THE ESOLATEGN PM) PE‘fi’SZCAL-CEEMECAL
CHARAC'E'ERIZA’WN (3!: A, {SLYQDF‘ROTEIN FROM THE
PROTEOSEPEPTONL FRACTEGN 0F COWS MfiLK

Thesis: fer the Degree cf Ph. 0.
MICHIGAN STATE UNIVERSITY
WESU C. MG
1%?

 

   
  

‘— m hug-L

LIBRARY 3
Michigan State 2

University ' rt

THEE!»

This is to certify that the

thesis entitled

The Isolation and Physical-Chemical
Characterization of a Glyc0protein from the
Proteose-Peptone Fraction of Cow's Milk

presented by

Wesu C. Ng

has been accepted towards fulfillment
of the requirements for

Ph.D. degree inFood Science

Major professor

9

7
/fl gMfi/A Lanvkkx

Date June 16, 1967

ABSTRACT

THE ISOLATION AND PHYSICAL-CHEMICAL
CHARACTERICATION OF A GLYCOPROTEIN FROM THE
PROTEOSE-PEPTONE FRACTION OF COW'S MILK

by wesu c. Ng

The proteose-peptone fraction of milk is a group of
heat-stable, minor proteins Which are not precipitated by
heating skimmilk to 95° C for 30 minutes and subsequent
acidification to pH 4.7. Three main components of the
proteose-peptone fraction have been designated as components
3, 5 and 8 in free-boundary electrophoretic patterns. Com-
ponent 3 referred to in this study is the slowest moving,
electrophoretically homogeneous component in acrylamide gel
electrOphoretograms.

Procedures were developed for isolating component 3
from both heated and unheated skimmilk. An enriched prepara-
tion of component 3 was obtained from the acid-whey superna-
tant of heated skimmilk by fractionation with ammonium sulfate
(55% saturation). The precipitate obtained contained component
3 in high concentration. Similarly, an enriched component 3-
preparation was obtained from unheated skimmilk by fractiona-
tion of the crude globulin fraction with ammonium sulfate.
Electrophoretically homogeneous preparations were isolated
from the enriched preparations by a preparative-scale acryla-

mide gel electrophoretic technique. ‘

Nesu C. Hg

A distribution study of the proteose-peptone components
in whey and casein by ultracentrifugal methods and isoelectric
precipitation indicated that component 3 was present only in
whey and mainly in the classical lactoglobulin fraction.

Chemical analysis showed that component 3 was low in
nitrogen (13.1%) and high in carbohydrate (17.2%). The carbo-
hydrate portion contained 7.2% hexose, 1.0% fucose. 6.0%
hexosamine and 3.0% sialic acid. The carbohydrate moities
were identified by paper chromatography and consisted of
galactose. mannose, glucosamine. galactosamine and sialic
.aoid. The phoSphorus and sulfur contents were 0.5 and 0.59
per cent reSpectively. Amino acid analyses revealed that
component 3 was low in the aromatic amino acids tyrosine and
phenylalanine: in the sulfur containing amino acids (no
cysteine and cystine, lowmethionine): and high in glutamic
acid, lysine and leucine. The chemical compositions of com-
ponent 3, from heated and unheated skimmilk, were similar.
The ionic mobility of component 3 in veronal buffer (pH 8.6,
ionic strength = 0.2) was 3.5 Tiselius units in the descending
pattern. The estimated isoelectric point from free-boundary
electrophoretic eXperiments was pH 3.7. The sedimentation
coefficient in veronal buffer (pH 8.6, ionic strength = 0.1)
was estimated at 4.0 S units at infinite dilution. The dif-
fusion coefficient in the same veronal buffer was estimated
at D30 = 1.8 x 1.0-7 cmZ/sec at infinite dilution. Sedimenta-
tion-equilibrium studies showed that the weight-average molecu-

lar weight of component 3 in veronal buffer (pH 8.6, ion

Wesu C. Ng
strength = 0.1) was concentration dependent. The polymer-
monomer equilibrium was shifted toward the light component
in the presence of a dissociating system such as 5 M
guanidine hydrochloride. The equilibrium molecular weight
in veronal-5 M guanidine hydrochloride was estimated at 40,000

at infinite delution.

THE ISOLATION AND PHYSICAL-CHEMICAL
CHARACTERIZATION OF A GLYCOPROTEIN FROM THE

PROTEOSE-PEPTONE FRACTION OF CON'S MILK

By

Wesu C. Ng

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Food Science

1967

ACKNOWLEDGEKENT

The author eXpresses his most sincere appreciation to
Dr. J. R. Brunner for his guidance, counsel and interest dur-
ing this study.

Grateful acknowledgement is due to Dr. B. S. Schweigert.
Chairman of the Department of Food Science, for providing
financial support.

The author is also indebted to Mr. K. C. Rhee for
performing the ultracentrifugal eXperiments and to Mr. C. W.
Kolar who has contributed to this study.

11

TABLE OF

INTRODUCTION . . . . . . . .

C OLE TE

\ Y

l‘xl

TS

REVIEW or LITERATURE . . . . . . . . . . . . . . . .

PART I

The Preparation and Isolation of

Heated’and Unheated Skimmflk

INTRODUCTION TO PART I . . .
EXPERIRENTAL EETHODS . . . .
Apparatus . . . . . . .
Chemicals and Buffers .

Preparatory Procedure .

Procedure for Enriched

heated skimmilk

Procedure for Enriched
Unheated skimmilk .

Procedure for Checking

Componentg3gfrom

Component

Component

b)

3 from

from

the Effect of Gel
Buffer pH on the Carbohydrate Content
of the Purified Component 3 Preparation

Isolation Procedure for Component 3 from
the Enriched Fraction by Preparative

Acrylamide Gel ElectrOphoresis

Distribution of Component 3 in Casein

Protein Fractions .

Ultracentrifugation

Isoelectric Precipitation

RESULTS AID DISCUSSION . . .

Preparation of Enriched Component

and Unheated Skimmilk

3

and Whey

from Heated

Effect of Gel Buffer pH on the Carbohydrate

Content of the Purified Component 3

Preparation . . . .

iii

18

20

23
23
24
26

26

27

Isolation of Component 3 from the Enriched
Fraction . . . . . . . . . . . . . . .

Distribution of Component 3 in Casein and

 

SUI'IAIIY TC P111132? I o o o o o o o o o o o

 

PART II

Properties of Component!) . . . . . . . . . .
IETBODUCTICQ TC PART II . . . . . . . . . . .
EXPERILEHTAL . . . . . . . . . . . . . . . . .
Apparatus . . . . . . . . . . . . . . . .
Chemicals . . . .'. . . . . . . . . . . .
Chemical Lethods . . . . . . . . . . . .
Amino Acid Analysis . . . . . . . .

litro:en . . . . . . . . . . . . . .
PhoSphorus . . . . . . . . . . . . .

Sulfur . . . . . . . . . . . . . . .
Tryptophan . . . . . . . . . . . . .

Cysteine . . . . . . . . . . . . . .

Rexose . . . . . . . . . . . . . . .

Eucose . . . . . . . . . . . . . . .
Hexosanine . . . . . . . . . . . . .

Sialic Acid . . . . . . . 3 . . . .

Paper Chromatographic Identification

Carbohydrates . . . . . . .

. . .
Physical Hethods . . . . . . . . . . . .
Free-Eonndary Electrophoresis . . .
bltracentrifugation . . . . . . . .

iv

of‘
C 0
O O
. O

‘\
I“

'~.;\ K it Kn
kn \n \Jt

"Q

VJ“

Sedimentation-Velocity . . . . . . .
Sedimentation-Equilibrium . . . . .

Diffusion Coefficient . . . . . . .

RESULTS AND DISCUSSION . . . . . . . . . . . . . .
Chemical Composition . . . . . . . . . . . . .
Physical Properties of Component 3 . . . . . .
Electrophoretic Characteristics . . . . .
“Ultracentrifugal Characteristics . . . .
Diffusion Coefficient . . . . . . .

Sedimentation Coefficient . . . . .

Sedimentation-Equilibrium Studies of
Molecular Weights . . . . . . . .

SUl‘iltARY TO PART II o o o o o o o o o o o o o o o o
LIPERATLTRE CITED 0 o o o o o o o o o o o o o o o o

81

82

83
86

109

TABLE

1.

10.

LIST OF TABLES

Colorimetric values for hexcse content of
purified component 3 exposed to buffers
of various pH values as determined by
the Orcinol-Sulfuric acid method . . . .

Folin-Ciocalteu colorimetric values for
"total protein" content to determine
the number of precipitation steps
required for effective removal of
acrylamide from protein extract . . . .

Amino acid composition of component 3
(heated and unheated preparations) . . .

Elementary analysis of component 3 from .
heated and unheated skimmilk . . . . . .

The carbohydrate contents of component 3
from heated and unheated skimmilk . . .

Minimum molecular weights of component 3
(heated preparation) estimated from
chemical analysis . . . ,,, . . . . . .

Electrophoretic properties of component 3
(heated preparation) in various buffers

Diffusion coefficient of component 3 (heated
preparation) in veronal and veronal-5 M
GU buffers O O O O O O O O O O O O O I O

Sedimentation coefficient of component 3
(heated preparation) in veronal and
veronal-5 M GU buffers o o o o o o 6.0 o

Equilibrium molecular weight data for com-

ponent 3 (heated preparation) in veronal
and veronal-5 M GU buffers o o o o o o 0

vi

Page

33

3t
88
89

9O

91

92
93
94

9'5

LIST OF FIGURES

FIGURE _ Page

1. Procedure followed for obtaining enriched com-
ponent 3 from heated skimmilk . . . . . . . . 35

2. Procedure followed for obtaining enriched com-
ponent 3 from.unheated skimmilk.. . . . . . . 36

3. Colorimetric values (Orcinol-Sulfuric acid
method) for hexose contents of purified
component 3 exposed to buffers of various
pH values 0 O O O O O O O O O O O O O O O O 0 38

u. Folin-Ciocalteu colorimetric values for "total
protein" content to determine the number of
precipitation steps required for effective
removal of acrylamide from protein extract . 39

5. Procedure followed for obtaining protease-
peptone components from casein and whey by
ultracentrifugation . . . .w. . . . . . . . . #0

6. Procedure followed for obtaining proteose-
peptone components from casein and whey by
isoelectric precipitation at pH h.6 . . . . . hi

7. Acrylamide gel electrophoretic patterns of
protecse-peptones obtained from the prepara-
tive scheme (Figure 1) . . . . . . . . .,. . #2

8. Acrylamide gel electrophoretic patterns of
' proteose-peptone and various whey protein
.fractions as designated in the isolation
SChemegFimezooeeooooooooooM

9. Acrylamide gel electrophoretic patterns of
enriched component 3 (heated preparation)
eXposed to buffers of various pH for 12
hours 0 O O O O - O O O O O O O O O O I O O O O “6

10. The preparative electrophoretic sell used in
the isolation of component 3 from the
enriched fraction . . . . . . . . . . . . . . “7

11. -Acry1amide gel electrOphoretic patterns of

purified component 3 from two side strips
of the preparative gel . . . . . . . . .1. . 48

vii

FIGURE Page

12. Acrylamide gel electrophoretic patterns of
component 3 isolated from the preparative-
scale acrylamide gel electrophoresis . . . . 49

13. Acrylamide gel electrophoretic patterns of
proteose-peptones obtained from casein
and Whey o o o e o o o o o o o o o o o o o o 51

14. Amino acid prOfiles of component 3 from heated
and unheated skimmilk . . . . . . . . . . . 96

15. Paper chromatogram of the hydrolysis-released
carbohydrate moities of component 3 (heated
preparation).. a o o e o o o o o o o o e c o 97

16. Free-boundary electrophoretic patterns of com-
ponent 3 (heated preparation) in buffers
Of various pH 0 o o o o o o e o o o o c a o .99

17. A plot of electrophoretic mobilities (descend-
ing values) of component 3 as a function of

pH 0 C O O O O C O O O O O C O O O O O O O O 100

18. Plot showing concentration dependence of the
apparent diffusion coefficient of component
3 (heated preparation) in veronal and
veronal-5 M guanidine hydrochloride buffer . 101

19. Plot showing the concentration dependence of
the sedimentation coefficient of component
3 (heated preparation) in veronal and
veronal;5 M guanidine hydrochloride buffer . 102

20. Plot showing concentration dependence of equi-
librium molecular weight of component 3
(heated preparation) in veronal buffer . . . 103

21. Plot showing concentration dependence of equi-
librium molecular weight of component 3
(heated preparation) in veronal-5 M
guanidine hydrochloride buffer . . . . . . . 104

22. Sedimentation-velocity and sedimentation-
equilibrium patterns for component 3
(heated preparation) in veronal buffer
(pH 8.6: ionic strength = 0.1) . . . . . . . 105

23. Sedimentation-velocity and sedimentation-
equilibrium patterns for component 3
(heated preparation) in veronal-5 M
guanidine hydrochloride buffer . . . . ... . 107

viii

LIST OF APPENDICES
Page
APPENDIX PART I o o o o o c o o o a c e e o e o o o o 118

Preparation of the classical proteose-peptone
fraCtion O O O O I O O O O O O O O O O O 0 O O O 118

Composition of the buffers employed in thiS~
Study 0 O O O O O O O O O O O O O O O O O O O O O 119

Formulation for StarCh-urea gel 0 o o e c o o o c o 120
Preparations of discontinuous buffers, staining

solution and destaining solution for prepara-

.tive acrylamide gel electrophoresis and starch-

urea gel electrophoresis . . . . . . . . . . . . 120
Reagents used in Orcinol-Sulfuric acid method . . . 1211;

Reagents used in Folin-Ciocalteu colorimetric
reaction . . . . . . . . . . . . . . . . . . . . 121

APPENDIXPARTII.........'.......... 122
Standard curves for the colorimetric determina-
tions of phoSphorus, tryptophan. cystein.
hexose, hexosamine and sialic acid . . . . . . . 122

Buffer preparations used in free-boundary elec-
trophor9818 e o o o c o o o o o o o o o o o o o o 129

Composition of veronal buffer used in ultracen-
trifugal and diffusion runs . . . . . . . . . . . 130

PrOperties of the solvents used for molecular
weight calculations and correction of the ,
sedimentation and diffusion coefficients to
water 0 O O O O O O O O O O O O I O O O .- O O O O. 130

Correction of the observed 8 to standard condi- . .
tions 0 o o o o o o o o o o o o o o e o o 0’. o c '131

Correction of the observed diffusion coefficient '
to standard conditions . . . . . . . . . . . . . '131

Calculation of partial Specific volume . . . . . . 132

Sedimentation equilibrium method for molecular
weight determination . . . . . . . . . . . . . . 134

ix

NOtation O O O O O O O O O O O O 0 O O O O O O 0
Example calculation . . . . . . . . . . . . . .

Calculation of molecular weights from the sedimen-
tatlon'dlffu810n data 0 o o e o o e o o o o o o

INTRODUCTION

The proteose-peptone fraction of milk is a group of
heat stable, minor proteins remaining in the supernatant
after skimmilk has been subjected to heat treatment (95° C
for 30 minutes) and subsequent adjustment to pH h.6. The
protein fraction occurs in low concentration compared with
the principle milk proteins such as casein or B-lactoglob-
ulin.’

Classically. proteose-peptone is referred to as a
secondary or derived protein formed by hydrolysis of pro-
tein. whether heating of skimmilk results in the hydrolysis
of peptide bonds of the native milk proteins is not well
established. The prevalent concept of the effect of heat
on protein concerns the rearrangement of the tertiary
structure of the protein molecule, rather than cleavage of
the peptide bonds. Hence, the term proteose-peptone, as
described in this study, is used to designate a particular
milk protein fraction and is not intended to imply that it
is a group of low molecular-weight, breakdown products of
milk proteins resulting from heat treatment.

Previous studies indicated that proteose-peptone is
a group of heterogeneous proteins. In order to best study
the proteose-peptone components, a reliable analytical
technique needs to be developed for detecting the individ-
ual components. ‘Acrylamide gel electrophoresis and starch-

urea gel electrophoresis were found to be quite satisfactory

for this purpose.

For my study, I chose to work with the slowest moving
component of proteose-peptone in gel electrophoresis. This
component may be analogous with the component 3 observed in
free-boundary electrophoretic pattern of the proteose-pep-
tone fraction. In later discussions, component 3 is referred
to as the slow-moving component of the proteose-peptone
group. Since it has not been established if component 3
exists in the native skimmilk, attempts were made to isolate
component 3 from heated and unheated skimmilk.

In view of the above discussion, the present study
seeks to work on the following objectives: 1) to prepare
workable quantities of component 3, both from heated and
unheated skimmilk; 2) to obtain chemical compositional data
of both; and 3) to obtain physical parameters such as molecu-
lar weight, sedimentation Coefficient, diffusion coefficient
and others. For the purpose of Organization, the thesis has
been divided into two parts: Part I describes the prepara-
tion of component 3 while Part II evaluates the physical-

chemical parameters of the preparations.

