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ELECTROPHORETIC CHARACTERIZATION OF THE PROTEOSE-

PEPTONE FRACTION OF BOVINE MILK

BY

Gary Allan Kasper

A THESIS

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

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1978

ABSTRACT

ELECTROPHORETIC CHARACTERIZATION OF THE PROTEOSE-
PEPTONE FRACTION OF BOVINE MILK

BY

Gary Allan Kasper

Classical acid-soluble and heat-stable proteose-peptone
specimens were prepared from skimmilk, casein—free, centrifuged serum,
and micellar casein. As many as 38 protein-stained zones were evidenced
in PAGE—discontinuous. Sixty—two zones could be accounted for by
excising zones from 7.5% PAG and reelectrophoresing them in 17.5% PAG.

Twenty-two zones, ranging in molecular weight from <3000 to
~90,000, were revealed by SDS-PAGE, with a large number of Species
£31,000 daltons.

As many as 23 zones, ranging in pI from 3.8 to 5.3, were
demonstrated in IEF-PAG.

Zones stained for phosphorus and carbohydrate verified that
phosphOproteins, glycoproteins, and phosphoglycoproteins exist in this
fraction.

Previous observations that component 3 is of serum origin and
that components 5 and 8 are casein-associated and distributed between
the casein micelle and the serum were substantiated. All three
principal components were confirmed to be electrophoretically

heterogeneous.

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to his
major professor, Dr. J. R. Brunner, for his advice, patience, and
guidance during the course of this study and for his aid in the
preparation of the thesis manuscript. Appreciation is also extended
to Dr. L. R. Dugan and Dr. R. C. Chandan of the Department of Food
Science and Human Nutrition and to Dr. H. A. Lillevik of the Department
of Biochemistry for reviewing the thesis manuscript and for serving on
the examination committee.

The author also acknowledges the fine interaction and aid of
the many fellow graduate students during the course of this study.

The author also wishes to express appreciation to the Department
of Food Science and Human Nutrition and to the Ralston-Purina Company
for the financial assistance granted to him for the duration of this

study.

ii

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES.
INTRODUCTION.
REVIEW OF LITERATURE . . . . .
EXPERIMENTAL.
Chemical Analysis
Nitrogen.
Physical Methods.
Preparation of Proteose- -Peptone Specimens. .
Polyacrylamide Gel Electrophoresis in Glass Tubes .
Polyacrylamide Gel Electrophoresis in a Discontinuous
Buffer System . . . .
Polyacrylamide Gel Electrophoresis in Gradient Pore Gels.

Sodium Dodecyl Sulfate Polyacrylamide Gel ElectrOphoresis
IsoeIectric Focusing in Polyacrylamide Gel

Excision of Classical Regions from 7. 5°o Polyacrylamide Gels.

Densitometric Scanning of Stained Polyacrylamide Gels.
Phosphoprotein Staining of Polyacrylamide Gels .
Glycoprotein Staining of Polyacrylamide Gels.

RESULTS AND DISCUSSION

Electrophoretic Methods

Polyacrylamide Gel Electr0phoresis in Single Pore-Size Gels.

Excision of Classical Regions from 7.5% Polyacrylamide
Gels and Their Reelectrophoresis in 17.5% Polyacrylamide
Gels .. .

Differential Staining of 17. S°o Polyacrylamide Gels for
Phosphorus and for Carbohydrate . . . .

iii

Page

vi

15
15
15
15

15
17

19
19
20
20
21
21
22
22

23
23

23

30

34

Page

Polyacrylamide Gel Electrophoresis in 10—25% and 15-30%

Gradient Pore Gels . . . . . 38
Polyacrylamide Gel Electrophoresis Containing Sodium
Dodecyl Sulfate . . . . 40

Excision of Classical Regions from 7. S°o Polyacrylamide Gels
and Their Reelectrophoresis in 17. 5% Sodium Dodecyl Sulfate

Polyacrylamide Gels. . . . . . . . . . 54

IsoeIectric Focusin in Polyacrylamide Gel . . . . . . . 59

Origin of the Proteose- -Peptone Fraction . . . . . . 62
Suggested Experiments to Further Examine the Proteose-

Peptone Fraction . . . . . . . . . . . . . . . 71
SUMMARY. . . . . . . . . . . . . . . . . . . . 73
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . 74
APPENDIX . . . . . . . . . . . . . . . . . . . 80
ADDENDUM . . . . . . . . . . . . . . . . . . . 89

iv

Table

A-5.

LIST OF TABLES

Physical and chemical parameters of components 3, 5,
8-slow, and 8-fast of the proteose-peptone fraction .

Molecular weights of proteose-peptone specimens determined
from 5.0%, 7. %, 10.0%, 12.5% and 17.5% SDS-PAGE .

Molecular weights of regions 3, 3+5, 5, 5+8, and 8 excised
from 7.5% PAG determined from 17.5% SDS-PAG.

Comparison of amino acid compositions of peptide 1-107 of
B-casein A2 and component 5 . . . . . . .

Comparison of amino acid composition of peptide 1-28 of
B-casein A2 and component 8-fast (unheated).

Comparison of amino acid compositions of peptide 1-28 of
B-casein A2.and component 8-fast (heated)

Comparison of amino acid composition of peptide 29-107 of
B—casein A2 and component 8-slow (unheated).

Buffer solutions used in this study .

Formulation of the polyacrylamide gels used in this study.
Staining solutions used in this study

Staining procedures used in this study .

Chemicals used in this study and their sources

Addendum. Amino acid analyses (mol/mol protein).

Page

13

41

57

64

66

67

69
80
82
85
86

87

91

 

Figure

1.

LIST OF FIGURES

Procedure for the preparation of proteose-peptone from
skimmilk . . . .

Procedure for the preparation of proteose-peptone from
micellar casein and serum

Electrophoretic patterns of proteose-peptone specimens
obtained from serum (A), micellar casein (B), and skimmilk
(C) electrophoresed in 12.5% (1) and 7.5% (2) PAG

Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 7.5% PAG .

Electrophoretic patterns of proteose-peptone specimens
obtained from serum (A), micellar casein (B), and skimmilk
(C) electrophoresed in 10% (l) and 5.0% (2) PAG .

Electrophoretic patterns of proteose—peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 15.0% (1) and 17.5% (2) PAG .

Densitometric scanning patterns of proteose—peptone specimens

obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 17.5% PAG. The corresponding
electrophoregrams are beneath the pattern . .

Electrophoretic patterns of proteose-peptone specimens
obtained from skimmilk (A), region 3 (B), interstitial
region 3+5 (C), region 5 (D) interstitial region 5+8
(E), and region 8 (F) excised from 7.5% PAG and re-
electr0phoresed in 17.5% PAG . . . . .

Diagram of electrophoretic patterns of proteose—peptone
specimen obtained from skimmilk (A), region 3 (B),
interstitial region 3+5 (C), region 5 (D), interstitial
region 5+8 (E), and region 8 (F) excised from 7.5% PAG
and reelectr0phoresed in 17.5% PAG . . . . .

vi

Page

16

18

24

25

27

28

29

31

32

Figure Page

10. Electrophoretic patterns of proteose-peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 17. 59a PAG stained for protein (1)
and for carbohydrate (2) . . . . . . . . . . 35

11. Electrophoretic patterns of proteose-peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 17.5% PAG stained for protein (1)
and for phosphorus (2). . . . . . . . . . . . 36

12. Diagram of electrophoretic patterns of protease-peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 17.5% PAG demonstra-
ting those zones which are proteins, phosphoproteins
(p), glycoproteins (g), and phosphoglycoproteins (pg) . 37

13. Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 10-25% linear
gradient PAG (1) and 15-30% linear gradient PAG (2) . . 39

14. Standard curve for molecular weight determination in 5.0%,
7.5%, 10.0%,12.5°o, and 17.5% SDS-PAG. . . . . . . 42

15. Electrophoretic patterns of BDH standard proteins, 53,000-
265,000 (B), l4,300-71,500 (C), RNA polymerase (E. coli)
(A), bovine serum albumin (A), and proteose-peptone
specimens obtained from micellar casein (F), serum (E),
and skimmilk (D) electrophoresed in 5.0% SDS-PAG . . . 43

16. Electrophoretic patterns of BDH standard proteins, 53,000-
265,000 (E), l4,300-71,500 (D), bovine serum albumin (F),
ribonuclease (G), ovalbumin (H), B-lactoglobulin B (H),
and proteose-peptone specimens obtained from micellar
casein (A), serum (B), and skimmilk (C) electrophoresed
in 7.5% SDS—PAG . . . . . . .. . . . . . . . 44

17. Diagram of electrOphoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 7.5% SDS-PAG . . . 45

18. Electrophoretic patterns of BDH standard proteins, 14,300—
71,500 (D), L-glutamic dehydrogenase (E), ovalbumin (E),
B-lactoglobulin B (E), ribonuclease (E), insulin (E), and
proteose- peptone specimens obtained from micellar casein
(A), serum (B), and skimmilk (C) electrophoresed in 10. 0%
SDS PAG. . . . . . . . . . . . . . . 46

vii

Figure

19.

20.

21.

22.

23.

24.

25.

26.

27.

Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 10.0% SDS-PAG .

Electrophoretic patterns of BDH standard proteins, 14,300-
71,500 (D), L-glutamic dehydrogenase (E), ovalbumin (E),
B-lactoglobulin B (E), ribonuclease (E), insulin (E), and
proteose-peptone specimens obtained from micellar casein
(A), serum (B), and skimmilk (C) electrophoresed in
12.5% SDS-PAG . . . . . . . . . . . . .

Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 12.5% SDS-PAG .

Electrophoretic patterns of proteose-peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 17.5% SDS-PAG

Diagram of electrophoretic patterns of proteose—peptone
specimens obtained from micellar casein (A), serum (B),
and skimmilk (C) electrophoresed in 17.5% SDS-PAG .

Electrophoretic patterns of region 3 (A), interstitial
region 3+5 (B), region 5 (C), interstitial region 5+8
(D), and region 8 (E) excised from 7.5% PAG and reelectro-
phoresed in 17.5% SDS-PAG .

Diagram of electrophoretic patterns of region 3 (A),
interstitial region 3+5 (B), region 5 (C), interstitial
region 5+8 (0), and region 8 (E) excised from 7.5% PAG
and reelectrophoresed in 17.5% SDS-PAG. . .

Standard curve of pH 3-6 linear gradient in IEF-PAG .
Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B),

and skimmilk (C) electrophoresed in pH 3-6 linear
gradient IEF-PAG . . . .

viii

Page

47

48

49

50

51

55

S6

60

61

INTRODUCTION

The proteose-peptone fraction of cow's milk is an uncharacterized
group of acid—soluble (pH 4.6) and heat-stable (95 C for 30 min)
proteins found in the plasma. This fraction is believed to consist
primarily of minor caseins that are distributed between the casein
micelle and the serum. Certain components are believed to be of serum
origin. Electrophoresis has been the primary means of characterization
of this fraction, with only four components--3, 5, 8-slow, and 8-fast--
being identified with suspected heterogeneity in each. The presence of
phosphorus and carbohydrate has been detected.

Zonal electrophoresis in polyacrylamide gel simultaneously
exploits differences in molecular size and molecular net charge for
purposes of fractionation. The mobility of a macromolecule in poly-
acrylamide gel electrophoresis (PAGE) is directly proportional to its
intrinsic charge, the pH of the buffer system employed, the magnitude
of the electric field applied, and inversely prOportional to the
frictional resistance encountered by the macromolecule due to its size
and the density of the gel.

