 0.06N HCl
(ndnntes) KOH ml. KOH ml. HCl/ml.

 

Whole Casein

0 0 0 0 0 o 0
15 0.03 1.35 0.03 1.35 0.02 1.20
30 0.07 3.15 0.065 2.92 0.03 1.80
60 0.13 5.85 0.11 b.95 0.05 3.00
120 0.16 7.20 0.13 5.85 0.06 3.60
210 0.20 9.00 0.1h 6.30 0.09 5.10
Alpha Casein
0 0 0 0 0 0 0
15 0.06 2.7 0.03 1.35 0.07 h.20
30 0.18 6.3 0.065 2.93 0.105 6.30
60 0.21 9.h5 0.115 5.20 0.1h 8.h0
120 0.28 12.62 0.16 7.22 0.17 10.20
2u0 0.32 11.13 0.20 9.02 0.191 11.u6
Beta Casein
0 0 0 0 or 0 0
15 0.02 0.90 0.00 0.00 0.015 0.90
30 0.03 1.35 0.00 0.00 0.03 1.95
6C) 0.07 3.15 0.03 1.35 0.0h5 2.7
120 0.09 b.05 0.06 2.70 0.055 3.30
2h£> 0.11 h.95 0.08 3.6 0.075 h.5

-c_v‘—

 

 

TABLE II

LIBERATION OF ACID SOLUBLE PHOSPHCRUS DURING PROTEOLYSIS OF 3%

WHOLE CASEIN WITH CHXMOTRYPSIN

 

 

Digestion Total Acid Soluble
Time Pho horus Inor 'c Phos horus Or c Phos horus
(mimtes) W W W
of total of total of total
Protein P. Protein P. Protein P.
0.0165 mg. Chmtgpsin per ml. of Digest
0 O 0 0 O 0 0
15 5.95 5.511 5.91. 5.53 0.01 0.01
30 9.86 9.19 7.39 6.87 2.117 2.32
g 60 10.36 9.65 7.58 7.05 2.78 2.60
‘ 120 11.11 10.61 7 .18 6.96 3 .93 3 .65
21.0 11.82 11.00 7.91 7.36 3.91 3.61»
i 0.010 mg. CMotgpsin per ml. of Digest
,1 O O O O O O O
; 15 11.1 3.82 2.1 1.96 2.00 1.86
30 7.5 7.0 11.13 3.35 3.37 3.15
60 10.58 9.85 6.2 5.78 14.38 11.07
120 11.8 11.0 6.9 6.1111 11.9 11.56
.2110 12.00 11.2 7.1 6.62 11.9 11.56

 

   

110

TABLE III

LIBERATION OF ACID SOLUBLE PHOSPHORUS DURING PROTEOLYSIS OF 3%
ALPHA CASEDI WITH CHYMOTRYPSIN

 

__f V iv

Digestion Total Acid Soluble

Time Pho horus Inor c Phosphorus Organic Phosphorus
(minutes) 31§7ml. Percent .11 ml. Percent ngfll. Percent

 

 

 

 

 

 

of total of total of total
Protein P. Protein P. Protein P.
0.0165 mg. CWsin per ml. of Digest
0 0 0 0 0 0 0
15 '5.5 3.8 3.0 2.07 2.5 1.73
30 10.0 6.92 7.6 5.26 2.1 1.66
60 15.16 10.18 10.810 7.5 1.32 2.98
120 15.1 10.65 12.10 8.58 3.0 2.07
210 15.1 10.65 12.6 8.6 2.8 2.05
0.010 mg. C sin per ml. of Digest
0 0 0 0 0 0 0
215 2.8 1.935 2.1 1.66 0.1 0.275
:30 6.5 1.19 1.56 3.16 1.91 1.33
60 11.0 7.60 8.2 5.67 2.8 1.93
12C) 11.0 9.67 10.3 7.11 3.7 2.56
210 11.0 9.67 10.6 7.33 3.1 2.31

fi‘oa».

u»...-

 

11

TABLEIV

LIBERATION OF ACID SOLUBLE PHOSPHORUS DURING PROTEOLYSIS OF 3%
BETA CASEIN WITH CHYMOTRYPSIN

 

 

Digestion Total Acid Soluble

Time Pho horus c Phos horus Or anic Phos horus
(mimtes) 1127111. Percent1 ,ng7ml. Percent 4137M. Percent

 

of total of total
Proteinl P. Protein P. Protein P.
0.016 m . C sin er ml. of Di est
0 O 0 O O O O

