ABSTRACT

ISOLATION AND CHARACTERIZATION OF PROTEOSE—
PEPTONE COMPONENTS 8—FAST, 8-SLOW
AND 5 FROM COW'S MILK

by Charles W. Kolar, Jr.

The proteose-peptone fraction of cow's milk is
defined as that portion of the proteins not precipitated by
heating at 96—100 C for 20 minutes and subsequent acidifi-
cation to pH 4.6. This fraction contains a group of heat
stable proteins which account for about 18% of the whey
proteins or about 4% of the total proteins in milk.
According to data obtained by free-boundary electrophoresis,
three peaks called components 3, 5 and 8 are present in the
fraction.

The purpose of this study was to develop procedures
suitable for the fractionation of proteose—peptone into its
various components from both heated and unheated cow's milk
and to elucidate the chemical and physical characteristics
of these components.

Exploratory isolation experiments resulted in the
development of optimal procedures for the isolation of highly
enriched fractions of components 3, 5 and 8 from heated and
unheated skimmilk. The enriched component-5 fraction was
further purified by ammonium sulfate and pH adjustments.
Data were presented illustrating the electrophoretic separa-

tion of component 8 into components 8-fast and 8—slow on

Charles W. Kolar, Jr.

acrylamide gels employing a continuous buffer system. These
components were subsequently separated by gel filtration on
Bio-Gel P—l0.

Chemical analyses were performed on components 8-fast
and 8-slow isolated from heated and unheated skimmilk and
component 5 isolated from heated skimmilk. These proteins
were low in nitrogen (l2.3l-l3.83%) and high in phosphorus
(0.97-3.29%). The total carbohydrate content (hexose,
hexosamine, and sialic acid) varied from a high of 10.27%
for component 8-slow to a low of 1.45% for component 5.
These proteins were prepared from heated skimmilk. Paper
chromatography, employed for the tentative identification
of the carbohydrate moieties, showed the presence of
glucosamine, galactosamine, galactose, glucose, mannose and
fucose.

Amino acid compositions of these proteins were charac-
terized by relatively high contents of glutamic acid,
aspartic acid, serine and threonine and relatively low
contents of glycine, methionine and tryptophan. Cysteine
was absent. Component 5 contained a high content of proline
(10.55%).

An isoelectric pH of 3.25 for component 8 was deter-
Huned by free—boundary electrophoresis employing various
buffers of 0.2 ionic strength. The average descending and
ascending electrOphoretic mobility measurements for com-

ponent 8-fast from heated and unheated skimmilk in veronal

Charles w. Kolar, Jr.

buffer at pH 8.6, I = 0.1, were -9.00 and -9.31 Tiselius
units, respectively. The average mobilities for component
8—slow from heated and unheated skimmilk were -9.15 and
—9.21 Tiselius units, respectively, whereas a value of
-u.80 Tiselius units was observed for component 5.

The crystallization of a preparation of component
8-fast from unheated skimmilk was observed in veronal buffer
at pH 8.6, I = 0.1.

The sedimentation coefficients, S° , for components

20,w
8-fast and 8-slow isolated from unheated skimmilk and for
component 5 isolated from heated skimmilk were 0.78, 1.35,
and 1.22, respectively, in veronal buffer at pH 8.6, I = 0.1.

Sedimentation-equilibrium weight-average molecular
weights for components 8—fast and 8-slow (prepared from

unheated skimmilk) and component 5 (prepared from heated

skimmilk) were ”,100, 9,900 and 14,300, respectively.

ISOLATION AND CHARACTERIZATION OF PROTEOSE-
PEPTONE COMPONENTS 8-FAST, 8—SLOW

AND 5 FROM cow's MILK
By

Charles W. Kolar, Jr.

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Food Science

1967

 

ACKNOWLEDGMENTS

The author expresses his most sincere appreciation
to Dr. J. R. Brunner, Professor of Food Science, for his
counsel, encouragement and inspiration during this study
and for his assistance in the preparation of this manu-
script.

Grateful acknowledgment is due Dr. B. S. Schweigert,
Chairman of the Department of Food Science, and the Public
Health Service for providing the research facilities and
funds necessary for this research.

The writer is indebted to Mr. Khee Choon Rhee for
the art work shown in this thesis and to the author's
fellow graduate students for their many contributions.

The author thanks his wife, Diane, for typing the

rough drafts and for her help and encouragement.

ii

TABLE OF CONTENTS

ACKNOWLEDGMENTS

LIST OF TABLES

LIST OF FIGURES . . . . . . . . . . .
INTRODUCTION

LITERATURE REVIEW

Historical.
Contemporary

EXPERIMENTAL . . . . . . . . . .

Apparatus and Equipment .

Chemicals and Materials .

Preparative Procedures for Proteose-Peptone
Fractions.

Exploratory Procedures for the Preparation and
Fractionation of Proteose— —Peptone from
Heated and Unheated Skimmilk . . . . . .

Preparation of proteose- peptone .

Preparation of sigma proteose .

Preparation of component 5 as adapted from
the method described by Jenness (1959)

Preparation of washed casein micelles and
the material released from micellar
casein at pH A. 6.

Preparation of casein micelles and the
material released from micellar casein
at pH A. 6 . . . .

Preparation and ammonium sulfate fractiona-
tion of proteose-peptone

Effect of calcium on sigma proteose

Release of component 8 from whole casein
with EDTA .

Release of component 8 from whole casein
by heating.

Release of component 8 from whole casein
with ammonium sulfate .

Fractionation of proteose—peptone with
ethanol. . .

iii

Page
ii
vi

vii

U100

16

l6
18

2O
2O
20
21

21

23

24

2A
25

26
26
27
27

Page

Preparation of a proteose-peptone-rich
fraction from unheated skimmilk. . . . 28
Sodium chloride and ammonium sulfate
fractionation of a proteose-peptone—
rich fraction prepared from unheated
skimmilk . . . . . . . . . . . 29
Sodium chloride and ammonium sulfate
fractionation of proteose-peptone
prepared from heated skimmilk . . . . 30
Ammonium sulfate fractionation of a
proteose-peptone—rich fraction at pH A.9
prepared from unheated skimmilk. . . . 31
Ammonium sulfate fractionation of a
proteose-peptone—rich fraction at pH 7.0
prepared from unheated skimmilk. . . . 31
Optimized Procedures for the Fractionation of
Proteose- -Peptone from Heated and Unheated

Skimmilk. . . . 32
Isolation of component 8 by preparative
acrylamide gel electrophoresis . . . 32

Ammonium sulfate fractionation ofaiproteose-
peptone- -rich fraction at pH A. 9 and 7.0
prepared from unheated skimmilk. . . . 3A

Ammonium sulfate fraction of proteose-
peptone at pH A. 9 and 7.0 prepared from

heated sk’ mmilk . . . . . 35
Isolation of component 5 from proteose—
peptone obtained from heated skimmilk. . 35
Separation of components 8-fast and 8—slow
by gel filtration . . . . . 36
Preparation of Samples for Chemical and
Physical Analysis. . . . . . . . . . 37
Chemical Methods. . . . . . . . . . . . 38
Nitrogen . . . . . . . . . . . . . 38
Phosphorus. . . . . . . . . . . . . 39
Hexose . . . . . . . . . . . . . . A0
Hexosamine. . . . . . . . . . . . . Al
Sialic Acid . . . . . . . . . . . . A3
Sulfhydryl Groups . . . . . . . . . . AA
Amino Acids . . . . . . . . . . . . A6
Tryptophan. . . . . . A7
Paper Chromatography (Carbohydrates) . . . . A8
Physical Methods. . . . . . . . 50
Electrophoresis . . . . . . . 50
Free- -boundary electrophoresis . . . . . 50
Starch-urea gel electrophoresis . . 52
Acrylamide gel electrophoresis with a dis-
continuous buffer system . . . 5A
Acrylamide gel electrophoresis with a con—
tinuous buffer system . . . . 56
Two- dimensional acrylamide gel electro-
phoresis . 58

Staining of glyc0proteins of the proteose-o
peptone— —rich fraction after electro-
phoretic separation in acrylamide gels . 58

iv

Ultracentrifugation. .
Protein concentration.
Sedimentation velocity .
Sedimentation equilibrium
Densities and partial specific volume

RESULTS AND DISCUSSION.

Preparative Procedures.

Exploratory Procedures for the Preparation and.
Fractionation of Proteose- -Peptone from
Heated and Unheated Skimmilk . . .

Optimized Procedures for the Fractionation
of Proteose- -Peptone from Heated and
Unheated Skimmilk. . . .

Chemical Composition . .
Physical Properties. . .
Electrophoretic Properties
Ultracentrifugal Properties .
Sedimentation coefficient
Sedimentation- -equilibrium molecular weights

SUMMARY.
BIBLIOGRAPHY

8A

91

93
100
100
102
102
10A

1A3
1A6

Table

LIST OF TABLES

Summary of compositional data describing sigma
proteose, proteose-peptone, milk component-5
concentrate, Weinstein's minor-protein
fraction, and milk whey phosphoglycoprotein

Composition of components 8, 8—fast, 8—slow
and 5. .

Amino acid composition of component 8-fast
prepared from heated and unheated skimmilk

Amino acid composition of component 8-slow
prepared from heated and unheated skimmilk
Amino acid composition of component 5 prepared

from heated skimmilk. . .

Electrophoretic mobilities of component 8
purified by acrylamide gel electrophoresis

Electrophoretic mobilities of components
8-faSt, 8-SlOW, and 5 o o o o

Sedimentation coefficient and equilibrium
molecular weight data of components 8—fast,
8-slow, and 5 in venoral buffer at pH 8.6,
I = 0.1 . . . . . . . . . . . .

vi

Page

15

107

108

109

110

111

112

113

Figure

1.

10.

11.

Procedure

LIST OF FIGURES

for the preparation of proteose—

peptone, adapted from the method
described by Rowland (1938a)

Procedure for the preparation of sigma proteose,

adapted

from the method described by

Aschaffenburg (19A6).

Procedure
adapted
Jenness

Procedure

for the preparation of component 5,
from the method described by
(1959).. . . . . .

for the preparation of the washed

casein micelles and the material released
from micellar casein at pH A.6

Procedure

for the preparation of casein

micelles and the material released from
micellar casein at pH A.6 . . . . .

Procedure
peptone
sulfate

Procedure

for the preparation of proteose—
and its fractionation with ammonium

for the isolation Of EDTA—released

components from isoelectric precipitated

casein

Isolation

of heat-released material from

isoelectric precipitated casein . . .

Procedure

for the isolation of the ammonium

sulfate-released fraction from isolectric
precipitated casein . . . . . .

Schematic
peptone

Procedure
rich in

for the fractionation of proteose—
with ethanol.

for the preparation of a fraction
proteose- peptone from unheated skim—

milk and the preparation of additional
fractions by heating. . .

vii

Page

65

66

67

68

69

70

71.

72

73

7A

75

Figure Page

12. Schematic for the fractionation (sodium
chloride and ammonium sulfate) of a proteose
peptone-rich fraction prepared from unheated
skimmilk. . . . . . . . . . . . . 76

13. Schematic for the fractionation (sodium
chloride and ammonium sulfate) of proteose—
peptone prepared from heated skimmilk . . . 77

1A. Procedure for the ammonium sulfate fractiona-
tion at pH A.9 of a proteose—peptone-rich
fraction prepared from unheated skimmilk . . 78

15. Procedure for the ammonium sulfate fractiona—
tion at pH 7.0 of a fraction richixlproteose-
peptone prepared from unheated skimmilk . . 79

16. Optimized procedure for the ammonium sulfate
fractionation of a fraction richin.proteose-
peptone prepared from unheated skimmilk or
of proteose-peptone prepared from heated
skimmilk. . . . . . . . . . . . . 80

17. Procedure for the isolation of component 5
from proteose-peptone obtained from heated
skimmilk. . . . . . . . . . . . . 81

18. Plexiglas gel- electrophoresis bed used in this
study for horizontal acrylamide gel
electrophoresis . . . . . . 83

19. Starch-urea gel electrophoretic patterns of
proteose-peptone, sigma proteose and
fractions from the Jenness component 5 prep—
aration. Column A: 1—proteose—peptone,
2-sigma proteose, 3—sigma—proteose super;
natant. Column B: fractions from Figure 3;

1-Rl, 2—Rl, 3-R3, A-Ru, 5-R5, 6—s . . . . 11A

1

20. Starch-urea gel electrophoretic patterns of
fractions obtained by washing of casein
micelles and acidifying washed casein
micelles Sas outlined in lg‘igure7DR A: 1- -Sfl,

5- R , R
9-c8seinsmice11AsR l10-wa8hed céseinu
micelles R , ll-wasfiedl casein micelles

(different preparation) R6’ 12- -R7, 13- S 115

5

viii

Figure Page

21. Starch-urea gel electrophoretic patterns of
fractions obtained in the preparation of the
casein micelles and the material released at
pH A.6 according to the schematic in Figure
5: l-Rl, 81,3-R2, A-R3, 5-S2 . . . . . 116

22. Starch-urea gel electrophoretic patterns of
fractions obtained in the preparation and
ammonium sulfate fractionation of proteose-
peptone as outlined in Figure 6: 1-80%
residue, 2-55% residue, 3-35% residue . . . 116

23. Starch— urea gel electrophoretic pattern of
component 8 purified by acrylamide gel
electrophoresis . . . . . . . 116

2A. Starch-urea gel electrophoretic patterns of
fractions obtained from whole casein by
treatment with EDTA, heating, and ammonium
sulfate as outlined in Figures 7, 8,and 9,
respectively: 1-supernatant in Figure 8,
2—residue in Figure 8, 3-R1, in Figure 7,
A-Sl in Figure 7, 5-R2 in Figure 7, and
6-81, 7-R3, 8-R2, 9-Rl, all in Figure 9 . . 117

25. Free—boundary and starch-urea gel electro-
phoretic patterns of proteose—peptone
fractions. Free—boundary (veronal buffer
pH 8.6, I = 0.1): A-proteose-peptone, B-
component 8 purified by acrylamide gel
electrophoresis, C-a mixture of proteose-
peptone and component 8 purified by
acrylamide gel electrophoresis. Starch-
urea gels: D—slot 1, component 8 removed
from Tiselius cell; slot 2, the components
remaining in the Tiselius cell after re-
peated electrophoretic analyses; slot 3, .
fraction R (Figure 3) before separation
in the Tisglius cell. . . . . . . . . 118

26. Starch-urea gel electrophoretic and acrylamide
gel electrophoretic (continuous buffer system)
patterns of proteose—peptone (Figure 1),
sigma-proteose supernatant (Figure 2), and
component 8 (Figure 16). Acrylamide gel:
l—component 8, 2—sigma—proteose supernatant,
A-proteose—peptone. Starch—urea gel: 3—
sigma—proteose supernatant, 5-proteose-
peptone . . . . . . . . . . . . . 119

27. Two—dimensional acrylamide gel electrophoretic

pattern of sigma-proteose supernatant
(Figure 2) . . . . . . . . . . . . 120

ix

Figure Page

28. Acrylamide gel and acrylamide—urea gel
electrophoretic (discontinuous buffer
system) patterns. Acrylamide gel without

urea (Column A): 1-sigma proteose, 2-
proteose-peptone. Acrylamide gel with urea
(Column B): l-sigma proteose, 2—proteose-

peptone . . . . . . . . . . . . . 121

29. Starch—urea gel electrophoretic patterns of
alcohol—fractionated proteose—peptone as
outlined in Figure 10: l-supernatant S1
and residues Z-RA’ 3-R3, A—R2 and 5-Rl. . . 122

30. Starch-urea gel electrophoretic patterns of
fractions obtained by the procedure out-
lined in Figure 11. The fractions are from
different preparations. Column A: 1—Rl,
2—R2, 3-R3, A—Ru, 5—Sl. Column B: 1-Sl,
2—Rl, 3-R2, A—R3, 5-w8ey, 6-8”.

31. Starch—urea gel electrOphoretic patterns of
fractions obtained by sodium chloride
fractionation of proteose—peptone concentrate
from unheated skimmilk as outlined in
Figure 12: 1-S (component 8), 2—R , 3-RA’
A-R3, 5-R2, 6—Rl (enriched componeng 5) . 12A

32. Starch-urea gel electrOphoretic patterns of
fractions obtained by sodium chloride and
ammonium sulfate fractionation of proteose—
peptone from heated skimmilk as outlined in
Figure 13: 1—R , 2—Sl, 3-R2, A—R3, S—RA’
6-S2 (component 8) . . . . . . . . 125

33. Starch-urea gel electrophoretic patterns of
fractions obtained by ammonium sulfate
fractionation at pH A.9 of proteose—peptone
from unheated skimmilk as outlined in
Figure 1A: 1-Sl, 2—R5, 3-RA’ A—R3, 5-R2,
6-H . . . . . . . . . .

1
3A. Starch—urea gel electrophoretic patterns of
fractions obtained by ammonium sulfate
fractionation at pH 7.0 of proteose-peptone
from unheated skimmilk as outlined in

Figure 15: 1—Rl, 2—R2, 3—R3, A—Sl . . . . 126

126

Figure
35.

36.

37.

38.

39-

A0.

A1.

Starch-urea gel electrophoretic (Column A)

and acrylamide gel electrophoretic (Column B)
Patterns of fractions obtained by ammonium
sulfate fractionation at pH A.9 and 7.0 of
proteose-peptone from unheated skimmilk as
outlined in Figure 16. A continuous buffer
system was used in acrylamide gel electro-
phoresis. Column A: 1-25% residue, 2-A0%
residue, 3-50% residue, A-65% residue, 5-80%
supernatant, 6-80% residue. Column B:

l-25Z residue, 2-AO% residue, 3-50% residue,
A-SSZ residue, 5-80% residue, 6-80% super-
n‘tant 0 0 0 0 O O 0 I O I I O O

Acrylamide gel electrophoretic patterns

(continuous buffer system) of some of the
fractions obtained by fractionation of
proteose-peptone from unheated skimmilk as
outlined in Figure 17: l-Rl, 2-R2, 3-R3,
u-R” e e e e e e e o e e e e e

Acrylamide gel electrophoretic patterns of

the fractions collected by gel filtration
through Bio-Gel P-lO: l-first fraction,
2—second fraction, 3-third fraction,

A-fourth fraction, 5-fifth fraction. . . .

Vertical acrylamide gel electrophoresis

patterns (continuous buffer system) of
various proteose-peptone fractions: 1-
component 8-fast from heated skimmilk,
2-component 8-fast from unheated skimmilk,
3-component 8-slow from heated skimmilk,
A-component 8—slow from unheated skimmilk,
S-proteose-peptone, 6-component 5 from
heated skimmilk, 7-lambda casein prepared

by Swaisgood (1963) . . . . . . . . .

Amino acid profiles of component 8-fast

prepared from heated and unheated skimmilk .

Amino acid profiles of component 8-slow

prepared from heated and unheated skimmilk .

Vertical acrylamide gel electrophoretic

patterns (continuous buffer system) of
fractions obtained as designated in Figure 16
and of component 8-slow and proteose-peptone.
The gel (Column A) was stained with amido
black and the gel (Column B) was developed
for carboh drates. Columns A and B: 1-
component -slow, 2-25% residue, 3-AO%
residue, A-50% residue, 5-65% residue, 6—80%
residue, 7-80Z supernatant, 8-proteose-
peptone . . . . . . . . . . . . .

xi

Page

127

128

128

129

130

131

132

Figure
A2.

A3.

AA.

A5.

A6.

A7.

A8.

A9.

Paper chromatograms of authentic carbohydrate
mixtures and of carbohydrates released from
various proteose-peptone components.
Chromatogram: 1—samp1e on left from
component 8—slowa and sample on right from
component 8-slowb, 2-sample from component
8-fasta, 3—samp1e from component 8-fastb,
A-sample from component 53. Legend:
A-galactosamine, B-glucosamine, C—galactose,
D—glucose, E—mannose, F-fucose, G-unknown,
possibly neuraminic acid. aPrepared from
heated skimmilk. bPrepared from unheated
skimmilk. . . . . . . . . . .

A plot of the average ascending and descending
electrophoretic mobilities of component 8 at
various pH values.

Free—boundary electrophoretic patterns of
proteose—peptone fractions which were
analyzed chemically and physically in this
study: A—component 8—fast from unheated
skimmilk, B-component 8-fast from heated
skimmilk, C-component 8-slow from unheated
skimmilk, D—component 8-slow from heated
skimmilk, E-component 5 from heated skimmilk.
Electrophoresis was performed in veronal
buffer at pH 8.6, I = 0.1 . . . . .

A plot showing the concentration dependence of
the sedimentation coefficient for component
8—fasta in veronal buffer at pH 8.6, I = 0.1.
aPrepared from unheated skimmilk. . . .

A plot showing the concentration dependence of
the apparent molecular weights for component
8-fasta in veronal buffer at pH 8.6, I = 0.1.
aPrepared from unheated skimmilk. . . . .

A plot showing the concentration dependence of
the sedimentation coefficient for component
8—slowa in veronal buffer at pH 8.6, I = 0.1.
aPrepared from unheated skimmilk. . .

A plot showing the concentration dependence of
the apparent molecular weight for component
8-slowa in veronal buffer at pH 8.6, I = 0.1.
aPrepared from unheated skimmilk. . .

A plot showing the concentration dependence of
the sedimentation coefficient for component
5a in veronal buffer at pH 8.6, I = 0.1.
aPrepared from heated skimmilk. .

xii

Page

133

13A

135

136

136

137

137

138

Figure Page

50. A plot showing the concentration dependence of
the apparent molecular weight for component
58 in veronal buffer at pH 8.6, I = 0.1.
aPrepared from heated skimmilk . . . . . 138

51. Sedimentation-velocity and sedimentation—
equilibrium patterns for component 8-fast in
veronal buffer at pH 8.6, I = 0.1. All
experiments were performed at 200. A—
sedimentation velocity at 59,780 RPM,
schlieren diaphragm angle of 600 and protein
concentration of 7.0 mg/ml. B—sedimentation—
equilibrium patterns with a schlieren
diaphragm angle of 650 . . . . . . . . 139

52. Sedimentation—velocity and sedimentation-
equilibrium patterns for component 8-slow in
veronal buffer at pH 8.6, I = 0.1. All
experiments were performed at 20C. A—
sedimentation velocity at 59,780 RPM,
schlieren diaphragm angle of 550 and protein
concentration of 7.1 mg/ml. B-sedimentation
equilibrium at 23,150 RPM and schlieren
diaphragm angle of 650 . . . . . . . . 1A0

53. Sedimentation-velocity and sedimentation-
equilibrium patterns for component 5 in
veronal buffer at pH 8.6, I = 0.1. All
experiments were performed at 20C. A-
sedimentation velocity at 59,780 RPM,
schlieren diaphragm angle of 500 and protein
concentration of 6.1 mg/ml. B—sedimentation
equilibrium at 23,150 RPM and schlieren
diaphragm angle of 650 . . . . . . . . 1A1

5A. Photomicrographs of component 8—fast (unheated
skimmilk) crystallized from veronal buffer
and of sodium barbital crystals. A-component
8—fast, Mag. 225x, phase optics, B-component
8-fast, Mag. 26X, Rheinburg illumination,
C—sodium barbital, Mag. 26X, Rheinburg

illumination 1A2

xiii

INTRODUCTION

The proteose-peptone fraction of cow's milk is defined
as that portion of the proteins not precipitated by heating
at 96—100 C for 20 minutes and subsequent acidification to
pH A.6. This fraction contains a group of heat stable pro-
teins which account for about 18% of the whey proteins or
about A% of the total proteins in milk.

