Pages 1881-1894 MILK PROTEINS Heterogeneity, Fractionation and Isolation

MILK PROTEINS
Heterogeneity, Fractionation
and Isolation
K F Ng-Kwai-Hang, McGill University, Quebec,
Canada
Copyright 2002, Elsevier Science Ltd. All Rights Reserved
Introduction
The milk from approximately 150 of the estimated
4000 mammalian species has been analysed in dif-
ferent degrees of detail and, in all cases, this secretion
has been shown to contain a protein component
which varies from 1% in human to >20% in rabbit
milk. Berzelius, in 1814, described the ®rst method
for the separation of casein, the major protein com-
ponent of cows' milk. With the development of more
sophisticated analytical techniques over the years,
more than 200 types of protein have been char-
acterized in bovine milk. The proteins in cows' milk
are the most widely studied in terms of their isola-
tion, characterization, structural properties, functions
and biosynthetic pathways. Hence, unless otherwise
stated, the term `milk proteins' in this article refers to
the bovine species.
Milk protein is a very heterogeneous group of
molecules and, for ease of description, could be
classi®ed into ®ve main categories: caseins, whey
proteins, milk fat globule proteins, enzymes and
other miscellaneous minor proteins. Among the
several factors which contribute to the heterogeneity
of milk proteins, this article will focus on methods
used for isolation, molecular structure, degree of
posttranslational modi®cation, self-association and
association between different types of protein, dif-
ferences in the amounts and relative proportions of
individual proteins, origin of the proteins, diversity
of functions, and the presence of homologues across
species. The heterogeneity of milk proteins is further
complicated by the presence of genetic variants which
have been identi®ed in several species apart from
bovines. With the developments in molecular biology
and the improvements in cloning techniques, it is
possible to increase further the heterogeneity of milk
proteins by site-directed mutagenesis, controlling
the levels of expression of proteins indigenous to
milk and of novel proteins that are foreign to the
mammary gland.
Contents
Heterogeneity, Fractionation and Isolation
Casein Nomenclature, Structure and Association Properties
Caseins, Micellar Structure
Caseins, Functional Properties and Food Uses
Caseins, Industrial Production and Compositional Standards
Alpha-Lactalbumin
Beta-Lactoglobulin
Minor Proteins, Bovine Serum Albumin and Vitamin-Binding Proteins
Lactoferrin
Immunoglobulins
Whey Protein Products
Bioactive Peptides
Analytical Methods
Functional Properties
Protein Coprecipitates
Nutritional Quality of Milk Proteins
MILK PROTEINS/Heterogeneity, Fractionation and Isolation 1881
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The heterogeneity of milk proteins has contributed
to dif®culties associated with their fractionation and
isolation and, as we acquire more information about
themacromolecules involved, these problems are being
overcome. For more than 50 years, it was thought
that the casein fraction prepared by Hammarsten in
1883 was a pure protein. The application of moving-
boundary electrophoresis by Mellander in 1939 dem-
onstrated the heterogeneity of the casein fraction
with three electrophoretic components denoted as a-,
b-, and g-. Present-day knowledge con®rms that each
of the three components represents more than one
protein. The primary structure and gene sequence
of the four caseins (a
S1
-, a
S2
-, b-, k-) and three of
the whey proteins (b-lactoglobulin, a-lactalbumin,
bovine serum albumin) are now established. The
fractionation and isolation of protein components
depend on the intrinsic physicochemical properties of
the individual proteins. Because some of the proteins
tend to self-associate or associate with other proteins,
a denaturing reaction is required prior to the frac-
tionation stage. Techniques of ultracentrifugation,
size-exclusion chromatography, ultra®ltration and
sodium dodecyl sulphate±polyacrylamide gel elec-
trophoresis (SDS±PAGE) could be adapted for the
separation of certain proteins on the basis of mo-
lecular mass differences. Variability in net electrical
charge, sensitivity to ions, e.g. calcium, and solubility
in the presence of denaturing agents, e.g. urea, could
be exploited for the isolation of some proteins by
precipitation with different concentrations of salt
(ammonium sulphate, calcium chloride), solutions of
alcohol under different pH and temperature condi-
tions. Based on the charge and ionic strength prop-
erties of the proteins, several chromatographic and
electrophoretic procedures have been developed for
their fractionation and isolation. The most appro-
priate procedures for the isolation of milk proteins
depends on the level of purity and the amount (ana-
lytical, preparative, industrial) of protein required.