The possibility exists that a larger protein, more highly
charged, and a relatively smaller protein, less highly charged, may

migrate at the same rate, and appear as a single zone. Thus the

appearance of a single zone in PAGE should not be interpreted as
unequivocal evidence of homogeneity. Different gel concentrations should
be used to determine whether a single zone is actually homogeneous
(Hedrick and Smith, 1968). Increasing the percentage.of monomer in

PAGE, while keeping the ratio of monomer to crosslinker constant, re-
duces the pore size of the gel matrix and enables one to separate zones
that may be moving together even though they differ in size and charge.

Discontinuous buffer systems in PAGE have been demonstrated
to afford maximal resolution due to the ultra-thin starting zones
achieved (Ornstein, 1964).

Zonal electrophoresis in polyacrylamide gels containing sodium
dodecyl sulfate (SDS—PAGE), in which the proteins have been heated to
aid in SDS binding, has been demonstrated to separate proteins solely
on the basis of size (Shapiro, Vinuela, and Maizel, 1967; Weber and
Osborn, 1969). This is accomplished by the proteins unfolding, binding
the same amount of the highly negatively charged SDS (1.4 g SDS/g
protein), assuming a rod-like form which is directly proportional to
their molecular weight (Reynolds and Tanford, 1970), and moving through
the gel matrix as simulated cylinders (Svasti and Panijpan, 1977).

Electrophoresis in polyacrylamide gels containing carrier
ampholytes, in which a linear pH gradient is produced, has been
demonstrated to separate proteins solely on the basis of charge. The
proteins migrate to, and reside, at their isoelectric points (Finlayson
and Chrambach, 1971). This procedure is referred to as isoelectric
focusing in polyacrylamide gel (IEF-PAG).

Cognizant of the ability of each of the above electrOphoretic

methods to separate proteins on entirely different characteristics,

the author utilized PAGE in a discontinuous buffer system, SDS-PAGE,
and IEF-PAG to better assess the number of components comprising the
proteose-peptone fraction. By isolating the proteose-peptone com—
ponents from skimmilk, casein—free centrifuged serum, and micellar
casein the distribution of these components in the milk protein system
was investigated. The presence of phosphorus and carbohydrate in this
fraction was examined by differential staining of the electrophoretic

zones for these substances.

REVIEW OF LITERATURE

Osborne and Wakeman (1918) were among the first workers to
suggest the presence of an acid-soluble and heat-stable protein in milk.
They observed that some proteinaceous material still remained in
solution after the albumins and globulins of acid whey were heat-
denatured. They were uncertain as to whether this protein was indige-
nous to milk or an artifact resulting from the heat treatment.

Palmer and Scott (1919), using tannic acid to precipitate
protein from casein-free, heat-denatured whey, postulated that lactal-
bumin and lactoglobulin were not the only proteins present in a casein-
free filtrate of milk.

Kieferle and Gloetzl (1930), and Kieferle (1933) employed
phosphotungstic acid to precipitate soluble proteins from heat-
coagulated milk, classifying these components as proteoses and peptones.

Jones and Little (1933) reported the presence of considerable
amounts of proteose in milk, designated as the material precipitated by
10% trichloroacetic acid (TCA), but not by 5% TCA.

Moir (1931) reported that approximately 70% of the soluble
proteins of casein-free filtrate of milk were removed by heat coagula-

tion.

 

Rowland (1938a) designated the protease-peptone as a protein
fraction which did not precipitate at pH 4.7 after heating skimmilk at
95 C for 30 min but was precipitated by 12% TCA. He (1938b) reported
the nitrogen distribution of normal milk as follows: 78.5% casein N,
9.2% albumin N, 3.3% globulin N, 4.0% proteose-peptone N, and 5.0% non-
protein N. He (1937) also found that the non-protein nitrogen content
was not affected by heating up to 100 C and that on continued heating
at 95 and 100 C only minute amounts of proteose resulted from the
hydrolysis of protein.

Harland and Ashworth (1945) noted that casein obtained by
saturating skimmilk with NaCl accounted for more nitrogen than con-
ventionally obtained casein. The whey from this fractionation, when
acidified to pH 3.0, contained 17.7% less nitrogen than that released
by Rowland's precipitation method. In addition, 95% of the whey pro-
teins in this preparation were coagulated by heating to 95 C for 10 min
compared with 76% by Rowland's method.

Aschaffenburg (1946) isolated a surface-active material, which
he called sigma proteose, by heating skimmilk to 90—95 C for 15 min,
followed by coprecipitation of the casein and denatured serum proteins
by acid or rennet coagulation. Treatment of this fraction with one-
half saturated ammonium sulfate resulted in a precipitate, which was
called sigma proteose. This fraction contained significantly less
nitrOgen than other principal milk proteins. In free-boundary electro—
phoresis in phosphate buffer at pH 8.0, three components were seen in
decreasing order of mobility, accounting for 10.5%, 82.5%, and 7.0%,
respectively, of the total protein in Sigma proteose, demonstrating

this fraction's heterogeneity.

Ogston (1946) examined this fraction by ultracentrifugation,
finding that it was heterogeneous with 49% of the material consisting
of a single, well—defined constituent of molecular weight 4900 and a
component comprising 11% of the fraction with a molecular weight of
23,000. The remaining 40% of the fraction was unaccounted for.

Weinstein, Duncan, and Trout (1951) isolated a protein fraction
from heated, rennet whey by a procedure similar to that of Aschaffen-
burg, calling it the ”minor-protein fraction," capable of being photo—
sensitized to produce the solar—activated flavor of homogenized milk.
From elementary analysis, the minor-protein fraction was different from
sigma proteose. The nitrogen content of this fraction, 10%, was low
compared to the 13.95% for Aschaffenburg's sigma proteose. In the
Tiselius cell, at least two components were present in the minor-
protein fraction, with the isoelectric zone of the major components at
pH 3.7 to 4.4, based on electrophoretic mobilities at various pH values
(Weinstein, Lillevik, Duncan, and Trout, 1951).

Gordon, Jenness, and Geddes (1954) reported that both casein
and whey depressed the loaf volume of bread when used in the dough
formulation. Jenness (1959) reported that component 5 isolated from
the proteose—peptone fraction of raw milk was the heat-labile loaf—
volume depressant. Volpe and Zabik (1975) isolated a loaf-volume
depressant, which they attributed to proteose-peptone component 5, from
acid whey and from whey ultrafiltrate. Sodium dodecyl sulfate poly-
acrylamide gel electrophoresis yielded a molecular weight of 14,000-
15,000 for this component. Fuchsin-sulfite dye staining of the gel

component indicated that the loaf-volume depressant was a glyc0protein.

Larson and Rolleri (1955), studying the effect of heat treat-
ment on free-boundary electrophoretic mobilities of whey proteins,
attributed peaks 1, 2, 4, 6, and 7 to euglobulin, pseudoglobulin, a»
lactalbumin, B-lactoglobulin, and serum albumin, respectively, with
peaks 3, 5, and 8 being proteose-peptones. Increasing heat treatments
were employed, until at 95 C for 30 min, there were only peaks 3, 5,
and 8 remaining, which accounted for 4.6%, 8.6%, and 5.7%, respectively,
of the whey proteins. The electrophoretic mobilities of components 3,
5, and 8, calculated from the descending boundaries, were -2.9, -4.5,
and -7.9 x 10"5 cm2 volt.l sec-1, respectively. Since proteose-peptone
components appeared in the electrOphoretic pattern of unheated whey,
Larson and Rolleri suggested that the proteose-peptone fraction was
indigenous to milk.

Jenness (1957) isolated a proteose-peptone fraction, precipir
tated by saturation with NaCl, but not by acid, which contained the
principal constituent of proteose—peptone. This fraction was partially
purified by fractional precipitation at varying values of pH and
(NH4)ZSO4 concentrations. Over 90% of this material had an electro-
phoretic mobility of -4.5 x 10"5 cm2 volt-1 sec.1 at pH 8.6 (Veronal)
and contained 1.2-l.3% phosphorus. The fraction was soluble in the
presence of CaCl2 and not affected by rennin. This is interesting in
that in Jenness' procedure, the proteose-peptone components were ob-
tained from fractions associated with micellar casein, while pre—
viously, the proteose-peptone components were shown to be present in
the whey protein fraction.

Aschaffenburg and Drewry (1959) noted six bands in paper

electropherograms of acid filtrates derived from heat-treated skimmilk.

The prominent band corresponded to peak 5 and the five minor bands
corresponded to peak 3. The same six bands were found in unheated
skimmilk, further indicating their presence in native milk. By salting-
out with sodium sulfate at a concentration of 12 g/100 ml, the hetero-
geneous fraction could be separated from the other whey proteins

present in the casein-free filtrate at pH 4.6. They also observed that
the proteose-peptone stained yellow with bromophenol blue on the filter
paper strips, whereas other whey proteins formed normal bluish-green
bands, possibly indicating the presence of carbohydrates.

Thompson and Brunner (1959) were the first workers to demon-
strate that the proteose-peptone fraction contained glyc0proteins. They
identified hexose, hexosamine, fucose, and sialic acid in several minor.
proteins of milk-~the soluble membrane protein, Weinstein's minor-
protein fraction, and proteose-peptone. A high hexose and sialic acid
content was a common characteristic of these fractions. They suggested
that the glycoproteins of the proteose—peptone fraction might originate
from blood serum.

Brunner and Thompson (1961) reported chemical composition and
physical parameters of five minor protein fractions, namely: Rowland's
proteose-peptone, Aschaffenburg's sigma proteose, Jenness' component 5,
Weinstein's minor-protein fraction, and the soluble fat globule membrane
protein. The chemical composition of these five protein fractions were
very similar in that they contained low amounts of sulfur-containing
amino acids, contained carbohydrate, and were high in phosphorus. Some
similarity was observed in their electrophoretic and ultracentrifugal
properties. They concluded that a common component existed in all the

fractions.

 

Marier, Tessier, and Rose (1963) indicated that 17% to 28% more
sialic acid was present in proteins precipitated with 12% TCA than in
casein precipitated at pH 4.5. Proteose—peptone contained 1.8% sialic
acid which accounted for the entire difference between the sialic acid
content of acid casein and the milk proteins precipitated with TCA.

Bezkorovainy (1965) isolated orosomucoid and M-2 glycoproteins
from bovine serum and colostrum whey, and M-2 glyc0protein from whey.
Milk whey contained no orosomucoid and only trace amounts of the M-2
glyc0protein. Furthermore, both milk and colostrum whey contained a
phosphoglycoprotein which had practically no absorption at 280 nm, thus
reflecting the low concentration of aromatic amino acid residues.
Physical and chemical properties of this phosphoglycoprotein corresponded
to the major component of the milk proteose-peptone fraction. Bezkoro-
vainy concluded that close relationships could exist between colostrum
glyc0proteins and the proteose-peptone fraction.

Ganguli, Gupta, Joshi, and Bhalerao (1967) determined the sialic
acid and hexose contents of the proteose-peptone fraction of milk.
Proteose had higher concentrations of sialic acid and hexose than the
corresponding proteose—peptone sample. Even though the casein fraction
contributed the largest share of sialic acid to the sialic acid distri-
bution in milk proteins, the concentration of sialic acid in the
proteose-peptone fraction was four times that found in the casein
fraction. The sialic acid found in the protease-peptone and proteose
fractions was identified as a neuraminic acid derivative, similar to
that found in k-casein.

