ISOLATION AND CHARACTERIZATION OF
LACTEAL IMMUNOGLOBULINS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
KHEE CHOON RHEE
1969

   
 

  
   

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thesis entitled

ISOLATION AND CHARACTERIZATION OF

LAC TEAL IMMUNOGLOBULI NS

presented by

Khee Choon Rhee

has been accepted towards fulfillment
of the requirements for

_Eh..._ll._ degree in___FOOd Science

 
    

Major professor

Date Sept. 25, 1969

 

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ABSTRACT

ISOLATION AND CHARACTERIZATION OF
LACTEAL IMMUNOGLOBULINS

by Khee Choon Rhee

The immunoglobulin fraction of cow's milk contains two
principal components; a water-insoluble euglobulin and a
water-soluble pseudoglobulin. Euglobulin consists mainly-of
IgGZ-like globulins and small amounts of the IgGl-like glob-
ulins, IgA, and IgM, whereas pseudoglobulin conSists mostly
of IgG1 and a small amount of "secretory IgA". Each of
these protein groups comprises about three per cent of the
total whey proteins.

This investigation was directed toward a better under-
standing of the role of euglobulin in the creaming phenome-
non of cow's milk, using both chemical and physical methods.
of assesément. In addition, some of the physico-chemical
properties of euglobulin and pseudoglobulin were determined
to reevaluate or confirm the existing data on these prote—
ins. To isolate electrOphoretically pure euglobulin and
pseudoglobulin fractions from other milk components, gel
filtration in Sepharose 6B and DEAR-cellulose anion ex-
change column chromatographic techniques were employed.

. Enrichment of the starting material was achieved by salting-
out with ammonium sulfate.

Chemical analyses revealed that euglobulin and pseudo-

globulin are identical in composition. They contained

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Khee Choon Rhee

fucose, galactose, galactosamine, glucosamine, mannose, and
sialic acid in their carbohydrate moiety. Phosphorus was
absent in both proteins. Both proteins were similar in
amino acid content and quite like other milk proteins except
for lower glutamic acid and proline and higher serine and
threonine contents.

The electrOphoretic mobilities of euglobulin and pseudo-
globulin were -l.82 and -2.02 x 10"5 cm2 sec“l volt-l, re-
Spectively. The isoelectric point of euglobulin was pH 6.03
and that of pseudoglobulin was pH 5.54. Euglobulin had a
partial specific volume of 0.712 ml/g while pseudoglobulin
had a value of 0.710 ml/g. The diffusion coefficients of
euglobulin and pseudoglobulin in veronal buffer (pH 8.6,

u = 0.1) were 3.20 and 4.20 x 10-7 cm2 sec-1, respectively.
Euglobulin contained two sedimenting species whose sedimen—
tation coefficients were 6.248 and 19.048, while pseudo-
globulin showed a single sedimenting boundary of 6.008, as
determined in veronal buffer at pH 8.6 with an ionic
strength of 0.1. The weight average molecular weights of
these two proteins were approximately 175,000 in veronal
buffer and 90,000 in 6 M guanidine hydrochloride solution
containing 0.02 M 2—mercaptoethanol.

Both the sedimentation coefficient and the weight
average molecular weight of euglobulin increased at low
temperatures. The weight average value in milk salt solu-

tion at 5° C was approximately equal to a dimer weight of

350,000. The hydrogen ion concentration and ionic strength

sn-

Khee Choon Rhee

of buffers also had significant effects on the polymeriza—
tion of euglobulin. As the hydrogen ion concentration or
ionic strength decreased, both the sedimentation coeffi-
cient and the weight average molecular weight decreased
owing to the slow dissociation of the euglobulin molecules.
The necessity of the euglobulin for the "creaming" of
cow's milk was demonstrated in experiments with model
systems. As the concentration of euglobulin was increased,
up to about 0.04%, the creaming ability was improved grad—
ually close to that of normal milk. At constant euglobulin
concentration, the creaming ability was significantly im-
proved by lowering the creaming temperature to 5° C. The
effects of hydrogen ion concentration and ionic strength of
the system on the creaming ability of the model system were
similar to those on the sedimentation coefficient and

weight average molecular weight of euglobulin.

ISOLATION AND CHARACTERIZATION OF

LACTEAL IMMUNOGLOBULINS

BY

Khee Choon Rhee

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Food ScienCe

1969

EB

(ye/775
7/~27~70

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation
to his major professor, Dr. J. R. Brunner, for his guid-
ance, encouragement, and patience throughout the course of
this study.

He also wishes to thank the other members of his
guidance committee, Dr. P. Markakis, Dr. C. M. Stine, and
Dr. C. H. Suelter for their confidence and suggestions in
preparing this manuscript.

Grateful acknowledgment is due to Dr. B. S. Schweigert,
Chairman of the Department of Food Science, and the Nation-
al Institutes of Health for providing the research facili-
ties and funds necessary for this research.

Finally, special gratitude is expressed to his wife,
Ki Soon, for her understanding, encouragement, and valuable
assistance throughout his graduate program and in the

preparation of this manuscript.

ii

TABLE OF CONTENT

ACKNOWLEDGMENTS . . . . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . . . .

Nomenclature of Immunoglobulins in
Cow's Milk and Colostrum . . . . . . .

Isolation Procedures of Immunoglobulins
from Cow's Milk and Colostrum . . . . .

Characteristics of Immunoglobulins in
Cow's Milk and Colostrum . . . . . . .

The Role of Immunoglobulins on the Clustering of

Fat Globules in Creaming of Cow's Milk
EXPERIMENTAL . . . . . . . . . . . . . . . .
Apparatus and Equipment . . . . . . . . .
Chemicals and Materials . . . . . . . . .

Preparative Procedures . . . . . . . . . .
Preparation of Euglobulin and
Pseudoglobulin . . . . . . . . .
Precipitation of Immunoglobulins
with Ammonium Sulfate . . . . . .
Separation of Euglobulin and Pseudo-
globulin by Gel Filtration . . .
Purification of Euglobulin and
Pseudoglobulin Fractions . . . .
Preparation of Washed Cream . . . .

Chemical Methods .
Nitrogen . . .
PhOSphorus . .
Carbohydrate .

iii

Page
ii
vi

viii

13
19
19
21
23
23
23
25

26
27

28
28
29
30

RF

Fucose . . .
Hexose . . .
Hexosamine .
Sialic Acid .
TryptOphan .
Amino Acid .

Physical Methods . . . . . . . . . . . . .
Free-boundary Electrophoresis . . . . .
Polyacrylamide Gel ElectroPhoresis in

a Discontinuous Buffer System
Ultracentrifugation . . . . . .
Protein Concentration .
Density . . . . . . . .
Viscosity . . . . . .
Partial Specific Volume .
Diffusion Coefficient . .
Sedimentation Coefficient .
Low-speed Sedimentation—equil'brium

1

Creaming Studies . . . . . . . . . . .
Creaming PrOperties of Normal Milk

Cluster Index . . . . . . . . . . .

Cream Volume . . . . . . . . . . . .

Creaming Properties of Recombined Milk
Creaming Properties of Model System . .

RESULTS AND DISCUSSION . . . . . . . . . . .

Preparative Procedures . . . . . . . . .
Preparation of Euglobulin and
Pseudoglobulin . . . . . . . . . . .
Preparation of Washed Cream . . . . .
The Effects of Washing the Cream . .
The Effects of Antibiotics on
Fat Globule Clustering . . . . .

Chemical Analyses . . . . . . . . . . . .

Physical Properties . . . . . . .
Electrophoretic Characteristics
ElectrOphoretic Mobility . .
Isoelectric Point . . . . .
Ultracentrifugal Characteristics
Partial Specific VOlume . .
Diffusion Coefficient . . .
Sedimentation Coefficient .
Molecular Weight . . . . . .

Creaming Studies . . . . . . . . . . . . .

The Effects of Euglobulin Concentrations

on Creaming . . . . . . . . . . . .

iv

Page

32
33
34
35
36
37

39
39

40
42
42
42
43
44
45
47
49

51
51
51
52
52
53

54
54

54
58
58

61
62

72
72
72
74
74
74
78
86
93

101
101

a

Page

The Effects of Temperatures on Creaming . . . . 103
The Effects of Electrolyte

Concentrations on Creaming . . . . . . . . . 104
The Effects of Hydrogen Ion

Concentrations on Creaming . . . . . . . . . 108
Interpretation of the Creaming Phenomenon

in Relation to the Physical Properties

of Euglobulin . . . . . . . . . . . . . . . 108

SUMMRY O O O O O C C O O C O O O I O O O C O O O O O 112

BIBLIOGRAPHY 0 0 O O O O O O O O O O O O O O O O O O 115

APPENDIX C Q .0 O O O O O O O I O O O O O O O O C O O 124

KS-

LIST OF TABLES

Table Page

1. The effect of antibiotics on the creaming
properties of milk . . . . . . . . . . . . . 62

2. Chemical compositions of euglobulin and
pseudoglobulin . . . . . . . . . . . . . . . 63

3. Amino acid composition of euglobulin . . . . . 67
4. Amino acid composition of pseudoglobulin . . . 68

5. ElectrOphoretic mobilities of euglobulin
and pseudoglobulin . . . . . . . . . . . . . 72

6. Electrophoretic mobilities of euglobulin and
pseudoglobulin in various buffer systems . . 75

7. The apparent diffusion coefficients of
euglobulin and pseudoglobulin in veronal
buffer, milk salt solution and 6 M
guanidine-HCl plus 0.02 M 2-mercapto-
ethanol solution . . . . . . . . . . . . . . 80

8. The effects of various hydrogen ion
concentrations on the diffusion coeffi-
cients of euglobulin . . . . . . . . . . . . 83

9. The effects of electrolyte concentrations
on the apparent diffusion coefficients
of euglobulin . . . . . . . . . . . . . . . 84

10. The effects of temperatures on the apparent
diffusion coefficients of euglobulin in
veronal buffer and milk salt solution . . . 85

11. The sedimentation coefficients of euglobulin
and pseudoglobulin in veronal buffer,
milk salt solution and 6 M guanidine
hydrochloride solution containing 0.02 M
2-mercaptoethanol . . . . . . . . . . . . . 87

vi

Table
12.

l3.

14.

15.

16.

17.

18.

19.

20.

21.

22.

The effects of temperatures on the sedimen-
tation coefficients of euglobulin in
veronal buffer and milk salt solution . . .

The effects of various hydrogen ion concen-
trations on the sedimentation coeffi—
cients of euglobulin . . . . . . . . . . . .

The effects of electrolyte concentrations
on the sedimentation coefficients of
eugIObulin C O O O O O C I O O O O O O O O 0

Molecular weights of euglobulin and pseudo-
globulin in veronal buffer, milk salt
solution and 6 M guanidine hydrochloride
solution containing 0.02 M 2-mercapto-
ethanol . . . . . . . . . . . . . . . . . .

The effects of temperatures on the apparent
weight average molecular weights of
euglobulin in veronal buffer and milk salt
solution . . . . . . . . . . . . . . . . . .

The effects of hydrogen ion concentrations
on the apparent weight average molecular
weights of euglobulin . . . . . . . . . . .

The effects of ionic strengths on the apparent
weight average molecular weights of euglob-
ulin in veronal buffer, pH 8.6, at 20° C . .

The effects of euglobulin concentrations on
creaming O O O O O I O O I O O C O O O O O O

The effects of temperatures on the creaming
properties 0 O O O O O O O O I O O O I O O O

The effects of electrolyte concentrations on
creaming phenomenon . . . . . . . . . . . .

The effects of hydrogen ion concentrations
on the creaming properties . . . . . . . . .

vii

Page

90

91

92

94

98

100

101

102

105

106

109

Tigris:

 

LI ST OF FIGURES

Figure Page

1. Schematic for the fractionation of
euglobulin and pseudoglobulin from
cow's milk . . . . . . . . . . . . . . . . 24

2. Elution diagram of crude immunoglobulin
fraction through Sepharose GB column with
0.01 M tris-HCl buffer, pH 7.0 . . . . . . 56

3. Polyacrylamide gel electrophoretic patterns
of skim milk, whey, crude immunoglobulin
fraction and purified euglobulin and
pseudoglobulin . . . . . . . . . . . . . . 57

4. Free-boundary electrophoretic patterns of
purified euglobulin and pseudoglobulin
in veronal buffer, pH 8.6, ionic strength
of 0.1. . . . . . . . . . . . . . . . . . . 59

5. Polyacrylamide gel electrophoretic patterns
of skim milk, whey and cream washings . . . 60

6. Paper chromatogram of authentic carbohydrate
mixtures and of carbohydrates released
from euglobulin and pseudoglobulin . . . . 66

7. Amino acid profile of euglobulin and
pseudoglobulin . . . . . . . . . . . . . . 70

8. Free-boundary patterns of euglobulin and
pseudoglobulin at various time intervals
in veronal buffer, pH 8.6 and ionic
strength of 0.1 . . . . . . . . . . . . . . 73

9. Electrophoretic mobility as a function of
pH for the euglobulin and pseudoglobulin . 76

10. Interferograms obtained in high-Speed
sedimentation-equilibrium experiments
with euglobulin and pseudoglobulin in
H20 and D20 solutions . . . . . . . . . . . 77
11. Sedimentation equilibrium of euglobulin and

pseudoglobulin in H20 and D20 solutions . . 79

viii

55-19;:

Fiau.
12.

 

l3.

14.

15.

 

Figure Page

12. Diffusion properties of euglobulin and
pseudoglobulin in veronal buffer, milk
salt solution, and 6 M guanidine hydro-
chloride solution containing 0.02 M
2-mercaptoethanol . . . . . . . . . . . . . 81

13. Sedimentation behavior of euglobulin and
pseudoglobulin in veronal buffer, milk
salt solution, and 6 M guanidine hydro-
chloride solution containing 0.02 M
2-mercaptoethanol . . . . . . . . . . . . . 89

14. Concentration dependency of the apparent
weight average molecular weights of
euglobulin and pseudoglobulin in veronal
buffer, milk salt solution, and 6 M
guanidine hydrochloride containing 0.02 M
2-mercaptoethanol . . . . . . . . . . . . . 96

15. The effects of temperatures on the weight

average molecular weight of euglobulin
in veronal buffer and milk salt solution . 99

ix

.llllil ! I.

an]:

INTRODUCTION

The lactoglobulin fraction of cow's milk is classical-
ly defined as that portion of the whey proteins precipi-
tated by saturation with magnesium sulfate or half-satura—
tion with ammonium sulfate. Upon dialysis this fraction
separates into two components, a precipitate called eu-
globulin and a protein remaining in the solution called
pseudoglobulin. These proteins possess the immunological
properties and are often called the "immunoglobulins".
They each comprise about three per cent of the total whey
proteins and are present in much higher concentration in
coloStrum than in normal milk.

Previous studies indicated that euglobulin might have
an important role in the fat globule clustering or creaming
of milk, but little has been elucidated in this regard.
Very little information is available presently on the tem-
perature directed association and dissociation phenomenon
which euglobulin is believed to undergo. This phenomenon
has been thought to be one of the fundamental causes of
the "creaming" of cow's milk.

The main purpose of this research is to elucidate the
role of miAK euglobulin in the creaming of milk. Partic-
ular emphasis has been devoted to exploring the nature of

association and dissociation of this protein in model

1

531:1:

2
systems at different protein concentrations, temperatures,
ionic strengths, and hydrogen ion concentrations in rela—
tion to fat globule clustering and the creaming abilities
of this protein fraction.

Although reports have been made of the procedures for
isolating the immunoglobulins and of their physical and
chemical prOperties, they differ widely depending on the
purpose of the experiments, the methods of analyses, and
the source of the milk, both in the chemical and physical
prOperties. Therefore, an extensive reinvestigation on the
prOperties of these particular protein fractions is needed

for elucidating the physico-chemical characteristics.

REVIEW OF LITERATURE

Nomenclature of Immunoglobulins
in Cow's Milk and Colostrum

 

 

The Committee on Milk Protein Nomenclature, Classifi-
cation and Methodology of the Manufacturing Section of
American Dairy Science Association (Rose 32 31., 1969)
recommended a complete revision of classical bovine immuno-
globulin nomenclature to correspond with that used for more
extensively studied Species in accord with the World Health
Organization Report (1964). The term "immunoglobulin" re-
places terms like "immune lactoglobulin", "gamma globulin",
"euglobulin", "pseudoglobulin", and "T-globulin" which can
be found in the early dairy science literature.

Accordingly, three distinct classes of immunoglobulins
occur in the lacteal secretions of the cow: IgG, IgA, and
IgM. IgG immunoglobulins which have sedimentation constant
of 78 can be further divided into at least two subclasses,
IgG1 and 1962. IgGl, with a sedimentation coefficient of
6.68, is the principal immunoglobulin of milk and colostrum
while 1962, with a value of 6.38, is primarily a serum compo-
nent. IgG immunoglobulins are relatively low in carbohy-
drate (Smith gt al., 1946; Nolan and Smith, 1962; Groves
and Gordon, 1967; Kickhoefen gt al., 1968). IgA of the

lacteal secretions is a 10-128 molecule probably composed

 

 

“Ls:

4

of a dimer of subunits plus glyCOprotein-a. It is best
referred to as "secretory IgA". IgM occurs in milk and
colostral whey. This immunoglobulin has a sedimentation
constant of 198, is sensitive to 2-mercaptoethanol and has
been reported to contain 12.3% carbohydrate (Gough gt a£.,
1966).

The new nomenclature of immunoglobulins can be corre-
lated with the early preparations of Smith (1946a) in the
following manner. Although antigenically heterogeneous,
Smith's pseudoglobulin and plasma T-globulin contain mostly
IgGl. The pseudoglobulin fraction also contains “secretory
IgA". Smith's euglobulin consists of IgG2-like globulins,
slower IgGl globulins,IgAq and IgM. His serum gamma globu-
lin corresponds to the IgGZ subclass.