For a better appreciation of this article, some
familiarity with the nomenclature used for the milk
protein system, as proposed by a committee of the
American Dairy Science Association, is recommended.
Classification and Nomenclature of
Milk Proteins
When raw skim milk is adjusted to pH 4.6, a pre-
cipitate containing approximately 80% of the protein
of cows' milk is formed. This precipitation procedure
has been used as the basis for the classi®cation of
milk proteins into two main groups, with casein in
the precipitate and whey protein (noncasein protein)
inthesupernatant.Table1givesasummaryofsome
of the characteristics of the four types of casein and
the four major proteins in the whey protein fraction.
The primary structure (amino acid sequence) and the
gene sequences of the four caseins, ranging in mol-
ecular mass from 19 038 for k-casein to 25 388 for
a
S2
-casein, have been established. Among the major
whey proteins, a-lactalbumin is the smallest (mol-
ecular mass 14 175 Da) and the immunoglobulins are
the largest (143 000±1 030 000 Da). The complete
amino acid sequences of b-lactoglobulin, a-lac-
talbumin and serum albumin, with molecular masses
of 18 277, 14 175 and 66 267 Da, respectively, are
also known. Unlike b-lactoglobulin and a-lactal-
bumin, serum albumin and some of the immuno-
globulins are not synthesized in the mammary gland.
The immunoglobulins are extremely heterogeneous
and their identities are based on immunochemical
properties. Five classes of immunoglobulins (IgG,
IgA, IgM, IgE, IgD) have been identi®ed in bovine
milk. Their basic structure is similar to other im-
munoglobulins in that they have two heavy and two
light polypeptide chains covalently linked by disul-
phide bonds. The molecular mass varies from 50 to
70 kDa for the heavy chains, depending on the type,
and is about 25 kDa for the light chains. In addition to
Table 1 Characteristics of the major proteins in cows' milk
Protein Molecular mass
a
No. of AA residues No. of
PO
4
Presence of
CH
2
O
Concentration
(g l
ÿ1
)
Genetic variants
detected
Total Pro Cys
a
S1
-Casein 23 164 199 17 0 8 0 10 A, B, C, D, E, F, G, H
a
S2
-Casein 25 388 207 10 2 10±13 0 2.6 A, B, C, D
b-Casein 23 983 209 35 0 5 0 9.3 A
1
,A
2
, A
3
, B, C, D, E, F, G
k-Casein 19 038 169 20 2 1 3.3 A, B, C, E, F
s
, F
I
, G
S
, G
E
, H, I, J
b-Lactoglobulin 18 277 162 8 5 0 0 3.2 A, B, C, D, E, F, H, I, J
a-Lactalbumin 14 175 123 2 8 0 0 1.2 A, B, C
Serum albumin 66 267 582 28 35 0 0 0.4 Ð
Immunoglobulin 1 430 000±1 030 000 8.4% 2.3% Ð 0.8 Ð
a
Molecular mass is for genetic variants in bold.
1882 MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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differences in the organizational structure, there are
also differences in amino acid sequences and carbo-
hydrate groups present in the immunoglobulin mol-
ecules. More than 60 enzymes have been identi®ed
in milk and, as a group, they account for less than
1% of the total milk protein. Enzymes are hetero-
geneously distributed in milk, e.g. catalase, lacto-
peroxidase, ribonuclease and glucosaminidase are
found mainly in the whey fraction while proteinases
and lipase are associated with the casein micelles,
and xanthine oxidase is found in the milk fat globule
membrane. The fat globules of milk are stabilized by
a complex membrane consisting of proteins and
phospholipids; these proteins are designated as milk
fat globule membrane proteins. The major proteins
of the milk fat globule membrane were recently re-
viewed. Proteins that do not fall into the classi®ca-
tion of caseins, major whey proteins, enzymes or
milk fat globule membrane proteins are categorized
as miscellaneous minor proteins and include trans-
ferrin, lactoferrin, ceruloplasmin, lactollin, glyco-
protein-a, kininogen, M-1 glycoprotein, epidermal
growth factor, glycolactin, angiogenin, etc. A wide
range of biological functions has been assigned to
those minor proteins present at concentrations in the
mg kg
ÿ1
range.