Joshi, Ganguli, and Bhalerao (1971) found that proteose-peptone

of colostrum differed significantly in its concentration, its sialic

10

acid content, its molecular size, and its electrophoretic pattern from
the proteose-peptone in milk. They contended that proteose-peptone in
milk is likely to originate from mammary function whereas proteose—
peptone in colostrum probably appears from the blood. Joshi, Ganguli,
and Bhalenao (1971) observed that blood serum proteose-peptone has
higher concentrations of sialic acid compared to that of milk proteose-
peptone. The proteose-peptone from the blood shows more marked
similarity with colostrum proteose-peptone than proteose-peptone from
milk. They concluded that proteose-peptone in milk does not come from
the blood.

Ganguli's group (1966, 1967, 1968a,b) made an extensive study
of the effect of storage, trypsin action, rennet action and heat treat-
ment on the proteose-peptone content of milk. The proteose-peptone
content increased upon heating milk to 25 C, presumably derived from
other milk protein fractions such as casein and the whey proteins. Upon
heating at 30 C and 37 C, some proteose-peptone apparently had also
undergone degradation since the non-protein nitrogen increased while
the proteose-peptone decreased. The proteose-peptone content of milk
and casein was increased by exposure to trypsin, supposedly by release
from casein and eventually changing into non-protein nitrogen by
further enzymatic degradation. The newly released "proteose-peptone”
contained more components, possessing lower electrophoretic mobilities
and lower sialic acid contents, than the native proteose-peptone con-
tent. The action of rennet on milk nearly doubled its proteose—peptone
content. They suggested that a proteose-peptone—like material and non-
protein fractions were released from casein, increasing with increased

rennet concentrations. Proteose-peptone-like material from casein

11

and proteose-peptone from milk showed similarities in their electro—
phoretic patterns, sialic acid content, and gel filtration patterns on
Sephadex G—75. By gradually removing casein from milk by ultracentri-
fugation, the corresponding milk serum showed a linear increase in its
proteose-peptone content. Utilizing a synthetic model system comprising
proteose—peptone and micellar casein, the protease-peptone level
decreased upon heating whereas a similar system containing d-lactalbumin
and B—lactoglobulin in place of micellar casein did not affect the
proteose-peptone level. They postulated that a possible heat-induced
interaction existed between micellar casein and proteose-peptone.

Joshi and Ganguli (1972a) treated K-casein with 2-mercapto-
ethanol, using 8% TCA to precipitate the proteose-peptones, and found
an increase in the proteose-peptones. They concluded that proteose-
peptones could arise as a result of a selective cleavage of K-casein at
the disulfide bridges by indigenous reducing agents in the milk or the
mammary glands.

Joshi and Ganguli (1972b) employed gel filtration chromato-
graphy to separate the proteose-peptone into fractions of different
molecular weights, the leading peak evidencing the presence of sialic
acid.

Bezkorovainy, Nichols, and Sly (1976) isolated proteose—peptone
from both human and bovine milk. The proteose-peptone from bovine milk
evidenced molecular weights of 30,000, 18,000, and 12,000 in SDS—PAGE.

Brunner's group has done the most detailed work on the distri-
bution and composition of the proteose-peptone fraction. Kolar and
Brunner (1965) found that component 8 was present in both casein

micelles and in whey, and was a tenacious contaminant of K-casein

 

 

12

preparations and a leading zone in discontinuous starch-urea gel
electrOpherograms. Kolar and Brunner (1968) further isolated components
5 and 8 from both heated and unheated skimmilk. Component 8 was
fractionated into components 8-fast and 8-slow by gel filtration
chromatography on Bio-Gel P-lO. Component 5 was characterized by its
high proline and relatively low carbohydrate contents. Components 8-
fast and 8-slow contained relatively higher concentrations of phosphorus.
All three components were void of cysteine and cystine, and contained
low concentrations of methionine.

Kolar and Brunner (1969) reported that components 5 and 8 existed
in equilibria between micellar casein and the serum, whereas component
3 was not present in micellar casein. Ng, Brunner, and Rhee (1970)
substantiated this convention by isolating lacteal serum component 3
from both heated and unheated skimmilk. Component 3 was characterized
by its high content of carbohydrates and low content of sulfur-containing
amino acids.

Physical and chemical parameters of the proteose-peptone
fraction derived from Kolar (1967) and Ng (1967) are listed in Table l.

Kang (1971) observed that the proteose-peptone fraction
accounted for 18—25% of the whey proteins. Component 3 migrated as a
single zone in both continuous and discontinuous polyacrylamide gels.
Component 5, homogeneous in continuous gels, was resolved into five
closely migrating zones when examined with a discontinuous buffer
system. Component 8, migrating as two very close zones near the ion
front in discontinuous gels, was resolved into multizonal areas-~8-slow

and 8-fast--in continuous gels. Double diffusion immunoelectrophoresis

 

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14

demonstrated homology between some of the proteose-peptone components

and those of bovine serum.

 

EXPERIMENTAL

Chemical Analysis

 

Nitrogen

Nitrogen analyses were performed according to the semiemicro
Kjeldahl method in which the ammonia is steam-distilled into 4% boric
acid (Swaisgood, 1963). Selenium dioxide and cupric sulfate were the
catalysts employed. Tryptophan was used as a standard, with an average

recovery of 94.1%.

Physical Methods

 

Preparation of Proteose-Peptone Specimens

 

Five gallons of raw, uncooled milk was obtained from Holstein
cows of the Michigan State University dairy herd. The milk was brought
up to 37 C and separated immediately. Approximately four liters of the
skimmilk was heated in a 95 C water bath for 40 min. After this time
the milk was promptly cooled to 20 C. One normal HCl was added until
pH 4.6 was reached. The milk was left to coagulate overnight at 4 C.
The coagulum and serum was filtered through.Whatman #1 filter paper.
The filtrate was collected, dialyzed, pervaporated, lyophilized, and
then stored at O C. Figure 1 depicts the isolation procedure used to

prepare proteose-peptone from skimmilk.

15

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The remaining four gallons of skimmilk was cooled to 20 C and
centrifuged at 105,000 x g for 50 min. The supernatant was decanted,
heated at 95 C for 40 min, cooled to 20 C, and acidified to pH 4.6 with
1N HCl. Centrifugation at 1000 x g for 20 min was performed for clari-
fication purposes. The supernatant was decanted, dialyzed, pervaporated,
lyophilized, and then stored at 0 C. Figure 2 portrays the isolation
procedure used to prepare proteose—peptone from milk serum.

The pellet from the 105,000 x g centrifugation was collected,
redispersed two times in Koops' simulated milk ultrafiltrate, and then
heated at 95 C for 40 min. After cooling to 20 C, the caseins were
isoelectrically precipitated by adjustment to pH 4.6 with 1N HCl. The
solution was left to further coagulate overnight at 4 C. The coagulum
and serum was filtered through Whatman #1 filter paper. The filtrate
was collected, dialyzed, pervaporated, lyophilized, and then stored at
0 C. Figure 2 shows the isolation procedure for preparation of proteose-

peptone from micellar casein.

Polyacrylamide Gel EleCtrophoresis in Glass Tubes

 

All electrOphoretic studies were performed in 6 mm I.D., 2 mm.
walled, and 75 mm length glass tubes. The tubes were detergent washed,
immersed in chromic acid, treated with Photoflo, and rinsed in distilled,
deionized water after use. The acrylamide and bisacrylamide used were
of the highest purity grade or were recrystallized from acetone. The
acrylamide to bisacrylamide ratio was kept in a 19:1 ratio to yield 5%
crosslinked gels, which afford minimal pore size at that total gel

concentration (Rodbard, Levitou, and Chrambach, 1971). All polymerized

gels were not used until at least 18 hours had transpired after

18

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19

polymerization to insure that all gels had achieved the same polymeriza-
tion state. Electrophoresis was performed in a jacketed Buchler
electrophoresis apparatus that was cooled with tap water.

Polyacrylamide Gel Electrophoresis in a
Discontinuous Buffer System

 

 

Polyacrylamide gel electrophoresis in a discontinuous buffer
system was performed according to the method of Melachouris (1969).
Modifications employed were the following:
1. A 1/10 dilution of running buffer with distilled, deionized
water.
2. Protein staining was by Coomassie Brilliant Blue R (Weber and
Osborn, 1969) or Coomassie Brilliant Blue G (Reisner, Nemes,
and Buchalty, 1975).
3. Five percent, 7.5%, 10.0%, 12.5%, 15.0%, and 17.5% gels were
employed.

Polyacrylamide Gel Electrophoresis in
Gradient Pore Gels

 

 

Gradient gels, utilizing the discontinuous buffer system of
Melachouris (1969), of 10-25% and 15—30% were used as a means of
characterization. A sucrose gradient of 2-15% was used to stablize the
polyacrylamide gradient.

The less concentrated gel solution in 2% sucrose was added to
the left-hand chamber of the gradient maker, while the more concentrated
gel solution in 15% sucrose, was added to the right-hand chamber. After
equilibration, mixing was begun, the outlets opened, and the gradient

dispensed into the gel tubes. The linear gradient was pushed up into

20

the apparatus containing the gel tubes with a 60% sucrose solution con-
taining bromophenol blue.

The rest of the electrophoretic procedure was as that for single
pore-size gels.

Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis

 

 

Sodium dodecyl sulfate electrophoresis in polyacrylamide gel
was performed according to the method of Weber and Osborn (1969).
Modifications employed were the following:
1. Protein samples were dissolved in 3% SDS, 1% 2-mercaptoethanol,
in .01M phosphate buffer, pH 7.2. Incubation was at 100 C for
5 min.
2. Five percent, 7.5%, 10.0%, 12.5%, and 17.5% gels were employed.
The molecular weight standard proteins used were the following:
BDH standard molecular weight proteins (14,300, 28,600, 42,900, 57,200,
71,500 and 53,000, 106,000, 159,000, 212,000, and 265,000) which are
synthetically crosslinked, RNA polymerase (E, coli) B, B'4subunits
(155,000 and 165,000), bovine serum albumin (68,000), L-glutamic
dehydrogenase (53,000), ovalbumin (43,000), chymotrypsinogen (25,700),
B-lactoglobulin B (18,276), ribonuclease (13,700), and insulin (3396

and 2337).

IsoeIectric Focusing_in Polyacrylamide Gel

 

Isoelectric focusing in polyacrylamide gel was performed
according to the method of Josephson (1972). The following modifica-

tions were employed:

21

l. Gels of 12.5% total concentration and 1% crosslinkage were
used.

2. Sucrose was eliminated from the gel formulation.

3. Protein was layered at the gel surface, rather than incorporated
into the gel.

4. Staining was by the method of Reisner et a1. (1975).

Ampholine of pH 3.0—5.0 and pH 4.0-6.0 was used. The pH
gradient in the gel was determined by slicing four identical gels into
5 mm sections, determining the pH of these sections by penetrating the
gel surface with a microelectrode, and plotting pH versus distance in
the gel.

Excision of Classical Regions from 7.5%
Polyacrylamide Gels

 

 

Classical regions 3, 5, and 8, and the interstitial regions
were excised from 7.5% PAG. The gels were sliced into the above
mentioned regions by comparison with an identical gel stained by the
method of Reisner et a1. (1975).

The gel sections were triturated, dissolved in sample buffer
overnight at 4 C, and then centrifuged at 1144 x g for 20 min. After
filtering, the filtrate was collected andapplied onto appropriate gels

as protein solution.

 

Densitometric Scanning of Stained
Polyacrylamide Gels

 

A Gilford model 1140 XL gel scanning spectrophotometer was
used to scan 17.5% PAG stained with Coomassie Brilliant Blue R at

560 nm.

22

Phosphoprotein Staining of Polyacrylamide Gels

 

Phosphoproteins present in the gels were detected by the method
of Green, Pastewka, and Peacock (1973). Proteins containing no

phosphorus were also stained as a control.