However, the old nomenclature is used throughout this
thesis for the following reasons: (a) the new system recom-
mended by the committee has not yet been announced formal-
ly and (b) more importantly, the nature of this study was
so designed that the classical euglobulin and pseudoglobu-
lin classification of immunoglobulins was more suitable for
comparing with the presently available information and in
explaining the gross effects of euglobulin on the fat
globule clustering.

Isolation Procedures of Immunoglobulins
from Cow's Milk and Colostrum

 

 

Until the 1880's, milk was known to contain casein as

the predominant protein and smaller quantities of other

5
protein with properties similar to those of blood serum

albumin and blood serum globulin. It was found that after
casein was removed by isoelectric precipitation at pH 4.6,
the whey contained about 0.5% protein or about 20% of the

total protein of skim milk. The milk serum proteins were

then classified as lactalbumin and lactoglobulin according
to their solubilities in one-half saturated ammonium sul-

fate or saturated magnesium sulfate.

The presence of globulin in milk was first recognized
by Eugling (1880). He isolated globulin from milk by pro-
longed treatment of diluted whey with carbonic acid. A few
years later, Hammersten (1883) observed that after sepa-
rating casein from milk the filtrate contained albumin and
a substance separated by saturation with magnesium sulfate.
He suggested that this precipitate was a globulin.

Sebelien (1885) introduced the name "lactoglobulin"
for a flocculent precipitate obtained by saturating whey
with magnesium sulfate. This material possessed character-
istics identical with serum globulin. He proved that
albumin was not precipitated by saturating whey with magne-
sium sulfate, but that it remained in solution from where
it could be precipitated by the addition of acetic acid.
Halliburton (1890), to the contrary, claimed the substance
isolated from milk by saturating with magnesium sulfate was
lactalbumin. Schlossmann (1896-1897) observed that globu-
lin, after prolonged standing at room temperature, col-

lected on the surface of the liquid. The procedures of

6
Sebelien (1885) and Schlossmann (1896-1897) were reexamined

by Simon (1901) and the globulin fractions obtained proved
to be identical in their compositions and solubilities.

Storch (1897) isolated globulin from freshly drawn milk
by saturating it with sodium sulfate. The salt concentra-
tion was about 14-19% at this temperature (ca. 38° C).

Crowther and Raistrick (1916) employed anhydrous magne-
sium sulfate for precipitating globulin. They observed
that, like serum globulin, milk globulin could be separated
into water-soluble pseudoglobulin and water-insoluble eu-
globulin by exhaustive dialysis.

Applying the sodium sulfate fractionation procedures of
blood proteins to milk proteins, Howe (1921: 1922) found
that the material precipitated up to 14.2% salt at 34° C was
euglobulin. He designated the material precipitated between
l4.2-18.4% salt as pseudoglobulin I and that precipitated at
18.4—21.5% salt as pseudoglobulin II. The yield of the last
fraction was very small and there was no positive evidence
that this was a separate protein.

Smith (1946b) observed that globulin separated by
repeated precipitations with saturated magnesium sulfate
gave preparations which showed complex electrophoretic
patterns in moving-boundary electrophoresis. Thus, he
devised a method of preparing an electr0phoretica11y homo-
geneous globulin by stepwise fractionation with ammonium
sulfate. With his method, the crude globulin was precipi-

tated by half-saturation of the whey with ammonium sulfate.

7

This fraction was redissolved at about 3% protein concentra-
tion, the pH was adjusted to 4.6, and ammonium sulfate added
to 0.25 saturation. After removing the precipitate by cen-
trifugation, the immunoglobulins were precipitated from the
supernatant at 0.4 saturation with ammonium sulfate at pH
6.0. The precipitate was reworked by dissolving in water

at 1° C, adjusting to pH 4.5, and removing the insoluble
residue by filtration. The supernatant was brought to 0.3
saturation with solid ammonium sulfate. The resulting pre-
cipitate was removed. A final adjustment to pH 6.0 and 0.4
saturation with ammonium sulfate resulted in yet another
salted-out fraction. Upon dialysis, this fraction separated
into electrOphoretically homogeneous Specimen of euglobulin
and pseudoglobulin.

To isolate immunoglobulins from colostrum, a slightly
different procedure was described (Smith, 1946a; Smith and
Greene, 1948). After casein was removed by isoelectric
precipitation at pH 4.5, a globulin fraction was salted-out
from the acid whey with ammonium sulfate added to 0.3 satu-
ration, and another fraction was obtained when the-salt
concentration was raised to half-saturation. These two
fractions contained most of the colostral protein. By
salting-out with ammonium sulfate at 0.4 saturation and pH
6.0, followed by resolution and precipitation with ammonium
sulfate, he was able to obtain electrOphoretically pure
globulin from colostral serum. This protein fraction com-

pletely accounted for all the immune preperties of the

en!

8

colostrum. Apparently this procedure is not applicable to
the isolation of the globulin from normal milk.

Another method for isolating lactoglobulins from milk
and colostrum was introduced by Kenyon gt gt. (1959), as an
adaptation of a method developed by Horejsi and Smetana
(1956) for the isolation of gamma-globulin from the blood
serum. In this method, rivanol (2-ethoxy-6,9-diamino—
acridine lactate) was used to form a metal-combining globu-
lin complex which remained in solution. The precipitated
albumins were filtered off. Rivanol was removed from the
supernatant by adsorption on activated charcoal, leaving a
crude globulin fraction in solution.

A column chromatographic technique employing anion ex-
change cellulose for the fractionation of the blood proteins
(Sober gt gt., 1956) was modified by Yaguchi gt gt. (1959)
to fractionate milk proteins. Filtered acid whey was ad-
justedto pH 6.8 with dilute sodium hydroxide and the traces
of casein were removed by reacidifying to pH 4.6. The whey
was filtered, adjusted to pH 7.0 and dialyzed against a
large volume of phosphate buffer for 48 hr at 2-3° C. At
least eight whey protein components were eluted from the
column deve10ped with a linear gradient of sodium chloride
(0 to 0.2 M) in phosphate buffer. The immunoglobulin frac-
tions were eluted in the first and second peaks (Yaguchi gt

3;., 1961).

Kai

9

Characteristics of Immunoglobulins
in Cow's Milk and Colostrum

 

 

In comparing milk with colostrum, the marked difference
in the physical and biological properties of the colostrum
can be ascribed primarily to its high protein content, par-
ticularly globulins, and atypical protein distribution
(Sebelien, 1885; Bauer and Engel, 1921; Smith, 1946a).

Crowther and Raistrick (1916) did a comparative study
of the proteins of the colostrum and milk of the cow and
their relationship to the blood serum proteins. They ar-
rived at the following conclusions: (a) casein, lactoglobu-
lin and lactalbumin are distinct proteins and have the same
composition whether prepared from colostrum or normal milk;
(b) globulin fractions obtained from colostrum and milk,
although occurring in small amounts in milk, are alike and
closely allied to or identical with serum globulin; (c) eu-
globulin and pseudoglobulin are identical insofar as their
composition is concerned. Dudley and Woodman (1918) and
Woodman (1921) suggested that euglobulin and pseudoglobulin
were structurally identical. They investigated the optical
rotational properties of these two proteins in alkaline
solution and made a comparative study of the optical proper—
ties of the amino acids derived from racemized euglobulin
and pseudoglobulin by hydrolysis.

A series of outstanding studies and contributions to
our knowledge of the lactoglobulin fraction was made by

Smith (1946a; 1946b; 1948). The globulin character of the

sad

10

isolated protein fractions was indicated by their precipi-
tation at low concentrations of ammonium sulfate, low solu-
bility near the isoelectric point, and by a marked increase
in solubility in the presence of neutral salts. Smith also
reported that exhaustive dialysis of the lactoglobulin
fraction resulted in the separation of a water insoluble
euglobulin and a water soluble pseudoglobulin and that the
immune activity was associated with both of these proteins.
Furthermore, he found by electrophoresis that the lactoglob—
ulin of milk was identical to that found in colostrum. He
also reported the absence of phosphorus and the presence of
sulfur and carbohydrates in lactoglobulin fractions.

Hansen gt gt. (1947) reported high contents of proline and
glutamic acid, while Smith and Greene (1947) reported high
threonine content and that cystine and methionine accounted
for all the sulfur of these proteins. The carbohydrate
content of lactoglobulins in milk and colostrum was contri—
buted by the protein-bound hexose and hexosamine (Smith gt
gl., 1946; Smith, 1946b).

Smith (1946a; 1946b) was the first to use electropho-
resis and ultracentrifugation as well as chemical analysis
in comparing the properties of lactoglobulin fractions.

He reported values of pH 6.05 and 5.60 for the isoelectric
points of euglobulin and pseudoglobulin, respectively. He
recorded the electrOphoretic mobilities (Tiselius units) of
euglobulin and pseudoglobulin of milk and colostrum of -l.7

and -2.5, and -l.9 and-2.2, respectively. Murthy and

an:

 

11
whitney (1958) reported values of —l.76 and -2.04 for simi-

lar protein fractions isolated from milk.

The sedimentation coefficient of the principal compo-
nent (about 84-89%) of milk and colostral immunoglobulins
was reported to be approximately 7 Svedberg units (Smith,
1946a8il946b; Smith and Brown, 1950). The identity of
these molecules to those of the serum immunoglobulin IgG
was confirmed by many investigators (Larson and GilleSpie,
1957; Carroll, 1961; Dixon gt gt., 1961; Micusan and Buzi-
la, 1965; Pierce and Feinstein, 1965; Milstein and Fein-
stein, 1968). However;some evidence was given that a small
change might occur in the molecule during its transport
from serum to milk (Kickhoefen gt gt., 1968). The carbohy-
drate content of these proteins seems to be relatively low--
approximately 2-3% (Smith gt gl., 1946; Nolan and Smith,
1962; Groves and Gordon, 1967; Kickhoefen gt gt., 1968).
The second most concentrated component had a sedimentation
coefficient of 10-128 and was very sensitive to 2—mercapto—
ethanol (Smith 2E.El°' 1946; Jenness gt gt., 1965). This
component had a carbohydrate content of 8-9% (Gough gt gl,,
1966) and was reported to be identical to serum immunoglob-
ulin IgA (Hanson and Johansson, 1959; Murphy gt gt., 1964;
Jenness gt gt., 1965; Baglioni and Fioretti, 1967). A
third component which was found only in the euglobulin
fraction had a sedimentation coefficient of 19-208 (Smith
et a1., 1946; Gough SE.E£°' 1966), was sensitive to 2-mer-

captoethanol, and was reported to contain 12.3 per cent

aw

 

12
carbohydrate (Gough gt_gt., 1966). This macroglobulin was

reported to have the same physico-chemical and biological
properties as the IgM immunoglobulin from the blood of other
species (Murphy gt gl., 1964; Jenness gt gt., 1965; Gough gt
gt., 1966; Baglioni and Fioretti, 1967; Coffey and Reithel,
1968).

The molecular weight of immune lactoglobulin was re-
ported at about 160,000 to 190,000 by Smith gt_gt. (1946)
while the much higher values of 252,000 for euglobulin and
289,000 for pseudoglobulin were reported by Murthy and
Whitney (1958). Smith (1946a) reported a diffusion coeffi-
cient of 3.6 x 10.7 cm2 sec”1 for the immunoglobulin
fraction.

The'ultravioletuabsorption spectra of electrophoreti-
cally homogeneous immunoglobulins were obtained by Smith
and Coy (1946). No evidence was found for the presence of
light-absorbing groups other than the three aromatic amino
acids, phenylalanine, tyrosine and tryptOphan.

The effect of heat treatment of milk on the denatura-
tion of globulin fractions had been studied by many inves-
tigators. Hetrick gt gt. (1950) observed that the extent of
denaturation of globulin with time at constant temperature
of 170° F followed closely the first order law but the
first order law was no longer obeyed at 230° F. Steam-
injection produced more severe effects at short-time and
low-temperature combinations than have been reported for

other heat exchange methods, but less severe-effects than

13
other methods at high temperatures (Dill gt gt., 1964).

Globulins showed a rapid increase of denaturation even at
low temperatures of heating, showing that this is the most
heat-labile protein fraction of milk (Melachouris and
Tuckey, 1966).

Changes in the whey protein fraction during cold
storage of raw milk were studied by Lindquist and Storgards
(1966). They observed some changes on pseudoglobulin and
blood albumins during the first 24 hr of storage of raw
milk at 2° C. The decomposition products of both proteins
appeared in the alpha-lactalbumin peak in free—boundary
electrOphoresis diagrams. However, they could not determine
at what stage this change took place.

The Role of Immunoglobulins on the Clustering of
Fat Globules in Creaming of Cow's Milk

 

 

The process by which fat globules rise to the tOp of
milk upon standing, forming a fat-rich layer, is known as
”creaming". This phenomenon represents one of the fundamen—
tal physical prOperties of the fat emulsion. The formation
of a cream layer on milk involves two steps: (a) clustering
of the fat globules and (b) rising of the clusters to form
the cream layer. The manner in which fat clusters rise and
pack in the cream layer has been extensively investigated
and is well understood. While it has been established ex-
perimentally that the clustering of fat globules is essen-
tial for the rapid creaming and the formation of deep cream

layer on milk, the fundamental mechanism involved is still

(an

 

14

a matter of controversy.

Babcock (1889) was the first to study the phenomenon of
fat globule clustering in freshly secreted milk. He ad-
vanced the theory that clustering resulted from coagulation
of fibrin, which he thought to be a normal constituent of
milk, and that the coagulated fibrin entangled the fat
globules and weighed them down enough to prevent the "clots"
from rising. Although his conclusions were proved incor-
rect, many investigators give him credit for being the
first to indicate the significance of fat globule clustering
in the creaming of milk.

Since the work of Babcock in 1889, several theories
were proposed to explain fat globule clustering: (a) gravi-
tational rise of fat globules; (b) electrokinetic potential
of fat globules; (c) fat-serum interfacial tension;

(d) stickiness and state of hydration of the adsorbed fat
globule membrane; and (e) fat clustering considered as an
agglutination process.

Dunkley and Sommer (1944) reviewed extensively all the
pertinent references concerning the above proposed theories,
performed a series of carefully designed experiments, and
drew the following conclusions: (a) that the gravitational
rise of the fat in milk favors the formation and increase
in size of fat globules, but the variability in creaming
properties of milk can not be explained on the basis of
differences in the rates of rise of fat globules and clus-

ters; (b) that the salts normally present in milk are

15

sufficient to reduce the surface potential on the fat glob-
ules below the critical level which permits cluster forma—
tion, thus the creaming prOperties of milk are not deter-
mined by the variability of the charge on the fat globules;
(c) that the interfacial tension at the fat-serum interface
does not determine the creaming prOperties of milk and that
the free energy at the surface of the fat globules is not
the cause of clustering; and (d) that milk euglobulin
promotes the clustering of fat globules.

There are many publications favoring the theory that
the mechanism for the clustering of the fat globules in
milk is the same as that<xfa.bacterial agglutination reac-
tion in which euglobulin is involved.

Babcock (1889) was the first to report that the addi-
tion of blood serum to milk caused an aggregation of the
fat globules. Van Dam gt gt. (1922) and Hansson (1949)
reported similar results. However, when blood serum,
heated to above 62°C, was added to milk no improvement in
creaming was observed (Van Dam gt gt., 1922; Hekma and
Sirks, 1923; Rowland, 1937). Therefore, it was suggested
that the substance responsible for the enhancement of
creaming is thermolabile at the same temperature as are
bacterial agglutinins.

The substance responsible for the improvement in
creaming in milk was precipitated with the globulin frac-
tion (Hekma and Sirks, 1923). Brouwer (1924) fractionated

the globulin into euglobulin and pseudoglobulin and

i?“

 

16

demonstrated that the euglobulin was effective in improving
the creaming prOperties of milk but that the pseudoglobulin
had only a slight beneficial effect. Palmer gt gt. (1926)
and Troy and Sharp (1928) also observed that the agglutinin
material followed the globulin fraction when the proteins
of milk were separated.

Sharp and Krukovsky (1939) considered the clustering of
fat globules as an agglutination process and observed that
the agglutinating substance normally present in milk was
adsorbed on the surface of solid fat globules but not on
liquid fat globules. They also found that the agglutinin
was concentrated in cream separated at low temperatures and
was relatively absent in the corresponding skim milk. The
Opposite was true for milk separated at high temperatures.

Dunkley and Sommer (1944) prOposed a possible explana-
tion for the clustering of fat globules based on Marrack's
theory of the mechanism of bacterial agglutination (Marrack,
1939). Accordingly, clustering would be promoted by: (a) a
partial dehydration of the adsorbed membrane on the fat
globules as affected by a Specific polar adsorption of the
euglobulin; (b) an aggregation of fat globules resulting
from the adsorption of a single globulin molecule by two or
more fat globules; and (c) a maintenance of the surface
potential of the fat globules below the critical level by
the presence of salts.

Samuelsson gt gt, (1954) observed that the agglutinin

responsible for normal creaming in milk was separated at

17

2° C as a yellowish powder from rennet whey which had been
heated to 60° C. The agglutinin formed Opalescent solu-
tions in warm whey or water, from which it was precipitated
by adding sodium chloride or gum arabic. This material
consisted of two components, one inactivated by homogeniza-
tion and the other by heating in water, whey or milk to 70-
75° C for 15 seconds. In systems containing cream mixed
with water, why or separated milk, creaming would occur if
one portion of the available agglutinin had been inacti-
vated by homogenization and the other by heating, but if
all the agglutinin had undergone either one or the other
of these treatments, no creaming resulted. Similar obser—
vations were made by Kenyon and Jenness (1958) and Jenness
and Patton (1959). They observed that either homogeniza-
tion or heat treatment of skim milk prevented agglutina-
tion of fat globules, but a mixture of equal parts of
homogenized and heated skim milk produced normal creaming.
They also found that the addition of very small amounts of
colostral euglobulin to treated skim milk restored the
creaming ability of the heated but not that of the homo-
genized.

