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A COI"i?AI?ATIV:G S'l'UDY OF TH:S CJ�.S£IN I·liCELL:SS OF CAI-'RINE, OVI NE iU'ID BOVIN:E I1ILKS

A thes i s pr8sented in partial fulfilment

of the requirem ents for the degree of

Doc tor of }'hi losophy in rhys iology a-t

I'Ias s ey University.

Barry Charles Hichards6n

Novemb er , '1974

ll

Several oifferent hypotheses of casein micelle st ructure

hav8 been pro pos eQ from the data obt2ined in the investi�utions

carried out on micelles over the past twenty years. The present comparative st udy of c�princ (�oat) and ovine (sheep)

casein micelle sy:::otems v1ith that of the bovine 1:.'as made i?:l an

attempt to demonstrate the validi t�r of t;hos c ll;ypothcs cs.

The casein contents, minero.l levels, o_nd Ca:phosphate

ratios in the skim milks �aried between species, but no

significant differences in the Ca:phosphate r�tios in the

casein micelles or the sera were found. There were major dif-

ferences betv;een the electrophoretic p2tterns of the casein

saoples from the different sneci es. Both caprine and ovine

caseins cont�ined two prominent bands in the � -ca sein re gion ,

v1hile ca-orine casein nlso contained ;:1 snmller proportion of its

casein in more mobile components (ao-caseins) than the bovine. �)

Three major �-caseins were observed in ovine casein, and one

major a -casein in the mo_jorit;y of the caprine casein samy;les. s

Bovine, caprine a_nd ovine cuseins, isol ated from the

whole milks, were separated int o th eir major components by

ion exchane;e chromatography. The K-, (J-, c-J.nd a -cas eins s

which were isolated accounted for 15;j, 35;� and 50)j , respect-

ively of bovine casein, 10;;, 60'); and 2:;%, respectively of capri:te casein and 10�), Lt-5% and 35�;,s, res pect ively of ovine

cas ein . The caprine and ovine cas cins were identified as

�-, �- and K-type caseins by their chemical and physical

characteristics.

11he bovine, caprine and ovine K-cas eins v:ere readily

hydrolysed by rennin and were able to stabilize the Ca sensi­

tive as- or ,8-cas eins. Caprine 131- and �2-cas eins which •.-:ere

present in equimolar amounts, had nearly identical amino acid

compositions, and Here similar to the.t of bovine t3-casein A2• The ,e1 component, however, contained an additional phosphate

residue.

11he temperatm'e dependent Ca sensitivities and the

temperature dependent polymerization of the cuprine � -ca s eins

were similar to that of bovine t3-cas ein. Hov1ever, � -casein

J..lJ..

app ear e d to as s ociat e more easily than ��-casein , des pi t e c

i t s higher net negative charge . Visc os ity measurement s indicated that the conformation of the two caprine �-caseins was s imilar t o that of b ovJ..ne �-cas ein , b o th at 4°C and at 25°C .

The maj or caprine a -cas ein was more s imi lar t o the s minor b ovine as3-cas e in than to bovine as 1 -cas ein. This was demons trat ed by their b eb.avi our on gel e l ectrophore s i s with Mg b uffers , C a s ens i tiviti es , amino aci d compos i t i ons a�d m o lecular wei ght s .

The two ovine �-cas eins were s imilar t o each other , and t o the caprine and b ovine �-cas eins . The relationship b etween the ovine �-cas eins was s imi lar to that of the caprine �-cas eins . Two of the thrE.e maj or ovine a -cas eins s were i s o l at e d . Thes e were both s imilar t o e ach o ther , and t o b ovine as1 -cas ein .

The proper t i e s o f t h e a -cas eins from the thr e e s peci e s s were mor e vari ab l e than tho s e of the f1-cas eins, which in turn were more variab l e than the K-cas e ins .

