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SYMPOSIUM: GENETIC PERSPECTIVES ON MILK PROTEINS:

COMPARATIVE STUD1

ES

AND NOMENCLATURE

Review and Update of Casein ChemIstryl,*

HAROLD E. SWAISGOOD

Southeast Dairy Food

Research

Center

Department of

Food

Science

North

Carolina

State

University

Raleigh 276957624

ABSTRACT

Of all food proteins, bovine milk pro-

teins are probably the most well charac-

terized chemically, physically, and ge-

netically. The primary structures are

known for most genetic variants of

a s l - ,

as2- ,

0-.

and K-caseins, &lactoglobulin,

and a-lactalbumin. Secondary and ter-

tiary structures of the whey proteins have

been determined, and secondary struc-

tures of the caseins have been predicted

from spectral studies. The caseins, al-

though less ordered in structure and

more flexible than the typical globular

whey proteins, have significant amounts

of secondary and, probably, tertiary

structure. The amphipathic structure of

the caseins is especially noteworthy;

thus, these proteins most likely are

divided into polar and hydrophobic do-

mains. The presence of anionic phos-

phoseryl residue clusters in the calcium-

sensitive casein polar d omains is particu-

larly significant because of their interac-

tion with calcium ions, or calcium salts,

or both, and the formation of micelles.

Flexibility of casein structures is

reflected by their susceptibilities to

limited proteolysis, which dramatically

changes functionality.

Received August 24, 1992.

Accepted December 16, 1992.

'Paper Number FSR92-27 of the Journal Series

of

the

Department

of

Food Science, Noah Carolina

State

Univer-

sity,

Raleigh 27695-7624.

The

use of trade names in this

publication does not imply endorsement by

the

North

Carolina Research Service of products named or criticism

of similar ones not mentioned.

2Support for the

presentation

of this symposium papcr

was

paaially provided by

che

California

Dairy Foods

Research Center.

(Key words: casein chemistry, milk pro-

teins, nomenclature, symposium)

Abbreviation

key:

FPLC

= fast protein liquid

chromatography.

INTRODUCTION

A great opportunity for increased utilization

of milk rests with increased use of milk pro-

teins as highly functional food ingredients.

Functional properties are directly related to the

physicochemical characteristics of the protein.

Optimal functionality requires different phys-

icochemical properties for specific functions,

such as water binding, gelation, emulsification,

and foaming. Therefore,

an

understanding of

the

relationship between structure and func-

tionality will be essential for selection of in-

gredients and ultimately for design of function-

ality by bioprocessing operations or genetic

modification. Many of the physicochemical

characte ristics of individual milk p roteins are

now known, and much of the current research

is focused on understanding the protein inter-

actions that occur among themselves, with

other proteins, and with other food ingredients.

The brief review presented herein summa-

rizes some areas of milk protein chemistry that

provide the basis fo r future investigation of the

relationship between structural and phys-

icochemical characteristics and functionality.

A number of reviews have appeared previously

(6,

50, 51).

Therefore, the present article at-

tempts to summarize several fundamental

characte ristics and to review a few m ore recent

studies.

IDENTIFICATION. COMPOSITION,

QUANTITATION, .AND

ISOLATION

OF CASEINS

This review uses the revised nomenclature

recommended by the ADSA milk protein

1993

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3056 SWAISGOOD

quence and by cDNA or genomic DNA se-

quences. The latest corrections have been

presented in a recent review (51). These revi-

sions allow more accurate compositions and

characteristics

to

be calculated. Composition

for the major variants of the four individual

caseins is listed in Table 1. The unique

fea-

tures of these compositions compared with

those of typical globular proteins

are

the pres-

ence and numbers of phosphoseryl residues

and the high frequency of prolyl residues. Be-

cause of absence of cysteinyl residues

in asl-

and &casein, these proteins cannot participate

in sulfhydryl-disulfide interchange crosslinking

reactions. Accurate compositions also permit

calculation of a number of physicochemical

parameters, such as the molecular charge

characteristics given in Table 2. Because the

tertiary structures of caseins are apparently

more flexible than those of typical globular

proteins, calculated values agree well with

those measured experimentally (Table 2). For

example, their rates of electrophoretic migra-

tion and order of ion-exchange chromato-

graphic elutions, with a few exceptions, are

consistent with their net charges.

A unique feature of the primary structures

of caseins, which are most likely responsible

for their unique functional properties,

s

the

distinct amphipathic nature of their sequences.

These structures suggest that their tertiary

structures are organized into polar and

hydrophobic domains

(50).

