ABSTRACT

STUDIES ON THE ISOLRTION OF -CASEIN
AND THE INACTIVATION OF NNIN

By John Callaghan Owicki

The isolation of g-casein from whole bovine casein by
the 1965 method of Fox and Lillevik was attempted. In addi-
tion, the fractionation of casein in Ca++-urea and Quadrafos—
urea solutions was investigated. The final fi-casein study
involved the iSOIation of’p-casein from whole casein which
had previously been renneted to specifically degrade
contaminating k-casein. Moderate success was achieved in the
p-casein isolation studies, and several promising new avenues
for investigation were Opened.

In the second portion of this research the kinetics of
the inactivation of rennin by urea were characterized. The
order of the inactivation rate in urea and hydrogen ion
concentration and the apparent Arrhenius activation energy
were determined, and substrate protection was tested for.
Finally, experiments were performed to elucidate the mechanism
of the inhibition of rennin milk clotting activity by anionic
detergents.

At condition near room temperature and neutral pH, an
approximate rate equation for the inactivation of crystalline

_ d Eiennirfl

rennin by urea is dt aCfiifl ’1'“ Elma] 2'5 Eiennirfl .

The kinetics are somewhat complicated by the inhomogeneity of

 

John Callaghan Owioki
crystalline rennin. The experiments with detergents suggested,
but did not prove, that the observed inhibition was caused by

a detergent-substrate complex.

STUDIES ON THE ISOLATION OF -CASEIN
AND THE INACTIVATION OF CRYSTA LINE RENNIN

By

John Callaghan Owicki

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry

1968

BIOGRAPHY

The author was born on June 25, 1947 in Rochester,
New York. After living in Alaska for nine years, in 1958
his family moved to Niles, Michigan, where he attended
Brandywine Senior High School, graduating in l96h. There-
upon he matriculated at Michigan State University and received
a B. 8. degree in biochemistry there in 1968. After finish-
ing research for the M. 8. degree in biochemistry under
Dr. Hans A. Lillevik at Michigan State, in the fall of 1968
he began Ph. D. study in the chemistry department of Cornell

University as a National Science Foundation Fellow.

ii

ACKNOWLEDGEMENT

The author is very happy to acknowledge the advice and
guidance of Dr. Hans A. Lillevik throughout this study.
Thanks also go to Dr. J. R. Brunner for his stimulating '
interest and for providing certain of the chemicals utilized
in this study; to Professor Jewell Jensen for his donation
of detergent materials and literature; and to Mr. Dennis
Armstrong, manager of the MSU dairy herd, for his excellent
cooperation in providing the raw milk from which the casein
used in these experiments was isolated.

This study was supported in part by funds granted by
the Michigan Agricultural Experiment Station under Hatch
Project #966.

iii

I.
II.

III.

IV.

TABLE OF CONTENTS
Page
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . l
HISTORICAL. . . . . . . . . . . . . . . . . . . . . . 2

A. Whole Casein and Its Component Proteins . . . . . 2
B9 Bennino o o o o o o o o I o o o o o o o o o o o o 11

EXPERIMENTAL. O O O O O O O O O O O O O O O O O O O O 15

A. Materials 0 o o o o o o o o o o o 15
B. Starch Gel Urea ElectrOphoresis (SGUE). . . . . . 18
Ce fi-casein Isolation StfldieSo o o o o o o o o o o o 21

10 Preparation Of Whole Casein o o o o o o c o o 21

2. Preparation of B-casein by the 1965 Fox-
LillCVik Technique. 0 o o o o o o o of. o o 22
3. Precipitation of S-casein with Ca++ and Urea. 26
4. Casein Fractionation in the Presence of
Quadrafos (Sodium Tetraphosphate) . . . . . . 27
5. The Isolation of e-casein from Henneted Whole
Casein. o o o o o o o e o e o o o o o o o o o 29

D. Rennin Inhibition and Inactivation. . . . . . . . 33
10 The Milk ClOtting Time Assay. o o o o o o o o 33
2. The Inactivation Of Rennin by Urea. p o o c o 35

3. The Inhibition of the Milk Clotting Activity
of Bennin by Anionic Detergents . . . . . . . 36
RESULTS AND DISCUSSION. 0 O O O O O O O C C O O O O 0 no
A. p-Oasein StUdieSo o o o o o o o o o o o c 40

1. Preparation of B-casein by the Modified
1965 Procedure of Fox and Lillevik. . . . . . 4O
2. Precipitation o§+9-casein from Aqueous
Solutions of Ca and Urea. . . . . . . . . . 44
3. Casein Fractionation in the Presence of
Quadrafos o o c o a o e o o o o o o o o o o o “6
4. The Isolation of B-casein from Henneted Whole
Casein. o o o o o o o o o o o o o o o o o o o 51

B. Studies on the Inactivation and Inhibition of

Rennin. o o o c o o n o o e o o o o o o 54
l. Linearity of the Milk Clotting Assay. . . . 9 54
2. Inactivation of Rennin by Urea. . . . . . . . 56
3. Inhibition of Rennin by Anionic Detergents. . 76
4. Suggestions for Further Research. . . . . . . 81

iv

TABLE or CONTENTS (continued) Page
.v. smmmmcwcmsxons.........,.... 86
VI, BIBLIOGRAPHY o o o o o o o p o p 0 o o o o t o o o 0 89

APPENDIX I: Observed Milk Clotting Activities from
Studies on the Inactivation of Hennin by
Urea. o q o o o o o o o o o o o o o o o o o 96

APPENDIX II: Derivation of the Equation Relating fines;
and tzo o o o o o o o o o o o o o o o o o o 105

APPENDIX III: Derivation of the Equation relating Ea,
Temperature, and t2 0 c o o o o c o o o a 9 106

Table
I.
II.

III.
IV.

V.

VI.

VII.

VIII.

LIST OF TABLES

Physical and Chemical Data on B-oasein . . . . .

Solubilities of the Caseins during Isolation
Procedures 0 e e e e e e e e e e e e e e e e e 0

Test for Linearity of Milk Clotting Assay. . . .

Index of Experimental Data on the Urea Inactiva-
tion of Bennin, Data Compiled in Appendix I. . .

tz Data for the Determination of Ea’ the Order
in Urea Concentration, and the Order in H
Concentration. e e e e e p e e e e e e e e e e e

The Order of the Inactivation Hate in Urea
Concentration. e e e e e e e e e e e e e e e e e

The Order of the Inactivation Rate in H+
Concentration. e e e o e e e e e e e e e e e e 0

Results of the Clotting Activity Study on the
Inhibition of Bennin by Anionic Detergents . . .

vi

Page

40
54

57

67
68
7o

73

LIST OF FIGURES

Number

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

Scheme for the isolation of S-casein according to
FOX and L1113V1k, 1965. . e e . . . . . . . e . . .

Modification of the Fox-Lillevik e-casein isolation
procedure Of 1965 . . . . . . . . e . . . . . . . .

Flow sheets for the studies on the fractionation
of fi-casein rich solutions with urea and
Quadrafos . . . . e . . . . . . e . e e e . e e e e

The isolation of S-casein from rennin treated whole

casein. . . . . . . . . . . . . . . . . . . . . . .

pH 8.6 SGUE patterns of the precipitates and
supernatants from steps 1-5 in the modified 1965
Fox-Lillevik: fi-casein isolation procedure. . . . .

pH 3.1 SGUE patterns of the final S-casein products
from the modified F-L isolation procedure and the
isolation of’ @-casein from renneted NaCas. . . . .

pH 8.6 SGUE patterns of precipitates and superna-
tants obtained by the treatment of o.5;;.o%
fi-casein rich solutions with 0.2 M Ca in 1.7 M
urea, pH 3.6-3.7, 25°C. . . . . . . . . . . . . . .

pH 8.6 SGUE patterns of precipitates and super-
natants obtained from a fi-casein rich solution,
1.7 M in urea, pH 4.6, with Quadrafos, by warming
from 4 C to 30°C. . . . . . . . . . . . . . . . e .

pH 8.6 SGUE patterns from fractions obtained during
the dilution of a 4.7 M urea, 0.079% Quadrafos
solution of a. S-casein rich fraction derived

from renneted NaCas . . . . . . . . . . . . . . . .

pH 8.6 SGUE patterns from casein fractions obtained
by diluting aoB-casein rich Quadrafos-urea solution
at pH 3.0, 25 C, keeping the Quadrafos concentration
OODStant. . . . . . e . . . . . . . . . e . . . . .

pH 8.6 SGUE patterns from representative fractions
obtained during the isolation of fi-casein from

renneted NaCas. . . . . . . . . . . . . . . e . . .
Linearity check for the clotting time assay . . . .

vii

Page

23

25

28

30

41

41

45

47

49

5O

52
55

LIST OF FIGURES (continued)

Number Page
13. The inactivation of rennin by urea; the effect of

rennin concentration on the inactivation rate. . . . 61
14. pH 8.6 SGUE pattern of the crystalline rennin used

in the inactivation and B-casein isolation studies. 63
15. The inactivation of rennin by urea; the data of

Cheeseman. . . . . . . . . . . . . . . . . . . . . . 6“
16. The inactivation of rennin by urea; the effect of

urea concentration on the inactivation rate. . . . . 66
.17. The inactivation of rennin by urea: determination

of the order of the inactivation rate in urea

concentration. . . . . . . . . e . . . . e . . . . . 69
18. The inactivation of rennin by urea: the effect of pH

on the inactivation rate . . . q . e . e . . e . . e 71
19. The inactivation of rennin by urea: determination

of the order of the inactivation rate in hydrogen

ion concentration. . e 9 . . . . . . . . . . e . e . 72
20. The inactivation of rennin by urea: the effect of

temperature on the inactivation rate . . . . . . . . 74
21. The inactivation of rennin by urea: determination

of the Arrhenius energy of activation Ea for the

reaction . . . . . . . . . . . . . . . . . . . . . . 75
22. The inactivation of rennin by urea: the effect of

BubStrate on the inactivation rate . . . . .w. . . . 77
23. Possible mechanisms for the inhibition of rennin

milk clotting acitivity by anionic detergents. . . . 78
24. pH 8.6 SGUE patterns from the experiment to deter-

mine whether SDS inhibits the primary phase of milk

CIOtting by rennin . . . . . . . . . . . . . . e e . 80
25. Ultraviolet absorbance spectra of native rennin,

80% inactivated rennin, and the difference spectrum. 83

viii

I. INTRODUCTION

The initial objective of this study was the isolation of
highly pure p-casein from bovine milk. After several frac-
tionation and isolation methods had been investigated, it
became apparent that alternate means of eliminating contamina-
ting k-casein from the preparation would be highly desirable.
Treatment with the enzyme rennin (the specific substrate at
which is k-casein) seemed a likely way to accomplish this.

It was necessary, however, to be able to inactivate
this enzyme, for it slowly degrades all the milk proteins
after its initial rapid destruction of k-casein. Urea is
known to inactivate rennin and detergents have been found to
inhibit it, but very little systematic study of the processes
has been heretofore carried out. A more complete knowledge
of the characteristics of the inhibitory action of each was
necessary before either could be utilized with confidence in
the isolation procedure.

This investigation, then, is divided into two major
sections: one deals with the isolation of B-casein, and the
other concerns the inactivation and inhibition of the milk
clotting activity of rennin by urea and anionic detergents.
The second tOpic, in the beginning only a tool for the study
of the first, developed into a series of experiments of high
scientific interest in their own right.

1

II. HISTORICAL

A. Whole Casein and Its Component Proteins.

The Nomenclature Committee of the American Dairy Science

 

Association defines casein as ”a heterogeneous group of
phosphoproteins precipitated from skimmilk at pH 4.6 and
20°C” (1). The system has been the object of active study
since its rigorous isolation in 1883 (2), although its
existence has been known since the acid precipitation studies

of Mulder (3) in 1838.

1. The Eractignation 9f_Casgin

Until about 1918 casein was believed to be a homogeneous
protein; in that year Osborne and Wakeman (4) isolated a
casein fraction of much higher alcohol solubility than whole
casein. During the 1920's its heterogeneity was confirmed,
primarily through the classical work of Linderstrdm-Lang at
the Carlsberg Laboratories (5 , 6, 7). He succeeded in ='
obtaining chemically and physically distinct fractions by‘
the manipulation of pH in aqueous ethanolic solutions of
casein.

Although by 1930 it was well demonstrated that casein
was a complex of proteins, there existed no analytical method
with which to monitor a separation into its components. Such
a technique was provided in 1939 by Mellander (8), who

2

3

demonstrated the presence of three casein components upon
Tiselius moving boundary electrOphoresis of casein in
alkaline solutions. Mellander named the component proteins
dg,[33 and chaseins, in order of diminishing mobility;
this classification formed the basis of modern casein
nomenclature.

In 1944 Warner (9) separated the casein fractions giv-
ing Mellander's dand E electrophoretic peaks by the
precipitation of<fl~casein from solution at pH 4.4 and 2°C.
Q-casein was obtained by raising the pH of the supernatant
to 4.9 and warming to room temperature. This was the first
observation of the temperature dependent aggregation of
@-casein. It was recognized that although Warner's frac-
tions were almost mutually exclusive, they were not homoge-
neous.

The next important advance in the isolation of casein
components was made in 1952 by Hipp and coworkers (10) in
their deve10pment of urea and ethanolic precipitation tech-
niques. It was found thatCi-casein was insoluble in 4.6 M
urea at room temperature and at pH's near the isolectric
point, While B- and1XFcaseins were soluble.l After removal
' of theci-casein precipitate and dilution of the solution to
1.7 M urea, E-casein precipitated, leaving Zicasein in solu-
tion. Repetition of the above procedure on the separated
fractions resulted in electrOphoretically pure components'as
analysed by the moving boundary technique.

In the ethanolic method, casein was dissolved at room

temperature in 50% aqueous ethanol, 0.2 M ammonium acetate,

1+ ,

at pH 7.0. Upon the adjustment of the pH to 6.5, a precip-
itate (fraction A), predominantly dL-casein, was obtained.
Further acidification of the supernatant solution to pH 5.7
resulted in a mixed precipitate (fraction B) of d- and @-
caseins, and cooling the supernatant solution from this step
to 2°C precipitated a mixture (fraction C) rich in g-casein.
Pureci-casein was obtained by reworking fractions A or B in
ethanolic solutions, and B-casein was isolated from fraction
C by a method similar to that of Warner (9). K-casein was
separated from fractions obtained in the @-casein workup.

The isolation of kappa-casein (k—casein) by Waugh and
von Hippel (11) in 1956 fulfilled the prediction made by
Linderstrom-Lans (7) in 1929 that there existed a casein
component which stabilized the other fractions against
precipitation by Ca++. The k—casein itself proved to be
non-precipitable by Ca++ and was shown to be the primary
substrate for the enzyme rennin (11, 54, 55). The studies
also extended the findings of Warner (9) in the discovery
that while B-casein is precipitable (in the absence of
k-casein) by Ca++ at neutral pH and room temperature, it is
soluble near 0°C. The isolation of k-casein necessitated a
change in terminology, for what had been termed<h~casein was
found to be a complex between k-casein and another fraction,
termed<is-casein. The subscript 's' denotes the sensitivity
of the protein to Ca++ precipitation at all temperatures
studied.

The early 1960's saw two major developments in casein

fractionation research. The first was the advent of starch

5
gel urea electrophoresis (SGUE) as an analytic method; the

second was the perfection Of anion exchange chromatography

as a separation technique. Wake and Baldwin (12) showed by
SGUE that casein, previously resolved into only three frac-
tions by moving boundary electrOphoresis, contained a whole
spectrum Of major and minor components. Their method, provid-
ing superior dissociating conditions, was thus a much more
sensitive embodiment of the criterion of electrOphoretic
purity than was Tiselius' electrophoresis, and it Opened the
way for correspondingly more sensitive purification procedures.

A number of workers laid the groundwork for DEAE cel-
lulose chromatographic resolution Of casein (13, 14, 15, 16).
Groves and coworkers (17) published a procedure for the purifica-
tion of @-casein by elution with a stepwise salt gradient;
Thompson (18) described DEAE cellulose chromatography in the
presence Of 2-mercaptoethanol, the latter chemical proving to
be useful in promiting the elution of reduced k-casein from
other adsorbed fractions. The chromatographic method gives
fractions of high homogeneity, but it is ill-adapted to large
scale preparative work and Often requires a relatively great
deal Of time to perform.

A recent fractionation development is the use Of poly-
phosphate anions to precipitate casein components selectively
from urea solutions at pH 3 to 4 (19, 20). Binding of the
polyanion reduces the solubility Of the caseins to different
extents, thus allowing clean isolations OftiS-casein (20)

and k-casein (19) from whole casein.

6
2. Physical_and_0h.emip_al_ Propertigs_o_1: evgagein_

Ashcaffenburg (21) showed by paper electrOphoresis that
d-casein exists in at least three protein polymorphs or
genetic variants (denoted A, B, and C). The discovery was
confirmed by Thompson and coworkers (22) with polyacrylamide
gel electrOphoresis (PAGE) at alkaline pH, and more recently
Peterson and KOpfler (23) Observed that p-casein A was inhomoge-
neous upon gel electrOphoresis at pH 3.1. Coupling these
Observations with peptide mapping and amino acid analyses Of
tryptic hydrolysates (24), it was concluded that there exist
at least four variants in what had been termed the @-casein A
variant, differing slightly among themselves in amino acid
composition and electrOphoretic mobility. Q-Caseins B and C
have not been found to be heterogeneous. Physical and chem-
ical data on e-casein should be Obtained from samples Of
single variants; these can be Obtained from homozygous cows or
by DEAE cellulose chromatography (24).