Glycoprotein Staininggof Polyacrylamide Gels

Glycoproteins present in the gels were detected by the method
of Zacharius, Zell, Morrison, and Woodlock (1969). As a control, gels
were subjected to all treatment steps except for the periodic acid

treatment .

 

RESULTS AND DISCUSSION

Electrophoretic Methods

 

Polyacrylamide Gel Electrophoresis in
Single Pore-Size Gels

 

 

Figures 3 and 4 demonstrate the electrophoretic patterns
obtained in 7.5% PAG in a discontinuous buffer system of proteose-
peptone specimens obtained from micellar casein, casein-free centrifuged
serum, and skimmilk. The patterns show the classical 3, 5, and 8 zones,
with zone 3 notably lacking in the micellar casein specimen. This
supports the work of Ng et a1, (1970) in that component 3 is not present
in the proteose-peptones liberated from washed casein micelles, but is
present in large quantities in the serum. This also corroborates the
work of Kolar and Brunner (1970) in that components 5 and 8 are casein-
associated and are distributed between the casein micelle and the
serum.

Region 5 in each specimen appears to be nearly identical except
for different concentrations of the zones as evidenced by the intensity
of staining. Region 8 appears as an intense, sharp zone moving with
the bromophenol blue tracking dye, its mobility unimpeded by the gel
matrix. Zones with relative mobilities between those of regions 3 and
5 and between regions 5 and 8 are present in minor concentrations in

in each specimen. The casein4free, centrifuged serm specimen showed

23

 

PLEASE NOTE:
Dissertation contains glossy photographs that will not reproduce well on
microfilm. Filmed best way possible.

UNIVERSIT{ MICROFILMS

 

 

24

 

 

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Figure 3. Electrophoretic patterns of proteose-peptone specimens
obtained from serum (A), micellar casein (B), and skimmilk (C)
electrophoresed in 12.5% (1) and 7.5% (2) PAG.

 

   

 

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 4. Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 7.5% PAG.

 

 

 

 

26

profuse zones in the region between 5 and 8. Appearing at the t0p of
the running gel for each specimen is a protein-staining material that
did not enter the gel matrix. Apparently a very high molecular weight
specie or species is present in the proteose-peptone fraction. Aggre-
gation of smaller molecular weight species is a possible contributory

factor to this phenomenon.

When the gel concentration was increased to 10.0% and 12.5%,
additional zones were apparent as shown in Figures 3 and 5. RegiODS 3
and 5 did not migrate as far in these gels, with zone 3 still being the
slowest moving zone. More zones, with greater staining intensity, were
detected between the 3 and 5 regions and between the 5 and 8 regions.
Zones that were moving unimpeded in the 7.5% gels, that is with the
tracking dye, appear to have been hindered in their migration by the
decreased pore size of the 10.0% and 12.5% gel matrix.

Figures 6 and 7 demonstrate that as gel concentration was
further increased to 15.0% and 17.5%, even more zones are evidenced,
with as many as 31, 33, and 29 zones being present in the skimmilk,
casein-free centrifuged serum, and micellar casein Specimens, respec-
tively, in 17.5% PAG. Because of the dramatic resolution obtained as
gel concentration was increased, correlations between resulting
electrophoretic patterns becomes tenuous. Gel concentrations greater
than 17.5% demonstrated fewer zones due to the inability of the slower
moving zones to enter the gel. Thus 17.5% gels were used to maximally
resolve this heterogeneous protein fraction. However, it must be
recognized that by increasing the gel concentration beyond 17.5%,
resolution of zones that may be moving together in 17.5% gels even

though they differ in size and charge, could be achieved.

27

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.0 4111111115:

 

Figure 5. Electrophoretic patterns of proteose-peptone specimens
obtained from serum (A), micellar casein (B), and skimmilk (C)
electrophoresed in 10% (l) and 5.0% (2) PAG.

28

I ~
’ 1
x
N
-4

1A 1B 1C 2A ZB 2C

 

Figure 6. Electrophoretic patterns of proteose-peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk (C)
electrophoresed in 15.0% (1) and 17.5% (2) PAG.

.:uoupwm may numocon one manhwouonm
nonuoofio wcflvcommoypoo 6:9 .u<m ww.- :4 pomouoamonpoofio mow xafleefixm can .mmu ESHom .n<v aflommo
Hwfifioows scum eocflmpno mcoefloomm ocoumomuomoouonm mo mahouuwm mcwccmom owhuoEOpfimcoa .h ensued

 
 
 

 

29

 

 

 

 

 

 

30

Excision of Classical Regions from 7.5%
Polyacrylamide Gels and Their Reelectrophoresis
in 17.5% Polyacrylamide Gels

 

 

 

The most plausible way to verify from which regions the multiple
zones were arising was to excise regions 3, S, and 8, and the inter-
stitial regions from 7.5% PAG and reelectrophorese them in the re-
solving l7.5% gels. The proteose-peptone specimen obtained from skim-
milk was selected as the protein sample because it represented the
classical preparation. The electrophoretic patterns obtained are
revealed in Figures 8 and 9.

Excised region 3 accounted for 10 zones in the 17.5% gels with
the principal zones being a slow—migrating doublet. The three fastest
migrating zones had greater relative mobilities in the 17.5% gels than
the corresponding relative mobilities of region 3 in the 7.5% gels.
This appears to be contrary to all principles of electrophoretic
mobility in polyacrylamide gels. The most specious argument is that
some association of the proteins was diminished as the frictional
resistance encountered increased while moving through the 17.5% gel
matrix. Ng (1967) demonstrated the tendency of component 3 to undergo
concentration dependent association—dissociation phenomena. Margolis
and Kenrick (1968) found that if "shearing" stress is excessive during
gel electrophoresis, loose molecular complexes may be torn apart.

Thus it is plausible that component 3 is actually being dissociated by
the movement through the 17.5% gel. The law of mass action would
increase the tendency for the complex to dissociate further once

dissociation commenced.

 

31

 

 

Figure 8. Electrophoretic patterns of proteose—peptone specimens
obtained from skimmilk (A), region 3 (B), interstitial region 3+5
(C), region 5 (D), interstitial region 5+8 (E), and region 8 (F)
excised from 7.5% PAG and reelectrophoresed in 17.5% PAG.

 

 

 

 

 

 

 

 

 

 

 

   

 

32

.u<m 4m.nfi Ce womoposmoewoofloop can w<m ww.m Eowm vomfloxo nmv w :0fimoh
vcm .mmv w+m coamop Hmfluflpmhopcfl 4nov m :oamoh .nuv m+m :oammg Hmwufiumyoucfi 4mmv m cofiMop 44<U
xfifieeflxm Eopw cocflwpno seafloomm.ocoumomlomoowopm mo mcpouuwm ofluopocmompoofio mo Empmoflo .m ohzwflm

 

J

J
l

+J

 

 

 

lUlIHIWHlIl j

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I!
lefll I IIL Ll
I
“111111 I

 

 

 

 

 

33

The excised region between regions 3 and 5 accounted for 10
closely migrating zones and one faster migrating zone when reelectro-
phoresed in 17.5% PAG.

Excised region 5 appeared as three prominent, closely migrating
zones and two major, faster migrating zones. Eight other zones of minor
concentration were also apparent. The two fastest migrating zones had
very nearly the same relative mobility in 17.5% PAGE as in 7.5% PAGE.
This characteristic attests to their low molecular weight.

The excised region lying between regions 5 and 8 appeared as 16
zones in 17.5% PAGE. A minor zone showed an exceptionally fast relative
mobility.

Excised region 8 migrated as 12 zones with great dispersity in
relative mobilities. Two zones appeared to be greatly hindered in their
migration by the high gel concentration. Three zones moved nearly as
far as the bromophenol blue tracking dye, evidencing their peptidyl
nature.

The proteose-peptone specimen obtained from skimmilk, which was
the origin of these 62 zones evidenced from the excised regions, showed
only 33 zones when electrophoresed in 17.5% gels. Thus one must
realize that the highly resolved electrophoretic patterns obtained from
17.5% PAG underestimates the number of zones actually present. An
explanation may be that closely migrating zones may not be distin-
guishable from each other. The concentration of multiple zones in an
area of the gel could result in a dark, wide zone where, indeed, it

represents numerous, closely migrating zones.

34

One cannot exclude the possibility that several of the 62 zones
evidenced arise from improper slicing of the gels so that overlapping
of the excised zones occurred.

Even the 62 zones demonstrated by electrophoresis in the 17.5%
gels may underestimate the number of components comprising this
fraction. Genetic variants which differ in histidine residues may move
together in alkaline PAGE, but could be separated in acidic PAGE.
Peterson and Kopfler (1966) demonstrated this for the genetic variants
of B-casein A. Also one must realize that the proteinaceous material
which did not enter the gel matrix could account for additional zones.

Differential Stainihg of 17.5% Polyacrylamide
Gels for Phosphorus and for Carbohydrate

 

The results from staining 17.5% gels for carbohydrate and for
phosphorus are represented in Figures 10 and 111 Figure 12 indicates,
utilizing the results of the staining procedure, those zones which are
classified as proteins, phosphoproteins, glycoproteins, and phospho-
glycoproteins. The two slow migrating zones of region 3 stained for
both phosphorus and carbohydrate, verifying the work of Ng 3E_§l,

(1970) in which they found component 3 to be a phosphoglycoprotein.

Three prominent zones, which canbe attributed to region 5,
stained for phosphorus in the proteose-peptone specimens obtained from
skimmilk and casein-free, centrifuged serum. In each of these Specimens
one of the zones stained for carbohydrate, but these zones did not appear
to have the same relative mobilities. The proteose-peptone specimen
obtained from micellar casein demonstrated six zones in the 5 region,
with three containing phosphorus; carbohydrate was not detected. In

proteose-peptone Specimens obtained from both casein-free, centrifuged

35

 

 

1A 2A 18 23 1C 2C

Figure 10. Electrophoretic patterns of protease-peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk (C)
electrophoresed in 17.5% PAG stained for protein (1) and for
carbohydrate (2).

33.13,.

36

     

4 4 I '1
V3 p...
.0 /
1A 2A 13 23 1c 2c

Figure 11. Electrophoretic patterns of proteose~peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk (C)
electrophoresed in 17.5% PAG stained for protein (1) and for
phosphorus (2).

37

 

 

 

 

 

 

 

 

 

 

 

_
99
.______ 99
2 P99
,.__..B 3
p
‘P
—P
E p pg
~—_ p
‘—
p
-——.. p — p
——p —p
~P
pg — P9
———P
P ——P
p —-—_.._..p
P —p
"—P — p
l——-—— p r p
.—_.
*— _____
——__. .—_—..
"—' —P

Figure 12, Diagram of electrophoretic patterns of proteose—peptone
specimens obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in 17.5% PAG demonstrating those zones which are
proteins, phosphoproteins (p), glycoproteins (g), and phospho-
glycoproteins (pg).

38

serum and micellar casein, a phosphoglycoprotein was detected with a
relative mobility greater than either of the phosphoglycoproteins found

in the 5 region.

The proteose-peptone Specimens obtained from both casein-free,
centrifuged serum and skimmilk showed glyc0proteins moving slightly
ahead of the two primary zones of region 3. One of these zones from
the serum specimen appeared to be a phosphoglycoprotein.

The fast migrating zones in 17.5% gels appear to be void of
phosphorus. Such swift moving zones, without deriving a great Share of
their mobility from phosphorylated residues, must be very acidic and/or
very small in Size.