In protein nomenclature, the proteose-peptone is
usually considered as the hydrolysis products of primary
proteins. But the proteose-peptone fraction of milk refers
to a group of proteins in milk and does not necessarily
imply that they are hydrolytic products of other milk pro—
teins since they are present in unheated milk. However,
this concept is not well established. One should isolate
individual components from heated and unheated milk for com—
parison of their chemical and physical properties.

Studies of the proteose—peptone fraction have been few
in number but indicate that the proteose-peptone fraction is
a complex group of proteins. According to data obtained by
free-boundary electrophoresis, three peaks called components
3, 5, and 8 are present in the fraction. Of these components,
only component 5 has been studied in a partially purified
state. Consequently, the information concerning the separa—

tion and properties of these individual components is

incomplete. Also, information indicating whether components
5 and 8 exist in the whey or casein portion of milk is
lacking.

To study the individual proteins of the proteose—
peptone fraction, the individual proteins must be separated
from the mixture. Since procedures for separating components
3, 5, and 8 are unavailable, suitable methods of separation
must be devised. The development of a method for the iso-
lation of the individual components requires an investigation
of the solubility characteristics of the various components
at various values of pH and in the presence of salting—out
concentrations of various salts and solvents. Also, studies
of gel filtration, ion exchange techniques, and electro-
phoretic resolution in various gels are indicated.

Seemingly, the proteose-peptone fraction should present
a challenging system for further elucidation. The isolation
of some of its components in homogeneous form in sufficient
quantity to perform chemical and physical analyses would
provide new descriptive information about these heretofore
obscure proteins present in milk.

Thus, this study was conducted with the objectives of
(a) developing procedures suitable for the fractionation of
proteose-peptone into its various components from both
heated and unheated cow's milk and (b) to elucidate the

chemical and physical characteristics of these components.

LITERATURE REVIEW

Historical

 

Until the 1880's, milk was known to contain casein as
the predominate protein and smaller quantities of other
proteins with properties similar to those of blood serum
albumin and blood globulin. Most investigators assumed
these proteins were blood serum proteins which had passed
into the milk from the blood. Later, the milk serum pro-
teins were classified as lactalbumin and lactoglobulin
according to their solubility or insolubility, respectively,
in one-half saturated solutions of ammonium sulfate.

Osborne and Wakeman (1918a) found that some protein
always remained in solution after the acid filtrate of milk
was heated to remove lactalbumin and lactoglobulin. The
properties and prOportions of these proteins varied greatly
in different eXperiments. They (1918a) thought that these
proteins were largely derived from other milk proteins
through the action of reagents used in fractionation and
were not the result of enzymes or bacteria degradations.
Palmer and Scott (1919) stated that the estimation of lactal-
bumin, based on heat coagulation of the casein filtrate, did
not represent all of the protein in the filtrate, since
after the heat coagulation step it was possible to recover
protein from the albumin filtrate by the addition of tannic

acid.

Kieferle and Gloetzl (1930a, 1930b) and Kieferle (1933)
referred to the soluble proteins not coagulable by heat but
precipitated by phosphotungstic acid as proteose and peptone.
Also, they concluded that proteose and peptone were present
in the residual fraction of milk after protein precipitation,
since the filtrate gives a biuret reaction. Moir (1931)
reported that about 70% of the soluble protein of casein—
free filtrate of milk was removed by heat coagulation.

Jones and Little (1933) used 5% trichloroacetic acid to
precipitate unhydrolyzed proteins from casein—free filtrate
and then 10% trichloroacetic acid to precipitate the re-
maining unhydrolyzed proteins and proteose.

Rowland (1937b) stated that the soluble—protein nitrogen
fraction of normal fresh milk contained approximately 76%
albumin and globulin and 2A% proteose—peptone substances.
Further, Rowland (1937a) reported no change in the non-
protein nitrogen content of milk on heating at temperatures
up to 100 C. Also, he found that on continued heating at
95 and 100 C that only extremely small amounts of proteose
could be attributed to the hydrolysis of protein and con-
cluded that proteose-peptone substances were present in
fresh milk (Rowland, 1937b). Accordingly, the proteose-
peptone fraction of milk is defined as that fraction remain-
ing in solution after heating milk to 95-100 0 for 20
minutes, followed by the addition of acid to pH A.7 to pre—
cipitate the casein and denatured whey proteins (Rowland,

1938a). Alternatively, the proteose-peptone fraction

remained soluble when the pH of the casein—free filtrate
was adjusted to A.75 and boiled to denature and coagulate
the whey proteins. The soluble fractions could be precipi-
tated by the addition of trichloroacetic acid to 12%.
According to Rowland (19380), the average nitrogen distri-
bution in normal cow's milk was as follows: 78.5% casein,

3.3% globulin, A.0% proteose and 5.0% non-protein nitrogen.

Contemporary

 

Aschaffenburg (19A6) isolated a surface-active material
which he called sigma proteose by heating skimmilk to 90-

95 C for 15 minutes, followed by co-precipitation of casein
and the denatured proteins by acid or rennet coagulation.
Sigma proteose was precipitated from the supernatant by
half-saturation with ammonium sulfate. His report did not
state clearly whether the sigma proteose he used for analysis
studies was obtained from acid whey or rennet whey.

Larson and Rolleri (1955), using the free—boundary
electrophoretic technique, reported three discernible
gradient boundaries present in classically prepared proteose-
peptone. These were designated as components 3, 5, and 8
in ascending order of electrOphoretic mobility. Euglobulin,
pseudo-globulin, a—lactalbumin, B-lactoglobulin, and serum
albumin were designated as components 1, 2, A, 6, and 7,
reSpectively. Larson and Rolleri (1955) reported values of
A.6, 8.6, and 5.7% concentration for components 3, 5, and 8,

respectively. The electrophoretic mobilities of components

.7
.1

”-

. .Ld

'V

'9

 

3, 5, and 8 calculated from the descending boundaries were

2 l

-2.9, -A.5, and -7.9 x 10’5 cm volt- sec’l (lxlO-S cm2

volt-lsec-l = Tiselius unit), respectively. These three
components were considered to be present in unheated milk.

Aschaffenburg and Drewry (1959) were able to distin-
guish the "true" proteins of milk from the proteose—peptone
components by staining paper electrophoretic strips with
bromophenol blue and washing with dilute acetic acid. The
proteose—peptone components formed yellow bands rather than
the bluish-green bands characteristic of "true" proteins.
Six yellow bands were found in the proteose—peptone con-
taining filtrates prepared according to the method of
Rowland (1938b). One of these bands corresponded to com-
ponent 5 while component 3 was separated into five small but
dietinct bands. These investigators stated that the proteose—
peptones were native constituents of milk since the same six
bands were observed in the ultrafiltrates of unheated milk.

According to Harland and Ashworth (19A5), the pre-
cipitation of casein from raw skimmilk by saturation with
sodium chloride resulted in a 17.3% loss of whey-protein
nitrogen when compared with recoveries by the method of
Rowland (1938b). Thus, some of the heat-labile whey pro-
teins or the proteose-peptones were co-precipitated by
saturation with sodium chloride.

Jenness (1957) found a correlation coefficient of
+0.82 between the quantity of protein precipitated by salt

but not by acid and the concentration of proteose—peptone in

skimmilk. A protein which appeared to be the principal
constituent (probably component 5) of the proteose—peptone
fraction was partially purified from the fraction which was
precipitated by salt but not by acid. Later, Jenness (1959)
isolated component 5 from the precipitate obtained by
saturating skimmilk with sodium chloride. The precipitate
was redispersed and the casein removed at pH A.6. An ion
exchange resin (IRC 50 H+) was used to remove lactoperoxidase.
The filtrate was concentrated and component 5 was removed by
adjusting the pH to approximately A.5. In the highly puri-
fied fraction more than 90% of the material had an electro—
phoretic mobility equivalent to that reported previously for
component 5 by Larson and Rolleri (1955).

Weinstein, Duncan,and Trout (1951a) isolated a minor-
protein fraction from rennet whey which was capable of being
photosensitized to produce the typical solar—activated flavor
of homogenized milk. The fraction was prepared by a method
similar to that used in preparing sigma proteose.

Gordon, Jenness, and Geddes (195A) reported that both
casein and whey depressed the loaf volume of bread when used
in the dough formulation. Jenness (1959) reported that com—
ponent 5 isolated from the proteose—peptone fraction of raw
milk was the heat-labile loaf volume depressant.

Shahani and Sommer (1951) observed an increase in the
proteose-peptone fraction when milk was pasteurized at 155 F
for 30 minutes followed by homogenization. Menefee, Overman,

and Tracy (19Al) reported that the pasteurization (1A5 F for

30 minutes) and homogenization of milk at both normal and
abnormally high pressures produced no significant changes
in the classical nitrogen distribution of milk.

The occurrence of carbohydrate-containing proteins has
been recognized for many years. Glycoproteins are defined
by Gottschalk (1966) as conjugated proteins containing as
a prosthetic group one or more heterosaccharides with a
relatively low number of sugar residues, lacking a serially
repeating unit, and bound covalently to the polypeptide
chain. The carbohydrate moiety in proteins usually contains
amino sugars like glucosamine, galactosamine, and/or sialic
acid and usually hexoses like galactose, mannose or fucose.

Several workers have shown the presence of glycoproteins
in milk (Bezkorovainy, 1965, 1966; Blanc, Bujard, and Mauron,
1963; Brunner and Thompson, 1959, 1961; Groves, 1960; Hipp,
Groves, and McMeekin, 1961; Jackson, Coulsen, and Clark,
1962; Alais and J611es, 1961; Nitschmann, Wissman, and Henzi,
1957; and Thompson and Brunner, 1959, 1960). Storch (1897)
reported that the fat globule membrane contained carbohydrate
thus indicating the presence of glycoproteins in milk. The
presence of hexose and hexosamine in the lactoglobulin
fraction (euglobulin and pseudoglobulin) of milk was shown
by Smith (19A6) and Smith, Greene, and Bartner (19A6). The
composition of a high-carbohydrate component released from
casein by the action of rennin was determined by Nitschmann

et a1. (1957). Lactotransferrin, a3—casein, K-casein, red

protein, and interfacial protein have been reported by Blanc
9.12.9.1- (1963); Hipp 223.1.- (1961); Alais and J611es (1961);
Groves (1961); and Jackson gt_al. (1962), respectively, to
be carbohydrate—containing proteins in milk.

Aschaffenburg (19A6) reported a negative Molisch test
for sigma proteose. He did not indicate whether the sigma
proteose was obtained from acid or rennet whey. A positive
Molisch test was obtained by Weinstein et_al. (1951a) thus
indicating the presence of carbohydrate in the minor—protein
fraction isolated from heated rennet whey. The minor-
protein fraction was prepared using an adaptation of the
method described by Aschaffenburg (19A6).

Brunner and Thompson (1961) reported selected physical
and chemical characteristics of four minor-protein fractions
obtained from the same skimmilk and a fifth fraction, the
soluble membrane protein, obtained from washed cream. The
four minor-protein fractions were prepared by the methods
reported by Rowland (1938b) for proteose—peptone, by
Aschaffenburg (19A6) for sigma proteose, by Weinstein et_al.
(1951a) for the minor—protein fraction and by Jenness (1959)
for component-5 concentrate.

Colostrum whey contained appreciable amounts of the
two well-characterized bovine serum acid glycoproteins,
orosomucoid and M-2 glycoprotein (Bezkorovainy, 1965).

Milk whey contained no orosomucoid and only trace amounts
of the M-2 glycoprotein. But, it contained a phospho—

glycoprotein that was apparently less acidic than

10

orosomucoid and behaved like the serum M-l fraction in
isolation procedures. The orosomucoid-containing fraction
from colostrum whey also contained an additional component
which behaved like the milk phosphoglycoprotein in cellulose
acetate electrOphoretic experiments. This milk phospho—
glycoprotein was the major acid glycoprotein of milk. Its
properties, i.e., carbohydrate and phosphorus content,
electrophoretic mobility, and ultracentifugal behavior,
corresponded to the major component of the proteose—peptone
fraction (Bezkorovainy, 1965).

A summary of the nitrogen contents of sigma proteose,
proteose-peptone, milk component-5 concentrate, Weinstein's
minor-protein fraction and milk whey phosphoglycoprotein
reported by various researchers is shown in Table 1.
Bezkorovainy (1965) stated that the nitrogen value of the
milk whey phosphoglycoprotein corresponded to the major
component in the proteose-peptone fraction.

Also, shown in Table l is a summary of the phosphorus
and sulfur contents of several minor-protein fractions.

All of the protein fractions contained phosphorus.

Ganguli, Gupta, Agarwala, and Bhalerao (1965) reported
5.0% cysteine and A.9A% methionine from the amino acid
analysis of proteose-peptone while Bezkorovainy (1965) found
only trace amounts of cysteine and 0.2% methionine in the
milk whey phosphoglycoprotein. Weinstein et_al. (1951a)
reported 0.86% cycteine and 1.3A% methionine. Bezkorovainy's

milk whey phosphoglycoprotein had practically no absorption

11

at 280 mu thus reflecting the low concentration of aromatic
amino acid residues, i.e., a trace amount of tyrosine and
1.3% of phenylalanine. However, Ganguli et_a1. (1965)
reported values of 2.96% and 3.19%, respectively for tyrosine
and phenylalanine in proteose—peptone while Weinstein £3431.
(1951a) obtained a value of 2.3A% for the phenylalanine
content of the minor—protein fraction. Obviously, the amino
acid analyses reported by the above researchers are not in
agreement.

The following information concerning carbohydrates is
shown in Table 1: the hexose, hexosamine, fucose, and
sialic acid content in proteose-peptone and Weinstein's
minor-protein fraction; the hexose content in sigma pro—
teose, milk component-5 concentrate and milk whey phospho—
glyCOprotein; the N-acetyl hexosamine and sialic acid con-
tents of the milk whey phosphoglycoprotein.

Bezkorovainy (1966) isolated a M-1-type glycoprotein
from both milk and colostrum which had similar character—
istics except that the colostrum material contained 25%
carbohydrate while the milk fraction contained 7% carbohy—
drate.

Larson and Rolleri (1955) reported electrOphoretic
mobilities of -2.9, -A.5, and -7.9 Tiselius units for com-
ponents 3, 5, and 8, respectively, in veronal buffer at pH
8.6, I = 0.1. They obtained a mobility range of -5.0 to
-8.2 Tiselius units for component 8. A Tiselius value of

-A.5 in the same buffer system was obtained by Jenness (1959)

12

for purified component 5. Weinstein, Lillevik, Duncan and
Trout (1951b) stated that the minor-protein fraction was
composed of at least two components or complexes but did

not report any electrophoretic mobilities. Brunner and
Thompson (1961) reported free-boundary electrophoretic data
for proteose-peptone, sigma proteose, milk component-5
concentrate, and Weinstein's minor-protein fraction using
veronal buffer at pH 8.6, I = 0.1 and HCl-NaCl buffer at pH
2.A, I = 0.1. Some differences existed between each of
these fractions but the major peak in each fraction appeared
to be similar. Bezkorovainy (1965) obtained electrophoretic
mobilities of —3.0 and —7.1 Tiselius units at pH A.5
(acetate buffer, 1 = 0.1) and pH 8.6 (barbital buffer,

I = 0.1), respectively, for the phosphoglycoprotein isolated
from normal milk. The mobility reported at pH 8.6 was much
higher than the mobility reported previously for component

5 or the major component of the proteose-peptone fraction.
Bezkorovainy (1965) presented cellulose acetate electro-
phoretograms of the milk whey phosphoglycoprotein which
indicated that it was homogeneous.

According to Ogston (19A6), sigma proteose had two
distinct components with S20 (8 = 1 x 10—13 sec) values of
0.96 and 2.75 in sedimentation-velocity experiments. For
this analysis, the sigma proteose (concentration = 1.35%)
was dissolved in pH 8.6 buffer consisting of 0.2 M sodium
chloride, 0.017 M Na2HPOu and 0.017 M KH2POM. Assuming

that the molecules were spherical and that the partial

13

specific volumes were 0.75, then the minimum molecular
weights calculated for the two components were A,900 and
23,900.

The major component in the sedimentation-velocity
patterns of proteose-peptone, sigma proteose, milk component-
5 concentrate, and Weinstein's minor-protein fraction had
S2O values of 0.77, 0.83, 0.88, and 0.8A, respectively
according to Brunner and Thompson (1961). They reported
820 values of 2.6A, 2.86, 5.88, and 2.83 for the other com—
ponent present in proteose—peptone, sigma proteose, milk
component—5 concentrate and Weinstein's minor-protein
fraction, respectively. The ultracentrifugal analysis was
performed in veronal buffer at pH 8.6, I = 0.1 and a protein
concentration of 1.5%. The 820 values for sigma proteose
obtained by Brunner and Thompson (1961) corresponded to those
obtained by Ogston (19A6). The milk whey phosphoglycoprotein
isolated by Bezkorovainy (1965) had a sedimentation coeffi-
cient of 0.8 S at 20 C at a protein concentration of 1% in
phosphate buffer at pH 7.0, I = 0.1. This sedimentation
coefficient correspondedix>that reported previously for the
major component present in the sedimentation—velocity
pattern of proteose—peptone (Brunner and Thompson, 1961).

Bezkorovainy (1966) isolated the M-l-type glycoprotein

from milk and colostrum. Previously, Bezkorovainy (1965)

called these fractions the milk whey phosphoglycoprotein.

1A

The milk and colostrum glycoproteins were homogeneous in

the ultracentrifuge and 80 values of 1.0 and 1.1 were

20,w
obtained for the milk and colostrum glycoproteins,

respectively. From the 8020 w values, molecular weights of
3

about 10,000 were reported for both proteins. The milk and

colostrum glycoproteins had diffusion coefficients of 9.66

2 —1

and 8.68 x 10"7 cm sec , respectively.

15

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EXPERIMENTAL

Apparatus and Equipment

The milk used in this study was collected in five- or
ten-gallon stainless steel cans and separated with a DeLaval
disc-type separator (Model 9). Stainless steel, plastic,
or pyrex containers were used for performing all experiments.
A Beckman Zeromatic or Photovolt, Model 115, pH meter,
equipped with glass electrodes, was used to measure pH
values. Low—speed centrifugations were performed with
International Model V, size 2, and International Model U
centrifuges. Intermediate-speed centrifugations were per—
formed with Sorvall, type SS-l; Sorvall, type SS—A; and
I3eckman, Model L, centrifuges. The Model L preparative
(lentrifuge, equipped with a type 50 fixed-angle rotor, was
Ilsed for high—speed centrifugation.

Laboratory—constructed Plexiglas electrophoretic cells
Were used for starch—urea, acrylamide, and acrylamide—urea
€361 electrophoretic analyses. Vertical acrylamide-gel
€31ectrophoresis was performed with an electrophoretic cell
nlanufactured by E. C. Apparatus Company powered by a Beckman/
E5Il>inco Constant power supply. Other power sources used were
61 Reco Model E-800—2, a Savant Model 5000, and a Heathkit
Model IP—32. A Polaroid Land Camera (Model MP—3) was used

to photograph the electrophoretograms. Saran Wrap was used

16

17

to cover the gel during the electrophoretic analysis. The
excess stain was removed from all gels by means of an
electrolytic destainer.

A Perkin-Elmer, Model 38-A Tiselius electrOphoresis
apparatus, using circulating refrigerated water to maintain
the bath temperature at 2 C, was used for the free-boundary
electrophoretic analyses. Buffer resistances were determined
with an Industrial Instruments, Model RC, conductivity
bridge.

Protein solutions were dried from the frozen state by
a laboratory-constructed 1yophilizer.

The eluate from gel filtration columns was monitored
at 280 mu by Gilson Medical Electronics' Absorption Meter
and recorded by a recording milliammeter manufactured by
fisterline Angus Instrument Company. Also, the eluate at
‘times was monitored at 25A mu with a recording ultraviolet
armlyzer manufactured by Instrumentation Specialties Company,
IInc.

Proteins and chemicals were dried in a temperature—
<Zontrolled Cenco vacuum oven in which the vacuum was obtained
With a Cenco Pressovac A vacuum pump.

For most laboratory weighings, a top-loading, direct-
Ixeading, Mettler type K—7 balance was used. For analytical
Vi“Eighings, Mettler type H—16 and Voland and Sons type 750-D
analytical balances were used.

Amino acid analyses were performed with a Beckman/

Spinco Model 120 C amino acid analyzer. A Mini-Shaker

l8

supplied by Fisher Scientific Company was used for stirring
small samples in test tubes and for stirring the reaction
mixture in the hexose determination.

A Spinco Model E enalytical ultracentrifuge equipped
with a RTIC temperature control unit and a phase plate as
a schlieren diaphragm was used for sedimentation-velocity
and sedimentation—equilibrium studies. A capillary-type,
synthetic boundary cell was used for sedimentation studies
and initial concentration determinations. A capillary-type,
double—sector synthetic boundary cell was used for the
equilibrium experiments. In all determinations, 12 mm cells
equipped with quartz windows were used. An An—D Duralumin
rotor was used for centrifuging and a General Electric
lUi-6 Mercury lamp served as the light source.

Kodak metallographic glass plates were used for
Iwecording the schlieren patterns and a Nikon Model 6 Shadow-
Earaph microcomparator was used for measuring the plates.
Tnle microcomparator will move 250 mm on the Y—axis and 500
"UH on the X—axis and can be read directly to 0.002 mm.

A Beckman DK—2A ratio recording spectrophotometer,

eCUJipped with quartz cells with a 1 cm light path, was used

fklr spectrophotometric examinations.