Heterogeneity of Milk Proteins
In addition to the high degree of heterogeneity which
exists between the ®ve different classes of milk pro-
teins, there is also heterogeneity among proteins
within each class. This is to be expected when one
considers that milk proteins range from 10 to
>1000 kDa in molecular mass and have different
amino acid compositions and sequences which ulti-
mately determine the structures and physicochemical
properties of the molecules. The degree of posttrans-
lational modi®cation (proteolysis, phosphorylation,
glycosylation, formation of disulphide bridges) and
the existence of genetic variants further contribute to
the observed heterogeneity.
Caseins
Bovine milk contains four types of casein denoted as
a
S1
-casein, a
S2
-casein, b-casein and k-casein, all of
which are products of speci®c genes. However,
alkaline PAGE of whole casein preparations resolves
in excess of 20 protein bands. Several of the electro-
phoretic bands represent posttranslational products
of one of the four caseins (see below).
The caseins, which are synthesized in the
mammary gland, are proteins containing ester-bound
phosphate and due to their relatively high content of
proline(seeTable1),theytendtohaveverylittle
secondary structure. The primary structure of a
S1
-
casein, containing 199 amino acid residues, is shown
inFigure1.Ithasnocysteineresidueandeight
phosphates attached to serines. A minor a
S1
-casein
with a faster electrophoretic mobility, denoted earlier
as a
S0
-casein, has been characterized and is slightly
different from the former by having nine phos-
phorylated serines and hence contain one extra
negative charge at alkaline pH. Three hydrophobic
regions are located in the sequences residues 1±44,
90±113 and 132±199. The sequence of residues
41±80 is very polar due to the presence of seven seryl
phosphates, eight glutamates and three aspartates.
10 20
H.Arg ± Pro ± Lys ± His ± Pro ± Ile ± Lys ± His ± Gln ± Gly ± Leu ± Pro ± Gln ± Glu ± Val ± Leu ± Asn ± Glu ± Asn ± Leu ±
30 40
Leu ± Arg ± Phe ± Phe ± Val ± Ala ± Pro ± Phe ± Pro ± Gln ± Val ± Phe ± Gly ± Lys ± Glu ± Lys ± Val ± Asn ± Glu ± Leu ±
50 60
Ser ± Lys ± Asp ± Ile ± Gly ± S
j
P
er ± Glu ± S
j
P
er ± Thr ± Glu ± Asp ± Gln ± Ala ± Met ± Glu ± Asp ± Ile ± Lys ± Gln ± Met ±
70 80
Glu ± Ala ± Glu ± S
j
P
er ± Ile ± S
j
P
er ± S
j
P
er ± S
j
P
er ± Glu ± Glu ± Ile ± Val ± Pro ± Asn ± S
j
P
er ± Val ± Glu ± Gln ± Lys ± His ±
90 100
Ile ± Gln ± Lys ± Glu ± Asp ± Val ± Pro ± Ser ± Glu ± Arg ± Tyr ± Leu ± Gly ± Tyr ± Leu ± Glu ± Gln ± Leu ± Leu ± Arg ±
110 120
Leu ± Lys ± Lys ± Tyr ± Lys ± Val ± Pro ± Gln ± Leu ± Glu ± Ile ± Val ± Pro ± Asn ± S
j
P
er ± Ala ± Glu ± Glu ± Arg ± Leu ±
130 140
His ± Ser ± Met ± Lys ± Glu ± Gly ± Ile ± His ± Ala ± Gln ± Gln ± Lys ± Glu ± Pro ± Met ± Ile ± Gly ± Val ± Asn ± Gln ±
150 160
Glu ± Leu ± Ala ± Tyr ± Phe ± Tyr ± Pro ± Glu ± Leu ± Phe ± Arg ± Gln ± Phe ± Tyr ± Gln ± Leu ± Asp ± Ala ± Tyr ± Pro ±
170 10
Ser ± Gly ± Ala ± Trp ± Tyr ± Tyr ± Val ± Pro ± Leu ± Gly ± Thr ± Gln ± Tyr ± Thr ± Asp ± Ala ± Pro ± Ser ± Phe ± Ser ±
190 199
Asp ± Ile ± Pro ± Asn ± Pro ± Ile ± Gly ± Ser ± Glu ± Asn ± Ser ± Glu ± Lys ± Thr ± Thr ± Met ± Pro ± Leu ± Trp .OH
Figure 1 Primary structure of bovine a
S1
-casein B. (Reproduced with permission from Mercier JC, Grosclaude F and Ribadeau-
Dumas B (1971) Structure primaire de la case
Âine a
S1
-bovine. European Journal of Biochemistry 23: 41±51.)