Furthermore, the

polar domains of the calcium-sensitive caseins

contain anionic clusters comprising phos-

phoseryl residues, but the polar domain of K-

casein, although strongly anionic, does not

have phosphoseryl residue clusters. In addition

to determination of the solubility in the pres-

ence of calcium ion, t h i s structural characteris-

tic is undoubtedly responsible for the unique

interactions with calcium salts that define

micelle structure.

Secondary

Structure8

Because a direct observation of secondary

structures of caseins by X-ray crystallography

TABLE 1. Chemical composition of the

commonly

Occurring caseins

(CN).

a,l-CN Q s 2 - a K-CN &CN

Acid B-8P A-1 lP B-1P AZ-5P

ASP 7

4 3 4

Asn 8

14 8 5

Thr

5

15 14 9

Ser 8 6 12 11

SerP 8

11 1 5

Giu 25 24 12

19

Gln 14 16 14 20

Pro

17 10 20 35

9 2 2 5

Ala 9 8 15

5

GlY

Half

Cys

0

2 2 0

Val

11 14 11 19

Met

5 4 2 6

Ile

11 11 13 10

Leu

17 13

8

22

5 r

10 12 9 4

Phe

8 6

4

9

Trp

2 2 1 1

LYS

14 24 9 11

His

5 3 3 5

6 6 5 4

0

0

1

0

Arg

Pyr

or

Glu

Total

residues 199 207 169 209

Molecular weight 23,623 25,238 19,006 23,988

H kJ

per

residue2 4.89 4.64 5.12 5.58

'Based on their

primary structures.

ZAverage values for

individual

residues taken from Bigelow

5).

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SYMPOSIUM: GENETIC PERSPECTIVES ON MILK PROTEINS

3057

TABLE 2. Physicochem ical chara cteristic s of casein

(CN)

calculated from composition.

ch rgt

at

Isoionic

protein

pH 6.61

P*

as

-CN

A-8P -21.0 4.94

B-8P -21.9 4.94

C-8P -20.9 4.97

D-9P -23.5 4.88

A-1OP -12.2 5.45

A-11P -13.8 5.37

A-12P -15.5 5.30

A-13P -17.1 5.23

A3-5P -13.8

5.07

A2-5P -13.3 5.14

A1-5P -12.8 5.22

B-5P -11.8 5.29

C-4P -9.2 5.46

A-1P -3.0 5.61

B-1P -2.0 5.90

aS2-CN

8-CN

K-CN

'Calculated using

the

rela tion ZH

=hh

, where

f =

ci

(IljkjhHY(1 + k,hH). and ZH s the net p t O ~ C

charge. The apparent pKj used were a-carboxyl, 3.6 (41);

phosphoseryl, pK1 = 1.5, pK2

=

6.4 (10); 8-, y-carboxyl,

4.9 (23); histidyl. 6.6 (41); and a-amino, 7.4 (41) exce pt for

x-casein, where the

more

common value for

b-

y-carboxyl

of 4.6 w as used (41). If

an

apparent pK of 4.9

is used,

the

isoionic point is 5.84 for

K-CN

A and 6.06 for K-CN B.

T a lcd a ted

as

for charge at pH 6.6, but

Z

0

at

the

isoionic pH.

is not possible, the amount of the various

structures in caseins has been estimated from

measurements using various spectral tech-

niques and using algorithms to predict secon-

dary structure from the established primary

TABLE 3. Secondary structure of the caseins o.

structures (Table 3).These results suggest that

the Erequent statem ent, that ca seins lack secon-

dary

structure, is incorrect (50).Furthermore,

prediction methods suggest that several struc-

tural motifs may

be

present [e.g., YO structure

in the hydrophobic domain of K-casein (45) nd

a turn-8-strand-turn motif in the chymosin-

sensitive region

(28)].

Also, the calcium-

sensitive caseins may contain helix-loop-helix

motifs centered on the sites of phosphorylation

28).

Tertiary Structures

Obviously, the three-dimensional structures

of the caseins re not known because caseins

apparently are not crystallizable. Nevertheless,

recent attempts have been made to predict the

tertiary struc tures of caseins from their primary

structures by molecular modeling (33, 34).The

predicted structures are compatible with the

gross features of division into hydrophobic and

polar domains as predicted from their am-

phipathic primary structures and their phys-

icochemical properties (50). The predicted

structures suggest that the hydrophobic do-

mains of individual caseins may interact

through formation of extended secondary

structures leading to formation of submicelles.

Physicochemical properties indicate that the

tertiary structures of these proteins are more

open and flexible than tho se

of

typical globu lar

proteins (50). Greater flexibility than that of

typical globular proteins is also indicated by

analysis of proteolysis rates

(9, 52).