Chemicals and Materials

The principal chemicals used in this research and

'their*suppliers are given below.

l9

Tris-hydroxymethyl aminomethane (Sigma 121) was used
as a primary standard and Sigma 7-9 was used to prepare
buffers. Both were obtained from Sigma Chemical Co.

Reagent grade ammonium sulfate was purchased from Fisher
Scientific Company. Orcinol was obtained from the Matheson
Company. Phenol was acquired from Mallinckrodt and redis-
tilled prior to use. Acetylacetone was purchased from
Fisher Scientific Company and redistilled. The p—
dimethylaminobenzaldehyde, 2—thiobarbituric acid and N,N,N',
N'—tetramethylethylenediamine were obtained from Eastman
Organic Chemicals.

Cyanogum Al used in acrylamide gel electrophoresis
was purchased from E. C. Apparatus Company. Cleland's
reagent, 5—5' dithiobis (2—nitrobenzoic acid), was obtained
from Calbiochem. Hydrolyzed starch especially prepared for
gel electrophoresis, was acquired from Connaught Medical
IResearch Laboratories.

Bio-Gel P—2, Bio—Gel P—10, and a 1% solution of
(iimethyldichlorosilane in benzene was obtained from Bio-Rad
lLaboratories. The ion exchange resins, Dowex 1 x 8 (20-50
Inesh) and Dowex 50 x 8 (20—50 mesh) were obtained from
J. T. Baker Chemical Co. and the Dow Chemical Co., respec—
txively. Mannose was acquired from Fisher Scientific Company
Enid galactose was obtained from Pfanstiehl Laboratories, Inc.
(Germany). Glucosamine hydrochloride, galactosamine, and

fucose were purchased from Nutritional Biochemicals Corp.

20

Triphenyltetrazolium chloride was obtained from Eastman
Organic Chemicals. N—acetyl neuraminic acid, tryptophan,
and streptomycin sulfate were obtained from Calbiochem.
Glutathione and penicillin "G" were obtained from Nutri-
tional Biochemical Corp. Basic fuchsin was purchased
from Allied Chemical Corp.

The other organic and inorganic chemicals used in this
research were of reagent grade.

Preparative Procedures for Proteose-
Peptone Fractions

Exploratory Procedures for the Preparation
and Fractionation of Proteose—Peptone
from Heated and Unheated Skimmilk

The milk used in this study was obtained from the
Iflichigan State University dairy herd which consisted of
I3rown Swiss, Jersey and Holstein cows. All milk was
(obtained immediately after milking and separated as soon
as possible. The fresh skimmilk was used as the starting
Inaterial in this study.

Preparation of_proteose-peptone.-—The proteose—
IDeptone fraction was prepared in a manner similar to the
13rocedure described by Rowland (1938a). Fresh skimmilk in
Staainless steel containers was heated at 96—100 C in an
Eiutoclave for 20—30 minutes. The milk was removed and
Cooled to 20 C. The pH of the milk was adjusted to A.6

With 1 N HCl and the precipitated casein, in addition to

the denatured serum proteins, was removed by filtering

21

through E & D No. 512 filter paper. Rowland's (1938a) final
concentration step with 12% trichloracetic acid was not used.
The supernatant was adjusted to a pH of 3.0 and dialyzed at

A C against three or four changes of deionized water adjusted
to pH 3.0 with acetic acid. By dialyzing at a low pH, the
calcium and phosphate was removed. Next, the pH was adjusted
to 7.0 and dialyzed at A C against three or four changes of
deionized water. Dialysis time was from 12 to 2A hours for
each change of water. The dialyzed supernatant was pervapor-
ated and 1y0philized. The dried proteose-peptone was stored
at =-1O C.

The isolation procedure for proteose-peptone is pre-
sented diagrammatically in Figure 1.

Preparation of sigma proteose.——Aschaffenburg (19A6)
iscflated sigma proteose using a procedure similar to that
Ilsefl.for the isolation of proteose-peptone. Some question
e)(ists as to whether he used rennet or acid whey in obtaining
$5iigma proteose for analysis. In this procedure the casein
EUHd heat denatured serum proteins were precipitated by acid-
ifying heated skimmilk to pH A.6. The supernatant was adjusted
PC) pH 6.7; then, ammonium sulfate was added until the super-
natant was one-half saturated with respect to ammonium sulfate.
Tr“? flocculated protein was removed by centrifuging and the
PGBSidue (sigma proteose) was treated as shown in Figure 2. The
fraction soluble in one—half saturated ammonium sulfate was
:prepared for storage as described for the sigma proteose.

Preparation of component 5 as adapted from the method

Skfififlzgped by Jenness (1959).--Component 5 was prepared as

22

described by Jenness (1959) and by Brunner and Thompson
(1961) except for slight modifications. A schematic diagram

of this procedure is shown in Figure 3. After washing the
casein with a saturated sodium chloride solution, it was
slurried in deionized water and dialyzed to remove the salt.
The solution was adjusted to pH A.6 with 1 N HCl to precipi-
tate the casein. The supernatant was adjusted to pH 8.0

and a flocculate formed which was removed by centrifuging.
The supernatant was discarded.

At this point, the isolation scheme differed from that
of Jenness (1959) and Brunner and Thompson (1961). They
treated the pH A.6 supernatant with a IRC-50 H+ resin. In
this study, a pH 8.0 precipitate was obtained from the pH
A.6 supernatant and was slurried in deionized water; the pH
'was adjusted to A.6, At first the precipitate dissolved, then,
another precipitate formed which was removed by centrifuging.
The residue was saved and called residue Rl as shown in
Figure 3. The pH of the supernatant was adjusted to pH 6.0
and another precipitate formed which was removed by centri-
fuging. The precipitate was called residue R2. This pro-
cedure was repeated three times by adjusting the supernatants
‘to pH 7.0, pH 9.5, and pH 11.5 with the resulting precipi—

‘tates called residues R3, Ru,and R respectively. The

5’
final supernatant Sl contained material which could be pre-
cipitated when the solution was made to one-half saturation

With ammonium sulfate. These fractions were dialyzed, con-

centrated, and lyophilized.

23

Preparation of washed casein micelles and the material

released from micellar casein at_pH A.6.-—A protein-free

 

milk system (milk dialysate) was prepared by dialyzing

3,000 ml of deionized water against nine gallons of skimmilk
for 2A hours. The skimmilk was changed at 2A, A8, and 72
hours. The milk dialysate was used for resuspending the
casein micelles. Streptomycin and penicillin "G" were

added to the skimmilk and milk dialysate at the rate of

50 mg/l and 5,000 units/1, respectively.

Skimmilk was sedimented at 18,000 RPM or 32,600 x G
(middle of tube) at A C for 1A hours to obtain a A0 S pellet.
The Beckman Model L ultracentrifuge was used with the No. 21
rotor and polypropylene tubes. The supernatant (whey) was
adjusted to pH A.6 and centrifuged 1000 x G for 30 minutes
to remove the soluble casein. The supernatant solution and
the precipitated soluble-casein were dialyzed and lyophilized.

The residue (A0 S pellet) was slurried with a volume
of milk dialysate equal to the volume of whey removed from
the skimmilk. The mixture was stirred at A C for 8-12 hours
to resuspend the casein micelles. The described procedure
‘Nas repeated three additional times.

The final A0 S pellet was suspended in deionized water,
Eidjusted to pH A.6 and the precipitated casein removed by
centrifuging. The residue and resulting supernatant were
neutralized to pH 7.0, dialyzed and lyophilized. A

SChematic diagram of the above procedure is shown in Figure

A.

2A

Preparation of casein micelles and the material

released from micellar casein at pH A.6.-—Skimmilk was sedi-

 

merited at 160,000 x G (middle of tube) for 1.5 hours at A C
to obtain a A0 S pellet. The skimmilk was equilibrated at

A C prior to centrifugation. The supernatant, containing whey
and soluble casein, was adjusted to pH A.6 and centrifuged

to remove the flocculated casein. The resulting residue R1

and supernatant S were treated as shown in Figure 5. The

1
‘40 S pellet was slurried in deionized water equal to twice

_..r._~,_ .L.‘

the volume of the removed whey. The slurry was stirred for
2’4 hours at A C and adjusted to pH A.6. The precipitated ¥——'
Casein was removed by centrifuging. The pH of the supernatant
was adjusted to pH 6.8 and a flocculate (residue R3 in Figure
5) occurred which was removed by centrifuging. A11 residues
and supernatants were treated as described in Figure 5.
Prpparation and ammonium sulfate fractionation of
Wose-peptonem-Fresh skimmilk was heated at 96-100 C for
2C3‘30 minutes in stainless steel containers which were placed
in an autoclave. Then, the skimmilk was removed from the
autoclave and immediately cooled to 20 C. The pH of the
heat ed skimmilk was adjusted to A.6 and the casein and
denatured whey proteins were removed by filtering with E & D
NO - 512 filter paper. Ammonium sulfate was added slowly to
the supernatant with constant agitation. At the point of
25% Saturation with the salt, a precipitate began to form.
Additional ammonium sulfate was added until the solution

Wa
S 35% saturated. The salted-out protein was removed by

25

centrifugation. The residue (35% residue, Figure 6) was

slurried in deionized water at pH 7.0, dialyzed and

lyophilized. The supernatant was made 55% saturated with
respect to ammonium sulfate and allowed to stand for 30
minutes. The flocculated protein was removed by centrifu-
ggaition and treated as already described. The remaining
supernatant was made to 80% saturation with ammonium sulfate
and the flocculated protein removed by centrifugation. The
residue was treated as described in Figure 6. All lyophilized
s amples were stored at 2—10 C.

Effect of calcium on sigma proteose.——Sigma proteose
was prepared as shown in Figure 2. Two grams of the prepara—
tion were dissolved in 100 m1 of deionized water. The pH
Was adjusted to A.6 and the residue removed by centrifuging
at 1000 x G for 30 minutes and discarded. The cloudy-
aF3pearing supernatant was adjusted to pH 7.0 followed by the
a~<i<iition of acid to pH A.6 at which point it became clear.
The pH was maintained at 7.0 during the addition of calcium
Chloride to a final concentration of 0.25 M calcium. A
precipitate was not obtained at this concentration. While
maintaining the pH at 7.0, calcium chloride was added to
g1 Ve a final concentration of 0.50 M calcium. Again, a
pr’Ei‘Cipitate was not obtained. Then, the pH was lowered to
A ° 6 and no visual change occurred. An equal volume of 95%
eth3211101 was added to the solution without any change in

appearance of the solution. This solution was discarded.

26

Release of component 8 from whole casein with EDTA.-—
Di—sodium ethylenediaminetetraacetic acid (EDTA) was used to
chelate the calcium in casein with the idea of releasing
the minor components.

Approximately 1 g of EDTA was added to 100 m1 of a 2%
solution of whole casein. The pH was maintained at 7.0; then,
the pH was adjusted to A.9 and the precipitate removed by
centrifugation. The residue was dispersed in deionized
water at pH 7.0, dialyzed and lyophilized. The supernatant
was adjusted to pH A.1 at which point a precipitate formed
Which was removed by centrifugation and processed as described
above. The pH of the resulting supernatant was adjusted to
pH 7.0, dialyzed and lyophilized. A schematic representation
Of the above procedure is shown in Figure 7.

Release of component 8 from whole casein by heating.——
Because proteose-peptone was prepared from skimmilk, it
Seems reasonable to conjecture that casein may contain some
of the proteose-peptone components. Both whole casein
( 1%) and sodium chloride (1%) were dissolved in deionized
Water at pH 6.8 and heated at 96—100 C for 30 minutes. The
SOlution was cooled to 20C, adjusted to pH A.3, and the
ensuing precipitate removed by centrifugation. The residue
(Figure 8) was dissolved in deionized water with the addition
of l N NaOH to pH of 7.0. The pH of the supernatant was
adjusted to 7.0. Both the residue and the supernatant were

dialyzed and lyophilized. A schematic diagram of the isola—

t a
101') procedure is shown in Figure 8.

27

Release of component 8 from whole casein with ammonium
sulfate.--Isoe1ectric casein was dissolved in deionized
vvater at pH 7.0 to a concentration of 1.5%. Ammonium
ssulfate was added with constant stirring to this solution
Lintil it was 10% saturated with respect to the salt. The
ssolution was allowed to stand for about 10 minutes, the pH y

vvaas adjusted to A.6 and the flocculated protein was removed

by centrifugation. The residue R1, Figure 9, was slurried

. ._.__ A. ,‘L_.l___u .___
1

in deionized water at pH 7.0, dialyzed, and lyophilized.
'IPIJe pH of the supernatant was adjusted to 7.0, made to 25%
sseaturation with ammonium sulfate, adjusted to pH A.6 and the
flocculated protein removed and treated as described above.
'1311e pH of the solution was changed to 7.0 and the solution
Visas made to 55% saturation with ammonium sulfate. After the
Eilrunonium sulfate was dissolved, the pH of the solution was
€1Cijusted to A.6. The precipitated protein was allowed to
551:6and about 10 minutes and was removed by centrifugation.
TPluee residue was treated as the previous residues and the
ESl—Ipernatant was treated as shown in Figure 9.

Fractionation of proteosejpeptone with ethanol.-—
I5’I’<:>teose-peptone prepared as shown in Figure l was used as
'tliei starting material in this fractionation. One—half gram
C3f‘ LDroteose—peptone was dissolved in 50 ml of deionized
water. The solution was made 0.1 M with respect to potassium
chlAbride and was cooled to 0-A C. Thirty-five milliliters

CDf‘ 595% ethanol (O—A C) were added with constant stirring to

28

give a concentration of 39% ethanol. Fractionation was

carried out at O-A C. The aggregated protein was removed

by centrifugation at 12,000 x G for 10 minutes. The first

residue R1, Figure 10, was dispersed in deionized water,

dialyzed and lyophilized. To the supernatant 55 m1 of 95%

ethanol were added with constant stirring to give a concen-
This solution was allowed to

The

tration of 61% ethanol.
$313and overnight and centrifuged the next morning.

second residue R2, Figure 10, and succeeding residues were

treated the same as the first. Forty milliliters of 95%

ethanol were added to the resulting supernatant to give a

Concentration of 68.5% ethanol. The precipitated protein

was removed by centrifugation. Forty milliliters of 95%

ethanol were added to the supernatant with constant stir—

ring to a concentration of 73.5% ethanol. The resulting

flocculated protein was removed. Forty milliliters of

95% ethanol were added to the supernatant to give a final

Concentration of 77% ethanol. The protein remaining in the

Supernatant was not precipitated. Therefore, the super—

natant was dialyzed, concentrated by pervaporation and

lyophilized. A schematic diagram of this procedure is shown

in Figure 10 .
Preparation of a proteose-peptone-rich fraction from

wated skimmilk.--The casein was removed from skimmilk by

adJ usting the pH of skimmilk to A.6 and centrifuging 1000 x

G for 30 minutes or by filtration. If the whey was cloudy,

it Was cleared by filtering with Celite and a Buchner funnel

29

operated with an aspirator. The pH of the clear whey was
adjusted to pH 6.8 and the flocculated calcium-phOSphate—
garotein complex was removed by centrifugation at 1000 x G

Ifor 30 minutes. The residue R1 was treated as shown in

IPigure 11.

An aliquot of the clear whey (pH adjusted to 7.0) was
Iaeeated for 30 minutes at 96—100 C. After cooling, the pH
vvzas adjusted to A.6 and the aggregated protein was removed
t>3l centrifugation. The residue was discarded. The super—
rlaitant was adjusted to pH 3.0 and treated as the first
I‘e sidue .

Additional precipitates were removed from the super—
rleatant at pH 8.0, 10.0 and again at pH A.6 and treated as

Tilie residue R in Figure 11.

1
The final supernatant was adjusted to pH 3.0 and
tllreated the same as the residues. Also, an aliquot of the
ISjgnal supernatant was heated at 96-100 0 for 30 minutes at
Ibfi 6.8 and cooled with flowing tap water. Then, the pH was
Eicijusted to A.6 and the coagulated protein was removed by
Cleentrifugation. The residue was discarded. The supernatant
531,, Figure 11, was adjusted to pH 7.0, dialyzed and
:Lsfophilized. This procedure is outlined in Figure 11.
Sodium chloride and ammonium sulfate fractionation of
EL‘jgroteose-peptone-rich fraction prepared from unheated
Siklinmnlk.-—About 2 g of a proteose-peptone-rich fraction R1,

\—_—_
Fj—E§ure 11, were dissolved at pH 7.0 in A00 ml of deionized

30

water. The solution was saturated with sodium chloride
(A0 g/100 ml). The saturated sodium chloride solution was
adjusted to pH A.A. The ensuing precipitate was removed by
centrifugation, dispersed in deionized water at pH 7.0,
dialyzed and lyophilized. An aliquot of the supernatant
was removed and the pH was changed to determine whether
protein could be precipitated from the saturated sodium
chloride solution after the removal of the precipitate at
pH A.A. The precipitate R2, Figure 12, at pH 3.5 was
removed and examined electrOphoretically. The pH of the
Supernatant (pH A.A) was adjusted to 7.0 and the supernatant
dialyzed and concentrated about 2.5:1 by pervaporation. By
the addition of ammonium sulfate during constant agitation,
the pervaporated solution was made to A5% saturation. The
aggregated protein was removed by centrifugation and treated
as described in Figure 12. Percipitated protein fractions
Were collected at 65 and 80% saturation with ammonium
SLllfate. Figure 12 shows a schematic diagram of the above
procedure.

Sodium chloride and ammonium sulfate fractionation of
mteose—peptone prepared from heated skimmilk.—-In the
previous fractionation, Figure 12, the sodium chloride super-
natant was dialyzed, pervaporated and further fractionated
with ammonium sulfate. In this procedure, the sodium
chloride supernatant was adjusted to pH 3.1 and centrifuged

to remove proteins which were soluble at pH A.A.

.1 __..- ‘--“.‘-—v

31

The precipitate was dispersed in deionized water at
pH 6.7. A final pH of 6.2 was reached after the addition
of ammonium sulfate to A5% saturation. The salted-out
fraction was removed by centrifugation. Two additional
fractions were obtained at 65 and 80% saturation with

ammonium sulfate. These residues and the final supernatant

new--.l).

were treated as shown in Figure 13.

"u...

Ammonium sulfate fractionation of a proteose-peptone-
P1 ch fraction at pH A.9 prepared from unheated skimmilk.-- E

A proteose—peptone-rich fraction R Figure 11, from unheated

l,

Skimmilk was dissolved in deionized water to a concentration P

of 0.5% and fractionated as shown in Figure 1A. The solution
Was adjusted to pH 7.0 prior to the addition of ammonium
Sulfate to make a 25% saturated solution. Then, the pH of
tl'le solution was adjusted to A.9. The precipitated protein
Was removed by centrifugation. After removing the first
residue, the pH of the solution was maintained at A.9 and
precipitates were removed when the solution was made to 37,
55 , 65, and 80% saturation with ammonium sulfate. These
Sa.ZLted--out residues were dispersed in deionized water at
IDPi 7.0, dialyzed and lyophilized. The pH of the supernatant
was adjusted to 7.0 and the solution dialyzed, pervaporated
and lyophilized.

Ammonium sulfate fractionation of a proteose-peptone—
13:31:} fraction at pH 7.0 prepared from unheated skimmilk.—-

The proteose—peptone-rich fraction R Figure 11, prepared

1,
f170m unheated skimmilk was dissolved in deionized water to

A...

‘r

Pk a
V .-

 

I.

an.

e

. .
a.-

 

32

a concentration of 0.5%. The solution was adjusted to pH
7.0 and ammonium sulfate added slowly with constant agita—
tiion until a flocculate formed—-at 52% saturation. The
onrecipitate was removed by centrifugation. Additional
ifxéactions were obtained at 65 and 80% saturation with
ainnmonium sulfate. The residues and the supernatant were
tzlreated as shown in Figure 15.
Optimized Procedures for the Fractionation
of Proteose-Peptone from Heated and
Unheated Skimmilk
Isolation of component 8 by preparative acrylamide
E55321 electrOphoresis.--In 1960 Raymond and Wang described
tzluee use of acrylamide gel as a medium for zone electro-
E>11<3resis. A method described below using acrylamide gel
6Electrophoresis was devised for the isolation of the com-
EDCDrient 8 from proteose—peptone. A discontinuous buffer
E3§lsstem similar to the one used by Wake and Baldwin (1961)
“V5155 employed. The tris—citrate stock buffer solution con—
ESisted of 92.0 g of tris-hydroxymethyl aminomethane and
112 -1 g of citric acid per liter. The gel solution consisted
C>f‘ 210 m1 of stock buffer, 75 g acrylamide (Cyanogum Al),
2’40 g urea and enough deionized water to make a total
VOlume‘of 1000 ml. After filtering, 1 m1 of N,N,N',N'-
tet3Pamethylethylenediamine (TMED) and l g of ammonium per—
Slll~fate were dissolved in the gel solution. The solution
”as poured into the gel bed of the electrophoretic cell and

allOwed to polymerize under an atmosphere of nitrogen. The

A -u a. *‘u

 

u ‘

i»

n50

; 1
A."-

33

borate stock buffer consisted of 881 g boric acid and 190 g
sodium hydroxide diluted to 19 liters. Fourteen hundred
milliliters of stock buffer diluted with 2100 ml of deionized
water served as the final solution. Approximately 1600 ml
of“the diluted buffer were poured into each of the two
buffifer tanks connected to the gel bed. Five milliliters of

a.1L% solution of proteose-peptone, prepared as shown in

m. “I; '0‘... ‘l

Fignlre l, were placed in the sample slot. The gel was
coveered with Saran Wrap and platinum electrodes were placed
in eeach of the buffer tanks. The negative lead of the power
supqoly was connected to the electrode closest to the sample
slxyt. Electrophoresis was conducted at 150 volts and 180 ma
for' approximately 1A—16 hours or until the brown band
norunally observed with a discontinuous buffer system had
Ufl4grated.approximately 16 cm. The gel was approximately 25
CHI long, 25 cm wide and 1 cm thick.

The section of acrylamide gel containing the brown
barui which also contained component 8 was removed and placed
in 51 glass tube 7 cm in diameter x 16 cm in length with a
fufiitted glass at one end. The tube was placed in a beaker
”1131 the fritted glass about 1 cm from the bottom. Tris—
citrate buffer (70 m1 of tris-citrate stock + 280 ml of
deiflniized water) was added until the buffer covered the
aCI‘Illamide gel to a depth of about 2 cm. The negative
electrode was placed inside the glass tube and the positive
electrode was placed on the outside of the glass tube. A

current of 30-50 ma was applied for 20-2A hours. Then, the

3A

buffer solution containing the extracted protein was
filtered to remove any large particles of the acrylamide
gel. Next, the protein was separated from soluble acryla-
rnide by making the solution 90% saturation with ammonium
Slllfate and by adjusting the pH to A.6. The aggregated
Ixrotein was removed by centrifuging 1000 x G for 30 minutes.
TTlis residue was dissolved in deionized water at pH 7.0 and
prnecipitated again with ammonium sulfate. The final residue
was dialyzed and lyophilized.