15 0.73 0.83 0.18 0.51 0.25 0.29
30 1.1 1.25 0.911 1.07 0.16 0.18
60 1.3 1.175 1.0 1.11 0.3 0.31
120 1.5 1.7 1.1 1.25 0.1 0.15
210 1.88 2.11 1.3 1.18 0.58 0.66

0.010 mg. Gmtmsm per ml. of Digest

0 0 0 0 0 0 0
: 15 0.60 0.68 0.30 0.314 0.30 0.3).;
3 30 1.0 1.135 0.50 0.57 0.5 0.565
’ 60 1.1; 1.59 0.80 0.91 0.6 0.68
1 120 1.56 1.77 1.0 1.11 0.56 0.63

210 2,6 2,915 1.26 1.13 1.31 1.515

 

 

fl“,—

.1 ”'7.—

 

TABLEV

 

12

CHANGE IN CONDUCTIVITY DURING PROTEOLYSIS 0F 3% WHOLE, ALPHA, AND BETA
CASEINS, WITH 0.0165 MG. OF CHYMOTRYPSIN PER ML. 0F DIGEST

A

—__._

 

 

 

 

Digestion Whole Casein f_ Alpha Casein Beta Casein
Time Resist- Specific Resist- Specific Resist- Specific
( mirmtes) anc e C onduct- ance Conduct- ance C onduct-
(ohms) ance (ohms) ance (ohms) ance
(mhos) (mhos) (mhos)
0 150 1.085 326 1.5 180 1.02
15 110 1.11 321 1.52 161 1.06
30 130 1.137 316 1.515 150 1.085
60 120 1.16 311 1.57 118 1.09
120 120 1.16 309 1.58 1117 1.092
2140 119 1 .163 309 1 .58 1117 1 .092

 

 

13

TABLEVI

NON-PROTEIN NITROGEN DURING PROTEOLYSIS 0F
3% WHOLE CASEIN WITH CHYMOTRYPSIN

 

 

Digestion Kjeldahl N Eaa a
Time ,ug./ml. Percent of Total Absorbancy
(minutes) Protein N Units

 

0.016 m.C t sin erml. ofDiest

0 0 0 0
15 6.16 3 .19 0 .102
30 10.78 5.57 0.71
.. 60 11.56 7.58 1.21
i 120 21.08 10.93 1.93
210 28.21 11.7 ,0
0.010 mg. Chmflpsin per ml. of Digest
0 0 0 0
15 5.65 2.92 0.257
30 9.10 1.7o 7 0.568
60 13.77 7.12 0.89
120 20.30 10.19 1.5
1 210 26.09 13.18 > 2.0

 

 

 

TABLE VII

 

 

11

NON-PROTEIN NITROGEN DURING PROTEOLYSIS OF

3% ALPHA CASEIN WITH CHYMOTRYPSIN

 

_*

 

 

 

 

 

 

Digestion K71 eldahl N E
Time mg. ml. Percent of Total W
(mirmtes) Protein N Units
0.0165 mg. Wen per ml. of Digest
0 0 0 O
15 6.11 2.82 0.503
30 11.16 1.92 1.02
60 16.31 7.20 1.92
120 25.07 11.05 0‘)
210 35.21 15.16 co
0.010 mg. Wain per ml. of Digest
0 0 0 0
15 1.51 1.99 0.308
30 8.60 3.78 0.635
60 12.71 5.60 1.20
120 18.80 8.27 2.00
210 27.60 12.15

 

 

7m ww'tr‘vv'

 

 

TABLE VIII

NON-PROTEIN NITROGEN DURING PROTEOLYSIS OF
3% BETA CASEIN WITH CHYMOTRYPSIN

 

 

 

Digestion Kielfiahl N E
Time A}g.7ml. Percent of Total Absorbancy
(minutes) Protein N Units
0.0165 mg. Wain per ml. of Digest
0 0 0 0
15 2.58 1.15 0.139
30 1.82 2.15 0.238
60 7.76 3.16 0.358
120 11.96 5.31 0.522
21.10 17.98 8.02 0.790
0.010 mg. CWsingper ml. of Digest
0 0 0 0
15 1.93 0.86 0.0148
30 3.50 1.56 0.107
60 5.60 2.19 0.195
120 8.15 3.76 0.31
210 13.30 5.92 0-173

-g-—_.r—._._o-—-v —’—

I;

 

 

 

 

 

16

TABLEDI

ELECTROPHQIETIC MOBILITHS OF A CASEIN DERIVATIVE PRODUCED
BY CHIMOTRYPSIN AND PRECIPITABLE AT pH 5.6
AT DIFFERENT HYIROGEN ION CONCENTRATIONS

 

 