REVIEW OF LITERATURE

The term proteose-peptone was first designated by
Rowland (1938) as the milk protein fraction which was not
precipitated by heating skimmilk at 95° C for 30 minutes
and‘subsequent acidification at pH n.7, but precipitated
by trichloroacetic acid at a concentration of 8%. Rowland
referred to this group of proteins as the "secondary"
proteins of a proteose-peptone nature which are present.
together with the albumins and globulins, in the whey frac-
tion of normal cow's milk. He reported a procedure for
estimating the proteose-peptone content of milk based on
nitrogen determination. The proteose-peptone nitrogen was
calculated as the difference in the nitrogen contents
between casein-free, non-coagulable heated-sera and that
portion of the sera remaining in the supernatant at 85
trichloroacetic acid concentration, i.e., the non-protein
substances. He reported that the soluble protein fraction
or whey of normal milk was composed of approximately 76%
albumin and globulin and 2h% proteose-peptone substances.

Aschaffenburg (1946), studying the surface activity
of milk proteins, obtained a protein fraction from acid or
rennet whey by salting-out the sera obtained after the
removal of casein and heat-coagulable proteins with ammonium
sulfate at half saturation. He called this protein fraction
Sigma proteose because of its pronounced surface activity.

A nitrogen analysis of sigma proteose showed that it had a

4
markedly reduced nitrogen content compared with other prin-
ciple milk proteins. He first reported the heterogeneity of
the fraction when it was shown that the fraction contained
three electrophoretic peaks in phOSphate buffer at pH 8.0 in
the Tiselius cell. The three components, in decreasing
order of mobility, and based on peak area, accounted for
10.5, 82.5 and 7.0%, reSpectively, of the total protein in
sigma proteose. The ultracentrifugal data showed that this
protein fraction was heterogeneous.

Weinstein,uuncan, and Trout, (1951) isolated a pro-
tein fraction from heated, rennet whey by a procedure simi-
lar to that of Aschaffenburg which they called the "minor-
protein" fraction. They indicated from elementary analysis
that the minor-protein fraction was different from sigma
proteose. The nitrogen content of the minor-protein frac-
tion, 10.0%, was low compared with the reported value of
13.95% N for sigma proteose. Their electrophoretic data
showed that at least two components were present in the
minor-protein fraction. They estimated the isoelectric
zone of the major components of the minor-protein fraction
at pH 3.7 to 9.”, based on electrophoretic mobilities at
various pH values.

Larson and Rolleri (1955) made a systematic study
of the effect of heat treatment on the electrophoretic
mobilities of serum whey proteins. They noted that the
unheated whey proteins on free-boundary electrophoresis

showed a series of peaks, some barely visible due to the

5

predominance of other peaks. However, with increasing heat
treatment, they observed a progressive decrease in the rela-
tive peak areas of the heat-denaturable whey proteins and an
increase in the peak areas of the heat-stable whey proteins;
until at 95° C for 30 minutes, only 3 components appeared in
the electrophoretic pattern. By measuring the electro-
phoretic mobilities of the components appearing in the
electrophoretic patterns of both heated and unheated whey,
they accounted for a total of eight peaks, designated as
component 1 through 8, in an increasing order of mobility.
The three heat-stable components correSponded to component
3, 5 and 8 in the electrophoretic pattern, with component

5 constituting the major fraction. Components 3, 5 and 8,
by nature of the preparation, are similar to the Rowland's
proteose-peptone fraction. In the electrophoretic pattern
of unheated whey, component 3 was obscured by the immune
globulins (component 1 and 2); component 5 was partially
obscured and appeared as a small peak or asymmetry on the
side of R-lactoglobulin peak (component 6); component 8 was
also partially obscured by B-lactoglobulin and serum albumin
(component 7). The calculated electrophoretic mobilities of
Component 3, 5 and 8 in veronal buffer (ion strength = 0.1:
PH 8.6) were -3.0, -4.6 and -7.9 x 10.5 cm2 volt.1 sec'l,
reSpectively. Since proteose-peptone components appeared in
the electrophoretic pattern of unheated whey, Larson and
ROlleri (1955) suggested that the proteose-peptone fraction ‘

was present in unheated skimmilk.

6
Jenness (1959) obtained a protein fraction rich in
whey component 5 from unheated skimmilk. Casein and pre-
sumably the proteose-peptone fractions were salted-out of
skimmilk saturated with sodium chloride. The enriched
component 5 obtained with this procedure showed a major
peak with an electrophoretic mobility of -#.5 x 10"5 cm2

-1 at pH 8.6 in veronal buffer, which agreed

volt"1 sec
well with the electrophoretic mobility of component 5 of

the proteose-peptone fraction. It is interesting to note
that in Jenness' procedure, the proteose-peptone components
were obtained from fractions associated with micellar casein,
while previously, the proteose-peptone components were shown
to be present in the whey protein fraction.

Thompson and Brunner (1961) characterized several
minor protein fractions of bovine milk, following previ-
ously known procedures for preparing the fractions. These
included the proteose-peptone fraction of Rowland's,
Aschaffenburg's sigma proteose, the minor protein fraction
of Neinstein, Jenness' milk component 5 concentrate, and
the soluble membrane-protein. They showed that these frac-
tions were characteristically low in nitrogen (10-1u%),
high in ash (3-7%) and phOSphorus (0.6-1.5%), and contained
hexose sugars. Free-boundary electrophoretic patterns and
sedimentation constants for the above fractions were mea-
sured. Their data showed that the four minor-protein frac-
tions prepared from skimmilk were heterogeneous systems and

'bhat a major electrophoretic and ultracentrifugal peak

7

appeared to be a common component in these fractions.
Whether the different procedures for preparing the minor-
protein fractions yielded one or more identical components
has not been elucilated.

Thus, from the above discussions, it is apparent that
the proteose-peptone fraction is a group of heterogeneous
proteins, composed essentially of three major components.
However, to date, no individual components of the proteose-
peptone fraction have been isolated and characterized. Also,
since proteose-peptone has been reported to be obtained from
the casein system (Jenness, 1959), as well as from whey, it
would seem appropriate, in studying the distribution of
proteose-peptone components, particularly component 3, to
investigate their occurrence both in the casein micelle and
the whey.

The presence of carbohydrate in protein has been
recognized for years (Gottschalk, 1966). The association of
carbohydrate with protein may be weak, as in the form of
diSSOCiable ion binding of mucopolysaccharides to protein,
or it may be tightly bound in the form of a covalent link-
age. A glyCOprotein is defined as a protein-carbohydrate
complex in which the carbohydrate is covalently bonded to
the polypeptide and can only be dissociated by<iestruct1ve
methods such as acid hydrolysis. The carbohydrate portion
contains amino sugars such as glucosamine and galactosamine,
Sialic acid and hexoses such as galactose, mannose and/or

fucose. Some well known glycoproteins that have been

8
studied are egg ablumin (Johansen g§_§;,. 1961), ovomucoid
(Chartterjee and Montgomer, 1962), al-acid glycoprotein
(Eylar and Jeanloz, 1962), submaxillary mucoproteins (Graham
and Gottschalk, 1960) and others. Glycoproteins have been
found in milk proteins (Hipp 22, §;.. 1961: Jolles gt. a;..
1962; Gordon 23, 31., 1963: Thompson and Brunner, 1959).
Kappa casein is the main glycoprotein of the casein micelle
and contains about 5% carbohydrate (Alais and Jolles, 1961).
In whey proteins, the immune globulins, i.e., englobulin and
pseudoglobulin are known to contain amino sugars and sialic
acid (Smith 22, 2;., 19h6). That proteose-peptone of milk
may be a group of glycoproteins is not surprising in view of
its low nitrogen content. That it falls into the definition
of a glycoprotein would also seem valid since in the course
of its preparation, high salt concentration, i.e., salting
out with ammonium sulfate, is employed which may have caused
dissociation of the ionic binding between carbohydrate and
protein, but would not affect the covalent linkage in a gly-
coprotein.

There are generally two principal types of covalent
linkage between carbohydrate and polypeptide. One type
involves the glycosidic linkage between the hydroxyl groups
of threonine and serine of the polypeptide chain and the
hexose or hexosamine of the carbohydrate moiety. This type
of binding is alkali labile by the mechanism of s-elimina-
tion with the resultant cleavage of carbohydrate from pro-

tein (Neuberger 22, §;,, 1966). Hence, in the course of

9

preparation of a glycoprotein, care should be taken to avoid
prolonged exposure to alkaline pH. If an alkaline condition
is achieved, one should check the protein for its carbohy-
drate content before and after treatment. Another type of
linkage involves the N-acylglycosylamine bond between the
amide nitrogen of aSparagine or glutamine and hexose. This
type of linkage is alkali stable. With regards to the
sequence of carbohydrate moiety, it has generally been

known that sialic acid occupies the terminal position in

the carbohydrate chain, and that either hexose or hexosamine
is linked to the protein moiety (Gottschalk, 1966).

Thompson and Brunner (1959) in their study of the
carbohydrates of some minor protein of cow's milk, reported
the concentrations of hexose, hexosamine, fucose and sialic
acid in the proteose-peptone fraction and Weinstein fraction.
Further, they indicated the presence of galactose, mannose,
fucose, galactosamine (weak) and glucosamine in the proteose-
peptone fraction. However, since individual components of
the proteose-peptone fraction have not been isolated, no
relative concentrations of carbohydrate in the various com-

jponents of proteose-peptone have, so far, been studied.

PART I

THE PREPARATION AND ISOLATION OF COMPONEMT 3

FROK HEATED AND UNHEATED SKIMHILK

INTRODUCTION TO PART I

The preparation of proteose-peptone was described
previously (Thompson and Brunner, 1961). Our aim in devising
a somewhat similar procedure was to obtain an enriched frac-
tion of component 3 suitable for further purification. Tech-
niques tried‘included fractionation with ethanol, pH adjust-
ment and salting-out with ammonium sulfate. Ammonium sulfate
fractionation was, by far, the most satisfactory method for
obtaining enriched component 3. In this procedure, ammonium
sulfate was added at various concentrations to heated whey at
pH 9.6 following the removal of casein and heat-denatured whey
proteins.

In the preparation of component 3 from proteose-peptone,
it has been implied that component 3 from heated skimmilk was
present mainly in the whey protein fraction since casein was
removed at pH 4.6 after heat treatment. Hence, our initial
step in locating component 3 from unheated skimmilk would be
to fractionate the whey proteins. These fractions were examined
by gel electrophoresis, together with the classical proteose-
peptone, to see if a band correSponding in mobility to that of
component 3 of proteose-peptone appearel. If such a band
appeared in any of the whey protein fractions, its relative
1mibilities both before and after heat treatment were compared.

Once enriched component 3 from both heated and unheated
Skimmulk:was obtained, several techniques seemed to offer
POSSiluJities for its purification. These included diethy-
lamilloethyl (DEAE) cellolose and carboxymethyl cellulose or

11

12

CI~1C ion exchange chromatography, Sephadex or Bio-Rad gel fil-
tration, preparative-gel electrophoresis and others. DEAR
ion exchange chromatography and Bio-Rad gel filtration were
tried with some degree of success, though a satisfactory
isolation would require refinement of the techniques. Pre-
viously, the author had some experience in preparative-gel
electrophoresis as a means of obtaining a highly purified
casein component (Ng and Brunner, 1966). The analytical gel
electrophoretic run of the enriched component 3 showed that
component 3 was well separated from the other protease-pep-
' tone components. Hence, the method of preparative-gel elec-
trophoresis seemed to present the best hOpe for obtaining
small amounts of electrophoretically homogeneous component 3.

However, certain possible shortcomings of the tech-
nique need to be considered. The major concern here is the
possible cleavage of the carbohydrate moiety from glycopro-
tein when eXposed to solution of alkaline pH. For satisfac-
tory separation, the preparative acrylamide gel electrophor-
resis was usually conducted at buffer and gel pH values of
8.6 for a period of 10 to 14 hours at‘( 100 C. During such
treatment, the type of carbohydrate-protein linkage known to
be labile to alkali hydrolysis, i.e., the threonine or serine
type of polypeptide linkage to carbohydrate might have
cuzcurred. Though, at present, we do not know the type of
protein-carbohydrate linkage in proteose-peptone. Conse-
CDMhdtly, it seemed necessary to check the effect of the

buffer pH exposure on the carbohydrate content of enriched

13
component 3. Once this question was answered, the technique
could be used with some degree of confidence. The prepares
tion isolated from the gel contained monomeric species of
acrylamide which should be removed before any analysis was
performed on the fraction. A convenient method of removing
acrylamide from the extracted component 3 was by salting-
out with ammonium sulfate, coupled with pH adjustment. The
procedure was repeated several times to obtain component 3
essentially free of acrylamide.

Component 3 has been obtained from the whey protein
fraction. However, we do not know as yet if part of the
component 3 may be associated with micellar casein. Jenness
(1959) reported the isolation of an enriched milk component-
5 which was associated with the casein micelles in the prep-
aration. Hence, it seems appropriate to investigate the
distribution of proteose-peptone components, specifically
component 3, in both casein and whey fractions. Two proce-
dures of separating casein from whey proteins were used.

One method was by preparative ultracentrifugation, which
removed casein micelles in the form of a pellet, leaving

Whey proteins and the soluble casein in the supernatant.

The other method was by isoelectric precipitation at pH 4.6,
in.which the casein, micellar as well as soluble, was removed,
.Leaying the whey proteins in the supernatant. The four frac-
t1Ons, i.e., ultracentrifuged casein micelles and ultracen-
trif‘uged "whey" supernatant, isoelectric precipitated casein
andl‘whey supernatant were analyzed for the distribution of

InTTbeose-peptone components by gel electrophoresis.

EXPERIMENTAL METHODS
Apparatus

Raw milk was obtained from the university herds, both
of Holstein and Jersey breeds, in ten gallon stainless steel
cans. The raw milk was separated in a De-Laval, disc-type
separator. Heating was conducted in an autoclave made by
American Sterlizer Company. A Beckman Zeromatic pH meter
equipped with glass electrode was used to measure pH values.
Low-Speed centrifugation (1000 x G) was performed in a
model IV International centrifuge. Celite filter aid on a
Buchner funnel was used for clarification of the supernatant
when required. Dialyzing was done in Visking tubing obtained
from Union Carbide Company. Intermediate-Speed centrifuga-
tion (20,000 x G) was performed in a Servall (Type SS-i)
centrifuge. Spectrophotometric measurements were made with
a Cenco-Sheard-Sanford photelometer equipped with filters
transmitting light at appropriate wavelengths. The horizon-
tal gel electrophoretic cell was made from Plexiglass. A
typical preparative cell had a dimension of 30 x 50 cm, with
a gel bed thickness of about one cm (Figure 9). The buffer
compartments held approximately one liter of buffer each and
‘were fitted with 26-gauge platinum wire electrodes. A high-
voltage power pack for preparative gel run was obtained from
Savant Instrument Company (Range: 0-500 ma, 0-5,000 volts).
13“? electrolytic destainer was constructed from plexiglaSS.
Enki was fitted with stainless steel plate electrodes. A

14

15
battery charger with a range of 12 volts and operated at
about 3 ma was used as power pack for the destainer. Ultra-
centrifugation was performed in a Beckman Model I Prepara-

tive Ultracentrifuge.
Chemicals and Buffers

Chemicals and their suppliers are given as follows:
ammonium sulfate (certified reagent) from Fisher Scientific
Company; Cyanogum-41 gelling agent from E-C Apparatus Cor-
poration; N, N, NC Hatetramethylethylenediamine (TMED)
from Eastman Organic Chemical; ammonium persulfate (reagent
grade) from Baker Chemical Company; starch-hydrolyzed for
gel electrOphoresis from Connaught Medical Research Labora-
tory; urea (reagent grade) from Baker and Adamson; boric
acid (reagent grade) from Fisher Scientific Company; Tris
(trimethylhydroxymethan, primary standard) from Sigma Chem-
ical Company; citric acid (monohydrate, reagent grade) from
Fisher Scientific Company; Folin-Ciocalteu reagent from
Fisher Scientific Company; cupric sulfate (CuSOu - 5H20,
certified reagent) from Fisher Scientific Company; potasium
tartrate (certified reagent) from Fisher Scientific Company;
orcinol (monohydrate, reagent grade) from Fisher Scientific
Company.

