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Some Minor Com ponents o f Case in and O therPhosphoproteins in M ilk A ReviewM L GROVES

E as te rn U t i l i za t i on Research and Deve l opm en t D i v i s i on US D AP h i l ade l ph i a P ennsy l van i a ] 9 ] ] 8

b s t r a c tT - C a s e in a n d o t h e r m i n o r p r o t e i n s t e n -t a t i v e l y d e s i g n a t e d T S - , R - , a n d S - c a s e i n ,h a v e b e e n i d e n t i fi e d i n u n f r a c t i o n a e d c a -s e i n b y d i s c g e l e l e c t r o p h o r e s i s . T - C a -s e i n i s p o l y m o r p h i c a n d l i k e f l - e a s e i n i ts h o w s t h r e e v a r i a n t s o f t h e A t y p e a n d

o n e B t y p e . S o f a r , n o T -C c a s e i n h a s b e e no b s e r v e d. T h e A 2 a n d B p o l y m o r p h s o fT - a n d f i - c a s e in s h o w t h e s a m e a m i n o a c i ds u b s t i t u t i o n s . T h e R - c a s e i n i s f o u n d i nA s a m p l e s ; t h e S - c a s e i n i s a s s o c i a t e d w i t hc a s e i n s t y p e d B w i t h r e s p e c t t o T - , f l -c a s ei n . T S - C a s e i n s o c c u r i n a l l s a m p l e sa n d a r e p o l y m o r p h i c . T h i s r e v i e w w i l la l s o d is c u s s r e c e n t d a t a o n o t h e r m i n o rc a s e in s , th e c o m p o n e n t s o f t h e p r o t e o s e -p e p t o n e f r a c t i o n , a n d t h e p h o s p h o g l y c o -p r o t e i n f r a c t i o n o f w h e y.

C a s e i n h a s b e e n d e f i ne d a s t h a t p r o t e i nf r a c t i o n p r e c i p i t a t e d b y a c i d i f y i n g r a w s k i m -m i l k t o p H 4 .6 t o 4 . 7 (1 8 ) . M o r e r e c e n t l y , t h i sd e f i n i t i o n h a s b e e n m o d i f i e d s o t h a t c a s e i n i sd e s c r i b e d a s a h e t e r o g e n e o u s g r o u p o f p h o s -p h o p r o t e i n s p r e c i p i t a t e d f r o m s k i m m i l k tp H 4 .6 a n d 2 0 C ( 3 8 ) . C a s e i n e x is t s i n m i l ki n c o m p l e x p a r t i c l e s ( m i c e l le s ) c o n t a i n i n g c a l -c i u m a n d p h o s p h a t e , w h i c h c a n b e s e d i m e n -t a r e d b y u l t ra c e n t r i f u g a t i o n . I t i s k n o w n t h a tt h e m i c e l le s a r e s t a b i l i z e d b y u n i q u e c o m p l e x e so f K - c a s e i n w i t h a S- a n d f l - c a s e i n ( 3 3 , 4 1 , 4 2 ) .T h e s e t h r e e p r o t e i n s c o m p r i s e m o s t o f t h ec a s e i n in t h e m i c e l l e , y e t t h e r e a r e a n u m b e ro f o t h e r c a s e i n s p r e s e n t i n t h e m i c e l l e w h i c ha l s o p r e c i p i t a t e w i t h t h e c a s e i n o n a c id i f i c a t io no f m i l k . W h e t h e r t h e s e m i n o r c o m p o n e n t s a r ea n e s s e n t i a l p a r t o f t h e c o m p l e x h a s n o t b e e ne s t a b l i s h e d ; h o w e v e r , a n u m b e r o f t h e m h a v eb e e n i s o l a t e d . N o t a l l o f t h e p h o s p h o p r o t e i n si n m i l k a re p r e c i p i t a t e d a t p H 4 .6 w i t h t h ec a s e in . I t a p p e a r s t h a t s o m e r e m a i n in s o l u-t i o n w h i l e o t h e r s a r e d i s t r i b u t e d b e t w e e n t h ec a s e in a n d w h e y f r a c t i o n s . I n t h i s r e v ie w t h em o r e r e c e n t s t u d i e s o n t h e s e m i n o r c a s e i n sa n d o t h e r p h o s p h o p r o t e i n s w i l l b e d i s c u s s e d .

Rece iv ed f o r p u b l i ca t io n Mar ch 2 6, 1 9 6 9.