The cas ein mice l l es from caprine , ovine and bovine milks wer e all highly s o lvat ed and roughly s pherical . Alth ouc;h their s i z e distribut i ons varied , the micel l es were in each cas e compos ed of sub-unit s ab out 1 2 nm in diamet er . Gel chroma tography o f s ub -mice l l ar cas e in aggregat e s from the three s peci es indicat ed that they had s imi lar hydrodynamic s i z es and appear e d to b e in equi l ibrium Hith th eir component cas eins .

The finding that s imilar s ized sub-unit s ar e ·formed in the cas e in micel l es in the milks of the various s p eci es , s ugges t s that the di ffering proport i ons of the vari ous component cas eins must compens at e for the differ ences obs erv e d in t h e proper t i e s o f t h e cas eins . I t appeal'S l ikely that n o t enough emphas i s has b e en given t o the rol e of �-cas ein in micel l e s truct ur e , and �- and as -cas eins may b e int er­changeab l e .

I am grateful to the'fol lowing:

Professor H. E. I'1unford, for supervision and for the

collection of samples of sheep milk.

iv

Dr L.K. C reamer, ·N.l. Dairy Research Institute, for

guidance and continuing interest, and for obtainin� the

el ectron rnicror;raphs used in this thesis ( F.'igs .4.2 and 4.3 ) .

Dr K.N. Fearce, N.Z. Dairy Research Institute, for

measuring the cation concentrations and activities in the

milks.

Analytical Chemistry IJaboratory, N. 6. Dairy Hesearch

Institute, for Kjeldahl nitrogen determinations.

I"Ir B. Le ask, 'l'e Arakura, for supplying the goat milk.

Mrs A. Halligan, N.Z. Dairy Research Institute, for

assistance with the diagrams.

Mr C.K. Drew, r-lrs P.A. Leyland, l'iiss G . F . Evans and

Mr A.R. Matheson, N.3. Dairy Research Institute, for their

technical assistance at different p€�iods throughout the

course of the work.

I'Ty wife, Joy, for her considerabl e assistance during

the preparation of this thesis.

N .Z. Dairy Research Institute, for employment and the

use of the facilities during the course of the work.

.

'l'ABL.t.; 01•' C G.l�'l'LJ:LrS

CHAPl'ER 1 G:c.'NE.HAL INTHOlJUCTION

1 . 1 1 . 2

1 . 3

C omposi tion of Milk

The CG.seins

Aim of the Investigation

PAHT I A COI"'lf'A:RISON ·o:l!' CAPHI NE, OVI NE AND BOVINE MILKS Al�D 'rJ:fEIH CASEIN MICELLES

CHAPTER 3

2.1 2.2

2 . 3

C omposi t i on of Milk Size Dis tribution of Casein Mic ell es

Mol ecular Wei�1t of C as ein Mic elles

2.4 Voluminos ity and Solvat i on of

Cas ein Mic el l e s 2.5 Temperatur e Dependent Dis s oc i at ion

of Cas ein Mic el l es

EXFCRIMENT ... �L

3.3

Pr eparat ion of Skim l'1ilks

Nitrogen Distribution

InorGanic Phosphat e Det erminati on

3.4 Det ermination of Cat i on C onc entra­

t i ons

3-5 3 . 6 3-7 3 . 8 3 . 9

Cat i on Act ivity

C itric Ac id C ont ents El ec trophores is

Fre e z e- etch El ec tron MicroGraphs \mo l e Mic e l l e Electron Mic ro graphs

3 . 1 0 Solvation o f Cas ein Mic el l e s :

Ultrac entrifugat i on

3 . 1 1 Solvat i on o f C as ein Mic el l es :

Page No .

1

1 1 4

7

8

8

9

1 1

1 1

1 2

1 4

1 4 1 4 1 5

1 5 1 5 1 6 1 6 1 6 1 6

1 7

Vis c os ity Measur ements 1 8 3 . 1 2 Temperatur e Dependent Dis s oc i at i on

of C as e in Mic ell es

3 . 1 3 Gel Chromatography of C�s ein

Mic e l l e s at 6°C and 37°C

20

Table of Cont2nts ( Contd.)