Ammonium sulfate fractionation of a proteose—peptone—
rixzh fraction at pH A.9 and 7.0 prepared from unheated skim—
mg£LE,—-The proteose-peptone-rich fraction was fractionated
atnzording to the scheme shown in Figure 16.

Fourteen grams of the proteose-peptone-rich fraction
RJ_, Figure 11, were dissolved in 1A00 ml of deionized water
allij 7.0. Ammonium sulfate was added to the solution with
Ccnistant stirring to produce a solution of 25% saturation.
TTHB pH was lowered to A.9 and the flocculated protein was
Penuived by centrifugation. A11 centrifugations in this pro-
CeChire were conducted at 8000 x G for 20-25 minutes.

Precipitates were collected from solutions which were
madeto A0, 55, 65, and 80% saturation with ammonium sulfate.

The residues were dispersed in deionized water,
adJllsted to pH 7.0, dialyzed against deionized water and

lycDIDhilized. The final supernatant was dialyzed free of

Salt and lyophilized.

35

Ammonium sulfate fractionation of proteosejpeptone at
pH A.9 and 7.0 prepared from heated skimmilk.--Proteose-
peptone was prepared according to the procedure outline in
.Rigure 1 and fractionated similarily to the proteose-peptone—
ricfli fraction obtained from unheated skimmilk. A schematic
dizagram of this procedure is Shown in Figure 16.

Isolation of component 5 from proteose—peptone obtained
frcnn heated skimmilk.—~Component 5 was isolated from proteose-
peptone according to the diagram shown in Figure 17.

Proteose-peptone was prepared according to the proce-
durwe shown in Figure 1 and dissolved in deionized water at
pH '7.0 to a concentration of 1%. Ammonium sulfate was added
witrl constant stirring to 25% saturation. The pH was adjusted
to 24.9 and the flocculated protein was removed by centrifuga—
‘tiorl. The supernatant was fractionated further to obtain
COHuoonent 8 as described in Figure 16. The residue was
dissnolved in deionized water at pH 7.0 and dialyzed. After
dieilysis, the solution was adjusted to pH 7.0 and the
insmxluble material was removed by centrifugation and dis—
calThEd. The pH of the supernatant was adjusted to A.6 and
the? flocculated protein was removed by centrifugation. The
reSLllting supernatant was discarded. The residue was dis—
perwfled in deionized water to a concentration of about 0.5%
at FHA 7.0. The solution was adjusted to pH A.6 and the
flocCulated protein removed by centrifugation. The pH of
the’ Supernatant was adjusted to 7.0 and ammonium sulfate

w
35‘ added to make a solution of 25% saturation. Next, the

36

pH was adjusted to A.6; the precipitated protein was
removed by centrifugation and designated as component 5.
The supernatant was discarded.

The residue precipitated at pH A.6 in the absence of

ammonium sulfate was dispersed in deionized water at pH 7.0.

After stirring for 20-30 minutes, the pH was adjusted to

A.6 and the precipitated protein removed by centrifugation.
The supernatant was treated as described above to obtain
component 5. Again, the residue precipitated at pH A.6 in
the absence of ammonium sulfate was dispersed in deionized
water and component 5 extracted as described previously.

Separation of components 8—fast and 8-slow by gel

.filtration.——Gel filtration through Bio-Gel P—10 was used

 

:for'the separation of component 8 into components 8-fast

and 8-s low .
Bio—Gel P—10 has a molecular weight exclusion limit

c>f 10,000 and an operating range of 5000—17,000. The beads

vvere hydrated by stirring with deionized water for about

tzhree hours. Chromatographic columns with inside dimensions

c>i'2.8 cm in diameter and approximately 60 cm in length

‘vwere coated with dimethyldichlorosilane to prevent water

-fVPom flowing faster at the glass-bed interface. A 1% solu—

tSion of dimethyldichlorosilane in benzene, heated to

ElIDproximately 60 C, was poured into a clean column. The

S(Dilution was removed and the benzene evaporated in a drying

C"’€fll. This procedure was repeated. The chromatographic

ccDillimn was filled with water and a small amount of glasswool

I
uu’

37

placed at the bottom of the column. A wide-stem funnel was
attached at the tOp. The hydrated beads were poured into
the funnel with constant stirring to prevent blockage of
the funnel stem while the gel beads settled. Following the
formation of a small layer of beads at the bottom of the
column, the outlet was opened to allow the bed to pack more
tightly and to complete the packing. The final column
dimensions were 2.8 cm by A0 cm. Void volume (50—60 ml)
was determined by eluating a solution of Blue Dextran 2000
through the column. The flow rate was approximately 150
Inl per hour. The eluating solution was 0.2 M sodium
chloride and in most cases, deionized water was used.

The sample was applied to the column in approximately
53—6 ml of a 2% protein solution. The column eluates were
rnonitored at 280 mu in one case and at 25A mu in another. Frac—
t:ions were collected at intervals of 25 ml, 20 ml, 30 ml, 30 m1,
61nd 50 ml after appearance of the initial peak. Each frac—
tsion was lyophilized and analyzed by acrylamide gel electro—

I>horesis. Eluate fractions 3 and A were rechromatographed.

Preparation of Samples for Chemical
and Physical Analysis

Gel filtration was used for desalting the proteins
tDEEfore any chemical or physical analyses were conducted.
criie column was prepared in the same manner as described
fer the Bio-Gel P-10 column except that Bio-Gel P-2 beads

'“’€3:re used. The flow rate for the desalting column was about

38

200 ml per hour when developed with deionized water. The

proteins were passed through the P-2 columns; then 1y0phi1ized.

This process was repeated.

The samples were dried in a vacuum oven (over P205) at

 

approximately 25 C for 2A—A8 hours before weighing. Dry
weights were used as the size basis for all samples.
Chemical Methods
Nitrogen
A micro—

Nitrogen analysis was performed in duplicate.

Kjeldahl apparatus with round bottom flasks and round, ground

.glass joints was used. The digestion mixture consisted of

55.0 g CuSOu-5H2O and 5.0 g SeO2 in 500 m1 of concentrated

I12SOM. Ten to 50 mg of dried protein were digested with A
Inl of the digestion mixture over a gas flame for approximately

c>ne hour. The digestion mixture was cooled, 1 ml of 30%

I1202 was added and digestion was continued for one hour.
IXfter cooling, the sides of the digestion flasks were rinsed

vwith 10 m1 of deionized water. The digestion mixture was

rleutralized with approximately 25 ml of a A0% sodium hydroxide

ssolution after the digestion flask was connected to the dis—

tzillation apparatus. The released ammonia was steam dis—

‘tiilled.into 15 m1 of a A% boric acid solution with A to 5

C11?0ps of indicator. The indicator consisted of A00 mg

t311."omocresol green and A0 m1 methyl red in 100 m1 of 95%

eTZInanol. The distillation was completed when a total volume

C’JT' approximately 60 ml was present in the receiving flask.

39

The ammonia was titrated with 0.019A N HCl. Tris-

hydroxymethyl aminomethane (Sigma 121) was used as the

primary standard to determine the normality of the hydro-

chloric acid.

The average recoveries of ammonium sulfate and

tryptophan standards were 99.6A% and 98.75%, respectively.

Phosphorus
Phosphorus was determined by a colorimetric pro—

cedure adapted from the method used by Sumner (19AA).

Phosphorus analyses were performed in duplicate. Dried

Iarotein (1 to 11 mg) was digested with 2.2 ml of 50% H280“

:in a test tube. The digestion was carried out for 20

rninutes on a sand bath heated by an electric heater main-

tlained at 160 to 170 C. After cooling, 16 drops of 30%

P1202 were added to the test tube and heated for 15 minutes.

ikfter cooling, 16 drops of 30% H2O2 were added and the

Ciigestion was continued for an additional hour. If the

Ciigestion mixture was not clear at this point, additional

Ii: 0 was added and the digestion was continued. The accuracy

2 2
Cbi‘the determination depends upon the complete removal of

After cooling, the digestion
Then,

El]1.the hydrogen peroxide.

ITilixture was transferred to a 50 m1 volumetric flask.

55 nfl of a 6.6% (NHA)6 Mo7O2u-AH20 solution and enough water

‘VTEEIe added to give a total volume of approximately A0 ml.

Pqeecxt, A ml of a solution of 5 gm FeSOu-(H2O)7 in 50 m1 of

water plus 1 ml of 7.5 N sulfuric acid were added and mixed.

A0

This solution was prepared immediately before use. The
volumetric flask was made up to volume with water and mixed.
After standing for 30 minutes, the transmission (%) was read
at 660 mu with a Beckman DK-2A spectrophotometer.

Ten milliliters of a stock solution containing 1.3613
g of KH2POu dissolved in 1000 ml were diluted to 100 ml
(0.031 mg/ml). A standard curve was prepared covering the

range of 0.0 to 0.31 mg of phosphorus.

Hexose

The method of Winzler (1955) was used for determining
the hexose content of the purified component 8 extracted
from acrylamide gel. All other hexose analyses were
determined by the colorimetric method reported by Dubois,
Gilles, Hamilton, Rebers, and Smith (1956).

A weighed amount of protein was placed in a test tube
and 1 ml of water was added. One milliliter of 5% phenol
in water was added and mixed. Reagent grade phenol was
redistilled before preparation of the 5% solution. Next,

5 m1 of concentrated sulfuric acid was added with a fast-
<jelivery 5 ml pipette with a portion of the tip removed.
{The acid stream was directed against the liquid surface and
tflde liquid was stirred simultaneously with a Mini—shaker
CDbtained from Fisher Scientific Company. The mixing of
EBech tube equally was critical to reproducibility. Finally,
tSk'ie tubes were allowed to stand 10 minutes, stirred again

Eirfid placed for 20-30 minutes in a water bath at 25 to 30 C.

Al

The transmission (%) was read at A90 mu with a Beckman
DK—2A spectrophotometer.

A blank was prepared by omitting the protein from the
reaction mixture. The standard curve was prepared covering
the range of 0.0 to 90 ug of galactose dissolved in 1 ml of

water.

Hexosamine

The method of Cessi and Piliego (1960) was used for
determining the hexosamine content of component 8 purified
by acrylamide gel electrophoresis. The hexosamine content
of the other purified proteins was determined according to
the method described by Johansen, Marshall and Neuberger
(1960) with some modification.

Samples (1 to A mg) were weighed directly into 5 m1
ampoules to which 1 m1 of A N HCl was added. The samples
were frozen in a dry ice—ethanol bath, evacuated, allowed
to melt to release air from the frozen solution, refrozen
and sealed while under vacuum. One milliliter of standard
glucosamine (25 ug) was added to each of two ampoules and
dried. One milliliter of A N HCl was added to the ampoules
which then were treated the same as the protein samples.
These standard samples were designated to serve as recovery
standards.

Hydrolysis of the protein and the standard glucosamine
was carried out for six hours at 100 C. After cooling, the

hydrolyzed samples were transferred to distillation flasks

A2

and the ampoules rinsed with 1 m1 of A N sodium hydroxide,
followed by two 1 m1 rinsings with water.

The blank consisted of 1 m1 of A N HCl, 1 ml of A
N NaOH and 2 m1 of deionized water. Glucosamine was used
to construct a standard curve covering a range from 0 to
50 ug in 5 ug increments.

Acetylacetone reagent was prepared by dissolving 1 m1
of freshly distilled acetylacetone in 25 m1 of l M Na2CO3
solution and 20 ml of water. The pH, if different from 9.8,
was adjusted to that value and the volume made up to 50 m1.
This solution was used within 30 minutes. Ehrlich's reagent
was prepared by dissolving 2 g of p—dimethylaminobenzalde—
hyde in absolute ethanol containing 3.5% of concentrated
HCl and diluted to a final volume of 250 ml. This solution
was stored at O-A C. The reagent was recrystallized from
alcohol (Rondle and Morgan, 1955).

Five and five-tenths milliliters of the acetylacetone
reagent were added to each of the distillation flasks con-
taining the hydrolyzed sample, blank, and standard solutions.
The final pH of the solution was maintained within the limits
of 9.5 to 10.0. The flasks were stoppered tightly and
heated in a vigorously boiling water bath for 20 minutes.
Next, the flasks were cooled in an ice—water bath and then
connected to the distillation apparatus. Each flask was
heated with a Bunsen burner and the steam—volatile chromogen
was distilled into a 10 ml volumetric flask containing 7.5

m1 of Ehrlich's reagent. The volumetric flask was stoppered

A3

and mixed. After at least 30 minutes, the trans-
mission (%) of the solution was read at a wavelength of

5A8 mu with a Beckman DK—2A spectrophotometer.

Sialic Acid

The method of Aminoff (1961) was used for determining
the sialic acid content of component 8 extracted from
acrylamide gel. For the other isolated proteins, Warren's
thiobarbituric acid method (1959) as modified by Marier,
Tessier, and Rose (1963) was adopted. A detailed descrip-
tion of the method follows. 2-Thiobarbituric acid was
recrystallized four times from hot water (25 gm in 600 ml).
The solutions used in the analysis were as follows: 0.1 N
H280”, 0.2 M sodium periodate (meta) in 9 M phosphoric
acid, sodium arsenite—-10% in a solution containing 0.5 M
sodium sulfate and 0.1 N H280“, and 2-thiobarbituric acid—-
0.6% in 0.5 M sodium sulfate.

Samples of dried protein of known weight (0.5 to 3 mg)
were placed in test tubes followed by the addition of 0.5
ml of 0.1 N H280“. Digestion was performed at 80 C for A0
minutes to release the sialic acid. A standard curve was
prepared covering the range (0 to 20 ug) of N—acetyl_neuraminic
acid. To the hydrolyzed sample and to 0.5 m1 of the N-
acetyl-neuraminic solution,0.l m1 of the periodate solution
was added, mixed, and incubated at room temperature for 20

minutes. After the addition of 1 ml of the arsenite solution,

the tubes were shaken until the yellow—brown color disappeared.

AA

Following the addition of 3 ml of the thiobarbituric acid
solution, the tubes were shaken; covered tightly with
aluminum foil; and heated in a vigorously boiling water
bath for 15 minutes. Next, the tubes were removed and
placed in a cold—water bath for five minutes. After cool-
ing, A m1 of cyclohexanone were added to the tubes and they
were shaken vigorously twice before centrifugation for

five minutes in a clinical centrifuge. In some cases, an
increased time of centrifugation was required since a clear
cyclohexanone phase was essential to reproducibility. The
transmission (%) of the cyclohexanone layer was read at

5A9 mu with a Beckman DK-2A spectrophotometer.

Sulfhydryl Groups

Ellman's reagent (5,5'—dithiobis [2—nitrobenzoic] or
DTNB) was used for the determination of free sulfhydryl
groups in the proteins (Ellman, 1959).

In preparing a standard curve, glutathione (0.1
umole to 1.0 umole) was added to a series of test tubes with
enough deionized water to give a total volume of 1 ml.
Then, 8 m1 of phosphate buffer (pH 8.0, I=0.1) containing
8 M urea were added and mixed. The solution was allowed to
stand for ten minutes; then, 3 ml were removed and placed
in the spectrophotometeric cell followed by the addition of
0.02 ml of the DTNB reagent. The reagent was prepared by
dissolving 39.6 mg of recrystallized DTNB in 10 m1 of 95%

ethanol. After the solution was mixed and allowed to stand

A5

for five minutes, it was read at A12 mu (%T). A reagent
blank was treated in the same manner except that 1 ml of
water replaced the glutathione solution.

The time limitations imposed on the procedure
resulted from preliminary observations which indicated
that a good standard curve could not be obtained with
glutathione when it was in contact with the urea—phosphate
buffer for an extended period of time. Also, low values
for the sulfhydryl groups present in B-lactoglobulin were
obtained when B—lactoglobulin was exposed to the same
solution. The eXperimental conditions selected produced a
maximum of reproducibility in preparing the standard curve
and a minimum of sulfhydryl destruction in the proteins.

Ten to 13 mg of dried protein were transferred to
test tubes to which 1 ml of deionized water was added to
dissolve the protein. Eight milliliters of phosphate buffer
containing 8 M urea were added, mixed and allowed to stand
for ten minutes. Three milliliters of the solution were
removed and placed in a spectrophotometric cell and read at
A12 mu (%T) to obtain a value attributed to the protein.
Then, 0.02 ml of DTNB reagent was added, mixed and allowed
to stand for five minutes. A final reading was made at A12
mu indicating the presence of —SH groups.

To standardize the instrument, reagent blanks were
prepared by mixing 1 ml of water and 8 m1 of the urea—

phosphate buffer. Three milliliters of this solution were

A6

transferred to a spectrophotometric cell and 0.02 ml of

DTNB reagent was added.

Amino Acids

Amino acid analyses were performed on 20 and 70 hour
acid (hydrochloric) hydrolysates of the proteins using a
Beckman Amino Acid Analyzer Model 120 C (Moore, Spackman,
and Stein, 1958). About 3 mg of dried protein were weighed
directly into 10 or 20 ml ampoules. Five or 6 m1 of 6
N HCl were pipetted into the ampoules. The contents of the
ampoules were frozen in a dry ice—ethanol bath. The
ampoules were evacuated with a high vacuum pump, removed from
the dry ice-ethanol bath and allowed to melt slowly in order
to remove any gases that were in the frozen samples. The
ampoules were again placed in the freezing-mixture and,
while under vacuum, sealed with an air—propane flame. The
sealed ampoules were placed in an oil bath in a forced—draft,
recirculating oven regulated at 110 C. The proteins were
hydrolyzed for 20 and 70 hours. The ampoules were removed
from the oven, cooled and stored in a deep freeze until used.

The hydrolysate was transferred quantitatively to a
25 ml pear-shaped flask fitted with a ground glass joint.
In some cases, nor-leucine was added to the hydrolysate
before transferring in order to check for transfer losses.
The hydrolysate was evaporated to dryness on a rotary
evaporator. The residue was taken up in a small amount of

deionized water and redried. Five milliliters of citrate-

A7

HCl buffer (pH 2.2) was added to the dried hydrolysate.
When all the soluble material dissolved, an aliquot was
removed for analysis on the amino acid analyzer. Standard
amino acid mixtures were analyzed with the same ninhydrin

solution within four or five days.

Tryptophan

Tryptophan may be determined microbiologically or
chemically by alkaline hydrolysis or by colorimetric analy—
sis of the unhydrolyzed proteins.

In this study tryptophan was determined by methods
described by Spies and Chambers (19A8, l9A9). A combina—
tion of procedure N (Spies and Chambers, l9A9) and pro-
cedure B (Spies and Chambers, 19A8) was used.

In preparing the standard curve, a water solution of
tryptophan (6 to 120 ug) was added to each test tube and
made up to 1 ml. Following the addition of 8 m1 of 23.8 N
sulfuric acid to each test tube, 1.0 m1 of 2 N sulfuric
acid containing 30 ug of p—dimethylaminobenzaldehyde (pre—
pared immediately before use) was added. This solution
was mixed and cooled to 25 C; then allowed to stand at this
temperature for 12 hours in the dark. After the addition
of 0.1 m1 of a 0.0A5% sodium nitrite solution, the reaction
mixture was shaken and the color was allowed to develop for
30 minutes in the dark at 25 C. The solution was read at

590 mu (%T).

A8

Proteins (2 to 10 mg) were treated the same as trypto-
phan except that the samples were weighed directly into a
test tube and 1 m1 of water was added to dissolve the protein.

B-Lactoglobulin was used as a control material.

Paper Chromatography (Carbohydrates)

The identity of the hexoses and hexosamines in the
proteose—peptone fractions was determined by paper chromato—
graphy. Approximately 50 mg of protein were placed in a
20 ml ampoule and 10 ml of 2 N hydrochloric acid were
added. The solution was frozen in a dry ice—ethanol bath
and the ampoules were evacuated with a mechanical vacuum
pump. While under vacuum, the ampoules were removed from
the bath and were allowed to warm slowly in order to
release any dissolved air present in the frozen solution.
Then, the solution was refrozen and the evacuated ampoules
were placed in an oil bath maintained at 100 C for five hours.

The hydrolysate was transferred to a 50 m1 round-
bottom flask/ evaporator assembly and dried. The residue
was taken up in deionized water and again dried. This
process was repeated two more times.

The final residue was dissolved in deionized water
(5 to 10 ml) and passed through tandem columns of 2 x 20 cm
filled with Dowex 1 X 8 (220-A00 mesh, acetate form) in the
top unit and with Dowex 50 X 8 (20-50 mesh, H+ form) in the

lower unit.

V
1 1

.4

“J-

r‘u

m-

ll

p
a

fin;
J.J

ea.-

b u i

A.“ h.u
n1.

 

A9

Dowex l X 8 in the Cl— form was changed to the OH-
form by passing 1 liter of l N sodium hydroxide through the
column. Then, the resin was converted to the acetate form
by passing 1 liter of 1 N acetic acid through the column.
Next, the column was rinsed with deionized water until the
pH was greater than A.8 but less than 9.0. Dowex 50 X 8
was converted to the H+ form by passing 1 liter of l N
hydrochloric acid through the column and then rinsing with
deionized water until the pH was greater than A.8 but less
than 9.0.

About A00 m1 of the eluate were collected and taken to
dryness with a rotary evaporator and then dried for about
two hours on a 1yophilizer. The dried hydrolysate was
extracted with dry pyridine according to the method described
by Block, Durrun, and Zweig (1958). In this procedure,
the dried residue was extracted with 5-10 ml of redistilled
pyridine at 100 C for 10 minutes. The resulting mixture was
cooled, filtered and the pyridine removed with a rotary
evaporator at a temperature not exceeding A0 C. The residue
was dissolved in a small amount of 10% iSOpropanol. The
unknown carbohydrate solution was spotted on Whatman No. l
(10 x A2 cm) chromatography paper and run as a descending
chromatogram for 16—20 hours with a butanol-pyridine—water
(6:A:3) solvent system (Bezkorovainy, 1963). Authentic
standards of glucosamine, galactosamine, galactose, mannose,

and fucose were analyzed concurrently with the unknown

50

samples. The carbohydrate constituents were detected by
Spraying the dried chromatogram (600 for 15 minutes) with
a mixture of equal volumes of a 2% aqueous solution of
triphenyltetrazolium chloride and 1 N sodium hydroxide
(Block gp_a1., 1958). The sprayed chromatogram was placed
in a moist atmosphere at about 60—70 C until the red spots
appeared. Storage at =-10 C aided in preserving the

chromatograms.