 

pH Buffer 0.1 M Mobility
(Tiselius Units)
3 .50 Acetate 5 .11
1 .00 Acetate 1 .55
8.60 Veronal -5 .30
9 .00 Veronal -6 .05
TABLE X

APPROXIMATE SEDIMENTATION COEFFICIENTS OF THE CASEIN DERIVATIVE,
ALPHA CASEIN AND BETA CASEIN AT 21 DI
0.1 M VFRONAL BUFFER pH 8.6

 

 

 

Protein Concentration Sedimentation Coefficient
(Svedbergs)

Casein derivative 3%

Peak 1 5.96

Peak 2 18.01
Casein derivative 1.5%

Peak 1 6.73

Peak 2 27.3
Alpha casein 3% 1.11
Alpha caSein 1.0% 1.58
Beta casein 3% 1.9

Beta Gas
'41:], 1.5% 7 .8

 

 

TABLE XI

 

 

17

SOME PHYSICOCHEMICAL PROPERTIES OF THE CASEIN DERIVATIVE PRODUCED
BY CHIMOTRYPSIN AND PRECIPITABLE.AT pH 5.6

 

Nitrogen
PhOSphorus

ElectrOphoretic mobility in
0.1M veronal buffer pH 8.6

Isoelectric point
Sedimentation coefficient in

O.lMWgeronal buffer pH 8.6,
at 21 .

15.10%
0.30%

5.3 Tiselius units
pH 6.10 (Figure 23)
Peak 1. 7.15 (Svedbergs)

(Figure 21)
Peak 2. 36.1 (Svedbergs)

 

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CURVE OF THE CASEIN DERIVATIVE FROM WHOLE CASEIN.
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75

IV. DISCUSSION

1. Age; Stabililof We Stock Solution.

Jacobson (142) for chymotrypsinogen conversion studies stored the
alpha chymotrypsin stock solution at pH 3.2 and 2° c, and noticed no
decrease in clotting activity after 25 days. Christensen (13) prepared
stock solutions in 0.025 N hydrochloric acid and stored them in the
refrigerator to avoid any loss in activity.. To keep the system as
simple as possible, it was thought desirable to make stock solutions
of the enzyme in distilled water and to store these in the cold room

at around 5° C. Thus, if such were possible and if no appreciable
loss in activity occurred, no change in pH would be necessary.

In orienting experiments, the activity of a one month old enzyme
Solution as measured by E280 absorbancy of trichloroacetic acid (TCA)
s011.1.b1e products was found to be the same (within experimental error)
as that of a new solution. This observation is supported by the work
or KU-nitz and Northrop (71), who in the preparation of their beta and
gamma chymotrypsins stored alpha chymotrypsin at pH 8.0 and 50 C. for
over- ‘bwo weeks. They also showed that beta and gamma chymotrypsins have
the Same activity and specificity as alpha. Thus storing of stock
solution for about three weeks at 50 C. was regarded as not effecting

the Specificity and activity, although alpha chymotrypsin might have
Change(i to beta or gamma forms during this period.

 

 

76

2. Method for Inorganic Phosphate 9.1%.

Even today the most frequently employed procedure for inorganic
phosphate analysis is that of Fiske and Subbarow (23). Nicholson (68)
while trying this method upon the TCA filtrate from casein proteolysate
with taka-diastase, observed that addition of the molybdate reagent
produced a white precipitate. This precipitate contained most of the
phosphorus. She tried numerous modifications (68) in treatment of the
filtrate but without much success. Finally she adopted a modification
by Norberg (70) of the Fiske and Subborow method (2h). According to
this procedure, the inorganic phosphate is separated from interfering

substances as a precipitate (hydroxy apatite) with a reagent, consist-
ing of calcium chloride dissolved in ammonium chloride buffer of pH

9 .0 and saturated with calciiun hydroxide. This precipitate is dissolved
in O -05 N sulfuric acid and using Fiske and Subborow reagents, analyzed
for ortho-vphosphate. As is evident, any other substance which forms
811 insoluble compound with calcium will also precipitate along with
i“Organic phosphate. Organo-dphosphorus compounds like diphenyl phos-
Phate, were found, during this investigation, to precipitate on
treatment by the Norberg modification. High results were then found
for inorganic phosphorus due to hydrolysis of the organic phOSphorus
°°mP°und in the presence of sulfuric acid. As further difficulty, the
“lei-urn precipitate from TCA filtrates of casein digests with
chymotrypsin either did not dissolve completely or else dissolved with

great difficulty in 0.05 N sulfuric acid.