MILK PROTEINS/Heterogeneity, Fractionation and Isolation 1883
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AsshowninFigure2,a
S2
-casein contains 207
amino acids. It has 10 prolines, more phosphoserines
(seeTable1)andmorelysinesthantheothercaseins,
and has two cysteines at positions 36 and 40. Several
forms of a
S2
-casein are discernible by PAGE due to
different degrees of phosphorylation which range
from 10 to 13 phosphate groups. These forms have
been identi®ed as a
S2
-, a
S3
-, a
S4
-, a
S5
- and a
S6
-casein
(a
S5
- is a dimer of a
S3
- and a
S4
-). The 13 phosphates
(12 on serine, one on threonine) are located in three
regions of the molecule: residues 7±31, 55±66 and
129±143. Among the caseins, a
S2
-casein is the least
hydrophobic with regions of hydrophobicity located
at residues 90±120 and 160±207.
The sequence of the 209 amino acids in b-casein is
showninFigure3.Itisthemosthydrophobiccasein,
10 20
H.Arg ± Glu ± Leu ± Glu ± Glu ± Leu ± Asn ± Val ± Pro ± Gly ± Glu ± Ile ± Val ± Glu ± S
j
P
er ± Leu ± S
j
P
er ± S
j
P
er ± S
j
P
er ± Glu ±
30 40
Glu ± Ser ± Ile ± Thr ± Arg ± Ile ± Asn ± Lys ± Lys ± Ile ± Glu ± Lys ± Phe ± Gln ± S
j
P
er ± Glu ± Glu ± Gln ± Gln ± Gln ±
50 60
Thr ± Glu ± Asp ± Glu ± Leu ± Gln ± Asp ± Lys ± Ile ± His ± Pro ± Phe ± Ala ± Gln ± Thr ± Gln ± Ser ± Leu ± Val ± Tyr ±
70 80
Pro ± Phe ± Pro ± Gly ± Pro ± Ile ± Pro ± Asn ± Ser ± Leu ± Pro ± Gln ± Asn ± Ile ± Pro ± Pro ± Leu ± Thr ± Gln ± Thr ±
90 100
Pro ± Val ± Val ± Val ± Pro ± Pro ± Phe ± Leu ± Gln ± Pro ± Glu ± Val ± Met ± Gly ± Val ± Ser ± Lys ± Val ± Lys ± Glu ±
110 120
Ala ± Met ± Ala ± Pro ± Lys ± His ± Lys ± Glu ± Met ± Pro ± Phe ± Pro ± Lys ± Tyr ± Pro ± Val ± Gln ± Pro ± Phe ± Thr ±
130 140
Glu ± Ser ± Gln ± Ser ± Leu ± Thr ± Leu ± Thr ± Asp ± Val ± Glu ± Asn ± Leu ± His ± Leu ± Pro ± Pro ± Leu ± Leu ± Leu ±
150 160
Gln ± Ser ± Trp ± Met ± His ± Gln ± Pro ± His ± Gln ± Pro ± Leu ± Pro ± Pro ± Thr ± Val ± Met ± Phe ± Pro ± Pro ± Gln ±
170 180
Ser ± Val ± Leu ± Ser ± Leu ± Ser ± Gln ± Ser ± Lys ± Val ± Leu ± Pro ± Val ± Pro ± Glu ± Lys ± Ala ± Val ± Pro ± Tyr ±
190 200
Pro ± Gln ± Arg ± Asp ± Met ± Pro ± Ile ± Gln ± Ala ± Phe ± Leu ± Leu ± Tyr ± Gln ± Gln ± Pro ± Val ± Leu ± Gly ± Pro ±
209
Val ± Arg ± Gly ± Pro ± Phe ± Pro ± Ile ± Ile ± Val.OH
Figure 3 Primary structure of bovine b-casein A
2
. (Reproduced with permission from Ribadeau-Dumas B, Brignon G, Grosclaude F
and Mercier JC (1972) Structure primaire de la case
Âine bbovine: se Âquence comple Áte. European Journal of Biochemistry 25: 505±514.)