Physical Methods

 

Electrophoresis

Free-boundary electrophoresis.-—A Perkin—Elmer Model

 

38—A electrophoresis apparatus was used to estimate the

electrOphoretic mobilities of the protein fractions.
Electrophoretic mobilities were calculated for the

descending and ascending patterns using the following

equation:

('an

ehm
5??

where p is the electrophoretic mobility in l x 10_5 cm2

volt-l sec—l, d is the distance the boundary migrated from
the initial boundary in cm, a is the cross-sectional area
of the electrophoretic cell in cm2, k is the conductivity
cell constant, p is the time of electrophoresis in seconds,

i is the current in amperes, p is the resistance of the

buffer in ohms determined in a conductivity cell at the

51

same temperature as the electrOphoretic run, and m is the
magnification factor of the optical system.

The buffers described by Miller and Golder (1950) and
veronal buffer, pH 8.6, I = 0.1, served as the solvent
systems for the electrOphoretic mobility experiments. The
mobilities obtained employing the veronal buffer were used
as references. Electrophoretic mobilities determined at
pH 2.0, 2.5, 3.5, A.5, 5.5, 6.5, 7.5, and 8.5 (I = 0.2) in
the buffers described by Miller and Golder (1950) were used
for the isoelectric point determination. From a plot of
the average ascending and descending mobilities and the pH,
the isoelectric point was estimated.

In determining the mobilities for the isoelectric
point determination of component 8 purified by acrylamide
gel electrophoresis, the protein solutions were dialyzed 2A
hours against A00 m1 of the buffers. Some of the protein
(component 8) probably passed through the dialysis membrane.
Dialysis was not conducted when the mobilities were deter-
mined for the purified proteins—-components 8—fast and 8—slow
—-since these proteins would pass through the dialysis
membrane. All free—boundary electrophoretic analyses were
performed at a temperature of 2 C.

In one experiment, the fastest migrating peak
(component 8) was removed from the ascending channel of the
electrophoretic cell and analyzed by starch—urea gel

electrophoresis. The location of the peak in the Tiselius

52

cell was determined photographically at the conclusion of
the electrophoretic run. The cell was shifted to avoid
mixing the gradients. That portion of the cell contents
containing component 8 was removed with a syringe assembly
connected to a trap with an applied vacuum.

Starch-urea gel electrOphoresis.--Starch-urea gel

 

electrophoresis was one method of zonal electrophoresis
used in this investigation. The procedure used in pre-
paring and performing starch—urea gel electrOphoresis was
adapted from the procedure described by Wake and Baldwin
(1961) .
The Plexiglas gel bed had the same dimensions as the
one used by Wake and Baldwin (1961). Eaton & Dikeman No.
652 filter paper wicks cut to the dimensions of A.5 x A.75
inches were inserted at the ends of the Plexiglas bed
before pouring the gel. The gel bed had a small chamber
underneath the bed surface for the circulation of cooling
water. Electrophoresis was performed in the laboratory
with circulating tap water to keep the gel cool (about 15 C).
The gel was prepared as follows: to a one-liter
beaker, 70 ml of stock tris-citrate buffer (92.00 g of
tris-hydroxymethyl aminomethane and 12.1 g of citric acid
per liter or the tris—hydroxymethyl aminomethane was titrated
to pH 8.6 with citric acid), 170 ml water and A5 g hydrolyzed
starch were added with constant stirring and heated over an

open flame to 65 C. Then, 1A7 g urea were added slowly and

53

dissolved with continued stirring and heating to 90 C.

The solution was poured into a two—liter filter flask and
the air bubbles were removed by the application of vacuum.
The degassed solution was poured into the gel bed and a
slot former placed in position near the cathodic end of the
cell. The gel was covered with Saran Wrap but not allowed
to come in contact with the gel solution. After aging for
approximately 2A hours, the gel was prepared for the
electrophoretic run.

Sample solutions (0.5 to 5% protein) were prepared by
dissolving the protein in an aliquot of a buffer consisting
of 70 ml of tris-citrate stock buffer, 170 ml water and 1A7
g urea. The slot former was removed and the protein samples
were introduced into the slots with capillary tubes. Each
slot was covered with a layer of mineral oil and a cover—
glass. Again, the gel was covered with Saran Wrap to prevent
evaporation.

The buffer tanks, connected to the gel through the
filter paper wicks, were filled with boric acid—sodium
hydroxide buffer. Stock boric acid—sodium hydroxide buffer
was prepared by dissolving 881 g boric acid and 190 g sodium
hydroxide in 19 liters of water, adjusted to pH 8.6. The
stock buffer was diluted 1:1.5 with deionized water and about
800 m1 of the diluted buffer were added to each buffer tank.
This system represents the discontinuous buffer referred to
in this manuscript. Platinum wire electrodes were inserted

in each buffer tank and 200—250 volts (d.c.) were applied

5A

across the system for 16—20 hours or until the brown band
characteristic of this system had migrated about 12 cm past
the starting slots. In some cases, a constant current of
25 ma was maintained.

The gel was sliced transversally after electrophoresis

was completed. The middle and bottom slices were stained
with an amido black staining solution consisting of 250 ml
water, 250 m1 methanol, 50 m1 glacial acetic acid and 2 g
amido black. The gel was placed in the staining solution
for about 15-30 minutes, removed and rinsed with water.
The unbound stain was removed from the gel in an electro-
phoretic destaining cell containing 7% acetic acid as an
electrolyte. The electrophoretogram was photographically
recorded.

Acrylamide gel electrophoresis with a discontinuous

 

buffer system.—-The application of Cyanogum A1, a mixture of
acrylamide and N,N'—methy1enebisacry1amide, as a zone
electrophoresis medium was reported by Raymond and Weintraub
(1959) and Raymond and Wang (1960). The Cyanogum Al was
polymerized to form a transparent gel which served as the
medium for zone electrophoresis.

In this research, several methods of acrylamide gel
electrophoresis were used: discontinuous and continuous
buffer systems and gels with and without urea. Also,
electrophoresis was conducted in horizontal and vertical
gel beds. In this technique, the electrophoretic cells

were designed to provide a gel to buffer contact.

55

With the discontinuous buffer system, the gel consisted
of Cyanogum Al (8%) dissolved in the tris-citrate buffer and
the buffer tanks were filled with borate buffer. Both
buffers were described previously for starch—urea gel
electrophoresis. The gel solution was prepared by dissolving
20 g of Cyanogum A1 in 50 m1 of stock tris-citrate buffer
and enough water to make a total volume of 250 m1. For an
acrylamide gel with A.5 M urea, the gel solution was pre-
pared by dissolving 68 g of urea, 20 g of Cyanogum Al in
50 ml of stock tris-citrate buffer, and enough water to make
a total volume of 250 m1. When the E. C. Apparatus vertical
cell was used, 150 m1 of gel solution was required. While
vigorously stirring the filtered gel solution, 0.30-0.A0 m1
of N,N,N',N'-tetramethylethylenediamine (TMED) and 0.30-0.A0
g of ammonium persulfate were added. After about 20—30
seconds, the solution was poured in a gel bed as illustrated
in Figure 18. The slot former was introduced and the gel
bed was placed under an atmosphere of nitrogen until the
solution was polymerized. The slot former was removed and
0.10-0.15 ml portions of the protein solutions (0.5 to 5%)
were introduced into the slots. Again, the gel was covered
with Saran Wrap to reduce evaporation.

The stock borate buffer described for starch-urea gel
electrophoresis was diluted 1:1.5 parts with deionized water
and poured into the buffer tanks to complete the discon—
tinuous buffer system. Platinum electrodes were introduced

into the buffer tanks and the current was applied until the

56

brown band had migrated 12 cm past the sample slots. The
negative electrode was closest to the sample slots.

When electrophoresis was completed, the gel was removed
from the gel bed and stained with a dye solution consisting
of 250 ml water, 250 ml methanol, 50 m1 of glacial acetic
acid and 5 g of amido black (naphthol blue black). The gel
remained in the dye solution for one hour or longer. The
unbound dye was removed electrophoretically in an electro-
phoretic destaining cell containing 7% acetic acid. The
electrophoretogram (destained gel) was photographed for a
permanent record.

Acrylamide gel electrophoresis with a continuous buffer
system.--Except for a different buffer system, the procedure
followed for acrylamide gel electrophoresis with continuous
and discontinuous buffer systems were similar. In the con—
tinuous buffer system, the same buffer was used in both the
gel and the buffer tanks. The buffer was prepared by mixing
one part of the stock borate buffer described for starch—urea
gel electrophoresis with two and one-half parts of deionized
water. The gel solution was prepared by dissolving 160 g of
Cyanogum A1 in approximately 1.5 liters of the borate buffer.
This solution was filtered and made to a total volume of two
liters with borate buffer. To 250 ml of this gel solution,
0.3-0.A m1 of TMED was added. While the solution was being
vigorously stirred, 0.3-0.A g of ammonium persulfate was

added. Within 30 seconds, the solution was poured into the

57

gel bed shown in Figure 18. Immediately, the slot former
was placed in the bed and the entire assembly placed in a
nitrogen atmosphere until the gel solution polymerized.

The gel bed was connected to the electrode vessels by
gel—filled legs which rested in tanks each of which con-
tained about 1800 ml of borate buffer prepared as described
above. The slot former was removed and 0.1—0.15 ml of a
1.0-5.0% protein solution was introduced into each slot. The
gel was covered with Saran Wrap and platinum electrodes were
inserted into each buffer tank. Electrophoresis was per—
formed for approximately 16 hours with a voltage of 110—120
volts (d.c.) and a current of 100 ma. A drop of bromo phenol
blue solution was introduced into one slot to serve as an
indicator for observing the electrophoretic migration since
bromo phenol blue usually migrates with the fastest moving
proteins.

When performing vertical acrylamide gel electrophoresis
with the E. C. Apparatus cell, the amount of gel solution
was reduced to 150 ml. Also, the TMED and ammonium per-
sulfate were reduced to 0.2 m1 and 0.15 g, respectively.
Protein samples (= 3%) were dissolved in borate buffer con—
taining 8% sucrose and enough bromo phenol blue to give a
dark blue color. A 100 pl syringe with a curved needle was
used to introduce 10 to A0 pl of the protein solution into
each slot. A constant current of 100 ma was applied for
approximately six hours or until the bromo phenol blue indi—

cator had migrated about 11 cm.

58

After completion of electrophoresis, the gel was
removed and stained as previously described.

Two-dimensional acpylamide gel electrophoresis.--

 

Acrylamide gel with a continuous buffer system was used in
the first dimension. Each slot contained the same protein
sample. Upon the completion of the run, a strip of the gel
containing the resolved proteins was removed, stained, and
destained. An analogous section of the gel was removed and
placed across a horizontal gel bed as shown in Figure 18.

A gel solution prepared according to the procedure described
for acrylamide gel electrophoresis with a discontinuous
buffer system was poured around the strip of gel and allowed
to polymerize. Thus, in the second dimension, electrophoresis
was conducted with a discontinuous buffer system. After
completion of the electrophoretic run, the gel was removed,
stained, destained, and photographed. The purpose of this
procedure was to determine which proteins migrate with the
brown band in the discontinuous buffer system.

Staining of glycoproteins of the proteosejpeptone—rich

 

fraction after electrophoretic separation in acrylamide

 

gglg.—-Acrylamide gel electrophoresis was performed accord—
ing to the method described for acrylamide gel electro-
phoresis with a continuous buffer system. But in this
instance, the gel was developed for glycoproteins according
to the method of Keyser (196A). The E. C. Apparatus cell

was used for this experiment because the gel was much

59

thinner and, thus, responded more satisfactorily to the
reagents employed.

The reagents employed are given below. A methanol-
water—acetic acid solution was prepared by mixing together
eight parts methanol, ten parts water and one part glacial
acetic acid. Periodic acid reagent was prepared by dis-
solving 3.0 g of periodic acid (H5106) and 1.66 g of sodium
acetate-3 H20 in 500 ml of deionized water. This solution
was stored in the refrigerator. Six volumes of this
solution were diluted with four volumes of 95% ethanol
before using. The thiosulfate—metabisulfite reagent was
prepared by dissolving 10 g of potassium metabisulfite and
60 g of sodium thiosulfate in two liters of water. This
solution was diluted with an equal volume of ethanol before
using. The fuchsin—sulfite reagent was prepared by dis-
solving 16 g of potassium metabisulfite in two liters of
water and then adding 21 ml of concentrated hydrochloric
acid. Next, 8 g of finely—powdered basic fuchsin was added
to the solution and stirred with a magnetic stirrer for
three hours. The solution was treated with decolorizing
charcoal and filtered. In some cases, the solution was not
colorless; thus, the treatment with charcoal was repeated.
This reagent was stored at O—A C.

The ethanol-sulfite wash solution was prepared by
dissolving 5 g of potassium metabisulfite in one liter of
deionized water. One liter of 95% ethanol and 9 m1 of

concentrated hydrochloric acid were added.

60

After completion of electrophoresis, the gel was
removed from the gel bed and placed in a shallow teflon—
coated glass dish with 200 mlwlf 95% ethanol. The gel was
kept in motion by slowly shaking the dish. After ten
minutes, the gel was immersed in 200 ml of the methanol-
water-acetic acid solution for another ten minutes. Next,
the gel was treated with 150 ml of the periodic acid
reagent for five minutes, rinsed in water for three to
five minutes, treated with 150 ml of fuchsin—sulfite reagent
for 25 minutes and washed with the ethanol-sulfite wash
solution until the gel was clear. The gel was photographed
for a permanent record. In many cases, the stain was too

light to record photographically.

Ultracentrifugation

Protein concentration.—-The dried protein was weighed
and dissolved in the desired amount of veronal buffer at
pH 8.6, I = 0.1. In some cases, the protein concentration
was determined by measuring the area of the schlieren
pattern formed in the synthetic boundary cell and then by
reading from a previously constructed plot of the peak area
(cm2) versus protein concentration.

Sedimentation velocity.-—Sedimentation—velocity
eXperiments were performed at a rotor speed of 59,780 RPM
(259,700 x G) and a temperature of 20 C. These proteins
were presumed to have low sedimentation coefficients. Con—

sequently, the synthetic boundary cell was used as recommended

61

for substances with sedimentation coefficients below 3 S
(Schachman, 1957). The ultracentrifuge analyses were
performed in veronal buffer solutions with an ionic strength
of 0.1 or greater to dampen the charge effects inherent to
proteins in solution (Schachman, 1957).

The sedimentation coefficient is defined as the
velocity of the sedimenting molecules per unit field as

shown by the equation:

where x is the distance of the boundary in centimeters
from the axis of rotation, p is the time in seconds, and p
is in radians per second (Schachman, 1957). Thus, by
rearranging the above equation and integrating, the follow—

ing equation was obtained:

§§_= w2sdt ‘*ln x = Est-+0 * 2.303 log x = wZSt + C
By measuring the distance x and plotting log x versus
p, the sedimentation coefficient may be obtained from the

slope of the line using the following constants:

 

S (Svedberg unit) = s x 10'13 sec = (slope) (2'303/69)
2w 59,780 C

62

Sedimentation coefficients are usually reported as

S which is the value the protein would have in a solvent

20,w
with the density and viscosity of water at 20 C. The
observed sedimentation coefficients were corrected to this

standard state according to the following equation:

 

S20 w = obs Eu; 8— T : ;:2O,w
: ° 20 0 1:
nt
where the first term :20 is the viscosity of water at the
experimental temperature relative to that at 20 C; the second
term %; is the relative viscosity of the solvent to that

of water; and the last terms p20’w and pt are the densities
of water at 20 C and the solvent at the experimental tem—
perature, respectively. The partial specific volume V in
the reference solvent was assumed to be the same as in the
solvent used in the experiment.

The 8°20,w at infinite dilution was obtained by

plotting the S versus concentration of protein in the

20,w

test solution and extrapolating to zero concentration.

Sedimentation equilibrium.—-Molecular weights were

 

determined using the short—column sedimentation-equilibrium
technique described by Van Holde and Baldwin (1958). The
analyses were conducted at 20 C in a double—sector cell
using column depths of l to 2 mm. The solutions were con-

sidered to be at equilibrium after 2A hours of centrifugation.

63.

The eXperiment was conducted as follows: first, a
small amount of fluorocarbon oil was introduced 1nto one
sector of the cell to provide a false bottom. Secondly,
enough protein solution was introduced to give a column
depth of l to 2 mm. Finally, the solvent was introduced
into the other sector of the cell to provide a column of
solvent greater in depth than the protein solution. The
solvent column extended above the air/solution meniscus
and below the oil/solution meniscus of the protein solu-
tion. The solvent, solution, and fluorocarbon oil were
introduced with a Hamilton 100 pl syringe.

The double-sector synthetic boundary cell was used
to obtain optical areas which were used to estimate the
protein concentration for the purpose of making molecular
weight calculations.

Densities andgpartial specific volume.—-Solvent den-

 

sities were determined at 20 C with 25 ml pycnometers. The

following relationship:

0 =0 (l-If—D )C

solution solvent solvent

was used for the calculation of solution densities (Fujita,
1962). The E corresponds to the partial specific volume
of the protein and 9 corresponds to the concentration of
the protein in g/ml. The pycnometers were calibrated with

freshly—boiled, redistilled water.

6A

The apparent partial specific volume was estimated
from amino acid and carbohydrate composition data using the

equation:
ZWiVi

2W.
1

<l
n

 

where i is the partial specific volume of the protein, V1

is the specific volume of the ifih amino acid residue, and

W1 is the weight percentage of the iFh amino acid residue

(Schachman, 1957).

65

FRESH SKIMMILK

Heat to 96—100 C for 20-30 min
Cool to 20 C

Adjust to pH A.6

Filter

 

 

I 1
CASEIN + DENATURED ' SUPERNATANT
SERUM PROTEINS

(Discard) Adjust pH to 3.0

Dialyze at A C against 3 or A
changes of deionized water
adjusted to pH 3.0

Adjust pH to 7.0

Dialyzevat A C against 3 or A
changes of deionized water

Pervaporate

Lyophilize

 

PROTEOSE—PEPTONE

Figure l.——Procedure for the preparation of proteose—peptone,
adapted from the method described by Rowland (1938a).

FRESH SKIMMILK

 

Heat to 96-100 C for 20-30 min
Cool to 20 0

Adjust pH to A.6

Filter

 

r
CASEIN + DENATURED
SERUM PROTEINS

SUPERNATANT

 

 

(Discard) Adjust pH to 6.7
Make to 50% satr with
(NHu)2SO
Allow to settle
Centrifuge 1000 x G for
20 min
I
RESIDUE SUPERNATANT

Disperse in water
Adjust pH to 3.0
Dialyze at A 0 against
3 or A changes of
deionized water
adjusted to pH 3.0
Adjust pH to 7.0
Dialyze at A C against
3 or A changes of
deionized water
Pervaporate
Lyophilize

 

SIGMA PROTEOSE

Adjust pH to 3.0
Dialyze at A C against
3 or A changes of-

deionized water
adjusted to pH 3.0
Adjust pH to 7.0
Dialyze at A C against
3 or A changes of
deionized water
Pervaporate
Lyophilize

 

SIGMA-PROTEOSE SUPERNATANT
(Enriched component 8)

Figure 2.--Procedure for the preparation of sigma proteose,
adapted from the method described by Aschaffenburg (19A6).

l’
SUPERNATANT
(Discard)

67

SKIMKILK

I

Satr with RaCl at A0 C
Let stand overnight

Centrifuge 1000 x G for 30 min

RESIDUE
Wash A—Sx with satr NaCl

Centrifuge 100C x G for 3t min

 

r
SUPEEXATAUT

(Discard)

RESIDUE

Disperse in deionized water
Dialyze to remove salt
Adjust pH to A.6
Centrifuge 1000 x G

 

 

 

r’
SUPERNATAET IICIEDE
Adjust rH to 8.0 (Casein)
Centrifuge 10JH x G for 30 min (Discard)
r —1
SL’PLAK :\V V‘

 

 

.‘

Disperse in deionized
Adjust pH to A.b
Centrifuge 1000 x G

water

for 30 min

 

 

 

 

 

 

 

 

 

 

r _I
SUPTEGJATAHC‘ PESIllfii (R1)
Aoju t pH to t.0 Disperse in deionized water
Centrifuge lOth x G lor 3C min Adjust pH to 7.0
Dialyze
Lyoyhilize
(Enriched components 5 and ’)
SUIfliPhACVHYT Pix IDUI (R,)
L
Adjust pH to 7.0 Disperse in deionized water
Centrifuge lUEO x G for 30 min Dialyze
yophiiize
(Enriched components 5 and t)
SCPHRHAT NT RESIIUE (R,)
Adjust pH to 9.5 Disperse in deionized water
Centrifuge lCDO x G for 30 min Adjust pH o 7.0
Dialyze
Lyophilize
(Enriched components 5 and 8)
' _ j
SUPERNATANT RESIDUE (RA)
Adjust pH to 11.5 Disperse in deionized water
Centrifuge 1000 x G for 30 min Adjust pH to 7.0
Dialyze
LyOphilize
(Enriched components 5 and 8)
I ”'——1

SUPERNATANT ($1)

Adjust pH to 7.0
Dialyze
Lyophilize

xESIDUE (a:

Disperse in deionized water
Adjust pH to 7.0

Dialyze

LyOphilize

Figure 3.--Procedure for the preparation of component 5,
adapted from the method described by Jenness (1959).

 

EKIMHILK
I

 

 

 

Cool to A C
fiiElnl gen' tube
Centrifuge 32,800 x G for 1A hours at A C
r” " i
SUPERNATANT RESIDUE (PL)
Adjust pH to A.6 Disperse micelles in milk dialysate t0
Centrifuge lOLU x G for 30 min original volume of skimmilk
stir at A C for 8—12 hours
Centrifuge 32,6C0 x G for 1A hours
(Whole casein micelles)
I j
SUPERNATAhT (3,) RESIDUE (all
Adjust pH to ,.0 Diaperse in deionised water
Dialyze Adjust rh to 7.0
Lyophilize Dialyze
(Whey proteins) Lyophilize
(Enriched s-casein)
r .
SUTEFVATANT RESIDUE
Adjust :1 to A.‘ Lispersc micelles in milk dialysate to
Centrlfugc lélf ( C fo* %’ r‘n original volume of skimmilk

sure harp”? (

 

I'—

nv. ~.-.,.nrfi,,.,fi
K, )4“ E
g‘uFLiuNiil .1..'.