 

77

Berenblum and Chain (8) investigated in 1938 the interfering
substances of non-phosphate character such as those which form
molybdenum complexes, e.g., fluorides, citrates, oxalates, etc. To
avoid such interference, they offered a new procedure for inorganic
phosphate analysis by taking advantage of the extreme solubility of
pheaphomolybdic acid in organic solvents. Their method was simplified
in 1919 by Martin and Doty (59) {as improved the choice of solvent and
decreased the time of analysis. This simplified procedure when tried

on TCA filtrates obtained from casein digests with chymotrypsin proved
utterly unsuccessful. Even known quantities of phosphate added to the
filtrates could not be recovered (see appendix Table I). Hence it
demonstrated that some interfering material was released from casein
during digestion. However a combination of the precipitation feature
of Norberg's method with the simplified organic solvent extraction
teChnique of Martin and Doty was then tried and found to be quite
Successful. The procedure in detail, as adOpted in its final form is
given on pages 31 and 32 of this dissertation.

Quantitative analytical recoveries of the known amounts of phos-
phate solutions added to the filtrates were obtained (see appendix
Table II) by this method. Another advantage of the method adopted in
this W'Ork was the small amount of TCA filtrates required for each

determination .

3 ' A*--_.._.1<3(>1101, Acetone and Water Media Titrations.
Acoording to Green and Neurath (27), titration methods give more

Si .
gnMileant information about the course of the proteolytic reaction

 

78

than do the other methods, but they are more tedious to use and less

suited to routine work. The Willstatter and Waldschmidt-Leitz (95)
modification of Foreman's (214a) alcoholic sodium hydroxide titration,
which determines the total acid group's liberated during proteolysis,
is very sensitive to carbon dioxide contamination (h2). To minimize
this error all the aliquots were titrated as quickly as possible and
under identical conditions. The time interval between the removal of
aliquots and completion of titration was most carefully controlled in
the case of titration in water medium. The period was the same for
each sample, in view of the fact that chymotrypsin was expected to
continue its action even in very dilute solutions.

Linderstrdm-Lang‘s acetone titration (53) determines the basic
$011133 released during digestion. Incidently and as also reported by
Jacob sen (h2) it was observed that acetone titrations were less tedious
to Perform as compared to the alcohol titrations.

From theoretical considerations, if during proteolysis there is
only peptide bond cleavage, the moles of hydrochloric acid consumed in
an a~<=etone titration should equal the moles of potassium hydroxide
°°n3umed in an alcoholic titration and there should be practically no
consul'Ip'tion of either base or acid in water medium when using the same
indicator.

Titration data for whole, alpha, and beta caseins (see Table I
and Figure 1) clearly show that consumption of base in alcohol was
higher as compared to the consumption of hydrochloric acid in acetone. .

Also: there was definite and noticeable consumption of base in

 

79

aqueous medium and it was more pronounced in the digests of whole and

alpha caseins.

From these results it is quite logical to submit that the action
of chymotrypsin was not limited to the hydrolysis of peptide bonds
only, but in addition some other acid groups were also liberated. More
such acid groups which must be considered as other than those from
peptide bond cleavage were liberated during the early stages of
digestion because then the aqueous medium titration corresponded more
closely to the alcohol titration values. It may also be deduced from
the same observation that during later stages of proteolysis most of
the additional acid groups now came from principally peptide bond
cleavage.

From all methods of determination employed the rate and extent of
Proteolysis with respect to beta casein appeared considerably less than
that for either of the other two preparations. Beta casein titration
Values, although low, are significant enough to also support the con-
t9111321011 that acid groups other than those from peptide bond cleavage
are released during proteolysis.

The aqueous titration values in the digests from whole casein are
higher than the corresponding acetone titration values. This might be
attributed to the effect of presence of one casein fraction upon the
other in a mixture as shown by PerlmaImUh) in connection with phos-
phoms liberation by certain enzymes. Thus it could be the result of
more PhOSphodiester cleavage from the beta fraction or/and less phos-

h
p Omnide bond hydrolysis from within alpha casein. According to

l—ww “gm-2‘

 

 

80

Perlmann (73) alpha casein is practically devoid of phosphodiester groups
whereas ca 80 per cent of the beta casein phosphorus is regarded to be

phosphodiester linked.
It may be remarked that the water medium titration results obtained

from beta casein digests were not as reliable as others, on account of

the solutions being quite milky.

Lt . Liberation of Phosphorug.