CHAPTER 4 CONPOBJI:PION OF C/JRINE, OVIN� AND

BOVIN:t; I'1ILKS

4 . 1 Nitrogen, Inorganic Phosphorus and

Citric Acid Contents

4 . 2 Cation Concentrations and Activities

Electrophoresis of the Caseins

Page No.

23

23 25 28

A COI'1PAR IS ON OI•' T HB PHOl-l�HTIES 01? THE CASEIN I''IICELLES 28

CHAPI'ER 5

4 . 4

4 . 6

4 . 7 4 . 8

Electron Micrographs of Intact

T'Iicelles

Electron Micrographs of Freeze-etch

I?.eplicas

Ultracentrifugation of Casein

I1icelles

Ca and Phosphate Distribution

Discussion

'fEI"ll-).t!:R,V11UHJ� DBFENDEJ:f'r IJRCPEH'l'IBS CF C J0SEI N

l'UCELLES

Ultracentrifugation

Voluminosity and Solvation of Casein

I'1icelles

5 . 3 Gel Chromatography of Bovine, Caprine

and Ovine Casein Micelles

5 . 4 Discussion

PART II ISOLATION AND CHARACTERIZATION OF THE I'1AJOR

CAFHINE AND OVINE CAS:EINS AND A COJ."lPARISON 1./ITH

THE BOVINE C ASEINS

CHAPT:ER 6 IN'rRODUCTION

BOVINE CASEINS

6 . 1 Historical

6 . 2 Identification of the Major Casein

Fractions

28

30

30 32 34

37

37

39

47

54

55

56

56

56

57

Tab l e of Cont ents ( C ontd . )

�;-CASEIN

.8-CASEI N

a -CASEINS s

6. 3

6 .4

6 .5

6 .6

6 .7

Genetic Variant of Bovine C as eins

I solation of �-Cas ein

Phys ical Properti es of K-C as ein Act i on. of Rennin on K-Cas ein

Nature o f the Carbohydrat e Residues

in K-C as ein

6 . 8 Amino Ac id C ompositiJn and Primary Struc ture of K-Cas ein

Page No .

58

61

61

6 3

6 3

64

64

68

6 .9 I s olation of #-Ca s e in 66

6 .10 Physical Properti es of /3-Cas eins 68

6 .11 Amino Ac i d Compos ition and Sequenc e of /3-C as ein 72

6 .12 �-Caseins 74

6 .13

6 .14

6 .15

6 .16

6 .17

I s o lat ion of as1 -Cas eins

Physical Properties of a -Cas e ins s Amino Aci d C ompos ition and Primary

Structur e of as 1 -C as eins

I-linor a -Cas eins s Homol o gi es Within the C as eins

6 .1 8 A Pos s ib l e Mechanism of Cas ein Fhosph0rylation

6 .19 Phylogenetic R e l at i onships of the Bovine Cas eins

75

75

'75

78

80

81

81

83

CASEINS FROM OTHER SPECIES 87

6 .20 C aprine Cas eins

6 .21 Ovine Cas eins

6 .22 Buffalo Cas eins

6 .2 3 Human C as eins

6 .24 Oth er Cas eins

87

89

9 3

93

96

Table of Cont.o�ts (Contd. )

EXrERI I'1ENrl'AL

7 . 1

7 . 2

7.3

7 • Lj.