The small pore Size of the 17.5% gels would indicate that the
separation achieved is based more on size than on charge. The electro-
phoretic pattern obtained is a fairly representative reflection of the
molecular weight of the proteins in the zoneS--higher molecular weight
species at the top of the gel and smaller species nearer the anodic
end of the gel. This is not to infer that a larger, highly charged
protein cannot migrate farther than a smaller protein possessing a
lower charge. Electrophoretic resolution on polyacrylamide gels still
leaves open the question of whether two proteins are separated because

they differ in charge and/or size.

Polyacrylamide Gel Electrophoresis in 10—25%
and 15-30% Gradient Pore Gels

 

 

Electrophoretic patterns obtained when the three-proteose-
peptone specimens were electrophoresed in 10-25% and 15-30% linear
gradient gels are represented in Figure 13. Proteose-peptone specimens

obtained from skimmilk, casein-free, centrifuged serum, and micellar

 

 

 

IIIIIIIIIIIIIII M HIM] Hill I! 18

1C

 

 

_—

mmmmm “mm m H m l:
|—* +
[m Ill um 11mm .4

(U

:45
"-1-

UMr-x

' V)

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E

——

 

 

 

ll IIHHIHIMIUHLMIJ

4O

casein demonstrated 24, 24, and 26 zones, respectively, in 10-25%
polyacrylamide linear gradient gels and 33, 38, and 26 zones, respec-
tively, in 15-30% polyacrylamide linear gradient gels.

The rationale for employing gels consisting of decreasing pore
size was to decrease the migration rate of the proteins, so that a
particular protein would reach its pore limit, and very little altera—
tion in zonal pattern would result with further passage of current
(Margolis and Kenrick, 1968). This effect was not achieved totally
since the leading zones were still moving with the ion front at the end
of electrophoresis, obviously being unimpeded by the gel matrix.

Rodbard, Kapadia, and Chrambach (1971) dispute the idea of
"dead-stop electrophoresis" as proposed by Margolis and Kenrick (1968).
They maintained that a limiting pore size cannot be attained and that
gradient gels are, at best, useful for initial "mapping" of complex
mixtures. Because of the large variation of pore size in polyacrylamide

gels at any given concentration, increased resolution cannot be obtained.

Polyacrylamide Gel Electrophoresis Containing

 

Sodium Dodecyl Sulfate

 

The proteose—peptone specimens obtained from skimmilk, casein-
free, centrifuged serum, and micellar casein revealed 12, 17, and 15
zones, respectively, of differing molecular weights. Twenty-two differ-
ent molecular weight species were found collectively in the Specimens,
ranging in molecular weight from <3000 to ~90,000, as shown in Table 2.
Different gel concentrations were required to resolve these diverse
molecular weight species because a single gel concentration could not
separate adequately both large proteins and small peptides. (See

Figures 14—23.) High molecular weight components (>38,000) were

41

Table 2.-—Molecular weights of proteose—peptone Specimens determined
from 5.0%, 7.5%, 10.0%, 12.5% and 17.5% SDS-PAGE.

 

 

 

Skimmilk Micellar Casein Serum

90,000 120009“b

80,000 12000991”c

74,000 1200093”C 74,000 izoooa’b’C
68,000 :1000b’c 69,000 :1000b’C 71,000 izooob’C

66,000 :1000b’C 66,000 110001”c
61,000 ilooob’c’d 61,000 110009“d 61,000 ilooob’c’d
55,000 1.1000“d 56,000 :1000c’d 55,000 11000“d
51,000 _4;1000"’d 51,000 :1000b’c’d
44,000 11000“d 44,000 :1000b’c’d
37,000 :1000C’d’e 37,000 11000998 38,000 _4_~__1000‘:’d’e

31,000 :1000‘1’e

28,000 :1000‘1’e

25,000 :_1oood’e 24,000 :1000‘1’e 25,000 :1000‘1’e

22,000 :1000‘1’e 23,000 :1000‘1’e
20,000 :1000‘1’e 19,000 iloood’e
14,000 :1000‘1’e 14,000 :1000‘1’e

12,000 :1000‘1’e 11,000 :1000‘1’e
8,200 13008 8,200 1300e
7,200 i300e 7,200 :3008

5,700e

4,600e

<3,000e <3,000e <3,000e
b

Cdetermined from 10.0% SDS-PAGE;

6determined from 17.5% SDS-PAGE.

adetermined from 5.0% SDS-PAGE;

determined from 7.5% SDS-PAGE;

ddetermined from 12.5% SDS-PAGE;

42

20.04

10.0-
801
6.0-

40‘

-4

MOLECULAR WEIGHT X IO

2 .04

I .04

0.3-

 

 

 

(>2 on} 0.6 0.8 ”-"fio

RE LATIVE MOBILITY

SDS—PAG
SDS-PAG
SDS-PAG
SDS-PAG
SDS-PAG

o\° o\° o\° o\° o\°

.00 F o
HHH

\INONU'I
. O C O .
mmomo

Figure 14. Standard curve for molecular weight determination in
5.0%, 7.5%, 10.0%, 12.5%, and 17.5% SDS-PAG.

 

43

ll' ‘1‘“ “

1

mh’ in! 1
if,“

n“” Mil, III

A B C D E F

Figure 15. Electrophoretic patterns of BDH standard proteins,
53,000-265,000 (B), l4,300-71,500 (C), RNA polymerase (E. coli) (A),
bovine serum albumin (A), and proteose-peptone specimens obtained
from micellar casein (F), serum (E), and skimmilk (D) electrophoresed
in 5.0% SDS-PAG.

   

44

 

~_tmm’g

ill-W ll—
” 111—11011

,- . ‘7 mfi

| Q

and

1m

C)
U
m
~11
C)

A B H.

Figure 16. Electrophoretic patterns of BDH Standard proteins,
53,000-265,000 (E), l4,300—7l,500 (D), bovine serum albumin (F),
ribonuclease (G), ovalbumin (H), B—lactoglobulin B (H), and proteose-
peptone specimens obtained from micellar casein (A), serum (B), and
skimmilk (C) electrophoresed in 7.5% SDS-PAG.

 

45

.o<a-mom 4m.e CH eomonoeaoepomem mow xHHEehxm 4:4 .mmv 5:466 .fl<v chommo “weeooes
Eogm pocfimuno mCoEfioomm ocoumomnomoououg mo mahouumm ofiuohonmoyuoofio mo Emgwmflo .AH ogsmfim

+

5'
2!“

a0
%<
I.
1‘2

7

553’

/

é
7/4
9
9'
1H9|3M
Winoaiow

 

 

 

 

-III
L I” ll
Ill
66

,01 x

46

.. III
" Ill
*’ ;
. l
A B 'C D E

Figure 18. Electrophoretic patterns of BDH standard proteins,
14,300-71,500 (D), L-glutamic dehydrogenase (E), ovalbumin (E),
B-lactoglobulin B (E), ribonuclease (E), insulin (E), and proteose-
peptone specimens obtained from micellar casein (A), serum (B), and
skimmilk (C) electrophoresed in 10.0% SDS—PAG.

47

.o<a-wom $0.04 :fl womohocm09uooao mow xHMEEMxm cum .mmv Enhom .m<v :fiommo HmHHoofiE
EOAM wocflmpno mcoefloomm ocoumomuomoopOHm mo mahopumm ofluohonmoauoofio mo Emgmmflo .mH ounwflm

<

l—I

/m
EV
|
e

A

7/

"
4’

/ ION
I

7
A

7
I
IHSIEIM
EIV'IHDEI'IOW

4

I
o
co

 

IllIIIIl I Z 7A
"III-
I
O
V

|
8_Ol X

48

1 Mi
III?

 

mil—w-
. Cl 1.
rum - ‘iiaili

 

Figure 20. Electrophoretic patterns of BDH standard proteins,
l4,300-71,500 (D), L-glutamic dehydrogenase (E), ovalbumin (E),
B-lactoglobulin B (E), ribonuclease (E), insulin (E), and proteose—
peptone Specimens obtained from micellar casein (A), serum (B), and
Skimmilk (C) electrophoresed in 12.5% SDS-PAG.

49

.u<a-mam 4m.me :4 eomweoeeonpuoeo flow 944e54¥m 4:4 .flmv 5:466 .m<v aeommo Haeemoes
Eoum pocflmpno mzoEfioomm ocopmomuomoopOHm mo mahoppmm ofipOHonm09uooHo mo Ednwmflo .HN ohsmflm

o m

+

I <

 

 

IHQIEIM
EIV'InDEI'IOW

4
I u ..
I “ low
H :8
“ Ion X
I _ m lom I0...
I . 8

 

 

"' '- r 0 "‘

i
I
I
I ——— —T’ 4..
I ‘\
I

A B C D E F

Figure 22. Electrophoretic patterns of proteose—peptone specimens
obtained from micellar casein (A), serum (B), and skimmilk (C)
electrophoresed in 17.5% SDS-PAG.

 

51

.64a-mam 4m.ee :4 eomoeoeeoeuooeo flow adheaexm 6:4 .mmv 5:466 .m<v :46666 Hwefiouae
Eogm wocwwuno mcoefioomm ocoumomnomoouonm mo mcaoupwm ofiuohocmoupooao mo,empwmfio .mm onsmflm

o m . <

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IHSIEIM
UV'IODEI'IOW

 

 

 

 

 

 

 

 

 

 

 

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CD
‘3

 

 

 

I
301 x

 

   

52

estimated from 5.0%, 7.5%, and 10.0% SDS-PAGE, while molecular weight
species of 538,000 were determined from 12.5% and 17.5% SDS-PAGE. To
separate peptides of £8200 daltons, electrophoresis in 17.5% gels was
required.

Segrest and Jackson (1972) have shown that glycoproteins behave
anomalously in SDS-PAGE. Evidently glycoproteins bind less SDS than
simple proteins. Thus their molecular weight is often overestimated
because of a slower rate of migration. However, by increasing gel con-
centration, molecular sieving predominates and the anomalously high
apparent molecular weight of glycoproteins decreases, approaching in an
asymptotic manner, values close to their real molecular weights.

The proteose-peptone fraction contains glycoproteins; thus the
five gel concentrations employed in this study would be advantageous
in detecting anomalous electrophoretic behavior of the protein compo-
nents. The results of this study indicated that no anomalous behavior
was apparent.

The presence of molecular weights of components >38,000 is
surprising, in that previously no completely dissociated Species with
molecular weights of >40,000 have been demonstrated to be present in
this fraction. Ng (1967) found component 3 to have a molecular weight
of 200,000 at infinite dilution in veronal buffer in the ultracentri-
fuge. This component showed concentration-dependent association
phenomena. In 5M guanidine hydrochloride component 3 demonstrated a
molecular weight of 40,000. Several milk proteins have been Shown to
have molecular weights >40,000, but they have not been demonstrated to
be stable to heating at 95 C for 40 min. Possibly the proteose-peptone

fraction contains components that are rendered heat—stable by

 

 

53

glycosylation. A thorough carbohydrate staining of the electrophoretic
zones in SDS-PAGE would document this. It is also plausible that
associated smaller molecular weight species have not been dissociated
by the SDS and heat treatment employed in preparing the protein sample
for electrophoresis. However, the SDS treatment used in this Study was
typical of the manner in which proteins are treated prior to SDS-PAGE.
Also, the 1% Z-mercaptoethanol used Should have reduced any existing
disulfide bonds.

The zones of molecular weight 531,000 appeared as diffuse zones
extending from the 31,000 dalton region to the region immediately
behind the marker dye. Gels prepared with increasingly higher con—
centrations of polyacrylamide were required to resolve closely migrating
zones in this region. Zones differing in molecular weight of ~1000-2000
could be distinguished from each other in 12.5% and 17.5% SDS-PAG.