The liberation of 10 per cent TCA soluble total phosphorus from

whole and alpha casein during proteolysis by chymotrypsin did not cor-
respond to the release of non-protein nitrogen. Where there was
progressive increase in nitrogen release, the phOSphorus reached a
maximum and then practically levelled off (cf. Tables II, III, and IV
and Figures 2 and 3), especially the organic phosphorus. The maximum
POIL‘nt in the case of whole casein represented solubilization of about
11 per cent of the total protein phosphorus and in the case of alpha
casean it amounted to about 10 per cent.

The rate and extent of total acid soluble phosphorus liberation
from beta casein was very low and the same was true for its nitrogen
liberation. Only about 2 per cent of its total protein phosphorus was
released during the 1; hours of digestion by chymotrypsin.

Heat of the phosphorus so released was found to be in the form of
inorganic phosphorus. More than three-fourths of the total phosphorus
liberated from alpha casein and about two-thirds the phosphorus from
Whole casein was found as inorganic phosphorus (See Tables II and III

and Figures 2 and 3).

 

 

81

More organic phosphorus as compared to inorganic phosphorus was
released from whole casein rather than from alpha casein. This may be
attributed to the presence of the beta fraction contained in whole
casein and absent in pure alpha. Furthermore, as shown by Perlman ('23)
the presence of one fraction in a mixture may possibly effect the
behavior of enzymes upon the other. In all three cases organic phos-
phorus liberated as compared to inorganic was greater with lower
concentration of the enzyme than that with higher concentration
(Tables II, III and IV).

Simultaneous conductivity change curves (Figure 1;) did not support
the suggestion made by Nicholson (68) that inorganic phosphorus might

be determined by the much simpler resistance measurements. It may be
noted that the liberation of inorganic phosphorus during digestion was
a maximum (Figures 2 and 3) from alpha casein, whereas conductivity

change was the least when compared with whole and beta caseins (Table
V, Figure )4).

S . Nitrogen Products in the Trichloroacetic Acid Soluble Portion.

a) Acid soluble nitflen products.

As determined by measurement of absorption at 280 mu or Kjeldahl
nfitrOgen of their TCA filtrates, these products showed with all three
caseins and both chymotrypsin concentrations, a progressive increase
during h hours of digestion. (Tables VI, VII and VIII, Figures 5 and 6.)

The rate and extent was highest with alpha casein and lowest with beta
casein.

 

     

 

82

b) Paper chromatogapm.

For further specific characterization of the acid soluble nitrogen
products resulting from proteolysis, TCA filtrates from various casein

digests were examined after prescribed treatment by paper chroma-

tography (cf. page 33). The solvent systems successfully employed

1) butano : acetic acid : water (14:15) and 2) water saturated
2,6 lutidine : collidine :

were:
phenol. Another solvent system tried was:

water (1:1:1), but was found to be unsuitable as no movement of the ;
-/"

peptides could be observed.
With respect to proteolysates of whole casein, only two ninhydrin

positive spots of high Rf values (presumably from peptides) and of close

proximity could be detected (Figures 7 and 8). It may be noted that

the same two spots could be obtained from early stages and upwards to

h hours of digestion. Such was true for both concentrations of the

enzyme. This observation may be taken to support the work of Tiselius
and Eriksson-Quensel (88) and others such as Haugaard and Roberts (141),

Bellof and Anfinsen (6), etc., who have used various enzyme-substrate
Systems. On the basis of their work these investigatbrs have stated
that protein molecules are broken down one by one to the ultimate
Peptide stage without the accumulation of intermediate products.
Similar filtrates from alpha casein by the same treatment also
Showed 2 spots (Figure 8) of Rf values identical to those from whole
casein Spots whereas with beta casein (Figure 8) only one spot corres-

pending to the faster of the two in the other cases could be detected.

-:-" s

 

 

83

When filtrates from whole casein digests were first hydrolyzed
with 6 N hydrochloric acid and then examined in the same manner by one
dimensional paper chromatog‘aphy, they showed 8 spots which were identi-
cal for all the samples resulting from early stages and up to 14 hours
of digestion. This again proved that the same peptides were liberated
throughout the digestion period under investigation.

Products from filtrates of alpha and beta casein digests when
similarly hydrolyzed and treated, also showed identical 8 spots. The
only difference was that the TCA soluble products from whole and alpha
caseins during acid hydrolysis formed humin showing the presence of
tryptophan whereas no humin was observed when beta casein products were
involved, showing the absence of typtophan (51;).

This demonstrated that the faster moving peptide which was a
common product of all three proteins did not contain tryptophan;

whereas tryptophan was present in the slower moving peptide(s) which
came only from alpha and whole casein. In whole casein this slower
tI'yptophan containing spot might originate from alpha casein since beta
casein did not give the said spot. It may also be remarked that apart
from tryptophan, the two acid soluble peptides had some amino acids in
Common.