I The following buffer preparations are given in the
Appendix: discontinuous buffer systems used in analytical
and preparative acrylamide gel electrophoresis; discontinu-

ous buffer systems used in starch-urea gel electrophoresis;

16
buffers used in checking the effect of buffer pH exposure on
the hexose content of purified component 3; Folin-Ciocalteau

reagents and buffers; orcinol-sulfuric acid reagents.
Prepgratory_Procedure

Procedure for Obtaining Enriched Component 3

from Heated Skimmilk

Whole raw milk obtained from the university herd was
maintained at temperatures between 35° to 40° c until sepa-
rated. The skimmilk was placed into several 5-liter stain-
less steel beakers and covered with Aluminum foil. Heating
was conducted in an autoclave, with steam pressure of less
than 5 psi, at a temperature slightly above 100° C, for a
period of 30 minutes. The heated skimmilk was immediately
cooled with running tap water, and allowed to come to room
temperature. Casein and denatured whey proteins were pre-
cipitated by adjusting the pH to 4.6 with 1 N HCl. The
precipitate was removed by centrifugation at 1,000 x G for
10 minutes in the International centrifuge. On occasion,

A the supernatant remained turbid after centrifuging, neces-
sitating filtration through a Buckner funnel covered with
Celite filter aid. The clear supernatant contained the
proteose-peptone components. Fractionation with ammonium
sulfate was carried out at pH 4.6. Solid ammonium sulfate
was added to 35% saturation. The solution was left at room

temperature for 30 minutes to allow the precipitate to form

 

 

 

pr:

pet

'11

t4-

17
and settle. The solution was centrifuged at 1,000 x G for
20 minutes. The precipitate was rediSpersed in deionized
water, pH adjusted to 6.5, dialyzed under several changes
of deionized water and freeze dried. Then, more ammonium
sulfate was added to the 35% saturated (NHg)2804 supernatant
until another precipitate was formed at 55% saturation. The
solution was centrifuged at 1,000 x G for 20 minutes. The
precipitate was treated as before. The 55% saturated
(NHh)2304 supernatant was made to 80% saturation and another
precipitate was formed. The final precipitate was redis-

h

persed as before.

Procedure for Obtaining Enriched Component 3

from Unheated Skimmilk

Whole raw milk was separated at 35° to 40° C. The
skimmilk was acidified with 1 N HCl to pH 4.6. The preci-
pitated casein was filtered on cheese cloth, and the whey
filtered through’a Buchner funnel-covered with Celite filter
'aid. The pH of the clear whey was adjusted to 6.5 with 1 N
NaOH. Solid ammonium sulfate was added to whey to 50% satu-
ration. The precipitate containing the crude globulin frac-
tion was removed by centrifugation at 1,000 x c for 20
minutes and saved for further working. The supernatant,
designated as 31 (Figure 2), containing the crude albumin
fraction, was discarded. The precipitate was redissolved
in distilled water to about 3% protein concentration, and

the solution was adjusted to pH 4.6. Ammonium sulfate was

 

 

18
added to 25% saturation. The precipitate, P1, was removed
by centrifugation at 1,000 x G for 30 minutes. The super-
natant was adjusted to pH 6.0 with 1 I? NaOH and ammonium
sulfate was added to 40% saturation. The solution was held
at 37° C for 30 minutes. The precipitate, P2, was removed
by centrifugation at 1,000 x G for 20 minutes. More ammon-
ium sulfate was added to the supernatant bringing the salt
concentration to 45%, saturation. The solution was held at
37° C for one hour and was centrifuged at 1,000 x G for 20
minutes. The final precipitate was redissolved in water,
the solution was adjusted to pH 6.5, dialyzed and freeze
dried. To check the heat stability of the fraction, a
small amount of the supernatant (after removal of P2 pre-
cipitate) was dialyzed against deionized water to remove
most of the salt, and was pervaporated to the same original
volume before dialysis. The solution was heated at 100° C .
for 30 minutes and cooled to room temperature. The pH was
8‘d.‘)usted to 4.6 followed by centrifugation at 1,000 x G for
20 minutes. The clear supernatant was adjusted to pH 6.5,

dialyz ed and freeze-dried .

Procedure for Checking the Effect of Gel Buffer pH on the
CarbOhydrate Content of the Purified Component 3 Preparation

The buffer system used for the preparative-acrylamide
gel electrophoresis was a discontinuous one, with borate
buffer (pH 8.6) in the electrode compartments and Tris-
citrate buffer (pH 8.6) in the gel. To assess the effect

 

1‘

 

19
of buffer pH on the carbohydrate content of purified compo-
nent 3, buffers ranging in pH from 4.6 to 8.6 were employed.
The buffer preparations are described in the Appendix, Part
I. Purified component 3 (heated preparation) was dissolved

and eXposed in the buffers for a period of 12 hours at room
temperatures. Then, the pH of the buffers were adjusted to
4. 6. Solid ammonium sulfate was added to 80% saturation to
salt-out the protein which was collected by centrifugation
at 20,000 x G for 15 minutes in a Servall (Type SS-i) cen-
tri fuge. The precipitate was redissolved in deionized water
and. the solution was adjusted to pH 7.0, dialyzed and freeze
dried. The treated, purified component 3 preparations were
analyzed for total hexose by the orcinol-sulfuric acid
procedure (Ninzler, 1955). The preparation of the orcinol-
sulfuric acid reagents is given in the Appendix, Part I.
Two to ten milligrams of the buffer-eXposed component 3
samples were dissolved in one ml of 0.1 N.Na0H. A blank
(one ml of water) was prepared. Eight and one half milli-
liters of the orcinol-sulfuric acid reagent was added to
the tube and the solution mixed by inversion. The tube was
capped with a glass marble to minimize evaporation and
placed in a water bath at 80° C for 15 minutes. The tube
was cooled in tap water and readings (% transmission) were

taken in a Cenco-Sheard-Sanford photelometer equipped With

a mlter transmitting light at 540 mu.

CU

 

Fv

20
Isolation Procedure for Component 3 from the Enriched Fraction

by Preparative Acylamide-Gel Electrophoresis

Both continuous and discontinuous buffer systems can
hue used for gel electrophoresis. However, the discontinuous
system has the advantage of showing a sharp visible, frontal
band, enabling one to follow the progress of electrophoresis
arui hence was adopted for this purpose. The buffer prepara-
tions, Tris-citrate buffer in the gel and borate buffer in
the electrode compartment, were described in Appendix, Part
I. For the gel buffer a 1:10 dilution of the stock Tris-
citrate solution (pH 8.6 to 8.9) was used. For the elec-
trcmle buffer, two parts of stock borate to three parts of
water, at pH 8.6, was used. Cyanogum—41 gelling agent was
dissolved in the Tris-citrate buffer to make an 8% acryl-
amide solution (w/v). This solution was filtered to remove
resixlual.insoluble particles. N, N, N, N-tetramethylenedi-
amine: (0.1 ml/100 ml gel solution) was added to the clear
Sel Solution, followed by the addition of ammonium persul-
fate (0.2 gm/100 ml gel solution) with constant stirring.
The solution was poured immediately into the preparative gel
bed 81nd a continuous slot former was positioned approximately
3 cm from the cathodic end of the bed. Electrical contact
between the buffer and gel was provided by means of one?-
quarteIII'--1nch holes in the gel/buffer partition. The holes
were Seeled temporarily with electrical insulation tape

placed on the buffer side. This strip of tape was removed

 

:y IL. -
L0 .Nu rw :1. N r v . 1 . -
.f. n .3 e O nu "a C cw O .1. .n . «he . t .
a a a . J .. V IL A a S 1 be. S a . . . «J .C. 8 an O. h. nu «2.
x 14
.11. , In.

21

after the gel formed. A plastic cover, placed over the cell

and flushed with nitrogen, provided the necessary low-oxygen
atmOSphere required for the gelling agent to polymerize.

Gelling was completed in one minute but the gel was allowed

to stand for 20 minutes before using. Cold water, circulat-

ing under the gel bed, served to dissipate the heat generated

during the run. A 5 ml portion of -a 5% component 3 (enriched)

solution (250 mg of protein in borate buffer) was added

to the sample slot. The entire assembly was covered with

Saran wrap to retard loss of moisture. A high-voltage

power supply provided the current flow with a current den-

sity of from 8 ma to 10 ma per cm2 of gel cross-sectional.

area. Electrophoresis was conducted for a period of from

10 to 12 hours during which time the current dropped to

3 ma/cmz. No attempt was made to maintain constant current

during: the run.

At the end of the electrophoretic run, 2-cm wide
strips were removed from each side of the gel bed, stained
With amido black and destained electrOphoretically for about
one hour to locate the position of the migrating zone of
component 3. The stained sections were replaced to their
original positions in the gel and a cross-sectional strip
corresponding to the component 3 zone (unstained portion)
was I‘emoved in preparation for the protein extraction step-
The eXcised gel was macerated in a Haring“ blender With
deionized water to make a gel slurry. Flore water was added

t
O the gel slurry and the pH was adjusted to 5.0 with 1 I»:

22
HCl. The diluted gel slurry was stirred in the cold for one
day to allow diffusion of protein from gel particles. The
equilibrated gel slurry was centrifuged at 1,000 x G for 20
minutes to separate the gel particles from the aqueous
pmase. The supernatant was saved. The gel particles were
again extracted twice with deionized water. The supernatants
from the three extractions were combined, adjusted to pH 6.5,
dialyzed, pervaporated, and freeze-dried.

Since monomeric Species of the gelling agent were
recovered with the protein in the extracted solution, addi-
tional steps were taken to recover gel-free protein. For
component 3, salting-out with sodium chloride, coupled with
pH adjustment, was used. The procedure was as follows:
Extracted component 3 was dissolved in deionized water to
1;: solution. Solid sodium chloride was added to the solu-
tion until a saturation concentration was reached (36 gm
33aC1/100 ml solution). The prf the solution was adjusted
to 2.0. A flocculent precipitate was observed which was
collected by centrifugation at 20,000 x G for 15 minutes.

The Precipitate was again redissolved in deionized water to

a cone entration of approximately one percent. The solution
was 8aturated with sodium chloride and the pH adjusted to
2'°° Again, a flocculent precipitate was obtained. This
pr°°eSS was repeated two to four more times to insure the
complete removal of acrylamide. To determine the number of
precipitation cycles required for effective removal of acryl-

am -
ide, a colorimetric Folin-Ciocalteu procedure for "total

me e

L.
of ,.
V. ,-
3.148.

‘1‘

A31

IVJ.‘

‘1. r
”U AU
. we hell

 

 

M
9'4.

23

protein" content was performed on the precipitate obtained

from the four precipitation cycles. The preparation of

Folin-Ciocalteu reagents was given in Appendix, Part I.

The colorimetric procedure was as follows: Five milliliters

of the mixed sodium carbonate-potasium tartrate reagents was
added to one milliliter of sample solution containing 0.5 mg
of isolated component 3. The solution was allowed to stand

at room temperature for 10 minutes prior to adding 0.5 ml of

Folin-Ciocalteu reagent. This solution was allowed to stand

for 30 minutes before making transmission readings at 525 ml

with a Cenco-Sheard-Sanford photelometer.

Distribution of Component 3

in Casein and Whey Protein Fractions

Proteose-peptone components were obtained from casein
and whey protein fractions by ultracentrifugation and iso-

electric point precipitation as depicted in the following

sections.
Ultracentrifugation

The preparative scheme is shown in Figure 5. Fresh

raw milk was separated at 40° C in a De-Laval cream separa-
tor. The skimmilk phase was ready for ultracentrifugation.
Ultracentrifugation was performed in a Beckman I-iodel L pre-

parative ultracentrifuge, equipped With a tYpe 50 rotor. run

at
a 8Deed of 48,000 rpm for 90 minutes at room temperature.

Th
e ca-Sein pellet was dispersed in deionized water with

21+
stirring and was made up to the original skimmilk volume.
Both ultracentrifuged casein and ultracentrifuged "whey"
supernatant were heat treated at 100° C for 30 minutes in

After cooling to room temperature, the pH

of the solution was adjusted to 1+.6 with 1 N HCl. The

an autoclave .

solutions were centrifuged at 1,000 x G for 20 minutes.

The supernatants of both fractions, containing the proteose-
peptone components, were adjusted to pH 6.5 with 1 N NaOH,

d1 alyzed and freeze-dried .

Isoelectric Precipitation

The preparative scheme is shown in Figure 6. Skim-
milk was adjusted to pH 4.6 with 1 N H01. The solution -:as
centrifuged at 1,000 x G for 15 minutes. The whey super-
natant was adjusted to pH 6.7 before heat treatment. The
isoelectric casein was redissolved in deionized water, with a
small addition of 1 N NaOH, and made up to original skimmilk
volume. Since small amount of whey protein may be occluded
in the first casein precipitate, another IEP precipitation
was undertaken. The casein solution was adjusted to pH 11.6.
Ehe SOlution was centrifuged at 1,000 x G for 20 minutes.
The Precipitated casein, which was almost free of whey
prOtein, was redissolved in deionized water with small
addition of 1 N HaOH. The casein solution was adjusted to
pH 6. 7 and made up to original volume of skimmilk. The whey
and the twice IEP precipitated casein solution were heat

tr
eated at 100° C for 30 minutes in an autoclave. After

25

cooling to room temperature, the solutions were adjusted to

pH.u.6 with 1 N HCl. The solutions were centrifuged at 1,000
:x G for 20 minutes. The supernatants from the two fractions,

(containing the proteose-peptone components, were adjusted to

:pfi 6.5, dialyzed and freeze-dried.

ESULTS AND DI SCU SS ION 3

Preparation of Enriched Component 3

from Heated and Unheated Skimmilk

Three protein fractions from the proteose-peptone
fraction were obtained by the ammonium sulfate fractiona-
tion procedure when applied to heated skimmilk as outlined
in Figure 1. Acrylamide gel electrophoretic patterns of
the three fractions and the classical proteose-p'eptone
fraction are shown in Figure 7. The preparation of the
classical proteose-peptone is outlined in the Appendix,
Part I. Gel electrophoretic patterns of these fractions
revealed some interesting observations. Evidently, enrich-
ment of certain components of proteose-peptone was obtained
in the various ammonium sulfate cuts. The proteose-peptone
fraction corresponding to the 3553 saturated ammonium sulfate ,
cut shows an enrichment of component 5; the 55:5 saturated
ammonium sulfate out, an enrichment of component 3; and the
30.5 saturated ammonium sulfate cut, an enrichment of com-
ponent 8. Hence, the enriched component 3 fraction corres—
ponding to the 55% saturated ammonium sulfate cut was used
as Starting material for the isolation of component 3.

To locate component 3 in unheated skimmilk, whey was
fractionated as outlined in Figure 2. The various protein

fractions, designated 3 P1, P2, and P3, were examined

19
electrophoretically in acrylamide gels. together with the
pI'oteOSe-peptone fraction (see Figure 8A)- As observed.

26

27

the P fraction contained a component similar in mobility to

3
component 3 of the proteose-peptone fraction. The P:3 frac-
tion was heated to 1000 C for 30 minutes as a check on heat
alteration. The electr0phoretic pattern of the heated P3
fraction showed a component similar in electrophoretic char-
acteristics to component 3 in proteose-peptone (Figure 8B).
Thus, the component in the P3 fraction appeared to be com-
ponent 3 in unheated skimmilk. The gel electrophoretic
patterns of enriched component 3 from heated and unheated
skimmilk is shown in Figure 8C. Judging from the concen-
tration of component 3 relative to other proteose-peptone

components as they appeared in the gel electrophoretogram,

the enrichment procedures can be considered satisfactory.

Effect of Gel Buffer_pH on the Carbohydrate Content

of the Purified Component 3 Preparation

The hexose contents of the glyCOprotein that was
eXposed to buffers of various pH are shown in Table 1. Here,
the hexose content was used as an index of the carbohydrate
content of purified component 3. The colorimetric value or
absorbance readings at 540 mm was a measure of the hexose
cnsntent. The hexose contents of purified component 3
eXposed to neutral buffer (pH 7.0) and deionized water were
used as "standards," with presumably no loss of hexose from
the glycoprotein in these solutions. As observed from
Figure 3, the purified glycoprotein that was eXposed to the

acidic and basic buffers (citrate-phoSphate, pH #.6; Tris-

28
citrate, pH 3.6; borate, pH 8.6) showed the same relative
hexose contents as those of the glyc0protein eXposed to the
neutral buffer (Tris-citrate, pH 7.0) and deionized water.
Thus, eXposure to the buffers ranging in pH from 4.6 to 8.6
did not markedly affect the hexose content of the prepara-
tion. Exposure to 0.1 N NaOH (pH )>12) decreased the hexose
content slightly (Figure 3).

That the hexose contents of the glycoprotein exposed
to buffers of various pH values remained essentially
unchanged does not preclude the possibility that the gly-
coprotein might undergo physical alterations in molecular
association during such exposures. Hence, the various
buffer-eXposed component 3 samples were run on acrylamide
gels at pH 9.6 to check their electrophoretic characteris-
tics. Again, the gel patterns of the treated, purified
component 3 samples remained the same (Figure 9). Thus,
it can be assumed that exposure to the buffer systems used
in preparative acrylamide gel electrophoresis, i.e., Tris-
citrate and borate buffer at pH 8.6, for the duration of
the electrophoretic run at temperature less than 10° C,

did not result in drastic cleavage of carbohydrate from

the glycoprotein.
Isolation of Component 3 from the Engiched Fractions

Enriched component 3 samples were applied on the
preparative acrylamide gels for the isolation of the elec-

trOphoretically homogeneous component. A typical electro-

29
phoretogram from the two strips of the preparative gel is
shown in Figure 11. The resolution of enriched component 3
in the preparative acrylamide gel was quite satisfactory and
comparable to the resolution obtained in analytical gel elec-
trophoresis. Also, component 3 was well separated from the
other proteose-peptone components and, hence, was conven-
iently removed from the gel.