W a k e a n d B a l d w i n ( 4 0) , us i n g s ta r c h g e le l e c t r o p h o r e s i s i n u r e a a t a n a l k a l i n e p l = [ ,d e m o n s t r a t e d t h a t t h e p a t t e r n f o r c a s e i n c o n -t a i n s a b o u t 2 0 p r o t e i n b a n d s , a m o n g w h i c ht h e a - c a s e i n [ n o w c a l le d a s l - c a s e i n ( 3 5 ) ] a n df l -c a s e i n b a n d s a r e m o s t p r o m i n e n t . B e c a u s ec a s e i n f r o m p o o l e d s k i m m i l k w a s u s e d , s o m eo f th e s e b a n d s p r o b a b l y re p r e s e n t e d p o l y -m o r p h s , p a r t i c u l a r l y t h o s e o f th e f l- a n d a s l -c a s e in s . T h e y a l s o f o u n d t h a t x - c a s ei n s h o w sa b r o a d s t a i n i n g a r e a o f s l ow e r m o b i l i t y t h a nt h a t o f f l- a n d a s l - c a s e in . T h e o t h e r b a n d s r e p -r e s e n t t h e m i n o r p r o t e i n s f o u n d i n t h e c a s e i nf r a c t i o n . N u m b e r s w e r e a s s i g n e d to e a c h b a n dd e p e n d i n g o n i t s p o s i t i o n r e l a t i v e t o a z o n ed e s i g n a t e d a s 1. 00 w h i c h h a d a m o b i l i t y s l i g h t l ys l o w e r t h a n t h a t o f a s l - ca s e i n . U s i n g t h i ss y s t e m a n d d e p e n d i n g o n th e g e n e t i c t y p e s ,t h e a s l - c a s e i n s w e r e f o u n d t o r a n g e f r o m 1 .0 7t o 1 .1 8 a n d f i - e a s e i n s f r o m 0 .7 0 t o 0.8 0 ( 3 8 ) .S o m e o f t h e s l o w - m o v i n g c o m p o n e n t s , i d e n -t i f i e d a s B a n d s 0 .1 8 , 0. 3 4, a n d 0 . 4 1 , w e r e r e -f e r r e d to a s t h e T - c a s e in f r a c t i o n ( 2 6 ). T h e s eb a n d s w e r e r e p r e s e n t a t i v e o f t h e f i r s t f r a c t i o no f r e d u c e d a n d a l k y l a t e d c a s e i n e lu t e d f r o m aD E A E - e e l l u l o s e c o l u m n a t p H 7 .0 w i t h a 2. 0b o r at e ~ 0 .1 8 ~ p h o s p h a t e b u f f er , c o n t a i n i n g2 0 d i m e t h y l f o r m a m i d e . T h i s f r a c t i o n a l sos h o w e d a n e l e c t ro p h o r e t ic b a n d c o r r e s p o n d i n gt o o n e o f t h e r e d u c e d a n d a ] k y l a t e d x - ca s e in s ,t o g e t h e r w i t h s o m e m a t e r i a l w h i c h m o v e dt o w a r d t h e c a th o d e . O n e o f t h e p r o t e i n b a n d s ,N u m b e r 0 .8 6, w i t h a m o b i l i t y b e t w e e n a ~ l- a n df l -c a s e in , d i s a p p e a r e d o n r e d u c t i o n a n d a l k y l a -t i o n o f t h e c a s e i n .

T Caseins. M e ] l a n d e r ( 2 8 ) o b s e r v e d t h a t c a -s e i n i s c o m p o s e d o f t h r e e e l e c t r o p h o r e t i c c om -p o n e n t s , w h i c h h e n a m e d a - , f l - , a n d ~ , - e a s e i ni n t h e o r d e r o f d e c r e a s i n g m o b i l i t y i n m o v i n g -b o u n d a r y e l e e tr o p h o r e s i s . H i p p e t a l . ( 14 , 1 5 )i s o l a t e d T - c a s e i n b y c h e m i c a l f r a c t i o n a t i o n a n de s t im a t e d t h a t i t a m o u n t s t o a b o u t 3 o f t h eu n f r a c t i o n a t e d c a s e in . T h e y al s o f o u n d ac o n s i d e ra b l e a m o u n t o f m a t e r i a l a s s o c i a te dw i t h t h e T - c a s e i n f r a c t i o n w i t h a m o b i l i t ys l o w e r t h a n t h a t o f T - c as e in . A l t h o u g h t h ep u r i f i e d T - c a s e i n s h o w e d a s i n g l e e l e c t r o -p h o r e t i c c o m p o n e n t a t p t I 8 .6, a s d e t e r m i n e db y m o v i n g - b o u n d a r y e l e c tr o p h o re s i s , i t p r o b a -