Adjust ph to t

Centrifuge lluu x a r

 

 

for

E .‘17
A... k. LAA
/ I
.1 '-
‘\ .7.- 1
11',
r— _|
r gkww—I,VIA
...... Lt
. - 1
L1 yw
I131“? LAL
1.1311“:
Lyutu
v< -
(inrl

 

l

I

SUPERNATANT (Bu)
Adjust pH to 7
Dialyze
Lyophilize

.0

‘_"'1

Nci

,, .).

 

RESIDUE (FA)

9:;
Adjust

 

 

 

(Washed tasein micelles)

in deionized water

pH to A.6 ‘
Centrifuge 1000 x G for 30 min

V'\ I
'1 k‘ V“ 4‘6!“ "
L/ltj‘t.6., Gt?

Disperse in deionized water
Adjust pH to 7.0

Dialyze
Lyophilize
(Enriched B-casein)

 

i

Stir at A C for 8—12 hours
Centrifuge 32,600 x G for 1A hours
in url_ni.eq ’fittr
to 7 i
'.--C, 4 I
RESIDUE
Digger e mi;e;_ws in milk dialysate to
\ri‘ihal «with of Skimmilk
ilr a ” frr 5—12 hours
.,,..'~,l.,‘ .rq , 11,.
(entriilpr j.,CW- i G for 14 hours
\.
/
in lilChi; d hater
L‘IJ {.k‘
é—cacein)

 

I

SUPERNATANT
Adjust to
Dialyze

Lyophilize
(Components 5 and 8)

85)
H 7.0

(
P

Adjust pH to )
Dialyze
Lyophilize

 

RESIDUE (R7)

Disperse in deionized water

,7

.0

(Washed casein micelles precipitated at

pH A.6)

Figure A.-—Procedure for the preparation of the washed
casein micelles and the material released from micellar

casein at pH A.6.

69

FRESH SKIMMILK

sUPERNATANT

Adjust pH to A.6
Centrifuge 1000 x G
for 30 min

 

 

SUPERNATANT (SI)

Adjust pH to 7.0
Dialyze
Lyophilize

(Whey proteins)

 

1
RESIDUE (R1)

Disperse in deionized
water
Adjust pH to 7.0
Dialyze
Lyophilize
(Enriched B—casein)

Cool to A C

Centrifuge 32,600 x G for
1.5 hours to obtain No S
pellet

l
RESIDUE

Disperse in deionized water
(volume equal to twice
the amount of supernatant
removed from skimmilk)

Stir at A C for 2H hours to
suspend the micelles

Adjust pH to 4.6

Centrifuge 1000 x G for
30 min

 

 

r
SUPERNATANT

Adjust pH to 6.8
Centrifuge 1000 x G
for 30 min

RESIDUE (R2)

Disperse in deionized water
Adjust pH to 7.0
Dialyze
Lyophilize
(Casein micelles precipitated
at pH A.6)

 

 

SUPERNATANT (S2)

Adjust pH to 3.0
Continue same as for
residue (R3)
(Residual
whey proteins)

RESIDUE (R3)

Disperse in deionized water

Adjust pH to 3.0

Dialyze at A C against 3
changes of deionized water
adjusted to pH 3.0

Adjust pH to 7.0

Dialyze at A C against 3
changes of deionized water

Lyophilize

(Enriched component 5 and 8)

Figure 5.--Procedure for the preparation of casein micelles
and the material released from micellar casein at pH 4.6.

7O

FRESH SKIMMILK

 

Heat to 96—100 C for 20-30 min

Cool to 20 C

Adjust pH to 4.6

Filter with E & D No. 512
filter paper

 

l
SUPERNATANT

Adjust pH to A.6

Make to 35% satr with
(NH ) SO

Let s agd 0 min

Centrifuge 1000 x G
for 30 min

 

_j
RESIDUE
(Discard)

 

SUPERNATANT

Adjust pH to A.6

Make to 55% satr with
(NHA) SO,4

Let stagd 30 min

Centrifuge 1000 x G
for 30 min

 

SUPERNATANT

Adjust pH to A.6
Make to 80% satr with

l
35% RESIDUE

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Lyophilize

(Enriched component 5)

J

I
55% RESIDUE

Disperse in deionized water
Adjust pH to 7.0

 

 

(NH ) SO“ Dialyze
Let stadd 30 min Lyophilize
Centrifuge 1000 x G (Enriched component 3)
for 30 min
j
SUPERNATANT 80% RESIDUE
(Discard)

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Lyophilize

(Enriched component 8)

Figure 6.——Procedure for the preparation of proteose—
peptone and its fractionation with ammonium sulfate.

71

CASEIN

 

Dissolve 2 g in 100 ml of
deionized water

Adjust pH to 7.0

Dissolve approximately 1 g
of di-sodium EDTA

Adjust pH to A.9

Centrifuge 1000 x G for 30 min

 

r
SUPERNATANT

Adjust pH to A.1
Centrifuge 1000 X G
for 30 min

,1
RESIDUE (R1)

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Iyophilize

(Casein)

 

 

SUPERNATANT (Sl)

Adjust pH to 7.0

Dialyze

Lyophilize

(EDTA-released components)
(Enriched component 8)

j
RESIDUE (R2)

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Lyophilize

(Casein)

Figure 7.-—Procedure for the isolation of EDTA-released
components from isoelectric precipitated casein.

72

CASEIN

Dissolve 10 g casein in 1000
ml of deionized water at pH
6 8

Add 10 g sodium chloride
Heat to 96-100 C for 30 min
Cool to 20 C

Adjust pH to 4.3

Centrifuge 1000 x G for

 

 

30 min
gUPERNATANT RESIDUE
Adjust pH to 7.0 Disperse in deionized water
Dialyze Adjust pH to 7.0
Lyophilize Dialyze
(Heat released material) Lyophilize
(Enriched component 8) (Casein)

Figure 8.--Isolation of heat—released material from iso-
electric precipitated casein.

73

 

CASEIN

Make a 1.5% solution of casein
Adjust pH to 7.0

Make to 10% satr with (NHA)2SOA
Adjust pH to A.6
Centrifuge 1000 x G for 30 min

 

 

I
SUPERNATANT

Adjust pH to 7.0
Make to 25% satr with

1
RESIDUE (R1)

Disperse in deionized water
Adjust pH to 7.0

 

(NHu) SO Dialyze
Adjust SH 80 A.6 LyOphilize
Centrifuge 1000 x G (Casein)
for 30 min
SUPERNATANT RESIDUE (R2)

Adjust pH to 7.0
Make to 55% satr with

(NH ) SO
Adjust EH 80 A.6

Centrifuge 1000 x G
for 30 min

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Lyophilize

(Casein)

 

 

SUPERNATANT (Si)

Adjust pH to 7.0

Dialyze

Lyophilize
(Ammonium sulfate
released material)

_j
RESIDUE (R3)

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Lyophilize

(Enriched component 8)

Figure 9.--Procedure for the isolation of the ammonium
sulfate-released fraction from isoelectric precipitated
casein.

7A

PROTEOSE—PEPTONE

 

Dissolve 0.5 g proteose—
peptone_per 50 ml of
deionized water

Dissolve 0.37 g KCl (.lM)

Cool to O-A C

Conduct all operations at O-A C

Add 35 ml of 95% ethanol with
constant stirring

Centrifuge 12,000 x G for 30 min

 

sUPERNATANT
Add 55 ml of 95% ethanol

I
RESIDUE (R1)
Disperse in deionized water

 

 

with constant stirring Dialyze
Allow to stand overnight LyOphilize-
Centrifuge 12,000 x G (Enriched component 3)
for 10 min
I
SUPERNATANT

Add A0 ml of 95% ethanol

 

RESIDUE (R2)
Disperse in deionized water

 

with constant stirring Dialyze
Centrifuge 12,000 x G Lyophilize
for 10 min (Enriched components
3 and 8)
I
SUPERNATANT RESIDUE (R3)

Add A0 ml of 95% ethanol
with constant stirring

Centrifuge 12,000 x G
for 10 min

 

Disperse in deionized water
Dialyze
Lyophilize
(Enriched components
5 and 8)

 

SUPERNATANT (81)

Add AO ml of 95% ethanol
with constant stirring

Precipitate not formed

Dialyze

Pervaporate

Lyophilize

(Components 5 and 8)

l
RESIDUE (RA)

Disperse in deionized water
Dialyze

Lyophilize

(Enriched component 5)

Figure lO.--Schematic for the fractionation of proteose-

peptone with ethanol.

75'

FRESH SKIEMILK

Adjust pH to A.6

Centrifuge or filter to obtain clear supernatant

If not clear, filter with Celite filter aid and
Buchner funnel

 

r fl 1

SUPERNATANT (Si) SUPERNATANT (81) RESIDUE

Adjust pH to 6.8 ‘ Adjust pH to 7.0 ‘ (Casein)

Centrifuge 1000 x G heat 96—100 C for 30 min (Discard)
for 30 min Adjust pH to A.6

Centrifuge 1000 x G for 30 min

 

f l
SUPERNATANT (Sq) RESIDUE
Adjust pH to 3.0 (Discard)
Treat same as for
residue (R )
(Proteose—peptone)

r.___ll

 

 

v

0-

 

 

 

 

 

sUPEENATANT RESIDUE (R1)
! Adjust pH to 8.0 Disperse in deionized water
} Centrifuge 1000 x G for 30 min Adjust pH to 3.0
| Dialyze at A C against 3 or A changes of
‘ deionized water adjusted to pH 3.0
Adjust pH to 7.0
Dialyze at A C against 3 or A changes of
deionized water
| Lyophilize
L (Enriched components 3, 5 and 8)
ECPEHNATANT RESIDUE (R2)
l
I Adjust pH to 10.0 Treat same as for residue (. )
Centrifuge 1000 x G for 30 min (Enriched components 3, 5 and 8)
.__ ____.l_l i-l .-W,,,, _l_ l_-l.-._"ll_isl_r ._l 1
SUPERNATANT RESIDUE (R3)
Adjust pH to A.6 Treat same as for residue (R )
Centrifuge 1000 x G for 30 min (Enriched components 3, 5 and 8)
I """‘ "" ‘“‘ “ ’ ' ’ ' ‘ ‘ ”“‘ “ """ I
SUPERNATANT (S3) ' RESIDUE (RA)
Adjust pH to 3.0 Disperse in deionized water
Treat same as for residue (R1) Adjust pH to 7.0
(Whey proteins) Dialyze
Lyophilize

(Enriched B—lactoglobulin)

SUPERNATANT (s3)

Adjust pH to 6.8

Heat 96-100 C for 30 min

Cool to 20 C

Adjust pH to A.6

Centrifuge 1000 x G for 30 min

 

 

l
SUPERNATANT (SQ) RESIDUE
Adjust pH to 7.0 (Discard)
Dialyze
Lyophilize

(Enriched component 3 and B-lactoglobulin)

Figure ll.-—Procedure for the preparation of a fraction
rich in proteose-peptone from unheated skimmilk and the
preparation of additional fractions by heating.

76

PROTEOSE-PEPTONE-RICH FRACTION FROM UNHEATED SKIMMILK
(Residue, H1, in Figure 11)

Dissolve 2.2 g in AOO ml of deionized water
Make to satr with sodium chloride

Adjust pH to A. A

Centrifuge 12 ,000 x G for 15 min

 

I 1

SUPERNATANT SUPERNATAIIT (10 ml) RESIDUE (R1)
Adjust pH to 7.0 Adjust pH to 3.5 Disperse in deionized water
Dialyze out salt Remove precipitate Adjust pH to 7.0
Pervaporate by centrifuging Dialyze
Final volume = 160 ml Lyophilize
Make to A5 % satr with (Enriched component 5)

(NH )
Fina 2pHu= 6.0
Centrifuge 12,000 x G
for l5 min

 

 

 

 

 

 

 

l
SUPERNATANT RESIDUE (R2)
Dialyze Disperse in deionized water
Lyophilize Adjust pH to 7.0
Not enough for Dialyze
electrophoretic Lyophilize
analysis
I
SUPERNATANT RESIDUE (R3)
Hake to 65% satr with ( ‘HA )2SOu Treat same as for residue (H )
Adjust pH to 7. 0 (Component 3 and B—lactoglobulin)
Centrifuge 12,000 x G for 15 min
ij
SUPERNATANT RESIDUE (RA )
Make to 80% satr with (NHu)2SOu Treat same as for residue (H1)
Adjust pH to 7. O
Centrifuge 12, 000 x G for 15 min
|
SUPERNATANT (Sl) RESIDUE (R5)
Dialyse Treat same as for residue (R1)
Pervaporate (Enriched component 8)
Lyophilize

(Component 8)

Figure 12.~—Schematic for the fractionation (sodium
chloride and ammonium sulfate) of a proteose—peptone—
rich fraction prepared from unheated skimmilk.

77

PROTEOSE—PEPTONE FROM HEATED SKIMMILK

 

Dissolve 1.2 g in
deionized water
Adjust pH to 7.0
Make to satr with
Adjust pH to A.A
Centrifuge 12,000

200 ml of

sodium chloride

x G for 15 min

 

I
SUPERNATANT

Adjust pH to 3.1
Centrifuge 12,000 x G
for 15 min

 

I

RESIDUE (R1)

Disperse in deionized water
Adjust pH to 7.0

Dialyze

Lyophilize

(Enriched component 5)

 

SUPERNATANT (Sl)
Adjust pH to 7.0

1
RESIDUE
Disperse in deionized water

 

 

Dialyze Adjust pH to 6.7
Lyophilize Make to A5% satr with
(NHA) 803%“
Final pEi 6.2
Centrifuges 12,000 x G for 15 min
SUPERNATANT RESIDUE (R2 )
Make to 65% satr with Treat same as for residue (R1)
(NHu)2 SO“ (Enriched component 3)

Adjust pH to 7. O
Centrifuge 12, 000 x G
for 15 min

 

 

SUPERNATANT

Make to 80% satr with
(NH )2SOA

Adjusg pH to 7. O

Centrifuge 12,000 x G for
15 min

1
RESIDUE (R3)

Treat same as for residue (R

)
(Enriched component 3) l

 

 

SUPERNATANT (s2 )

Dialyze free 2of (NHu)2 SOLl
Pervaporate

Lyophilize

(Component 8)

l
RESIDUE (RA)

Treat same as for residue (R1)
(Enriched component 8)

Figure 13.--Schematic for the fractionation (sodium chloride

and ammonium sulfate)
heated skimmilk.

of proteose-peptone prepared from

78'

PROTEOSE-PEPTONE-RICH FRACTION FROM UNHEATED SKIMMILK
(Residue, R in Figure 11)

 

 

 

 

l,
Dissolve 0.5 g in 100 ml of
deionized water
Adjust pH to 7.0
Make to 25% satr with (NHu)2SOu
Adjust pH to A.9
Centrifuge 12,000 x G for 15 min
I T
SUPERNATANT RESIDUE (R1)
Make to 37% satr with Disperse in deionized water
(NHu)2SOu Adjust pH to 7.0
Adjust pH to A. 9 Dialyze
Centrifuge 12,000 x G LyOphilize
15 min (Enriched component 5)
11
SUPERNATANT RESIDUE (R2 )
Make to 55% satr with Treat same as for residue (R1)
(NH ) 2“SO (Enriched components
Adjusg2 pH to A. 9 3 and 5)
Centrifuge 12 ,000 x G for
15 min
11
SUPERNATANT RESIDUE (R3 )
Make to 65% satr with Treat same as for residue (R1)

(NH )2s0

Adjusg pH“ to A. 9

Centrifuge 12, 000 x G
for 15 min

(Enriched components
3 and 8)

 

SUPERNATANT

Make to 80% satr with

(NH )ZSOA

Adjusfi pH to A. 9

Centrifuge 12, 000 x G
for 15 min

I
RESIDUE (RA )

~Treat same as for residue (R1)

(Component 8 and whey
proteins)

 

 

SUPERNATANT (81)

Adjust pH to 7.0
Pervapoate
Dialyze
Lyophilize

1
RESIDUE (R5 )

Treat same as for residue (R1)
(Whey proteins)

Figure lA.—-Procedure for the ammonium sulfate fractionation
at pH A.9 of a proteose—peptone-rich fraction prepared from

unheated skimmilk.

"I, 9

PROTEOSE-PEPTONE-RICH FRACTION FROM UNHEATED SKIMMILK
(Residue, R1, in Figure 11)

Dissolve 0.5 g in 100 m1 of
deionized water

Adjust pH to 7.0

Make to 52% satr with (NHu)2SOu

Centrifute 12,000 x G

 

 

 

 

 

 

for 15 min
If 1
SUPERNTANT RESIDUE (R1)
Make to 65% satr with Disperse in deionized water
(NH )Zsou Dialyze
Adjusé pH to 7.0 Lyophilize
Centrifuge 12,000 x G (Enriched component 3)
15 min
1
SUPERNATANT RESIDUE (R2).
Make to 80% satr with Treat same as for residue (R1)
(NHu)2SOu (Enriched components
Adjust pH to 7.0 3 and 5)
Centrifuge 12,000 x G
for 15 min
” I
SUPERNATANT (Sl) RESIDUE (R3)
Dialyze free of Treat same as for residue (R1)
(NH ) SO (Enriched whey proteins)
Pervapo ate
Lyophilize

(Enriched component 8)

Figure 15.——Procedure for the ammonium sulfate fractionation
at pH 7.0 of a fraction rich in proteose—peptone prepared
from unheated skimmilk.

80

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81

 

 

 

 

 

 

 

 

 

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RESULTS AND DISCUSSION

Preparative Procedures

 

Exploratory Procedures for the Preparation
and Fractionation of Proteose-Peptone
from Heated and Unheated Skimmilk

Larson and Rolleri (1955) assessed the effect of heat
on the whey proteins in milk by analysis with free-boundary
electrophoresis. However, an analysis of the proteose-
peptone or the sigma-proteose fraction with the more recent
techniques of gel electrophoresis has not been reported.
Because of the superior resolving power of the gel
electrophoretic technique, there existed the possibility
that more than three components could be detected in the
proteose—peptone fraction.

In Figure 19, column A, the starch-urea gel electro—
phoretic patterns show the similarity between proteose-
peptone and sigma proteose. In the proteose—peptone pattern,
component 5 separated into two zones. The sigma-proteose
supernatant contained an increased amount of the electro-
phoretic frontal material (component 8) and decreased
amounts of components 3 and 5. Component 5 was again
separated into two zones.

Figure 23 shows a starch-urea gel electrophoretic

pattern of component 8 purified by electrophoretic resolution

8A

85

resolution in preparative-size acrylamide gels. Component
8 migrated with the electrophoretic frontal zone in starch-
urea gel electrophoresis as well as in acrylamide gels from
which it was isolated. A free-boundary electrOphoretic
pattern of this material is shown in Figure 25, row B. The
top pattern (A) shows the separation of proteose-peptone.
In this pattern, component 8 had a mobility of -7.8

Tiselius units (= l x 10"5 cm2 volt—l sec-l).

Component 8
isolated by acrylamide gel electrophoresis is shown in the
middle pattern (B) and its mobility was -8.A Tiselius units.
The lower pattern (C) showed an increase in the area of the
peak attributed to component 8 when proteose—peptone and
component 8 were mixed in equal amounts. In this system,
the component—8 peak decreased in mobility to -7.8 Tiselius
units——equal to its mobility in the proteose-peptone fraction.
This implicates the electrophoretic frontal material as
component 8. The increased mobility of purified component 8
was due, possibly, to the absence of interaction between
component 8 and other proteins in the proteose-peptone
fraction.

Again in Figure 25, row D, slot 1 shows the starch-
urea gel electrOphoretic pattern of component 8 isolated
from the ascending channel of the Tiselius cell. Again,
component 8 migrated in the electrophoretic frontal zone.

In Figure 19, column B, the electrophoretic patterns
indicate the presence of material similar to components 5

and 8 as shown in column A for proteose-peptone. These

86

fractions were obtained from unheated skimmilk by a pro-
cedure (Figure 3) similar to the method described by Jenness
(1959). In these starch—urea gel patterns, component 5
separated into two zones.

Figure 20 shows the starch-urea gel patterns of
fractions obtained in the preparation of washed casein
micelles (Figure A). Slots l—A show the soluble proteins
remaining in the A0 S supernatant after acidification to
pH A.6. These patterns indicate that nearly all of the
whey proteins are removed after the first washing of the
casein micelles. Slots 5-8 are the electrophoretograms of
the soluble casein removed at pH A.6 from the A0 S super-
natant. These fractions contain a very high percentage of
B-casein, thus indicating the removal of B-casein from the
micelles upon washing with milk dialysate at O-A C. The
starch-urea gel electrophoretic pattern of the casein micelles
before washing are shown in slot 9; while slot 10 shows the
casein micelles following the washing treatment. Accordingly,
the gel patterns of the washed casein micelles show that B-
casein was removed during the washing process.

In a different preparation, slot 11 shows the pattern
of washed casein micelles. Slot 12 shows the same washed
casein micelles after adjusting the pH to A.6; while slot 13
shows the proteins released into the supernatant. These
proteins, as shown by the electrophoretic patterns, migrate
similar to components 5 and 8 in the proteose-peptone

fraction.

87

Similarly, components 5 and 8 were released from a
preparation of unwashed micellar casein (Figure 5) when
adjusted to pH A.6. However, an additional array of zones
(Figure 21) was observed in the gel patterns which pre-
sumably were whey proteins carried into the intermicellar
spaces of the casein pellet.

Component 3 was noticeably absent from the protein
fraction released from the washed casein micelles upon I
acidification to pH A.6. Since component 3 was present in
the proteose-peptone from heated skimmilk, one would surmise
that component 3 exists only in the serum. These results
agree with those observed by Ng (1967).

The preparation and fractionation of proteose-peptone
with ammonium sulfate as outlined in Figure 6 gave enriched
fractions of components 3, 5, and 8. Most of component 5
was precipitated when the solution was made to 35% satura—
tion with ammonium sulfate. Component 3 was precipitated
at 55% saturation. Thus, the supernatant contained compon—
ent 8 which was precipitated with ammonium sulfate at 80%
saturation. ElectrOphoretic patterns of these fractions
are shown in Figure 22.

Sigma proteose at a concentration of 2% was not pre-
cipitated at pH 7.0 or A.6 in the presence of calcium ions
(0.5 M). Also, upon the addition of ethanol to A7.5%,

there was no change in the appearance of the solution.