Component 3 was extracted from the excised gels by
the process of equilibrium-diffusion, using several changes
of fresh, deionized water. The excised gels was macerated
into a slurry to provide greater efficiency in extraction.
The extracted component 3 contained monomeric Species of
the acrylamide gelling agents, which must be removed from
the sample. The protein content of the extracted component
3 increased with subsequent removal of acrylamide by the
salting-out technique (see Figure 4). Here, the "total
protein" content was measured by the absorbence values
obtained with the Folin-Ciocalteu reaction. Component 3
was essentially free of acrylamide after the second pre-
cipitation (Figure 4). Hence, three time precipitations
were adopted for the removal of acrylamide. The procedure
d1d.not preclude the possibility that small amount of acryl-
amide may be tightly complexed with the protein and cannot
be removed by further precipitation. The gel electrophoretic
patterns of the isolated component 3, together with their
enriched fractions, both from heated and unheated skimmilk,

are shown in Figure 12A. The starch-urea gel electrophoretic

30
patterns of the isolated component 3 are shown in Figure 12B.
The incorporation of urea in the gel increased the mobility
of component 3. The isolated component 3 was essentially
electrophoretically homogeneous and free of other proteose-

peptone components.
Distribution of Component 3 in Casein and Whey

Proteose-peptone components were obtained from casein
and whey by ultracentrifugation and isoelectric precipita-
tion. The term proteose-peptone is applied to the protein
fraction that remains in the supernatant after heat treat-
ment and subsequent acid precipitation at pH 4.6. The gel
electrophoretic patterns of proteose-peptone obtained from
1) ultracentrifuged "whey" supernatant; 2) ultracentrifuged
casein micelle: 3) whey after_isoelectric precipitation of
skimmilk at pH 4.6 and 4) isoelectric (pH 4.6) precipitated
casein, are shown in Figure 13. It is interesting to note
that component 3 is present in the proteose-peptone fraction
from whey, but is conSpicuously absent in the proteose-
peptone fraction from casein. This is rather interesting
from the conjecture that if component 3 is associated with
<rther proteose-peptone components, one would normally eXpect
that part of it would also be found in the casein fraction,
since the other proteose-peptone components appear to be
present, in part, in the casein fraction. Yet the gel elec-
trophoretic patterns show that component 3 is found only in

the proteose-peptone obtained from whey. Previous isolations

31
of component 3 from unheated skimmilk showed that component
3 resides mainly in the classical lactoglobulin fraction of

whey.

SUMMARY OF PART I

An ammonium sulfate fractionation procedure was
developed for obtaining enriched component 3, both from
heated and unheated skimmilk. Component 3 was isolated
from the enriched fractions by a preparative acrylamide
gel electrophoretic technique. The buffer system used
in preparative acrylamide gel electrophoresis did not
produce apparent cleavage of carbohydrate from the gly-
coprotein. The component 3 preparations, from heated and
unheated skimmilk, were electrophoretically homogeneous
and essentially free of other protein components. A study
of the distribution of proteose-peptone components in
casein and whey indicated that component 3 was present

only in the whey and mainly in the lactoglobulin fraction.

32

TABLE 1

Colorimetric values for hexose content
of purified component 3 exposed to buffers
of various pH values as determined by
the Orcinol-Sulfuric acid method (see Figure 3)

 

 

 

Buffer Sample Weight Absorbance

(ion strength = 0.1) (mg/ml) (at 540 mu)
Citrate-phosphate 2.2 0.216
pH 4.6 4.7 0.403
6.7 0.582
Citrate-phosphate 2.6 0.252
pH 7.0 605 00569
902 - 00796
Deionized water 2.0 0.197
PH 6.5 4.4 0.377
8.5 0.745
Tris-citrate 3.1 0.310
9.1 0.796
Borate 2.3 0.280
8.5 0.788

0.1 N NaOH 2.0 0.16

pH>\12 5.0 0.393
7.7 0.553

 

1
1

1

34

TABLE 2

Folin-Ciocalteu colorimetric values
for "total protein" content to determine
the number of precipitation steps required
for effective removal of acrylamide from protein extract
(see Figure 4)

 

 

 

Samples Absorbance
(at 525 mu)
Extracted component 3*
(contains acrylamide) 0.292
ist Precipitate 0.648
2nd Precipitate 0.721
3rd Precipitate 0.721
4th Precipitate 0.721

 

 

*Concentration of sample = 0.5 mg/ml

35

SK II‘EI-ZILK

 

Heat at 100° c for 30 min
Cool to room temperature
Adjust to pH 4.6 with 1 N HCl
LCentrifuge (1,000 x G;

10 min)

 

i

SUPERNATANT (Proteose-peptone) PHECIPI1A‘E

At pH 4. 6
Add solid (143114).)
to 35% saturation

 

(Casein plus
denatured whey
proteins; dis-

 

Allow to stand for 30 min card)
Centrifuge (1,000 x G;
20 min)
. T
SUPERHATANT

Add (NHu)ZSO to
55% saturation
Allow to stand for
30 min
Centrifuge (1,000 x G;
20 min)

 

PBECIPITATE
(Enriched component 5)

Redissolve in
water

Dialyze

Freeze-dry

 

SUPERfiATAUT

Add (IH “)280 to
80p satura ion
Allow to stand for
30 min
Centrifuge (1,000 x G;
20 min)

 

PRECILITATE
(Enriched component 3)

Hedissolve in
water

Dialyze

Freeze-dry

 

SULEBNATAHT
(Discard)

PBECIPITACE
(Enriched component 8)

Redissolve in
water

Dialyze

Freeze-dry

 

 

Figure 1. Procedure followed for obtaining enriched compo-
nent 3 from heated skimmilk.

36

 

 

 

Acidify with 1 N HCl to DB 4.6
Filter
. . . I- - .
‘31 iii: 1 CASEIN
(Discard)
Adjust pH to 6.5
with 1 N NaOH
Add (NH “)2804 to 505
saturation
Centrifuge (1, 000 x G;
20 min)
1' :15 ’3 IP I‘; SUPEIMATANT
(Crude globulin fraction) (8 , crude albumin
rAction; discard)

Redissolve at 33
protein conc.

Adjust to pH 4. 6

Add (NHu ) son to 25p
saturat on

Centrifuge (1, 000 x G;
30 min)

 

 

SUPERKATANT PHECILITATE
(P1, discard)
Adjust to pH 6.0
Add (NH ) sou to 405
saturat on
Hold at 37° C for
30 min
Centrifuge (1,000 x G
20 min)

 

 

SUPJREATAKT PHECIPITATE
(P2, discard)
Add (NI ) son to 45m
satur t onu
Hold at 37° C for 1 hr
Centrifuge (1,000 x G;
20 min)

 

(Continued on Page 37)

 

SUPERNATANT PBECIPITATE
(Discard) (P3, enriched component 3)

Redissolve in
water

Dialyze

Freeze-dry

 

 

Figure 2. Procedure followed for obtaining enriched com-
ponent 3 from unheated skimmilk.

Absorbance (540 mu)

 

 

 

 

1.0 *-«---~ —~ ..._._1..,._... - 1 11 1 11 - p
I
I
0.8 .’/ ;
P éy/Cé :
//)d
0.6 _ ,
. 20
i Buffers A,
,/ i
o/ l
0.4 #— [CA
/‘7 0.1 N NaOH
0/
0.2 i
i
I
l J -.Jw_1_l1.- l1. 1
2 4 6 8 10

Edgme3.

Protein Concentration (mg/m1)

Colorimetric values (Orcinol-Sulfuric acid

method) for hexose contents or purified

component 3 eXposed to buffers of various pH

values. Legend: E] , citrate phOSphate

pH 4.6; [S . citrate phOSphate pH 7.0; V7 ,

deionized water; 0 , Tris-citrate pH 8.6;
8 , borate pH 8.6; A , 0.1 N NaOH pH 12.

Absorbance (525 mu)

39

 

 

 

 

 

 

0.8
n A
V U
0.6
0.4
0.25q
J J L l
o 1 2 3 a
Number of Precipitation
Figure 4. Folin-Ciocalteu colorimetric values for

"total protein" content to determine the
number of precipitation steps required
for effective removal of acrylamide from
protein extract.

no

SKIhMILK

Ultracentrifugation
(48,000 rpm: 90 min
Beckman Type 50 rotor)

 

 

 

 

 

CASEIN PELLET "WHEY" SUPERNATANT
Hedissolve in water Heat at 1000 C for
to original volume 30 min
Heat at 1000 C for Cool to room tem-
30 min perature
Cool to room tempera- Adjust to pH 4.6
ture Centrifuge (1,000 x G;
Adjust to pH 4.6 - 20 min)
Centrifuge (1,000 x G;
20 min)
PBECIPITATE SUPERNATANT PRECIPITATE SUPERNATAKT
(Discard) (Proteose-peptone (Discard) (Proteose-peptone
components) components)
Adjust to pH 6.5 Adjust to
Dialyze pH 6.5
Freeze-dry Dialyze
Freeze-dry

 

 

Figure 5. Procedure followed for obtaining proteose-peptone
components from casein and whey by ultracentrifu-
gation.

41

SKILMILK

Adjust to pH 4.6 with 1 N HCl
Centrifuge (1,000 x G; 15 min)

 

 

 

 

 

WHEY PHECIPITATE (Casein)
Adjust to pH 6.7 Hedissolve in water to
Heat at 1000 C for original volume with
30 min 1 N NaOH
Cool to room tempera- Adjust to pH 4.6
ture Centrifuge (1,000 x G;
Adjust to pH 4.6 20 min)
Centrifuge (1,000 x G;
20 min)
PRECILITATE SUPEHNATANT PRECIPITATE SUPERNATANT
(Discard) (Proteose-peptone (Casein) (Discard)
components)
Hedissolve in water
Adjust to to original volume
pH 6.5 with 1 N NaOH
Dialyze = Adjust to pH 6.7
Freeze-dry Heat at 100° c for 30 min

Cool to room temperature
Adjust to pH 4.6
Centrifuge (1,000 x G;

 

 

20 min)
PHECIPITATE SUPERNATANT
(Discard) (Proteose-peptone
components)

Adjust to pH 6.5
Dialyze
Freeze-dry

 

 

Figure 6. Procedure followed for obtaining proteose-peptone

components from casein and whey by isoelectric
precipitation at pH 4.6.

.' ’1

Figure 7.

L',

 

Acrylamide gel electrophoretic patterns of proteose-
peptones obtained from the preparative scheme
(Figure 1): '1) classical proteose-peptone (Appendix,
Part1), 2) enriched component 5, 3) enriched com-
ponent 3, 4) enriched component 8. Discontinuous
buffer systems (Appendix, Part I).

:m:

 

:m:
a 5-2.0 a E o o

 

2w:

1"18111‘3 63' o

44

Acrylamide gel electrophoretic patterns of proteose-
peptone and various whey protein fractions as
designated in the isolation scheme, Figure 2.
Discontinuous buffer system (Appendix, Part I).
Colume A: 1) P3 fraction, 2) P fraction, 3) P1
fraction, 4) S fraction, 5) prgteose-peptone.
Column 3: 1) fraction following heat treatment,
2) P fraction prior to heat treatment. 3) roteose-
pept ne. Column C: 1) proteose-peptone, 2) en-
riched component 3 fraction from the preparative
procedure as outlined in Figure 1, 3) enriched com-
ponent 3 fraction from the preparative procedure as
outlined in Figure 2,

 

45

 

.: phdm .NadSoaai Hopmhm Homage.
naoasdpsooman .sogaaom moaz z H.o an .36 m3 Novas pouasoaod A
.35 m3 wagon Am .36 m3 openpdoludfia AN .Sémav octagon
13.933 A." ”mason N.“ you ma 26.73», mo 95.33 on domoauo 218381393
condos: m psosoaaoo doe—3.96 .wo mahoppa caponosaonuooac How ovdaddhngw .m onswdm

V;-

 

\

%

_ m
. m
N

.sodpomnm cosoango map scum
m psosoaaoo mo sodpwfloma on» ad poms HHmo oapmaoaaoapooao mbdpwndAona was .OH mammam

.jmu m> :53:me

 

48

.How obapmamamaa on» no maaapm mean
03» Scam m psosoaaoo doamdmsa no announce oapmaosaoapomam How oedemaaaod

 

 

.aa masmaa

 

 

Figure 12.

49

'-

Acrylamide gel electrophoretic patterns of com-
ponent 3 isolated from the preparative-scale
acrylamide gel electrophoresis. Colume A.
Acrylamide gel electrophoretic patterns of:

1) isolated component 3 (unheated preparation)
obtained from the preparative gel, 2) enriched
component 3 (unheated reparation) applied to
the preparative gel, 3 isolated component 3
(heated pre aration) obtained from the prepara-
tive gel, 4) enriched component 3 (heated
preparation) applied to the preparative gel,

5) proteose-peptone. Discontinuous buffer sys-
tem (Appendix, Part I). Colume B. Starch-urea
gel electrophoretic patters of: 1) isolated
component 3 (heated preparation), 2) isolated
component 3 (unheated preparation). Discontin-
uous buffer system (Appendix, Part I0.

50

 

51

.AH phdm .Navzoaa<v aopwhm acumen

msossdusooman =.hosx= dowamnnusoomapaa .Umudo: A: .snommo woman
Iaausooanpas .eopmos Am .505: condo: AN .naomdo mmH condos AA sachu
cwsdcpno monopaoAIomoopona ho ushoppda capouosaonpooao How oddfiwahnod

.ma ohsmdm

 

PART I I

THE CHEI-LICAL Al‘-."D PHYSICAL PROPERTIES OF

COIQPOLJEIIT 3 FROl-Z SKIICI‘EILK

NTRODUCTION TO PART II

The proteose-peptone fraction obtained from bovine
milk has been reported to be low in nitrogen and to contain
carbohydrate (Thompson and Brunner, 1959). This would sup-
port the classification of proteose-peptone as a glycopro-
tein. However, to date, no individual components of the
proteose-peptone fraction have been isolated and their com-
positions studied. It is possible that within the proteose-
peptone group, components vary in the type and amount of
carbohydrate.

In the following eXperiments, chemical analyses were
performed on component 3, obtained from heated and unheated
skimmilk. The results of such analyses would reveal if com-
ponent 3 isolated from proteose-peptone is similar to the
component 3 isolated from unheated skimmilk. The amino acid
composition was determined with the use of Beckman Model 120C
Amino Acid Analyzer. Independent analyses were performed on
the amino acids tryptophan and cystein. For quantitative
determinations of the carbohydrate, colorimetric procedures
were adopted, using standard curves constructed from known
carbohydrate substances. The carbohydrate moities of com-
ponent 3 fraction were identified by means of paper chroma—
tography. Physical characterization, i.e., sedimentation-
velocity, equilibrium molecular weight, ionic mobility, and
diffusion coefficient, was performed on component 3 from

heated skimmilk.

53

EXPERIMENTAL
Apparatus

Amino acid analyses were performed on a Beckman
(Spinco) model 120C Amino Acid Analyzer. Spectrophotomet-
ric measurements were made with a Beckman DK-Z ratio-
recording Spectrophotometer. A micro-Kjeldahl distilla-
tion apparatus was used for hexosamine determination. The
hydrogen ion concentrations of various buffers and solu-
tions were determined with a Beckman Zeromatic pH meter
using a glass electrode. Materials were dried in a tem-
perature controlled Cenco vacuum oven.

Sedimentation-velocity and sedimentation-equilibrium
studies were performed in a Spinco Model E analytical ultra-
centrifuge equipped with an HTIC temperature control unit
and a phase plate as a schlieren diaphragm. Centrifugation
was performed in an An-D dural rotor and a General Electric
AH-6 mercury lamp served as the light source. A Starrett
microsyringe-burette was used to fill the ultracentrifuge

cell for short-column equilibrium eXperiments.
Chemicals

Unless otherwise Specified, all chemicals were rea-
gent grade quality. The chemicals and their suppliers are
given as follows: tris (hydroxylmethyl) amino methane from
Signmi<30mpany; tryptOphan from Nutritional Biochemical Com-
pany; p-dimethylaminobenzaldehyde from Eastman Kodak Company;

54

55
Ellman's reagent, 5-5' di-thiobis (2-nitrobenzoic acid) from

Sigma Company; L-cysteine from Nutritional Biochemical Com-
pany; acetylacetone from Fisher Scientific Company; gluco-
samine-HCl, galactose, fucose from Nutritional Biochemical;
thiobarbituric acid from Eastman Kodak Company; synthetic
N-acetylneuraminic acid from Carbiochemical Corporation;
triphenyltetrazolium chloride from Eastman Kodak Company;
sodium barbital (veronal) from Fisher Scientific Company;

guanidine HCl from Eastman Kodak Company.
Chemical Methods
Amino Acid Analysis

Component 3 was analyzed for amino acids with a Beck-
man (Spinco) Hodel 120C amino acid analyzer. The protein
was first hydrolyzed with strong mineral acid. During
breakdown of the protein polypeptide chain into amino acids,
reactions involving degradation of amino acids, interactions
with sugar residues in glycoprotein and rearrangement of
bonds might occur. Amino acids differ in their ease of
release from polypeptide chain. Thus, amino and imido acids
having no side chain groups, i.e., glycine, alanine, valine,
leucine. isoleucine, phenylalanineland proline are rather
stable in hot mineral acid solution and require longer time
of hydrolysis. 0n the other hand, the hydroxy amino acids,
serine and threonine, are gradually degraded during hydro-

lysis. Consequently, the choice of the variables time and

56
temperature is a compromise between sufficiently complete
hydrolysis of the protein and minimizing the loss of certain
amino acids. Two sets of hydrolytic conditions, i.e., 6 N
HCl at 1000 C for 20 hours and for 70 hours, were employed
in these analyses. In case of a glycoprotein, intermediate
sugar degradation products are formed during mineral acid
hydrolysis. These are capable of reacting with the amino
acid residues, although the rate of reaction is very slow
in strong acid solution. This type of interaction can be
minimized by using a dilute concentration of the glycopro-
tein (0.1% in 6 N HCl) in the hydrolysis step.