5 5

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] 1 5 6 ~ O V E Sbly conta ined o ther p ro te in impur i t i es o fsl ight ly different mobil i t ies .The i so la t ion of r -case in us ing co lumnchromatography has a l so been descr ibed (13) .Ch r o ma t o g r a p h y o n D E A E - c e l l u l o s e o f t h a tp a r t o f a c i d - p r e c i p i t a t e d c a s e i n e x t r a c t e d b ydilute acid at 3 C yielded a protein elutedf r o m t h e c o l u mn w i t h t h e s t a r t i n g b u f f er o f0 .005 ~ phospha te , p H 8 .3 . I t w as des igna tedtempe ra ture -sens i t ive (TS ) , because i t gave ac lear so lu t ion a t pH 8 , 3 C, bu t became turb idwi th warming . The T-case in was e lu ted a t0.02 ~ p hosp hate, p i t 8 .3. The T-casein puri -f ied by th i s method showed phys ica l andchemica l p roper t ies s imi la r to the T-case ini so la ted by prev ious wo rkers (8, 14 , 15) . I tconta ined very l i t t l e impur i ty , based on s ta rchge l e ]ec t rophores i s in 7 M urea . The T S f rac -t ion showed, by zone e tec t rophores i s a t pH8.6 , a ma jor ban d of mobi l i ty s lower than tha tof r -case in .Three gene t ic types o f fl -casein , name ly A,B, and C~ have been f oun d in milk by gele lec t rophores i s a t a lka l ine pH in the presenceof urea (1, 2 , 30, 36) . Type A of f l-caseinshows fur ther complex i ty by e lec t rophores i s a tan acid pH , and i t is su bdivide d into A 1, A e,an d A a var ian ts (19, 31, 32) .Aschaf fe nburg (1) in 1961 foun d some ev i-dence for gene t ic po lymorphism in r -case in ,us ing paper e lec t rophores i s. Recent ly , E1-l goum y (6) p rep ared T-casein f rac t ions f rom

milk of ind iv idua l cows by a modi f ica t ion oft I i pp ' s method (14 , 15) . These f rac t ion sshowed polymorphism by s ta rch ge l e lec t ro-phores i s and the var ious bands were iden t i -fied as T~ to T~.Polymorphism in T-case in has a l so beendemons t ra ted , by us ing the h igh reso lv ingpower of disc gel e lectrophoresis , 4 ~ ureaat pI-I 9 .6, to exam ine caseins isolated fro mindiv idua l cows (12) . To es tab l i sh whichbands would correspond to T-casein, as de-f ined by chromatography and descr ibed ear l ie r( 1 3 ) , u n f r a c t i o n a t e d c a s e i n w a s c h r o ma t o -graphed on a DEAE-ce l lu lose co lumn, and aTS f rac t ion was e lu ted a t 0 .005 ~ phospha tewhile the r-casein was eluted at 0 .02phosp hate. Fig ure 1 shows the disc gel e lectro-

phore t ic pa t te rns of case ins typed wi th re -spect to f l -casein, AC (Gel a) , A (Gel b) , AB(Gel e) , and B (Gel d) . The zones somewhats lower than the f l -case in bands a re des igna tedr -case ins. The i r re la t ive mobi l it i es cor respondin types to the f l -caseins, with the exceptionof the AC type . W i th th i s sample , on ly T-ca-sein A is observed. The protein in the banddes igna ted R, wi th a mobi l i ty be tween tha tof T-casein A and B, is not a r-casein by thes ta ted def in i t ion , s ince i t i s eh ted f romDEAE-ce l lu iose in the TS f rac t ion , wi th theTS- and S-caseins. These prot eins w il l bediscussed. Th ey are cal led caseins becausethey occur in ac id-prec ip i ta ted case in , can be

:FIG. :l. Disc ge] electrophoretic patterns, at p H 9.6 and 4 M urea, of caseins typ ed: a, fl-casein AC, 7-easeln ; b, fl-easein A, T-casein A; c, fl-easein AB, ~/-easein A B ; d, fl-casein B,v-casein B.J. DAI~y SCIENCE ~oL . 52 No. 8

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C A S E I N C O M P O N E N T S 1 1 5 7