A number Of the physico-chemical prOperties Of‘Q-casein,
such as its urea solubility, temperature dependent aggregation,
and precipitation by Ca++, have been applied to its isolation
from the other casein fractions. Detailed study of these and
other characteristics of the purified protein have shown that
it is a most interesting compound. ”Purified" here must be
taken reservedly, for all preparations made before the advent
of SGUE in 1961 must be considered to be Of questionable
homogeneity. .

In 1949 Gordon and coworkers (25) published an amino acid

analysis Of e-casein which established three important facts:

7
1) fi-casein contains no cysteine and, therefore, no disulfide
bridges; 2) the proline content of p—casein is extremely high
(over 15 mol %); and 3) the content of nonpclar residues is
absormally high, about 50 mol %. These Observations are
significant for an analysis and mediation Of the conformation
and aggregation behavior Of the protein. A later amino acid
analysis was reported by Pion and coworkers (26) in 1965.
Mellon and coworkers (28) presented a DNFB alkylation study
which implicated arginine and lysine as amino terminal residues.
A recent study on purer material (29) revealed only arginine

at the N-terminal and suggested that the C-terminal sequence

is either ileu-ileu-val or ileu-val-ileu.

The literature abounds with physical data and elemental
analyses Of p-casein: nearly every paper presents some such
measurement as corroborative evidence to the electrOphoretic
purity Of the preparation under consideration. Representative
data are collected in Table I.

Reynolds and coworkers (36) assign e-Oasein a galactose
content Of 0.8 mg/g protein. This is extremely low; assuming
a monomer molecular weight of 25,000, it is equivalent to 0.11
mol galactose per mol OfB-casein. It is not lmown whether a
great deal Of galactose was lost during purification or whether
the reported carbohydrate resided on a contaminant (perhaps

k-casein).

3 . Chemicalfiiractusal Characteristics 9L9 :09. 821:1.
Results from several lines Of study indicate that the

secondary and tertiary structure of e-casein is extremely

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9

unordered, lacking disulfide bonds and appreciable amounts Of
alpha helix and beta structure. Herskovits (37) analyzed the
OED spectrum ofiarcareins A and B and concluded that the
helical content was less than 10% in aqueous solutions. The
same conclusion was reached earlier by Kresheck (38) and
Jirgensons (39), whose proteins may have been mixtures Of
variants.

Whole casein and.AS-casein appear to behave as random
coils in solution (34), but the data pertaining to the gross
conformation Of fi-casein monomers and polymers may be inter-
preted, in general, tO'show that these are either random coils
or rod shaped structures Of axial ratio about eight (92).
Most Of the evidence is hydrodynamic in nature (32,43) and
suffers from the ambiguity Of restricted model choice. Two
ways Of distinguishing between the proposed conformations are
a measurement Of the radius Of gyration (e.g., from a Zimm
plot (40) ) and the use of flow birefringence. Kresheck and
coworkers (34) were unable to Obtain a radius Of gyration on
the polymerized protein because Of unreliable extrapolation
of light scattering data to zero concentration, although the
data (such as they were) seemed to fit the spherical random
coil model. As far as is known, no flow birefringence studies

have been attempted.

4- its {Energetics Eéhaxbarlof fi-seseim
Except at extreme pH's there is a direct relationship

between the state of aggregation Of'e-casein and the tem-

perature Of the solution in which it is dissolved. This has

10

been most thoroughly studied by Payens and van Markwijk (32),
who concluded from ultracentrifugal measurements that at pH
7.5 the protein exists asra monomer (MW ca. 25,000) at 4°C 2
and as a polymer Of degree close to 22 at 8.50. Light
scattering studies by Kresheck and coworkers (34) in pH 6.5
buffer gave a weight average molecular weight Of 2.94 x 106
at 30°C and 2.50 x 107 at 90°. The system appears to be
paucidisperse, with monomers in equilibrium with a narrow
range Of polymers. In addition to this self-aggregation,
association between as- and S-caseins has been described (41).

The Dutch workers (32) discussed the slow equilibration
of @-casein aggregation and concluded that Gilbert theory (42)
is inapplicable. There is also as yet an inexplicable
viscosity anomaly, best exemplified in their data, wherein
the specific viscosity Of a polymer solution does not, at
infinite dilution, approach that Of a monomer solution. One
would expect progressive dissociation of the polymer to give
a monomeric solution in the limit.

Sullivan and coworkers (43) have concluded that increas~
ing ionic strength dissociates p-casein aggregates; Halwer
(44) found the Opposite effect, but his data were tur-
bidimetric rather than true molecular weight measurements.

The high proline content Of’S-casein is probably respon-
sible for its lack Of alpha and beta structure and consequent
resemblance tO denatured proteins (44). It is likely that
because Of this, the abnormally strong hydrophobic forces

present in this predominantly apOlar protein are not localized

ll
internally, but instead result in intermolecular interactions.
The well-known stabilization of p— by k-casein against
precipitation by Ca++ is caused by a rather tight complex
formed between the two proteins. The main workers in the

++ affects on caseins have been Zittle and his

area of Ca
colleagues. Zittle and Pepper (45) reported on the influence
Of Ca++ and other factors on the rate of aggregation Of
B-casein, and Zittle and Walter (46) described the k—casein
stabilization phenomenon. A decrease in the Ca++ precipé
itability of photooxidized p-casein was noted by Zittle

and coworkers (47).

B. Rennin
Rennin, a protease from the fourth stomach of the calf,
was first Crystallized by Hankinson and Palmer (48) and
Berridge (49). Such preparations have been found to contain
at least three enzymatically active components (designated
AP, B-, and C—rennin) resolvable by DEAE cellulose chromatog—
raphy (50) and by electrophoresis at low ionic strengths (51).
Rennin's milk clotting reaction has been shown to involve
three phases (52, 53): 1) the primary phase, a rapid and
specific enzymatic attack on catalyzed limited proteolysis
Of casein; 2) the secondary phase, a nonenzymatic coagulation
Of the modified casein; and 3) the tertiary phase, a slow
general proteolysis. Waugh and von Hippel (11) and Wake
(54, 55) discovered that it is k-casein which is the specific
substrate for the primary reaction, products being a

glycomacropeptide soluble at pH 4.7 and an insoluble portion

12
termed para-k-casein (56). It is thought that the hydro-
philic sugar residues on k-casein counteract the nonpolar
aggregative forces on the highly hydrOphObic casein molecules;
when the sugar residues are lost with the glycomacroPeptide,
the stabilizing power Of k-casein is destroyed (57).

That as- and e-caseins are not attacked in the primary
phase, although both are degraded slowly in the tertiary
phase, has been indicated by the work Of several authors
(53, 58, 59, 60). These conclusions are based on the rate
Of liberation from the casein fractions of non-protein
nitrogen (NPN) soluble in 12% TCA. ElectrOphoresis is a
more sensitive criterion of incipient proteolysis, since the
initial stages Of degradation do not always lead tO the
liberation Of 12% TCA soluble protein fragments. Degrada-
tion products have, however, electrOphoretic mobilities which
are almost invariably different from that Of the parent com-
pound (61, 62).

The tertiary phase of rennin action has a pH activity
maximum at about 3.8 (63). This is nearly two pH units lower
than the Optimum for clotting activity due tO limited
proteolysis (64).

Milk clotting time has long been used as an assay for
the enzyme rennin as well as for other proteases. Starch and
Segelcke (65, 66) found the relationship BRTO = K to hold,
where R is the rennin concentration, To the clotting time,
and K is a system constant. Holter (67) found deviations from

this; as a correction he introduced a lag time t for the

13
secondary (clotting) phase Of the reaction, so that the
equation then read B'(T°-t) = K. Foltmann (63) found that
this relationship in its turn was valid only under limited
conditions. It appears that the clotting reSponse is extremely
sensitive to variations in the experimental situation.

The influence Of pH on the stability of rennin has been
studied by Foltmann (63) and Mickelsen and Ernstrom (68).
These workers concluded that there is a relative maximum Of
instability near pH 3.8, an area Of stability between pH 5 and
6, and rapidly increasing instability as pH 7 is approached.
The latter authors found a large temperature dependence Of
the inactivation rate in the higher pH range and also a
specific Cl’ destabilization Of the enzyme at low pH. It was
tentatively concluded that the inactivation below the iso-
electric point (about 4.5) was primarily autOproteOlytic,
while that at higher pH's was due to general alkaline denature-
tion.

Cheeseman (69) associated the loss Of rennin enzymatic
activity with the reduction Of disulfide bonds, although his
reductions were carried out at high pH, which in itself may
have denatured the protein or desulfurized it (70). He also
noted irreversible inactivation of B-rennin (as defined by
Foltmann (50) ) by aqueous urea at pH 5.4 and 37°C.

Inhibition Of the milk clotting activity Of rennin by
detergents such as ”sodium Olein methyltaurinate" and sodium
dodecyl sulfate (SDS) has been noted (71, 72). It was thought
that the inhibition was a result Of binding of the detergent

14
to the substrate rather than to the enzyme, although little

direct evidence for this view was presented.

III. EXPERIMENTAL

A. Materials
1. Equipment

Balances; For general gravimetric work, Ohaus Centogram
and CencO Agate-Bearing Trip Scale single pan balances were
used. Where great accuracy was required, a Mettler Type H15
analytical balance was employed.

Timers; A wristwatch and General Electric spring wound
120 minute alarm laboratory timer were suitable for most
purposes. Milk clotting time was measured with a ”Time-It"
timer (Precision Scientific Co.) graduated in seconds and
tenths Of seconds.

pH_M§a§urement§: A line Operated Beckman Zeromatic pH
meter equipped with a Beckman #39142 combination electrode
was used for all pH adjustments. The pH meter was standardized
with Mallinckrodt #0029 buffer (pH 4.01), Beckman #3581 buffer
(pH 7.00), and Mallinckrodt #0032 buffer (pH 10.00).

Centrifugagign;~ Solutions to be centrifuged at room
temperature were run in an International Centrifuge, Universal
Model UV, equipped with a 800 ml capacity swinging bucket
rotor. Refrigerated samples were centrifuged in a Servall
Refrigerated Centrifuge with type 881 angle head. Unless

otherwise stated, centrifugation was carried out for 10-15

minutes at about 1000 x g (about 2000 rpm) in the International
35

l6
and for the same period Of time at near 3000 x g (about 5000
rpm) in the Servall.

Qialysis; Solutions were dialyzed in Union Carbide
dialysis tubing against H20 at 4°C.in an external liquid
rotation dialyzer designed by Djang, Lillevik, and Ball (73).
Occasionally samples were dialyzed by suspension of the sac
in water agitated by a magnetic stirrer.

-. Lygphillgafiignl, Lyophilization was carried out with a
Virtis Freeze-Dryer cooled by an ethanol-dry ice bath and
powered by a Cenco Hyvac Model 14 vacuum pump. An ethanol-
dry ice water vapor trap was inserted between the freeze-
dryer and the pump to help prevent the corrosion Of internal
pump components.

Elgcprgphoregig: Starch gel urea electrophoresis (SGUE)
was performed with parts from an LKB zone electrOphoresis
apparatus and 0-300 Volt unregulated power supply, LKB Model
3290A. A.more detailed description of the electrophoresis
equipment is contained in section III-B.

Temperature Control: 00 I 0.5°C temperature control for
the.rennin inactivation experiments was achieved with an ice-
water bath. 25.2° i o.2°c was held in a 70 1 water bath
(thermostat) regulated by a mercury expansion relay in cir-
cuit with a 125 W Cenco knife blade heater. The bath water
was agitated with a stirring shaft attached to a 50 W rheostat-
adjusted speed reducing Bodine motor.

Qprutgr;_ Rennin inactivation data were analyzed on the

Michigan State University CDC 3600 computer. A routine from

17
the MSU Computer Institute library ("Least Squares Curve
Fitting With Orthogonal Polynomials," program #39) was used
to fit first through fourth degree curves to the data and
to Obtain extrapolation to the clotting activity Of fully
active enzyme. A second program, written by the author,
converted raw clotting time to both percent of initial

activity and logarithm Of percent of initial activity.

2- Seassnia_

Unless otherwise stated, all commercial chemicals were
analytical reagent grade, used without further purification.

Quadrafos (sodium tetraphosphate), in large white gran-
ular crystals manufactured for detergent and water softener
use, was kindly donated by the Rumford Chemical 00., Rumford,
R. I.

Urea (Baker or Mallinckrodt) was tested for cyanate by
the procedure of Warner (74); no contaminant was detected in
any Of the lots used in these experiments. The cyanate,

spontaneously formed by the reaction
fl;
(NH2)2CO NH40CN

can carbamylate free amino and carboxyl groups Of proteins
(75. 76, 77).

Crystalline rennin was Obtained from the Sigma Chemical
CO. (Lot R78—052). This was the same preparation used by
Broomfield (78); it had at that time been found to contain

14.15% nitrogen and to have an activity Of 15.7 Ru/mg according

18
tO the definition Of Berridge (79). The enzyme had been
stored in a dessicator at —2o°c until the advent of the
present study. SGUE at pH 8.6 revealed one major component
in the crystalline rennin as well as two minor ones which
were present in much smaller amounts. As noted by Broomfield,
the enzyme was in the form of thin plates ordered in a mica-

1ike structure.

B. Ge ea El t 0 ho esis SGUE
Procedures followed in this study are, with minor altera-
tions, those of Wake and Baldwin (12), as modified by Neelin

(80), and Of Groves and coworkers (17).

apparatus

Unregulated 0 to 300 Volt power supply (LKB Model 32902)

LKB zone electrOphoresis base, tanks, and electrodes

12 x 24 x 0.6 cm gel frame; slotformer for six samples
(0. 2 ml/sample)

Eaton-Dikeman #652 paper for wicks

1.5 amp battery charger; destaining tank and gel frame

gel slicer (fine stainless steel wire stretched in a
coping saw frame)

shaft prOpeller stirrer

hydrator (closable container to keep the gel in a humid
atmosphere)

BCHSEnES.
For pH 8.6 gel:
Gel formulation:

22? ml distilled, de-ionized (DDI) water
23 ml stock tris-citrate buffer (0.76 M tris
(hydroxymethyl)-aminomethane (sigma) titrated
to pH 8. 6 with citric acid)
45- 50 g hydrolyzed potato starch (COnnaught)
100 g urea
0.25 ml 2-mercaptoethanol (Eastman)

"1
Taskbafferu

0.3 M boric acid, titrated to pH 8.6 with sodium
hydroxide.

19
For pH 3.1 gel:
961 formulation!
225 ml DDI water
25 m1 stock formate buffer (0.5 M formic acid,
0.1 m NaOH)
100 g urea
45-50 g hydrolyzed potato starch (Connaught)
Electrgdg_tank_bgf§eg:
stock formate buffer diluted 1 —9.10 with DDI water
Protein sample solvent (for both pH 3.1 and pH 8.6 SGUE):
2% hydrolyzed starch in 7 M aqueous urea, heated to
70°C to burst starch grains and cooled to room
temperature.
Staining solution:
2 g Amido Black 108
250 m1 methanol
10 m1 glacial acetic acid
250 ml water

Destaining solution:

7% (W/v) aqueous glacial acetic acid

ETQCQGErQ

While agitating with the prOpeller shaft stirrer,
dissolve all of the urea in the total volume Of stock buffer
and all but 100 m1 of the water. With a stirring rod or
spatula disperse the starch in the remaining water, taking
care to break up all lumps; add the suspension tO the urea
solution. If the starch is added dry, lumping often occurs.

Gently warm the solution to 70°C and pour it into a 2 l
suction flask immersed in a 70°C water bath. Apply negative

pressure with a water driven aspirator until the gel comes

to a rolling boil with large bubbles (caution--the pH 8.6 gel

20
tends tO froth at first). Release the pressure and, if
appropriate, add the 2-mercaptoethanol dropwise and with
swirling.

Have the plastic gel frame and its paper buffer wicks
assembled (with maSking tape) and leveled horizontally in
the hydrator. Add a little water to the bottom of the
hydrator to produce a humid atmosphere. Pour the gel into
the frame and insert the slotformer (a thin coating Of
mineral Oil on the slotformer will facilitate its removal).
Close the hydrator and let the gel set for 12-24 hours.

Dissolve the protein samples to concentrations Of 1-5%
(w/v) in the starch urea sample solvent and pipette into the
slots in the gel. The higher sample concentration (5%) was
used routinely for casein electrophoresis because of the
superior resolution of minor components so Obtained. After
applying the samples, layer mineral Oil over the slots to
prevent sample overflow between slots and cover the whole gel
with Saran Wrap or Parafilm to prevent evaporation. Then
place the gel and frame in the electrophoresis setup in the
cold room at 4°C for one hour tO equilibrate. After equalizing
the liquid levels in the two electrode buffer tanks to avoid
a pressure head, apply voltage at 150-275 V. The resultant
currents will be roughly in the range Of 30-50 ma and may
decrease to as little as 50% Of the initial value during the
course Of a run.