88

The electrophoretic frontal material (component 8) as
detected by starch-urea gel electrophoresis was released
from isoelectric precipitated casein when treated as out-
lined in Figures 7, 8, and 9. The starch—urea gel electro-
phoretic patterns in Figure 2A show that component 8 was
released from whole casein by treatment with EDTA by heating
and by the addition of ammonium sulfate. One could conclude
that component 8 was released from the casein by acidifying
to pH A.6 since component 8 was shown to be released from
casein micelles at pH A.6.

The fractions obtained by the fractionation of
proteose—peptone with ethanol were analyzed by starch-urea
gel electrOphoresis and the patterns are shown in Figure 29.
Most of component 3 was precipitated with A0 and 60% ethanol
as shown by slots 5 and A, respectively. Component 5 was
soluble in 60% ethanol and component 8 was distributed
throughout the fractions. Component 5 could possibly have
been a portion of the fraction Osborne and Wakeman (1918b)
isolated from casein and also from the filtrate obtained
after the boiling of acid whey. Their protein fraction was
soluble in 50-70% alcohol.

In the preparation of a proteose—peptone-rich fraction
from unheated skimmilk as outlined in Figure 11, a pre-
cipitate R1 was obtained which contained increased concen-
trations of components 3, 5, and 8 in addition to some of

the other whey proteins. Residues R1, R2, and R3 obtained

89

at pH 6.8, 8.0, and 10.0, respectively, were similar
according to the zonal patterns obtained on starch-urea
gels. An aliquot of supernatant S1 was heated to denature
the heat labile proteins. These proteins were removed by
centrifugation. The resulting supernatant 82 was analyzed
by gel electrophoresis and the pattern was characteristic
of the proteose-peptone prepared as described in Figure 1.
Thus, the residue R1 was considered to be a concentrated
fraction of components 3, 5, and 8 which served as the
starting material in the preparation of component 8 from
unheated skimmilk. Starch-urea gel electrophoretic patterns
of these fractions are shown in Figure 30.

The starch—urea gel electrophoretic patterns of the
fractions obtained from the fractionation of a proteose-
peptone—rich fraction from unheated skimmilk by the addition
of sodium chloride and ammonium sulfate are shown in Figure
31. The first fraction, precipitated with saturated sodium
chloride, was enriched in component 5. It was separated into
two zones by gel electrophoresis. The final supernatant

S an 80%—saturated solution of ammonium sulfate, was

1’
highly enriched in component 8.

Figure 32 shows the starch-urea gel electrOphoretic
patterns of the fractions obtained from the fractionation
of proteose—peptone from heated skimmilk by the addition
of sodium chloride and ammonium sulfate. According to

these patterns and the patterns shown in Figure 31, the

proteose-peptone-rich fraction from unheated and heated

9O

skimmilk can be fractionated in the same manner. Component
8 was concentrated in both of the final supernatants. The
fractions obtained from the proteose-peptone-rich fraction
isolated from unheated skimmilk contained some of the heat
labile whey proteins as contaminates.

In the fractionation of the proteose-peptone-rich
fraction from unheated skimmilk with ammonium sulfate at
pH A.9, the first two residues collected showed enrichment
in component 5. The isolation procedure is outlined in
Figure 1A and the corresponding starch-urea gel electro-
phoretic patterns are shown in Figure 33. The remaining
fractions collected contained components 3 and 8 and some
of the whey proteins.

The procedure for fractionating the heated-skimmilk
proteose-peptone with ammonium sulfate at pH 7.0 is shown
in Figure 15. Corresponding starch—urea gel electrophoretic
patterns of these fractions are shown in Figure 3A.
Components 3 and 5 were present in residues R and R and

l 2

the final supernatant S was rich in component 8. Thus,

1
component 5 was the first proteose-peptone component to
precipitate in the salting—out procedure. In general,
component 8 was not salted—out unless high concentrations

of ammonium sulfate in excess of 60% saturation were

employed.

91

Optimized Procedures for the Fractionation
of Proteose—Peptone from Heated
and Unheated Skimmilk

By combining the procedures outlined in Figures 1A
and 15, an optimized procedure, Figure 16, was deveIOped
for the separation of components 3, 5, and 8. In this
manner, component 8 was isolated in a highly purified
state. The starch-urea gel and acrylamide gel electro—
phoretic patterns of these fractions are shown in Figure
35. Component 5 was concentrated in the ammonium sulfate
(25% saturation) salted-out residue while the 50% residue
was enriched in component 3. The 80%—saturated super-
natant contained component 8. This procedure was success-
fully employed for the fractionation of proteose-peptone-
rich fractions originating from both unheated and heated
skimmilk.

Component 5, in relatively purified state, was isolated
from the 25% residue (Figure 16) or proteose—peptone
(Figure 1) according to the schematic shown in Figure 17.
Figure 36 shows the acrylamide gel electrOphoretic patterns
of residues R1, R2, R3,

composed essentially of component 5. The chemical and

and RA' These four residues were

physical characterizations reported herein were performed
on a composite sample of these residues.

Sigma-proteose supernatant and component 8 (Figure
16) were analyzed by acrylamide gel electrophoresis employ-
ing a continuous ion buffer system. In this system,

component 8 separated into two components as shown in

92

Figure 26, slot 1. The faster migrating component was
named component 8-fast while the slow component was called
8-slow. In slot 2, the acrylamide gel patterns show the
presence of component 8-fast and component 8-slow in the
sigma-proteose supernatant fraction. Figure 26 also shows
an electrophoretic comparison of proteose-peptone in starch—
urea gel employing a discontinuous buffer system and
acrylamide gel employing a continuous buffer system.
Further evidence of the presence of components 8-fast and
8-slow in sigma proteose is shown by the two—dimensional
gel electrophoretic pattern in Figure 27. From this pattern,
one can see that the electrophoretic frontal band observed
in discontinuous buffers contains both components 8-fast
and 8—slow.

When urea was incorporated into acrylamide gels, some
of the protein zones were changed and became diffuse. The
zones ascribed to components 3 and 5 appeared to dissociate
and migrate in different positions, see Figure 28.

Acrylamide gel patterns of the five fractions col-
lected from the Bio-Gel P—lO column are shown in Figure 37.
The first two fractions contained component 8-slow and the
last fraction contained component 8-fast. The third and
fourth fractions contained a mixture of component 8—fast
and 8-slow. These two fractions were rechromatographed to
further separate the components. Acrylamide gel electro-
phoresis was used to monitor the fractions collected from

the Bio-Gel P—10 column.

93

Vertical, acrylamide gel electrophoretic patterns of
the protein fractions which were analyzed in this study are
shown in Figure 38. Obviously, these fractions are not

pure proteins but represent highly-purified preparations.

Chemical Composition

 

The chemical composition of components 8 purified
by acrylamide gel electrophoresis, 8-fast, 8—slow and 5
are shown in Table 2. The nitrogen value obtained for
component 8 was similar to that reported by Brunner and
Thompson (1961) for proteose—peptone and sigma proteose.
There were differences in the phOSphorus and carbohydrate
contents of component 8 and the similar values reported
for proteose—peptone and sigma proteose. This is to be
expected because the preparation of component 8 analyzed
here represented a purified fraction obtained from the
gross proteose-peptone fraction. The composition of com-
ponent 8 does not compare favorably with components 8-fast
and 8—slow. This could result from contamination or the
loss of carbohydrates and phosphorus during purification
by gel electrophoresis.

The nitrogen values of component 8-fast prepared from
heated and unheated skimmilk were similar. In the case
of component 5, the nitrogen value (13.83%) was similar to
the value (13.6%) reported by Bezkorovainy (1965) for the

milk whey phosphoglycoprotein.

9A

The phosphorus contents of components 8-fast and 8-
slow are high when compared with the phosphorus content
(0.5%) of component 3, another component of the proteose-
peptone fraction (Ng, 1967). Component 3 approximates the
phosphorus value (0.AA%) reported for the milk whey
phosphoglycoprotein (Bezkorovainy, 1965). The phosphorus
content of component 5 was 0.97%. In general, components
prepared from heated skimmilk had a lower phosphorus content
than those prepared from unheated skimmilk. Some of the
phosphorus could have been cleaved from the protein during
heating. Or, the unheated preparations may have had a high—
phosphorus contaminate not present in the heated prepara-
tions; and, conversely, the heated preparations may have
complexed with a low phosphorus protein contaminate.

The hexose contents of the heated preparations of
components 8-fast and 8-slow were higher than the hexose
contents of the unheated preparations. In the case of
hexosamine, the unheated preparation of component 8-fast
contained more hexosamine than the heated preparation. The
opposite was true for component 8—slow.

Sialic acid contents of component 8-fast from heated
and unheated skimmilk were approximately similar. In the
case of component 8—slow, the preparation obtained from
heated skimmilk had a higher sialic acid content than the
preparation prepared from unheated skimmilk. These dif-

ferences reflect heterogeneity between the preparations.

95

The carbohydrate content (l.A5%) of component 5 was
the lowest of any of the isolated components. The carbo-
hydrate content (10.27%) of component 8-slow from heated
skimmilk was the highest. This value was close to the
value (9.5%) reported by Bezkorovainy (1965) for the milk
whey phosphoglycoprotein. However, the carbohydrate con-
tent was not as high as that reported by Ng (1967) for
component 3 (17.3%) or by Alais and Jollés (1961) for the
caseino-glyCOpeptide (28.1%) released from kappa—casein
by treatment with rennin.

Paper chromatograms of the carbohydrates released
from components 8-fast, 8—slow and 5 are shown in Figure A2.
Component 8-slow contained glucosamine, galactose, glucose,
mannose, and fucose. Component 8-fast from heated skimmilk
contained galactose, glucose, and mannose. The component 5
fraction contained galactose, mannose and galactosamine.
However, the chromatogram obtained from the amino acid
analyzer on the 20-hour hydrolysate showed that component 5
contained more glucosamine than galactosamine. Again, the
chromatogram from the amino acid analyzer showed that com—
ponent 8—fast contained only traces of galactosamine and
glucosamine; while component 8-slow prepared from unheated
skimmilk contained more galactosamine than glucosamine.
Galactosamine and glucosamine were detected for component 8-
fast by the amino acid analyzer but not by paper chromato-

graphy. The indication of glucose could possibly be due to

96

an ion effect, since glucose has not been reported in
glycoproteins previously.

The carbohydrate-containing proteins were detected in
acrylamide gels with the periodic acid—Schiff technique
reported by Keyser (196A). A comparison of acrylamide gel
electrophoretic patterns stained with amido black and
developed with periodic acid-Schiff reagents is shown in
Figure Al. As seen in these patterns, the 50% residue
(component 3) was heavily colored whereas the 25% residue
(component 5) was only lightly colored. Component 8-slow,
slot 1, contained one dark band when stained with amido
black. However, when deveIOped with periodic acid-Schiff
reagents, a slower migrating zone was detected which appeared
to contain a high content of carbohydrate. Thus, some
carbohydrate-containing proteins are not stained with amido
black but are detected with the periodic acid-Schiff
reagents. The active sites which react with amido black
could‘Mainaccessible to the stain, but the carbohydrate of
the protein could react with the periodic—Schiff reagents.

The amino acid compositions of component 8—fast from
heated and unheated skimmilk are shown in Table 3. These
values were based on nitrogen values of 13.33% and 13.16%
for component 8—fast prepared from heated and unheated
skimmilk, respectively. The values for serine, threonine,
and tyrosine were corrected for destruction during hydroly—
sis according to the equation given by Hirs, Stein, and

Moore (195A):

97

log A0 = E__—- log A

-t log A

i‘t-t 2

where Al, A2, and A

t

O are the quantities of amino acids

after t 2, and t0 hours of hydrolysis, respectively.

1’
Actually, they estimated that about 5% of the threonine

and tyrosine and about 10% of the serine were lost in 20
hours of hydrolysis.

Tryptophan was determined independently by the method
of Spies and Chambers (19A8, 19A9). Neither cysteine nor
any of its hydrolytic products was detected with the amino
acid analyzer. Also, free sulfhydryl groups were not
detected with Ellman's reagent.

The amino acids were reported as g amino acid residues
per 100 g protein and in number of amino acid residues per
1000 residues. The g residues per 100 g protein is actually
the weight percentage of the residue. In Figure 39, amino
acid profiles of component 8—fast from heated and unheated
skimmilk indicate the similarities of their amino acid
distribution. One characteristic of component 8—fast was
that it contained approximately 25% glutamic acid as shown
in Table 3.

Using the compositional values obtained for amino
acids and carbohydrates, the apparent partial specific
volume of component 8-fast (unheated preparation) was

calculated to be 0.719 cc/g.

98

The amino acid composition of component 8-slow from
heated and unheated skimmilk is shown in Table A. These
values are based on the nitrogen content as determined by
micro-Kjeldahl and corrected for the destruction of serine,
threonine, and tyrosine as described earlier. As shown in
Table A and by the amino acid profiles in Figure A0, the
two components are quite similar. Component 8-slow was
high in glutamic acid and aspartic acid contents. However,
the amount of these amino acids which exist as the amide was
not determined. Similarities in amino acid compositions
appear to exist between component 8-slow and the whey
phosphoglycoprotein reported by Bezkorovainy (1965);
especially with respect to the residues of glutamic acid,
aspartic acid, serine, threonine, glycine, and cysteine.
However, their respective phosphorus contents were not at
all in agreement. From the amino acid and carbohydrate
composition of component 8—slow, the apparent partial
Specific volume was calculated to be 0.700 cc/g.

The amino acid composition of component 5 isolated
from heated skimmilk is shown in Table 5. These values are
expressed as described for the previous proteins. Both
component 5 and component 8—fast contain approximately 25%
glutamic acid; while the caseins contain from 22 to 23%. Of
interest is the observation that whole casein, d-casein,
B-casein, and y—casein contain 11.3, 8.2, 16.0, and 17.0%
PPOJine, respectively; whereas, the whey proteins,

B-iéactoglobulin and d—lactalbumin contain only A.1 and 1.5%

99

proline, respectively (Jenness and Patton, 1959). Com-
ponent 5 was found to contain 10.55% proline. The whey
phosphoglycoprotein contains 9.0% proline (Bezkorovainy,
1965). Components 8-fast (heated preparation) and 8-slow
(heated preparation) contain 3.67 and 3.1A% proline,
respectively. Like the caseins, component 5 precipitates
from a solution made to saturation with sodium chloride
and appears as a constituent in casein micelles. These
similarities in composition and characteristics lend support
to the conjecture that component 5 should, indeed, be
classified as a casein.

The apparent partial specific volume (0.736 cc/g)
of component 5 was calculated from its amino acid and carbo-
hydrate composition.

Based on the limiting amino acids--histidine, arginine,
methionine, and tyrosine--a minimum molecular weight of
about 7,300 was calculated for component 5.

In general, the amino acid composition of proteose-
peptone reported by Ganguli §t_§g, (1965) was not similar
in composition to component 8—fast, component 8—slow, or
component 5 reported in this study or to the composition of
component 3 as reported by Ng (1967). If proteose-peptone
contained 5.0% cysteine, A.9A% methionine, and A.2l%
tryptophan as reported by Ganguli e£_a1. (1965), then com-
ponent 3, component 5, component 8-slow, or component 8—
fast would be expected to contain a relatively high percent—

age of these amino acids. But, actually, the reverse

100

situation exists. The amino acid analysis reported by

Ganguli gt_§1. (1965) was conducted on a proteose—peptone
fraction which quite possibly contained a large amount of
the heat-labile whey proteins notoriously rich in sulfur-

bearing amino acids.
Physical Properties

Electrophoretic Properties

Free—boundary electrophoretic analyses of component
8 purified by acrylamide gel electrophoresis were performed
in buffers covering a pH range of 2.0 to 8.5 at an ionic
strength of 0.2. The results are shown in Table 6. At pH
8.5, the large, single peak began to separate into one
large peak and one small peak, possibly, 8-fast and 8-slow.
At the other values of pH, only one large peak was observed.
From the plot of the average ascending and descending
electrophoretic mobilities versus the corresponding pH, an
isoelectric point of pH 3.25 was determined at the point of
zero mobility, see Figure A3. The electrophoretic mobility
of component 8 was -8.A Tiselius units in veronal buffer
at pH 8.6, I = 0.1. However, when in the presence of com-
ponents 3 and 5, its mobility was -7.8 Tiselius units.
This value agrees with that (-7.9 Tiselius units) reported
by Larson and Rolleri (1955). The lower value results, no
doubt, from the interaction of component 8 with other

proteins in the mixture.

101

Free—boundary electrophoretic analyses of component
8-fast, component 8-slow, and component 5 were performed
in veronal buffer at pH 8.6, I = 0.1. The results are
shown in Table 7 and the electrophoretic patterns are
shown in Figure AA. The mobility of the ascending channel
varied greatly from the descending channel. This disparity
reflects the absence of ionic equilibrium between the
solution and the buffer. Dialysis was restricted to mini—
mize the loss of the low—molecular weight protein from the
dialysis sac.

The average electrophoretic mobilities for component
8—fast from heated and from unheated skimmilk were -9.00
and -9.31 Tiselius units, respectively. Corresponding
mobilities for preparations of component 8-slow were —9.15
and -9.21 Tiselius units, respectively. These mobilities
are about one Tiselius unit higher than that reported by
Larson and Rolleri (1955) for component 8.

The average mobility for the component 5 studied here
was —A.80 Tiselius units. Larson and Rolleri (1955)
obtained a value of -A.5 Tiselius units for component 5 in
the descending channel of the Tiselius cell. Likewise,
Jenness (1959) reported an electrophoretic mobility of —A.5
Tiselius units in veronal buffer at pH 8.6, I = 0.1, for
purified component 5.

When component 8—fast (unheated skimmilk) was added
to 0.1 ionic strength veronal buffer in preparation for

free-boundary electrophoresis, interesting observations

102

were encountered. The protein first appeared to go into
solution during stirring; then, 1-2 hours later, it
appeared to come out of solution in the form of a dense
precipitate. Because of its unusual appearance, the
precipitate was examined under the microscope which
revealed the presence of irregular rhombic crystals, see
Figure 5A. Crystals of sodium veronal formed by evapora-
tion of the buffer were needle-like and in no way resembled
the "protein" crystals. Presumably, the crystals were
entirely protein or a combination of protein and sodium
veronal.

Because the formation of crystals was unexpected, a
second attempt to crystallize the protein was made. Twenty
milligrams of the dried protein were weighed into a small
vial and one milliliter of veronal buffer was added. The
vial was rotated gently to allow the protein to dissolve.
The solution was placed at O-A C overnight. The next
morning, crystals had formed at the bottom of the vial.

A fraction of a drop of crystal violet was added to
a drop of the suspension of crystals and observed under
the micrOSCOpe. The crystals appeared to take up the dye,
thus indicating they were protein in nature (Sumner and

Somers, 19A9).

Ultracentrifugal Properties

Sedimentation coefficient.-—The sedimentation co-

 

efficients for component 8—fast (unheated skimmilk) at

103

various protein concentrations are shown in Table 8.

Veronal buffer at pH 8.6, I = 0.1 was used as the solvent.

A plot of sedimentation coefficients and protein concentra—
tions is shown in Figure A5. Corresponding sedimentation—
velocity patterns are shown in Figure 51. The sedimentation
coefficient was increased with decreasing protein concen—

tration and an S° value of 0.78 (S = Svedberg =

20,w
l x 10"13 sec) was estimated by extrapolation to infinite

dilution. Ogston (19A6) reported a S value of 0.96 for

20
one of the components of sigma proteose in phosphate buffer
(pH 8.6). Also, a value of 0.77 S was reported for one of
the components of the proteose—peptone fraction in veronal
buffer at pH 8.6 (Brunner and Thompson, 1961). A value
of 0.8 S was obtained for the milk whey phosphoglycoprotein
in phosphate buffer at pH 7.0, I = 0.1 (Bezkorovainy, 1965).
The sedimentation coefficients for component 8—slow
(unheated skimmilk) at various protein concentrations are
shown in Table 8. The sedimentation eXperiments were per—
formed with veronal buffer at pH 8.6, I = 0.1. Correspond—
ing sedimentation—velocity patterns are shown in Figure 52.
Figure A7 shows a plot of the sedimentation coefficients
against protein concentrations. As the protein concentra-
tion was increased, the sedimentation coefficient decreased
and at infinite dilution a SO20,W of 1.35 was estimated.
Thus, component 8-slow appears to be a larger molecule or

has a more spherical configuration than component 8—fast.

Bezkorovainy (1966) isolated a M-l—type glycoprotein from

10A

milk and colostrum for which he reported S values of

20,w
1.0 and 1.1, respectively.

The sedimentation coefficients for component 5
(heated skimmilk) at various concentrations in veronal
buffer are shown in Table 8. Figure 53 shows the
sedimentation-velocity patterns of component 5 at a
protein concentration of 6.1 mg/ml. A plot of protein
concentrations against sedimentation coefficients is
shown in Figure A9. According to this plot, the sedi-
mentation coefficient appears to be independent of con—

centration. At infinite dilution, the S° = 1.22.

20,w

This value approximated the S values of 1.0 and 1.1 S

20,w
reported by Bezkorovainy (1966) for the milk and colostrum
glycoprotein, respectively.

The proteins, components 8-fast, 8—slow and 5,

sedimented as single homogeneous boundaries.

Sedimentation-equilibrium molecular weights.--The

 

apparent molecular weights of component 8-fast at various
protein concentrations are shown in Table 8. All of the
sedimentation—equilibrium experiments were performed in
veronal buffer at pH 8.6, I = 0.1. The experimental
sedimentation—equilibrium patterns for component 8—fast
are shown in Figure 51. The plot of apparent molecular
weights and protein concentrations (Figure A6) for com—
ponent 8-fast showed evidence of concentration dependence.
A weight—average molecular weight of A,100 was estimated

by extrapolating to infinite dilution as shown in Figure A6.

105

Ogston (l9A6) reported a molecular weight of A,900
for one of the components of sigma proteose. He assumed
that the molecules were Spherical and the partial specific
volume was 0.75.

The apparent molecular weights of component 8-slow at
different concentrations are shown in Table 8. Sedimenta—
tion—equilibrium patterns are shown in Figure 52. Figure A8
presents a plot of the apparent molecular weights at various
concentrations of protein. Component 8-slow was more con—
centration dependent than either component 8—fast or com—
ponent 5. From the plot in Figure A8, a weight-average
molecular weight at infinite dilution was estimated to be
9,900. Bezkorovainy (1966) reported molecular weights of
about 10,000 for the M-l—type glycoprotein isolated from
milk and colostrum. More SOphisticated centrifugation experi-
ments should be performed to achieve a more accurate estima—
tion of the molecular weight.

The apparent molecular weights of component 5 are
shown at various protein concentrations in Table 8.
Sedimentation-equilibrium patterns are shown in Figure 53.