When two dimensional chromatograms were run with the hydrolyzed
Peptides of the filtrates, 12 spots could be detected (Figures 11 to
1h) . These spots were positively identified by comparing and running

them in mixture with known amino acids (Figures 15 and 16).

 

 

8h

Thus it may be said in summry that the faster moving peptide (or
peptides) was common to all three protein digests (in reality common to
both alpha and beta caseins) and contained 12 amino acids of which 5 or
6 are nutritionally essential. These are all named upon Figures 11 to
16. The slow moving peptide (or peptides) absent from beta casein
contained tryptophan as one of the amino acid residues and other amino
acid residues contained herein were also present in the fast moving

peptide (or peptides) .

6. The Acid Soluble Nitrogen and Titration Data flamed to Phoahorus
ye 3.

Comparing the titration data with phosphorus liberation results
(previously mentioned separately), it may be proposed that the acid
groups found released during digestion, other than those from peptide
bond cleavage, were the result of phosphate bond cleavage.

Examining the phosphorus data along with the chromatography find-
ings the following observations can be made. Two identical spots were
Obtained from the filtrates of whole and alpha casein and only one
Spot corresponding to the faster was given by beta casein filtrates.
Thus one spot (the faster one) was common to all three proteins. All
three proteins released organic phosphorus and the proportion of in—
Organic to organic phosphorus was the highest in alpha casein (compared
to the total phosphorus liberated). With all three proteins there was
Progressive increase of acid soluble nitrogen throughout the 14 hour
digestion period. Organic phosphorus did not follow the same pattern.

It leveled off substantially during the same period in the case of alpha

 

85

and whole casein but did not go as far with beta casein. (See Figures

2 and 3, Tables II, III and IV.)
Now, there are two possibilities offered as to course of reaction,

either the organic phosphorus was associated with both the spots or

perhaps the faster moving one. If the organic phosphorus was associated

with both the spots, there should have been no leveling off of phos-
phorus in any protein digest and its liberation should always have
corresponded to the release of nitrogen, which was not the case. Thus
this possibility of both spots containing phosphorus cannot be justi-
fied on the basis of the data obtained.

The second alternative possibility; namely, that the organic
phosphorus was associated with one spot (evidently with the faster mov-

ing one which was common to all) is the only choice left and needs

further direct confirmation. To justify this choice, one assumption

will have to be made-~that the liberation of the fast moving peptide(s)
will level off simultaneously with the organic phosphorus in the case

01‘ alpha casein. Then if the continued release of nitrogen is to be
accounted for it may be due to the continuous liberation of slow moving

non-phoSphorus peptide( 3) .

7 - lphibition of ohmsm Activity with casein.
Stopping the chymotrypsin action was found necessary for studying

 

the electrOphoretic changes produced during different stages of

Proteolysis of casein. Jansen _e_t_ 3.3;. (mi) studied the inhibition of

this enzyme in its pure form and in the absence of any substrate.

 

 

 

 

 

86

They found that chymtrypsin ms irreversibly inhibited by di-isopropyl
fluorOphosphate (DFP) . The reaction was fast but not instantaneous.
This reagent is highly toxic and very reactive. Additionally, the
groups with which DFP is supposed to react, i.e., hydroxyl of serine
and/or imidazole (60) are also present in casein. Thus the use of this
inhibitor reagent was undesirable.

Gergely and coworkers (25) while studying the digestion of mosin
with chymotrypsin used DFP to stop the reaction but made no mention
about the effect of DFP on the substrate, if any.

Li at al. (50) in their paper pertaining to the action of
chymotrypsin on hypophyseal growth hormone stated without any further
details that one drop of glacial acetic acid stopped the reaction.

A. number of possible inhibition treatments with glacial acetic acid as
reported on page were tried but without any such success.

According to the data of Kunitz and Northrop (71), chymotrypsin
should be practically inactive at pH below 5.9. When casein-
chymotrypsin mixture after bringing to pH 5 .6 by the addition of acetate
buffer were allowed to dialyze over night at 5° 0. against the same
buffer, a white precipitate appeared, showing that chymotrypsin was
Still active. Although it did not lead to successful inhibition, this
Observation opened a new avenue of investigation as to proteolytic
(maages on casein. The precipitate thus. obtained was further examined
and findings are discussed in later paragraphs.

High alkalinity produced by saturated potassium carbonate as
mentioned by Schwert at él- (81) was not tried due to the fact that

alkali dephoSphorylates casein (76).