The amino acid analyses were performed on 20 and 70
hour acid hydrolysate. A protein concentration of about
0.06% in 6 N HCl was used in hydrolysis which were conducted
at 110° C for 20 and 70 hours in vacuum sealed tubes. The
hydrochloric acid was removed by evaporation at reduced
pressure in a warm water bath. The dried amino acid residue
was dissolved in dilutor buffer (pH 2.2) and an aliquot
applied to the appropriate resin column of the analyzer.
Standard chromatograms of known amino acid composition were
obtained one to two days prior to the actual runs of the

samples, using the same buffer and ninhydrin solutions.
Nitrogen

The digestion mixture consisted of 5 gm. SeO2 and
5 gm. CuSOu'5H20 in 500 m1 of concentrated H2804. The
protein (5-10 mg) was digested with 3 to 4 ml of the

57

mixture in a digestion flask. This operation was performed
over a gas flame until the mixture was clear. Then, after
cooling, one ml of H202 (30%) was added and the mixture was
heated for one hour. The solution was cooled and diluted
with 10 ml water. The digestion flask was connected to the
distillation apparatus through ball and socket fitting.
Twenty-five milliliters of a 403 NaOH solution was added to
neutralize the acid. Ammonia was steam distilled into 15
ml of a 4% boric acid solution containing 3 drops-of the
indicator mixture (400 mg bromcresol green and 40 mg methyl
red in 100 ml of 95% ethanol). Standard 0.01974 N HCl was
used to titrate the ammonia-borate complex. The normality
of the standard HCl solution was determined by titration
with standard tris (hydroxymethyl) amino methane. Deion-
ized water was used as a blank. Tryptophan with a known

nitrogen content, was used to evaluate the recovery.
PhOSphorus

Phosphorus was determined colorimetrically by the
method of Sumner (1944). Protein was dried in a vacuum

oven over P20 and weighed (5 go 10 mg) into a test tube.

5
“Two and two tenths milliliters of a 50% H SO was added.

2 4
The mixture was digested in sand bath over an electric
burnern until SO3 fumes appeared. It was heated for another
20 minutes. After cooling, 4 drops of 30:75 H202 solution
was added and heating was continued for 20 minutes. The

H202 treatment was repeated, if necessary, until the

58
solution was clear. The solution was heated for one hour to
remove all H202. The digestion mixture was transferred quan-
titatively to a 50 ml volumetric flask.~ Five ml of a 6.63
ammonium molybdate solution was added. Then four ml of
FeSOu solution [5 gm Fe804(H20)7 in 50 ml water containing
1 ml 7.5 N stoél was added. The final volume was made up
to 50 ml with deionized water. Transmission values were
read with a Cenco-Sheard-Sanford photelometer equipped with
a filter with maximum transmission at 650 mu. The standard
curve was constructed from values obtained with anhydrous
potasium phoSphate (KZHPOQ) as a source of phOSphorus. The
standard was dissolved in 5 m1 of 7.5 N H2804 solution
before addition of ammonium molybdate. Deionized water was

used as a blank.
Sulfur

Total sulfur was determined by Spang Nicroanalytical
Laboratory, Ann Arbor, Michigan from 10 mg samples by com-

bustion in a Parr bomb.
Tryptophan

Tryptophan is unstable under the usual conditions of
acid hydrolysis employed in amino acid determination. Con-
sequently, a separate method of analysis for tryptOphan is
required. Generally, three methods are available. One
involves an alkaline hydrolysis in which tryptophan is

relatively stable and subsequent analysis on the amino acid

59

analyzer (Noltmann, 1962). Also, tryptophan can be deter-
mined by a direct spectrophotometric method at wavelengths
between 280 and 315 mu in the ultraviolet region (Beaven
and Holiday, 1952). This method has the advantage of avoid-
ing hydrolysis, however, a clear protein solution is required.
Third method consists of a colorimetric determination of
tryptophan with p-dimethylaminobenzaldehyde (Spies and
Chamber, 1949). This method is uncomplicated, sensitive
and reproducible, thus it was the method adopted for the
tryptophan analysis made here.

Protein (5 to 10 mg) was dissolved in 10 ml of 0.1 N
NaOH. Eight milliliters of 24 N H2304 and 1.0 ml of 2 N
H280“, containing 30 mg of p-dimethylaminobenzaldehyde, were
mixed and cooled to 25° C. The sample solution was added to
this solution. The solution, after shaking and cooling to
25° C, was kept in the dark at 25° C for one hour. To this
solution, was added 0.1 ml of 0.04% sodium nitrite solution.
The solution was shaken, and the color was allowed to develop
for 30 minutes at room temperature in the dark. Per cent
transmission was read at 580 mu with a DK-Z Spectrophoto-
meter. A standard curve was prepared from commercially

available tryptophan.
Cysteine

A large number of methods are available for the esti-
mation of the sulfhydryl groups in proteins. This is mainly
due to the diversity of reactivity of -SH group with various

60
chemical reagents. The chemical reagents generally used for
-8H group estimation include: 1) mercaptide forming agents,
such as p-chloromercuribenzoate (PCMB); 2) oxidizing agents,
i.e., performic acid; 3) alkylating agents, such as iodo-
acetamide, which form a S-carboxymethyl cysteine derivative;
and 4) sulfhydryl-disulfide interchanging agent such as
Ellman's reagent, 5,5-di-thiobis (2-nitrobenzoic acid) (DTNB).
.For the quantitative estimation of ~SH groups in protein,
several methods should be tried to check on their agreements.
The agreements between the various methods can be determined
by estimating the -SH content of a well characterized protein,
such as B-lactoglobulin, which is commercially available in
chromatographically homogeneous form. The PCNB-dithizone
method (Sasago and others, 1963) and the DTNB method (Ellman,
1959) were tested by the author for estimating -SH group in
bovine B-lactoglobulin. Both methods gave approximately
Z-SH groups per molecule when determined in the presence of
urea. The DTNB method had the advantages of simplicity and
high sensitivity, and was the method selected for the detec-
tion and estimation of cysteine.

Protein (5 to 10 mg) was dissolved in 1 ml of deion-
ized water. Ellman's reagent, 5-5' di-thiobis (2-nitroben-
zoic acid) was prepared by dissolving 39.6 mg of DTNB in 10
m1 of phoSphate buffer (pH 7.0). A phoSphate buffer, pH 8.0
and containing 8 M urea, was also prepared. To one ml of
the sample solution, was added 9.0 ml of the phoSphate-urea

buffer. The solution was shaken and allowed to stand at

61
room temperature for ten minutes. A three ml aliquot was
pipetted into a test tube, to which, was added 0.02 ml of
DTNB reagent. The solution was shaken and the per cent
transmission was immediately read at 412 mu, using a 03-2
Spectrophotometer. The yellow color formed faded with time.
After 24 hours, it was completely colorless. A standard

curve was prepared from commercially available L-cysteine.
Hexose

A number of colorimetric procedures are available
for the estimation of hexose, which depend on properties
other than their reducing behaviors and which do not require
prior hydrolysis of the sample. Most of these involve the
reaction of sugar with sulfuric acid to give a furfural
derivative, yielding colored condensation products with a
phenolic base compound. Among these are the orcinol-sul-
furic acid method described by Nenzler (1955), the phenol-
sulfuric acid method developed by Debois (1956), et a1.
These methods give optical densities that are proportional
to the amount of hexose present over a certain range of
hexose concentrations. Other substances present in the
saxzple, such as protein, amino acids, hexosamine and sialic
aCid, do not interfere with the reaction. Since optical
densities vary with the type of hexose, a standard curve
Should.be constructed which contain hexoses in the same
. Propcxrtions as they occur in the sample. We found'that

the I>henol-sulfuric acid method was more sensitive than

62
the orcinol-sulfuric acid method, in addition to being simple
and reproducible. Hence, it was adopted for the hexose
analyses of the protein.

Protein was dissolved in deionized water to a concen-
tration of approximately one mg per ml. Two ml aliquot of
the sample solution was pipetted into a test tube, to which,
was added 0.05 ml of a 80% redistilled phenol solution.
Then, 5 ml of concentrated sulfuric acid was added rapidly,
the stream of liquid was directed against the liquid sur-
face rather than against the side of the test tube in order
to obtain good mixing. The tubes were shaken and allowed
to stand at room temperature for 10 minutes. Then, they
were placed inma water bath at 25° C for 20 minutes. The
per cent transmission of the characteristic yellow orange
color was measured at 490 mu, using a Beckman DK-2 Spectro-
photometer. Two standard curves were prepared from commer-

cially available galactose and mannose.
Fucose

Fucose, a methylpentose, is a normal constituent of
the tflood-group substances and is present in many glycopro-
teied‘.jFucose has been reported to be present in the proteose-
Peptone fraction of boVine milk (Thompson and Brunner, 1959).
A colorimetric methodfor fucose determination involves heat-
1r1C3'the sample with sulfuric acid, followed by addition of
Cysteine hydrochloride to give a green-yellow color (w'enzler,

1955) . The optical densities are read at two wavelengths and

63
the difference between these values is compared with that
given by standard fuCOSe solution. Hexose, hexosamine and
pentose do not interfere with the reaction.

Protein (5 to 10 mg) was dissolved in 10 ml of deion-
ized water. A fucose standard solution, containing approxi-
mately 20 mg per ml, was prepared. A sulfuric acid-water
mixture was made up of six volumes of concentrated sulfuric
(acid and one volume of water. One milliliter aliquots of
the sample solutions were pipetted into test tubes. To
these tubes (and to one ml of water for blank and one ml of
the fucose standard), were added 4.5 ml of ice cold H2304 -
H20 solution. The solutions were mixed while maintained in
an ice bath. The solutions were heated for exactly three
minutes in a boiling water bath and cooled in tap water.
Cysteine-hydrochloride solution (0.1 ml of a 3% w/v solu-
tion) was added and mixed immediately. The cysteine rea-
gent was omitted from one of the samples to correct for
non-specific color development. The solutions were allowed
to stand at room temperature for 60 minutes and the per cent
transmission was read at 396 and 430 mg, with a Beckman DK-Z

spectrophotometer.
Hexosamine

The analysis of hexosamine in glycoprotein involves,
firstly, the hydrolysis of hexosamine from the glycoprotein,
followed by the determination of hexosamine in the hydro-
lysate. The usual hydrolytic conditions employed in the

64

hexose analysis (1 to 2 N HCl at 100° c for 4 to 5 hours)
might result in incomplete liberation of hexosamine. The
hydrolytic condition employed in the amino acid analysis
(6 N HCl at 110° C for 20 hours) would result in appreci-
able destruction of hexosamine. The hydrolytic condition
usually employed for hexosamine determination, i.e., 4 N
HCl at 100° C for 4 to 6 hours, represents a compromise
between maximum release of the amino sugars with minimum
destruction. A common procedure for the determination of
hexosamine is the method developed by Elson and Morgan,
(1933), whereby acetylacetone is allowed to react with
the amino sugar in a hot, mildly alkaline solution. A
mixture of pyrroles is obtained, giving a pink color with
Ehrich reagent (p-dimethylaminobenzaldehyde). Several
modifications of the Elson and Morgan method have been
reported (Rondle and Morgan, 1955; Kraan and Muir, 1957;
Exley, 1957). One of these, the Cessi method (Cessi and
Piliego, i960) involves the steam distillation into
Ehrlich's reagent of 2-methylpyrrole, a volatile compound
that is produced among the mixture of pyrroles. The Cessi
method was reported to be highly reproducible (Johansen
gt, al.. 1960) and was the method employed for the hexo-
samine analysis.

The acetylacetone reagent was prepared by dissolv-
ing one ml of colorless, redistilled acetylacetone (boil-
ing point, 138 to 140° C) in 100 ml of 0.5 N sodium

carbonate-sodium bicarbonate buffer, containing 0.1 L

65
sodium chloride. The pH of the buffer should be close to
9.8. Ehrich reagent was prepared by dissolving 80 mg of
p-dimethylaminobenzaldehyde in 100 ml of absolute ethanol
containing 3.5 ml of concentrated hydrochloric acid.
Hydrolysis proceeded as follows: ,protein (5 to 10 mg) was
dissolved in 5 ml of 4 N HCl and hydrolyzed at 100° c for
6 hours under an atmOSphere of nitrogen. the hydrolysate
was dried in vacuum over NaOH in a dessicator maintained
at room temperature. The dried hydrolysate was dissolved
in 10 ml of water, a two-milliliter aliquot was pipetted
into a small micro-Kjeldahl distillation flask, to which,
was added 5.5 ml of acetylacetone reagent (pH 9.8). The
solution was heated in a boiling water bath for 20 minutes.
After cooling in tap water, the digestion flask was con-
nected to a steam distillation apparatus. Heating was
done with a microburner. Portions (2 ml) were distilled
into 10 ml volumetric flasks containing 8 ml of the
Ehrich reagent. Per cent transmissions were determined
30 minutes later at 545 mm, with Beckman DK-2 spectro-
photometer. A standard curve was prepared from.commer-
cially available glucosamine-HCl, which was treated with

the acetylacetone reagent as described above.
Sialic Acid

Sialic acids comprise the various N-acetylated and
N-acylated-O-acetylated neuraminic acids widely distributed

in animals in predominantly bound forms. Among the carbo-

 

 

66

hydrate moities of glycoprotein, sialic acid is the most
labile to acid hydrolysis. This is related to the struc-
ture of sialic acid which resembles a deoxy-sugar, the
glycosides of which have been reported to be hydrolyzed
much more readily than other glucose derivatives (Overend
§§,'gl.. 1962). The sialic acid appears to occupy a non-
reducing terminal position in the heterosaccharides chain
and is linked ketosically either to hexose or hexosamine
(Kuhn, 1960). In the determination of total sialic acid,
the uSual procedure is to perform the hydrolysis first to
release sialic acid from its bound form. The condition
employed is a mild acid treatment in 0.1 N 32304 at 80° c
for 30 minutes. The free sialic acid can be determined by
' several colorimetric procedures, among which the thiobar-
bituric acid method (Warren, 1959) is the most sensitive
and reproducible. The method is based on the periodate
oxidation of sialic acid to form cleavage products which
react with thiobarbituric acid to give a color compound
with an absgrption maximum at 549 mu.

Protein (5 to 10 mg) was dissolved in 10 ml of 0.1 N
H280“ solution and hydrolyzed at 800 C in a water bath for
30 minutes. A four-tenths m1 aliquot was pipetted into a
test tube, to which was added 0.1 ml of sodium periodate
solution (0.2 M sodium meta-periodate in 9 M phosphoric
acid). The tubes were shaken and allowed to stand at room
temPerature‘for 20 minutes. One milliliter of sodium

arsenite solution (10% sodium arsenite in a solution of

67
0.5 M sodium sulfate-0.1 N H2304) was added and the tubes
shaken until the yellow-brown color disappeared. Three
milliliters of thiobarbituric acid solution (0.6% in 0.5 N
socium sulfate) was added. The tubes were shaken, capped
with marbles, and heated in a vigorously boiling water
bath for 15 minutes. The tubes were removed and placed in
cold water for 5 minutes, followed by the addition of 4.3
m1 of cyclohexanone, which was used for the extraction of
the chromophore. The tubes were shaken and the contents
were transferred to conically shaped tubes and centrifuged
for 3 minutes in a clinical centrifuge. The clear, upper
cyclohexanone phase was red and more intense than in the
aqueous phase. Per cent transmission of the organic phase
was measured at 549 mu with a Beckman DK-Z Spectrophotometer.
.A.standard concentration curve was prepared from commer-

cially available, synthetic N-acetylneuraminic acid.
Paper Chromatographic Identification of Carbohydrate

The colorimetric methods for hexose and hexosamine
dc> not differentiate between the types of hexose and hexo-
samine in glycoproteins. Hence, a qualitative analysis of
the; carbohydrate moities was required. Several methods are
avelilable for the identification of sugars in biological
sut>stances. These include paper chromatography, thin-layer
Chrxamatography, gas chromatography of sugar derivatives,
and, others. The carbohydrate was hydrolyzed from the sample

Wit?! 1-2 N HCl. The acid and other charged groups, such as

68

amino acids or peptides that may be present in the hydrolysate,
should be removed before the sample is applied on the chromato-
graphic paper. This operation can be accomplished in several
ways, such as ion exchange chromatography, repeated lyophiliza-
tion (in case of HCl). extraction with pyridine or a combina-
tion. Paper chromatography was adopted for the tentative
identification of hexose and hexosamine in component 3. A mix-
ture of known sugars was chromatographed as a standard.