A plot of the apparent molecular weights and protein con—
centrations showing the concentration dependence of com—
ponent 5 is presented in Figure 50. From this plot, the
weight-average molecular weight at infinite protein dilution
was estimated at 1A,300. As was discussed earlier, a molecu-
lar weight of 7,300, based on four limiting amino acids,

was calculated. However, if two residues of each of

these amino acids were present in the molecules, the

106

molecular weight would be 1A,600. This would agree with
the sedimentation equilibrium molecular weight of 1A,300.
Component 5 was separated into two zones by starch—
urea gel electrophoresis. The two zones could be individual
proteins with a molecular weight of 7,300 and differing
in only one or two amino acid residues which would account
for the difference in the electrOphoretic migration. In
the veronal buffer, the two proteins may be strongly
associated in a stable dimer to give a molecular weight of
1A,300. On the other hand, each zone could be an individual
protein with a molecular weight of about lA,300; the two
individual proteins differing in only one or two amino acid
residues. Then, a molecular weight of 1A,300 would be
obtained if the individual proteins existed in the mono-
diSpersed state. Possibly, the molecular weight could be
elucidated by studying component 5 in the presence of
dissociating agents such as 5 M guanidine or sodium dodecyl

sulfate.

107

.xHHEEme ompmmncs Scum cosmomnmo

.xHHEEflxm condos soak commomnm

n

.mfimmgonoonpomam How oUfiEmHAnom an omHMHLSQ pcocoosoo UmdeOHpooAMCDm

 

 

mm.o mm.m mm.m As.o 03.0 m.A sfisd afiadfim
mm.o mA.A ms.m mm.o wm.o o.A defiedmoxdm
mm.o mm.m mm.s mm.o Am.A w.A mmoxdm
mm.m ss.m AA.m sm.s sm.s A.m dssfimss momma mm s
Am.o mm.m mm.A mm.m o:.m m.A assosdmosd
mm.mA mo.ma Am.mA 0A.MA mm.mA m.mA admossaz
ARV Ass Ass Asv ARV Ass
homomom encompass powwow ooopmosso powwom poopmom pcmzpfipmcoo

 

m uQmQOQEoo

BOHmIm pamcoosoo

pmmwlm pcocoosoo

 

ow pcmcoosoo

 

.m use .zoame .pmmmnw «m monocoosoo no cofipfimoosoonn.m mumSN

TABLE 3.--Amino acid composition of component 8—fast prepared

108

from heated and unheated skimmilk.

 

Residue

Grams residue/
100 g proteina

Number residues/

1000 residues

 

Unheated Heated

Unheated Heated

 

Unidentified peak
Lysine

Histidine
Unidentified peak
Arginine

Aspartic acid
Threonine

Serine

Glutamic acid
Proline

Glycine

Alanine

Valine

Methionine
Isoleucine
Leucine

Tyrosine
Phenylalanine
Tryptophan
Cysteine

Weight of amino acid
residue (weight %)

Total carbohydrate

(weight %)

P as H2PO3

Total (weight %)

(weight %)

0.05
7-92
1.AA
0.13
3.63
8.20
A.A8
12.17
23.96
2.51
1.21
0.80
5.57
1.07
8.Al
A.27
1.A5
0.73
0.06

88.06

1.81

8.59

 

98.A6

5.22
0.82
0.25
5.A2
8.11
5.0A
10.00
25.30
3.67
1.19
0-93
5.18
1.10
7.36
7.16
0.70
1.0A
0.23

88.72

2.03
6.26

 

97.01

0.51
79.09
13.A2

1.12
29.72
91.22
56.67

178.88
237.6A

32.9A
26.92
1A.A0
65.5A
10.2w
95.16
u8.28
11.36

6.11

0.39

ZN=999.81

51.73
7.53
2.01

AA.19

89.53

63.35

1A5.9l
2A9.03

A7.95

26.A2‘

16.57
66.39
10.6u
82.63
80.38
5.A5
8.91
1.AA

1000.06

 

aWeight percentage of each amino acid residue based
on a nitrogen content of 13.16% and 13.33% for component
8—fast prepared from unheated and heated skimmilk, respec-

tively.

b

Values corrected for destruction during hydrolysis

according to the equation given by Hirs, Stein and Moore

(195”)-

‘-.u ‘o-u -‘fl

109

TABLE A.--Amino acid composition of component 8—slow
prepared from heated and unheated skimmilk.

 

Residue

Grams residue/
100 g proteina

Number residues/
1000 residues

 

Unheated Heated

Unheated Heated

 

Unidentified peak
Lysine

Histidine
Unidentified peak
Arginine

Aspartic acid
Threonine

Serineb

Glutamic acid
Proline

Glycine

Alanine

Valine

Methionine
Isoleucine
Leucine

Tryosine
Phenylalanine
Tryptophan
Cysteine

Weight of amino acid
residue (weight %)

Total carbohydrate
(weight %)

P as H2PO3

Total (weight %)

(weight %)

0.88
8.79
5.63
.36
.3A
.A9
.67
.A0
.38
.53
.13
.A0
.67
.90
.27
.68
.A2
.23

[—1

I-‘
ONOONONNOWU‘ICDKU‘H—J

82.17

7.65
5.79
95.61

 

.38
.A2
.92
.13
.35
.98
.A5
.A3
.62
.1A
.50
.0A
.A9
.37
.8A
.85
.65
.AA
.19

EJOtmO

H

l-‘
ONOU‘INOI’JNOUJSCDJZ".1‘

77.19

10.27
5-17
92.63

 

9.A9 A.28
9A.25 95.81
56.36 52.33

-— 1.A0
11.75 12.65

183.17 177.21
61.11 6A.1A
136.82 1A1.21
16A.03 165.21
A7.79 A7.16
12.6A 12.70
Al.l9 A1.95
33.28 36.60
6.9A A.1A
35.19 36.60
76.15 75.35

5.72 5.77
22.61 2A.1A

1.5A 1.35

£N=1000.03 1000.00

 

aWeight percentage of each amino acid residue based on
a nitrogen content of 13.03% and 12.31% for component 8-slow
prepared from unheated and heated skimmilk, respectively.

b

Values corrected for destruction during hydrolysis
according to the equation given by Hirs, Stein and Moore

(195A)-

110

TABLE 5.——Amino acid composition of component 5 prepared
from heated skimmilk.

 

 

 

Grams residue/ Number residues/
Residue 100 g proteina 1000 residues

Lysine 7.55 69.55
Histidine 1.92 16.59
Arginine 2.60 19.58
Aspartic acid 5.81 60.55
Threon neb A.72 55.05
Serine 5.77 78.12
Glutamic acid 2A.89 227.29
Proline 10.55 128.07
Glycine 1.23 25.A0
Alanine 1.92 31.83
Valine 6.60 78.A1
Methionine 1.68 15.17
Isoleucine 6.1A 63.97
Leucine 7.33 76.33
Tyrosineb 1.9A lA.05
Phenylalanine A.86 38.93
Tryptophan 0.20 1.12
Cysteine -— --
Weight of amino acid

residue (weight %) 95.71 £N=1000.01
Total carbohydrate

(weight %) 1.A5
P as H2PO3 (weight %) 2.53
Total (weight %) 99.69

 

aWeight percentage of each amino acid residue based
on a nitrogen content of 13.83% as determined by micro-
Kjeldahl determination.

bValues corrected for destruction during hydrolysis
according to the equation given by Hirs, Stein and Moore
(195A).

A

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111

 

 

 

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:H.HHI mm.mal N>.ml ooom m.H 3.5 mumnawocm
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112

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.xfifiesfixm ompmon Song omnmomnmo

 

 

 

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mA.mI mm.m I Ho.mI comm m.A s.m dsoamIm paddedsoo
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nzpfiafinos ofipononoonpomam

.m one «soamlw .pmdMIm mucoQOQEoo Mo mofipfiafinoe capouonmospomHMII.n mqm<e

113

TABLE 8.--Sedimentation coefficient and equilibrium mole-
cular weight data of components 8-fast, 8—slow, and 5 in
veronal buffer at pH 8.6, I = 0.1.

 

 

Protein Sedimentation
concentration coefficient Molecular weight
Sample (mg/ml) S2O w (weight average)
3
Component
8—fasta 5.2 .7u A,ooo
6.1 —- A,100
7.0 .73 3.500
10.0 -- A,100
10.1 .70 3,500
0.00, .78 A,100
Component
8-slowa 5.0 1.27 8,700
7.1 1.16 7,100
10.1 1.16 7,000
0.00 1.35 9,900
Component 5b A.1 1.21 13,700
5.1 1.19 l3,A00
6.0 1.21 —-
6.1 1.19 l3,A00
0.00 1.22 1A,300

 

aPrepared from unheated skimmilk.

bPrepared from heated skimmilk.

CValues extrapolated to infinite dilution.

114

 

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"m essaoo

mIm .mmIm .smIs .mmIm .mmIm .AmIH mm dsswfis Boss msofipodsu
IH u< CEsHoo

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meHm .mCOpomQIomoopono no monoppmo ofipmnozoonpooam How moLSI£onmmeI.mH osswfim

 

.I<||1

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r

+Il|||l|l|_h:lllllllll$_

A
4 = = V

   

115

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an oochuno chHpomnm mo mcnmppmo ofipmnonaonpooao How monsnnosmpmln.om onswfim

 

116

 

(19
° "8"”. I
| "5" ~-
.ll3ll .-
- to
1
Figure 21 Figure 22 Figure 23

Figure 21.--Starch-urea gel electrophoretic patterns
of fractions obtained in the preparation of the
casein micelles and the material released at pH A.6
according to the schematic in Figure 5: 1—R1, 2-81,
3-R2. A-Rg. 5-s2.

Figure 22.--Starch-urea gel electrophoretic patterns
of fractions obtained in the preparation and ammonium
sulfate fractionation of proteose-peptone as outlined
in Figure 6: 1-80% residue, 2-55% residue, 3-35%
residue.

Figure 23.--Starch-urea gel electrophoretic pattern
of electrophoretic frontal material (component 8)
purified by acrylamide gel electrophoresis.

117

 

Figure 2A.--Starch—urea gel electrophoretic patterns
of fractions obtained from whole casein by treatment
with EDTA, heating, and ammonium sulfate as outlined
in Figures 7, 8, and 9, respectively: l-supernatant
in Figure 8, 2-residue in Figure 8, 3—R1 in Figure 7
A—Sl in Figure 7, 5-R2 in Figure 7, and 6-Sl, 7-R3,
8-R2, 9-Rl, all in Figure 9.

118

-—ASCEND|NG-—> FDESCENDING—

An.

3800 sec. F = 10. 79 v cm'1

MM

3400 sec. F = 10. 70 V cm—1

 

 

 

Figure 25. --Free-boundary and starch- urea gel
electrophoretic patterns of proteose- peptone fractions.
Free- -boundary (veronal buffer pH 8.6, I = 0.1): A-
proteose- peptone, B-component 8 purified by acrylamide
gel electrophoresis, C—a mixture of proteose-peptone
and component 8 purified by acrylamide gel electro—
phoresis. Starch-urea gels: D-slot 1, component 8
removed from Tiselius cell; slot 2, the components
remaining in the Tiselius cell after repeated electro-
phoretic analyses; slot 3, fraction R (Figure 3)
before separation in the Tiselius celT

119

. , (4.)
38:: A)
"8-fast" 5 ‘ ’-
"3H
"8—sl0w" “
7 (—I

 

Figure 26.--Starch—urea gel electrophoretic and
acrylamide gel electrophoretic (continuous buffer
system) patterns of proteose-peptone (Figure 1),
sigma-proteose supernatant (Figure 2), and component
8 (Figure 16). Acrylamide gel: l—component 8, 2-
sigma-proteose supernatant, A-proteose-peptone.
Starch-urea gel: 3—sigma—proteose supernatant, 5-
proteose-peptone.

120

Discontinuous Buffer System—9

 

— Continuous Buffer System—>

Figure 27.--Two dimensional acrylamide gel electro-
phoretic pattern of sigma-proteose supernatant
(Figure 2).

121

(+0
+_n8u_a,‘ll-”'HII

(_ll5ll

   

A.
00
l 2 1 2
I—A——I I——B——I

Figure 28.-—Acrylamide gel and acrylamide-urea gel
electrophoretic (discontinuous buffer system)
patterns. Acrylamide gel without urea (Column A):
l-sigma proteose, 2—proteose-peptone. Acrylamide
gel with urea (Column B): 1-sigma proteose, 2-
proteose-peptone.

. Lsd‘

122

(1')
G—-”8”

  
  
  

-(_II5II

e__u3u

..
.-‘.

Figure 29.--Starch-urea electrophoretic patterns
of alcohol-fractionated proteose-peptone as out-
lined in Figure 10: 1—supernatant S1 and resi-

dues 2'RA’ 3-R3, A-R2, and 5—Rl.

123

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.HmIm .mmIA "m sssHoo .mmIm .smIs .mmIm .mmIm .HmIH “a sssfioo .mdoapdsdd

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an omnHmpno mCOHpomsm mo mssmupoo OHpononoonpomHo How conglnonmumll.om onstm

 

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+:m: 0|:m:

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Al- .N- .

sz:

 
 

¢3

124

‘_I I8"
‘_Il5ll

(_.ll3ll

 

Figure 31.——Starch-urea gel electrophoretic
patterns of fractions obtained by sodium chloride
fractionation of proteose-peptone concentrate from
unheated skimmilk as outlined in Figure 12: l-Sl,

(component 8), 2-R , 3‘RA: A—R3, 5-R2, 6-R1
(enriched componeng 5).

125

(4')
‘___ll8ll
‘_II5II
6—-"3"
f»
.3
Ed

 

Figure 32.--Starch-urea gel electrophoretic patterns
of fractions obtained by sodium chloride and ammonium
sulfate fractionation of proteose—peptone from heated
skimmilk as outlined in Figure 13: l—Rl, 2-31, 3-R2,
A-R3, 5-Ru, 6-82 (component 8).

ANIILIIA'

126

 

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127

 

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128

e-"8-fast".

(—"8-slow"

      

1234 12345

Figure 36 Figure 37

Figure 36.--Acrylamide gel electrophoretic patterns
(continuous buffer system) of some of the fractions
obtained by fractionation of proteose-peptone from

unheated skimmilk as outlined in Figure 17: l-Rl,

2-R2, 3-R3, A-Ru.

Figure 37.--Acrylamide gel electrophoretic patterns
of the fractions collected by gel filtration through
Bio-Gel P—10: l-first fraction, 2-second fraction,
3—third fraction, A-fourth fraction, 5-fifth fraction.

129

(+1

<-—"8-fast"

<—"8-s|oW"

 

1' 345 "6 7

Figure 38.-~Vertica1 acrylamide gel electrophoresis
patterns (continuous buffer system) of various
proteose-peptone fractions: l-component 8—fast from
heated skimmilk, 2—component 8—fast from unheated
skimmilk, 3-component 8-slow from heated skimmilk,
A-component 8-slow from unheated skimmilk, S-proteose-
peptone, 6-component 5 from heated skimmilk 7-1ambda
casein prepared by Swaisgood (1963).

130

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133

 

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N

 
 

134

 

-l2 -

y
volt"1 sec”1)

retic Mobilit

l x lO="S cm2

Electropho

(u

 

 

 

 

pH

Figure u3.--A plot of the average ascending and descend—
ing electrophoretic mobilities of component 8 at various
pH values.

135

DESCENDING

 

2800 sec.

‘_

 

3100 sec.

 

3000 sec.

3200 sec.

 

4100 sec.

chemically and physically in this study:

ASCENDING A

  

F= |0.50V cm-

 

 

F - 10. 87 v cm'1

   

F - 10. 63 v cm'1

 

F - 10. 79 V cm'1

 

F - 10. 79 v cm‘1

Figure Mu.--Free-boundary electrophoretic patterns of
proteose-peptone fractions which were analyzed

A-component

8-fast from unheated skimmilk, B-component 8-fast from
heated skimmilk, C-component 8-slow from unheated
skimmilk, D-component 8-slow from heated skimmilk,

E-component 5 from heated skimmilk.

Electrophoresis

was performed in veronal buffer at pH 8.6, I = 0.1.

136

 

 

 

 

 

S“ 1.4

O

H

>4 1.2 -

‘5

(D 1.0 -

.r—1

0

a 0.8 : _________ _.._n
O 0 6 -

‘5

fl 0.14 —

4.)

Cd

2 0.2 ’

(I)

"E 0.0 1 ' ' I I

Protein Concentration (mg/ml)

Figure 45.--A plot showing the concentration dependenge
of the sedimentation coefficient for component 8-fast i
veronal buffer at pH 8.6, I = 0.1.

1'1

aPrepared from unheated skimmilk

 

 

 

 

 

5,000 -
13 13,000 I“----—--—___o 9 9
ho
H fl
5‘; o 0
$4
,1.“ 3,000 -
:3
0
<1)
H
O
2
2,000 . . ! l 1
2 u 6 8 10

Protein Concentration (mg/ml)

Figure H6.--A plot showing the concentration dependence
of the apparent molecular weights for component 8—fasta in
veronal buffer at pH 8.6, I = 0.1.

aPrepared from unheated skimmilk.

Sedimentation Coefficient X 1013

Figure
of the

veronal buffer at pH 8.6, I

137

 

~~
‘~
—~
~
‘~

 

l l l A l

 

 

u 6 8 10
Protein Concentration (mg/ml)

M7.--A plot showing the concentration dependen
sedimentation coefficient for component 8—slow
0.1.

aPrepared from unheated skimmilk.

12

-3
|—’
O

(I)

0\

Molecular Weight X 10

.L‘:

86

 

 

I l L _l

 

 

u 6 8 10
Protein Concentration (mg/ml)

ML.

Figure U8.--A plot showing the concentration dependence
of the apparent molecular weight for component 8—slow

veronal buffer at pH 8.6, I

0.1.

aPrepared from unheated skimmilk.

in

in

138

 

 

 

 

m
H
g 1.8
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p 1.6 b
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o l.“ '-
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2g 0.6 -
8
l
33 0.14 ' ' ' '

2 U 6 8 10

Protein Concentration (mg/ml)
Figure 49.-—A plot showing the concentration dependence
of the sedimentation coefficient for component 5a in
veronal buffer at pH 8.6, I = 0.1.

aPrepared from heated skimmilk.

 

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2 u 6 8 10
Protein Concentration (mg/ml)

Figure 50.-—A plot showing the concentration dependence
of the apparent molecular weight for component 5a in
veronal buffer at pH 8.6, I = 0.1.

aPrepared from heated skimmilk.

139

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vv-ry'rr'vc’ v-

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102

 

Figure SU.-—Photomicrographs of component 8-fast
(unheated skimmilk) crystallized from veronal buffer
and of sodium barbital crystals. A-component 8-fast,
Mag. 225x, phase optics, B—components 8-fast, Mag.
26X, Rheinburg illumination, C-sodium barbital, Mag.
26X, Rheinburg illumination.

SUMMARY

Several eXploratory studies were performed with
various salts, solvents and values of pH to obtain individ—
ual components from the proteose—peptone fraction and a
proteose-peptone—rich fraction prepared from unheated
skimmilk. These experiments resulted in the development
of optimal procedures for the isolation of components 3,
5, and 8.

The protein carried in the electrophoretic frontal
zone in both starch-urea gel and acrylamide gel
discontinuous—buffer electrophoresis was essentially com—
ponent 8. This fast—moving frontal material was isolated
from preparative—size acrylamide-urea gels. A salting-out
method employing ammonium sulfate was developed for the
fractionation of proteose—peptone fraction from heated
skimmilk and of a proteose-peptone—rich fraction from
unheated skimmilk into highly enriched fractions of com-
ponents 3, 5, and 8. The component—S—rich fraction was
further pruified by ammonium sulfate and pH adjustments.

Data were presented illustrating the electrophoretic
separation of component 8 into components 8-fast and 8-slow
by acrylamide gel electrophoresis employing a continuous

buffer system and by a two-dimensional acrylamide gel

lU3

1N4

electrophoresis system. These components were subsequently
separated by gel filtration on Bio—Gel P-lO.

Chemical analyses were performed on components 8—fast
and 8—slow from heated and unheated skimmilk and component
5 from heated skimmilk. These proteins were low in nitrogen
and high in phosphorus. Component 8-slow from heated
skimmilk was highest in carbohydrate (10.27%) and component
5 was the lowest (1.45%). Paper chromatography, employed
for the tentative identification of the carbohydrate moieties,
showed the presence of glucosamine, galactosamine, galactose,
glucose, mannose and fucose.

The amino acid compositions of these proteins were
characterized by relatively high contents of glutamic acid,
aspartic acid, serine and threonine and relatively low con—
tents of glycine, methionine and tryptophan. Cysteine was
absent. Component 5 contained a high concentration of
proline (10.55%). This value is higher than the proline
content of the whey proteins, B-lactoglobulin and
a—lactalbumin, and comparable to that found in the caseins,
e.g., aS-casein, B-casein, k—casein, y-casein.

The isoelectric pH (3.25) of component 8 was deter-
mined by free-boundary electrophoresis in various buffers
at 0.2 ionic strength. The average electrophoretic
mobilities of component 8—fast prepared from heated and
unheated skimmilk in veronal buffer at pH 8.6, I = 0.1,
were -9.00 and -9.3l Tiselius units, respectively. The

average mobilities for component 8-slow from heated and

145

unheated skimmilk were -9.15 and —9.21 Tiselius units,
respectively, while a value of —H.80 Tiselius units was
observed for component 5.

The crystallization of component 8-fast prepared
from unheated skimmilk was observed in veronal buffer at
pH 8.6, 0.1 ionic strength.

The sedimentation coefficients, S° for com-

20,w’
ponent 8-fast and component 8-slow isolated from unheated
skimmilk and for component 5 isolated from heated skimmilk
were 0.78, 1.35, and 1.22, respectively, in veronal buffer
at pH 8.6, I = 0.1.

Sedimentation—equilibrium weight-average molecular
weights for components 8—fast, 8-slow, and 5 were H,lOO,
9,900, and 10,300, respectively, in veronal buffer at pH
8.6, I = 0.1. Based on four limiting amino acids——histidine,
arginine, methionine, and tyrosine—-a minimum molecular
weight for component 5 was estimated as 7,300. Assuming
there were two residues of each of these amino acids in
the molecule, the molecular weight would be 10,600; a
close approximation with the weight-average molecular
weight of 14,300. Or, two proteins, each with a molecular
weight of 7,300, could be strongly associated in a stable

dimer to yield a molecular weight of 10,300.

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1146

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