 

 

87

Inhibition of chymotrypsin by strong urea solutions was tried in
1956 by Harris ( 33). He found that the enzyme was irreversibly
inhibited even in the presence of synthetic substrate, when. treated with
8 M urea. However, he made the following statement. "Nevertheless for
practical purposes it is possible to take advantage of the fact that
both enzymes (chymotrypsin and trypsin) are stabilized to a consider—
able extent against urea inactivation in the presence of a substrate
and can thus be used to degrade proteins which are themselves suscept-
ible to denaturation in urea but which resist digestion in their native
state." This statement was found to be valid as chymotrypsin was not
inhibited by 8 M urea in the presence of casein.

The only successful method for stopping the chymotrypsin activity
was found to be by isoelectric precipitation (adjusted to pH 14.6, with
acetate buffer) of the casein digests. The derived proteins thus
precipitated were washed practically free of enzyme (page 29) which
remained soluble under these conditions.

One disadvantage of this procedure was the loss of other digestion
products which were soluble at pH 14.6. Thus electrophoretic patterns
represent only those digestion products which were precipitable at pH
11.6. It may be stated that this procedure provided considerable
aSSurance that the changes produced were due to chymotrypsin action

011135 as no drastic treatment or reagent was involved.

8 . Electrophoretic Changes.
Electrophoretic analysis of proteins precipitated from digest

aliquots, removed at different time intervals showed (Figures lBIto 20)

.7 l, ‘—___l ...

 

 

 

88

that initially there was a split in the alpha peak. This we true for
whole as well as pure alpha caseins. Similar split in the alpha peak
by the action of rennin and pepsin was observed by Nitschmaxm and
Lehmann (69). With advancing digestion, both faster and slower moving
components started appearing from all three casein preparations. It
suggests that the first step involved a change in the alpha fraction
complex to produce the observed split in the alpha peak.

After this first step, the rate and extent of appearance of new
components was greater from the beta fraction substrate than those
with the alpha one. It may be noted that the concentrations mentioned
with the figures were based on the original protein concentrations in
the digest and the changes produced due to washing away of digestion
products soluble at pH 14.6 which would be greater with advanced

digestion were not taken into account.

On comparing the electrophoretic patterns of the digest precipi—

tates from the various caseins it may be remarked that a greater amount
of soluble products was produced from beta casein than from alpha

casein.

9 . A Casein Derivative Produced by cmtmsin and Precipitable at
P . .

As mentioned above, a white precipitate was observed when trying
t0 inhibit chymotrypsin around pH 5.6. This precipitate after washing
and reprecipitating showed, electrophoretically, at pH 8.6, a single

peak. Alpha and beta caseins similarly treated also gave a precipitate,

 

 

89

which on electrophoretic analyses again showed a single peak of about
the same mobility (Figures 5 and 6).

It may be remarked that from beta casein a white precipitate
appeared after 30 mirmtes of reaction but was not removed since the
aforementioned derivative could not be obtained in pure form. It was
only after the reaction proceeded for more than three hours, preferably
over night that the precipitate showing single peak could be obtained.

When a precipitate from alpha or beta casein was mixed with that
from whole casein and electrophoretically analyzed, again a single
pak was revealed.

V This evidence along with chromatography results discussed earlier,
suggests that both alpha and beta fractions may have some common units
or segments in their molecular chains. Complete proof of this postulate
requires further investigations.

For the determination of its isoelectric point, the precipitate
from whole casein was electrophoretically analyzed at different hydrogen
ion concentrations (Table IX, Figure 23). All the buffers used, con-
tained simple monovalent ions, as the polyvalent ions affect the
nrmbility of the compounds under investigation (2). Thus the mobility
of the derivative under discussion in phosphate buffer at pH 7.0 was
fOU-nd to be almost equal to its mobility in veronal buffer pH 8.6. The
cafieln derivative showed essentially a single peak at all the hydrogen
ion concentrations tried.

The same precipitate, when analyzed by the ultracentrii‘uge,

showed two peaks (Figure 25). Both the peaks had sedimentation rates

 

90

which were different from the sedimentation rates of pure alpha and
beta caseins, when run under identical conditions (Table X). This
indicated that the components present were different from the original
components of casein. Perhaps the faster moving component was a
polymer of the slower component but that their net charge was the same
thus accounting the single peak observed by electrophoresis.

Its phosphorus content (Table XI), mobility at pH 8.1; (Figure 22)
and minimum solubility show some resemblance to the properties of
alpha-2 casein reported by McMeekin (63). This similarity association
also needs further investigation. It may turn out that the kappa
casein of waugh (93) and alpha-2 casein of McMeekin (63) are the

enzymatic cleavage products of alpha and beta caseins, instead of being

regular casein components .