7 -5

7.6

7 -7

7 . 8

7. 9

Preparation of Acid Caseins

Polymorphism in Caprine nnd Ovine

Casoins

Chromatography of Casein on DEAE-

Cellulose

Chromatography of Casein on CM-

Cellulose

Polyacrylamide Disc Gel Electro-

phoresis

Polyacrylamide .Slab Gel Llectro-

phoresis

Gel Electrophoresis in Buffers

Containing I'lg

�rotcolytic Degradation of Caseins

with Hennet

Holecular .Jeight of Caprine

K-Casein

7 . 1 0 f'Tolecular ·,/eight Ang_lysis in 6fi

Guanidine.HCl

Page No. 9 7

9 7

98

98

99

99

1 01

1 01

1 04

1 0 5

1 06

7 . 1 ·1 �lln.ino .:kid Compositions 1 08

7.1 2 �hosphorus 1 1 0 7.1 3 :t.xtinction Coefficients 1 1 0

7.1 4 Viscosity f'1easurements on Co.prine

and Bovine P-Caseins 1 1 1

7 . 1 5 Sedimentation Coefficients 1 1 3

7.1 6 Calcium Sensitivity of Caprine,

Ovine and Bovine Caseins 1 1 3

7 . 1 7 Temperature Dependent Polymeriza-

tion of p-Casein 1 1 4

CASEIN COl"lPOSITION AND ISOLA'riON OF THE

Cm'lEONENTS

8 . 1 Chromatography of \·ihole Bovine

Casein on DEAE-Cellulose

8.2 Composition of Bovine Casein

1 1 6

1 1 6

1 1 6

Table of Contents (Contd. )

8.3

8.4

8.5

8.6

8.7

8.8

Chromatography of ,,/hole Caprine

Casein on DE/LE-Cellulose

Composition of Caprine Casein

Chromatography of \Jhole Ovine

Casein on DElLE-Cellulose

Coriposition of Ovine Casein

Polymorphism in the Caprine Caseins

Polymorphism in the Ovine Caseins

CHAPTER 9 CHARACTERL�N.riON OF CAPRINE CASEINS

CAPRINl� K-CASEIN

9.1

9.2

Purification of Caprine K -Casein

Molecular Weight of Caprine

K-Cnsein

9.3 Amino Acid Composition of Caprine

9.4

CAFRINE �-CASEINS

9.5

9.6

9-7

9.8

CAPRINE a -CASEIN s

9.9

9.10

9.11

9.12

K-Casein

Discussion

Isolation of Caprine �1

- and

�2-Caseins

Molecular Weights of Caprine

t3-Cas eins

Amino Acid Compositions of the

Caprine �-Caseins and Bovine

�-Casein B Discussion

Isolation of the a -Caseins s

Molecular Weights of the

a -Caseins s Amino Acid Compositions of the

a -Caseins s

Discussion

Page No.

121

124

126

129

131

135

141

141

141

144

146

148

149

149

152

156

160

161

161

165

165

169

C HAPTER 10 C HJ�J�, CTEHI �ii.TION OF THE 11AJOR OVINE C 1\SLINS

10. 1 Ovine �<-Cas ein

OVI NE {3 -C ASEINS

10.2

10. 3

10.5

OVIN:t; a -CASEIN B s

10.6

10.7

10. 8

10.9

I so lation of Ovin8 {3-C as eins Molec ular We i ghts of Ovine {3-C as eins Amino Ac i d C ompos i t i ons of the Ovine {3-C as eins Disc us s i on

I s olat i on of Ovine as 2-C as e in and as 3- cas ein Mol ec ul ar i.·le ie;hts of Ovine a -C as eins s Amino Ac i d C ompos i t i ons of th e Ovine as -C as eins D i s c uss ion

CHAPTER 1 1 PHYSIC AL CHARACTERI L:;A.T I ON OF THE C AFRI NE , OVINB 1\ND l30VIN.F CASEINS

1 1.1

1 1.2

1 1.3

1 1.4

U l trac entrifugation Viscos i ty Heas ur em ents on the Caprine and Bovine p-C as e ins The Effect of Temperat ure on the S ol ubility of the C aprine , Ovine and Bovine p-C a s e ins in t t.e Pres enc e of CaC12 C alcium S ens i tivity of the C aprine , Ovine and Bovine C as eins Stab il i z at i on of the Caprine, Ovine and Bovine a - and s P-Cas e ins in the Pr es enc e 0f C aC12 by K-Cas eins

Page N o .