Approximately 12—18 hours later turn off the current,
remove the gel from the LKB apparatus, and (with the gel

slicer) slice it into three 0.2 cm thick slabs. The middle

21

section is retained for staining, and the upper and lower
slices may be used for other types of assays if desired (81).
Mechanically unstable gels are more easily sliced under water.

Stain the middle slab with the Amido Black solution for
15 minutes, wash Off excess dye with running water, and
destain the gel in the electrolytic destaining apparatus for
45 minutes or more. The electrOpherOgram can then be photo—

0
graphed and/or stored in Saran Wrap at 4 C.

C. é;casein_lsolation Studies

In the casein experiments that follow, the pH of protein
solutions was adjusted by the drapwise addition Of HCl or
£301 with vigorous agitation on a magnetic stirrer. The
concentrations of the acid and base reagents varied between
0.1 M and 6 L; the concentration used in any particular pH
adjustment was dictated by the casein solution volume, the
size of the pH change desired, and the accuracy demanded by

the situation.

1. Preparation 9f_Whole_Casei;

Whole milk was obtained from Holstein cows of the Michigan
State University dairy herd and was immediately skimmed in a
De Laval centrifugal cream separator. In order to minimize
the number of genetic variants present, milk from different
cows was never mixed. Milk was not taken from newly lactating
animals.

Crude whole casein was precipitated from the skim milk

by adding 1 M HCl to pH 4.6 at room temperature. The precip-

itate was collected, washed briefly with DDI water, and

22
redissolved (to about 10% (w/v)) to pH 7.0 with l M NaOH.
It was precipitated as before at pH 4.6, separated from the
supernatant, and again dissolved to pH 7.0. Purified whole
sodium caseinate (NaCas) was Obtained by performing one more
cycle Of pH 4.6 precipitation and redissolution. The NaCas
solution was frozen and either lyOphilized or used directly

as starting material for the isolation Of p-casein.

2- Ereparatioa 2f_P:°éS.§1E sy_tb.e_12.62 for-Lillevik.Tscaniqs 6..

This investigator initially attempted to isolate R—casein
by the use Of a procedure develOped in this Laboratory by
Fox and Lillevik (35) during 1965. A flow sheet Of their
technique, hereafter called the F-L method, is presented in
Figure 1.

Step 1 is basically the Aschaffenburg (82) modification
of the 1952 Hipp et a1. (10) urea fractionation; it is
designed to precipitate dS-casein and kncasein. Steps 2 and
3 are also similar to the Hipp et a1. method, but Fox and
Lillevik found that conditions Of pH 4.6, 1.7 M urea, 4°C
precipitated as casein and some k-casein, leaving the solution
enriched in P-casein. The latter protein was flocculated
after the pH was raised to 5.2 and the temperature was brought
up to 30°C.

In Step 4 of Figure l e-casein is precipitated by 0.2 M
Ca++ at room temperature and pH 6.5 to 8.0, thus effecting a
separation from Ca++éinsensitive fractions such as k-casein.

Step 5 is a modification Of the 1952 Hipp et a1. (10) alcohol

precipitation. After performing this step, Fox and Lillevik

23

Starting material: Lyophilized whole sodium caseinate.

I

Step 1: Dissolve with stirring in 3.3 M urea to 10% (w/v),
lower pH to 5.0, allow to stand 1 hour, and cen-
trifuge.

l

SupIer-l '1 Ppg- 1

Step 2: Lower pH of Super-1 to 4.8, at 4°C dil. with cold
DDI water to 1.7 M urea, centrifuge.

I
Super-2 PBS-2

Step 3: Raise pH of Super-2 to 5.2, warm to 30°C.
1

T

Bpt-3 1 SHDEr-B

Step 4: Disperse or dissolve Ppt-3 in cold DDI water,
dialyze at 4°C until urea-free, adjust pH to 7-8,
make 0.2 M CaCl with cone. CaClz solution, warm
gradually to 33°C. Repeat this step on the
resulting precipitate (Ppt-4a).

l

 

PpH-4a -1 Super-4a
Ppg-4b j Super-4b

 

Step 5: Dissolve Pptu4b in DDI water, dialyze at 4°C until
ion-free, bring to room temperature, add NHquc to
0.21 M, and make 50% ethanol with 95% ethanol.
This gives 0.1 M NHuOAc. Lower H to 5.0. Repeat
on resulting precipitate (Ppt~5a and combine super-

 

 

 

 

natants.
I ll
F ‘1
Super-5a —1 Ppt-5a
Super;5b I PptLSb:First crOp Of
é-casein
Step 6: Cool the combinedJSuper-S fractions overnight at 4°C.
I .
Ppt-6 Super-6

Step-7: Purify Ppt-6 as in step 5 until pure (9-casein is
Obtained on SGUE J

l 1
Ppt-Y: Second crOp Of é-casein Super~7

FIGURE 1: Scheme for the isolation Of p-casein according to
Fox and Lillevik (35), 1965. In the flow sheet above, super =
supernatant and ppt = precipitate.

24
Obtained a highly pure Q-casein preparation which failed tO
reveal any contaminating proteins when it was analyzed at a
5% concentration on both pH 3.0 and 8.6 SGUE.

Steps 6 and 7 are auxiliary workups of the B-casein left
in solution after step 5. As such, they are concerned with
the quantitative rather than the qualitative aSpects of the
separation.

In the present investigation the procedure outlined in
Figure l (F—L method) was followed except in the following
respects: 1) no precipitate would form in step 3 unless the
pH was reduced from 5.2 to 4.6-4.8; 2) for the purposes Of
this study, the extra yield gained by performing steps 6 and
7 was not sufficient to justify their being carried out, so
only steps 1 through 5 were employed; 3) the first crOp Of
B-casein Obtained from step 5 was redissolved in urea and run
through the overall separation scheme a second time to help
clean out residual contaminants; and 4) the first time step 5
was performed three precipitations of B-casein from the
ethanolic solution were carried out instead Of the two
recommended in Figure l; the second time through the procedure
only one precipitation was done in step 5. A flow sheet Of
this modified F-L p-casein isolation method is presented in
Figure 2.

{Humughout the isolation small aliquots of all fractions
were dialyzed against water, lyOphilized, and analyzed by
SGUE. The weights Of the lyOphilized aliquots gave a rough
indication of the amount of protein in each fraction. An

overall yield Of approximately 3 g of final.$-casein product

25
Starting material: lyOphilized whole sodium caseinate

Step 1: As in Figure l. I'
Super-l Ppg-l

 

Step 2: As in Figure 1. -I
F:
Super-2

 

1
Ppt-2

Step 3: As in Figure 1, except at pH 4.6 (one preparation
done at 4.9).

 

 

 

 

 

 

a 1

Ppt-3 Sugar-3

Step 4: As in Figure 1.
Ppt-4a '] Super-4a
Ppt-4b .i Super-4b

Step 5: As in Figure 1, except perform three precipitations
instead of two. I
Ppt-Sa ‘1 Super-5a
Ppt-Sb -1 Supbr-5b
Ppé-5c «1 SupEr-Sc

Repeat Of step 1: Dialyze Ppt-5c ion-free at 4°C, treat as in
step 1 of Figure 1. 1

2-sJ;..-1 2-Ppt—1

 

Repeat of step_2: As in step 2, Figure l.
2-Super-2 2-Ppt-2

 

Repeat Of step 3: As in Figure 1, except at pH 4.6 (one
preparation done at 4.9).

r
2-Ppt-3 2-Sup3r—3

 

Repeat Of step 4: As in step 4, Figure 1.
l 1
2—Ppt-4a 'L_ 2-Super-4a
2-Ppt-4b 2-Sup3r-4b

Repeat Of step 5: As in step 5, Figure 1, except perform
precipitation only once1

2—Ppt-5: Final fi-Casein 2-Super~5
preparation (see Figs. 5 & 6)

 

 

FIGURE 2: Modification of the Fox-Lillevik B-casein isolation
procedure (16) of 1965. The above was utilized in the present
study; for the original method, see Figure 1. Super =
supernatant and ppt = precipitate.

26
(2-Ppt-5 Of Figure 2) per 100 g Of starting material was
obtained, and the purity Of the preparation can be assessed
from the SGUE patterns represented in Figures 5 and 6. The
former figure also includes pH 8.6 electrOpherOgrams from

representative fractions obtained during the isolation.

3 - aracipitatios at. e- ease in.wits Qai“‘..asd .Urea

In order to circumvent difficulties encountered with
step 3 (Figure 1) Of the F-L scheme, alternate methods Of
precipitating E-casein from 1.7 M urea were sought.

A sample Of NaCas was treated according to steps 1 and
2 Of Figure 1. The pH was raised to 4.9 and the temperature
to 30°C. This gave a small amount or Ppt-3, but the bulk or
the e-casein remained dissolved in Super-3, 1.7 M urea.

This fi-casein rich fraction (Super—3 Of Figure I) became the
starting material for the following investigation.

Super-3, 0.5-1.0% (w/v) protein, was made 0.2 M in CaClz,
and at 30°C its pH was gradually lowered with 1 N HCl while
the solution was stirred vigorously with a magnetic stirrer.
The precipitate which appeared at pH 3.7 was centrifuged away,
and the two resultant fractions (precipitate and supernatant
from Super-3) were dialyzed, lyOphilized, and examined by
SGUE. The electrOpherograms are shown in Figure 7a. The
experiment was repeated on Super-2 (Figure l) of a subsequent

isolation; this time pH 3.6 was attained before precipitation
occurred. The results Of this study are displayed in Figure 7b.

27

As a control, the precipitation was attempted (on the
Super-3 described above) without Ca++ and then with Ca++ but
at 4°C. In neither case did a precipitate form.
4. Qagein_F;agtiona§ign_in thg Prgsgnge~o§_guadrafog (Sgdium

leirsphoaosaiel

The following experiments were an attempt to apply the
desolubilizing prOperties Of Quadrafos to the isolation Of
6 -ca se in.

a. hg Effect_p§ 9.1 and Q.5%_Q_qad_ra_f_qs_on %-p_a§ein Rich
solupigns in_1_.,7_M_Agugo_us_Urea_ gt_pH 4’. and 3030_

A sample Of NaCas was put through steps 1 and 2 Of the
F-L scheme (Figure l), and the Super-2 fraction (0.5—1.0%
protein and very rich in e-casein) was divided into three
portions. As is described in Figure 3a, one was warmed tO
30°C, another was made 0.1% (w/v) with respect to Quadrafos,
and the third was made 0.5% Quadrafos. The pH's Of the latter
two were readjusted to 4.6, and both were warmed to 30°C.
Resultant precipitates were collected by centrifugation, and
samples Of all fractions were dialyzed, lyOphilized, and
analyzed by SGUE. The patterns so obtained are represented
in Figure 8.
b- The 9.1 luiianrf. e— caseinfiich Quad safes: Ersafiszlutios S.

936.33. loQa-25.°Q

A sample Of Ppt-3 derived from initially rennin treated
NaCas (see Figure 4) was dissolved to approximately 4% (w/v)
in 4.7 M urea containing 0.79% (w/v) Quadrafos and at pH 3.0,
25°C. This solution was diluted drOpwise (Figure 3b) with

DDI water to 3.7 M urea, 0.063% Quadrafos, and the resultant

28

 

 

 

 

 

 

 

3a: The Super-2 (Figure l)o

effect of 1.7 M urea, pH 4.6, 4 C

0.1 and o

0.5% Qua- 30 C 0.1% Qua—o 0. 5% Qua-

drafos on drafos 30°C drafos 30°C
- v 1 r

Elifisiéiu- 0.0% 0.0% 0.5% o-sfi
tions in ppt super ppt super
urea at pH 0 1 035

4.6 and 30°C ppt? supgr

Ppt-3 (Figure u)
3b: The effect

 

 

 

    
 
 

of Quadrafos on dissolve to 4% (w/v) in 4.7 H
the dilution of \lurea, 0. 093% Quadrafos at
é-casein'rioh pH 3 o, 250
331331;“: igdurea ldil. to 3.7 M urea with H20.
25°C. Quadrafos 3?§ M 3ffi M
concentration ppt‘ super
varying. Casein -_
solution derived 511- to 3.3 g
from renneted NaCas. urea with hzu

335 M 3T3 h

‘ppt super
3c: The effect ppt-3 (Figure 4)
0f Quadrafos on Dissolve to 10% (w/v) in
the dilution of 4. 5 M urea, o 10% Qua-
e-casein rich drafos, at pH 3. 0, 25° C
solutions at pH
3. o and 25° 0. dil. stepwise with 0.1%
Quadrafos concen- aQueous Quadrafos. pH 3.0,
tration constant. collecting the ppts which
Casein solution form. _
derived from s \\
renneted NaCas. 302 M (urea) PPté‘ \\

2.4 M ppt \\

2.2 M ppt

FIGURE 3: Flow sheets for the studies on the fractionation
of fi-casein rich solutions with urea and Quadrafos.

29
precipitate was collected. Further dilution of the decanted
supernatant to 3.3 M urea, 0.056% Quadrafos, produced a
second precipitate which was likewise harvested. The three
fractions were processed as usual for SGUE, and their elec-

tropherograms are seen in Figure 9.

c. The Dilution of flycasein Rich Quadrafos—Urea Solutions

In the third study of this series, a 10% (w/v) solution
of E-casein rich Ppt-3 (prepared identically to that above)
was made 4.5 M in urea at pH 3.0, 25°C. As a control to
determine whether it is indeed Quadrafos which causes the
precipitation, the solution was diluted to 3.3 M urea with
DDI water. No precipitation occurred, but a rich harvest was
obtained when Quadrafos was added to 0.1% (w/v), the pH being
readjusted to 3.0. This precipitate was collected, and the
supernatant was further diluted with 0.1% aqueous Quadrafos
at pH 3.0. As is outlined in Figure 3c, at each of the follown
ing urea molarities the precipitate was harvested and the
dilution was continued on the supernatant: 2.7 M, 2.4 M,
2.2 M, 2.0 M, 1.7 M, 1.5 M, and 1.2 M. No further precipitate
develOped from dilution of the 1.2 M supernatant. The protein
compositions of all fractions were viewed by SGUE, and sketches

of the electropherograms are compiled in Figure 10.

5. The Isolation offi-casein from Renneted Whole Casein

A flow sheet and identifying nomenclature of the fractions
isolated by the following procedure are presented in Figure 4.

Freshly prepared whole casein was dissolved to form a
12% (w/v) aqueous solution at pH 6.6 and about 25°C. This

30

Starting material: lyOphilized whole sodium caseinate

Rennin step: Dissolve NaCas to 1 % in DDI 320 at 25°C, adjust
pH to 6.6, add rennin to 10’ mg/ml (to give 15 min clot-
ting time if 0.05 M Ca were present). After 10 min
inactivate rennin by adding urea to 6.6 M and adjusting
pH to 7.5. Allow to stand 1 hr.

I
Step 1: Dilute to 3.3 M urea with DDI H20, proceed as in step
1 of Figure 1. I

 

 

I
Super-l Pp1t-l
Step 2: As in Figure l. u]
f ‘ I
Super-2 Ppt-2

Step 3: As in Figure l, but at pH 4.6.
r ' j
Ppt-3 Super-3

Repeat of rennin step: Dissolve Ppt-3 to 10% (w/v), repeat
rennin step described above.

Repeat of step 1: Dilute to 3. M urea with DDI H20, proceed
as in step 1 of Figure l. 1

I ‘3
2-Super-l ' 2-Ppt-l
Repeat of step 2: As in Figurell.

F '7
2-Super82 2-Ppt-2

 

Repeat of step 3: As in Figure l, but at pH 4.6. Wash 2-Ppt-3
with heat-inactivated 2-Super-3.

mg.-. I it“ 2- Sugar-3

Step 5: As in F1 ure 1, but on y precipitate once. Dialyze
ion-free at 00. I

Pp4-5 ”I , Super-5

Quadrafos step: Dissolve Ppt-5 to 3% (w/v) in 4 M urea, pH
3.0, 25°C. Make 0.08% Quadrafos, readjust pH to 3.0,
harvest precipitate (denoted 4.0 M ppt). With DDI H O
dilute successively to 3.6 M, 3.3 M, 2.9 M, and 1.2
urea, at each step collecting the precipitate formed.

400 M ppt
306 M ppt
3.3 M ppt
M ppt
super

 

  
 
   
 

  
     
 

  
  
  
 
 

1.2

 

   

; for
igures 6 & 12.

ppt--

    
 

  

  

see

y 89

FIGURE 4: The isolation of @-casein from rennin treated whole
casein. In the flow sheet above, super = supernatant and
ppt = precipitate.

31

mg/ml in crystalline rennin and was allowed to

3

was made 10-
incubate at the same temperature. Clotting time under these
conditions (but with 0.05 M Ca++) is approximately 15 minutes.