Payens (1964) and Payens gt_gt. (1965) reported that
I131-euglobulin was adsorbed on the surface of the fat
globules after homogenization and this adsorption was
accompanied by small amounts of casein, particularly K-
casein. With similar results, K00ps gt gt. (1966) specu-

lated that euglobulin-casein complex was formed during the

53*“

 

18

homogenization, which was still capable of adsorbing onto
the fat globule surface but unable to effect clustering,
and that the adsorption of K-casein would screen off the
clustering sites of the adsorbed euglobulin.

A model of the euglobulin-fat globule complex was
postulated by Dunkley and Sommer (1944) and Payens gt gt.
(1965). They reported that agglutination was brought
about by the formation of euglobulin bridges between two
fat globule molecules. The surface layer with the peptide
chains projecting into the plasma phase would fit best into
this picture. Kenyon gt gt. (1966) suggested the mechanism

for the Brucella ring test to explain the model; specific

 

agglutinins are adsorbed on the globules in such a way that

the reactive sites of the Brucella agglutinins are available

 

to combine with Brucella cells.

 

EXPERIMENTAL

Apparatus and Equipment

 

The milk used in this study was collected in five- or
ten-gallon stainless steel cans and separated with a DeLavel
disc—type separator. Stainless steel, plastic or Pyrex
containers were used for performing all experiments. A
Beckman Model 115 or a Sargent Model DR pH meter, equipped
with glass electrodes was used to measure pH values. For
weighings, tOp-loading, direct reading Mettler Type K-7
and H-16. a Sartorius series 2400 balance. and a Cahn
electrobalance were used.

Low-speed centrifugations were performed with an
International Model V, size 2 centrifuge. Intermediate-
Speed centrifugations were performed with a Sorvall RCZ-B
refrigerated centrifuge, using Type 88-34 and Type GSA
rotors. For high-speed separations, a Beckman Model L-65
refrigerated preparative ultracentrifuge, equipped with
Type 21, 30 and 65 fixed-angle rotors, was used.

Sephadex laboratory columns, Type K25/45, equipped
with cooling jackets were used for packing the supporting
materials. The eluates from the columns were monitored
either at 280 nm by Gilson Medical Electronics' Adsorption
Meter and recorded by a recording milliammeter manufac-

tured by Esterline Angus Instrument Company or at 254 nm

19

an?

 

 

20

with a recording ultraviolet analyzer manufactured by
Instrumentation Specialties Company, Incorporated.

Protein solutions were dried from the frozen state on
a laboratory-constructed lyophilizer. LyOphilized protein
samples as well as some chemicals were dried in a tempera—
ture controlled Cenco vacuum oven in which vacuum was ob-
tained with a Cenco PresSovac 4 vacuum pump.

Laboratory-constructed Plexiglas electrophoretic cells
were used for polyacrylamide gel electrOphoresis. Power
was supplied by a Heathkit Model IP—32 and a Beckman/Spinco
constant voltage supplier.

A Perkin—Elmer Model 38—A Tiselius electrOphoresis
apparatus was used for free—boundary electrOphoresis. The
resistances of all buffers and protein solutions were
determined with an Industrial Instruments' Model RC con-
ductivity bridge.

Amino acid analyses were performed with a Beckman/
Spinco Model 120 C amino acid analyzer. A Beckman DK-2A
ratio recording spectrophotometer, equipped with quartz
cells,was used for all colorimetric determinations. All
viscosity measurements were made using a Hewlett Packard
Model 5901B Auto-viscometer equipped with Thompson's ther-
mostatic water bath and circulator, Model TV-40. The
densities of buffers were measured pycnometrically at con-
trolled temperatures.

A Beckman/Spinco Model E analytical ultracentrifuge

equipped with a RTIC temperature control unit and phase

21

plates as schlieren and interference diaphragms was used
for the sedimentation coefficient, diffusion coefficient,
molecular weight, initial protein concentration, and par-
tial specific volume determinations. A capillary-type,
synthetic—boundary cell was used for sedimentation studies,
and a double-sector cell was used for determining the par-
tial Specific volume and molecular weight. A capillary-
type, double-sector synthetic-boundary cell was used for
the determination of initial protein concentration and
diffusion coefficient. In all determinations, 12 mm filled
Epon centerpieces with quartz windows were used. An-D and
An-J Duralumin rotors were used for centrifugation and a
General Electric AH-6 mercury lamp served as the light
source.

Kodak metallographic and Type II—G Spectroscopic glass
plates were used for recording the schlieren and interfer-
ence patterns, respectively. A Nikon Model 6 Shadowgraph
microcomparator was used for measuring the recorded patterns.

A Brice-Phoenix Differential Refractometer Model BP-
2000-V was used to adjust the refractive index of buffers
for interference centrifugation and to check protein

concentrations.

Chemicals and Materials

 

The principal chemicals used in this research and the
suppliers are given below.

Tris-hydroxymethyl aminomethane (Sigma 121 and Trisima

 

 

.ll‘ul ll'i .

22

Base) was used as a primary standard and Sigma 7-9 was used
to prepare buffers. Both were obtained from Sigma Chemical
Company. Diethylbarbituric acid and sodium diethylbarbitu—
ric acid for veronal buffer, monosodium phosphate and di-
sodium phOSphate for phosphate buffer, and monobasic potas-
sium phOSphate, potassium chloride, potassium sulfate, po-
tassium citrate, sodium citrate, anhydrous calcium chloride,
magnesium citrate hydrate, potassium carbonate and potassi-
um hydroxide for simulated milk salt solution were purchased
from Fisher Scientific Company.

p-Dimethylaminobenzaldehyde, 2-thiobarbituric acid and
N,N,N',N'-tetramethylethylenediamine were obtained from
Eastman Organic Chemicals. Reagent grade ammonium sulfate
and urea were purchased from Fisher Scientific Company.

Cyanogum 41 used in polyacrylamide gel electrOphoresis
was purchased from E. C. Apparatus Company. Ammonium per-
sulfate was obtained from Baker Chemical Company and 2-
mercaptoethanol was acquired from Fisher Scientific Company.

Bio Gel P-series and 1% solution of dimethyldichloro-
Silane in benzene were obtained from Bio-Rad Laboratories.
Sephadex G-series and Sepharose 6B were obtained from
Pharmacia Fine Chemicals. Cellulose N,N-diethylaminoethyl
ether was acquired from Fisher Scientific Company.

Fucose, galactosamine and glucosamine hydrochloride
‘were obtained from Nutritional Biochemicals Corporation.
Galactose was purchased from Pfanstiehl Laboratories,

Incorporated and mannose from Fisher Scientific Company.

 

A.“

 

 

 

State
Jerse
ately

while

Pare
me th
matc
PrOc
Ploy
Figu

fOll

After
was ac

23

Triphenyltetrazolium chloride was obtained from Eastman
Organic Chemicals and penicillin "G" from Allied Chemicals
Corporation. N—acetyl neuraminic acid, tryptOphan, and
streptomycin sulfate were acquired from Calbiochem.

All other chemicals used in this study were of reagent

grade.

Preparative Procedures

The milk used in this study was obtained from Michigan
State University dairy herd which consisted of Brown Swiss,
Jersey and Holstein cows. All milk was collected immedi-

ately at the afternoon milking and separated immediately

while still warm.

Prgparation of Euglobulin and Pseudoglobulin

The euglobulin and pseudoglobulin fractions were pre-
pared by combining the ammonium sulfate precipitation
method described by Smith (1946a; l946b) and column chro—
matographic technique using Sepharose 6B. The isolation
procedure for euglobulin and pseudoglobulin fractions em-
ployed in this study is presented diagrammatically in
Figure 1. Details of this procedure are described in the

following sections.

Precipitation gt Immunoglobulins with Ammonium Sulfate.

 

.After the fat phase was separated, the resulting skim milk
*was adjusted to pH 4.6 with 0.1 N HCl and the precipitated

casein was removed by filtering through double layered

 

m5

PRE

EUC

24

WHOLE RAW MILK

 

 

 

Separate
l '~l
SKIM MILK CREAM
Adjust to pH 4.6 with l N HCl
Filter
CASEIN ACID WHEY
Adjust to pH 6.5 with 1 N NaOH
Add ammonium sulfate to 0.5 satn
Centrifuge at 2000 x g for 15 min
CRUDE GLOBULINS SUPERNATANT

Make to 3% protein cone in deionized H20
Adjust pH to 4.6 with 1 N HCI

Add ammonium sulfate to 0.25 satn
Centrifuge at 25000 x g for 15 min

1

PRECIPITATE SUPERNATANT

 

 

Filter

Adjust to pH 6.0 with 1 N NaOH
Add ammonium sulfate to 0.4 satn
Centrifuge at 25000 x g for 15 min

 

 

I 7
CRUDE IMMUNOGLOBULINS SUPERNATANT
Make to 3% protein cone in Tris buffer, pH 7.0
Dialyze
Filter

Sepharose 68 column Chromatography

I I

EUGLOBULIN PSEUDOGLOBULIN

 

 

Figure 1. Schematic for the fractionation of
euglobulin and pseudoglobulin from cow's milk.

LIII? _ .

 

ti:

25
cheese cloth. The acid whey was adjusted to pH 6.5 with

0.1 N NaOH, and solid ammonium sulfate added to 0.5 satura-
tion to salt-out the crude globulin fraction. This precip-
itate was collected by centrifugation at 2000 x g for 15
minutes and redissolved to about 3% protein concentration
in deionized water. The pH was adjusted to 4.6 and ammo-
nium sulfate added to 0.25 saturation. After centrifuging
at 25,000 x g for 15 minutes to remove a suspended precip-
itate, the supernatant was filtered through a thick layer
of glass wool. The immunoglobulins were precipitated from
this supernatant with ammonium sulfate added to 0.4 satu-
ration at pH 6.0. The precipitated globulins were col-

lected by centrifugation at 25,000 x g for 15 minutes.

Separation gt Euglobulin and Pseudoglobulin ty Gel

 

 

Filtration. The principle of gel filtration employing

 

Sepharose 6B was used for separating euglobulin and pseudo-
globulin fractions from the enriched immunoglobulin
fraction.

Sepharose 6B has a molecular weight exclusion limit of
approximately 400,000. The column support was washed with
deionized water three times followed by equilibration with
0.02 M tris-HCl buffer, pH 7.0, before packing the column.
Chromatographic columns (2.5 x 45 cm) equipped with up-flow
adaptors were filled with eluting buffer. A funnel was
attached on tOp of the column and the equilibrated beads
were poured into the column with constant stirring. Fol-

lowing the formation of a thin layer of beads at the bottom

*i:

 

 

26

of the column, the outlet was Opened to allow the bed to
pack more tightly and to complete the packing. The final
column height was 30 cm. A void volume of 60 ml was deter-
mined by eluting a solution of Blue Dextran 2000 through
the column. The flow rate was approximately 200 ml/hr.

The immunoglobulin fraction obtained by salting-out
with ammonium sulfate was dissolved in 0.02 M tris-HCl
buffer, pH 7.0, at about 3% protein concentration and dia-
lyzed against a large volume (4 l) of the same buffer for
48 hr with constant stirring at 3° C. The dialyzing
buffer was changed every 12 hr.

The dialyzed protein solution was filtered through a
thick layer of glass wool and a 10 m1 aliquot of this solu-
tion was applied to the column by siphon action through the
bottom of the column. The column effluent was monitored at
254 nm. Each fraction was collected separately and concen-

trated by pervaporation to one quarter of its volume.

Purification gt Euglobulin and Pseudoglobulin Frac-

 

 

tions. The euglobulin and pseudoglobulin fractions sepa-
rated by gel filtration were purified using an anion ex-
change cellulose column chromatographic technique. N,N-
Diethylaminoethyl ether—cellulose (DEAE-cellulose) was
first soaked in 1 N NaOH for an hour to remove yellow im-
purities and to ensure complete regeneration. The regen-
erated adsorbent was washed with a large volume of deion-
ized water to remove NaOH, following several volumes of

0.02 M phosphate buffer (pH 7.0). The resulting suspension

27

was allowed to stand for 30 min to permit settling of large
cellulose particles. The fine particles remaining in the
supernatant were discarded. The suspension of sedimented
particles was poured as slurry into a Sephadex chromato-
graphic column (2.5 x 45 cm). The adsorbent was allowed to
settle under the flow conditions induced by gravity, a
filter disc was placed on the tOp of the column to prevent
erosion of the supporting material by the incoming buffer
solution. The adsorbent was further compacted by nitrogen
pressure at 5-6 p.s.i. until the cmflxmu1height.was constant--
approximately 30 cm. The column was washed with the eluting
buffer at pH 7.0 to obtain equilibrium.

Approximately 10 ml of the pervaporated protein solu-
tion was loaded on the column and eluted with 0.02 M phos-
phate buffer. The elution pattern was monitored at 254 nm
and the flow rate adjusted to about 200 ml/hr. The eluted,
purified proteins were desalted by passing the column cut
through a Bio Gel P—lO column (5.5 x 30 cm) equilibrated
with deionized water, 1y0philized, and stored at -15° C in
a tightly sealed brown bottle.

The 1y0philized proteins were dried in a vacuum oven
at room temperature over P205 for 24-48 hr before any
chemical and physical analyses were conducted. Dry weights

were used as the concentration basis for all samples.

Preparation of Washed Cream

Washed cream was prepared according to a method

28
described by Sw0pe (1968). After separating the cream, it

was diluted with warm (40° C) deionized water (1:3 dilution)
and reseparated. This process was repeated five times to
remove all of the whey proteins. Aliquotes of cream and
corresponding washings were collected after each separation
to check the amounts of whey proteins left in the cream.
This wash:d cream was preserved for subsequent experiments
by adding penicillin "G" and streptomycin sulfate at the
ratio of 50 mg and 5,000 units per liter, respectively.
Samples taken from skim milk, whey and each cream
washing were dialyzed against deionized water for 24 hr
with constant stirring to remove milk salts. Following
lyophilization, 5% suspensions of the residues were ana-
lyzed for whey proteins by polyacrylamide gel electropho-

resis according to a method described by Melachouris (1966).

Chemical Methods

 

Nitrogen

 

A micro-Kjeldahl apparatus was used for the nitrogen
determination. The digestion mixture consisted of 5.0 g

CuSO '5H 0 and 5.0 g SeO in 500 m1 of concentrated sul-

4 2 2

furic acid. Approximately 15 mg of dried protein in du-
plicate for each sample were digested with 4 ml of the
digestion mixture over a gas flame for 1 hr. After cooling,
1 ml of 30% H202

continued for one additional hour. Each digestion flask

was added to each flask and digestion

was cooled and rinsed with 10 ml of deionized water. Then,

29

each digested mixture was neutralized with approximately
25 ml of a 40% sodium hydroxide solution. The released
ammonia was steam distilled into 15 ml of a 4% boric acid
solution containing five drOps of indicator. The indicator
consisted of 400 mg of bromocresol green and 40 mg of
methyl red in 100 ml of 95% ethanol. Distillation was
continued until a final volume of 60 ml of the solution was
collectel in the receiving flask. The ammonia-borate com-
plex was titrated with 0.020 N HCl which had been standar—
dized with trishydroxymethyl aminomethane serving as a
primary standard.

The average recovery for a tryptophan standard was

98.83%.

Phosphorus

 

PhOSphorus was determined in duplicate for each sample
of protein according to the method described by Sumner
(1944). About 10 mg of dried protein were digested with
2.2 ml of a 50% sulfuric acid in Pyrex test tubes. Diges—
tion was carried out for 20 min on a sand bath heated by an
electric heater at a temperature of 160 to 170° C until the
protein was completely charred. After cooling, 1.0 ml of

30% H was added to each test tube and the heating con-

202
tinued for 15 min. This step was repeated to obtain color-
less solutions. The complete removal of all remaining H202

residue is very important to determine phosphorus content

correctly. The digested mixture was cooled and transferred

 

‘l

 

30

to a 25 ml volumetric flask. Five milliliters of a 6.6%

(NH MoO -4H 0 solution and enough deionized water were

4)6 24 2

added to give a volume of approximately 15 ml. Then 4 ml

4-7H20 1n 50 ml

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

of a freshly prepared solution of 5 g FeSO

quickly mixed. The volumetric flask was made up to volume
with deionized water and mixed. Color was allowed to
develOpe for 30 min. Percentage transmittance was read at
660 nm with a Beckman DK—2A spectrophotometer.

Ten milliliters of a stock solution containing 1.3613

g of KH PO4 dissolved in 1,000 ml of deionized water were

2
diluted to 100 ml to give 0.031 mg phosphorus/ml of stand-
ard solution. A standard curve covering the range of zero

to 0.28 mg phOSphorus was prepared.

Carbogydrate

 

The components comprising the carbohydrate moieties of
euglobulin and pseudoglobulin fractions were identified by
a paper chromatographic technique. Approximately 25 mg of
dried sample were placed in a 20 ml ampoule and 10 ml of
2 N hydrochloric acid were added. After the solution was
frozen in a dry ice-ethanol bath, the ampoule was evacuated
to remove dissolved air from the sample and immediately
sealed. The sealed ampoule was placed in a 110° C oil bath
for five hours.

The partial hydrolysate was transferred to a 50 m1

round-bottom flask fitted to a rotary evaporator and dried.

 

Ill x411}: llIIII

31

The residue was taken up in a small amount of deionized
water and redried. This process was repeated two more
times. The final residue was dissolved in 10 ml of deion-
ized water and passed through a 2 x 20 cm column of Dowex
resin. The column was prepared as follows: Dowex 1 x 8 in
C1- form was washed with l N NaOH to convert to the OH-
form. Then, the resin was converted to acetate form by
washing with l N acetic acid. Next, the resin was washed
with deionized water until the pH of the washing was
between 5.0 and 9.0. A second resin consisting of Dowex
50 x 8 was converted to the H+ form by washing with l N HCl
and rinsing with deionized water as above. The column was
packed with Dowex 50 x 8 (H+ form) in the bottom half and
with Dowex l x 8 (acetate form) in the tOp half.