172

1?2

1 74

174

176

176

178

179

179

1 82

182

1 85

1 87

1 87

1 89

195

200

206

Table of Contents (Contd.)

CHAPrER 12

PART III

CHAFrEH 13

REF:ERENCES

11.6 Discussion

Page No.

208

A C Ol"lPAHI SON OF THE PROPERTIES OF

CASEINS FROM THE l"liLK OF SEVEHAL

SPECIES

12.1

12.2

12.3

12.4

«-Caseins

(J-Caseins

a -Caseins s Conclusions

I'lODELS OF BOVINE CASEIN l"IICELLE STRUCTURE

nND THZI H RELATI ONSHIP TO CAI--RINE AND

213

213

218

223

226

OVTNE CASEIN l"ITCELLES 228

DISCUSSI ON

13.2

13.3

13.4

13.5

13.6.

13.7

Hydrophobic, Electrostatic and

Hydrogen Bonding

Role of Disulphide Bonds

Colloidal Calcium Phosphate

Ccat-Core Models

Internal Structure I·lodels

Sub-unit Models

Caprine and Ovine Casein Micelles

and their Relationship to the

Models of Bovine Casein Micelle

Structure

229

229

231

231

232

233

233

235

241

1 .1

1 . 2

4.1

The C ompos i t i on of Ty�ic al Bovine Skim Mi lk

Appr oximat e C ompos it ion o f the Serum and

Mic e lle 1:-has es of Typic al Bovine Skim Mi lk

at 20°C

C ompos ition of Mi lks

4.2 Conc e ntrat i o ns and Ac t iv ities of C at i ons in Ski mmed Mi lk fr om .Animals of Several Spec i es

4 . 3 Vari ation of Cas ein-Pe llet So lvat i on and Dry

Pe lle t \Jeight with Spec i es and C entri fugation T i me

4 . 4 Dist ribution o f Calc ium and Inor ganic Phosphat e

Bet vJeen f'1i ce llar and S erum Phas e s

5 .3

5 • L�

7 . 1

7 .2

7.3

7.4 7-5

7.6

7-7 8.1 8.2

8.3

8.4

Temperat ur e Dep endent Dissoc iati on of C as ein

Nic e lles

Re la t i ve D ens i t i es of f''Jilks and Ultrac entri fuge

s era Intrins ic Vis c os ities and Volumino s i t i es of

C aprine , Ovine and Bovine Cas e in l''lic e lles

S o lvation of C as ein Mic e lles

Stock So lut i ons

Working So lut i ons

St ock Ge l S o lut i ons

Ac id Gel So lut ion ( pH 4)

Po lyacr ylamide Ge ls C o rtaining J"Ig

Standard Prot e ins

Locarte J.�ino Ac id Ana lys er Program C o mpos ition o f Bovine C as ein C omposition of C aprine C as ein C omposition o f Ovine C as ein

R e lative Elec trophore t ic Mobi li t i e s o f Some

Bovine , C aprine and Ovine C as eins in Alkaline

Page No .

2

3

24

26

3 2

3 3

3 7

40

44 46

1 00

1 00 1 02

102

1 03

1 07 1 09 1 20 1 25 1 3 0

Ge ls 1 34 9 .1 Amino Ac id C ompos itions of C aprine , Ovine and

Bovine K-C as e ins

9 .2

9.3

9 .4

Mo lec ular Vei ght s o f C aprine and Bovine Cas eins

Amino Ac id C o mpos itions of /3-C as e ins

R e lative Nob i lities of C as ein C omponents in th e

Pr es enc e and Abs enc e of f'lg

1 47

1 55 1 58

1 64

'rables ( Contd. )