After 10 minutes of incubation1

, the rennin was inactivated
by the addition of solid urea to 6.6 M and the adjustment of
the pH to 7.5. In this step, as in all procedures which
involve urea solutions, it is essential to adjust the pH after
adding the urea, for urea significantly depresses the activity
of hydrogen ions and thus increases the pH (83). After allow-
ing one hour for the complete inactivation of the enzyme, the
para-casein solution was diluted to 3.3 M urea with DDI water,
and steps 1 and 2 of the F-L scheme (Figure l) were performed.
Ppt-3, rich in fi-casein, was collected by warming Super-2 (at
pH 4.6) to 30°C just as in the modified F-L scheme of Figure
2.

Ppt—3 was redissolved in water to about 10% (w/v), pH
6.6, 25°C, and was treated with rennin exactly as before.
This helped to eliminate residual k-casein. Again rennin was
inactivated as described above, and the solution was frac—
tionated according to steps 1 and 2 of the F-L procedure.
This time no precipitate appeared in step 1. Semipure fl-casein
was harvested from 2-Super-2 (see Figure 4) by warming the
solution to 3000.

The following technique helped to wash away any residual

rennin activity present in the Q-casein preparation. A portion

 

1Incubation 2/3 of the clotting time was overcautious; it
is probably safe to allow the rennin to act for the full clotting
time before inhibiting the proteolytic activity of the enzyme.

32
of the "mother liquor" (2-Super-3 of Figure 4) formed in the
precipitation of the semipure fi-casein (2-Ppt-3) from 2-Super—
2 was heated to 90°C for 5 minutes to inactivate any rennin
present. The e-casein rich 2-Ppt-3 was suspended in this
solution (now cooled to room temperature) and was collected
again by centrifugation. The e-casein could have been washed
with water, but material losses might have taken place because
of dissolution. This was avoided by washing the precipitate
in its own e-casein saturated mother liquor.

After dialysis until free of urea, the 2-Ppt-3 was sub—
jected to step 5 of Figure 4, the protein concentration before
precipitation from the ethanolic ammonium acetate solution
being about 2.5% (w/v). The s-casein rich fraction from this
step, Ppt-5, was dialyzed until ethanol and ion free.

The final step in the isolation procedure described in
Figure 4, an attempt to remove lingering contaminants, involved
the fractionation of Ppt-5 in a solution containing urea and
Quadrafos. The method used was adapted from that develOped
earlier in this section (page 22, heading 9) and outlined in
Figure 3b. Ppt-5 of Figure 4 was dissolved to 3% (w/v) in
4 M aqueous urea solution at 25°C, pH 3.0 and was made 0.08%
(w/v) in Quadrafos (the pH was readjusted to 3.0). The precip-
itate which formed (termed the 4.0 M ppt) was collected, and
the supernatant was diluted with DDI water successively to
3.6 M urea (0.072% Quadrafos), 3.3 M urea (0.066% Quadrafos),
2.9 M urea (0.058% Quadrafos), and to 1.2 M urea (0.024%
Quadrafos). At each stage the new precipitate was collected

and the supernatant was further diluted. The five precipitates

33

and the 1.2 M supernatant were dialyzed against water, then
against dilute (less than 0.02 M) NaCl solution, the Cl' serv-
ing as an anion to exchange for the bound tetraphosphate ion.
The fractions were finally lyOphilized and analyzed by SGUE,
with the resultant electrOpherograms being displayed in Figurer
11.

The 1.2 M Ppt proved to be the purest g-casein preparation
and was also the major product of the Quadrafos-urea fractiona-
tion. In addition to the pH 8.6 electrOpherogram in Figure
12, a pH 3.1 SGUE pattern of the s-casein product is displayed
in Figure 6. The protein appears to be a single genetic
variant, and its mobility is identical to that of therp-casein
isolated by the modified F—L procedure (which utilized Nacas

from a different cow).
D. Rennin Inhibition and Inactivation

1. The Milk-010£tins Tise_A§sey_

A good milk clotting time assay for rennin activity
should 1) give clotting times in a convenient measurement
range for the enzyme levels encountered and 2) relate clotting
time to rennin concentration in a mathematically simple manner.

To achieve these objectives, the following substrate
system was prepared: 3% (w/v) aqueous Carnation nonfat dry

skimmilk, 0.01 M in CaCl and at pH 6.6 and 25°C. For a

2
rennin activity measurement, 5°0°.Pl of aqueous crystalline

3

rennin solution (about 10- mg) was added to 1.00 ml of the
substrate with an Eppendorf micropipette. Clotting time on

the order of 500 seconds was obtained. This was a bit long,

34
so the pH was lowered to 610, thus reducing the clotting time
to about 100 seconds.

Results with this system were somewhat imprecise. To
improve the reproducibility of the assay, the substrate solu-
tion was centrifuged at room temperature to sediment a small
amount of undissolved material. Moreover, care was taken not
to use any substrate solution over 12 hours old. Due to the
action of bacteria and/or milk protease, the composition of
aged preparations differs from that of fresh ones (see, for
example, the work of Broomfield (78)). Data obtained (as in
Appendix I) were usually within : 5% of best fit curves,
although occasional erratic behavior was still encountered.

To check the clotting time vs. rennin concentration
relationship, known amounts of rennin were added to the stan-
dard substrate (5 p1 enzyme per 1 m1 skimmilk), and clotting
times were recorded. The data are presented in Table III.

A plot of l/Tc vs. R (i.e., 1/clotting time vs. rennin con-
centration) in Figure 13 indicates that, within experimental
error, the assay is linear for clotting times from somewhat
under 100 seconds up to several thousand seconds.

Since the assay is sensitive to compositional variation,
it is difficult to compare rennin activity measurements made
on different batches of substrate solution. This drawback is
not too serious for the purposes of these experiments, however,
because day-to-day comparison of enzymatic activities was not

nece SSBI‘Y.

35
2 - The Inec .121 rail 9.11.0.1: aesnin.bx area.

The basic eXperimental method used in this study involved
the incubation of a fairly concentrated solution of rennin in
defined concentrations of urea at various temperatures and
pH's, and the periodic withdrawal of 5.00 p1 aliquots with
an Eppendorf micrOpipette for activity determination in 1.00 ml
of the standard skimmilk substrate solution at 25.2°C.

The buffers employed for the rennin inactivation solu-
tions were 0.03 M in NaHZPOh, the pH being adjusted with NaOH
to 5.0, 6.0, or 7.0 after the addition of urea. Urea concentra—
tionswused for rennin inactivation were 0.0 M, 1.7 M, 3.3 M,
and 6.6 M; rennin was incubated in the phosphate-urea solu-
tions at 0°C and 25.2°C. To retard cyanate formation, the
buffers were stored at 4°C and those containing concentrated
urea were discarded no later than several days after they
were prepared.

To begin an inactivation experiment, crystalline rennin
was dissolved in the abovementioned buffers in concentrations
ranging from 0.133 to 0.266 mg/ml. This gave assay concentra-
tions from 6.7 x 10'° to 1.33 x 10°3 mg/ml and initial clotting
times on the order of 100 seconds. In order to effect com-
plete dissolution in minimum time, buffer solutions were
stirred vigorously with a magnetic stirrer for approximately
30 seconds after the addition of the crystalline rennin. To
maintain temperature control during this period, it was

necessary to temporarily place the rennin solution in a small

pan of water (25.2°C) or ice-water (0°C) atop the magnetic

36
stirrer. After dissolution, it was transferred to the appro-
priate large thermostat.

Each inactivation experiment was carried out either
until the clotting time became too long to conveniently
measure or until enough data had been collected to suffi-
ciently characterize the kinetics of the inactivation. The
data obtained from these studies are reported in Appendix I.

To determine whether denaturation by urea is easily
reversible, a solution of rennin inactivated by 6.6 M urea
at pH 7.0 and 25°C for several hours was dialyzed urea-free
against water at 4°C. As a control, a solution similar in
all respects, but without urea, was treated in the same
manner. After dialysis, the solutions were assayed for milk
clotting activity. The urea treated rennin showed very little
ability to clot milk, while the control retained its activity.
This demonstrated that the mere removal of urea is not (at
least under the conditions studied) sufficient to reactivate
the enzyme, a point which is crucial to the validity of the
milk clotting time assays on the urea-rennin inactivation
solutions.

3- The Ineieiiien.°£ ins Milkfilopfiins ACE111£y_0£ eesninm
Aniosie Qeiereeni 8..

These experiments were designed to help elucidate the
site of reaction of anionic detergents in their inhibition
of the milk clotting activity of rennin. Three mechanisms
seem most plausible: 1) the detergent binds to the enzyme e

itself, inactivating it; 2) it binds to the substrate, pre-

venting enzymatic degradation; and 3) the detergent binds to

37
the renneted casein (para-casein), preventing coagulation
but not actually inhibiting rennin.

In an attempt to determine whether enzyme or substrate
binding of the detergent was taking place, the following
experiment was tried. Utilizing the standard milk clotting
assay substrate solution and a solution of rennin (0.20 mg/ml)
in pH 6.0, 0.03 M phosphate buffer, the clotting time was
found for 5‘°°.P1 aliquots of rennin in 1.00 ml skimmilk sub-
strate-~the same procedure which was used for the urea
inactivation studies. An aliquot of the rennin solution was

° M in sodium dodecyl sulfate (SDS) and was

then made 5 x 10'
allowed to equilibrate 1-2 minutes. 5.0‘p1 was removed and
assayed for clotting activity. The resultant SDS concentra-
tion in the assay, about 10"6 M, was found to have no effect

on clotting time, so that any anticipated changes in activity
should have been due to the incubation of rennin with the

5 x 10’° M detergent. Similarly, 1.00 ml of the substrate

was made 5 x 10'° M in SDS, was equilibrated, and was clotted
as usual with 5.00/pl or rennin solution. The whole experiment
was repeated with sodium dodecyl sulfonate in place of $08.

The results of both studies are compiled in Table IX.

If it is the secondary (aggregative) phase of milk clot-
ting which is inhibited, the presence or absence of detergents
should not markedly influence the rate of formation of para-
k-casein. This rationale suggests a simple experiment to
determine which phase of milk clotting is inhibited.

First, to determine the extent of detergent inhibition,

the clotting time of 10% (w/v) NaCas, 0.05 M in CaClZ,

38
5 x 10"3 mg/ml in rennin, and at pH 6.6 and 25°C, was found
to be 200 seconds. The same assay, but with 1.5 x 10""2 M
303 present, gave a clotting time of 860 seconds. This
amounted to 24% of the full activity or 76% inhibition.

Then, to distinguish more precisely the event influenced
by the detergent, SGUE was employed to monitor the appearance
of para-k-casein, the direct product of the primary reaction
in casein clotting. Six samples (numbered 1 through 6) of
10% aqueous NaCas, pH 6.6 and 25°C, were prepared. Samples
1 and 6, serving as controls, were treated neither with SDS
nor with rennin.. Sample 2 was made 5 x 10.3 mg/ml in rennin.

2 M in SDS and then 5 x 10"3

Samples 3 and 4 were made 1.5 x 10—

mg/ml in rennin. Reactions were quenched after 200 seconds

by 1:1 dilution of the digest with a 1 to 10 dilution of stock

tris-citrate SGUE buffer, pH 8.6, which had been saturated

with urea at room temperature. The pH of the quenched solution

was near 8.0. Sample 5 was a control on the effectiveness

of the quenching; it was made 1.5 x 10°2 M SDS, quenched,

and afterwards incubated with rennin at the same concentra-

tion as were samples 2, 3, and 4. Poor quenching would

result in the formation of para-k-casein in sample 5. To

preserve uniformity, samples 1 and 6 were also quenched.
Finally, 0.2 ml of each sample, treated as described

above, was applied to a pH 8.6 SGUE slab, and electrOphoresis

was carried out for 5-1/3 hours at 300 V. The high voltage

involved heated the gel somewhat and caused streaking in the

electrOpherogram but did not affect the results of the

39
the experiment. Representations of the SGUE patterns so

obtained are given in Figure 25.

IV. RESULTS AND DISCUSSION

A. g-Casein Studies

1 - Ereperetios 2f.8:ca 8211.1. PLtheJIsd ifiesi. 19.6.5_Pr°sesi.ure-
gf_FQx_and_Lilleyik,

Figure 5 presents representative pH 8.6 starch gel-urea
electrOphoresis (SGUE) patterns from precipitates and superna-
tantstobtained as theimodified F L procedure (as in Figure 2)
progressed through steps 1-5 in the ultimate isolation of f3-
casein. Figure 6 includes a pH 3.1 electrOpherogram from
the final.3-casein product. In addition, the effects of the
various steps on the solubilities of the major casein compo-
nents throughout the isolation procedure are summarized in
Table II. This table was compiled both from the electrOphero- ;

grams of Figure 5 (and, for the Ca++murea precipitation of

TABLE II: The Solubilities of the Caseins during Isolation

 

 

 

 

 

 

Procedures
Step Casein Fractions
pre- a s ’3 k y para-k
F-L 1 Pa 3 s P P
F-L 2 B P S S P(Sl) P z
F—L 3" p p s
F-L 1+ s P P s P(s1) g
F-L 5 S P P S S )
Ca++-urea s P P s P
$1) = slightly

% P = precipitated S = soluble, (
performed at pH 4.6 4
O

Step 2

Step 3 -

Step 4

Step 5 ,

Final
product

FIGURE 5: pH 8.6 SGUE patterns of the preniwitate. and
steps l~-5 in the modifie d 1‘) 65 Fox and Lijlswik Rwon=£ip
procedure as outlined in Fi”ore 2.
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isolation From renneted NBC 2
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B

i—Cfsoin products From the
ation of R-casoin

L.--.i._.jL_..

42
[B-casein, from Figure 7) and from many others not included
in this report. The results are strictly an idealization of
the observed behavior, in which 100% dispersion of a fraction
into the supernatant or the precipitate is never completely
obtained.

This investigator was unable to isolate p-casein as
electrOphoretically pure as that reported by Fox and Lillevik
(35). The major contaminant in the current preparation was
a small amount of k-casein which tenaciously accompanied the
‘p-casein, doubtlessly combined in the type of complex that
the two proteins are known to form (46).

Steps 1 and 2 as described by Fox and Lillevik separated
aS-casein fromnp-casein effectively every time they were
performed. Step 5 (fractionation in ethanolic ammonium acetate)
was usually efficient in eliminating‘r-casein from the p-casein
preparation. The results of step 4 (the Ca++ precipitation)
were, however, erratic: at its best, the precipitation elim-
inated a great deal of k-casein from the preparation; at its
worst, virtually no fractionation was achieved and large
sample losses were sustained. The amount of k-casein present
should influence considerably the amount of p-casein left in
Super-4. This conclusion follows directly from consideration
of the ability of k-casein to stabilize‘fi-casein against
precipitation by Ca++. variation of the pH over the range
6.5 to 9.5 was found to have little effect--either qualitative

or quantitative--on the results of the step, although higher

_ ,, -- ....v--.-w~.~

pH seemed to favor the solubilization of k-casein slightly.

These conclusions were drawn from SGUE analysis of the

43
precipitates and supernatants obtained when step 4 was
performed at various pH's between 6.5 and 9.5.

Step 3, applied as outlined in the F-L method (35), was
found unworkable, for at no time did a precipitate form when
pH 5.2 was maintained. The difficulties were partly cir-
cumvented by loweringtthe pH, usually merely by leaving it
at 4.6 unadjusted from step 2. The essential utility of the
step was that it was a convenient way of concentrating the
fl-casein from a dilute urea solution; significant purifica-
tion was seldom obtained. Indeed, sometimes the Super-3 was
richer in p-casein than was Ppt-3.

Figure 6, electrOpherograms oan-casein preparations at
pH 3.1 in formate buffer, shows that isolated by the modified
F—L procedure (Figure 2) migrates as a single component,
probably the genetic polymorph A by Aschaffenburg's defini-
tion (21). It was not determined which of the fi-casein A
subwariants of Peterson and Kopfler (23) corresponds to the
protein isolated in the present investigation.

Judging by the results of this study, the F-L technique
of 1965 can be made to work only by the modification of
step 3 as described above and by extensive recycling of the
fi-casein product. Even then, the yield of highly pure
B-casein'may be prohibitively low.

In view of the success of the technique in the hands of
the original investigators, its relative failure here is
puzzling. It is possible--though not, it is believed, prob-
able--that the laboratory technique of the present investigator

may be at fault. A second possibility is that the use of DDI

44
water (deionized through a mixed bed resin column) rather
than the double distilled water used by Fox and Lillevik is
the cause; casein fractionation has been observed to be
sensitive to impurities in the water used to form the protein
solutions (84). The only other explanation which suggests
itself centers around the fact that different lots of casein
were used in the two isolation studies. Throughout the
present investigation, whole casein preparations from different
cows have, when used as starting materials for Pacesein isola-
tion, shown minor variations in their fractionation behavior.
Perhaps something of this sort, but more pronounced, caused
the discrepancies between the preceding isolations and that
reported here.

and Urea

2. Th$+Pr§cipitation;quflzcasgin frgm‘Aqugous_Sglgtions_o§
Ca

Figure 7 shows the SGUE patterns from fractions obtained
upon the treatment ofIB-casein rich solutions (Super-3 or
Super-2 of Figure 2), 1.7 M in urea, with 0.2 M Ca++ at pH
3.6-3.7. The composition of each of the two starting materials
and the derived supernatants and precipitates is pictured.
The effects of the step on the solubilities of the various
casein components are summarized in Table II.