Purified component 3 (approximately 50 mg) was dissolved
in 5 ml of 2 N HCl and hydrolyzed in a sealed tube at 100° c
for 6 hours. The hydrolysate was dried by evaporation under
reduced pressure in a warm water bath. The dried hydrolysate
'was dissolved in 5 ml of water and passed through a Dowex 50 8x
(B? form) column (2.2 x 30 cm) coupled with an Amberite 4B-(OH-
form) column (2.2 x 30 cm). The effluent (300 ml) was evapor-
zrbed to dryness under reduced pressure. The dried material was
«attracted with 5 ml of pyridine (redistilled) on a steam bath
for 5 minutes. Pyridine was removed by evaporation under
reduced pressure at temperature below 40° C. The extracted
mngar'residue was dissolved in a minimum amount of 10% propanol,
and. applied to the chromatographic paper (Whatman no. 1). A
starrdard mixture of known sugars was spotted alongside the
samp11e. The relative positions of the known sugars in the mix-
ture: were ascertained by spottings of individual sugars in the
Chrornatogram. The descending chromatogram was run for 18 hours,
at r<>om.temperature, using a solvent mixture made up of butanol-

PYridxtne-water (6:4:3, v/v). The chromatogram was dried at 1000 C

69 _
for 10 minutes and developed at 60° C in a moist atmOSphere

with 2% triphenyltetrazolium chloride in an equal volume of

1 N NaOH.

Ph sical Methods

 

Free-Boundary Electrophoresis

This technique is used to determine the ionic mobil-
ity of a purified protein preparation and to estimate its
isoelectric pH value. Electrophoretic mobilities are cal-
culated from measurements made from the position of initial
boundary on the descending and ascending patterns, as fol-

lows:

dAk
tIHm

 

H:

where u is the electrophoretic mobility in cm2 volt-1 sec-1,

d the distance measured from the initial boundary in cm, A
the cross-sectional area of the electrophoretic cell in cm2,
k the cOnductivity cell constant, t the time in seconds,

I the current in amperes, R the resistance of the buffer in
ohms, and m the magnification of the optical system. The
protein solution should be clear in order to obtain a good
photographic pattern. Also, the protein solution should be
ciialyzed against the buffer until ionic equilibrium is
eattained. For estimation of isoelectric point of protein,
21 series of electrophoretic runs with buffer pH ranging

from basic to acidic (so that the net charge on protein

<1hanges from negative to positive, reSpectively) are

70 ‘
employed. The isoelectric point of the protein is estimated
from a plot of ionic mobility against pH. The preparations
of the buffers used in free-boundary electrophoretic runs

are shown in the Appendix, Part II.
Ultracentrifugation

Sedimentation-Velocity; The sedimentation-velocity
method is used for the determination of the sedimentation
coefficient of the molecule and for providing information
about the purity of the material under investigation. The
sedimentation coefficient is defined as the velocity of
the sedimenting molecule per unit centrifugal field. The
ultracentrifuge is operated at top Speed, and the movement
of the boundary across the cell is recorded in the photo-
graphs which are taken at periodic intervals. Sedimenta-
tion coefficient is calculated from the following equation,

2.303 x
8212—108?

where S is the sedimentation coefficient in sec, x the dis-
tance of the boundary in cm from the axis of rotation, t the
time in secs and w the angular velocity in radians per sec.
Generally, the sedimentation coefficient is eXpressed in
units of 1 x 10'13 sec and the unit 1 x 10'13 sec has been
termed is, where S is the Svedberg. EXperimentally, a plot
of log x against t gives essentially a straight line, the
slape of which is used to calculate the sedimentation coef-

ficient. For many proteins, the sedimentation coefficient

71
is dependent on concentration (Schachman, 1959). Determina-
tions are made at different concentrations and the sedimenta—

tion coefficient is estimated at infinite dilution.

Sedimentation-Equilibriumi Sedimentation-equilibrium
is attained when the material migrating across a given sur-
face in a centrifugal direction is balanced by the tranSport
centripetally due to diffusion. Sedimentation-equilibrium
is used for calculating the molecular weight of a protein.
The classical sedimentation-diffusion equilibrium requires
a long running period for attainment of equilibrium (VanHolde
and Baldwin, 1958). Archibald (1947) has shown that molecu-
lar weights can be calculated from data obtained during the
early stages of a centrifugal run. He pointed out that the
solute does not leave the centrifuge cell either at the
meniscus or the bottom of the cell and therefore the condi-
tions for equilibrium are fulfilled at these two locations
in the cells at all times of the run. Thejnrchibald proce-
dure, generally known as the approach-to-equilibrium method,
has been frequently applied for molecular weight study
(Erlander and Foster, 1959). The method-requires knowledge
of the concentration of solute at the bottom and meniscus
of the cell. It should be noted that determination of
molecular weight by this method should be made over a range
of protein concentrations, since calculation based on one
concentration may be unsound unless the protein solution is

truly monodispersed. In the case of a polydiSperse system,

72
the apparent weight-average molecular weight is dependent on
concentration. The apparent weight-average molecular weight
is plotted against concentration and the h:,app at infinite
dilution is calculated by extrapolation to zero concentra-
tion. Where the apparent weight-average molecular weight
shows dependence on protein concentration, attempts are
made to determine molecular weight in the presence of a dis-
sociating agent, such as 5 M guanidine-HCl, in the hape of
obtaining the molecular weight of the light component that.
is nearly independent or shows a less degree of dependence
on concentration.

Sedimentation-velocity and sedimentation-equilibrium
studies were performed in a Spinco Model E, analytical untra-
centrifuge equipped with an RTIC temperature-control unit
and a phase plate as a schlieren diaphragm. A synthetic-
boundary cell was used in all sedimentation-velocity runs.
Equilibrium studies were performed in the double-sector
cell. Centerpieces were of the filled-Epon type.. A false
bottom of FC-u3 fléiocarbon oil was employed in the short-
column equilibrium eXperiments. The sedimentation-velocity
studies were performed at 200 C with rotor speeds of 59,730
r.p.t. The photographic plates were read with a Nikon.
microcomparator capable of measuring to less than 0.002 mm.
A sample calculation of the equilibrium molecular weight by
the sedimentation equilibrium method is shown in the Appendix,
Part II.

73

Diffusion Coefficignt: The diffusion coefficient is
the proportionality factor relating the rate of transfer of
material across unit cross section to the rate of concentra-
tion with reSpect to the distance (the concentration gradi-
ent) at the given cross-section. The diffusion coefficient
of a protein is a physical parameter relating to the size
and shape of the molecule. The diffusion coefficient can
be obtained employing the schlieren optics in Tiselius free-
boundary electrophoretic cell or the Beckman Model E ultra-
centrifuge. In the schlieren Optics, concentration gradient
is proportional to the peak height and area. Diffusion
coefficient can be calculated from measurements of peak
height and area at various time intervals, according to the
following formula,

mzA2

1+1TtH2

 

where D is the diffusion coefficient in cmZ/sec, A is the
area in cmz, H is the peak height in cm, t the elapsed time
in sec and m, the magnification of the optics. With
Tiselius free-boundary electrophoretic cell, the area is
measured by weighing the enlarged photographic patterns.
With Beckman Model E ultracentrifuge, the area is measured
from the photographic plates, using a Nikon microcomparator.l
The quantity mzAz/LLTIH2 is plotted against time. The dif-
fusion coefficient is obtained from the slope of the line.
The diffusion coefficient of a protein often depends on

protein concentration (Greenberg, 1951). Hence, diffusion

7Q
runs are made at various protein concentrations. In the
following diffusion runs, a Tiselius cell was used for the
protein in veronal buffer, pH 8.6, ion strength = 0.1 and
a Beckman Model B ultrancentrifuge was used for the protein
in veronal-5 H guanidine hydrochloride, since large quantity
of guanidine hydrochloride was not available at the time for

runs in the Tiselius cell.

RESULTS AND DISCUSSIONS
Chemical Composition

The amino acid compositions of component 3 from heated
and unheated skimmilk are shown in-Table 3. The analyses
were reported as g residue/100 g protein and as number of
residues/1000 residues. The weight percentages of the amino
acid residues were based on a nitrogen content of 13.1% and
13.2% for component 3 from heated and unheated skimmilk,
respectively, as determined by micro-Kjeldahl nitrogen deter-
mination. The second method of expression, i.e., number of
amino acid residues/1000 total residues, was used for compar-
ing the similarity of the protein isolated from heated and
unheated skimmilk. This method of presentation relates the
proportions of amino acid residues with reSpect to one
another, hence, is not affected by experimental errors such
as weighing or loéés during transfers.

A summation of the residue weights of the amino acids,
together with the values for hexose, hexosamine, sialic acid
and phoSphorus, accounted for approximately 95% of the protein.
The protein portion constituted about 80% by weight of the
glyc0protein, the remaining portion was essentially carbohy-
drate. Independent analyses on the amino acids tryptophan
and cysteine indicated that component 3 contained negligible
or trace amounts of tryptophan and cysteine. Analytical data
obtained with the Beckman Model 120C amino acid analyzer

revealed that component 3 was low in the aromatic acids

75

76
tyrosine and phenylalanine: low in the sulfur-containing
amino acids (no cystine and low methionine); and high in
glutamic acid, lysine and leucine. Two small unidentified
peaks (2% by weight of total) appeared in the chromatograms
from both basic and neutral columns. The chromatogram of
the 20 hour hydrolysate showed qualitative evidence of glu-
cosamine and galactosamine, the glucosamine being in higher
concentration than the galactosamine.

The amino acid compositions, in number of residues
per 1000 total residues, of component 3 from heated and
unheated skimmilk, are presented in the form of the amino
acid profiles as shown in Figure 1#. These profiles
matched surprisingly close to one another, hence their amino
acid compositions were very similar. This rendered strong
evidence that component 3 was present as a native glycopro-
tein in milk and that its amino acid composition was essen-
tially unaltered by the heat treatment employed in its prepa-
ration.

Elementary analyses of component 3 from heated and
unheated skimmilk are shown in Table 4. Again, their nitro-
gen and phoSphorus contents were similar. The nitrogen con-
tent of 13.1% for component 3 was low compared with the
nitrogen value reported for proteose-peptone, i.e., (13.7
to 13.9%) by Thompson and Brunner (1961). The phOSphorus
content of component 3, 0.5% was also low compared with
their value of 1.15 P. Elementary analyses of the other

proteose-peptone components, i.e., component 5 and 8 (Kolar,

77
Ph. D. Thesis, 1967), indicated that these components con-
tained higher concentrations of P than component 3. A sul-
fur analysis, performed by the Spang Analytical Laboratories,
identified 0.59% S for component 3. This value is higher
than can be accounted for from methionine sulfur.

The amino acid composition of component 3 indicated
that it was an acidic protein, i.e., the sum of glutamic
and aSpartic acid residues was greater than the sum of
lysine, histidine and arginine residues. The-low isoelec-
tric point, i.e., pH 3.7. estimated from free-boundary
electrophoresis studies supported this conclusion. Compar-
ing the chemical composition of component 3 with other minor
protein fractions, (i.e., Whitney, 1958; Thompson and
Brunner, 1962), component 3 had a lower nitrogen content
and contained a sizeable amount of carbohydrate, while most
of the minor protein fractions contained much lower concen-
trations of carbohydrate. The amino acid composition of
component 3 was characterized by its low aromatic amino
acid content compared with other known milk proteins.
Recently, a phOSphoglchprotein from bovine milk whose
chemical composition and ultracentrifugal prOperties cor-
responded to the major component of the proteose-peptone
fraction was reported (Bezkorovainy, 1965). Its amino acid
composition showed high glutamic acid, aSpartic acid and
isoleucine contents. It contained 9.5% carbohydrate and
had a sedimentation constant of 0.8 at pH 7.0 at in con-

centration. Although the amino acid composition, carbohydrate

73
content, and ultracentrifugal properties of the phOSphogly-
coprotein differ from that of component 3 prepared for this
study, it is conceivable that other proteose-peptone compo-
nents, in their predominant forms, or in varying associa-
tions with one another, might give a molecular Species
whose chemical and physical properties were similar to the
phoSphoglycoprotein reported. An acrylamide gel electro-
phoretic run of this phosphoglycoprotein, and compared with
various proteose-peptone components, would reveal if similar-
ities exist among these fractions.

The carbohydrate contents of component 3 from heated
and unheated skimmilk are shown in Table 5. The standard
curves for the carbohydrate analyses are shown in the
Appendix, Part II. Component 3 from unheated skimmilk
possessed a higher hexosamine and lower hexose content than
component 3 from heated Skimmilk. Possibly, a slight degree
of sugar degradation, such as deamination of the hexosamine,
occurred as a result of the heat treatment employed. The
sugar content of 17.3% in component 3 was among the highest
reported for milk glycoproteins. The caseino-glchpeptide
released from k-casein by treatment with rennet, had a
sugar content of 28.1% (Alais and Jolle's, 1961). k-Casein
was reported to contain 5.0% carbohydrate (Alais and Jolle's,
1961). Conceivably, the action of some proteolytic enzymes,
possibly rennin, pepsin and/or others, might release a gly-
cOpeptide from component 3 with a considerably higher sugar

content. The proteose-peptone fraction was reported to

79
contain 6.8% carbohydrate (Thompson and Brunner, 1959). A
comparison of the sugar contents of component 3 with those
of the other proteose-peptone components (Kolar, Ph. D.
Thesis, 1967) indicated that component 3 had the highest
carbohydrate content among the proteose-peptone components.
Paper chromatographic identification of the sugar moities
in component 3 is shown in Figure 15. The chromatograms
were interpreted to indicate the presence of galactosamine,
glucosamine, galactose, mannose and fucose as the major
carbohydrates in component 3.

Minimum molecular weights of component 3 from heated
skimmilk were calculated from chemical analyses of known
constituents, as Shown in Table 6. An independent deter-
mination of molecular weight by ultracentrifugal methods
permitted a calculation of the number of residues of each
constituent per mole of component 3. Assuming accurate
chemical analyses and a reliable molecular weight deter-
mination, the number of residues per mole of protein should
approach an integral value. The minimum molecular weights
calculated from determinations of Sialic acid (as N-acetyl-
neuraminic acid), hexosamine, phosphorus, sulfur and
tyrosine (the limiting amino acid in component 3) indicated
that the above constituents existed in roughly integral

ratios of 3:1:2:2:7 in that order.

Q
.5 0

Physical Properties of Component 3
Electrophoretic Characteristics

The ionic mobilities of component 3 from heated skim-
milk, calculated from the free-boundary electrophoretic
patterns, in buffers ranging from pH 8.6 to 2.0, ion
strength = 0.2, are Shown in Table 7. The free-boundary
patterns are Shown in Figure 16. The ionic mobility in
veronal buffer, pH 8.6 and ion strength = 0.2, is -3.5 cm2
sec"1 volt-1 x 105 from the descending channel. Thompson
and Brunner (1961) reported the mobility of component 3
from the electrOphoretic patterns of proteose-peptone, in
veronal buffer, pH 8.6 and ion strength = 0.1, to be -2.0
Tiselius units (from descending channel), while Larson
(1955), using similar buffer system, reported a value of
-3.0 Tiselius units (from descending channel). It is pos-
sible that ionic mobility of a protein component would
vary Slightly depending on whether it is present singly or
in association with other protein components. Also, equi-
librium dialysis is important in ionic mobility determina-
tion. In all the electrophoretic runs in buffers ranging
from pH 8.6 to 2.0, component 3 appeared as a single peak.

A plot of ionic mobilities (values from descending patterns)
against pH is shown in Figurel7. The mobility decreases to
a minimum as the pH of the buffer approaches the isoelectric
point of the protein. The isoelectric point, obtained from

the intercept of the curve on the pH-axis, was estimated at

 

1....