9.5 Amino 1\cid Compositions of Caprine and Bovine

� -Cas eins

10.1

10.2

11.1

11.2

s P�ino Acid Compositions of the Ovine P-Caseins

Amino Acid Compos itions of the Ovine a -Caseins s Sedimentation Coefficients of Caprine P-Caseins

Intrinsic Viscosities of Caprine and Bovine

.8-Cas eins

11.3 Temperature of Half Trans ition Vo.lues for the P-Caseins

11.4 Stabilization of as- and P-Caseins by the

«-Cas eins

12.1 Amino 1\.cid Compositions of K-Cas eins from

Different Species

12.2

12.3

Amino Acid Compositions

Different Species

Amino Acid Compositions

Different Species

of P-Cas eins from

of a -Caseins from s

Page No.

168

177

184

188

194

196

207

217

222

225

FIGURES

Page No .

4 . 1 Alkaline gel elec trophoresis o f bovine , c aprine

and ovine c as eins

4 . 2 Typic al el ectron mic rographs of shadowed b ovine ,

29

ovine and c aprine c asein mic elles 31

4 . 3 Fre eze-etch el ectron mic rograrhs o f the c as ein

5 . 1

mic e l l es in bovine , ovine and caprine milk

Alkaline gel el ectrophoresis of the cas e ins pre s ent in the ultrac entrifuge s era , ob tained

31

from the milks at different t emperatur es 38

5.2 Reduc ed vis c o s i ty plots for c aprine cas ein

mic e l l es

5-3 Reduc ed vis c o s ity plots for ovine c as e in

mic el l es

5 . 4 Reduc ed vis c os ity plo t s for bovine c as ein mic el l es

5 . 5 Elution of ovine sub -mic ellar cas ein aggregates

41

42

43

from a Sephadex G-1 0-Sepharos e 4B c olumn at 37°C 49 5 . 6 Alkal ine gel e l ec tropho�esis o: frac tions

obt ained by g e l chromat ography of ovine large

cas e in mic elles at 37°C 50 5 . 7 Elut i on of c aprine sub-micellar c as e in aggr e gat e s

obt ained from a Sephadex G-1 0-Sepharos e 4B c o lumn at 6°C

5 . 8 Alkaline gel e l ec trophores is of frac t i ons obtained by gel chromatography of c aprine small

52

c as e in mic e l l e s at 6°C 53

6 . 1 Relative electrophore t i c mob ilities of the

6 . 2

6 . 3

6 . 4

6 . 5

6 . 6

genetic variants o f b o vine as1 - , P- and «-c as eins

at pH 8 . 6 and pH 3 . 0 59 Frimary s t ruc ture of b ovine «-c as e in B Primary struc tur e of b o vine (1-cas ein A2

Primary s t ruc ture of bovine CXg1 -cas eins Homol o gi es in the sequenc es of bovine as1- and fJ-c as e in

Fhylogeny o f the "gene c ompl exes" formed by the

all e l es of the bovine as 1 -Cn and �-Cn l o c i

The model for the organiz ation of the

as 1 -Cn-P -Cn- K-Cn " c lus t er of genes"

65 73 79

82

85

85

Fie;ures (Contd.)

Page No.