The eluate collected from this column was lyOphilized
and extracted with 10 ml of redistilled pyridine at 100° C
for 20 min according to the procedure described by Block gt
gt. (1958). The resulting pyridine mixture was cooled,
filtered, and the solvent removed with a rotary evaporator
at 40° C. The resulting residue was dissolved in 2 ml of
10% isopropanol. This solution of unknown carbohydrates
was spotted on Whatman No. l (10 x 42 cm) chromatography
paper and developed as an ascending chromatogram for 20 hr
with a butanol-acetic acid-water (4:1:5) solvent system
(Bezkorovainy, 1963). Authentic standards of fucose,
glucose, galactose, mannose, glucosamine, and galactosamine

were analyzed concurrently with the unknown samples.

2

II IIl.l4

 

 

32

The carbohydrate constituents were detected by Spraying
the dried chromatogram (60° C for 15 min) with a mixture of
equal volumes of a 2% aqueous solution of triphenyltetra-
zolium chloride and 1 N NaOH (Block gt gt., 1958). The
Sprayed chromatograms were placed in a moist atmOSphere at

about 60-70° C until the red spots appeared.

Fucose

The fucose content of the protein samples was deter-
mined in duplicate with a method described by Dische and
Shettles (1948).

Approximately 2 mg of dried protein samples were
placed in test tubes (15 x 150 mm) and 5 ml of a 95% etha-
nol was added. After mixing, the tubes and their contents
were centrifuged for 15 min, the supernatant decanted and
the precipitates suspended in 5 ml of 95% ethanol. The
solutions of protein were again centrifuged and the super-
natants decanted. The precipitated proteins were dissolved
in 1 m1 of 0.1 N NaOH and 4.5 ml of ice cold H2S04-H20
mixture (6 volumes of concentrated c.p. H2804 and 1 volume
of H20) were added. The contents were well mixed while
maintaining a low temperature in an ice bath. Then, the
tubes and their contents were heated for exactly 3 min in
a boiling water bath, then cooled in tap water. To this
mixture 0.1 ml of the cysteine reagent (3 g of cysteine

hydrochloride in 100 ml of water) was added and the con-

tents mixed immediately. The addition of the cysteine

 

III‘ III.

33

reagent was omitted from one of the duplicate samples to
correct for nonspecific color develOpment. After 60 to 90
min at room temperature, Optical density readings were made
at 396 and 430 nm with distilled water set at zero.

The fucose content of the proteins was calculated from
the differences in the readings obtained at 396 and at 430
nm after correcting for the nonspecific color increment,

i.e., samples without cysteine added:

(OD396'OD430)5 "

(OD396-OD430)Std

(00 -00 )
396 430 b x 0.02 x 1000

 

= mg of fucose per 100 g of protein

The standard solution was prepared by dissolving 20 mg

of fucose per liter of distilled water.

Hexose

Hexose analyses were performed colorimetrically by the
method of Dubois gt gt. (1956).

Two to three milligrams of protein were placed in a
test tube and 3 ml of deionized water and an equal volume
of 5% phenol solution were added and mixed. Then, 15 ml of
concentrated sulfuric acid were added rapidly while stir-
ring simultaneously with a Mini-shaker to obtain maximum
mixing and heat generation which is critical to the repro-
ducibility of the assay. After 10 min of standing, the
tube was stirred again and placed in a water bath at 25 to

30° C for 30 min. The percentage transmittance was

34

monitored at 490 nm.
A standard curve was constructed covering the range of
zero to 200 ug of galactose-mannose (1:1, w/w) per 3 ml of

water.

Hexosamine

 

The method described by Johansen gt gt. (1960) was
used to determine hexosamine content of the proteins.

About 3 mg samples of protein were placed directly
into 5 ml ampoules. After adding 1 ml of 4 N HCl, the
samples were frozen in a dry ice—ethanol bath, evacuated,
refrozen and sealed under vacuum. The samples were hydro-
lyzed for 6 hr at 100° C in a hot air oven. After cooling,
the hydrolyzed samples were transferred to distillation
flasks. The empty ampoules were rinsed with 1 m1 of 4N
NaOH, followed by two 1 ml rinsings with deionized water.

The acetylacetone reagent was prepared by dissolving
1 ml of freshly distilled acetylacetone in 25 ml of 1 M
Na2CO3 solution plus 20 m1 of water. The pH was adjusted
to 9.8 and the volume was made up to 50 ml. This solution
was used within 30 min. Ehrlich's reagent was prepared by
dissolving 2 g of recrystallized p—dimethylaminobenzalde-
hyde (Rondle and Morgan, 1955) in absolute ethanol contain-
ing 3.5%cnfconcentratedHCl. This solution was diluted to
a final volume of 250 ml.

To each of the distillation flask containing the

hydrolyzed sample were added 5.5 ml of the acetylacetone

 

35

reagent. The final pH of this mixture must be maintained
between 9.5 and 10.0. The flasks were tightly stOppered
and heated in a boiling water bath for 20 min. Then, each
cooled flask was heated with a Bunsen burner until 2.5 m1
of the distillate containing the steam-volatile chromogen
was condensed in 7.5 ml of Ehrlich's reagent. The volu-
metric flasks were stOppered and the contents mixed.

After 30 min, the transmittance (%) of the solutions was
read at 548 nm with the spectrophotometer.

Standard glucosamine and galactosamine solutions (25
pg) were treated in the same manner to check the recovery.
The average recovery of the duplicate runs was 98.79%.

Equal amounts of glucosamine and galactosamine were
combined and used to construct a standard curve which spans
the range of zero to 50 pg carbohydrates per 3 ml. The
blank consisted of 1 ml of 4 N HCl, 1 ml of 4 N NaOH and

2 ml of deionized water.

Sialic Acid

 

Warren's (1959) thiobarbituric acid method as modified
for milk proteins by Marier gt gt. (1963) was adapted for
the sialic acid determination.

Approximately 1.5 mg of dried proteins were placed in
test tubes. Following the addition of 0.5 ml of 0.1 N
sulfuric acid to each tube, digestion was performed at 80°
C for 40 min to release the sialic acid. To each hydro-

lyzed sample was added 0.1 m1 of the periodate solution

36
(0.2 M meta-sodium periodate in 9 M phosphoric acid) and

the mixture was incubated at 25° C for 20 min. Following
the addition of 1 ml of arsenite solution (10% sodium
arsenite in a solution containing 0,6 M sodium sulfate and
0.1 N sulfuric acid), the tubes were shaken until the solu—
tion became colorless. Three milliliters of a 0.6% of re-
crystallized 2—thiobarbituric acid solution in 0.5 M sodium
sulfate were added and the tubes shaken, covered tightly
and placed in a boiling water bath for 15 min. After cool-
ing, 7 ml of cyclohexanone were added and the tubes shaken
vigorously. The contents were centrifuged in a clinical
centrifuge for 10 min to obtain a clear cyclohexanone phase.
The transmittance (%) was measured at 549 nm.

A standard curve was contructed using N-acetyl neura-

minic acid in the range of zero to 50 pg in 0.5 ml of water.

TryptOphan

 

TryptOphan content of a protein must be determined
separately from the rest of the amino acids since it is
labile to acid hydrolysis.

In this study tryptOphan was analyzed spectrOphoto-

metrically as described in Procedure W by Spies (1967).
A 3 mg sample of protein was weighed directly into a 1.5 ml
glass vessel. To each sample were added 100 pl of freshly
prepared pronase solution, containing 10 mg of pronase per
milliliter of 0.1 M phosphate buffer (pH 7.5), and a drOp

of toluene. Each vessel was placed in a vial, and the

 

I II: Jl‘ \\ .

37

vials were stOppered and then incubated for 24 hr at 40° C.
After cooling, the vessels were placed in 25 ml Erlenmeyer
flasks containing 9.0 m1 of 21.1 N sulfuric acid and 30 mg
p-dimethylaminobenzaldehyde which had been prepared immedi-
ately before use. To each vessel was added 0.9 ml of a
0.1 M phosphate buffer solution. The vessels were tipped
over and the contents were quickly mixed by gentle swirling.
The flasks were placed in the dark at room temperature for
6 hr. After adding 0.1 ml of 0.045% sodium nitrate solu—
tion, the reaction mixtures were shaken and the color was
allowed to develope for 30 min in the dark at room tempera-
ture. The transmittance (%) was read at 590 nm.

Duplicate samples of pronase solution, without sample
protein, were treated and analyzed as described above.
The tryptophan content of pronase solution was subtracted
from the total tryptophan content. The blank solution
contained everything but protein and pronase.

A standard curve was prepared using zero to 120 pg of
tryptOphan in phosphate buffer solution as described in

Procedure E and F by Spies and Chambers (1948).

Amino Acid

 

The amino acid analyses were performed on 20 and 70 hr
hydrolysates of the protein employing a Beckman Amino Acid
Analyzer Model 120 C (Moore and Stein, 1954; Moore gt gt.,
1958).

About 4 mg portions of the dried proteins were weighed

38
directly into 10 ml ampoules. Six milliliters of 6 N HCl

were added to the ampoules. The contents of the ampoules
were frozen in a dry ice-ethanol bath, evacuated with a high
vacuum pump, allowed to melt slowly to remove gases, re-
frozen and sealed with an air-prOpane flame. The sealed
ampoules were placed in an oil bath preheated to 110° C in
an oven regulated at this temperature. After either 20 or
70 hr hydrolysis the ampoules were removed from the oil bath
and cooled to room temperature (25° C).

In order to check the transfer losses, lml of 2.5 pM
norleucine solution was added to each ampoule before trans-
ferring the hydrolysate to a pear-shaped evaporating flask.
After evaporation to dryness on a rotary evaporator each
residue was taken up in a small amount of deionized water
and redried until all the remaining hydrochloric acid resi-
due was removed. Finally, the dried hydrolysate was dis-
solved in 0.067 M citrate—hydrochloric acid buffer, pH 2.2,
and the volume was made up to 5 ml. An aliquot of 0.2 ml
was removed from each sample for the analysis.

At least two or more standard amino acid mixture runs
were made with the same buffer and ninhydrin solution with-
in a week.

The amino acid composition of the protein samples was
expressed as gram residues of amino acids per 100 g protein,
number of amino acid residues per mole of methionine resi-
due, and moles of amino acid residues per 1000 moles of

total amino acid residues.

39

ngsical Methods

 

Free—boundary Electrophoresis

 

A Perkin-Elmer Model 38—A Tiselius electrOphoresis
apparatus was used to determine the electrOphoretic mobili-
ties of the protein fractions in free solutions.

ElectrOphoretic mobilities were calculated for the
ascending and descending patterns using the following

equation:

d a k

 

t i m r

where g is the electrophoretic mobility in l x 10'.5 cm2

sec-l volt-1, g the distance the boundary migrated from the
initial boundary in cm, g the cross sectional area of the
electrOphoretic cell in cm2, t the conductivity cell con-
stant, t the time of electrOphoresis in seconds, t the
current applied in amperes, t the resistance of the protein
solution or buffer used in dialyzing the protein solution
in ohms determined in a conductivity cell at the same
temperature as the electrOphoresis run, and g_the magnifi-
cation factor of the Optical system.

Veronal buffer, pH 8.6, p = 0.1, was used as the sol-
vent system for the electrophoretic mobility determinations.
Electrophoretic mobilities determined at pH 4.0, 5.0, 6.0,

7.0, and 8.6 (p = 0.2) in the buffers described by Miller

and Golder (1950) were used to determine isoelectric points.

 

40

The composition of these buffers is presented in Appendix.
The isoelectric points were estimated from a plot of the
average ascending and descending mobilities against the pH
values.

Protein samples were dissolved in the buffers at the
concentration of approximately 1.0 to 1.5% and the solu-
tions were dialyzed against 600 m1 of the same buffer for
24 hr at 2—3° C and with constant stirring. All free—
boundary electrOphoretic analyses were performed at 2'1° C.
The boundaries in the Tiselius cell were photographed both
at the beginning and termination of the run.

Polyacrylamide Gel ElectrOphoresis
in a Discontinuous Buffer System

 

 

The procedure for preparing and performing the discon-
tinuous acrylamide gel electrOphoresis was adOpted from the
method described by Melachouris (1969). The discontinuous
gel system, consisting of a running gel and a spacer gel,
was prepared by using two working solutions. The running
gel solution was prepared by dissolving 45 g of Cyanogum 41,
a mixture of acrylamide and N,N'-methylenebisacrylamide, in
0.38 M tris-HCl buffer, pH 8.9, and making up the volume to
500 m1. To this solution, 0.5 ml of N,N,N',N'-tetramethyl—
ethylenediamine (TEMED) was added. Cyanogum 41 at 5% con-
centration in 0.062 M tris—HCl buffer, pH 6.7, was prepared
as the Spacer gel. The catalyst, TEMED, was then added in
the ratio of 0.1 ml TEMED per 100 m1 gel solution. The

working gel solutions were filtered and stored at 2-3° C.

41

All solutions were brought to room temperature before use.

The running and spacer gels were cast in a Plexiglas
gel bed (26 x 12 x 0.4 cm). A Plexiglas divider was placed
15 cm from one end of the gel bed and silicon grease used
to prevent seepage of the gel-forming solution. The large
area of the gel bed was filled with 190 m1 of running gel
solution containing 2 ml of a freshly prepared 10% (w/v)
solution of ammonium persulfate in deionized water. The
divider was removed after polymerization of the running gel
solution. Then, the smaller area of the gel bed was filled
with 90 ml of the spacer gel solution containing 1 m1 of
the ammonium persulfate solution. The slot former (l x 0.1
x 0.3 cm) was inserted into the Spacer gel solution 0.5 cm
from the edge of the running and spacer gel interface.

The protein samples were dissolved in the spacer gel
solution diluted 1:1 with deionized water at approximately
5% protein concentrations. BromOphenol Blue was added to
each sample slot as a marker.

Approximately 15 pl samples were applied to the slots
with the aid of a micro-syringe and the slots were covered
with mineral oil. The gel was covered with Saran Wrap to
reduce evaporation and connected to electrode vessels
filled with 1,600 ml of 0.046 M tris-glycine buffer, pH 8.3.
Platinum electrodes were inserted into the buffer tanks and
electrophoresis was carried out at 15° C at a constant
voltage of 180 to 200 volts (instrument) until the buffer

front migrated 12 cm from the sample slots.

42

Upon completion of the electrOphoretic run, the gel
was removed from its frame and stained for 10 min in a dye
solution consisting of 250 ml of water, 250 ml of methanol,
50 ml of glacial acetic acid and 5 g of amido black 10B
(naphthol blue black). The excess dye in the gel was re-
moved electrically in an electric destaining cell contain-
ing 7% acetic acid.

The electrOphoretogram was photographed using a

Polaroid MP-3 Land Camera for a permanent record.

Ultracentrifggation

 

Protein Concentration. Protein samples were weighed

 

on a microbalance and dissolved in apprOpriate solvents.
Following dialysis against the solvent, the concentration
was rechecked by measuring the refractive index of the
solution with a differential refractometer. The protein
concentration was determined from a standard curve of re-
fractive index differences and concentration of protein.
In many cases, the concentration was determined by
measuring the area of the schlieren pattern for the initial
boundary formed in a double-sector cell. The amount of
protein was then determined from a plot of area versus
known concentration of protein from the data obtained in a

similar manner.

Density. The solvent densities were measured with

25 ml pycnometer at 20° C. The densities of solutions were

43

calculated according to the relation given by Fujita (1962):

psolution = psolvent + (l — Vpsolvent)°C

where g is the density in g cm-3, i the partial specific
volume of the protein in ml g-l, and 9 its concentration in
g cm-3. The pycnometers were calibrated with freshly

boiled, redistilled water at 20° C. The densities of the

solvent used are presented in Appendix.

Viscosity. The viscosities of solvent and Solution
were measured in a standard Ubbelohde Dilution Suspended
Level Type ASTM D 445 viscometer. The viscosity equation

applicable to capillary type viscometers is written as

follows:

_ Me

n = Apt t

where g is the absolute viscosity in g sec"1 or Poise, g
the viscometer constant, t the efflux time in seconds, g
the kinetic energy coefficient, and 8 the kinetic energy

constant.

To calibrate a capillary viscometer, the efflux times
of freshly boiled, redistilled water at two temperatures
were measured. With the efflux times measured and the
densities and viscosities known for the two liquids, the
viscometer calibration constants g and mt_were calculated

from the above equation.

44

The absolute viscosity for an unknown solvent or solu-
tion was then calculated from the factors g and gt and the
measured efflux time using the same equation. All solu-
tions were filtered through a fine sintered glass filter
before measuring the efflux time.

The relative viscosity was then calculated according

to the relation:

 

 

 

where g, t, g are the viscosity, efflux time, and density
of the solution and gt, t2, &2 represent the corresponding
values of the solvent. The viscosities of the solutions
used relative to those for solvent are presented in

Appendix Table 1.

Partial Specific Volume. The partial Specific volume

 

for the protein was determined by performing two parallel
sedimentation equilibrium experiments of a given substance
in H20 and D20 solutions at the same temperature and pro—
tein concentration (Edelsteine and Schachman, 1967).

Two double-sector cells were used for this purpose.
The solution containing 0.1 mg of protein in 0.1 M veronal

buffer in H 0, pH 8.6, was placed in one compartment of a

2
double-sector cell and the buffer in the other compartment.
The second cell contained the analogous solution of pro-

tein in the above buffer prepared with 90% D20 in one

compartment and the correSponding buffer in the other.

 

L.___ .

45

For this experiment an ultracentrifuge speed of 16,000 rpm
was used and the resulting interference pattern was photo-
graphed after 24 hr. The fringe measurements were made
from the recorded pattern and the data were plotted as the
logarithm of the fringe displacement against the square of
the distance, 5, from the axis of rotation. The partial
specific volume was then calculated according to the

equation:

2 2
k - [(dlnc/dr )DZO/(dlnc/dr )HZO]

o - 0 2 2
D20 H20 [(dlnc/dr )DZO/(dlnc/dr )HZO]

where t is the ratio of the molecular weight of the protein
in the deuterated buffer to that in the nondeuterated one

and the term, dlnc/drz, represents the slope of the plot.