10 20
H.Lys ± Asn ± Thr ± Met ± Glu ± His ± Val ± S
j
P
er ± S
j
P
er ± S
j
P
er ± Glu ± Glu ± Ser ± Ile ± Ile ± S
j
P
er ± Gln ± Glu ± Thr ± Tyr ±
30 40
Lys ± Gln ± Glu ± Lys ± Asn ± Met ± Ala ± Ile ± Asn ± Pro ± Ser ± Lys ± Glu ± Asn ± Leu ± Cys ± Ser ± Thr ± Phe ± Cys ±
50 60
Lys ± Glu ± Val ± Val ± Arg ± Asn ± Ala ± Asn ± Glu ± Glu ± Glu ± Tyr ± Ser ± Ile ± Gly ± S
j
P
er ± S
j
P
er ± S
j
P
er ± Glu ± Glu ±
70 80
S
j
P
er ± Ala ± Glu ± Val ± Ala ± Thr ± Glu ± Glu ± Val ± Lys ± Ile ± Thr ± Val ± Asp ± Asp ± Lys ± His ± Tyr ± Gln ± Lys ±
90 100
Ala ± Leu ± Asn ± Glu ± Ile ± Asn ± Glu ± Phe ± Tyr ± Gln ± Lys ± Phe ± Pro ± Gln ± Tyr ± Leu ± Gln ± Tyr ± Leu ± Tyr ±
110 120
Gln ± Gly ± Pro ± Ile ± Val ± Leu ± Asn ± Pro ± Trp ± Asp ± Gln ± Val ± Lys ± Arg ± Asn ± Ala ± Val ± Pro ± Ile ± Thr ±
130 140
Pro ± Thr ± Leu ± Asn ± Arg ± Glu ± Gln ± Leu ± S
j
P
er ± Thr ± S
j
P
er ± Glu ± Glu ± Asn ± Ser ± Lys ± Lys ± Thr ± Val ± Asp ±
150 160
Met ± Glu ± S
j
P
er ± Thr ± Glu ± Val ± Phe ± Thr ± Lys ± Lys ± Thr ± Lys ± Leu ± Thr ± Glu ± Glu ± Glu ± Lys ± Asn ± Arg ±
170 180
Leu ± Asn ± Phe ± Leu ± Lys ± Lys ± Ile ± Ser ± Gln ± Arg ± Tyr ± Gln ± Lys ± Phe ± Ala ± Leu ± Pro ± Gln ± Tyr ± Leu ±
190 200
Lys ± Thr ± Val ± Tyr ± Gln ± His ± Gln ± Lys ± Ala ± Net ± Lys ± Pro ± Trp ± Ile ± Gln ± Pro ± Lys ± Thr ± Lys ± Val ±
207
Ile ± Pro ± Tyr ± Val ± Arg ± Tyr ± Leu.OH
Figure 2 Primary structure of bovine a
S2
-casein A. (Reproduced with permission from Brignon G, Ribadeau-Dumas B, Mescier JC,
Pe
Âlissier JP and Das BC (1977) Complete amino acid sequence of a
S2
-casein. FEBS Letters 76: 274±279.)
1884 MILK PROTEINS/Heterogeneity, Fractionation and Isolation
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has no cysteine, and a high proportion of proline (35
residues), which has a profound effect on its struc-
ture. At milk pH, the N-terminal 21-residue segment
is highly negatively charged while the rest of the
molecule, which is very hydrophobic, has no net
charge. The amphipathic nature of the b-casein
molecule is the reason why it forms micellar ag-
gregates in solution. Beta-casein has been shown to
consist of six proteins with an identical amino acid
sequence, but with 0±5 phosphate groups attached to
serine residues. The g-caseins, which have been
known for a long time, are the hydrolytic products
of b-casein, produced by the action of plasmin.
Gamma-caseins corresponding to residues 29±209,
106±209 and 108±209 of b-casein are present in
the precipitate during isoelectric precipitation of
whole casein at pH 4.6. Other fragments of b-casein
(residues 1±28, 1±105 and 1±107) are found in the
whey and they constitute part of a fraction formerly
known as `proteose peptone'.