6 . 8 N-terminal sequences of bovine and ovine

K-casein A glycomacropeptides

6 . 9 Amino acid sequences of ovine and bovine

8 . 1

para- �-caseins

Chromatography of \tJhole bovine casein on DEAE­cellulose

8 . 2 Alkaline gel electrophoresis of whole bovine

8 . 3

casein and the fractions isolated by chromato­

graphy on DEAE-cellulose

Chromatography of whole caprine casein on

DEAE-cellulose

8 . 4 Alkaline gel electrophoresis of whole caprine

casein and the fractions isolated by chromato-

91

9 2

1 1 7

1 1 8

1 22

graphy on DEAE-cellulose 1 23

8 . 5 Chromatography of \vhole ovine casein on DEAE­

cellulose

8 . 6 Alkaline gel electrophoresis of whole ovine

casein and the fractions isolated by chromato­

graphy on DEAE-cellulose

8 . 8

�olyacrylamide zel electrophoresis (pH 8 . 4) of

samples of cap�ine casein

Polyacrylamide gel electrophoresis at pH 8 . 4

of samples of ovine casein from different

breeds

8.9 Polyacrylamide gel electrophoresis at pH 8 . 4 in

the presence of Mg of ovine caseins isolated

1 27

1 28

1 32

1 36

from different breeds of sheep 1 38

9 . 1 Chromatography of caprine K-casein on a CM­

cellulose column

9 . 2 Alkaline gel electrophoresis of purified caprine

K-casein

9 . 3 Acid gel electrophoresis of purified caprine

K-casein

9 . 5

l''Iolecular weight calibration curve obtained

using a Sephadex G-1 00 column

Ch::r:'om2.toe;raphy of crude caprine p2-casein on

CI'1-c ell ulos e

1 42

1 43

1 43

1 45

1 50

fic;ures ( C ontd . ) Page No .

9 . 6 Alkal ine gel electrophores is of the c aprine

p -c as eins in the abs ence and pres enc e of MG

9 . '7 A typical run used t o calibrat e the mol ecular

weicht c o lumn

9 . 8 Mol ecular weie;ht calib ration curve obtained

1 51

1 53

us ing a Sepharos e 6B c olumn 1 54

9 . 9 Amino ac id analysis chromatogram obtained for

c aprine p2- c as ein ·us ing a Loc art e Hk.IV analys er 1 5'7

9 . 1 0 Alkal ine gel elec trophores is of pur i fi ed c aprine as -cas ein and purifi ed bovine as 3-c as ein in the pre s enc e and abs enc e o f Mg 1 62

9 . 1 1 Chrom�t ography of a s ample c ontaining the major

c aprine a - c as ein and b ovine a �-c as ein B on - S SI the molecular weight c o lumn to c ompare their mol ecular wei ghts 1 66

1 0 . 1 Alkal ine gel e l e c trophor e s i s o f purified ovine

K-cas ein and the effec t of r ennet on this

frac t ion 1 0 . 2 Alkaline gel e l e ctrophores is o f the purif i ed

ovine P-cas eins in the abs enc e o f pres enc e of Mg

1 0 . 3 Chromat ography of ovine as 3- c as e in on DEAE-

c e llulos e

1 0. 4 Alkaline gel el ec trophoresis o f the ovine

1 1 . 1 1 1 . 2

1 1 . 3

1 1 . 4

cxs-cas eins in the abs enc e and pres enc e o f I'Ig Reduc ed vis c o s i ty plots for c aprine p1 -cas ein Reduc ed vis c o s ity plots for c aprine p2-cas ein

Reduc ed vis c o s i ty plots for bovine p -c as ein A1

The eff ect o f t emperature and ionic strength on the turb i dity o f c aprine p1 -cas e in

s olut i ons

11 . 5 The effect of temperature and ionic s trength on

1 73

1 75

1 80

1 81 1 90

1 91 1 92

1 97

the turbidity o f c aprine p2-c as ein s o luti ons 1 98

1 1 . 6 The effect o f t emperature and ionic s tr ength

on the turb idity o f b ovine p-cas ein B s ol utions 1 99

1 1 . 7 C alc ium s ens i�ivity o f the caprine ��c a�eins at

37°C 201

:B,ip;ures (Contd. )

'11.8 Calcium sensitivity of the caprine, ovine and bovine �-caseins at 37°C

Page No.

202

1'1.9 Calcium sensitivity of the as-caseins at 37°C 20L�

'11.'10 Calcium sensitivity of the caprine, ovine und

bovine a -caseins at 37°C 205 s