Diffusion Coefficient. The diffusion coefficient was

 

 

determined in a double-sector synthetic boundary cell at a

low centrifuge speed of about 4,000 rpm and computed by

using the relation:

2 ‘ 2
D = A H 1 4st 1 - w st
app (/)</ )< )
where Eapp is the apparent diffusion coefficient in cm2
sec-l, g the area enclosed by the sedimentation boundary

curve above its base line in cmz, g the maximum height of
the boundary in cm, t the time measured from the start of

centrifugation in seconds, and g the angular velocity of

46

rotation in radians sec-l (Lamm, 1929).

The sample was dissolved in apprOpriate solvents at
different concentrations. The cell was filled with 0.15 ml
of the solution in one compartment and the solvent in the
other. Schlieren photographs were taken at preset time
intervals. After measuring the plates, the data were plot-

ted as (l/4n)(A/H)2 versus t to obtain the apparent diffu-

 

sion coefficient (Dapp) which is equal to the slope of the
line. The observed diffusion coefficient (Dobs) was ob—
tained by plotting the values of apparent diffusion coeffi-
cient against the protein concentrations. The intercept of
this linear plot at infinite dilution is the observed
diffusion coefficient.

Since the value of observed diffusion coefficient ob-
tained as above relates to the given solvent and tempera-
ture, it is common practice to report the diffusion coeffi-
cient corresponding to a temperature of 20° C in a solvent

with the viscosity of water, The correction of the

D20,w'
Observed diffusion coefficient to this standard condition

was made by using the following equation:

D = Dobs. [293/(273 + t)]'(ns/nw)(nt/n20)w

20,w
where t is the temperature of the diffusion experiment in
degrees centigrade, (ns/nw) the relative viscosity of
solvent to that of water, and (nt/nzo)w the relative vis-

cosity of water at the temperature of the experiment to

47
that at 20° C.

Sedimentation Coefficient. The sedimentation-velocity
experiments were carried out at 20° C using a rotor speed
of 59,780 rpm (259,700 x g). The low sedimentation coeffi-
cients observed for the proteins in the presence of disso-
ciating agents necessitated the use of the synthetic bound-
ary cell in this study. Accurate sedimentation coefficients
are Obtained with this cell for solutes whose coefficients
are less than one Svedberg unit because the problem of
restricted diffusion at the meniscus is eliminated (Schach—
man, 1959). Furthermore, the concentrated guanidine hydro-
chloride solution forms gradients at the meniscus making
the observation of the protein boundary difficult unless a
synthetic boundary cell is used. Buffers with an ionic
strength of 0.1 or greater were used to dampen the charge
effects inherent to proteins in solution (Schachman, 1957).

The sedimentation coefficient is defined as the velo—

city Of the sedimenting molecules per unit field as shown

by the equation:

1 dx

 

wzx dt

where t is the distance of the boundary from the axis of
rotation in cm, t the sedimenting time in seconds, and g
the angular velocity in radians sec-1. By integrating the

above equation, the following relation is Obtained:

48

2.303 log x

s:
w2 t

 

Upon plotting the logarithm of the distance against
time, the sedimentation coefficient may be obtained from
the slope of the line using the following formula:

_ 2.303 60
S = 1 x 10 13 sec = / -slope

(2n-rpm/60)2

 

Sedimentation coefficients are usually reported as

S , which is the value the protein would have in a sol-

20,w
vent with the density and viscosity Of water at 20° C.
Therefore, the observed sedimentation coefficients were
corrected to this standard condition according to the

following equation:

 

S = S . ~' nt AI! ns ; 1 - szo'w\
20,w obs I ‘1 If /

l"20/\“° I1 - Got

where the first term (nt/nzo) is the ratio of the viscosity
Of water at the experimental temperature to that at 20° C,
the second term (nS/no) the relative viscosity of solvent
to that of water at any temperature, and the terms 920,w
and ot the densities Of water at 20° C and the solvent at
the experimental temperature, respectively. The partial
specific volume, 6, of the protein was assumed the same in

all solvent systems used in this experiment.

49
The standard sedimentation coefficient at infinite

dilution, 830 w' was obtained by plotting the values of

I

8 versus the concentrations of protein and extrapolating

20,w
to zero concentration (infinite dilution).

Low-speed Sedimentation-equilibrium. Molecular weights

 

were determined using the low-speed short-column sedimenta-
tion-equilibrium technique described by Van Holde and Bald-
win (1958). This method covers the protein concentration
range of 0.2 to 1.2%. Analyses were carried out in double-
sector cells using a column depth of l to 2 mm. The rotor
Speed was estimated from the relation:

M (1 - 6p) 02 b2 — a2

 

RT 2

where g'is the estimated molecular weight of the protein, 3
the gas constant, 3 the absolute temperature, and g_and g
the distances from the axis of rotation to the air-solution
meniscus and to the bottom of the solution, respectively.
The time required to achieve equilibrium condition was

approximated by the equation:

 

where g'is the diffusion coefficient and to 1% the time
required to achieve the concentration difference across the

solution column within 0.1% of the equilibrium value.

$5"!

50

The experiment was conducted as follows: first, an
amount of fluorovarbon FC 43 oil was introduced into both
sectors of the cell to provide a false bottom; secondly,
enough protein solution was added 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 solution. The solvent column
extended above the air-solution meniscus and below the oil-
solution meniscus of the protein solution.

The schlieren pattern was photographed and measured,
and the apparent weight- and z-average molecular weights

were calculated according to the equations:

 

 

_ c(b) - c(a) 2 RT
M =
w,app _
c° b2 — a2 w2 (l - Vp)
_ RT [AY(b)/b] - [AY(a)/a]
zlapp - 2 ..
w (l - Vp) c(b) — c(a)
where Ew,app and Ez,app are the apparent we1ght- and 2-

average molecular weights, respectively, c(b)-c(a) the

 

difference in protein concentration between the bottom of
the solution and the air-solution meniscus, g° the initial
protein concentration, and 92121 and gttgt the net vertical
displacement of the gradient curve at the bottom of the
solution and that at the air-solution meniscus. The values

of both apparent weight- and z-average molecular weights are

$31

51
plotted against the protein concentrations and extrapolated

to infinite dilution to obtain the corresponding true molec-

ular weights, M° and M°.
—w -z

Creaming Studies

 

Most of the creaming property determinations were made
on a series of samples in which the fat content was kept as
uniform as possible to 3.6% according to the procedures

described by Dunkley and Sommer (1944).

Creaming PrOperties of Normal Milk

 

The creaming prOperties of normal milk under various
conditions were determined as reference to those of recom-
bined milk samples as well as the model systems employed in
this study. Both the cluster index and cream volume were
used in expressing the creaming ability.

Cluster tgggt. The clustering ability of the fat was
expressed in terms of a "cluster index". Creaming cells
with the inside dimensions of 76 x 20 x 1 mm were con-
structed with thin microscope Slides. Two cells were
mounted on a thick glass base plate.

In order to eliminate the decrease in clustering
properties resulting from storage Of milk at low tempera-
tures, 2-4° C, the Samples were prewarmed to 40° C prior
to the cluster index determinations. The procedures used
for the prewarming treatment and for the determination of

cluster index were as follows: approximately 200 m1 of

52
fresh raw milk were measured into rubber stOppered bottles

and held in a warm water bath at 40° C for 30 min with
gentle agitation. The specially designed creaming cells
were filled with warm milk and placed in a water bath at
4° C. The milk in the creaming cells was examined by
transmitted light at one minute intervals until the
clustering of the fat first became visible. The cluster
index was calculated by dividing into 100 the time in
minutes required for clustering to become visible. The
milk samples which did not show clustering within 100 min
were arbitrarily given a cluster index of zero.

Cream Volume. The volume Of cream layer formed on

 

milk under standardized conditions was used as a measure of
the creaming ability of milk sample.

The milk was first prewarmed in the same manner as for
the cluster index determinations. The milk was then trans-
ferred to 100 ml graduated cylinders and placed in a water
bath maintained at 4° C. The creaming time was measured
from the time the cylinders were placed in the 4° C bath.
The cream volumes were divided by the percentage of fat in
the milk in order to express the results as cream volume

percentages per 1% fat.

CreamingAPrOperties of Recombined Milk

The creaming properties of recombined milk samples were
determined in the same manner as described for the normal

milk samples. The recombined milk samples consisted Of

ES".

53
three time-separated skim milk and five time-washed cream

mixed in various combinations.

Creaming PrOperties of Model System

A model system consisting of purified euglobulin
fraction and washed cream in simulated milk dialysates was
developed to study the effects of protein concentration,
temperature, hydrogen ion concentration and ionic strength
on the creaming prOperties under controlled conditions.
The cluster index and cream volume were determined as de-
scribed before. The composition of Jenness and Koops'

(1962) milk salt solution is presented in Appendix.

531

RESULTS AND DISCUSSION

Preparative Procedures

 

Preparation of Euglobulin and Pseudoglobulin

The isolation procedure for preparing a crude immuno-
globulin fraction from normal cow's milk (Figure l) was
adapted essentially from Smith's (1946a) ammonium sulfate
fractionation method. By following his original procedure,
an electrophoretically homogeneous pseudoglobulin fraction
was Obtained by prolonged dialysis of an immunoglobulin
fraction against large volume Of distilled water. However,
the euglobulin fraction always contained a small amount of
pseudoglobulin even after repeated dialysis (3X). In addi-
tion to this pseudoglobulin contamination in euglobulin
preparations, the dialysis procedure is so tedious that
there exists a possibility for the denaturation ofthese
two proteins during the purification process. Furthermore,
the yields Of these proteins were so small that large quan-
tities Of milk had to be processed to obtain sufficient
*working material. Therefore, his method was modified to
obtain workable amounts of electrophoretically pure euglob-
ulin and pseudOglobulin fractions in a relatively shorter
time.

In this study, the final three steps of Smith's

54

SS1

55
procedure, i.e., 0.3 saturation of the crude immunoglobulin

fraction (Fraction D) with ammonium sulfate at pH 4.6 to
remove pseudoglobulin and other components (Fraction E),
0.4 saturation of the supernatant with ammonium sulfate at
pH 6.0 to obtain immunoglobulin fraction (Fraction F), and
exhaustive dialysis to separate euglobulin and pseudoglob-
ulin, were replaced by Sepharose 6B, DEAE-cellulose and
Bio Gel P-lO column chromatographic techniques. Among the
buffer systems tested for their effectiveness in separating
and purifying these proteins, the following systems were
found to be most effective: 0.01 M tris-HCl buffer, pH 7.0,
for the fractionation of crude immunoglobulins into euglob-
ulin and pseudoglobulin through Sepharose 6B columns and
0.01 M phosphate buffer, pH 7.0, in purifying the separated
immunoglobulin fractions on DEAE-cellulose anion exchange
columns. The desalting process was carried out with deion-
ized water through a Bio Gel P-10 column.

The elution pattern of the crude immunoglobulin frac-
tion through Sepharose 6B column is presented in Figure 2.
The first peak corresponds to euglobulin and the second to
pseudoglobulin. The two small peaks were not characterized.
Polyacrylamide gel electrOphoretic patterns of skim milk,
whey, crude immunoglobulin fraction, and euglobulin and
pseudoglobulin fractions purified through DEAE-cellulose
columns are presented in Figure 3. The free-boundary
electrOphoretograms of the purified euglobulin and pseudo-

globulin in veronallbuffer, pH 8.6, p = 0.1, are shown in

53"

56

h’
.
G
I
3

d
-
G"
I
3

 

P
G"
+

 

0.0. at 254 nm
'9

 

 

 

__J M
0 100 200 300
ElIlTION VOLUME, ml

Figure 2. Elution diagram of crude immunoglobulin
fraction through Sepharose 6B column with 0.01 M tris-
HCl buffer, pH 7.0: Peak 1, euglobulin; Peak 2, pseudo-
globulin; Peaks 3 and 4, unidentified.

ES‘I

57

t
C

I III..

.0

1|.
So...

d
a. q

frac-

1n

lobul

whey, crude immunog
in and pseudoglobulin:
lobulin fraction; C-euglobulin;

ilk,
d euglobul

Polyacrylamide gel electrophoretic

immunog

a V
c???
nope-h?

 

\.

33.3..

Figure 3.
patterns of skim m
tion and purifie
A—whey; B-crude
D—pseudoglobulin.

58
Figure 4. ElectrOphoretically pure euglobulin and pseudo-

globulin fractions were obtained after once passing the
eluates containing peaks 1 and 2 from the Sepharose 6B
column through DEAE-cellulose column (see Figures 3 and 4).
The yields of purified euglobulin and pseudoglobulin were
approximately 20 and 30 mg per liter of Skim milk,

resPectively.

Preparation of Washed Cream

 

The Effects gt Washingthe Cream. Removal of the whey

 

 

proteins from the cream is of paramount importance for stud—
ying the role of immunoglobulins in the fat globule clus-
tering. Due to the differences in density between the whey
and the cream, the whey proteins were adequately removed by
diluting the cream successively with water (1:3 dilution)
and reseparating. Their removal was checked by polyacryl-
amide gel electrophoresis on each dried washing. The gel
patterns are shown in Figure 5. Although equal amounts of
a 5% concentration of the dried materials were applied to
the gel slots, the band intensities decreased progressively
to the fifth washing. Undoubtedly, the erosion products
from the fat globule membrane, principally membrane lipo-
‘protein complexes, made up the majority of the material
recovered in the fourth and fifth washings. This material
neither migrates in the gel nor stains well with amido
black dye.

.The concentration of the whey proteins decreased

59

 

 

l L
‘ ’

 

3,000 sec, r=1o.97 volt cm"

EUGLOBULIN

 

1

3,600 sec, F=10.95 volt cm"
PSEUDOGLOBULIN

Figure 4. Free—boundary electrophoretic patterns of
purified euglobulin and pseudoglobulin in veronal buffer,
pH 8.6, ionic strength of 0.1.

60

 

'lk;

1m m1

sk
—second washing; D—third washing.

A-
Samples were applied at 5% concentrations.

de gel electrophoretic patterns

i
1k, whey and cream washings

Polyacrylam

Figure 5.
1m mi

of sk

; D

1ng

B—whey; C-first wash

61
considerably from the second to third washing and then

became constant over the next several washings. Hence,
three washings were sufficient to obtain a whey protein-
free cream. The wash medium used was warm (40° C) deion-
ized water. With this temperature of the wash medium, the
separations were essentially carried out at the temperature
Of freshly secreted milk, minimizing induced lipolysis
(Thompson gt gt., 1961). Although a saline—sucrose wash
solution preserves the membrane integrity better than de-
ionized water (Swepe and Brunner, in preparation), the need
for prolonged dialysis and the threat of bacterial growth
in this suspension precluded its use.

SwOpe (1968) pointed out that the membrane is suscep-
tible to erosion when employing dilution techniques to
remove plasma materials. However, washing cream three
times with warm deionized water (1:3 dilution) removed
the plasma materials adequately while restricting erosion

to acceptable levels.

The Effects gt Antibiotics gg Fat Globule Clustering.

 

 

 

As mentioned previously in experimental section, the washed
cream was preserved by adding streptomycin and penicillin
”G" at the ratio of 50 mg and 5,000 units per liter, re-
spectively. The gross effect of these antibiotics on the
creaming phenomenon was tested by measuring the cluster
index and the cream volume Of milk samples with or without

added antibiotics. As evident from the results in Table 1,

62
these antibiotics did not alter the creaming prOperties of

these milk samples.
TABLE 1. The effect of antibiotics on the creaming
properties of milk

m
% Cream Volumea After

 

 

sample 8:22?
1/2 Hr 4 Hr
Normal Milk
Untreated 8.3 11.8 9.0
TreatedC 8.6 11.4
Recombined Milk
Untreated 7.7 8.3
Treatedc 7.4 8.1
b
Model System
Untreated 4.3 4.4 3.6
Treatedc 4.4 4.1 3.7

 

aPer cent cream volume per 1% fat.
bEuglobulin concentration was 0.01%.
cAntibiotics, penicillin "G" and streptomycin sulfate,

were added to samples in the ratio of 5,000 units and 50 mg
per liter, reSpectively.

Chemical Analyses

 

Various chemical constituents of the purified euglob-
ulin and pseudoglobulin fractions were analyzed and the
results are summarized in Table 2. In addition, the corre-

sponding values reported by other investigators are given

 

fl I..iilll. 1|!“

 

Ill—Huvhf... c... a». 9.24.9... 1.: u

63

.Aammav mmam>o

.Amemav mm mm_sua2m

n

.mmuwowamsp mo momuo>¢m

 

 

 

 

~H.m Inn: mv.m om.~ 1:11 mm.m baud Ufiamwm
mH.m h~.H ma.m wm.m mm.a oo.m museumoxmm
mH.N mv.m ma.m vm.m vm.~ Nh.m mmoxmm
mn.o nun: mm.o mm.o III: em.o mmoosm
uuun oo.a nuns III: Ho.H nun: usuaam
mcoc mcoc wcoc moo: mcoc wcoc monogamonm
mm.ma mm.mH VH.¢H mw.ma mo.oH mm.ma cmmouuaz
nnnnnnnnnnnnn vannnulnuuuulun nlunauulnlunvanunnluluunlnlu
ommflo> nauHEm mxooum mace ommflm> mmmflam mxcoum mace ucmauwumcou
cflasnon0©smmm cflHsnonsm

 

 

cflHsaoHoovsmmm pom cflasnonoo mo mcofluwmomeoo HmowEozo .m mqm<a

64
for comparison. The immunoglobulins prepared for this

study possessed lower nitrogen and higher carbohydrate con—
tents than those reported previously (Smith 33 31., 1946;
Veiss, 1961). A meaningful explanation for these differ-
ences is lacking but it should be mentioned that the source
of the milk and the methods employed for preparing and
analyzing these fractions were quite different. Surpris-
ingly, the euglobulin and pseudoglobulin fractions prepared
here show nearly identical chemical compositions.