 

81
pH 3.7. The low isoelectric point of component 3 would fit
its description as an acidic glycoprotein. This acidic
character agrees with its chemical constitution, which shows

an excess of the acidic residues over the basic residues.
Ultracentrifugal Characteristics

Diffusion Coefficient: The diffusion coefficients of
component 3 from heated Skimmilk in veronal and veronal-5 M
guanidine hydrochloride buffers, at various protein concen-
trations, are Shown in Table 8. The diffusion coefficients
were corrected to water at 20° C. A plot of diffusion coef-
ficients against protein concentrations is shown in Figure
18. The diffusion coefficients of component 3 increased
with increases in protein concentrations. Possibly, molecu-
lar aggregation decreased with an increase in protein concen-
tration, as would be evident in subsequent sedimentation-
equilibrium studies of molecular weights. (The smaller
molecular aggregate obtained at increased protein concentra-
tion would tend to give a higher diffusion coefficient. A
five-to-eight-fold increase in the diffusion coefficient of
component 3 was obtained in veronal buffer containing 5 M
guanidine hydrochloride, compared with straight veronal
buffer. Conceivably, in veronal buffer containing the
dissociating agent, a low molecular-weight Species or
monomer unit of component 3 existed. The light component
would tend to give a higher diffusion coefficient than a

large molecular polymer. The diffusion coefficient of

82
component 3, in veronal buffer, at infinite dilution, was

7

estimated at D20 w = 1.80 x 10' cmZ/sec, and in veronal-5
!
M guanidine hydrochloride, at infinite dilution, was esti-

mated at D20 w = 11.5 x 10'? cmZ/Sec.
O

Sedimentation Coefficient: The sedimentation coef-
ficients of component 3 from heated skimmilk in veronal
and veronal-5 M guanidine hydrochloride, at various protein
concentrations, are Shown in Table 9. A plot of the sedi-
mentation coefficients against protein concentrations is
shown in Figure 19. The sedimentation coefficient decreased
with increases in protein concentrations. The sedimentation
coefficient was considerably reduced in the presence of the
dissociating agent 5 M guanidine hydrochloride, indicating
that the polymer-monomer equilibrium was shifted toward the
low molecular-weight Species in a dissociating system. A
Single boundary appeared in the sedimentation patterns of
component 3 at protein concentrations greater than 5.mg/ml.
At dilute concentrations of less than 5 mg/ml, high molecu-
lar-weight contaminants, possibly some denatured euglobulin
or polymorphic species of component 3, appeared as spikes
(negligible areas), in the sedimentation patterns. The
sedimentation coefficient, at infinite dilution, in veronal
buffer, was estimated at 820’W = h.0 Svedberg, and that at
infinite dilution, in veronal-5 M guanidine hydrochloride,

was estimated at 820 w = 1.62 Svedberg.

33,3
Sedimentation-Equilibrium Studies of hglecular Weight

The equilibrium molecular weight of component 3 from
heated skimmilk, in veronal buffer, is dependent on protein
concentration as shown in Table 10. A plot of equilibrium
molecular weight against protein concentration is shown in
Figure 20 and 21. It is interesting to note that the molecu-
lar weight decreased with increases in protein concentra-
tions. k-Casein, a glycoprotein, also exhibitSSimilar
behavior (Swaisgood, 196k). The equilibrium molecular
weight of component 3, at infinite dilution, in veronal
'buffer, was estimated at 200,000. This value approximates
the calculated molecule weight of 207,000 obtained from
sedimentation and diffusion coefficients of component 3,
at infinite dilution, in the same veronal buffer (Svedberg
equation).

Presumably, the high molecular weight of component 3
in veronal buffer is due to polymer formation. To study the
molecular weight of the smaller unit or light component, a
dissociating agent, 5 h guanidine hydrochloride in veronal
buffer, was employed for the purpose of shifting the polymer-
monomer equilibirum toward the lower molecular-weight
species. The equilibrium molecular weight of component 3
in veronal-5 M guanidine hydrochloride at various protein
concentrations is shown in Table 10. The equilibrium
molecular weight of component 3 was considerably reduced

in the presence of the dissociating agent, indicating that

84
disaggregation of the polymeric species of component 3
occurred. A plot of equilibrium molecular weight of com-
ponent 3 against protein concentration in veronal-5 K
guanidine hydrochloride is shown in Figure 21. In the
dissociating system employed, the equilibrium molecular
weight again exhibited dependence on protein concentration,
although the degree of dependence of molecular weight on
protein concentration was less in the presence of the dis—
sociating agent. The protein was not monodispersed in
veronal-5 H guanidine hydrochloride buffer as evidenced by

an Rz/L ratio of greater than one, the Ez/hw ratio being a

w
measure of the degree of polydiSpersity (Tanford, 1961).
Possibly, other dissociating systems, such as 67$ acetic
acid-0.15 h NaCl, anhydrous formic acid, and others, should
be employed, which might further reduce the dependence of
molecular weight on protein concentration, and thus permit

a determination of the molecular weight of the monodis-
persed protein.

Alternately, the molecular weight of the light com-
ponent or small unit of component 3 might be obtained
according to Trautman's treatment of approach-to-equilibrium
method (Swangood, Ph. D. Thesis, 1963). Here, the sedimen-
tation equilibrium runs are conducted at various rotor
Speeds and at various protein concentrations. The molecular
weight of the small component is calculated from the slope
of the plot at rotor Speeds where only the small component

leaves the meniscus.

85

The equilibrium molecular weight of component 3, at
infinite dilution, in veronal 5 M guanidine hydrochloride
is estimated at 00,000 (Figure 21). A minimum molecular
weight calculated from the tyrosine determination (the
limiting amino acid in component 3) gave a value of approx-
imately 22,000. Since “0,000 does not represent the
molecular weight of the monodiSpersed protein, it is likely
that the molecular weight of the small component or the
monodiSpersed protein might lie in the range of 22,000 to
h0,000. .

SUMKARY TO PART II

Component 3 is a whey glycoprotein. Its amino acid
composition was characterized by a relatively high content
- of glutamic acid, leucine and lysine and a low content of
aromatic and sulfur-containing amino acids. Its carbohy-

drate content of 17.3% was high compared with other milk
glycoproteins. The carbohydrate moieties consisted of
galactosamine, glucosamine, galactose, mannose, fucose
and sialic acid.

Component 3 had an isoelectric point of 3.7, which
classified it as an acidic glycoprotein. The ionic mobil-
ity in veronal buffer, pH 8.6 and F‘/2 = 0.2, was 3.5
Tiselius unit. The sedimentation coefficient was 820,w =
4.0, at infinite dilution in veronal buffer, pH 8.6 and

(72 = 0.1. The diffusion coefficient was 1320’W = 1.8, at
infinite dilution, in veronal buffer, pH 8.6 and r"/2 - 0.1.
Sedimentation-equilibrium studies indicated that the molecu-
lar weight was concentration dependent. The equilibrium
molecular weight was estimated at 200,000, at infinite
dilution, in veronal buffer, pH 8.6 and r'/2 = 0.1. This
agreed.well with a molecular weight of 207,000 obtained
from sedimentation-velocity and diffusion runs. In the
presence of a dissociating agent, the polymer-monomer
equilibrium was shifted toward the low molecular-weight
component. The equilibrium molecular weight was estimated

at 00,000 in veronal-guanidine hydrochloride. However, even

87
in such dissociating agent, the molecular weight still
showed concentration dependence, indicating that the
protein was not completely monodiSpersed. A minimum
molecular weight estimated from the limiting amino acid
gave a value of 22,000. The molecular weight of the
monodispersed proteinwls estimated between 22,000 and

40,000.

O Q

J‘.)

TABLE 3

Amino acid composition of component 3
(heated and unheated preparations)

 

 

 

 

 

 

 

 

 

 

Grams Residue/ Number Residueg/
100 g Protein8 1,000 residues
Residue Heated Unheated Heated Unheated
Lysine 7.31 7.55 84.65 87.04
Histidine 3.27 3.19 35.33 34.44
Arginine 4.04 4.02 38.42 38.14
Unidentified Peak 1.79 1.85 18.70 19.15
ASpartic Acid 6.47 6.42 83.32 82.50
Threoninec 5.58 5.40 73.02 74.93
serinec 6.26 6.03 96.28 91.89
Glutamic Acid 14.40 14.22 165.91 161.98
Proline 4.79 4.82 73.31 73.57
Glycine ' 1.07 1.15 27.82 29.60
Alanine 2.16 2.16 44.16 45.11
Unidentified Peak 0.27 0.27 4.12 4.16
Valine 2.16 2.48 32.39 37.01
Lethionine 1.60 1.29 18.11 14.53
Isoleucine 4.82 4.99 63.30 65.17
Leucine 8.15 8.50 106.87 111.26
Tyrosine 0.74 0.74 6.18 6.36
Phenylalanine 2.28 2.29 23.00 23.09
Tryptophan Trace Trace -- --
Cysteine Trace Trace -- --
waa = 77.06 77.37‘
N1 = 1000.00 999.3
Total Carbohydrate
(weight 5) 17.30 17.20
P (as HZPOB) 1.30 1.31
2w = 95.66 95.87

 

 

aHeight percentage of the ith amino acid residue, based on a
nitrogen content of 13.1% and 13.2% for component 3 from
heated and unheated preparations reSpectively, as deter-
mined by micro-Kjeldahl nitrogen determination.

bObtained by summation of the moles of the 1th amino acid

from the chromatogram, corrected to 1000 total amino acid

residues.

CValues extrapolated to zero concentration.

TABLE 4

Elementary analysis of component 3
from heated and unheated skimmilk

 

 

 

Element Heated preparation Unheated preparation
P 0.5 0.5

S 0.59 ' --

 

 

9

TA:L

0

V1
N 5
u

The carbohydrate contents of component 3
from heated and unheated skimmilk

 

 

Unheated

 

Heated

Carbohydrate preparation preparation hethod

(i) (3)
Hexose 7.2 6.5 Phenol-sulfuric
Hexosamine 6.0 6.6 Cessi
Sialic acid 3.0 2.9 warren
Pucose 1.1 1.2 Dische
24"[1 17.3 1702

 

 

91

TABLE 6

Linimum molecular weights of component 3
(heated preparation) estimated from chemical analysis

 

 

 

Component Weight percentage hinimum molecular weight
Sialic acid 3.0 10,300
Hexosamine 6.0 2.990
Phosphorus 0.50 6,200
Sulfur 0.59 5.450

Tyrosine 0.75 21,800

 

 

.0 cm on 0 Ho chopdnoaaop no name

 

 

0:.m+ ma.m+ om.a+ m.a o.m Homuccaosac
mo.m+ H0.m+ dm.H+ m.a m.m HomumCHohHm
00.H+ ma.m+ sH.H+ m.H o.m Homnecdeaau
oe.o+ ms.o+ m:.o+ m.a m.m Hozuccacsao
m:.0i 53.0: mm.0| m.a 0.: mumpeo<
om.al mm.an om.ai m.a 0.0 opwpmo<
H0.NI mn.m| mw.a| m.H 0.0 endgamonm
em.mu Hm.ma sm.au m.a o.s mangancsm
mm.ma sfl.:u me.mu m.a m.m acconc>
ommao>< wnaemmom< wmaesoommm ARV Jim
A I> town So 10H x av scandapswosoo AN.0 u N\r_v
ammdaamoa 0H ego mohpomam Campohm ma .ampmhm nommsm

 

mammmsn msoaam> ma Acoapmamamaa condosv
m unecoasoo mo modpaoaoaa capoaosaoapomam

u mqm<9

93

TABLE 8

Diffusion coefficient of component 3
(heated preparation) in veronal and veronal-5 h 00 buffers

 

 

 

Buffer Protein concentration 020 w x 107
(mg/ml)
Veronal a
5.5 3.47a
6.0 3.76a
Veronal-S L 00 b
6.4 18.7
7.5 20.1b
8.9 22.0b
10.1 22.6b

 

 

8Obtained from ascending pattern of the Tiselius cell, run
at 3° C, corrected to water at 20° C.

bObtained from beckman Spinco Lodelo E ultracentrifuge, run
at 20°C, corrected to water at 20° C.

94

TABLE 9

Sedimentation coefficient of component 3
(heated preparation) from veronal and veronal-5 M GU buffers

 

 

 

Buffer Protein concentration 520 w x 1013
(mg/ml) ~ "

Veronal

4.4 ' 3.44

5.1 3.37

6.1 3.20
Veronal-S L 00 5.0 1.47

705 ' 1039

10.1 1.32

 

 

squilibrium molecular weight data for component 3

TAT-:1.-

95

[1“

' 10

(heated preparation) in veronal and veronal-5 h GU buffers

 

 

 

Buffer Protein concentration hw x 10'4 Lz/hw
(ms/m1)
Veronal
(pH 8.6, I"/2 = 0.1) 3.1 12.30 --
4.4 9.38 --
5.1 7.58 --
6.1 5.39 --
Veronal-B m GU 5.0 . 3.15 1.49
6.4 2.90 1.20
7.5 2.76 1.56
8.9 2.51 1.57
10.1 2.28 2.07

 

 

96

 

GLU
160L '

LEGEND:

140'— 0. HEATED
A. UNHEATED

120*-

 

 

100

 

80

RESIDUES/1000 RESIDUES

60

40

 

PHE
20r-

 

 

 

 

 

AMINO ACID RESIDUE

Figure 14. Amino acid profiles of component 3 from heated
and unheated skimmilk.

Figure 15.

97

 

Paper chromatogram of the hydrolysis-released
carbohydrate moities of component 3 (heated
preparation). Descending pattern: Whatman no. 1
chromatography paper: solvent system: butanol-
pyridine-water (6:4:3 v/v); stained with tri-
phenyltetrazolium chloride. Legend: A, galac-
tosamine; B, glucosaméneC, galactose; D, mannose;
E, fucose.

98
STANDARD

SAMPLE MIXTURE

A
9; i
Q 0

k

 

99
DESENDING ASCENDING

VERONAL
pH&5

60005196.: F= 7_.'182ch

 

 

   

PHOSPHATE
pH 6. D A

QQOua

 
  
  

5 F 7.82\_/le

“GNOME:

 

”005%. :F= 7.83ch1

GLYC I NE : HCI
pHZO

 

9200590.; F=7.84ch‘1

Fi re 16. Free-boundary electrophoretic patterns of com-
gu ponent 3 (heated preparation) in buffers of
lvarious pH.

. It}?! I )H:

100

 

 

 

+1.00 *—

 

+2.0 .—

Electrophoretic Lobility (u

.4--._.—.

_-.-——-.

+3.0 L_

Figure 17.

pH

A plot of electrophoretic mobilities
(descending values) of component 3 as a
function of pH.

6

 

- .1 -.—--— -“m- -—0—4

k m1-—_a44~.

 

101

 

1’4»__ /

10

 

U20.” x 107 CUZ sec'l
‘

k)
I
\

VT

 

 

‘o-n-o-m ma..-

 

 

:4 rotein Concentration ("18/ m1)

hixure 13. Plot honing concentration dependence of the
apparent diffusion coefficient of component 3
(heated preparation) in veronal and veronal-5
h guanidine hydrochloride. Legend: 0 , veronal
[3' veronal-5 H guanidine hydrochloride.

102

 

 

 

 

 

 

Figure 19.

430 -. -
\
\
\
\\
\
\
3.5 —’
\ i
\\
3.0 - \~\
\
\
\
\
\
\
\
\
52.5 d
N
"D
2.0 L—
5“
1.}; — ““S\SN—
1.0 J J J L 1 1 1 1 L
o 2 4 6 d 10

Protein Concentration (mg/ml)

Plot showing the concentration dependence of the

sedimentation coefficient of component 3 (heated

preparation) in veronal and veronal-5 M guanidine
hydrochloride buffer. Legend: 0 , veronal;

E], veronal-5 L guanidine hydrochloride.

 

 

103

.Homesé anacho> CH Acode mocha @opdmsv m ucocom:oo mo nguao:
Hoazooaoa nSHPLfiHHSWm mo mocmcfimmot Scandapmoozoo asflnczm poam .om casedm

AHE\wEv dofipnhpdmosoo Campoam

 

 

 

 

m m a m m H
_ a d w i. a o
/
/ ll.
/
In
x
aJOa mw
.1
All
/
/
/
/
/
/ i
/
/
/ ll
/
/
/
om

 

104

4 mladcomo> :H AcoHpmthmhc woudozv \

.a\ v»

.aciné odahoazoopfikx ozwzrgdzh

ms

pszogzoo go uxtfion
pdaaomaoa asfiunfiaazvo ho mcscfiroaoc Coapmaproo:oo wCazosm poam

AHE\n&v fiofideQCoosoo :HopOHA

.HN mhfiddm

 

ea ma 0a m my m m o
a a. . a _ a
|.o.H
w
i
//
/ Lo.m
.106
/

 

 

 

1‘1"

:isure 22.

105

Sedimentation-velocity and s

rium patterns for component
in veronal buffer (ph 6.6;

mentation-velocity patterns
elapsed time of 32 minutes,
the 3.1 ma/nl sanple, which
minut s.

edimentation-equilib-
a

) (heated preparation)
'/2 = 0.1). All

echriments were performed at 200 C. The sedi-

were taken at an
with the exception of
was taken at t = 16

 

106

SEDIMENTATION-VELOCITY SEDIMENTATION-EQUILIBRIUM
59.780 RPM. 9-55° ”,573 RPM, 6-600

     

 

6., I mglml

      
   
 

 

5. | mglml

5.l mglml

4.4m lml

 

 

3. I mglml

3. I mg/ml

Fiqure 23.

107

Sedimentation-velocity and sedimentation-equilib-
rium patterns for component 3 (heated preparation)
in veronal-5 M guanidine hydrochloride buffer.
All eXperiments were performed at 20° C. The
sedimentation-velocity patterns were taken at the
elapsed time of 32 minutes.