The high

frequency of prolyl residues may provide an

kquence prediction

CD spectra Raman spectra

Prokin a-Helix Structure 8-Turn a-Helix Structure a-Helix &Structure

Turn

31(35) w 35 ) 14 (35) 31 (35)

17(45) 1445)

m 33 ) 37(33)

2x34)

ll(1 1) 12.20 (11) 0 (11) 7-13

(8)

19-22 (8) 23-35 (8)

1x 1) 1-11 (1)313-16 (ly

7.3 1 1) 45(34) 13.20

(8)

17

(8)

13

(8)

20 (8)

34

(8)

20-26(22) 15-31(22) 7-10 (22) 17-33 (22)

'Circular dichroism.

2Numbers in parentheses

are the

reference

numbers.

30btained by analysis of optical rotatory dispersion

data.

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3058 SWAISGOOD

architectural stiffness, yielding an overall open

structure with typical flexibility around in-

dividual residues and a rapidly fluctuating

secondary structure. Thus, caseins may exhibit

a higher structural motility than typical globu-

lar proteins.

PRODUCTS

OF

LIMITED PROTEOLYSIS

OF

CASEINS

Because the structures of caseins are not

random coils

of

completely flexible chains, a

certain number of susceptible residues are

more rapidly hydrolyzed.

As

expected, these

residues occur in the most flexible regions of

the protein structure, which usually represents

residues in turns or regions between hydropho-

bic and polar domains (52). The structure of j3-

casein apparently is particularly open and flex-

ible in the region between the N-terminal polar

domain and the C-terminal hydrophobic do-

main. Thus, limited proteolysis by the natu-

rally occurring proteinase plasmin (18, 19)

results in the presence of y-caseins and

proteose-peptones in normal milks (Table 4).

Being derived from the hydrophobic domain,

y-caseins are extremely nonpolar and can be

extracted in organic solvents (46).However,

the proteose-peptones resulting from the polar

domain are highly charged and very heat

sta-

ble.

The ancient

art

of cheese making is based

on the liberation of the polar domain of K-

casein by cleavage of an apparently especially

flexible and exposed peptide bond joining the

hydrophobic and polar domains of this mole-

cule. The resulting loss

of

charge and increase

in

surface hydrophobicity lead to coagulation

of micelles. Upon further proteolysis by

chyrnosin during curd maturation, a flexible

region between the hydrophobic N-terminal

domain of a,l-casein and the polar domain is

hydrolyzed, yielding cusl-casein(f25-199) (12,

32). The increased hydrophilicity of this large

C-terminal peptide is correlated with changes

in the rheological characteristics of the curd

(12).

More extensive proteolysis of the calcium-

sensitive caseins by neutral or alkaline pro-

teinases frequently produces bitter peptides

(37, 39, 49). Such peptides usually are derived

from the C-terminal, 14-residue sequence of 0-

caseins (Table 4). Bioactive peptides derived

by in vivo digestion of the calcium-sensitive

caseins also have been obtained in recent

studies (36,

38, 60,

61).

CASEIN INTERACTIONS WITH CALCIUM

Casein interactions with calcium ions and

calcium salts (colloidal calcium phosphate) are

necessary for formation and maintenance of

casein micelles. The anionic clusters of phos-

phoseryl residues are the primary sites of cal-

cium binding (24, 26). Consequently, solubility

TABLE 4.

Some peptides or domains derived from casein (CN) y limited protealysis.

Enzyme Peptide or

domain

Functionality

8-CN X(f29-209) Hydrophobic

6-CN X(fl06-209) Hydrophobic

CN

X(fl08-209)

Hydrophobic

Roteose-peptones

8-CN X(fl-105 or

107)

8-CN X(f29-105 or 107)

8-CN X(fl-28)

Plasmin 7-CN

Heat stable, very soluble

Heat stable, very soluble

Heat stable, very soluble

Chymosin

Chymotrypsin

Para-K-CN; K C N X(fl-105)

Macropeptide; K-CN X(fl06-169)

asl -CN

I;

~ ~ 1 - 0 4C(f25-199)

B 0 4

X(fl-52) Possible amphipathic helix

Hydrophobic, low solubility

Very

polar,

very soluble

Increased hydrophilicity

Trypsin (more extensive hydrolysis)

Peptides

from

the region of

B-CN

Bitter peptides

X(fl96-209)

Pepsin, intestinal enzymes, or

both

Peptides from the region of CN

Opioid activity

X(f60-70)

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SYMPOSIUM:

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of the isolated proteins

as

a function of Ca2+

concentration is correlated with the number of

these clusters per molecule; thus, the

order

of

solubilities is c~~2-c~~1-c

-c

K-casein with

3,

2,

1,

and 0 clusters, respectively (2, 50, 56). A

common cluster sequence is SerP-SerP-SerP-

GluGlu. Glutamyl residues are a part of each

of the cluster sequences and are likely to be

an

integral part of the anionic site-binding cluster.