The results of this step differ from those of step 3 of
the scheme in Figure 2 in three respects: First,}3-casein is
more,completely precipitated in the presence of Ca++ and low
pH, a: is witnessed by the fact that the precipitation in

Figure 7a was obtained from the soluble fraction of step 3;

second, pre-d\components (those migrating ahead of’As-casein

45

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by the treatment of C. 5-1. W 8- “ca sin rich solutions (Super-a or Super- 3
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#6
during electrOphoresis at pH 8.6) are less precipitated by
the present method; and third,‘x-casein appears to be more
completely precipitated.

As regards its utility for13-casein isolation, then,
this step is quantitatively superior to step 3. Qualitatively,
it is more efficient in removing pre-a‘components but worse
for the removal of X—casein.

3. Casein Fractionation in the grgsgnge_0§,Quad;a§pg_£$gdium
'Tieirépfioénfiaie)’. " " " '7 " "

When an enriched fi-casein solution such as Super-2 (of
Figure 1), pH n.6, 1.7 M in urea, and at u°c is made 0.10%
with respect to Quadrafos and is warmed to 30°C, the precip-
itate and supernatant which form differ little from those
produced under the same conditions but without Quadrafos.

The precipitation of X;casein is promoted by the polyphosphate
anion, and a slight desolubilization of‘fi-casein can be seen
from the electropherograms of Figure 8. The addition of
Quadrafos to 0.50% instead of 0.10% had the surprising effect
of completely inhibiting precipitation. It is suspected

that faulty pH adjustment rather than Quadrafos might be the
cause of this phenomenon; the exPeriment bears repetition.

An enriched.p-casein solution (Ppt-3 of Figure h dis-
solved to #% (w/v) in 4.7 M urea, pH 3.0, at 25°C, and made
0.079% (w/v) with respect to Quadrafos) derived from renneted
whole casein was diluted with DDI water to 3.7 M urea and its
precipitate was harvested. The solution was further diluted
to 3.3 M urea, and again the new precipitate was collected.

The remaining supernatant and the two precipitates were

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Blow:

c {A
.11 um.” .- _,‘ P :' ' ‘ -,‘_..'<

.. . —_‘..¢--—4
.

#8
analyzed by SGUE. The resulting electropherograms of Figure
9 confirm the conclusion of Zittle (19) that the desolubiliza-
tion of casein fractions by polyphosphate anions is in the
order as) 8)k, and also indicates that Y—casein and the
pre—a.components are affected to an extent comparable to
As-casein.

ElectrOpherograms of fractions obtained in the second
Quadrafos study (which consisted of diluting a similar fi-casein
rich solution in urea at pH 3.0 and 25°C, keeping the Quadrafos
concentration constant at 0.10%) are depicted in Figure 10.

The results are essentially a repetition of those described

in the preceding paragraph. Two additional observations can,
however, be made: 1) the fastest pre-a_component is precip—
itated less readily than is p-casein, While the others tend

to be precipitated more easily, and 2) the solubility of
k-casein may vary in a complicated manner. Relatively more
k-casein appears in the 1.2 M urea supernatant and precipitate
and in the 2.2-3.2 M precipitates than in the 1.5 and 2.0 M
precipitates. Two explanations seem likely. First, the
behavior may be only apparent, since each SGUE sample contained
a fixed weight of protein and therefore only indicated the
relative prOportions of the casein fractions present. Thus

if 5 mg of k-casein are precipitated into each of the 2.0 and
2.2 M urea fractions but for p-casein the quantities are 30
and 60 mg respectively, upon SGUE it will appear that only
about 1/2 as much k-casein precipitated at 2.0 M urea as at

2.2 M. The second possibility is that k-casein is complexed

with the other fractions (as‘casein and fi-casein) and that its

49

Starting Material:

 
 
       

3.7 M urea Ppt

  

i,‘
i
(
x
e

 

 

‘3.3 M.urea Ppt

 

 

1
'1
s
2.
E

3.3 M urea Super

 

 

 

' . _, ‘ --‘v‘.. ' .i‘l. " " ;- 3 '. ‘3 97'. -“-'1" f h "2, I
C) ' iteratiuzugausm " 52...; advificfibfi.“ . , fl 4 .
\/ ,-~ ._- 3

FIGURE 9: pH 8.6 SGUE patterns of Fraciicms bidiuud jur’

ind the
dilution of a h.7 M urea, 0.07 % Quadrafos solution of a B—Casein rich
A

fraction (Ppt—B, Figure h) derived From renneted NaCas. flow sheet

of the procedure, Figure 3b, is reproduced below:

Ppt-3 (Figure h)

 

 

' «Dissolve to h% (u/v) in
' _ h.7 M urea, 060793 Quadrafos
at pH 3.0, 25 C.
' JDilute to 3.7 M urea with DDI H20.
ir'*- ~——..
3.7 M Ppt . 3.7 M‘Super
" [Dilute to 3.3 M urea
with DDI H D
j 2

 

 

3.3"M r3313 “ ‘ 32‘? M Super

 

   
    

 

 

 

 

 

 

 

 

. ' y ' . ” ‘
“5;,“ . .,:._._. w 1* ~ ' #2:; :7, a
.2 7 ; .V ._ ,
H ' € ; .
S‘altin ratesiel: Ppt~3 g 4 f : J
h i a ;
P! . . ._ ’_ -- ... . ~
i -‘ . a» .0 - ' 4‘ "I“ I
3.2 M urea Pet 9 f 4
s , , .-
k‘: 2.,- . 1...-..
gm ~~--~=-~‘—-~—~- -T”"‘““"""T7‘”‘“
g é i :E
" " - 5. ? g -.
2.7 M urea Ppt % i g E
- éflm «idiom ”rim: 1w“-.;iiil
E r‘ é .- ,
' g; s" ‘ ' 'A
' ,v t "I ‘
2.h M urea Ppt ; - g g 1
'i ’ i v’
:4 L- ‘ ”“5”“-.. v.~__-~_*v_ . _ ”~ _‘~ _M__._‘*m_
. "*~‘ F: ‘3 E ‘, .
I g é ‘2
2.2 M urea Ppt ‘ - g j g
i . i 2
E2, V,._.,..,,.. r 3'} i..-w,~~._._~.. ”was..- .‘ “ma —me-m‘~
2.0 M urea Ppt n ~ g f 5 ;Q
s ; g i
«A: r
‘1‘ --L.3--..._..,-..-.r_w-lh, _ z -...__,. “Min.--“ ,
', ' -- 3 I i: . 3
1.7 M urea Ppt E a - i 3 3 f: ii
‘. 9 '.~ 5 ~ ? I
2 ..~ ‘4- _ {hm-”N" ~-~. ”MN”- __:,___,-,,_‘ ‘ _ '1- - <5- -.. ”WV- '2‘,
"1'- ' ' L ! "
1.5 M urea Pot g g 5 ; }.
:2:- > , . I
Kiwi a i 1 -
r s:
. J -: 1 ‘6 .; I
1.2 M urea de ' .,- g .g
‘ P‘ f '5; g '
141......» ifi ~~-~m~~ "mm 1‘ ,l 4
" : .. '
1.2 M urea Super 5 -
é : .1 . V _ ~ _ ~.~ * h “42's.”:

 

’ISURE 10: pH W.S SGLE patterns from casein fractions obtained by
jiluting a B—sescin liCh QuedraFoS-urea solution at pH 3. U, 2508,
<eeping the Uuadr Woe c ncentreti on constant. Ppt— 3 ctdrtanc material
uas "U'u‘nflm rrom re nneted N386 s as in Figure A. Below is a flow sheet
if the Quadrafosnurea fractio"a'ir1n.

Ppt-3 (Fi ure h)

’ Dis- olve to 10% (u/v) in h.5 M urea,
n.1o5 Quadraros, pH 3.0, 2503. Dilute
stepwise with 0.18% aqueous Quedrefos,
, pH 3.0, collecting the ppts which form.

3.2 M Pot“ ~~m~ "~*4“\.

2.7 M Ppt’~-J—w»~”~~1~\
2.5 iiF>yt&“~"”"““M"-“”\\

p .
2.0 H Pptr*““”””‘";h”~l
1.7 M ppt+w~-w«~wf*~a\‘
1.5 H Ppt "“ idiiwlr

; ..__. ._-_..._.—..-——...
l

1.2 b f’p 1:2 Flihsper

A

 

Inn-'wo— 'o—s no. . ~\ — v----“-l

‘0‘-

o.-- q-

51
solubility will therefore be modified. An example of this
phenomenon is the precipitation of Ca++ insensitive k-casein
by Ca++ from a solution also containingls-casein (cf. Figure
5, step 4). The solubility behavior of k-casein in a solution
in which part of it was complexed would be difficult to predict.

4 - The lselstios 2f_B:G§. 821s iramfisnaeiei flanges

A study was made of the application of the specific
k-casein degradative activity of crystalline rennin to the
isolation of’3-casein from NaCas. it was hOped that treat-
ment of the starting material with rennin prior to the perform-
ance of the isolation procedure would eliminate the traces
of k-casein which had tenaciously adhered to all previous
fi-casein products (prepared as outlined in Figure 2).

Summarizing the procedure (which is also shown in Figure
h), the NaCas starting material was renneted, treated with
urea to inactivate the enzyme, and was fractionated in urea
solutions (steps 1-3, Figure 4). The renneting and urea
isolation steps were repeated on the semi-pure P-casein
obtained after step 3, and the product of the second cycle
of urea fractionation (2-Ppt-3 of Figure 4) was treated with
ethanolic ammonium acetate according to step 5, also of
Figure 1+. The p-casein precipitate so obtained was finally
fractionated in a Quadrafos-urea solution (Quadrafos step of
Figure 4). SGUE patterns of aliquots taken from the various
fractions throughout the isolation are displayed in Figure 11.

Comparison of the electrOpherograms in Figure 11 which

represent the products of steps 1-5 with those from the

fl“\ “‘3': I
\ I‘ i ‘ ". l [T 5

re

- we
'. , _ .
'1! *0»... _-o I\

u,-

I

gr’ZTJ-t‘s's. -\'Atlmmf;¢m‘$:' swag-guru..- “: 1‘3th w .

o o a (7' f. " i. 4.4, (‘3
Starting material: NaLas i ‘~3 f g p
. g :

 

rat-P2470! p

rw'wwnA } s- .2. . 3...“, ~ _-....~
_ f '3 f
Super-l é- 7% ii
0 H J . i
S t a D 1 . “.1“ :f::-‘....,.._~~:..:.:::: ; - -1‘: .‘r _ -_} ~"~':~J~-- - 7‘ ---~.-.~

’n,‘
23.

J..- ‘-
- -.

Ppt—l

 

”f4élz'lwhi

r

. .wuh.--

 

. -- -~“-~- .

Super-2

 

 

Step 2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pot-2
- Fpt-3

tep 3 _
Super-3
Z-Super-l E
2-Ppt-3
Ppt-S

‘ Step 5

Super-5 .
h.U M Ppt
3.6 M Ppt

 

Quadrefos

step . 3.3 M Ppt

 

2.9 M Ppt

 

 

Final g“ "
B—casein.a 1.2 M Ppt J
Product (

 

 

 

-.-‘9 '71s. sr‘v.. ,
. ‘ > ‘ i4N§,.* ~.,..
:1: ' ‘-" V

l .
. . I
- .- ‘ ' .- . 9.1"" Iva-.v‘w- w --*-4 . - M,“ ‘
.__,,,- I..a-_ ~ ea -- lulgfi‘;__~u ”Ania... '.-.. “3's...- thaw-two '- 9’33. "
I " l ’ '“/ "
. \a V ‘1 I '_,_ b ‘ \ 1 ('1 ,
V l t n 5

. ‘ .' .1: - - .. - _, ’ " ‘ I." Aha-‘Iy’ m3 V;
FthRE 11: pH 8.6 SGUE patterns From representative Fractions obtained
during the isolation oF Bucosein From renneted F8835. Fioure h, the

Flow sheet oF the isolation procedure, is reproduced on the Facing page.

. '53
corresponding steps performed beginning with non-renneted
Naaas (Figure 5) reveals that the two groups are quite similar
except that most of the k-casein in the renneted preparation
has been converted to para-k-casein. Examination of Figure
11 also reveals, however, that even the second treatment oft
the preparation with rennin failed to degrade the last traces.
of k-casein present (e.g., 2-Ppt-3). Longer enzymatic diges-
tion times would probably have eliminated this contaminant
(as suggested by the work of El-Negoumy (85)). but caution
must be exercised that general proteolysis does not become
important. Further work is necessary to establish the maximum
acceptable incubation time.

ElectrOpherograms from the fractionation of the ”p-casein
preparation in urea-Quadrafos solution (Quadrafos step, Figure
h) are also shown in Figure 11. The step served primarily to
reduce the amount of fecasein and fast migrating (pro-a) .
components contaminating the P-casein product. As noted
previously, these fractions tend to be less soluble in pH 3.0
urea-Quadrafos solutions that is p-casein and therefore.
precipitate at relatively higher urea concentrations. 'The 1
fraction Which was collected at 1.2 M urea was the purest
lung-casein. It also comprised the bulk of the precipitates
obtained in this step.

The yield of}3-casein (the 1.2 M urea precipitate) in the
overall procedure was 5-6% of the weight of the original NaCas,
calculations being based on lyOphilized weights. Analysis by
pH 3.1 SGUE (Figure 6) indicated that the product was composed

of a single P-casein genetic variant, probably the same one

5h
isolated by the modified F-L scheme (Figure 2), which
utilized starting material collected from a different cow.

The presence of pre-¢_components in the final prepara:
tion is in part due to the unusually high amount of them
present in the particular lot of NaCas used for this isolation.
The utility of the procedure must, hOWever, be Judged by its
effectiveness in eliminating k-casein from theep-casein product.
Its success was therefore only moderate, but with longer
enzymatic incubation times it may be that complete degradation

of k-casein can be achieved.

B. Studies on the Inactivation and Inhibition of Rennin

 

1- Lieearitx 2f.the_Mill<. elgtiiris_Ae say-
As was noted in the experimental section, the data of
Table III (and their plot in Figure 12) demonstrate that the

clotting time of the standard assay is indeed inversely

TABLE III: Test for Linearity of Milk Clotting Assay

 

 

 

t _

Rennin» T l/T ,

 

mg/ml x 10"3 secgnds (l/sec) g 10"3
0.05 1535 0.65
0.10 784 1.28
0.20 356 2.81
0.40 217 4.62
0.50 135 6.05
0.67 122 8.2
0.89 89 11.2
1.00 77 13.0
1.00 76 13.2
2.00 43 23.2

 

prOportional to rennin concentration. It is not necessary

 

25
; .
20
M
C)
r-I .
x 15
H
I
(n
’0
C
U
[‘4
(D
(n -
.. 10
0
F.
\.
H
5
'o
o 's 0.5 1.0 1 .5 2.0

. , . 3
Rennin Lonccntretion, mg./ml. x 10

FIGURE 12: Linearity check For the clogting time assay. Conditions:
tandard pH 6.0 skim milk substrate, 25 C.

56
to introduce a lag time for the secondary phase of the clot-

ting reaction.

2- The.Inaciiza£ien-°£.Beanin_br urea-

“(Table IV is an index to the seventeen series of experiments
performed to investigate the inactivation of rennin by urea.
The data from these studies, the enzyme incubation times and
observed associated milk clotting activity measurements (To)’
2) the fraction of the initial activity remaining (Ts/Tc) as
determined from the clotting time data, and 3) the logarithm
of the fraction of the initial activity remaining ( log (mg/Tc)).

The extrapolation to initial rennin activity, carried out
with a FORTRAN curve fitting program, was based on log (To)
data rather than on T0 itself because of the lesser curvature
of a plot of the logarithmic clotting time versus the rennin
time of incubation with urea. The more linear lepe permits
greater reliability in fitting the data points with low order
polynomials. The degree of the best fit curve used for the
extrapolation was primarily determined by the number of
measurements in the series under consideration. For example,
Series P consists of only four readings. A third degree curve
would fit the points perfectly and reveal practically nothing
about the general tendency, while a first degree curve is not
as close a fit but gives much better statistical information.
Series K, on the other hand, has 28 data points and can be best
treated by a fourth degree polynomial, the highest order func-
tion used. A second consideration in the selection of the

order of the best fit curve for a series was the rate of

57

TABLE IV: Indéx of Experimental Data on the urea Inactivation
of Rennin, Data Compiled in Appendix I

 

 

 

Series pH [urea], M Temp, 0C [rennin], Other
mg/ml

A 5.0 6.6 0.0 0.20

B 6.0 6.6 25.2 0.13

C 7.0 0.0 0.0 0.20

D 7.0 0.0 25.2 0.20

E 7.0 1.7 25.2 0.13

F 7.0 3.3 0.0 0.13

G 7.0 3.3 0.0 0.13

H 7-0 3-3 25-2 0.20

I 7.0 3.3 25.2 0.10

J 7.0 3.3 25.2 0.27

K 7.0 6.6 0.0 0.20

L 7.0 6.6 0.0 0.20

M 7.0 6.6 25.2 0.20

N 7.0 6.6 25.2 0.20

O 7.0 6.6 25.2 0.20

P 7.0 6.6 25.2 0.27 1% NaCaseinate

Q 7.0 6.6 25.2 0.27 1% NaCaseinate

 

58
inactivation observed. There can be no appreciable second,
third, or fourth order character to a study such as Series C,
where very little inactivation was observed. It is at all
times important to critically evaluate the statistical data
to insure that the mathematical manipulation has not divorced

the experimental results from physical reality.

a. The reversibility Qf_the_igagtivat;og

That the inactivation is not reversed by the removal of
urea from the enzyme solution was confirmed by the dialysis
experiment described in the eXperimental sections the dialyzed,
urea-inactivated rennin sample failed to regain appreciable

activity when the urea had been eliminated.

b. The order of the inactivation kinetics in rennin

The kinetics of protein denaturation by urea are typically
first order in native protein concentration (86), and first
order reactions give linear plots of logarithm of reactant
concentration versus time (87). Reference to Figures 13, 16,
18, 20, and 22, (in each of which log (TS/Tc) is plotted against
reaction time) shows that except for controls, only the exper~
iments performed at pH 7.0, 6.6 M urea, and 25°C fulfilled
this linearity condition. The deviations by the other series
are uniformly in the direction of an apparent order greater
than one, but in no case is there a curvature great enough to
indicate an order as high as two. Moreover, the curvature is
most apparent early in the reaction, and first order kinetics

are usually approached in the later stages. The apparent

59
linearity of Series M through Q (Figure 21) may thus be due
to a flattening of the curves before the first activity
measurements were made.