 

91

V. SUMMARY

1. A method for analysis of inorganic phosphate in trichloroacetic

acid filtrates of chymotryptic casein hydrolysates with chymotrypsin

was developed.

2. Alcohol, acetone and water media titration results showed that
acid groups, other than those coming from peptide bond cleavage, were
liberated during chymotryptic digestion of whole, alpha and beta

caseins.
3. These acid groups appeared to be coming from phosphate bond

cleavage.

h. The liberation of total acid soluble phosphorus from whole and
alpha casein did not parallel the liberation of non-protein nitrogen.

S. The rate and extent of liberation of acid soluble phosphorus
and non-protein nitrogen was the lowest with beta and highest with
alpha casein fractions.

6. It was noted that more organic phosphorus (of the total acid
soluble) was released from all three proteins with the lower concen-
tration of the enzyme.

7 . Paper chromatography of nitrogen products, soluble in 10%
trichloroacetic acid (TCA), from alpha and whole casein digests pro-
duced with chymotrypsin showed mainly two ninhydrin spots with high
Rf values .

8' Beta, casein TCA soluble products showed mainly one spot with

an Rf Value Corresponding to the faster of the two derived from whole

 

92

or alpha caseins.
9. Acid soluble nitrogen products from alpha and whole casein were

found to contain 13 amino acid residues, namely: 1) leucine, 2) valine,
3) tyrosine, )4) proline, 5) alanine, 6) threonine, 7) serine,

8) glycine, 9) glutamic acid, 10) aspartic acid, 11) arginine,

12) lysine and 13) tryptophan.

lO. Acid soluble digestion products from beta casein showed the
presence of all the amino acid residues, as mentioned above, with the
exception of tryptophan.

ll. It is postulated that the faster moving peptide(s) which was
common to all three proteins, contained the organic phosphorus.

12. Based on isoelectric precipitation procedures a method of
stopping chymotryptic action, for electrophoretic analysis, upon
casein digestion products was worked out.

13. At early stages of digestion a split in the alpha peak was
observed which was followed by increasing development of both faster
and slower peaks, from both the original components.

114. On treatment of whole casein with chymotrypsin at pH 5.6, a
white precipitate was obtained which on washing and reprecipitation
Showed electrophoretically a single peak at various pH.

15 . Alpha and beta caseins when similarly treated also gave
Precipitate which again showed a single peak of about the same mobility
as that from whole casein.

16. Some physicoehemical properties of the casein derivative ob-

tained from whole casein by the action of chymotrypsin at pH 5.6 are

 

   

93

reported (Table x1),

17. It is postulated that alpha and beta caseins have some common

units or segments in their molecular chains.

6. Bellof, A. and Anfinsen, C. B.
7 . Biffi, Virchow's Arch.

8. Berenblum, I. and Chain, E.

10. Cherbuliez, E. and Schneider, M. L.
11. Cherbuliez, E. and Jeannert, Recherches Sur la Caseine.

12. Cherbuliez, E. and Baudet, P. Recherches Sur la Caseins V.

 

9h

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II!‘ n

 

 

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102
APPENDIX I
TABLE I
RESULTS OBTAINED FOR ANALYSIS OF INORGANIC PHOSPHORUS DI
TRICHLOROACETIC ACID FILTRATFS OF CASEIN DIGESTS :
BY THE PROCEDURE OF MARTIN AND DOTY (59)
Casein
Digestion Phosphate Found
Time Phosphate Found 11g ..P/ml (added
(minutes) ug.P/t&. h. 0 ug. szl. )
Observed Calculated '
0 1.8 . — ..
15 1.2 h.h 5.2
30 1 .1 3 to 5.1
60 1 .8 3 .6 5 .8
120 0.1; 2.8 11.11
TABLE II
RESULTS OBTAINED FOR ANALYSIS OF INORGANIC PHOSPHORUS IN
'I'RICHLOROACETIC ACID FILTRATES 0F CASEIN DIGESTS
BY THE RECOMMENDED PROCEDURE
(i.e. used for the investigations)
Casein Phosphate Found
Digestion ug.P/m1. (added
Time PhOSphate Found 1.0 u LP/ml.)
(minutes) )1g.P/ml. Observed LCalculated
h5 9.20 13.10 13.20
120 10.10 111.00 114.10
2nd Run, Different Enzyme Concentration
30 10.80 111.9 111.80
120 11.00 15.20 15.00
: Beta Casein
‘ 30 1.85 5.90 5.85 ,

60 2.10 6.20 6.10

 

 

 

Warm