The preponderance of zones at molecular weights of £31,000
should be noted because they are in the molecular weight region of the
caseins. Possibly some of the minor glycosylated caseins in milk
survive acid precipitation and are recovered in the proteose-peptone
fraction. Also the possibility exists that zones assigned the mole-
cular weights of 31,000, 28,000, 25,000, 24,000, 23,000, 20,000, and
19,000 represent associated species of cleavage products of major
casein components.

The molecular weight species in the 14,000, 12,000,21nd 8200
regions are quite similar to the 14,300 and 9900 dalton Species of
component 5 and 8-Slow, respectively, reported by Kolar and Brunner

(1970).

54

By extrapolation from the standard curve (Figure 14), the
molecular weight of the zones near the bromophenol blue tracking dye
were estimated at <1000. Actually, the only conclusion that can be
drawn is that the molecular weights are less than that of insulin.
Excision of Classical Regions from 7.5%

Polyacrylamide Gels and Their ReelectrOphoresis in
17.5% Sodium Dodecyl Sulfate Polyacrylamide Gels

 

 

 

To better assess the molecular weights of the classical 3, 5,
and 8 regions, a proteose-peptone specimen from Skimmilk was electro-
phoresed first in 7.5% PAG. Regions of this gel containing the classi—
cal 3, 5, and 8 components, as well as the interstitial regions were
excised and reelectrophoresed in 17.5% SDS-PAG (see Figures 24 and 25).

Excised region 3 showed a major zone of 37,000 daltons and
minor zones of 31,000, 24,000, and 15,000 daltons (see Table 3). The
37,000 dalton zone correlates well with the molecular weight of com—
ponent 3 found by Ng (1970) in 5M guanidine hydrochloride in the ultra-
centrifuge (~40,000). The minor zones could be smaller proteins that
are associated with component 3.

The excised region between regions 3 and 5 showed molecular
species of 37,000, 30,000, 18,000, and 14,000 daltons, and a minor
zone at 24,000 daltons.

Excised region 5 contained a prominent zone of molecular weight
26,000, with minor zones of 14,000 and 12,000 daltons.

The excised region between regions 5 and 8 demonstrated a lone
zone of 25,000 daltons.

Excised region 8 Showed two closely migrating‘zones with

mobilities extending beyond that of the lowest molecular weight standard

55

 

Figure 24.

Electrophoretic patterns of region 3 (A), interstitial
region 3+5 (B), region 5 (C), interstitial region 5+8 (D),
and region 8 (E) excised from 7.5% PAG and reelectrophoresed
in 17.5% SDS-PAG.

56

o<a 4m.n Eoum pomfioxo Amy w :ofiwoa cam

44mg m+m :owmou HmwufiumHOpcfl

m a

 

 

- +

 

 

 

.u<mumom &

.A<S m scheme we magma»

o w m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m.>~ :fi pomouosmouuoofioon 6:6

.flav w+m seamen Hmfiuaumnooea .fiov m seamen

mm ofluoHocmouuooHo mo Emhwmwo .mN oH=MMm

<

 

iHQHM
UV'IODB'IOW

 

 

 

 

 

 

 

OlX

 

   

57

.m:0flpmcfishopov 039 we ommho><m

 

 

ooo.44
ooo.we ooo.m4
coo.me ooo.4~ ooc.4m
ooo.44 ooo.om coo.em
ooomv coo.mm coo.6m ooo.nm ooo.em
w sowwom w+m :oamom m coawom m+m seemom m :oflwom

 

m.o<mnmam wm.hH

Eoem pocflapouop o<a 4m.m Eoem nomfioxo w pew 4w+m .m 4m+m 4m mcofiwop mo magmaoz Hmflsooaozun.m oHan

 

 

58

used (insulin) with molecular weights estimated at <1000 by extrapola-
tion of the standard curve. This data reestablishes the possibility
that region 8 contains components of peptidyl nature.

The molecular weights obtained in this study are not to be
interpreted as unequivocal values. The only true criterion of mole-
cular weight is the amino acid sequence of homogeneous components.
Anomalous results have been reported in SDS—PAGE. Different proteins
do not bind the same amount of SDS (Nelson, 1972). Anomalous binding
of SDS by proteins, atypical conformation of the protein-SDS complex,
or unusual properties of the native protein-SDS complex maintained in
a SDS solution can affect the mobility of proteins (Banker and Cofman,
1972). Neville (1971) found that components of molecular weight
<15,000 are out of the linear region of the standard equation employed
to determine the mobility of macromolecules through the gel matrix.
Williams and Gratzer (1971) Showed that SDS complexes formed by all
proteins of molecular weight <6000 migrate with the same mobility.

This behavior is believed to occur because the frictional coefficients

of SDS complexes of this size are no longer dependent on their mole—
cular weight. Svasti and Panijpan (1977) explain this anomalous behavior
from the fact that the dimensions of the minor and major axes of the
SDS-protein complex are not sufficiently different, so that their
hydrodynamic behavior approaches that of a Sphere instead of a cylinder,
as is common for larger proteins. Swank and Munkres (1971) stated that
the anomalous behavior of small peptides in SDS-PAGE is due to three
reasons: (1) the intrinsic charge of the peptides becomes more impor-
tant at lower molecular weights, (2) the shape of the peptide-SDS com-

plex could cause it to move through the gel matrix in a manner that is

59

not a function of its molecular weight, (3) differential binding of
SDS by different protein species, that is, SDS may bind more to
hydrophobic peptides, and less to hydrophilic peptides.

The above reasons for anomalous behavior of peptides during
SDS-PAGE could assist in explaining the results of SDS-PAGE with that
of conventional PAGE for region 8. Region 8 may consist of many
differing sized Species distributed in the peptide region. Thus
region 8 proteins which Show only two zones by SDS-PAGE separate into

12 zones by PAGE.

IsoeIectric Focusing in Polyacrylamide Gel

 

Isoelectric focusing in polyacrylamide gel, employing pH
3.0—5.0 and pH 4.0-6.0 ampholytes, demonstrated that a linear gradient
was produced (see Figure 26), and that the proteose-peptone fraction
consisted of components in the pl 3.8 to 5.3 region. The proteose-
peptone Specimens obtained from micellar casein, serum, and skimmilk
showed 23, 20, and 13 zones, respectively in IEF-PAG (see Figure 27).
Most of the zones were found in the pl 4.3 to 4.8 region.

More zones may have been detected if the pH gradient covered a
narrower range.

Closely migrating zones were difficult to distinguish due to

background staining. The method of Reisner 23.21: (1975) was found to

be the best method of staining to prevent the ampholytes from staining.

However, some background staining was observed.
Some zones precipitated upon reaching their isoelectric point.
This was apparent by a white precipitation band which developed.

Electrofocusing was continued after the precipitation bands appeared

L I

6O

 

5000‘”
4070 TI—
pII
4.40-.
4010'"
I
3'80 1 : : 1 '4

 

10 20 30 4O 5O
DISTANCE IN GEL

(mm)

Figure 26. Standard curve of pH 3-6 linear gradient in IEF-PAG.

 

 

61

 

 

 

 

 

 

*
F 1 7‘
4075— __ -— ——
22:: iii; ____
____ ==== I..-
4050“ :
F)I-I hI-l ____
-- ——::
4.25— —— = F
—
4.00—
——-+
._._.I _._._. —J
A B C

Figure 27. Diagram of electrophoretic patterns of proteose-peptone
specimens obtained from micellar casein (A), serum (B), and skimmilk
(C) electrophoresed in pH 3—6 linear gradient IEF-PAG.

 

 

62

to insure that other proteins had sufficient time to reach their iso-
electric zones. Because the pH gradient undergoes a progressive
flattening with time of electrofocusing, called the "plateau phenomenon"
(Finlayson and Chrambach, 1971), it would be unreliable to determine
isoelectric points of precipitated proteins from pH measurements long
after they had precipitated. Thus, all that can be ascertained is that
the proteose—peptone fraction consists of components in the pl 3.8 to
5.3 region.

The isoelectric points of the caseins are in this isoelectric
pH region. Possibly some of the proteose—peptones are caseins or con-
sist of casein segments derived from the caseins.

There is the possibility in IEF—PAG that two proteins may have
the same isoelectric point, but actually be different in molecular
Size. Such proteins would be indistinguishable, appearing as one zone
in the electrophoretic pattern. The possibility that this phenomenon
was operative in this study is reinforced by the consideration that
the proteose-peptone fraction consists, in part, of proteolytic break-
down products of other milk proteins. Breakdown products derived from
the same region of the native protein, but with several more or less
uncharged amino acids, would be inseparable in IEF-PAG but separable
by 17.5% PAG. This could account for the better resolution by PAGE

then IEF-PAG.

Origin of the Proteose—Peptone Fraction

 

The proteose-peptone fraction of cow's milk derives its name
from the supposition that its components are heat-derived breakdown

products of major milk proteins, consisting of proteoses which are

63

precipitable by one-half saturated ammonium sulfate, and of peptones
which are precipitable by saturated ammonium sulfate. Since several
workers (Larson and Rolleri, 1955; Kolar and Brunner, 1970; Ng o£_§13,
1970) have demonstrated the presence of proteose-peptone components in
unheated skimmilk, the above mentioned supposition can be refuted.

With the increasing importance being ascribed to the role of
intrinsic milk protease (plasmin) in the milk protein system (Kaminogawa
and Yamauchi, 1972; Kaminogawa, Mizobuchi, and Yamauchi, 1972;

Yamauchi and Kaminogawa, 1972; Groves, Gordon, Kalan, and Jones, 1973;
Eigel, 1977a,b), one can postulate that this trypsin-like enzyme found

in milk could be a primary source of the protease-peptone components.
Groves 23.91: (1973) isolated the gamma caseins from Skimmilk,
demonstrating that they are indigenous to milk, and are actually the
106-209 and 108-209 segments of B-casein A2. Eigel (1977a,b) showed that
plasmin is responsible for gamma casein formation from B-casein A2.

The 1-105 and 1-107 N-terminal segments of B-casein have not
been discovered in the milk protein system. Could not these highly
acidic, phosphorylated peptides be present in the proteose-peptone
fraction? One worker has already made this hypothesis (Jenness, 1978).
By comparing the amino acid composition of component 5 of the proteose-
peptone fraction, as isolated by Kolar and Brunner (1970), with that
of the known amino acid sequence of the 1-107 peptide of B-casein A2,
Jenness uncovered an uncanny correlation in amino acid residues and in
phosphorus content. This comparison is shown in Table 4.