Many parallels can be drawn between the present investiga-
tions and the studies of Simpson and Kauzmann (88) on the
kinetics of the urea denaturation of ovalbumin. Using changes
in Optical rotation to monitor denaturation, they too observed
deviations from first order behavior. They suggested five
possible explanations, then showed that the fifth was the
true cause:

1) The order is truly greater than one.

2) A reactant other than protein is being depleted,

causing the reaction to slow down at a rate faster
than first order.

3) Reaction products are inhibiting the denaturation.

4) The protein preparation is inhomogeneous, with
different components denaturing at different rates.

5) The denaturation proceeds in discrete steps, as in
the scheme Native -7Denatured --> Denaturedz ——) . . . ,
each following first order kinetics.

To this list the present investigator would add:

6) Back reaction from the equilibrium Native Protein :éi
Denatured Protein is decreasing the overall reaction
rate.

All of the above phenomena would produce positive
curvature on the semilogarithmic activity graphs. Following
is an analysis of the six possibilities as they apply to the
inactivation of rennin by urea.

Phenomenon 1: If the order is truly greater than one,

plots of Tg/Tc or log (Tg/Tc) versus incubation time will be

sensitive to variations in initial rennin concentration. In

60
Figure 13 are plotted the data from three inactivation studies
(series H, I, and J) made under conditions which were iden-
tical except in initial rennin concentration. Note that the
points from series H (0.20 mg/ml rennin) and I (0.10 mg/ml)
coincide. This would be expected if a first order reaction
is Operating. The data from Series J (0.27 mg/ml) would seem
to support the presence of an order greater than one. It is
important, however, to note that Series J was performed with
a different set of reagents, two weeks later than Series H
and I, which were consecutively carried out. The complete
agreement of the latter two argues that the deviation of the
former is due to variations in experimental conditions and
that the reaction is truly first order, even though complica-
ting factors are present.

Phenomenon 2: No reacting species except the active and
inactive forms of rennin undergo significant changes in
concentration during the course of the reaction. The pH's
of the solutions were unchanged (within the accuracy of the
pH meter) after the incubation of rennin with urea. The
second possibility, then, is not promising.

Phenomenon 3: If, as is most likely, the mechanism of
inactivation is a conformational change in rennin, there
should be no reaction products which would inhibit the reac-
tion. Any release or uptake of protons during the reaction
would have no effect on the pH of the buffered solution.

Discussion of the fourth possibility will follow considera-

tion of the fifth and sixth.

131(1

Z/TC)

v1.2

-l.b

-1,6

61

\.

 

00,171." r/ f
a} y 1....

f, C‘I‘? ’1‘] I?

1P3? ETCU‘ 3CD-

Tn 5331111 T“'l_3, 3:300. .15

liu,tion nF rennin by ur-': the eFTec
inactivation rate. Conuitinnsz pH 7

e 1in as Follows: 3, 8.28 m“ nl Seri

es I);--»()——, ing/ml (FVerien

“/6

(,u 2“ ‘ fill-7“: ’

’7 ('2/0'17.‘

d

AGED

— J
L/ '

.9)

‘.- L

\
.C‘
\
25 2
"a
Vt
16 f.
\
k
§
\‘“o

H
C:
0

(.9

‘3“
a
W

N

.
I‘fi
h-J

61

11:8

 

\
.C§

25 5

\ . S

‘

X \ \ \ V

X\\\ , 01 r’. ("wax 1" ‘7" .’~‘-/ r“ \

‘\\\ .J' 7'... r' U l 3 I'd

\\\>‘ a C "”7" a E

”a \\ g
.ln:(| /T ) “; k4
C \ ~. 1:) 0
u. \ ‘ ‘ x c\

‘
L-J
o
N
I
I
I
CN
3
W

3 . \. ‘ - ‘
-1 . a. * . f \ . ~ 3 z. . n
0. .‘s? /_,’n:j‘/7 z fluvgtx

f2, (2" )2 m ,‘ 17

.. -- \
-ZQJ “’ '- ‘ .. , . - _ 1 r]
"‘ - r'. ' 'I' "" u‘ ' '4'.‘
U 1083 23LU 5GB; hGCG SQGU

FIGURE 13: Th9 inactivati
‘nn an :ne ind T

._... .r... I.“
{J'_'.|(42;51' ll). _'

0
:1 _., CC t L 1.,1 I UH '7 D, 3.3 M
urwa, 25.2 C, with “ennin a3 F01 GUS: 6, 8.25 mn n1 (befiies H);
x , 8.18 m /nl (3“1199 I);»——o—— 8.27 n*/ml (52:13” J).

62

Phenomenon 58 Whether a series of consecutive reactions
takes place cannot be determined on the basis of present data.
It should be emphasized that for this interpretation to apply
to rennin inactivation, the intermediate species must retain
some degree of milk clotting activity. Further conformational
changes on already inactivated enzyme would not be reflected
in activity assays, although they GOlld probably be monitored
by observing changes in Optical rotation or by some other
physical technique.

Phenomenon 6: Examination of the inactivation data
clearly indicates that the equilibrium between active and
inactive forms in the presence of urea lies far ().99,5%)
toward the inactive species, so back reaction should be
unimportant except, possibly, toward the latter part of the
inactivation process. A second point which militates against
this explanation is that deviation from linearity on the
semilOgarithmic activity graphs is in general greatest at
the beginning of the reaction. Bacx reaction effects would
be least anticipated during the initial stages.

Phenomenon 2: That this is the correct explanation of
the apparent deviation from first order behavior is suggested
by three independent lines of evidence. First, the crystalline
rennin used in these studies was known to contain small amounts
of proteins in addition to the major rennin component. This
was shown by SGUE analysis (see Figure 1% for a representation

of the electrOpherogram).

63

 

- f; +

FIGURE 1h: pH 8.6 SGUE pattern of the crystalline rennin
used in the inactivation and e-casein isolation studies.

 

 

 

 

tstar ting
S? . slot

Second, initial curvature followed by essentially linear
behavior is typical of semilogarithmic plots of data obtained
from a system in which several species are undergoing first
order reactions independently and at different rates. The
linear portion obtains after all but the slowest-reacting
component have been virtually exhausted; the conversion of
the final reactant then gives rise to simple first order
kinetics measurements.

The third indication that the heterogeneity of crystalline
rennin is the cause of apparent deviations from simple first
order inactivation comes from the studies of Cheeseman (69).‘
He utilized both purified gB-rennin and commercial rennet in‘
his brief study of the inactivation of rennin by uresj his
data are plotted semilogarithmically in Figure 15. It is
evident that the results for homogeneous {B-rennin are i011-
described by a linear plot, while those for commercial rennet
(inhomOgeneous, probably also containing ureaémnuuliiifti
pepsin and other inert proteins) exhibit high curvature- I
For comparison, data from Series H of the present study are

also presented in Figure 15.

0.0

-D.2

~U.h

I; ”0.6
'3

:3 ~e.8
0
('J

of -

3:3 “100
'5

i “102
43
‘0'"!
:3

2r: -1...
U
33

3‘ —1.6
I...

.1.8-

FIGURE 15: The inactivation of rennin by urea:

apt—.X':
f’“:
.. - ’6’:

I'd”:

20

la

hD

6h

 

L 7

60 80

’¢

Incubation time, minutes

\

0
B-rewnin, 6 M urea, pH 5.h, 37 Co
B-rannin, h.6 M urea, pH 5.h, 37 C
Commercial Hennat, 6 M u

Crystalline Rennin, Ser
pH 7.0, 3.3 H

urea,

25.

«.a, pH 5.u, 37°C

'es}{ of the nrssent study—4

8, 0.20 mg/ml rennin.

l

100

DU

63

#0

25

16

10

6.3

h.U

'2.5

1.6

the data of Cheeseman (69).-

activity

.1

36.1:uyt12..

65
In summary, it is highly probable that the apparent
deviations from first order inactivation of rennin are due
to inhomogeneity in the enzyme preparation. It is possible
that part of the deviation is due to consecutive steps in
the inactivation, but the linearity of Cheeseman's data when
plotted semilogarithmically suggests that any such contribu»

tions are not significant.

0» The influence stares concentration 9.11-1.riaetivatioa rate.
The time course of inactivation in the presence of various

concentrations of urea is shown in Figure 16. At pH 7.0 and

25.200 rennin is virtually stable in the absence of urea,

although an activity loss of approximately 3% is evident

after incubation under these conditions for one hour. The

rate of inactivation increases sharply with urea concentration.
The order of the reaction in urea can be determined as

follows: Given a number 2 between 0 and 1, define tz to be

that incubation time when the activity of rennin is 2 times

the initial activity. Thus, is the familiar half-life

t0.50
of the reaction, and t0°79 is the time when 79% of the full
activity remains. Under the assumptions that the inactiva-
tion rate is strictly first order in crystalline rennin
concentration and that the concentration of urea does not
change significantly during the reaction (both fairly good
approximations), a plot of the logarithm of tz versus the
logarithm of urea concentration should be linear. More

importantly, the lepe will be the negative of the reaction

rate order in urea. Although z = 0.50 is the customary choice

66

100
79
63

SD

% of
‘“AD Initial
Activity

0
109(TC/TC) 32
25
20

uD-fi

16

 

1 2 3 LL

FIGURE 16: Tie inactivation of rennin by urea: t
urea concentration on inactivation rate. pH 7.0,

urea as indicated:

ffect 3F
, rennin and

urea, 0.2 mg. ml. rennin; Series D

urea, 0.13 mC./m1. rennin; Series fi
urea, 0.2 mg./m1. rennin; Series H
urea, 0.2 mg./ml. rennin; lieries N

—-a"'

- o—‘a—l“ —

1

(Dull-JD
auqc
lzrnkzt:

’
’- "’-

log 1‘2

(seconds)

F'IFRJRE Jf7: ’Hwe i
the order of t
ifliis study to on

. ‘6? _ ‘ r-N'.”
S tUUVULuQi.) leI‘T . ‘ L’I.‘

.q-
b'G x.,-

a

 

f” f '1
3.2. {41"

37%.
: z =
: z =
: z =
: 7. =
t Z

67

U.79,

0.53,

9.50,
U.QU,
U.h3,

CA£€g {"33 ’1
230.90

8.h .3 0.8

loniyreg7, i1

vation of rennin by urea: determination of
"3tinn rate in urea concentration. Data From
9.13-5.27 mn/ml rennin. Cheeseman's

this study

H II
II II
.I I.

68
for such calculations, any other fraction between 0 and 1
will also serve. A mathematical derivation of the above
relationships is presented in Appendix II.

The tz data compiled in Table V was obtained graphically
from the log of activity versus time plots for the apprOpriate
series. The results given in Table V which pertain to studies
conducted at pH 7.0 and 25.2°c (from Series E, H, I, J, M,

N, and O) are plotted logarithmically in Figure 17 against
the logarithm of urea concentration. In the interest of
improved accuracy, several values of z are used for determina-
tions. For comparison, a calculation is also made there for
Cheeseman's (69) data (pH 5.h, 37°C). The order in urea is
calculated from the s10pes of the lines in Figure 17 accord-
ing to the equation develOped in Appendix II. A mechanistic
interpretation of the values obtained and compiled in Table
VI (2.5 order for this study, 3.9 for Cheeseman's) would be
extremely questionable. One can, however, note that the
calculated order is significantly lower than that calculated
by Simpson and Kauzmann (88) for the urea denaturation of

ovalbumin.

TABLE VI: The Order of the Inactivation Rate in Urea

 

 

 

Concentrationa
Data 2 Order
calC'do
This Stady 0029 203
" " 0. 2.
, n 0.5% 2,2 mean 2'5
a a 0040 206
Cheeseman 0.40 3.9

 

aDetermined from Figure 17, tz data taken from Table v.

69

.Aupv woa one mosam> mp on» Scamp mowerpnenmm ca moonsss ens m

 

 

 

 

 

Ae0N.m0 Ammo.mv Ammo.av Anme.av heee.av
mma mma m0 H0 om mm.m 0.5 ma0.0 0 .z .2
Ammm.mv Aflmmomv
0mHm one 00.m a
Ammm.m0 Ame0.mv Amoe.mv A0ea.m0
000 000 00m 03H 0H0.0 0
AeH0.mv Aenm.m0 Ame0.N0 Ammm.mv
oeoa owe 0e: 0am mam.0 H .m
AOH0.mv Amee.mv Asmm.00 Amem.mv
00H: memm ooea 00a Hmm.0 m
Aeeo.m0 Ammm.m0 mamma.mv
0mm: omHm 0H0 0.0 m
mm.0 02.0 0m.0 no.0 me.0 muoa M ameuau
n N you .mosoomm ea . HIMOJAIB mm moa mofinmm
soapmn

0cm .soapmnpsmosou men: SH Hmono exp

.6

m co zodpeeaesepea esp soc «pen p

unmosoo +3 SH H0090 om»
u> mqm<9

, 70
d- The inilseaca sf_p§ 2n.tbe_isastivatioa 2826.

Figure 18 shows the influence of pH on the inactivation
rate in 6.6 M urea at 25.2°C. Inactivation is favored by
high pH; to quantitate this observation, log (tz) is plotted
versus pH in Figure 19, the situation being mathematically
analogous to the determination of the orderin urea concentra-
tion. Data for Figure 19 were taken from Table V, and the
orders in hydrogen ion (given in Table VII) were calculated
in the same manner as were those for urea. The average order
thus obtained was -l.4. Because of the equilibrium present
between H+ and OH", this could as well be eXpressed as a +1.h
order in hydroxyl ions. Due to the small concentrations of
both of these species (about 10'7 M), it seems most probable
that they influence the inactivation rate by affecting the
state of ionization of amino acid side chains on the protein
molecule. A study of the effect of ionic strength on the
inactivation rate would help in evaluating the importance of

such charge phenomena in the reaction.

TABLE VII: The Order of the Inactivation Rate in 3*

 

 

 

 

...... Concentrationa '”
Order
Data 2 Calc'd.
This Study 0079 '103
II n 0050 ’1.“ mean ”104
" " 0.25 -1.4 J
aDetermined from Figure 19. ”/t
//

71

Mafia/TC) 41.5 - r - a. \"

 

0 1 . 2 3 h_ 5 6 7
. 3

Incubation Time, seconds x lD+

{fififlRE 18: The inactivation of rennin by urea: the effect of pH

1 ' " .' '-- 0 f' n m-r' D
on tne inscrivo.xon rate. Data taken in a.6 M urea at ae.2 C.

a : Series 8, pH 6.0, U.13 mg'nl rennin.
)(: Series N, pH 7.0, 6.20 m /nl rennin.

”100

79
63
SD
#0

32

.25

20

it

12.6

10.8

% Initial Activity

72

5.0 — “
3.5
3.0
2.5
log tz 2.0

1.5

 

1.0
8.5. - _ x . _ j

0.0 ‘ i. -
5.8 6.0 £.2 3.1. 6.6 3.8 7.0 7.2

TIUUHE 19: The inactivation CF rennin by urea: Determination of
the order of the inactivezion rate in hydro; n it”. Studies done
in 6.6 M urea, 25.208, on as indicated. The pH 6.0 run (Series 8)
gas 5.13 mg/ml in rennin, the pH 7.0 (Series E) 8.20 nv/ml.

o : Z =‘-' 0.79
x : Z = [3.50
A : Z = E.25

73

e . The inflae ace sttgmperaiure .011. the 28128.0: inaciizaii 2n.
2f.rsnsis sy.urea

The effect of temperature on the rate of the inactivation
of rennin by urea is shown in Figure 20. The reactions furnish-
ing the data plotted there were performed at 0°C and 25.2°c
in 6.6 M urea at pH 7.0. The apparent Arrhenius energy of
activation, Ea’ can be evaluated from a plot of log (tz) versus
T'l, the slope being Ea/2.33, where B is the gas constant.