The nitrogen contents of euglobulin and pseudoglobulin
were 13.86 and 14.14%, respectively, slightly lower than
those of most milk proteins which contain between 15.0 and
16.0% nitrogen. This difference is related to the presence
of higher concentrations of carbohydrates in the immuno-
globulins. The values of 13.86% for euglobulin and 14.14%
for pseudoglobulin are somewhat lower than the corresponding
values of 16.05 and 15.29% reported by Smith 33 a1. (1946)
and 15.53 and 15.58% by Veiss (1961). No phosphorus was
present in either of the preparations isolated in this
study.

The protein portion comprised 86.6% of euglobulin and
88.4% of pseudoglobulin, the remainder being essentially
carbohydrates. Thus, the total carbohydrate content ac-
counted for approximately 11% of these two glycoproteins.
Euglobulin contained slightly higher amounts of fucose,
hexose, and sialic acid and lower amount of hexosamine than

pseudoglobulin, but the differences were small.

65
A summation of the residue weights of the amino acids to-

gether with the total carbohydrate values accounted for
97.84 and 99.04% of the total weight for euglobulin and
pseudoglobulin, respectively.

Paper chromatogram of the carbohydrates released from
euglobulin and pseudoglobulin is shown in Figure 6. Both
protein fractions contained fucose, galactose, mannose,
galactosamine and glucosamine. Like most glyc0proteins, no
glucose was detected. The presence of galactosamine and
glucosamine in both euglobulin and pseudoglobulin fractions
was also confirmed in the chromatograms obtained from the
amino acid analyzer on 20 hr hydrolysates.

The amino acid compositions of the euglobulin and
pseudoglobulin fractions are presented in Tables 3 and 4,
respectively. The data are tabulated as gram residues of
amino acid per 100 gram of protein and the number of amino
acid residues per mole of methionine. The weight percent-
age of the amino acid residues was based on the nitrogen
content as determined by the micro-Kjeldahl method. The
latter method of presentation describes the molar ratios of
the amino acid residues present.

Under the conditions employed in preparing the samples
for amino acid analyses, about 5% of threonine, cystine and
tyrosine and about 10% of serine were lost in the 20 hr
hydrolysis. Therefore, the values for these amino acids
were corrected for the loss according to the equation given

by Hires 33 21' (1954):

66

GALAGTOS'AMINE
GLUCOSAMINE

G [0008 E
MANNOSE

 

Figure 6. Paper chromatogram of authentic carbohy-
drate mixtures and of carbohydrates released from
euglobulin and pseudoglobulin: A-authentic carbohydrate
mixture; B-euglobulin; C-pseudoglobulin.

67

 

 

 

 

TABLE 3. Amino acid composition of euglobulin a

. Gram Residues per Number of Residues per
ReSidue 100 g Proteinb Mole of MethionineC
Lysine 5.70 i 0.09 13
Histidine 1.79 t 0.06 4
Arginine 4.26 i 0.11 8
A3partic Acid 7.26 i 0.13 19
Threonine 7.92 i 0.07 23
Serine 9.36 i 0.09 33
Glutamic Acid 8.23 i 0.10 19
Proline 5.61 i 0.21 13
Glycine 3.24 i 0.01 10
Alanine 3.02 i 0.01 13.
Half Cystine 2.10 i 0.04 6
Valine 7.34 i 0.12 22
Methionine 0.44 i 0.02 1
Isoleucine 2.51 i 0.03 7
Leucine 6.32 i 0.08 17
Tyrosine 4.99 i 0.12 9
Phenylalanine 3.41 i 0.05 7
Tryptophan 2.90 i 0.10 5
Amino Acid Residue 86.60

(Weight %)
Total Carbohydrate 11.24

(Weight %)
Total (Weight %) 97.84

 

aAmino acid content based on four replicate analyses of
20 and 70 hr hydrolysates with values corrected for destruc-
tion during acid hydrolysis according to the equation given
by Hires gt 31. (1954).

bWeight percentage of ith amino acid residue, based on
the nitrogen content of 13.86%, with the standard deviation
from the mean for each value.

0Number of residues of amino acids based on a single
methionine residue per mole of protein.

 

68

 

 

 

 

TABLE 4. Amino acid composition of pseudoglobulina

. Gram Residues per Number of Residues per
ReSidue 100 g Proteinb Mole of Methioninec
Lysine 6.33 i 0.07 13
Histidine 2.02 i 0.10 4
Arginine 3.83 i 0.08 7
Aspartic Acid 6.99 i 0.10 17
Threonine 8.82 i 0.59 24
Serine 9.65 i 0.09 30
Glutamic Acid 8.91 i 0.11 19
Proline 6.06 i 0.25 17
Glycine 3.10 i 0.12 15
Alanine 2.97 t 0.07 11
Half Cystine 2.28 i 0.08 7
Valine 6.77 t 0.07 19
Methionine 0.48 i 0.03 1
Isoleucine 2.60 i 0.07 6
Leucine 6.51 i 0.19 16
Tyrosine 5.03 i 0.09 9
Phenylalanine 3.27 i 0.06 6
Tryptophan 2.78 t 0.01 4
Amino Acid Residue 88.40

(Weight %)
Total Carbohydrate 10.64

(Weight %)
Total (Weight %) 99.04

 

aAmino acid content based on five replicate analyses of
20 and 70 hr hydrolysates with values corrected for destruc-
tion during acid hydrolysis according to the equation given
by Hires gt_31. (1954).

bWeight percentage of ith amino acid residue, based on
the nitrogen content of 14.14%, with the standard deviation
from the mean for each value.

cNumber of residues of amino acids based on a single
methionine residue per mole of protein.

69

 

 

where A AZ, A0 are the quantities of amino acids after E

1' 1!

t and E0 hr hydrolysis, respectively.

-2'
These two immunoglobulins were similar in amino acid
content to other milk proteins except for lower glutamic
acid and proline and higher serine and threonine values.
As shown in Figure 7, there were no marked differences in
the amino acid composition of these two proteins when the
number of moles of each amino acid residue per 1000 moles of
total amino acid residues was compared. One of the charac-
teristic properties of the euglobulin was its insolubility
in the low ionic strength aqueous buffer systems commonly
employed to dissolve proteins. The quantity of hydrOphobic
amino acid residues in euglobulin was no greater than that
in pseudoglobulin which exhibited a high degree of solu-
bility. Therefore, it is presumed that the sequential
arrangement of amino acids and/or the conformational dif—
ferences in peptide chains might be the significant factors
in determining the solubility prOperties of proteins. It
should be noted, however, that proteins do not necessarily
show a marked difference in amino acid content in order to
reflect considerable differences in chemical and physical

properties.

These two immunoglobulins contained relatively high

70

 

 

 
 

. AldLmrv cflasnoHoomvsmmm 0cm T¢|®¢ cflflsnonsm mo mawmoum punom ocdsa .5 wusmflm
page 0:22
.1 d L g. I A D 9 d 9 S .1 V. V. H n.
q. n1 3 H S H I A
mmummmnamanaaasss.
_ 1 _ _ a : _ _ . _ _ W 4 _ _ A 4 . 1
mm, \...
,9 3 1mm
(./
Q
. w
, z 1am I
f c a
_ s
, < m.
C 1
6 1mm. T.
0
.. 0
( 0
N
c o
a 12: .a...
S
.\
JmNH
.oma

 

 

71

contents of hydroxyl amino acids as well as large amounts
of valine, proline, leucine, glutamic acid, aspartic acid,
and lysine. Methionine, histidine, isoleucine, phenyl»
alanine and tryptOphan represented the amino acid resi-
dues present in relatively small concentrations. Values of
the minimum molecular weights of these proteins were cal—
culated from the molar ratios of the amino acid residues
when one mole of methionine was used as the limiting resi-
due (Tables 3 and 4). The molar ratios of methionine,
fucose, hexose, hexosamine and sialic acid in euglobulin
and pseudoglobulin were approximately l:2:5:5:3 and 1:2:6:
5:3, respectively. Assuming that molecular weights of
about 175,000 for these two proteins are approximately
correct, the residue number per mole would be increased by
a factor of six.

The analytical data indicate that both euglobulin and
pseudoglobulin are acidic proteins as determined by the
ratio of the sum of acidic amino acid residues, aspartic
acid and glutamic acid, to that of basic amino acid resi-
dues, arginine, histidine and lysine. This ratio was
about 1.30 for both proteins. This value is much lower
than those for caseins which have the ratio of approxi-
mately 2.0 to 2.5.

Smith gt 31. (1946) reported limited amino acid data
for euglobulin and pseudoglobulin fractions which were
determined by microbial and chemical methods. Consistently

higher values than those found in this study were reported.

72

Physical PrOperties

 

ElectrOphoretic Characteristics

 

Electrophoretic Mobility. The electrophoretic mobil—
ities of the purified euglobulin and pseudoglobulin frac-
tions were determined in veronal buffer at pH 8.6, ionic
strength 0.1. A single migrating boundary was observed
for each of the proteins. The free-boundary patterns are
shown in Figure 8 and the mobilities calculated from the

ascending and descending channels are listed in Table 5.

TABLE 5. Electrophoretic mobilities of euglobulin and

 

 

 

 

pseudoglobulina
Mobility(u) x 105, cm2 sec_1 volt-1
Protein
Ascending Descending Average
Euglobulin -l.86 -l.77 -1.82
Pseudoglobulin -2.10 -1.93 -2.02

 

aAverage of duplicate runs. Each run consists of
three measurements at various time intervals.
The average mobilities of the ascending and descending
patterns were -1.82£nui-2.02 Tiselius units for euglobulin
and pseudoglobulin, respectively. The value for euglobulin
is in close agreement with that reported by Smith (1946),
i.e., -l.80 Tiselius units, but the value for pseudoglobu-

lin is somewhat lower than his value of -2.20 Tiselius

73

 

.H.o mo numcouum oflaofl paw w.m mm .uowwsn Hmcouo> ca mam>umucw mEHu
msoflum> um :flaanoHUOpsomm 0cm cwaznoaunm mo mcuouumm whopcsonlmoum .m ousmfim

 

 

use :2. and—fl“. use :3 3.3" .._
a: 8...» 2; 2:;
a: 2:2. I r- 2; 2.:
can 336 1 a: 2:..."

 

 

 

-az_=zmua< afazuummnw .aE-Euavé azazmuamn+
z_._=ne._aea=ma._ 21.—3.32:

74
units. It is, however, possible that the electrophoretic

mobility of a protein would vary slightly depending on the
purity of the preparation and the state of its occurrence,
i.e., dissociation or association with other protein compo-
nents. Equilibrium dialysis of the protein solution
against the buffer is also an important factor in deter-

mining the electrophoretic mobility.

Isoelectric Point. Isoelectric points of the immuno-

 

globulins were estimated from the electrophoretic mobili-
ties of the proteins in buffers covering a pH range of 4.0
to 8.6 at an ionic strength of 0.2. The results are tabu-
lated in Table 6. Mobility determinations were made in
duplicate with each run consisting of three measurements
at various time intervals. At all values of pH tested,
these preparations appeared as a single homogeneous bound-
ary. The isoelectric point of pH 6.03 for euglobulin and
that of pH 5.54 for pseudoglobulin were estimated from the
plot of the average of ascending and descending electro-
phoretic mobilities against the corresponding pH values as
shown in Figure 9.

The isoelectric points for both immunoglobulin frac-
tions are in close agreement with those reported by Smith
st 31. (1946),i.e., pH 6.05 for euglobulin and pH 5.60 for

pseudoglobulin.
Ultracentrifugal Characteristics

Partial Specific Volume. Figure 10 shows typical

75

um mpcmsmusmmoe owns» mo omumflmcoo can zoom

..o.w .mam>umuafi 05H“ ucoumMMflp
.mcsu mumoaamsw mo mmmum>¢o

.omm comb can oovm .oomm

.wmm.a mo coaumuucmocoo cflmuoumn

.N.o mo sumcwuum owcon

 

 

U

 

 

nm.~u ne.mu mm.mu mm.au om.an om.Hn m.m Hmcoum>umz
ev.au mm.au Hm.HI mm.ou Hm.on om.ou o.> mumnmmonmlmz
mm.o- Hm.o- mm.o- mo.o+ so.o+ oo.o+ o.m mumnmmondumz
mm.o+ vm.o+ om.o+ oa.a+ mo.a+ mH.H+ o.m mumumomnmz
mo.m+ mm.a+ no.m+ mH.N+ no.m+ mm.m+ o.v mumumomumz
mmmum>¢ omcflocwommo omcHocoomd ommmum>¢ omcflpcmommo omcflpcmom¢
CH 5 0 do new ca 5 0 m9
D .H o H p m Q.H n H m we muwmmsm
o> 0mm 80 x n m a a o
(HnuH an um (Moa A v u.H.n z

 

 

msofium> ca cHHsnonoosmmm 0cm ceasnonsm mo mmflufiawnoe oaumuosmouuomam

mEmumMm nommsn
. m "393.

76

 

 

 

 

 

 

 

pH

Figure 9. ElectrOphoretic mobility as a function of
pH for the euglobulin (—o—o—) and pseudoglobulin (H) .

77

EUGLOBULIN

 

PSEUDOGLOBULIN

 

 

Figure 10. Interferograms obtained in high-speed
sedimentation-equilibrium experiment with euglobulin and
pseudoglobulin in H O and D 0 solutions. Protein concen-

tration was 0.5 mg/ml. 2

Ss-

78
interferograms obtained in high-speed sedimentation equi-

librium experiments with euglobulin or pseudoglobulin in
H20 in one doublewsector cell and the same protein in D20
in the other. As shown in the patterns the concentration
change across the cell was much less for the cell containing
euglobulin or pseudoglobulin in 020. The representative
data from these experiments are illustrated in Figure 11.
The plots of the logarithm of net fringe displacement,

which is proportional to concentration, with reSpect to the
distance from the axis of rotation were of upward curvature.
This is a typical characteristic for specimens possessing x
molecular heterogeniety. The slopes of these plots for
euglobulin were 0.762 (H20) and 0.606 (020) and for
pseudoglobulin corresponding values were 0.789 (H20) and
0.628 (D20).

The ratios of the slope in D20 to that in H20 were
0.795 for euglobulin and 0.796 for pseudoglobulin.. The
value of k, the ratio of the molecular weight of deuterated
protein to that of the nondeuterated species, was assumed
as 1.0155 since the value should be relatively constant for
all proteins (Martin 25 31,, 1959; Hvidt and Nielsen,

1966). The values of G, the partial specific volume,
evaluated from the experiments as illustrated in Figures 10
and 11 were 0.712 ml/g for euglobulin and 0.710 ml/g for

pseudoglobulin.

Diffusion Coefficient. Apparent diffusion coeffi-

 

cients for euglobulin and pseudoglobulin fractions were

E3-

79

.caasnonocsmmmlm can ceasnonsmat uncowusHOm 0mm

 

 

 

 

 

can Cum cw seasnoflooosomo pom swasnoamao mo aswunflafisqm cofiumucmecmm .HH ousmwm
50 M ED
Am C N Am V «n
o.om m.mw o.mv m.mv o.om m.m¢ o.mv m.m¢
fi d d a .
OOH ooH
w w
-oo~ 1 com a
a a
1 1
... m.
u
. com .u can u
a m
-oov m. cow 9
m
o a w .m.
\ \ loom 2 com a
o o
\ .\ m m
\ a a
\ \ w. W
\ \ l l
\\ ~ coca n ,.1 oooe n
\ \ L \ j
\ o m \ommNQ
\
_.\ E 1 3

 

 

_ooo~ 1 .. .ooom

 

 

[3'

i
K.
w 1..

80
determined at various protein concentrations in veronal

buffer (pH 8.6, u = 0.1), milk salt solution (pH 6.6) and
6 M guanidine hydrochloride plus 0.02 M 2-mercaptoethanol
solution. The results of these experiments are recorded
in Table 7. The concentration dependency of the apparent
diffusion coefficients (Dapp ) of euglobulin and pseudo-

20,w
globulin is illustrated by the plots in Figure 12.

TABLE 7. The apparent diffusion coefficients of euglobulin
and pseudoglobulin in veronal buffer, milk salt solution and
6 M guanidine-HC1 plus 0.02 M 2-mercaptoethanol solution

 

 

 

Protein 01f£3§10n Coefficienta x10
Protein Concentration
(mg/ml) Veronal Milk Salt Guanidine
Euglobulin
0.0b 3.20 3.20 37.90
4.0 3.33 --—- 27.10
5.0 ---- 3.31 -----
7.0 3.40 ---- 18.43
7.5 --—- 3.40 -----
10.0 3.53 3.45 10.54
Pseudoglobulin
0.0b 4.20 3.62 30.00
4.0 ---- 3.20 24.04
5.0 3.62 ---- -----
7.0 -—-- 3.15 19.01
8.0 2.98 ---- -----
10.0 2.79 2.42 15.04

 

aValues corrected to water at 20° C.

b

using least squares linear regression method.