FIG. 2. V ertica l gel electrophoretic patt erns , at p H 8.7 and 4.5 ~ urea, of unfr actio natedcaseins, toge ther with % casein As and R-caseln. a, d, casein typed fl-, 7-casein Af ; b, e, caseintyp ed fl-, ~/-casein Af B; c, ~, casein typ ed fl-, /-casein B; g, y-casein A2; h, R-casein. Theprotein in d, e, f is half as concentrated as that in a, b, c.i d e n t i f i e d i n m ie e l l a r c a se in b y g e l e l e c t ro -p h o re s i s , a n d a r e s im i l a r t o o th e r c a se in s i nc o n te n t o f h ig h p e rc e n ta g e s o f p ro l in e , g lu -tamic ac id , va l ine , and leue ine , and in lack ofc y s t in e . I t i s n o t k n o w n , h o w e v e r , t h a t t h e ya re p h o sp h o p ro t e in s . F ig u re 2 sh ow s v e rt i c a la c r y l a m i d e g e l e l e c t r o p h o r e t i e p a t t e r n s , p t8 .7 and 4 .5 ~ urea~ of un f ra c t i on a te d case insa n d o f t h e i so l a t e d T -A 2 a n d R -c a se in s . U n d e rthese condi t ions , the S- , TS- , and T-B ca-se in s a r e c l e a r ly r e so lv e d , w h e re a s t h e R - a n dT-case in A 2 samp les show the same m obi l i t ies .G e l s a , b , a n d c r e p re se n t s a m p le s o f c a se in styp ed f l- , T -case in A 2, AfB, and B , resp ec t iv e ly ,whi le d , e , and a re o the same samples , bu ta t o n e -h a l f t h e c o n c e n t r a t i o n o f p ro t e in . Z o n eg shows the b and for v -case in A S, whi le h i sth a t o f t h e R -c a se im V e r t i c a l g e l e l e c t ro -

p h o r e t i e p a t t e r n s o f c a se i n s r e d u c e d w i t hm e re a p to e th a n o l sh o w th a t K -ca sein a n d v - c a -se in b a n d s a r e n o t c l e a r ly r e so lv e d . T h eK -ca sein A b a n d h a s a m o b i l i t y s l i g h t ly f a s t e rtha n tha t o f v -case in A 2, whe reas the K-case inB sh o w s th e s a m e m o b i l i t y as t h a t o f T -ca -se in B .T w o ty p e s o f v - c a se in , a s d e t e rm in e d b ydisc ge ls a t p H 9 .6 , a re d es ig na t ed A a nd B ,

and in a l l samples examined the T- and f l -e~-se in s sh o w th e s a m e ty p e s , w i th t h e e x c e p t io no f f l- c a se in C . N o v -c a se in w a s fo u n d fo rth e o n ly tw o c a se in s a v a i l a b l e c o n ta in in g th ehomoz ygous f l -case in C. The T-case ins in un-f r a c t i o n a t e d c a s e i n c a n n o t b e t y p e d b y d i s cg e l e l e c t ro p h o re s i s a t p H 4 .3 a n d 8 ~ u r e a d u eto in te r fe rence f rom o ther case ins . The v-ca-se in s i so l a t e d f ro m c a se in t y p e d f l - c a sein A 1,~. DAIRY SCIENCE ~OL . 52 ~0 . 8

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1158 GROVESA S, and A s show sl ightly fas ter mobilities tha nthose of the corresponding B-caseins and thedifferences in relative mobilities are consistentwith those found for the B-caseins (10). Fig ure3 shows the disc gel electrophoretic patterns,pH 4.3, 8 ~ urea , o f T-caseins typed B, A 1,and A s. The T-casein A ~ sample, with a mo-bil ity less th an tha t of T-casein A s, is no tshown. A simil ar pat ter n is obtai ned (Fig. 4)when the 7-casein polymorphs are compared,using the vertical acrylamide gel electropho-retie method of Peterson and Kopfler (31) atp i t 3.0 and 4.5 g urea. The T-casein band s,under these conditions, show mobilities nearthat of asl-easein. An unfractionated caseinsample, together with a mixture of the iso-lated S-, TS-A, TS-B, and R-caseins, whichwill be discussed, are also included for com-parison.