The derivation of this relationship is given in Appendix III.
The energy of activation was calculated from plots of this
type in Figure 21 using tz data from Table V, and the results
are listed in Table VIII below. A mean value of 21 keel/mol,
typical of many enzymatic inactivations (86), was obtained.
In view of the non-Arrhenius behavior of the temperature
dependence of the rate of urea denaturation of ovalbumin (88),

TABLE VIII: Results of the Clotting Activity Study on the
Inhibition of Rennin by Anionic Detergents.

 

‘

 

 

-Detergent Added To Conc., M 32:8 Inhibition
None (blank) ---------- 101 no
SDS Rennin 5 x 10'“ 102 1
SDS Substrate 5 x 10'“ 191 47
Sodium dodec- Rennin 5 x 10'“ 101 o
yl sulfonate
Sodium dodec- Substrate 5 x 10'”4 161 37

yl sulfonate

 

aAverage of several trials.

74

‘\ ’1 “' I ~1eo
-0.1 i. A ‘ I i i ' 79

f

 

L
-D.2 J 63'
l
3
ioo(T°/r ) ' as of
C c ~D.h »‘ #0 Initial
l ‘ Activity
0
43.5 I 32
-D.6 _ fi ’. . ‘. y _ . V ,_ I _ .i 25
"0.7 ‘ 20
i
I .

0 1 2 3 h
I D O -3
Incubation EIMR, sec. X 10
FTIGURE 28: The inactive'ion oF rennin by urea: *h” 8?

{M1 the inactivation rate. C nditions: pH 7.0, 6.6 M ure
Ing/ml :ennin.

cc; of temperature
a, 0.20

~vv-~_.’£-"-': USE. ’ F: ‘i-‘js L
_“_°__v-:25 8., Series N

75

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

III 01'

ILIH iII NIfL LII .ITL

nu
oIIJI 5 0
LL _ FLWLI “ __ 01. 1e _ -.o i . a. .
h. _ 02 due ttst. +L .H .H. H H JIIMM01,
:L0I-(fla-bz __.0 .++ i-in -fi- .0 1-1-Trfl
I»L_---.r._;.-_ ....r_0,:__.0+01. a 1:0 +_....
L... a. I: i 1041-

 

 

 

 

 

 

1
/
“I,
i
t_
f;
i.
L?
m

 

 

 

 

 

 

  

 

 

  

, PI
. . _ . U I
.7 .. . _ ” TV Irlt.“ IILLI.‘ .4. .40 t ,._-0HI ? LL .0
. X . -41...-LJIIIMI0 .Ir. n.l B
. . 0 ” .0.0...0p--q-|¢0.~0pl -IIA T 3 UJU t
h . . . I. +II >I 0..00I+I.| IO 4 U i n a
- w . 0 0 1 I 9 0M.¢I LII 4.” .QI_ 0 I 1 t O C
H o 4 « Ilduullu P0 V|_I0. ..o II “I «I. m C a
.--0. - .01-- rI e x. n
r _ ,d.n we..i - . m ..1
e r 0.-.. j. 0.. 3 h. r n
- -I. ..II I- 0 11-1.0.0 1.
. I. 1:- ; -3 a . r h -- o e
n a i. . . ...I_0."0 t .1 a
0 I u .II IrIHII 4 ”I IlnIfi IH I« D B t
w _ .. :0141. -.0.- I;+0i on C e
w . 4 . 1.70, e . 01— LIV . 00 I . 0'. , a r 9 D
r i 1. .II% .1 - 0I. a red 75
i. i - -110- 0. 7010. - - m. .. r . .
, I . _ . 1 . . no U 0
III a "It M0WI$IUI 0+I m IILI I FIN- r) m E 8 “II. D
_ II .I .-- , s. _ _ 0 . .- “in e ..=
1 i. 00 -_...-01..- - .. . .3 r u t n
,e. ._ I. .I“I~ . . “ III! 1 0|. . i 0t. ”W Z z
. . .HI. .. _ a I, M . may. V r E
0 , 404 I... T . I. I .60 _ .H b 0 (mlu
. . .. VII... . -. IO 0. “III m .0 I . . 8 pl .. ..
. a, i 0Ir.r+ 1.*. Iv.L . 1 cl n 0 .o.x
.“ ... H h .+ “I 7.“.0 III-eI04ml0r 0 I .1 an )
. . .- I- -.0.: .1. - nee Mm
.. . . n _ . _ _ . n
a t. . 0 .1, - - 0e. e n d
_ . r n. n
_ . it .1 -- 3 .. e
. _ H , .
. 0 M i . . II . . . .04 7 I9 . L w- I u «0 . 4.00 00- IIII I F L...
1 I. H n . I“ 0. ..... IL e w r i .0- - C uh
I I“ olol e 0. . II 0- I. I 0 o . II 9 II. III I
e0 . 4 .I .- _ I. I . .. ....I I. 000 . 0-- III “00 MD
I I0 I . II“ .I I n . . . ..0 0—0. I W PI I D e
. . .._. 4 m . 0 . / . . D/. II. _ .. . I II. 4 tel. .1
0 i. r ;._.. M _. _.0.- . I r
-. .. i .... M 8 e
. -I0. . . .

 

U
inactiv

the Arrhenius enerny of active

The

6.6 M urea, 8.28 mg/ml renni

data From Table V (5

log t
SURE 21
0H 7.5,
2

Fl
t

76
an activation energy calculation based on two data points
(as was that in Figure 21) is somewhat suspect. More credence
could be placed in a value obtained from data taken at several

temperatures.

f. Te§t_fgr_stabiligatign_by substrate

It is well known that some enzymes are stabilized by
their substrates against inactivation. Figure 22, comprised
of data from inactivations carried out at pH 7.0, 6.6 M urea,
25.200, and in the absence or presence of 1% (w/v) NaCas,
suggests that no such substrate protection is evident in the
case of the inactivation of rennin by urea. If anything, the
NaCas destabilized the rennin, for the curve for the eXperiments
run with substrate lies below the one for the case without
NaCas. The two sets of studies were performed using different
lots of reagents, however, and the discrepancy in reaction

rates might easily be related to experimental error.

3 - The Inbieiiien_0£ .R.eanin.bi Anya-19. QeierssnES.

Figure 23 pictures three possible mechanisms for the
inhibition of rennin milk clotting activity by anionic deter-
gents; the following discussion is in terms of the schemes
depicted there.

The first study, which involved exposure of anionic
detergents to the enzyme rennin or to the substrate casein,
failed to reveal which interaction was responsible for the
observed inhibition. The results, compiled in Table VIII,
indicate that the addition of SDS or sodium dodecyl sulfonate

to the enzyme solution failed to decrease the rennin activity

0.0
-u.1
-o.2 63
-C.3 so
.o.h to'

—0.5 32

iog<Tg/Tc)
20
16

12.6

10.0

 

. \1
C
\D

’1'

”1.3 D I ‘ I " 5.0
0 1rd 2cm 3:: «so 500.

e
Incubation Time, seconds
1:13035 22: The innotivetinn n? rennin by urea: tn: effect of sub brute

. e o L. k_ fl ';-“u > r‘ ’.F - r _ n
orI are lnECthnolnn rate. conditions: 0H 7.0, 0.c m urea, 25.2 C.

L

o : Tories M, 0.20 hq/ml rennin, without suhstrhte
B : deride h, 0.23 ug/ml rennin, without substrate
A : Q‘rir‘ 0, 0.70 m /nl rennin, hithout substrate
a : Fri .17 n‘n/iral i'rnnin, 1‘6 PIECES

x : ' ‘7 ”

J

(U ('5

'3 L.)

{—1 ('3 :
(\J 0\J "

S‘r- rrL"?1 rennin, lféIYnCas

7o {finial Acrfw‘f/

78

C
Scheme I:
\L B +1D;;:::::fIBD (inactive)
para-C
clot
Scheme II: C + D\:::_:\‘ CD (protected)
R
para-C
clot
Scheme III: C
LR
para-C +2D;;:::::i para~CD (non-aggregative)
clot

FIGURE 23: Possible mechanisms for the inhibition of rennin

milk clotting activity by detergents. R = rennin, D —
detergent, C = casein, para-C = para-Casein, the product of

the primary phase of rennin degradation.

79
appreciably, while the same concentration introduced directly
into the assay medium achieved significant inhibition.‘

These findings are easily compatible with schemes II and
III. They are also, however, compatible with Scheme I if the
stipulation is made that the enzyme + detergent complex
equilibrium is readily reversible. If this condition prevails,
then any rennin-detergent complex formed when the detergent
is added to the enzyme solution will dissociate upon the .
dilution of an aliquot of rennin in the substrate solution
during a milk clotting assay. The data of Table VIII indicate
that an upper limit of approximately one second exists for
the hypothetical dissociation process. Anything slower would
have been reflected in increased clotting times for determina-
tions in which the detergent had been added to the enzyme
solution. It should be noted that Just as the experiment
does not rule out any of the three schemes in Figure 23,
neither does it establish that one of them is Operating to
the exclusion of the others. In view of the fact that deter-
gent binding by proteins is a common phenomenon (89). it is
not implausible that more than one of the schemes is important
in the inhibition.

Figure 24 represents the SGUE patterns obtained from the
casein samples of the experiment designed to determine thather
anionic detergents inhibit the secondary (aggregative) phase
of milk clotting. As can be seen from patterns 1 and 6
‘(controls), no para-k-casein was produced in the absence of
rennin. Nor was para-k-casein produced in sample 5, the con-

trol on the quenching reaction (substrate + detergent,

80

,r ‘-- ’ ' ’ 3 ‘ r
1 ”u I .
‘
5‘ ‘4 I ( I I - I!
r: Manx? “5i"? :xs .1: c. I r“ ‘ ~*~ _ i : 72;- ‘V an: s‘" .L:{.;i~‘;£“
‘3 .. .f v, ,
r‘ _ ‘4 (fl .. ~‘. ‘ ;.‘ A g .
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Dumplg .L. 12': US i‘ Q ', ‘ g ,.«
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V ’ ’ i
«as —t¥u .7. .4.- w» a. ‘ _ V 3}“ 'w—u .A-n‘w -&--‘— “Wu-u:- ‘34- -~-- - »“ J A ‘d'i‘lL- — n W
1% i j ’1, "‘9
4‘3, 3 ; J’ 5; ‘3
[‘rlwy ‘ {3 20 rxir‘C'jr‘ r? F“ . f ‘1 I 3
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Ji‘lp .a e IC‘J .J + J J + \ + ‘, 1 ;
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E - i -
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mums-MW“, ~swi. : N:Mfi-~o’>LN--t'- n-uv horn-n ." ”~9‘ -—. ‘. ".me m-qu
s I . a
1",. ,. 1 6 . f”... [a 1 ~ » ,~ a .1 . .
JSMDLG . :QJUS + , " s i i >
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fi"."‘.’.“"~r""3::.~ “arr:.;;::“ . ”‘3’ i 3‘s. $3.4:
:\ J w- :.‘v- '
l‘ :‘ ll w:’-“ \. b—Wiw A ‘/

FIGURE 2“: pH 8.6 SGUE nattcrns From the axnnnimsnt catermininq whether
50% inhibits the primer J s F by rennin. NaCas =
snjium caseinate, } = rsnni _
inactivate rennin), 505 s. a. Reancnts and cnzym
were nixed to the $3355 snlu2‘nn5 in the orders indicated above.

 

k‘i}

81
quenched and then exposed to rennin). A great deal more
para-k—casein is evident in sample 2 (without SDS) than
in samples 3 and h (with SDS). It is therefore concluded
that the inhibition of the milk clotting activity of rennin
by anionic detergents is primarily the result of an inhibi-
tion of the primary phase of clotting, the limited proteolysis
of k-casein to para-k-oasein and glycomacropeptide. Any effect
of the detergents on the secondary phase (nonenzymatic aggrega-
tion) is of minor importance to the inhibition. Scheme III
of Figure 23 has thus been shown to be_inapplicable.

A decision between Schemes I and II is not possible at
present. Available evidence, however, seems to support Scheme
II more strongly. First, it seems that a complex as tight as
SDS usually forms with proteins would probably be dissociable
by dilution on a time scale greater than one second, the upper
limit required for Scheme 1. Second, SDS is known to bind
to k-casein, the primary substrate for rennin action (90);
its binding to rennin has not, to the present investigator's
knowledge, been investigated. It may be fairly easy to resolve
the question, and suggestions toward that and will be presented

in the next section of this discussion.

4. sussssiisns £0: Eartha; seseeram

In the investigations on the isolation of @-casein from
rennin-treated NaCas, it was necessary to inactivate the enzyme
after a reaction time approximately equivalent to the clotting
time in order to minimize the effects of general proteolysis.

It was not determined, however, at what point general

82
proteolysis becomes significant, and it may be that incuba-
tion with rennin for much longer periods can be allowed for
limited proteolysis without the degradation of @-casein.
The point at which general proteolysis becomes important could
be determined from SGUE analysis of samples taken from a
casein solution after various periods of rennin digestion;
alteration of the S—casein band would indicate tertiary
phase activity by rennin (general proteolysis).

The precipitation of casein fractions from urea-Quadrafos
solutions holds great promise as an isolation method. It
should be possible to find conditions of pH, temperature,
protein concentration, urea concentration, and Quadrafos
concentration which will allow a much sharper separation of
B-casein from the other casein components than was achieved
in this study. Precipitation with Quadrafos also has poten-
tial for the isolation of the minor casein fractions.

Further studies on the urea denaturation of rennin should
be performed on homogeneous enzyme (for isolation procedures,
see Foltmann (48)). Once a homogeneous fraction of rennin
has been prepared, the means of monitoring activity should be
improved. Although the milk clotting assay is reasonably
accurate, it is fairly awkward. Preliminary evidence indicates
that the inactivation of rennin is accompanied by a significant
increase in the ultraviolet absorption of the compound (see
Figure 25), and that long periods of inactivation (24 hours
or more) result in a general decrease in absorption. It may
be possible to relate the spectral shift to the degree of

inactivation, thus providing an exceedingly simple and exact

83

I
)5
l
7.
- O
O

. 1 C3 . , . '
Absorbence 9.h ’////. - _j
' o - . , 3

2am 260 ' "220 ' 300

CURE 25: U11
9

I ;revinlet abenrct nn snectra 0? na
Ffifl inenti at ' 3

.c enzguma (*k~9
("v.9. Inactiwnztinn n£n3 P 1

nhnentete, 8.h mn/ml rennin, 25
rctive enemies mes Lske r
Enectre cnsrected For t

i tive rennin (ro~) and
and the UifrerenC& s ectrum of the two
' d out “t pH 6.0 in :.b M urea, 0.03 M
, Fnr 105 minutes. Spectrum of the
the some cnnditinns, but ulttnut urea.
» F‘
I

l

L‘.

84

index of rennin specific activity. The same may apply to
any shifts in optical rotation shown during the inactivation
process, although this physical prOperty has not been studied
by the present investigator.

Differences between the ultraviolet absorption spectra,
optical rotatory dispersion, and viscosity (to mention only
a few properties) of solutions of native and inactive rennin
can be related to conformational changes taking place in the
protein molecule during inactivation. More detailed mechanis-
tic information could be obtained in this way.

An investigation of the denaturation by urea of the dif-
ferent forms of rennin described by Foltmann (48) might provide
insight into the relationships between protein structure and
conformational stability in closely related molecules.

Future studies could be made over a higher pH range, and
the temperature dependence might also be characterized in
greater depth. The variables investigated should be expanded
to include the contribution of ionic strength to the rate of
inactivation of rennin by urea.

Whether, anionic detergents indeed bind to rennin could
be determined by equilibrium dialysis or by the ultracentrif-
ugal method described by Swaisgood, Brunner, and Lillevik
(91). The following experiment may provide a direct answer
to the question of whether enzyme or substrate binding inhibits
the primary phase of the milk clotting reaction of rennin.

If SDS forms a stable complex with kmcasein, it may be pos-
sible to precipitate the complex (along with all the other

casein fractions) from a solution of NaCas and detergent.

85
Bedissolution and treatment of both this precipitate and a
control (without detergent) with rennin would test for
inhibition of clotting activity. Detergent concentrations
could be manipulated so that the free SDS in the clotting
solution would be below the level necessary for inhibition.
It would not be difficult to determine whether the SDS-k—
casein complex dissociated when it was redissolved for
renneting. If the detergent-substrate complex remained
associated and inhibition was observed, Scheme II of Figure
23 would be strongly supported. If association but no inhibi-
tion were observed, Scheme I would be supported (though less
forcefully), and if the complex dissociated, the experiment

would, alas, yield no information.

V. SUMMARY AND CONCLUSIONS

The first half of this study dealt with the isolation
cm‘ S-casein from whole bovine casein. Techniques investigated
included 1) a procedure developed by Fox and Lillevik in this
Laboratory in 1965 (35), 2) the precipitation of G-casein
from a crude casein solution containing urea and Ca++, 3) the
fractionation of casein in urea-Quadrafos solutions, and ”)
4) the treatment of whole casein with rennin to specifically
degrade k-casein, followed by the inactivation of the enzyme
with urea and the isolation of P-casein (from this renneted
starting material.