Since Kolar's component 5 was not pure, the presence of 1.5%
carbohydrate (which is minute) could result from a contaminating

glyc0protein or phosphoglycoprotein. In the author's study the 5

64

Table 4.-—Comparison of amino acid compositions of peptide 1-107 of
B-casein A2 and component 5.3

 

 

 

 

Peptide 1-107 of B-casein A2 Component 5b
Residue No. per Wt. per Wt. per moles

mole residue mole g/lOOg 12,469
Lys 8 128 1024 7.55 7.4
His 2 137 274 1.92 1.8
Arg 2 156 312 2.60 2.1
Asp 6 115 690 5.81 6.3
Thr 5 101 505 4.72 5.8
Ser 9 87 783 5.77 8.3
Glu 24 129 3096 24.89 24.0
Pro 15 97 1455 10.55 13.6
Gly 2 57 114 1.23 2.7
Ala 3 71 213 1.92 3.4
1/2 Cys — - - - —
Val 9 99 891 6.60 8.3
Met 2 131 262 1.68 1.6
Ile 7 113 791 6.14 6.8
Leu 8 113 904 7.33 8.1
Tyr 1 163 I 163 1.94 1.5
Phe 4 147 588 4.86 4.1
Trp - - - — -
H2P03 5 81 405 2.53 3.9
CorrectionC —1

Total 107 12,469

 

aFrom Jenness (1978).
bData from Kolar and Brunner (1970).

cAdd 18 for N-terminal H and C—terminal OH, subtract 14 for
difference between weights of -NH2 and -0H for 14 amides, subtract 5
for H's lost from serine by esterification with HZPOS'

65

region did demonstrate the presence of a phosphoglycoprotein. Con-
centration also plays an important role in the amino acid data. Minor
impurities in Kolar's preparations would not contribute significantly
to the data. Also assuming the impurities in component 5 to be caseins,
or casein—derived, less deviation could occur because of the quite
similar amino acid compositions of each of the caseins. Thus Kolar and
Brunner may have indeed isolated the 1-107 and/or 1-105 segments of
B-casein A2 which result from the action of milk protease (plasmin) on
B-Casein A2. Moreover, some of the zones in the 5 region evidenced in
this study appear to represent these two peptides. Possibly the gene-
tic variants ascribed to the N-terminal segments of B-casein are also
present in the 5 region. This is reinforced by the molecular weights
of 14,000 and 12,000 found for the excised 5 region by SDS-PAGE.

The possibility that segment 1-28 of B-casein, resulting from
the formation of Yl-Al, Yl—Az, Yl-AS, and Yl-B caseins from B-casein
by the action of milk protease, or plasmin, are present in the 8-fast
fraction, as isolated by Kolar and Brunner (1970), is plausible. A
good correlation results when the amino acid composition of Kolar's
8-fast is compared to the known sequence of segment 1-28 of B-casein,
as represented in Tables 5 and 6.

Data from the unheated Specimen shows a better correlation
between the residues of phosphorus of component 8-fast with that of
the 1—28 segment than does data from the heated Specimen. Perhaps
heating of the Skimmilk in the first step of the preparation of the
specimen may have cleaved some of the phosphorus from the serine mono-
phosphoesters. Since Kolar and Brunner's component 8-fast was not

pure, the same rationale can be used as that of component 5-—contaminating

66

Table 5.—-Comparison of amino acid composition of peptide 1-28 of
B—casein A2 and component 8-fast (unheated).

 

a
Peptide 1-28 of B-casein A2 Component 8-fast

 

 

 

 

(unheated)
Residue No. per Wt. per Wt. per moles
mole residue mole g/lOOg 3472
Lys 1 128 128 7.92 2.1
His - - - - -
Arg 2 156 312 3.63 0.8
Asp 2 115 230 8.20 2.5
Thr l 101 101 4.48 1.5
Ser 5 87 435 12.17 4.9
Glu 7 129 903 23.96 6.4
Pro 1 97 97 2.51 0.9
Gly 1 57 57 1.21 0.7
Ala - — — .80 0.5
1/2 Cys - - _ - _
Val 2 99 198 5.57 2.0
Met - - - - -
Ile 3 113 339 8.41 2.6
Leu 3 113 339 4.27 1.3
Tyr — — - 1.45 0.3
Phe - — - .73 0.2
Trp - - - .06 -
HZPO3 4 81 324 8.59 3.7
Correction +9
Total 28 3472

 

aData from Kolar and Brunner (1970).

bAdd 18 for N-terminal H and C-terminal OH, subtract 5 for
difference between weights of -NH2 and -0H for 5 amides, subtract 4
for H's lost from serine by esterification with H2p03'

67

Table 6.—-Comparison of amino acid compositions of peptide 1-28 of
B-casein A2 and component 8-fast (heated).

 

 

 

 

Peptide 1-28 of B-casein A2 Component 8-fasta
(heated)
Residue No. per Wt. per Wt. per mole§_

mole residue mole g/lOOg 3472
Lys 1 128 128 5.22 1.4
His - - - .82 0.2
Arg 2 156 312 5.42 1.2
Asp 2 115 230 8.11 2.4
Thr l 101 101 5.04 1.7
Ser 5 87 435 10.00 4.0
Glu 7 129 903 25.30 6.8
Pro 1 97 97 3.67 1.3
Gly l 57 57 1.19 0.7
Ala - - - .93 0.6
1/2 Cys - - - - _
Val 2 99 198 5.18 1.8
Met - - - 1.10 0.3
Ile 3 113 339 7.36 2.3
Leu 3 113 339 7.16 2.2
Tyr - - - .70 0.1
Phe — - - 1.04 0.2
Trp - - - .23 -
HZPO3 4 81 324 6.26 2.7
Correction +9

Total

28 3472

 

aData from Kolar and Brunner (1970).

bAdd 18 for N-terminal H and C-terminal 0H, subtract 5 for

difference between weights of -NH and -0H for 5 amides, subtract 4

for H's lost from serine by ester1fication with H2P03'

68

Species account for the small discrepancies in amino acid data and in
the presence of the ~2% carbohydrate.

Possibly one of the phosphorus staining zones migrating near
the tracking dye in this study represents this highly charged peptide.

Kolar and Brunner (1970) isolated proteose-peptone component
8-slow. Its molecular weight was determined to be 9900 in the ultra-
centrifuge. This might represent the 29-105 and 29—107 segments of
B-casein which would result if cleavage of the 1-105 and 1-107 segments
occurred at the Lys—Lys bond as demonstrated in the formation of yl-Al,
Yl-AZ, Yl-AS, and Yl-B caseins. However, this does not appear to be
the case. A comparison of the amino acid and phosphorus data of Kolar
and Brunner with that of the known sequence of this segment of B-casein A2
indicates a poor correlation (see Table 7). The high carbohydrate
content present in 8-Slow also supports the argument that 8—slow does
not represent the 29-105 and 29-107 segments of B-casein A2.

However, the possibility that component 8-slow could be derived
from the other caseins is a distinct possibility. Its high glutamic
acid and aspartic acid contents, along with a phosphorus content of
5.8% and a low proline content, suggests that it is derived from the
hydrophilic region of as-casein. Possibly milk protease, or plasmin,
cleaves aS-casein in a trypsin-like manner analogous to its action on
B-casein.

Eigel (1977b) demonstrated (12.31332) that as—casein is broken
down by plasmin, but not as readily as B—casein. He found the pre-

dominant breakdown species to have molecular weights at 20,500, 12,300,

and 10,300.

69

Table 7.--Comparison of amino acid composition of peptide 29-107 of
B-casein A2 and component 8-slow (unheated).

 

 

 

 

Peptide 29-107 of B-casein A2 Component 8-slowa
Residue (unheated)

No. per Wt. per Wt. per moles

mole residue mole g/100g 9015
Lys 7 128 896 8.79 6.2
His 2 137 274 5.63 3.7
Arg - - — 1.36 0.8
Asp 4 115 460 15.34 12.0
Thr 4 101 404 4.49 4.0
Ser 4 87 348 8.67 9.0
Glu 17 129 2193 15.40 10.8
Pro 14 97 1358 3.38 3.1
Gly 1 57 57 .53 0.8
Ala 3 71 213 2.13 2.7
1/2 Cys - - - — —
Val 7 99 693 2.40 2.2
Met 2 131 262 .67 0.5
Ile 4 113 452 2.90 2.3
Leu 5 113 565 6.27 5.0
Tyr l 163 163 .68 0.4
Phe 4 147 588 2.42 1.5
Trp - - - .23 0.1
HZPO3 1 81 81 5.79 6.4
Correction +8

Total 79 9015

 

aData from Kolar and Brunner (1970).

bAdd 18 for N-terminal H and C-terminal 0H, subtract 9 for
difference between weights of -NH2 and —0H for 9 amides, subtract 1 for
H lost from serine by esterification with HZPOS'

70

Reimerdes and Klostermeyer (1974) demonstrated that milk
protease is associated with casein micelles. Milk protease, with its
ready access to the caseins, could cleave susceptible casein substrates,
forming proteose-peptone components. These components could be held in
the calcium-apatite complex, being released upon acidification. Kolar
(1967) found that component 8 could be released from whole casein by
treatment with EDTA by heating and by the addition of ammonium sulfate,
processes which disrupt casein micelles.

Furthermore, the fact that the proteose-peptones liberated
from washed casein micelles contain phosphorus implicates their casein
origin. A large portion of the components comprising this fraction
seem to be located in the micelle, and are easily liberated upon
acidification to pH 4.6. Possibly they occupy periphery positions in
the micellar structure. This would explain the presence of proteose-
peptone components in both the serum and micellar casein, as demon-
strated in the respective electrophoretic patterns in this study.
B-casein seems to be loosely associated in the micellar structure since
it readily dissociates from the micelle at temperatures lower than
8.5 C (Garnier, 1966).

Conceivably milk protease could cleave the caseins further
than is presently acknowledged, forming very small peptides, such as
the extremely small peptides evidenced in this study.

Since proteose-peptone zones were present in the casein-free,
centrifuged serum specimen and not in the electrophoretic patterns of
the proteose-peptone specimen liberated from the casein micelle, not
all of the proteose-peptone components arise from micellar casein.

Ng oh 313 (1970) demonstrated that component 3 is solely a serum

 

71

constituent, which was verified in this study. Kang (1971), employing
double diffusion immunological techniques, showed homology between some
of the proteose—peptone components and those of bovine serum.

The heat stability of proteose—peptone components that are of
serum origin could be accounted for by postulating that they are
rendered heat-stable by virtue of glycosylation and/or phosphorylation.

Suggested Experiments to Further Examine
the Proteose-Peptone Fraction

 

 

Increased electrophoretic resolution might be achieved by two—
dimensional electrophoresis.

Casein could be treated with milk protease, or plasmin, and the
products subjected to heating at 95 C for 30 min, and then acidified to
pH 4.6. This would yield those breakdown products that are actually
proteose-peptone like. Examination of these products by electrophoretic
methods employed in this Study would verify whether they are, indeed,
analogous to the proteose—peptone components.

To assess whether proteose—peptone components are formed in the
mammary gland by milk protease, or plasmin, action or after milking has
occurred, milk could be stored for varying periods of time, and the
changes, if any, in the proteose—peptone fraction monitored by gel
electrophoresis.

Immunoelectrophoresis could be a further means of determining
whether the proteose~peptone components arise from micellar casein,
milk serum, bovine serum, and/or the plasmalemma.

The most important work to be done involves isolating the many
components, in pure form, so that structural work can be pursued.

Preparative gel electrophoresis and anionic exchangers could be used

72

as a means of isolation. Concanavalin A could be used, in an immobi-
lized form, to separate the carbohydrate-containing proteins from
those which are not glycosylated.

N— and C-terminal determinations and immunoelectrophoresis could
be used to test the homogeneity of isolated proteins, and once a pure
protein has been isolated, various Structural methods could be employed.
The determination of the amino acid sequence would be the ultimate,
structural method used, along with the determination of whether the
carbohydrate linkages to the native protein are N-glycosidic or O-
glycosidic, and the exact number and location of the linkages of the

polypeptide chain.

SUMMARY

The implementation of increasing gel concentrations in PAGE in
a discontinuous buffer system to fractionate the proteose-peptone
fraction of bovine milk demonstrated that at least 62 different
components comprise this fraction. This enhanced resolution is presumed
to be due to the separation of this fractions' components more on the
basis of size than on charge. The molecular weight species observed in
this fraction ranged from <3000 to ~90,000 daltons, with a majority of
components in the range of 31,000 daltons and below. AS many as 23
zones were observed in IEF-PAG with apparent pI's ranging from 3.8 to
5.3. Phosphoproteins, glycoproteins, and phosphoglycoproteins were
detected. Component 3 was found to be of serum origin, whereas com-
ponents 5 and 8 were shown to be casein—associated and distributed
between the casein micelle and the serum. All three classical com-
ponents were heterogeneous.