Values obtained by extrapolating to infinite dilution

81

gunman Hmcoum> cw ceasnoamocsmmm pcm ceasnoausm mo mmfluummoum scamsmmwo

.cflasnonopsmmm1m can cHHsnOHm9M1¢ ”A fl:hmv HocmzumoumuoumEIN 2 No.0
ocacwmucoo coHusHOm opfluoanooupmn mcwpficmsm z m can «1WImTV coflusaom pawn xafie .almlmlv

AHE\mEV sowumuucoocou cwmuoum

m m c N o

 

.. -7---— u

 

 

 

 

 

 

Amy C ow

 

 

(“é3201 L01 x nua10133600 “018n3310

 

.«H magmas

AHE\mEV cofiumuucoosou cwououm

 

 

m m e N o
_ _ . _ o
1 ed
A
_.,.// 1.. ON
.. ..on
/////
2V 1 3
. - 1. -.-11-....11I1L_

 

 

( égga) L01 x 3u870133900 natanJIa

'KS‘

82
In veronal buffer, the apparent diffusion coefficients

of euglobulin remained nearly constant over the entire
range of protein concentrations. Only a slight increase in
the diffusion coefficient with increasing protein concen-
trations was perceptible. Similar characteristics were
observed in the milk salt solution. Pseudoglobulin showed
somewhat different results. In both veronal buffer and
milk salt solution, the apparent diffusion coefficients
increased slightly with decreasing protein concentrations.
However, these slight changes in the diffusion character-
istics of both euglobulin and pseudoglobulin seemed insig-
nificant when compared to their behavior in the 6 M guani-
dine hydrochloride solution containing 0.02 M 2-mercapto-
ethanol dissociating system. Here, the apparent diffusion
coefficients of both euglobulin and pseudoglobulin showed
a high degree of dependency on the protein concentration,
increasing 5- to lZ-fold over the concentration range
observed. It seems that both proteins are extremely sen-
sitive to dissociating agent, particularly to 2-mercapto-
ethanol (Gough 22 31., 1966). These data indicate that
low molecular species or monomer units of these proteins
are present in this dissociating system. Smaller species
would tend to give higher diffusion rates than the larger
molecular polymers.

Corrected diffusion coefficients at infinite dilution
of euglobulin in veronal buffer, milk salt solution and

the 6 M guanidine hydrochloride solution containing 0.02 M

KS"

83
2-mercaptoethanol were 3.20, 3.20 and 37.90 x 10'7 cm2

sec , respectively. Corresponding values for pseudoglobu-

lin were 4.20, 3.62 and 30.00 x 10-7 cm2 sec-1. Values

.7

of 3.20 x 10.7 cm2 sec for euglobulin and 4.20 x 10-
for pseudoglobulin in veronal buffer are comparable with
reported values of 3.41 and 3.84 x 10"7 cm2 sec-1, respec-
tively (Smith 25 31., 1946).

The effects of various hydrogen ion concentrations on
the diffusion coefficients of euglobulin are shown by the
data presented in Table 8.

TABLE 8. The effects of various hydrogen ion concentra-
tions on the diffusion coefficients of euglobulin

 

   

Buffera pH Diffusion Coefficientb'c (Ficks)

 

Na-acetate 4.0 4.98
Na-phosphate 6.0 3.92
Na-veronal 8.6 3.53

 

aIonic strength of 0.1.
bProtein concentration of 10.0 mg/ml.

cValues corrected to water at 20° C.

The apparent diffusion coefficients of euglobulin at
10.0 mg/ml concentration increased as the hydrogen ion
concentration decreased. Euglobulin is quite stable within
the pH range of 6.0 to 8.6, as the differences in the

apparent diffusion coefficient at these pH values are not

IN.-

KS"

84
significant. The somewhat higher apparent diffusion coeffi-

cient obtained at pH 4.0 would indicate that euglobulin
might dissociate into smaller molecular species at low val—
ues of pH.

Effects of the electrolyte concentration of veronal
buffers on the diffusion rates of a euglobulin preparation
are illustrated by the data reported in Table 9.

TABLE 9. The effects of electrolyte concentrations on the
apparent diffusion coefficients of euglobulin

Ionic Strength Diffusion Coefficienta'b(Ficks)
0.01 4.63
0.05 3.94
0.10 3.53
0.20 3.38
0.50 3.31

aProtein concentration of 10.0 mg/ml.

bValues corrected to water at 20° C.

As the concentration of electrolytes in the buffers
decreased, the apparent diffusion coefficients of euglobu-
lin increased gradually. The rate of change was pronounced
in the lower ionic strength buffers than in the higher salt
concentrations. Timascheff (1964) stated that some pro-
teins tend to dissociate slowly in low ionic strength
buffer'systems.

The effects of various temperatures on the apparent
diffusion coefficients of euglobulin were studied in

veronal buffer and milk salt solution. The results are

summarized in Table 10.

TABLE 10. The effects of temperatures on the apparent dif-

fusion coefficients of euglobulin in veronal buffer and

milk salt solutiona

 

 

Diffusion Coefficientb(Ficks)

 

 

Protein
Buffer Concentration
(mg/ml) 5° c 20° c 32° c
Veronal
0.0c 3.01 3.20 3.41
4.0 3.17 3.33 3.59
7.0 3.29 3.40 3.75
10.0 3.41 3.53 3.89
Milk Salt
Solution
0.0c 2.93 3.20 3.36
5.0 3.09 3.31 3.51
7.5 3.23 3.40 3.62
10.0 3.37 3.45 3.67

 

aRotor was precooled or prewarmed to the temperature
slightly lower or higher than the desired.

b

Values corrected to water at 20° C.

cValues obtained by extrapolating to infinite dilu-
tion using the least squares linear regression method.

The diffusion coefficients of euglobulin in both

buffer systems decreased slightly with decreases in tem-

perature. However, the concentration dependency of the

apparent diffusion coefficients remained unchanged over

the range of all temperatures studied.

diffusion coefficients obtained in the milk salt solution,

The slightly lower

86
as compared to those in veronal buffer, are noteworthy.

Sedimentation Coefficient. The apparent sedimentation

 

coefficients of euglobulin and pseudoglobulin in veronal
buffer, milk salt solution and 6 M guanidine hydrochloride
solution containing 0.02 M 2—mercaptoethanol, at various
protein concentrations are summarized in Table 11.

The sedimentation-velocity studies in veronal buffer
revealed that the euglobulin preparation contained only two
components -- a 78 and a 198 molecular species. Approxi-
mately 85-90% of the protein consisted of the 7S component
while the 195 species accounted for from 10 to 15%. Smith
(1946a) reported three molecular species, 78, 108 and 19S
in the ratio of approximately 8:1:1, for a similar prepa-
ration obtained by ammonium sulfate precipitation.

The apparent sedimentation coefficients of the 7S
component were essentially independent of protein concen-
tration. The 19S component showed more of a concentration
dependency, increasing slightly with decreasing protein
concentrations (see Figure 13-A). This type of concentra-
tion dependency in the apparent sedimentation coefficient
is typical for a slow association-dissociation reaction of
a protein (Gilbert, 1959 and 1963; Gilbert and Jenkins,
1959). Nearly identical results were obtained in milk salt
solution. The sedimentation coefficients of these two
molecular species decreased considerably in the presence of
dissociating agents, indicating that the polymer-dimer

equilibrium was shifted toward the low molecular weight

87

TABLE 11. The sedimentation coefficients of euglobulin and

pseudoglobulin in veronal buffer, milk salt solution and

6 M guanidine hydrochloride solution containing 0.02 M
2-mercaptoethanol

 

 

 

 

Protein Sedimentation Coefficienta
Protein Concentration
(mg/ml) Veronal Milk Salt Guanidine
Euglobulin

0.0b 6.24 6.15 4.63
4.0 ---- 6.13 4.13
Slow Boundary 5'0 6'22 -—-- ----
7.0 ---- 6.06 3.75
7.5 6.21 --—- ----
10.0 6.20 6.03 3.37
0.0b 19.04 19.16 13.66
4.0 ----- 19.00 11.81
Fast Boundary 5'0 18'82 ----------
7.0 ----- 18.68 10.43
7.5 18.67 ----------
10.0 18.55 18.71 9.04

Pseudoglobulin
0.0b 6.00 5.91 4.61
4.0 ---- 6.33 4.10
5.0 6.53 ---- ----
7.0 ---— 6.75 3.72
8.0 6.89 ---- ----
10.0 7.10 7.04 3.34

 

aValues corrected to water at 20° C.

b

Values obtained by extrapolating to infinite dilution
using the least squares linear regression method.

53

 

 

88

species. The sedimentation coefficients, corrected for
density and viscosity of the solvent and at infinite dilu-
tion, were approximately 19.048, 19.168 and 13.668 for the
198 component in veronal buffer, milk salt solution and
dissociating system, respectively. For the 7S component,
the corresponding values were 6.248, 6.158 and 4.638.

The apparent sedimentation coefficients of pseudo-
globulin as function of protein concentrations are shown
in Figure l3-B. In both veronal buffer and milk salt
solution, the sedimentation values were concentration
dependent, increasing with increasing protein concentra-
tions. In the dissociating system, however, the usual
trend of concentration dependency of the sedimentation
coefficients was reversed, decreasing with increasing pro—
tein concentrations. The sedimentation coefficients ob-
tained in veronal buffer, milk salt solution and dissoci—
ating system were approximately 6.008, 5.918 and 4.618,
respectively. ’

The effects of various temperatures on the sedimenta—
tion behavior of euglobulin were studied in veronal buffer
and milk salt solution. The results are shown in Table 12.

The sedimentation behavior of the 7S component was
not affected significantly by the temperature changes.
However, the 198 component showed noticeably higher sedi-
mentation values as the temperature decreased, although not
as drastically as expected from the results of sedimenta-

tion-equilibrium experiments. Nevertheless, the noticeable

(a

89

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TABLE 12. The effects of temperatures on the sedimentation
coefficients of euglobulin in veronal buffer and milk salt
solution

 

Sedimentation Coefficientb

 

 

Protein
Buffer Component Concentration
(mg/m1) 5° C 20° C 32° C
Veronal
0.0C 6.40 6.24 6.19
75 5.0 6.33 6.22 6.17
7.5 6.26 6.21 6.21
10.0 6.22 6.20 6.18
0.0C 22.61 19.04 18.90
195 5.0 20.64 18.82 18.41
7.5 19.69 18.67 18.04
10.0 18.63 18.55 17.97
Milk Salt
Solution C
0.0 7.05 6.15 6.10
75 4.0 6.67 6.13 6.06
7.0 6.31 6.06 6.04
10.0 6.02 6.03 5.98
0.0c 23.55 19.16 18.84
195 4.0 21.67 19.00 18.57
7.0 20.73 18.68 18.49
10.0 18.88 18.71 18.11

 

a7S denotes the slow moving boundary and 198 the fast
moving boundary.

bValues corrected to water at 20° C.

cValues obtained by extrapolating to infinite dilution
using the least squares linear regression method.

$3

91
increase in the sedimentation coefficients at lower temper-

atures, particularly in milk salt solution, suggests that a
slow association or polymerization reaction of the 198
component occurred. In fact, slow association reactions of
proteins require a much longer time than the residence time
during a centrifuge run. Indeed, the detection of the
association phenomenon could escape during the short time
required for the sedimentation-velocity experiments.

The effects of various hydrogen ion concentrations on
the sedimentation behavior of euglobulin are represented by

the data in Table 13.

TABLE 13. The effects of various hydrogen ion concentra—
tions on the sedimentation coefficients of euglobulin

 

 

 

 

 

a Sedimentation Coefficientb'c
Buffer pH
78 Component 198 Component
Na-acetate 4.0 4.82 14.96
Na-phosphate 6.0 6.00 18.83
Na-veronal 8.6 6.20 ~ 18.55

 

annic strength of 0.1.
bProtein concentration of 10.0 mg/ml.

cValues corrected to water at 20° C.

The sedimentation coefficients of both the 7S and 198
components of euglobulin decreased with decreasing hydrogen

ion concentrations. However, the differences in

(a

92
sedimentation values between the pH 6.0 and 8.6 buffer

systems were not great. This observation suggests that
both the 7S and 198 components are stable in this pH range
but dissociate at pH values much lower than their isoelec-
tric points.

Table 14 summarizes data showing the effects of
ionic strength on the sedimentation behavior of euglobulin.

TABLE 14. The effects of electrolyte concentrations on the
sedimentation coefficients of euglobulin

 

 

Sedimentation Coefficienta'b

 

Ionic Strength

 

78 Component 198 Component
0.01 5.13 15.70
0.05 5.47 16.06
0.10 6.20 18.55
0.20 6.27 18.72
0.50 6.23 18.79

 

aProtein concentration of 10.0 mg/ml

bValues corrected to water at 20° C.

The concentration of electrolytes in veronal buffer
had only slight effects on the sedimentation coefficients
of the 78 and 198 components of euglobulin preparation
*which decreased with decreasing ionic strength. This
observation coincides with Timasheff's (1964) observation

that the dissociation of B-lactoglobulin is decreased by

Ea

93
increased ionic strength. Buffers with ionic strength

lower than 0.01 could not be tested because of the insolu-
bility of euglobulin in these systems. When the concen-
tration of the electrolytes reached a critical level, the
sedimentation coefficients were no longer affected by

further increases in the ionic strength.

Molecular Weight. The apparent weight average molec-

 

ular weights of euglobulin and pseudoglobulin were deter-
mined by the low—speed sedimentation-equilibrium method at
various protein concentrations in veronal buffer, milk salt
solution and 6 M guanidine hydrochloride solution con-
taining 0.02 M 2-mercaptoethanol. The results of these
experiments are summarized in Table 15. The ratios of the
apparent z— to weight-average molecular weights (MZPP/szp)
are also shown in the same table. It was extremely diffi-
cult to make accurate measurement of the apparent M2 Of
these samples, particularly euglobulin because of the
viscous layer of sedimented material which collected at the
bottom of the cell. It is very unlikely that the concen-
tration gradient curve would ever reach the bottom meniscus
during the sedimentation run. Considerably high Mz/Mw
ratios suggest that these proteins consist of highly hetero-
geneous molecular species. The extent of heterogeniety or
polydispersity increases considerably as the protein
concentration increases (Tanford, 1961). Polydispersity

is particularly evident in the dissociating buffer system.

83

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95
As illustrated in Figure 14, the apparent weight

average molecular weights of euglobulin and pseudoglobulin
were concentration dependent. In veronal buffer and milk
salt solution, euglobulin showed a stronger dependency on
the concentration than did pseudoglobulin. It is impor-
tant to note that in veronal buffer and milk salt solution
the apparent weight average molecular weights of euglobulin
decreased with decreasing concentrations of protein, where—
as the opposite behavior was observed for pseudoglobulin.
This behavior of euglobulin is typical for an interacting
system in contrast to a noninteracting system provided that

sufficient time is allowed for the attainment of equili—

brium at constant environmental conditions (McKenzie, 1967).

According to this theory, pseudoglobulin could be a non-
interacting material since the apparent average molecular
weights decreased as the protein concentration increased.

In the dissociating buffer system, both euglobulin
and pseudoglobulin behaved similarly. The apparent weight
average molecular weights of both proteins increased with
decreasing protein concentration.

The weight average molecular weights of both proteins
in various buffers were estimated by extrapolating the
apparent weight average values to infinite dilution. The
values obtained were approximately 175,000; 244,000; and
89,000 for euglobulin and 167,000; 174,000; and 86,000 for
pseudoglobulin in veronal buffer, milk salt solution and

dissociating buffer system, respectively. The values of

 

 

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97
175,000 for euglobulin and 167,000 for pseudoglobulin in

veronal buffer are in the range of 160,000 to 190,000 for
both proteins reported by Smith gt a1. (1946) but somewhat
lower than the values of 252,000 for euglobulin and 289,000
for pseudoglobulin reported by Murthy and Whitney (1958).

Why euglobulin exhibits a substantially higher weight
average molecular weight in the milk salt solution than in
veronal buffer is not clearly understood. Environmental
conditions such as hydrogen ion concentration, species and
amounts of salts in the milk salt solution possibly favor
the polymerization of euglobulin molecules during the long
equilibrium experiments, i.e., 220-30 hr.

The effects of various temperatures on the apparent
weight average molecular weights of euglobulin were studied
in veronal buffer and milk salt solution at three different
protein concentrations. The results are shown in Table 16.
The apparent weight average values for euglobulin in both
buffer systems were greatly affected by temperature
changes. At all temperatures studied, the pattern of the
concentration dependency of the apparent weight average
values in both buffers was not changed. As clearly illus—
trated in Figure 15, the weight average molecular weights
increased cosiderably as the temperature decreased.
Presumably, polymerization of euglobulin was greatly en-
hanced at low temperatures. The MW of euglobulin at 5° C
in veronal buffer was almost one and one-half times that

observed at 20° C in the same buffer, suggesting that a

SS

rEIII-illnc. .1.I

98

TABLE 16. The effects of temperatures on the apparent
weight average molecular weights of euglobulin in veronal
buffer and milk salt solution

 

 

 

Protein Molecular Weighta
Buffer Concentration
(mg/m1) 5° C 20° C 32° C

 

Veronal
0.0 275,918 175,250 104,027
4.0 294,013 179,639 117,663
7.0 323,781 190,047 138,819
10.0 337,619 194,968 144,492
Milk Salt
Solution
0.0 387,932 243,752 175,339
5.0 413,781 259,638 204,013
7.5 440,035 275,872 224,917
10.0 449,910 287,149 230,281

 

 

aApparent weight average molecular weight.

bValues obtained by extrapolating to infinite dilution
using the least squares linear regression method.
slow polymerization occurred at the lower temperature. The
MW at 5° C in milk salt solution was slightly more than
twice that observed in veronal buffer at 20° C. A dimeri-
zation reaction might have been favored by these conditions.
It is, therefore, presumed that the polymerization interac-
tion of euglobulin has a negative enthalpy and a large
negative entrOpy. Thus, the polymerization is favored by

low temperatures.

53

 

 

 

99

 

 

 

 

 

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50 _
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Protein Concentration (mg/ml)

Figure 15. The effects of temperatures on the weight
average molecular weight of euglobulin in veronal buffer
(—€P49-) and milk salt solution (15-Ara.

 

 

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100
The effects of hydrogen ion concentrations on the

apparent weight average molecular weights of euglobulin are
shown in Table 17.

TABLE 17. The effects of hydrogen ion concentrations on
the apparent weight average molecular weights of euglobulin

 

 

 

Buffera pH Molecular Weightb'C
Na-acetate 4.0 130,715
Na-phosphate 6.0 174,033
Na-veronal 8.6 194,968

 

annic strength of 0.1.
bProtein concentration of 10.0 mg/ml.

cApparent weight average molecular weight.