The inactivation of rennin by urea was then investigated,
and the dependence of the reaction rate on urea and rennin
concentrations, pH, temperature, and the presence of whole
casein substrate were determined. Finally, the inhibition
of rennin milk clotting activity by anionic detergents was
studied.

The following conclusions can be drawn from the results
of the experiments summarized above:

A. The present investigator was unable to achieve the
purity of the final product reported by Fox and Lillevik (35)
in their' g-casein isolation procedure of 1965. It was, in
addition, necessary to modify part of their Operation (step 3,

Figures 1 and 2) to effect the precipitation of a fi-casein
86

87
rich fraction which was not obtained under the conditions
given. Part of the deviation of the present results from
those of the previous workers may be due to differences in
the whole casein starting materials and purity of the distilled
water used in the two investigations.

B. The precipitation of B-casein from crude casein
solutions at pH 3.6-3.7, 25°C, 1.7 M urea, and 0.2 M Ca++
is a satisfactory alternative to the Fox-Lillevik step (step
3, Figure 1) with which difficulty was experienced.

C. The studies on the precipitation of ,et-casein from
urea-Quadrafos solutions at pH 3.0 confirmed the conclusion
of of Zittle (19) that the solubilities of the major casein
fractions under such conditions increase in the order ds-casein,
B-casein,k-casein. It was in addition found that X—casein
and the preix components have solubilities similar to that of
cis-casein. Fractional precipitation by gradually decreasing
the Quadrafos and/or urea concentrations of fi-casein rich
solutions at pH 3.0 proved to be a satisfactory method of.
eliminating X-gcasein and pre-a contaminants from the B-casein
preparation. The technique also holds promise for the isola-
tion of minor casein fractions.

D. The renneting of whole casein prior to the isolation
ofCB-casein by the procedure outlined in Figure h was moderately
successful; most of the k-casein present was degraded and
easily removed as parauk-casein, but a small amount of k-casein
remained unattacked by rennin and accompanied the e-casein

preparation even after two incubations with the enzyme.

Lenyxnning the rennin digestion time might well result in a

‘88
more complete degradation of the kuoasein.

E. The inactivation of rennin by urea proceeded at a
rate first order in rennin, although the heterogeneity of the
crystalline rennin preparation complicated the analysis of
the kinetics somewhat. At pH 7.0 and 25.200 the inactivation
rate is approximately 2.5 order in urea concentration. In
6.6 M urea and at 25.200 it is about 1.4 order in hydroxyl
concentration. The apparent Arrhenius activation energy for
the reaction in 6.6 M urea and at pH 7.0 was 21 kcal/mol,
typical of many enzymatic inactivations. No protection of
rennin by its substrate was observed.

F. It was shown that SDS inhibits the milk clotting
activity of rennin by blocking the degradation of k-oasein
to para-k-casein plus glycomaorOpeptide and not by interfer-
ing with the nonenzymatic aggregation of the para-casein.
Although it was not possible to determine whether the inhibi-
tion results from enzyme binding or suhstrate binding of the
detergent, available evidence indicates that substrate binding

is the more probably cause.

VI: BIBLIOGRAPHY

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APPENDICES

96

APPENDIX I

Observed Milk Clotting Activities from

§§udies on the Inactivation of Rennin by Urea

Series: A

T28 98 seconds

 

 

 

 

 

pH: 5.0 urea8 6.6 M
Temp: 0°C rennin: 0.2 mg/ml
Seconds To Tg/Tc log
Incubated (Seconds) (Tc/Tc)
180 97 1.013 0.006
330 99 0.993 -0.003
600 98 1.003 0.002
810 95 1.035 0.015
1200 97 1.013 0.006
1740 101 0.973 -0.011
2400 98 1.003 0.002
3360 100 0.983 -0.007
4130 102 0.964 -0.016
4530 98 1.003 0.002
5130 104 0.945 -0.024
6000 98 1.003 0.002
7560 102 0.964 -0.016
§eriesiaug T2: 127 seconds
pH8 6.0 urea8 6.6 M
Temp8 25.2°c _f rennin8 0.13 mg/ml
Seconds - Tc Tg/To log
Incubated (Seconds) (Tg/Tc)
42 128 0.994 -0.002
96 129 0.985 -0.006
258 138 0.921. -0.035
309 143 0.889 -0.050
629 158 0.804 -0.094
670 166 0.766 —O.115
715 171 0.744 -0.128
1040 183 0.694 -0.157
1415 205 0.620 -0.207
1765 227 0.560 —0.251
2090 253 0.502 -0.298
2372 270 0.:71 -0.326
2 2 0. -0.
.32. ,2 0.433 -388
3335 43 0°370 '0- 80
3751 392 0.324 -O.4 8

(continued on next page)

97
§§rie38s B (continued)

 

 

 

 

 

Seconds To Tg/Tc og
Incubated (Seconds) (Tc/Tc)
4043 441 0.288 -0.539
4552 486 0.261 -0.582
5260 569 0.223 -0.650
5883 673 00189 “00723
6627 779 0.163 -0.787
7494 908 0.140 -0.853
8540 1152 0.110 -0.956
Series: C T8: 104 seconds

pH: 7.0 urea8 0.0 M

Temp: 0°C rennin: 0.2 mg/ml
Seconds To Tg/T log

Incubated (Seconds) c (Tg/Tc)

195 103 1.010 0.003
345 104 1.000 0.000
870 99 1.050 0.020
1200 113 0.920 -0.037
1440 101 1.030 0.012
1800 106 0.981 -0.009
2400 101 1.030 0.012
3000 103 1.010 0.003
3900 107 0.972 -0.013
4800 100 1.040 0.016
6600 108 0.963 -0.017
7630 102 1.020 0.007
9000 123 0.846 -0.074

 

Series: D

98

T2: 62 seconds

 

 

 

 

 

pH: 7.0 urea8 0.0 M

Temp: 25.20C rennin: 0.2 mg/ml
Seconds T tug/Tc 1.3

Incubated (Sec8nds) (TS/Tc)

270 61 1.013 0.005
426 62 0.997 -0.002
600 64 0.066 -0.016
1080 62 0.997 -0.002
1560 62 0.997 -0.002
2345 64 0.966 -0.016
2790 63 0.981 —0.009
3325 63 0.981 -0.009
3860 66 0.936 -0.030
4750 69 0.896 -0.049
5630 70 0.883 -0.055
6720 71 0.870 -0.061
7220 72 0.858 -0.067
Series: E T28 91 seconds

pH: 7.0 urea8 1.7 M

Temp: 25.2°C rennin: 0.13 mg/ml
Seconds TC Tg/Tm log

Incubated (Seconds) V (Tg/Tc)

(+2 93 009716 "00008
67 102 0.890 -0.040
205 97 0.936 -0.018
261 101 0.899 -0.035
402 105 0.865 -0.052
540 112 0.811 -0.080
689 115 0.790 -0.092
835 118 0.769 -0.103
1039 127 0.715 -0.135
1244 135 0.673 -0.160
1614 142 0.639 -0.183
1674 142 0.639 -0.183
1938 153 0.593 ~0.216
1976 158 0.575 ~0.230
2288 163 0.557 —0.243
2327 166 0.547 ~0.251
2530 171 0.531 ~0.264
2737 180 0.504 -0.286
3063 188 0.483 «0.305
3373 203 0.447 -O.339
3649 207 0.439 -0.347

(continued on next page)

99

 

 

 

 

 

 

Series: E_(continued)

Seconds To Tg/T log
Incubated (Seconds) ° (mg/Tc)
3960 223 0.407 -0.379
3988 223 0.407 -0.431
4749 251 0.362 -0.540
5580 323 0.281 -0.514
6181 313 0.290 -0.527
6208 321 0.283 —0.538

Series: F 1:: 125 seconds
pH: 7.0 urea: 3.3 M
Temp: 0°C rennin: 0.13 mg/ml
Seconds TC Tg/TC log
Incubated (Seconds) (TE/Tc)
44 129 0.969 -0.014
76 125 1.000 0.000
235 122 1.025 0.011
313 122 1.025 0.011
520 126 0.992 -0.003
1028 127 0.984 -0.007
2318 120 1.042 “0.017
3409 113 1.106 70.044
4507 124 1.008 0.004
4783 113 1.106 0.044
5597 121 1.033 0.014
6792 125 1.000 0.000
8509 122 1.025 0.011
12165 129 0.969 -0.014

 

§§riesz G

100

o -
To: 72 seconds

 

 

 

 

 

 

pH: 7.0 urea:
Temp: 0°C rennin: 0.13 mg/ml
Seconds To 138/130 03
Incubated (Seconds) (Tc/Tc)
44 75 0.960 -0.019
74 70 1.029 0.011
203 73 0.986 -0.007
332 75 00960 -00019
6148 72 10000 -0001].
899 75 0.960 -0.019
1256 76 0.947 -0.025
1830 77 0.935 -0.030
2572 76 0.947 —0.025
3393 76 0.947 -0.025
4587 85 0.847 -0.073
6327 82 0.878 -0.058
Series: H T38 seconds
pH: 7.0 urea:

Temp: 0°C rennin: 0.13 mg/ml
Seconds To Tg/Tfl og
Incubated (Seconds) " (To/Tc)
77 62 0.886 -0.050
108 6)" 00858 “00061:"
213 66 0.833 -0.078
238 74 0.743 -0.128
273 81 00678 “00166
393 87 0.632 -0.198
420 86 0.639 -0.192
1041 142 0.387 -0.410
1083 151 0.364 -0.437
1372 163 0.338 -O.470
1407 171 0.322 -0.491
1724 205 0.268 o0.570
1822 225 0.244 m0.610
2087 269 0.204 ~0.688
2223 283 0.194 -0.710
2579 359 0.153 ~00813
2693 385 0.143 w0.843
4326 827 0.066 -1.176

 

 

101

O

 

 

 

 

 

Series: I To: 120 seconds
pH: 7.0 urea:
Temp: 25.2°C rennin: 0.10 mg/ml
Seconds T TO/T log
Incubated (Segonds) c c (Tg/Tc)
37 117 1.025 0.012
71 131 0.763 ~0.037
105 137 0.730 -0.057
271 157 0.647 -0.116
317 162 0.617 -0.130
359 167 0.599 -0.143
503 193 0.518 -0.206
623 212 0.566 -0.246
898 254 0.472 -0.325
1185 310 00387 -0.411
1362 352 0.298 -0.467
1817 453 0.264 -0.576
2084 611 0.196 -0.706
2445 705 0.170 -0.768
3185 937 0.128 -0.892
3528 1157 0.104 -0.983
3952 1461 0.082 -1.085
4873 2187 0.055 -1.260
4919 2184 0.055 -1.259
Series: J T2: seconds
pH: 7.0 urea:
Temp: 25.2°C rennin: 0.27 mg/ml
Seconds To To/T log
Incubated (Seconds) c c (Tg/Tc)
69 83 0.952 —0.021
136 96 0.821 -0.084
278 120 0.658 -0.181
469 147 0.537 -0.269
747 210 0.376 —0.424
1141 290 0.272 -0.564
2075 875 0.090 ~1.044
3977 3383 0.023 «1.631
4120 3327 0002!" “10622
4974 5086 0.016 -1.808

 

102

 

 

 

 

 

 

 

Series: K 1:: 125 seconds
pH: 7.0 urea: 6.6 M
Temp: 0°C rennin: 0.20 mg/ml
0
Seconds To Tc/Tc log
Incubated (Seconds) (TS/To)
180 129 0.959 -0.010
390 154 .31? -0.087
660 156 0.801 -0.092
900 157 0 o '(“71'1 -0 o 095
1170 186 0.672 -0.169
1410 195 0.641 «0.189
1800 210 0.595 -0.221
2100 210 0.595 -0.221
2‘360 2424' 0051.2- ”00286
3000 254 0.492 «0.304
3600 280 0.446 -0.346
4200 321 0.389 -0.406
5100 355 0.352 -0.449
6000 411 0.304 -0.513
16320 1800 0.069 -1.154
Series: L TS: 98 seconds
pH: 7.0 urea:
Temp: 0°C ren.1 8 0.20 mg/ml
Seconds Tc Tg/Tn og
Incubated (Seconds) ” (Tc/Tc)
180 93 1.048 0.013
330 116 0.841 -0.083
540 114 0.855 -0.078
720 125 0.780 ~0.116
900 128 0.762 -0.126
1320 129 0.756 -0.130
2130 162 0.602 -0.229
2490 187 0.521 m0.291
3000 213 0.458 -0.347
3720 221 0.441 w0.363
4230 243 0.401 ~0.405
4920 247 0.395 wfio412
5330 284 0.343 m0.477
6180 333 0.293 m0.541
6780 363 0.269 -0.579

 

103

 

 

 

 

 

 

 

 

 

Series: M T8: 150 seconds
pH: 7.0 urea: 6.6 M
Temp: 25.2°C rennin: 0.20 mg/ml
Seconds To Tg/Tc log
Incubated (Seconds) (Ts/Tc)
60 218 0.690 -0.116
105 322 0.466 -0.287
147 469 0.330 ~0.450
259 1124 0.133 -0.830
(475 3445 000-414“ -10316
Series: N T8: 127 seconds
pH: 7.0 urea: 6.6 M
Temp: 25.2°C rennin: 0.20 mg/ml
Seconds T TO/T log
Incubated (Segonds) c c (Ta/To)
49 179 0.711 -0.149
87 240 0.529 -0.277
117 361 0.352 -0.455
150 425 0.299 -0.525
183 537 0023? I"00627
258 949 0.134 -0.874
§g;;§§;__g T28 132 seconds
pH: 7.0 urea: 6.6 M
Temp: 25.20C rennin: 0.20 mg/ml
Seconds Tc Tg/To log
Incubated (Seconds) (Ta/To)
49 176 0.750 -0.124
77 215 0.614 -0.210
109 293 0.451 -0.345
143 352 0.375 -0.425
179 481 0.274 «0.560
323 966 0.137 m0.863
441 2784 0.047 al.323

 

Series: P

104

T2: 100 seconds

 

 

 

 

 

pH: 700 urea: 606 M
Temp: 25.2°C rennin: 0.27 mg/ml
Substrate present: 1%‘Nadas ' '
Seconds To To/Tc log
Incubated (Seconds) 0 (Tg/Tc)
50 172 Oefifil “00232
81 243 0.4;3 -0.382
112 350 0.28s -o.5uo
302 2710 0.03? -l.429
Series: 9 T2: 82 seconds
pH: 7.0 urea: 6.6 M
Temp: 25.2°C rennin: 0.27 mg/ml
Substrate present: 1% NaCas
Seconds To Tg/Tc 103
Incubated (Seconds) (TE/Ta)
an 134 0.61; -o.215
82 189 0 e 1‘: »“ “0.36“
112 285 0.2ifi -0.543
147 398 0.29: —0.688

 

105
APPENDIX II

Derivation of the Equation Relating Urea Concentration and tz

 

Define:

Assume:

Derivation:

deGwo’ww

N

NH
VV

The

a rate constant for the inactivation reaction
the initial rennin clotting activity (t = 0)
the rennin activity at time t

the urea concentration

the order of the reaction in urea

a number between 0.0 and 1.0

the reaction time when R = zRo

Inactivation is a simple first order reaction
in R.

Urea concentration does not change signif-
icantly in the course of any one reaction.

rate equation for the inactivation is then:

01'

dB/dt = -kUaR
dB/B = -kUadt

Integrating between t = 0 and t = t,

ln(B/Bo) = -kUat

Now, when t = tz, then R = zBo and we have

or

or

1n(z)

, a
ukU tz

~1n(z) kUafi,

ln(l/z) = kUatz

Taking logarithms of both sides, we get

01‘

log(ln(1/z)) = log(k) + alog(U) + log(tz)

1 (t ) = - 1 (U) + 1 (k) - 1 (1
(Ifizfij a og Cog og n

Differentiating with respect to log(U) and
noting that the bracketed term above is a
constant, we have

d [iog(tzi] = a

d [208(UXj—-

which is the desired result.

106
APPENDIX III

Derivation of theégguation Relating Arrhenius

Define:

Assume:

Derivation:

Activation Energy, Temperature. and t3

 

k

a rate constant for the inactivation reaction,
where k is of the form

k = A exp(-Ea/RT)

a constant

the apparent Arrhenius activation energy
the gas constant

the experimental temperature

the initial rennin activity (t = 0)

the rennin activity at time t

a number between 0.0 and 1.0

the reaction time when R = 230

dembm>
0 03

N

HHHHHHHH

Inactivation is a simple first order reaction in B,
and concentration variations of other reactants are
negligible.

The rate equation for the inactivation is

-dR/dt = kR
or
-dR/R = kdt

Integrating between t = 0 and t = t,
ln(Ro/R) = kt

Now, when t = t then R z 2R0 and we have

2’

ln(1/z) = ktz

or

t2 = ln(l/z) / k
Since

k = A exp(uEa/RT)

we can substitute for k, obtaining
tz == ELn(l/z)/1g exp(Ea/BT)
Taking logarithms of both sides,
log(tz) = log {in(l/z)/A] +-Ea/(2.3RT)

Then differentiating by l/T and noting that the
bracketed term above is a constant, we have

d §103(tz)| _ Ea
d 1 T ‘ 2.33

which is the desired result.