Studies of individual caseins (14, 15, 16, 21,

42, 43, 44) and of casein submicelles (30)

showed that more Ca2+ was bound than the

expected number of phosphoseryl residues

would indicate. Also, direct evidence of car-

boxylate participation has come from infrared

spectra (7, 42).

Analysis

of

Ca2+ binding to a,l-casein has

suggested several phases in the binding

equilibria as Ca2+ concentrations increased.

Thus below 1 mM Ca2+, exothermic binding

(27)-primarily to phosphoseryl residues (42)

accompanied by transfer of aromatic residues

from an aqueous to an apolar environment

(42)-s the major equilibrium. Between 1 and

3mMCa2+, a second exothermic phase occurs,

followed by an increasingly endothermic reac-

tion (27) with initiation of binding to carboxy-

late residues

(42)

and increasing self-

association (17, 27). Finally, above 3 mM

Ca2+, the reaction is very endothermic

(27);

binding is primarily to carboxylate residues

42),

accompanied by increasing aggregation

(27), and eventually leads to precipitation be-

tween 5 to

6

mM Ca2+ at the usual protein

concentrations. At a molecular level, these

results are interpreted to suggest that initial

binding causes conformational changes with

increased exothermic H-bonding, perhaps with

formation of extended @-sheet tructures, even-

tually leading to endothermic intermolecular

hydrophobic interactions. Continued binding to

carboxylate residues further reduces inter-

molecular electrostatic repulsion, and

hydrophobic interaction of the hydrophobic

domains leads to formation of large ag-

gregates.

The effects of pH, temperature, and ionic

strength on casein solubilities and on the sta-

bility of micelles are consistent with the effects

on the ch aracteristics of the binding equ ilibria

observed with individual caseins

(4, 14,

44)

and, more recently, with casein submicelles

see

Table 5) (30). Increased temperature in-

creases the affinity, and increased pH increases

the affinity and the number of sites; increased

ionic strength decreases the affinity. Similarly,

the solubilities of caseins in the presence of

Ca2+ decrease w ith increasing temperature and

pH and increase with ionic strength (14, 21,

44). The affinities for Ca2+ (Table

5

for sub-

micelles are slightly higher than those previ-

ously reported for individual caseins, perhaps

because the submicelle-bindin equilibria were

characterized below 1mM C$+ at which only

the highest affinity binding occurs

(30).

Alter-

natively, the structure of anionic clusters in

TABLE

5.

Effect of

pH

and temperature

on the

equilibrium dissociation

constant

and the

total

45C a bound in

the

analytical affinity column

of

immobilizcd

casein

submicelles.

Ca2+

' c a p 0 4 2 SMUF3

cc

w rtml) w

( W O I )

w OlmOl)

6.0

20 680 .46 670

.36

60

.43

6.7 20

88

.52 96 .61 30 .56

6.7 30 70

.44 78

.48 16 .59

6.7

40

52

.43 60

.46

9

.57

7.5 20 .8

.65 65 .88

.9 .73

*CaClz dissolved

in 25mM

imidazole buffer.

K m

The equilibrium

dissociation

constant for

45Ca2+. M =

The

total amount

of 45Ca-binding sites

in

the column of immobilized casein submicelles (42.9

m o l

of casein*using a

molecular mass average

of

23.3

kDa.)

X a C 4 and KH2PO4 dissolved

in

25 mM imidazole buffer.

Ratio

of

PO4:Ca was

maintained at 1.29.

3Simulated

milk

ultrafiltrate (31).

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3060 SWAISGOOD

submicelles may provide more ligand interac-

tions with Ca2+.

In the presence of inorga nic phosphates and

other ions

in

milk salts, such

as

citrates, the

interactions of calcium with casein are more

complex. Such interactions are of key impor-

tance to the structure and stability of natural

milk micelles. Results from studies of calcium

interaction with submicelles

(30)

in the pres-

ence of dilute milk salts suggest that a higher

affinity of binding occurs (Table

5).

van Dijk

(57,

58) has proposed the formation of ion

clusters, including Ca2+, inorganic phosphate,

and possibly other ions that interact with the

phosphoseryl residues. Perhaps extended

clusters are formed between submicelle anionic

clusters and milk salt clusters that optimize

liganding.

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1

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Journal of Dairy Science Vol. 76. No. 10. 1993