Kappa-casein is the only protein of the casein
family that is glycosylated. The different degrees of
glycosylation, as revealed by up to seven bands
during PAGE, are related to the number of negative
charges on the N-acetylneuraminic acid residues. The
primary structure of the carbohydrate-free portion of
the 169 amino acid-containing protein is shown in
Figure4.Kappa-caseinisthetargetforchymosinand
it is the most extensively studied milk protein. It
stabilizes casein micelles against calcium precip-
itation and loses this protective role when the
Phe105±Met106 bond is cleaved by enzymes to form
two peptides: para-k-casein (residues 1±105), which
is the hydrophobic portion and precipitates with the
casein micelles, and caseinomacropeptide (residues
106±169), which remains in solution due to its high
polarity and high content of negative charges. The
glycosyl moieties are bound to the caseinomacro-
peptide by O-glycosidic linkages with threonine at
positions 131, 133, 135 or 142. The two cysteine
residues of k-casein are located at positions 11 and
88 and the serine residue at position 149 and some-
times that at position 127 is phosphorylated. The
amphipathic character of k-casein encourages it to
form micelles in solution. Unlike other caseins, k-
casein does not bind calcium extensively and is not
sensitive to precipitation by Ca
2
.
Heterogeneity of the caseins also arises from their
interactions with one another and with other pro-
teins and small ions. With the calcium phosphate
present in milk, the caseins exist as micelles.
Whey Proteins
The whey protein fraction, which accounts for ap-
proximately 20% of total protein, comprises the
noncasein proteins that remain soluble when caseins
have been isoelectrically precipitated at pH 4.6.
Whey proteins are an even more heterogeneous
group of compounds than the caseins and share few
common characteristics, other than being soluble
under conditions that render the caseins insoluble.
Unlike the caseins, which lack secondary structures,
the whey proteins have more organized secondary
and tertiary structures and most are globular pro-
teins. Four major proteins, denoted as b-lactoglobulin,
a-lactalbumin, serum albumin and immunoglobu-
lins,asshowninTable1,accountfor>95%ofthe
noncasein proteins. Both b-lactoglobulin and a-lact-
albumin are synthesized in the mammary gland
whereas serum albumin is transported to the
mammary gland via the blood serum.
10 20
PyroGlu ± Glu ± Gln ± Asn ± Gln ± Glu ± Gln ± Pro ± Ile ± Arg ± Cys ± Glu ± Lys ± Asp ± Glu ± Arg ± Phe ± Phe ± Ser ± Asp
30 40
Lys ± Ile ± Ala ± Lys ± Tyr ± Ile ± Pro ± Ile ± Gln ± Tyr ± Val ± Leu ± Ser ± Arg ± Tyr ± Pro ± Ser ± Tyr ± Gly ± Leu ±
50 60
Asn ± Tyr ± Tyr ± Gln ± Gln ± Lys ± Pro ± Val ± Ala ± Leu ± Ile ± Asn ± Asn ± Gln ± Phe ± Leu ± Pro ± Tyr ± Pro ± Tyr ±
70 80
Tyr ± Ala ± Lys ± Pro ± Ala ± Ala ± Val ± Arg ± Ser ± Pro ± Ala ± Gln ± Ile ± Leu ± Gln ± Trp ± Gln ± Val ± Leu ± Ser ±
90 100
Asp ± Thr ± Val ± Pro ± Ala ± Lys ± Ser ± Cys ± Gln ± Ala ± Gln ± Pro ± Thr ± Thr ± Met ± Ala ± Arg ± His ± Pro ± His ±
110 120
Pro ± His ± Leu ± Ser ± Phe ± Met ± Ala ± Ile ± Pro ± Pro ± Lys ± Lys ± Asn ± Gln ± Asp ± Lys ± Thr ± Glu ± Ile ± Pro ±
130 140
Thr ± Ile ± Asn ± Thr ± Ile ± Ala ± Ser ± Gly ± Glu ± Pro ± Thr ± Ser ± Thr ± Pro ± Thr ± Ile ± Glu ± Ala ± Val ± Glu ±
150 160
Ser ± Thr ± Val ± Ala ± Thr ± Leu ± Glu ± Ala ± S
j
P
er ± Pro ± Glu ± Val ± Ile ± Glu ± Ser ± Pro ± Pro ± Glu ± Ile ± Asn ±
169
Thr ± Val ± Gln ± Val ± Thr ± Ser ± Thr ± Ala ± Val.OH
Figure 4 Primary structure of bovine k-casein B. (Reproduced with permission from Mercier JC, Brignon G and Ribadeau-Dumas B
(1973) Structure de la case
Âine k b bovine: se Âquence comple Áte. European Journal of Biochemistry 35: 222±235.)
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