The apparent weight average molecular weights of eu-
globulin, at 10.0 mg/ml concentration, decreased consider-
ably with decreasing hydrogen ion concentration in the
buffer. As mentioned previously concerning the sedimenta-
tion behavior of this protein, the significant reduction
of the apparent weight average molecular weight at pH 4.0
compared to those at pH 6.0 and 8.6 may be explained on
the basis that dissociation is favored by low values of
hydrogen ion concentration.

Results illustrating the effects of the ionic strength

of veronal buffer on the apparent weight average molecular

 

 

 

 

 

$3

101
weights of euglobulin (20° C) are found in Table 18.

TABLE 18. The effects of ionic strengths on the apparent
weight average molecular weights of euglobulin in veronal
buffer, pH 8.6, at 20° C

Ionic Strength Molecular Weighta'b
0.01 190,937
0.05 190,293
0.10 194,968
0.20 193,254
0.50 195,798

aProtein concentration of 10.0 mg/ml.

bApparent weight average molecular weight.

As was the case for the apparent sedimentation coeffi-
cients of euglobulin, the apparent weight average molecular
weights decreased slightly with decreasing ionic strength.
This slight tendency for decreasing MW values could be a

ramification of slow, apolar dissociation in the protein.

Creaming Studies

 

The Effects of Euglobulin Concentrations on Creaming

 

The effects of different concentrations of euglobulin
on the creaming abilities of normal and recombined milk
samples and model system are summarized in Table 19. The
model system was prepared by resuspending thrice-washed

fat globules (3.6%, w/v) in milk salt solution.

{a

102

TABLE 19. The effects of euglobulin concentrations on

 

 

 

 

creaming
a b
Sample Eugfgifilin C%:§::r Cream Volume '

(%) 1 Hr 4 Hr
Normal Milk

None 10.2 8.3

0-01 11.0

0.02 10.9 8.6
Recombined Milk

None 7.7 8.3 7,4

0.01 8.9 10,9

0.02 8.7 11.0 3,4
Model System

None 0.6 0.3 0,5

0.01 4.3 4.4 3,5

0.02 6.7 6.6 5,0

0.03 7,9 8.3

0.04 7.7 8.1 7,1

 

aAverage of duplicates.

bPer cent of cream volume per 1% fat (v/V).

In the model system, no measurable cream layer was
formed in the absence of euglobulin. The cluster index and
cream volume increased markedly when increasing amounts of
euglobulin were added, indicating that this protein plays a
definite role in the creaming phenomenon. The slight tend-

ency for fat globules to cluster in the absence of added

 

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$3

103
euglobulin may have been the result of small amounts of

adsorbed euglobulin on the surface of fat globules.

Even with the addition of 0.04% of euglobulin to the
model system, its net creaming ability seeded inferior to
that of normal milk. Obviously, then, the best simulated
milk salt solution does not provide the optimum conditions
for creaming when compared to natural milk.

The enhancement of the creaming ability of recombined
milk by the addition of euglobulin supports the contention
that euglobulin is a principal component in the creaming
phenomenon. The slightly poorer creaming ability of the
recombined milk minus added euglobulin suggests that the
wild agitation of the milk system during the process of
cream separation may have produced a partial denaturation
of the euglobulin. Additionally, the cream separation
process could have caused mechanical injury to the fat
globules, altering the plasma/lipid interfacial surface in
such a manner that the adsorption of euglobulin was impeded.

The addition of euglobulin to normal milk did not
enhance its creaming property significantly. There seems
to be a definite relationship between euglobulin concentra-
tion and fat globule surface area involved in the creaming

phenomenon.

The Effects of Temperatures on Creaming

The effects of various temperatures on the creaming

property of normal and recombined milk samples and model

 

53

 

101?]

 

104

systems are shown in Table 20.

In all three systems, creaming was greatly improved at
the lower temperatures, i.e., 25° C. This observation cor-
relates with the behavior of euglobulin in the sedimenta-
tion experiments. Recall that euglobulin, especially its
198 component, exhibited extensive intermolecular associa—
tion in the low temperature runs. However, no direct
evidence is available at present to claim that the cold
association of euglobulin molecules contributes signifi-
cantly to the enhancement of the creaming properties at low
temperatures. Further extensive studies are needed to

verify this hypothesis.

The Effects of Electrolyte Concentrations on Creaming

 

The creaming abilities of recombined milk and model
systems were studied at various electrolyte concentrations.
The results are shown in Table 21.

It was found that the dilution of the skim milk or the
milk salt solution improves the creaming properties of re-
combined milk and model system. For both systems, a maxi-
mum cream layer was obtained at 50% dilution with deionized
water. This observation supports the early explanation for
the improved creaming of diluted milk (Orla-Jensen, 1928;
Dunkley and Sommer, 1944). They conceded that the salts in
milk hinder the action of agglutinin and dilution favors
fat clustering by providing more favorable conditions for

the action of agglutinin even with its decreased

E3

I511? l. r

105

TABLE 20. The effects of temperatures on the creaming
propertiesa

 

 

Temperature Cluster Cream Volume (%)

 

Sample

 

(a C) Index 1 Hr 4 Hr
Normal Milk
3 - 4 8.5 10.2
12 - 15 8.6 9.8 8.3
23 - 25 7.2 8.1
32 — 35 3.2 5.3 4.8
42 - 45 1.3 3.1 2.2
Recombined Milkb
3 - 4 . . .
12 - 15 6.9 7.8 7.0
23 - 25 5.1 . 6.3 5.7
32 - 35 2.4 3.7 2.9
42 - 45 0.7 2.4 1.9
MOdel Systemc
3 — 4 .° . .
12 - 15 4.0 4.2 _ 3.5
23 - 25 3.1 3.3 2.9
32 - 35 1.9 2.4 1.6
42 - 45 0.3 1.7 1.2

 

aAfter the milk samples were warmed as described in the
experimental section, the creaming cells and the graduated
cylinders containing warmed samples were placed in water
baths maintaining the specified temperatures instead of
placing them in a 4° C bath.

bFive-time washed cream was redispersed in thrice-
separated skim milk at 3.6% fat concentrations (w/v).

cThe concentrations of euglobulin and fat were 0.01 and
3.6%, respectively, in milk salt solution (W/V).

S!

 

106

TABLE 21. The effects of electrolyte concentrations on
creaming phenomenon

 

 

 

 

Sample Dilutiona Cluster Cream Volume (%)
(%) Index
1 H{_ 4 Hr
Recombined Milk
None 7,7 8.3 7.4
25 11.3 8.8 3,1
50 20.8 9.3 3,9
75 20.2 9.0 3,5
100b 0.0 0.3 0,7
Model System ‘
None 4.3 4.4 3,5
25 4-8 4.7 4.0
50 5.8 5.0 4,0
75 5.5 3.8 3,0
100c 0.0 0.3 0,9

 

aThe electrolyte concentrations were adjusted by
diluting the whey or milk salt solution with deionized
water (v/v).

bCream was suspended in deionized water at 3.6% fat
concentration (w/v), and thus no euglobulin was contained
in this system.

cCream and euglobulin were suSpended in deionized
water at the concentrations of 3.6 and 0.01%, respectively,
(w/v).

H

107

concentration. However, it was not known exactly what
conditions would be changed favorably for creaming by dilu-
tion of the milk.

When salt ions were completely removed from the system,
no detectable creaming was observed in either system. It
appears that an Optimum combination of salts in an optimum
concentration is required to enhance euglobulin-fat globule
interactions. As discussed in a previous section of this
thesis, both the sedimentation coefficient and the molecular
weight of euglobulin decreased as the ionic strength of its
solvent was decreased. This behavior was explained as a
possible dissociation of euglobulin, affected by the low
electrolyte concentrations. It should be pointed out, how-
ever, that the concentration range of euglobulin used in
the ultracentrifuge studies was much higher, about five—
fold or more, than that used in the creaming studies. If
the creaming studies were carried out at sufficiently high
euglobulin concentration range, comparable to that of
centrifuge studies, it might be possible to observe slight
decrease in the creaming ability at lower electrolyte
concentrations. On the other hand, it might also be possi-
ble that the effect of slight denaturation of euglobulin
in low salt systems could have been overcome by the more
favorable conditions provided for the activity of

euglobulin.

 

 

 

[:11 1

108
The Effects of Hydrogen Ion Concentrations on Creaming

 

Table 22 contains the results of creaming studies per—
formed with normal and recombined milk as well as model
systems at various hydrogen ion concentrations. As the
hydrogen ion concentration of the systems departed from the
natural value of milk, about pH 6.6-6.8, the creaming abil-
ities of these milk samples decreased. The creaming abili-
ty was affected more adversely at low hydrogen ion concen-
trations than at the higher levels. The extremely poor
creaming observed in the model system at low hydrogen ion
concentrations may be related to the denaturation or
dissociation of euglobulin molecules previously observed
in the sedimentation studies.

Interpretation of The Creaming Phenomenon in
Relation to the Physical Properties of Euglobulin

 

 

That euglobulin is required in creaming of cow's milk
was demonstrated in the experiments with model systems.

No creaming was observed when euglobulin was completely
absent in the system, whereas creaming gradually improved
as the concentration of euglobulin was increased.

When the euglobulin concentration was kept at a
constant level of 0.01%, optimum creaming was observed at
temperatures ranging from 3 to 5° C in milk salt solution.
Interestingly, euglobulin itself undergoes significant
changes at 5° C, that is, the weight average molecular

weight in milk salt solution was approximately twice the

109

TABLE 22. The effects of hydrogen ion concentrations on the
creaming prOperties

 

Cream Volume (%)

 

 

5...... . 82:12:: ~
1 Hr 4 Hr.
Normal Milkb
5.0 --— 3.7 3.4
6.6 8.3 10.0 8.1
9.0 6.9 7.2 6.4
Recombined Milkb
5.0 --- 3.3 2.7
6.6 7.5 8.0 7.1
9.0 6.2 6.5 5.2
Model SystemC
3.0 0.9 0.6 0.9
5.0 2.4 2.9 2.1
6.6 4.3 4.6 3.9
9.0 3.9 3.2 2.4

 

apH of the skim milk or milk salt solution was
adjusted with 0.1 N HCl or 0.1 N NaOH.

bSatisfactory determinations of both cluster index and
cream volume were not possible at pH values lower than 5.0
since casein micelles in the normal and skim milk started
to aggregate.

chglobulin concentration of 0.01% (w/v).

110

value observed at 20° C in veronal buffer. Therefore, it
is conceivable that the enhanced polymerization of euglobu-
lin in milk salt solution at low temperatures could enhance
or even initiate the creaming of milk.

On the basis of the experimental results obtained in
this investigation and the available information regarding
the subject, it is concluded that the creaming phenomenon
is favored by; (l) a high concentration of euglobulin
available for the interaction with fat globules, (2) a
creaming temperature low enough to enhance the polymeriza-
tion of euglobulin, and (3) Optimum concentrations of
hydrogen ion and electrolytes to promote the association
of euglobulin molecules.

In the foregoing discussions, various chemical and
physical aspects of euglobulin and pseudoglobulin fractions
of cow's milk were reviewed with particular emphasis on the
association-dissociation phenomenon of euglobulin under
various conditions. Specially designed experiments were
also performed to assess the role of the euglobulin frac—
tion in creaming of normal cow's milk.

Both euglobulin and pseudoglobulin fractions must be
considered as highly heterogeneous molecules, containing
more than one molecular species in variable amounts. Un-
der'specific1conditions, these molecular species undergo
substantial changes, i.e., association, dissociation, or
aggregation. A typical example is the possible interac-

tion of euglobulin with fat globules to enhance fat globule

 

111

clustering and subsequent creaming. The precise opera-
tional function of these proteins and the mechanism of
euglobulin interaction with fat globules have not been
elucidated in specific terms, and thus further extensive
research is needed to unveil the mystery surrounding the

physical and chemical behavior of this interacting protein.

 

 

S!

, .I

S UMMA RY

Sepharose 6B gel filtration and DEAE-cellulose anion
exchange column chromatoqraphic techniques in conjunction
with ammonium sulfate precipitation method were employed to
isolate euglobulin and pseudoglobulin from cow's milk in
electrOphoretically pure form.

These two immunoglobulins were nearly identical in
chemical composition. The nitrogen content of euglobulin
was 13.86% and that of pseudoglobulin was 14.14%. Euglobu-
lin contained 11.24% carbohydrates and pseudoglobulin
10.64%. Both proteins contained fucose, galactose, galac—
tosamine, glucosamine, mannose and sialic acid. Both
contained less glutamic acid and proline and more serine
and threonine than other milk proteins. Neither protein
contained phosphorus.

The electrophoretic mobilities of euglobulin and
pseudoglobulin in veronal buffer at pH 8.6 were -l.82 and
-2.02 Tiselius units, respectively. Corresponding iso-
electric points were pH 6.03 and pH 5.54. The partial
specific volumes of euglobulin and pseudoglobulin were
calculated from ultracentrifuge data and were reported as
0.712 and 0.710 ml/g, respectively.

The diffusion coefficients of euglobulin in veronal
buffer, a milk salt solution and a guanidine hydrochloride-

112

113

mercaptoethanol dissociating system, as determined from
ultracentrifugal data, were reported as 3.20, 3.20, and
37.90 Ficks, respectively. Corresponding values for pseudo-
globulin were 4.20, 3.62, and 30.00 Ficks. The diffusion
coefficient of euglobulin decreased with decreasing temper-
atures,but.increased with decreasing hydrogen ion or elec-
trolyte concentrations.

The sedimentation coefficients of euglobulin in
veronal buffer, milk salt solution and the dissociating
system were 6.248, 6.158 and 4.638, respectively, for the
slow sedimenting component and 19.048, 19.168 and 13.668
for the fast moving component. The sedimentation values
of these two components increased with decreases in
temperature and decreased as the hydrogen ion or electro-
1yte concentrations were lowered. Pseudoglobulin exhibited
sedimentation coefficients of 6.008, 5.918 and 4.618,
respectively, in the above buffer systems.

The weight average molecular weights of euglobulin and
pseudoglobulin, determined by a low—speed equilibrium
technique, in veronal buffer, milk salt solution and the
dissociating system were 175,000; 244,000 and 89,000,
respectively, for euglobulin and 167,000; 174,000 and
86,000 for pseudoglobulin. As the temperature was
decreased, the weight average values of euglobulin in-
creased, but these values decreased as the hydrogen ion or
electrolyte concentrations were lowered. The weight

average molecular weights of euglobulin in veronal buffer

114
and milk salt solution at 5° C were approximately 276,000

and 388,000, respectively.

The requirement for euglobulin as a component in the
creaming phenomenon was evident from the experiments with
model systems of washed fat globule, milk salts and eu-
globulin. No creaming occurred when euglobulin was absent
in the system, whereas the creaming ability was im-
proved as the euglobulin concentration was increased. At
constant euglobulin concentration, approximately 0.01% by
weight, temperature affected the creaming property signifi-
cantly. The best creaming occurred in the temperature
range of 3—5° C. Ionic strength had little effect on the
creaming properties of both recombined milk and model
system. Approximately 50% dilution of the whey resulted
in the best creaming. The creaming ability decreased as
the hydrogen ion concentration departed from pH 6.0 to

pH 7.0.

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l I|lyrllbllr>h .nkilill

s,
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~‘\,____'

 

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APPENDIX

APPENDIX

Composition of the Buffers

 

Emp1oyed for Isoelectric Point Determinations

The following quantities were made to two liters with

deionized water for use solutions.

1. Sodium Acetate, pH 4.0, u = 0.2

72.0 ml of 5.0 M NaCl
20.0 ml of 2.0 M sodium acetate
33.7 ml of 3.5 M acetic acid

2. Sodium Acetate, pH 5.0, u = 0.2

72.0 ml of 5.0 M NaCl
20.0 ml of 2.0 M sodium acetate
3.7 m1 of 3.5 M acetic acid

0.2

3. Sodium Phosphate, pH 6.0, H

72.0 ml of 5.0 M NaCl
9.2 ml of 0.5 M NaZHPO4
6.6 m1 of 4.0 M NaHZPO4

4. Sodium PhOSphate, pH 7.0, u = 0.2

72.0 ml of 5.0 M NaCl
22.7 ml of 0.5 M NaZHPO4
1.6 m1 of 4.0 M NaHzPO4

5. Sodium Veronal, pH 8.6, u = 0.2

72.0 ml of 5.0 M NaCl
3.5 m1 of 2.0 M HCl
80.0 ml of 0.5 M sodium veronal

124

125

Composition of Jenness
and Koops' Milk Salt Solution

 

 

Solution I
The following quantities of salts were made to 900 ml

with deionized water:

KHZPO4 1.58 9
K3 citrate-H20 0.51 g
Na3 citrate-SHZO 2.12 g
K2804 0.18 g
M93 citrate-H20 0.50 g
KZCO3 0.30 g
KCl 1.08 9

Solution II

~2H O was dissolved in 50 ml of

A 1.32 g of CaCl2 2

deionized water.

Solution II was added very slowly to Solution I
while stirring the mixture vigorously, and the content

made up to one liter with deionized water.

126

Densities and Relative

 

Viscosities of the Buffers Used

APPENDIX TABLE 1. Densities and relative viscosities of
the buffers useda

 

 

 

Relative .
Buffer Viscosity Den51ty

Veronal, pH 8.6, u = 0.1 1.055 1.0039
Sodium Acetate, pH 4.0,

u = 0.1 1.058 1.0041
Jenness and Koops' Milk

Salt Solution, pH 6.6 1.074 1.0073
6 M Guanidine Hydrochloride

plus 0.02 M 2-Mercapto-

ethanol 1.443 1.1205

 

3The densities were determined in 25 ml pycnometers
and the viscosities in a Cannon-Ubelohde semi-micro
dilution viscometer at 20 : 0.0l° C.

 

 

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