The concept that proteose—peptone components arise from the
proteolytic action of milk protease or plasmin on B-casein was rein-

forced.

73

 

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Svasti, J. and Panijpan, B. 1977. SDS-polyacrylamide gel electro-
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Swaisgood, H. E. 1963. Ph. D. Thesis. Michigan State University.

Swank, R. T. and Munkres, K. D. 1971. Molecular weight analysis of
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Thompson, M. P. and Brunner, J. R. 1959. The carbohydrates of some
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Volpe, T. and Zabik, M. 1975. A whey protein contributing to loaf
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Weber, K. and Osborn, M. 1969. The reliability of molecular weight
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electrOphoresis. J. Biol. Chem. 244:4406.

 

79

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APPENDIX

APPENDIX

Table A-1. Buffer solutions used in this Study.

 

PAGE in a Discontinuous Buffer System
.062M Tris-HCI, pH 6.7
Dissolve 1.877 g Tris in 200 ml distilled, deionized water.

Add 1N HCl until pH 6.7 reached. Make to 250 m1.

.380M Tris-HCI, pH 8.9

Dissolve 46.018 g Tris in 900 ml distilled, deionized

water. Add 1N HCl until pH 8.9 reached. Make to 1000 m1.

.046M Tris-ggycine, pH 8.5
Dissolve 11.132 g Tris and 57.6 g glycine in 1800 ml

distilled, deionized water and make to 2000 m1.

SDS-PAGE
.40M phosphate buffer, pH 7.2, containing 0.2% SDS
Dissolve 15.6 g NaH2P04.H20 and 77.2 g NazHPO4.7H20, and

2.0 g SDS in 900 ml distilled, deionized water and make to

1000 ml.

.ZOM phosphate buffer, pH 7.2, containing 0.2% SDS I
- I
Dissolve 15.6 g NaH2P04.H20 and 77.2 g NazHPO4.7H20, and

4.0 g SDS in 1800 ml distilled, deionized water and make to

2000 ml.

80

 

81

 

.OlM phosphate buffer, pH 7.2, containing 3% SDS, % 2-mer-

 

captoethanol
Dissolve 6.00 g SDS and 200 pl 2-mercaptoethanol in 10

m1 .20M phosphate buffer, pH 7.2. Make to 200 ml with distilled,

deionized water.

 

 

82

Table A—2. Formulation of the polyacrylamide gels used in this study.

 

PAGE in a Discontinuous Buffer System

 

U1

% spacer gel solution

 

Dissolve 1.1875 g acrylamide and .0625 g bisacrylamide

in 20 ml .062M Tris-HCl buffer, pH 6.7, and make to 25 ml.

><
o\°

running gel solution

 

Dissolve .475X g acrylamide and .025X g bisacrylamide in

40 ml Tris-HCl buffer, pH 8.9, and make to 50 ml.

Polymerization of Gel

 

50 ml gel solution
50 ul TEMED

.20 ml 2.0% ammonium persulfate (freshly prepared)

SDS—PAGE

X% (5%—12.5%) gel formulation

 

10.00 m1 .20M phosphate buffer, pH 7.2

.80X m1 stock gel solution (23.750 g acrylamide and
1.250 g bisacrylamide dissolved in 100 ml).

1.00 ml 0.5% ammonium persulfate (freshly prepared)

20 ul TEMED

Y ml distilled, deionized water (to bring to 20 ml)

17.5% gel formulation

 

12.5 ml .40M phosphate buffer, pH 7.2
29.2 ml stock gel solution (28.500 g acrylamide and 1.500
g bisacrylamide dissolved in distilled, deionized water

I
I
and brought to 100 m1) 4

83

 

50 ul TEMED
1.50 ml 0.5% ammonium persulfate (freshly prepared)

Y ml distilled, deionized water (to bring to 50 ml)

IEF-PAG
8.00 ml gel solution (1.240 g acrylamide and .0125 g bisacrylamide
dissolved in distilled, deionized water and brought to 10 ml)
1.00 ml riboflavin (w/v)
.75 ml 0.8% TEMED (v/v)
125 pl ampholine pH 3.0-5.0

125 pl ampholine pH 4.0—6.0

PAGE-Gradient

X% in 2% sucrose--low concentration

 

Dissolve .95X g acrylamide, .05X g bisacrylamide, and 2.00 g
sucrose in 80 ml .380 M Tris-HCl buffer, pH 8.9, and make to

100 m1.

% in 15% Sucrose——high concentration

 

Dissolve .95Y g acrylamide, .05Y g bisacrylamide, and
15.00 g sucrose in 80 ml .380M Tris-HCl buffer, pH 8.9, and

make to 100 ml.

84

 

Gradient Formulation

 

75 m1 low concentration
75 ul TEMED
1.30 ml 1.0% ammonium persulfate (freshly prepared)

--into left-hand chamber of gradient-maker

70 ml high concentration

70 p1 TEMED

.60 ml 1.0% ammonium persulfate (freshly prepared)

--into right-hand chamber of gradient-maker

 

85

Table A—3. Staining solutions used in this Study.

 

Coomassie Brilliant Blue R-—.25% (w/v)

 

Dissolve 1.25 g Coomassie Brilliant Blue R in 46 m1 glacial
acetic acid and 227 ml methanol. Make to 500 ml with distilled,

deionized water.

Coomassie Brilliant Blue G--.04% (w/v) in 3.5% HC104

 

Dissolve .400 g Coomassie Brilliant Blue G in 35 g HC10 Make

4.
to 1000 ml with distilled, deionized water.

"Stains-all"--Stock Solution

 

Dissolve .100 g "Stains—all" in 75 m1 formamide. Make to 100 ml

volume.

"Stains-all"--Working Solution

 

Mix 10 ml of "Stains—all" stock solution with 10 ml formamide,

20 ml isopropanol, and 1.0 m1 3.0M Tris—HCl. Make to 200 ml with

distilled, deionized water.

 

86

Table A-4. Staining procedures used in this Study.

 

Coomassie Brilliant Blue R

 

Immerse gels in 5% TCA for at least 30 min. Stain in Coomassie
Brilliant Blue R for at least two hours. Destain in (5:7:88 :

methanolzacetic acidzwater).

Coomassie Brilliant Blue G

 

Immerse gels in Coomassie Brilliant Blue G. Zones evidenced within
1-2 min. No destaining necessary. Enhance zone intensity at expense

of background by immersion in 7% acetic acid.

Zacharius Method of Staining Glycoproteins

 

l. Immerse in 5% TCA for at least 30 min.

2. Rinse lightly with distilled, deionized water.

3. Immerse in H5106 (in 3% acetic acid) for 50 min.

4. Wash overnight with distilled, deionized water with several
changes.

5. Immerse in fuchsin—sulfite stain for 50 min in dark at 4 C.

6. Wash with 0.5% freshly prepared K2S205 3 times for 10 min
each.

7. Wash with distilled, deionized water until desired staining

level is achieved.

Phosphoprotein Stainingf—"Stains-all" Procedure

 

Fix gels in 25% isopropanol for at least two hours. Stain gels in
"Stains-all" overnight in dark, or until desired intensity achieved.

Excessive staining causes dark background.

 

 

Table A-5. Chemicals used in this study and their sources.

 

 

Chemical Source
Acrylamide Ames, BioRad
Bisacrylamide Ames
SDS (Sodium dodecyl sulfate) BioRad
TEMED (Tetraethylmethylenediamide) BioRad
Photoflo Eastman
Stains—all Eastman
Boric acid Fisher
Bromophenol blue Fisher
Glycine Fisher
Potassium metabisulfite Fisher
Schiff reagent Fisher

Periodic acid

BDH standard proteins
Ammonium persulfate
Sucrose

Ampholine

Acetic acid

Cupric sulfate
Hydrogen peroxide
Riboflavin

Selenium dioxide
Sodium hydroxide
Sulfuric acid

TCA (trichloroacetic acid)

G. Frederick Smith
Gallard-Schlesinger
J. T. Baker

J. T. Baker

LKB

Mallinckrodt
Mallinckrodt
Mallinckrodt
Mallinckrodt
Mallinckrodt
Mallinckrodt
Mallinckrodt

Mallinckrodt

88

 

2-mercaptoethanol
Chymotrypsinogen

Bovine serum albumin

RNA polymerase——B, 8'—subunits
Coomassie Brilliant Blue R
Coomassie Brilliant Blue G
L-glutamic dehyrogenase
Insulin

B-lactoglobulin

Ovalbumin

Phenol red

Ribonuclease

Tris (trishydroxymethylaminomethane)

Matheson, Coleman and Bell
Nutritional Biochemical Corp.
Pierce

Pierce

Sigma

Sigma

Sigma

Sigma

Sigma

Sigma

Sigma

Sigma

Sigma

 

ADDENDUM

 

ADDENDUM

Andrews (1978) concluded that proteose-peptone components 5 and
8-fast are, respectively, the 1-105 and 1-28 segments of B-casein A2
formed by the cleavage of B-casein to form Yz-casein and yl-casein.

Andrews heated skimmilk at 95 C for 30 min, adjusted the milk
to pH 4.6, collected the supernatant, and saturated with (NH4)ZSO4.
The precipitate was fractionated by dialysis to give the diffusible
proteose-peptone component 8-fast and by gel filtration to give com—
ponent 5 of the proteose-peptone fraction. The amino acid analyses of
components 5 and 8-fast demonstrated a very close correlation to the
1-105 and 1-28 segments of B-casein A2, respectively (see Addendum
Table).

Kanno and Yamauchi (1978) postulated that proteose-peptone
component 3 is identical to a soluble glyc0protein (SGP) of the milk
fat globule membrane. They employed Ouchterlony's double immuno-
diffusion and Scheidegger's immunoelectrOphoretic assays to assess any
antigenic Similarities between the SGP fraction and individual
proteose—peptone components 3, 5, and 8. Only component 3 contained
the anti—SGP reacting protein. Even though their SDS-PAGE patterns

were somewhat different, the SGP and component 3 each contained a major

89

90

glycoprotein of 20,000 daltons, which seemed to cause the identical

antigenicity of both protein fractions.

Andrews, A. T. 1978. Proteolysis in milk and the formation of
proteose-peptones. XX: Int. Dairy Cong.

Kanno, C. and Yamauchi, K. 1978. Antigenic Similarity between a major
soluble glyc0protein fraction of milk fat globule membrane and
proteose-peptone fraction of milk. XX: Int. Dairy Cong.

Table Addendum.--Amino acid analyses (mol/mol protein).

91

 

B-casein A2

B-casein A2

 

Amino acid Component 5 Component

(1-105) 8-fast (1-28)
ASpartic acid 6.0 6 2.1 2
Threonine 4.7 5 1.3 1
Serine 7.3 9 4.2 5
Glutamic acid 24.2 24 6.8 7
Proline 13.0 14 1.7 1
Glycine 3.0 3 1.0 l
Alanine 2.7 3 0.2 0
Valine 7.7 9 2.0 2
Methionine 2.3 2 0.3 0
Isoleucine 6.1 7 2.6 3
Leucine 8.3 8 2.9 3
Tyrosine 1.8 1 0.3 0
Phenylalanine 4.2 4 0.2 0
Histidine 1.8 l 0.2 0
Lysine 6.9 7 0.9 l
Arginine 1.9 2 1.9 2

 

aFrom Andrews (1978).

«1%;

 

"IIIIIIIIIIIIIIIIIIIII“