108
SEDIMENTATION-VELOCITY SEDIMENTATION-EQUILIBRIUM
59,780 RPM, 9=55° 9,34I_RP_M, 9=60°

  

ID. I mglml IO. I mglml

 

 

8. 9 mglml)

8.9 m'ghml

   
   

 

 

 

6-4 m9/ml 6.4 mglml

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(2)

(3)

(4)

(5)

(6)

(9)

(10)

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109

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APPERDIK

PART I

FRESH SKILLILK

Beat to 95° C for 30 min

Adjust to pH 4.6 with 1 N RC1

Let stand overnight at 4° C
and decant or centrifuge at
1000 x G for 20 min

 

 

l

SEPERAATART CASEIN + DENATURED SERUR PROTEIRS
(Discard)
Dialyze at 4° c
against several
changes of
distilled water
Pervaporate
Freeze-dry

 

PROTEOSE-PEPTONE

 

 

Figure I. Preparation of the classical proteose-peptone
fraction.

113

119

Composition of the Buffers Employed in This Study

The following buffer preparations were used in test-
ing the effect of buffer pH on the hexose content of purified
component 3:

1. Citrate phosphate; pB 4.6

A = 0.1 L solution of citric acid (19.21571n.1000 ml)

B = 0.2 M solution of dibasio sodium phOSphate (53.65 g

foogaéiPOu ° 7n20 or 71.7 g of AaZHPOQ ‘ 12H20 in

26.7 ml of A + 23.3 ml of B, diluted to a total of
100 ml

2. Citrate phosphate; pH 7.0
Same stocks buffers as A and B above.

6.5 ml of A + 43.6 ml of D, diluted to a total of
100 ml

3. Tris citrate; pH 3.60

Same as gel buffers used in acrylamide gel electro-
phoresis and starch-urea gel electrophoresis

Stock solution = 91.96 g Tris (solid) + 12.05 g citric
acid, diluted to a total of 100 ml

Use one part stock to nine parts deionized water
4. Borate; pH 3.60

Same as buffer used in electrode compartments in

acrylamide gel electrophoresis and starch-urea gel

electrOphoresis.

Stock solution = 881 g boric acid + 190 g NaOH,
diluted to a total of 19 liters

Use two Part stocks to three parts deionized water

120

Formulation for Starch-Urea Gel

Ingredients: 35 ml Tris stock solution; 205 ml dis-
tilled water, 40 g starch and 147 g urea. Heat Eris stock,
water and starch to 650 C with stirring, add urea, heat
rapidly with stirring to 900 C, degas and pour into gel

bed.

Preparations of Discontinuous Buffers4_StainingSolution
wand Destaining Solution for Preparative Acrylamide Gel

Electrophoresis and Starch-Urea Gel Electrophoresis

Buffers:
Tris citrate, pH 8.6

Stock solution = 91.96 g of Tris (solid) + 12.05 g
Citric acid, diluted to a total of 1000 ml

Lse one part stock to nine parts water
Borate, pH 2.6

Stock solution = 881 g boric acid + 190 g NaOH,
diluted to a total of 19 liters

Use two parts stock to three parts water

Staining solution:

250 ml water,.250 ml methanol, 50 ml glacial acetic
acid and 20 amino or buffalo black

Hashing solution:

200 ml glycerin, 1 liter water, 1 liter methanol. 200 ml
glacial acidic acid for mechanical destaining. 7L
acetic acid for electrolytic destaining

121

Reagents Used in Orcinol-Sulfuric Acid Method

Reagent A _ 60 ml of concentrated sulfuric acid and 40 ml water

Reagent B = 1.69 ml of orcinol (recrystallized from benzene)
in 100 ml water

7.5 ml reagent A is mixed with 1 ml of reagent B

Reagents Used in Folin-Ciocalteu Colorimetric:Reaction

Reagent A = 2% Na2C03 in 0.1 N NaOH

Reagent B 0.5} CuSOu ’ 5H20 in 1% Na or K tartrate
Reagent C = mix 50 ml A with 1 ml 3

Reagent D Folin-Ciocalteu reagent diluted with 1 N RC1

APPENDIX
PART II

Standard Curves for the Colorimetric Determinations
of PhOSphorus,_Trvntophan,,Cystein,»Rexose,

Hexnsamine and Sialic Acid Are Given Below

122

 

 

 

Absorbance(660 mo)

 

 

 

 

1.0

0.3e-
0.6»—

(9
0.4L_
0.2.—
0 L L i i
0.06 0.12 0.18
mg P

Figure II.

Standard curve for phOSphorus.

6:24

Transmissior (4’90 mu)

124

 

 

l 1 l__

 

 

Figure III.

“30-

40 6O 30
mg Rexose

Standard curve for galactose.

100

p Transmission(545 mu)

125

10

 

l,
I

O\
T

 

 

l 1 L
O L2°5 25 37.5 so

 

HS Bexosamine

Figure IV. Standard curve for glucosamine-RC1.

fl Transmission(549 mu)

126

 

O.)
T

O\ '\l
I

(\J

 

 

1

I...)
__
_.
1—
)—

 

Fixure V. Standard curve for H-actylneuraminic acid
(synthetic).

j Transmission(590 mu)

127

 

\\ (J

(J)

O\ —o
I

 

 

 

1 1 l 1 1
0 24 43 72 96 1

N“—
0

mg Tryptophan

Fisure VI. Standard curve for tryptophan.

' J
,9 Iran 53ml 3 s .1 o '. (41 2 mm)

128

 

 

 

l l l L J

 

 

0 0.2 0.4 0.6 0.3 1.0
u mole Cysteine

Figure VII. Standard curve-for cysteine.

129

Buffer Preparations Used in Free-Eoundary Electrophoresis

The following quantities were made to 2 liters with
redistilled water:
1. Sodium veronal; pR 2.6; Ifl/Z = 0.2

72.0 ml of 5.0 k MaCl

3.5 ml of 2.0 h BCl

30.0 ml of 0.5 L sodium veronal

2. Sodium phOSphate; on 7.0; (7/2 = 0.2
72.0 ml of 5.0 L AaCl
1.6 ml of 4.0 L waazpcu

3. Sodium nhOSphate; on 6.0; (”/2 = 0.2

72.0 ml of 5.0 h KaCl
9.2 ml of 0.5 h fiazfiPou
6.6 m1 of 4.0 h AaRZPOQ

4. Sodium Acetate; pH 5.0; ("/2 = 0.2
72.0 ml of 5.0 L LaCl
20.0 ml of 2.0 k JaAc
3.7 ml of 3.5 n acetic acid

5. Sodium acetate; pH 4.0; (w/Z = 0.2
72.0 ml of 5.0 R MaCl
20.0 ml of 2.0 L EaAc
33.7 ml of 3.5 A acetic acid

6. Glycine - 301; pH 3.5; F72 e 0.2
72.0 ml of 5.0 E KaCl
36.6 ml of 1.0 h glycine - 1.0 M NaCl
1.7 ml of 2.0 A BCl

130
0.2

7. Glycine - RC1; pH 3.0; ("/2
72.0 ml of 5.0 E EaCl
31.6 ml of 1.0 h glycine - 1.0 H UaCl
4.2 ml of 2.0 M RC1

Q. Glycine — BCl; pH 2.5; |”/2 = 0.2
72.0 ml of 5.0 k fiaCl
22.3 ml of 1.0 R glycine - 1.0 h NaCl
9.6 ml of 2.0 M RC1

9. Glycine - RC1; pH 2.0; ("/2 - 0.2
72.0 ml of 5.0 L NaCl
10.6 ml of 1.0 L glycine - 1.0 h NaCl

14.7 ml of 2.0 h HCl

Cogppsition_9f Verongl_Buffer Used‘in Ultracentrifugal

and Diffusion Runs

Veronal; pm a.6; ("/2 = 0.1
20.65 sodium barbiturate
2.797 cm barbituric acid

Lade up to a total of 1 liter with redistilled water

Properties of the Solvents Used for molecular Weight
Calculations and Correction of the Sedimentation

and Diffusion Coeffic$ents to Water

('- Q :1 fl
1 1'3 AJI—ra I

Densities and relative viscosities of some of the solvents

 

 

Solvent 0 o/U w F20o C

Veronal buffer 1.101 1.1034
Veronal - 5 h guanidine HCl buffer 1.437 1.1215

————i

 

 

131

Correction of the Observed S to Standard Conditions

The experimentally determined sedimentation coeffi-
cient was corrected to a value corresponding to sedimenta-

tion coefficient in water at 200 C. The equation commonly

 

used is
52 . =( ” w,t) (u o,t (1 - VIOZQJw ST
0’“: Uw,20 U w,t 1 - v Pt,o

where the first term is the viscosity of water at the
emperimental tenperature relative to that at 200 C, the
second term is the relative viscosity of the solvent to
that of water, and the last term is the relative buoyancy
term containing the partial Specific volume, the density
of water at 200 C, and the density of the solution at the

CXperimental temperature.

1

Correction of the Observed DiffusionfCoefficient

to Standard Conditions

The eXperimentally determined diffusion coefficient
Imis corrected to a value corresponding to diffusion coeffi-
cxient in water at 200 C. The equation used is

D20N=(‘27%2%‘t)(%ff ('gfl‘fg DI

vfluere QT is the experimentally measured diffusion coefficient
at 'temperature t, t is the temperature of the diffusion
GDUDerdnent in degree centigrade, (Uo,t/in,t) is the rela-
tiVTB viscosity of the solvent to that of water, 0 w,t is

the ‘viscosity of water at the temperature of the eXperiment,

and I] 20 is the viscosity of water at 200 C.
,w

-‘ . - — . w w» - , vv , ‘ ‘ - ’— -

‘ko partial SUGCLfic f.in e “as calculated fror
-,T-2 ‘. . ‘r. 1_‘ _' . ,“. k , ,1 ‘A f. vc,‘g \.‘ ‘n ‘ L ‘Q -
‘J.'IO 8.0a 1- 1‘83 3.130 191. lit/13 Zulu- “t: ..‘CJ.4..‘-A’1D Dcl‘CEi;lt’-1 CS

f'1qr‘» 9M1 V.~ ‘75..-$- ~xv~ ‘\" ,..,t-
I,.’\’ C\.-.; l,'O...'y\-,. & VG CO ')(.1.1:‘.;L--‘

.IL

I
M
LL:

,4:

 

o -- (.-
é‘ o.
l
where v : p.rtial specific volune of the prosoix
n th
1 = partial Speci‘ic vein”: of trc i coz-;r~o::e::t
.. x;- p aw th
'1 : “ei hr of use 1 PO'hoient
a 5a3ple tatulatio f snot on tno n It pate.
i».is be .ou, 51‘, of 0.71}.'ru:<jbtainei, £3.”.,
E -,‘ .‘ 07.1-va
~V' = —~ * = on“ : D.,/‘ch

133
TABLE III

Data and sample calculation of partial Specific volume

of component 3

 

 

 

 

 

Components wi V1 V1“;
Lysine 7.31 0.82 5.99
Histidine 3.27 0.67 2.19
Arginine 0.00 0.70 2.83
Unidentified peak 1.79 0.70 1.25
Aspartic acid 6.07 0.60 3.88
Threonine 5.58 0.70 3.91
Serine 6.26 0.63 3.90
Glutamic acid 10.00 0.66 9.50
Proline 0.79 0.76 3.60
Glycine 1.07 0.60 0.69
Alanine 2.16 0.70 1.60
Unidentified peak 0.27 0.86 0.23
Valine 2.16 0.86 1.86
methionine 1.60 0.75 1.20
Isoleucine 0.82 0.90 0.33
Leucine 8.15 0.90 7.30
Tyrosine 0.70 0.71 0.53
Phenylalanine 2.28 0.77 1.76
Hexose 8.20 0.613 5.03
Hexosamine 6.00 0.666 0.00
Sialic acid 3.00 0.580 1.73

2 W1 = 90.36 2V1W1 = 67.05

 

 

130
Sedimentation Equilibrium kethod for

holecular Weight Determination

The method used in the calculation of equilibrium
weight-average molecular weight from ultracentrifugal data
is presented here. The equations involved and sample cal-
culations are shown. For a thorough and extensive treat-
ment of the theory, the reader is referred to the following
references: Schachman (1959); Tanford (1961); Williams,
Van Holde (1960); Trautman (1956); Svedberg and Peterson

(1940); and Hales (1961)-

Notation

The following symbols appear in this section:
c - Concentration in gm/ml

c - Concentration of protein at the air-liquid
meniscus

cbr- Concentration of protein at the bottom of the
solution

c° - Initial concentration

D - Diffusion coefficient
ho - Magnification of the camera lens (radial)

R - Gas constant (8.310 x 107 ergs deg"1 mole'l)
r - Distance from the center of rotation

rb - Distance from the center of rotation to the
bottom of the solution

r - Distance from the center of rotation to the
air-liquid meniscus

S - Sedimentation coefficient

T - Absolute temperature

 

<:Id'
I I

:x,
l

VThe

135
time
Partial Specific volume of solute

no 1‘

I510 rm
Radial distance between measurements on the
photographic plate

Vertical distance between the solvent and
solution pattern

Density
Angular velocity of the rotor (radians sec-1)

Example Calculation

microcomparator readings and the calculationS*

for the equilibrium weight-average molecular weight are

presented in this section. The symbol‘fin refers to the

distance from the inner reference line to the designated

position as measured by the microcomparator on the photo-

graphic plate. For the Kodel E used, r

inner = 5.72 cm at

low speeds and no = 2.103

TABLE III

Data and example calculation from a Short-column

equilibrium pattern

 

 

 

Protein = Component 3 from heated skimmilk
Speed = 9,301 RPM Conc. = 5.0 mg/ml
Temperature = 20.00 C Buffer = Veronal-S M
guanidine-HCl
n Rn in in
0 3.032 0.361%
1 3.022 0.357 0.357
2 3.002 0.308 0.705

136

(Continuation of Table III)

 

3 2.932 0.300 1.005
0 2.962 0.332 1.377
5 2.902 0.326 1.703
6 2.922 0.320 2.023
7 2.902 . 0.310 2.337
8 2.882 . 0.307 ,2.600
9 2.862 0.301 2.905
10 2.802 x 0.295 3.200
11 2.822 0.289 3.529
12 2.802 0.233 3.812
13 2.732 0.276 0.083
10 ' 2.762 0.269 0.357
15 2.702 0.263 0.620
1 2.722 0.257 0.877
17 2.702 0.252 5.129
8 2.709 0.251*
Rb = 3.032 rb = “b + 5.72 = 7.162 rbz = 51.290
2.103
2
a = 2.709 r = Rm, _+ 5.7~ = 7.003 r = 09.112
m Ill 27103 In

 

*Values obtained by extrapolation

17
cb .. cm = TX 2 Yn, Arzem = 0.102
1.0 1

= 0.02 , _ .
I3 m 5.129 (0.142) X 1/1

= 0:02 X 20715
2,103

0.0258

0

c is obtained by similar measurements of a synthetic

boundary pattern obtained at the same schlieren diaphragm

angle, 1.8.

_ A ‘_; ‘VP - ‘
Co “ -l-’- E A ‘syn. boundary " 0‘ *051
*o

137

Other quantities necessary for the calculations are:

f9solution 1.1215
_____§2___, a (5.310 x 107) (293)
(1 - va“ (1 - 0.715 x 1.1215 0.97535 1: 10

= 1.2363 x 105

 

:- = 0.14533
"27""7T'
I‘b - I‘m

a) weight-average molecular weights for the total cell

contents is calculated from the following equation,

 

 

i‘lgpp = ? RT X 32—1-0211 X 1
<1 - 17pm)? c° rbz - rm2
= 2 (1.2068 x 105) (0.0258) (0.0533)
0.1051
(2.! 28,960

b) Z—average molecular weight for the total cell contents,

 

 

i = RT 7‘
(1-VF)W4 cb-c

 

0.1 1 _ 0.11 0
= (1.2363 x 105) ( 7.162 7.00%")
0.0253
2330.600

Calculation of Molecular weightsifrom the Sedimentation

Diffusion Data

Kolecular weights can be calculated directly from
the sedimentation and diffusion coefficients, at infinite

dilution, according to the Svedberg equation,

 

Y
. . ‘
n -

Here, R

FJ
I

F’:

for component
the molecular

is,

fitfl
., ‘

'S,D

133
RTs

D (1 - VfDS
8.310 x 107 ergS deg"1 mole".l
293°

0.0 x 10'13 sec (at infinite dilution, in
veronal buffer)

1.8 x 10'7 cmZ/sec (at infinite dilution,
in veronal buffer)

0.715
1.030 .

3 from heated Skimmilk, in veronal buffer,

weight calculated from theabove equation

(8.310 x 107) (293) (0.0 x 10'131
(1.8 x 10'?) (1 - 0.715 x 1.030)

207,000

This value agrees satisfactorily with a molecular

weight of 200,

000 of component 3, at infinite dilution, in

same veronal buffer, from sedimentation-equilibrium data.

 

 

 

H— '