Purified samples of ~,- and B-caseins wereprepa red from caseins typed either A 2 or Busing chromatography on DEAE-cellulose col-umn s (9, 39). Fig ur e 5 shows disc gel electro-phoresis, pH 9.6, 4 ~ urea , of the puri fied ~/-and B-caseins isolated from the two caseintypes. Table I compares the numbe r of aminoacid residues f ound for the A 2 and B t ypes ofT- and B-caseins. Values are based on molarratios, taki ng glycine and a lanine as 5 a nd 6,

FIG. 3. Disc gel e]ectrophoretic pat terns, at pt{4.3 and 8 ~ urea, of the isolated 5 -caseins typed]3, A 1, and A 2. A mixture of all three proteinsis shown in one gel.J. DAIRY SCI~NC~ ~OV,. 52, NO.

respectively, for the T-caseins and 5 for theB-caseins. The amino acid subst itut ions forthe A 2 and B types o f both T- and B-caseins(underlined in Table 1) are presumed to bearginine/serine and histidine/proline. Basedon the genetic code, each su bst itu tio n is con-sistent with one base su bstit ution in the DNAmolecule. The T-casein molecule is lar ger t hanB-casein, having seven more proline residuesand a net increase of seven other amino acids.Aside from proline, the other amino acidsof the 7- and B-caseins differ by no more thantwo residues. The similariti es in relative mo-bilities of the T-casein and B-casein poly-morphs, together with the common substi-tuti ons exhibited by the A 2 and B types,suggest a close relationship in the synthesisof T- and B-casein.R- S- and T~-caseins. The TS fraction, asmentioned earlier, is that fraction of caseineluted from a DEAE-cellulose column at pH8.3 with the st ar ti ng buffer of 0.005 ~ phos-phate. It contains the bands referred to inFigu re I as S-, TS-, and R-casein, togetherwith other caseins, including some K-casein.Reasonably pure preparati ons of these pro-teins can be obtained by rechromatographyof the TS fraction on DEAE-cellulose, eitherwith or wi thout urea. The A s type yields R-casein and TS-casein A; the B type gives S-casein and TS-cas ein B. Both TS-A and TS-Bsamples show the same mo bili ty by disc gelelectrophoresis at pit 9.6, 4 ~ urea (10, 11).However, at pH 4.3 and 8 M urea, the TS-Ahas a faster mobility than TS-B, Figure 6.The Gels a and e show S- and TS-ca sein Bobtained from casein of the B type shown inGel f. The Gels b and d show TS-casein A andR-casein obtained fr om the A 2 type casein shownin Gel g. The band in Gel f, corresponding inmobi lity to R-casein, is t hat of ~,-casein B.Casein samples of the AB type show by discgel electrophoresis both the TS-A and TS-Bcaseins. Ver tic al gel electropho resis at pl=[ 3.0and 4.5 ],~ urea of the partially purified minorcaseins and an unfractionated casein sampletype d B-, T-casein A s are shown in Fig ur e 7.Here the S-, TS-A, TS-B, and R-caseins arebetter separated from the T- and other caseinsthan in the disc gel runs (Fi gur e 6). This wasalso shown in Figure 4, b. The casein sample(Slot a) in both Fig ures 4 and 7 shows a fasterband corresponding to TS-A and a fai nt b andof slower mobility that corresponds to thatof the R-casein.Table 2 compares the various types of minorproteins with the B-casein types as found incasein of indivi dual cows. Fo r t he caseins

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0ASE IN COMPONENTS ] 15 9

FIG. 4. Ver tical gel eleetrop horetie patte rns, a t pH 3.0 and 4.5 ~ urea, of the r-caselnpolymorphs, together with un fractionated casein and a mixture of the isolated S-, TS-A, TS-B,and R-caseins . a, casein ty pe d fl-, y-casein A2; b, S-, TS-A, TS-B , and R -case in: e, y-caseinAS ; d, r-casein A2 ; e, '),-casein A~; f, r-casein B.e x a m in e d b y a lk a l in e t y p in g , t h o se c o n ta in in gf l - c a se in A , B , a n d C a l so h a v e th e c o r r e sp o n d -ing ~ ,-case in A and B bu t no C . As me nt io nedear l ie r , the TS-case in s g ive a s ing le band of thesa m e m o b i l i t y a t a lk a l in e p H v a lue s . S a m -ples . typed f l -ease in B conta in S-case in bn tno R-case in , whereas a l l o ther f l -case in sam-ples ty pe d A 1, A 2, A s , a nd C cont ain theR-case in bu t no S-case in . The ~-case ins inu n f r a c t io n a t e d c a se in , a s p r e v io u s ly s t a t e d ,c a n n o t b e t y p e d a t a c id p H , d u e to i n t e r -f e r e n c e f ro m o th e r p ro te in s . T h e l im i t e d n u m -ber o f ~ , -ease ins i so la ted do show re la t ivemobi l i t ies s imi l a r to those found in the cor -res po ndi ng f l-easeins A 1, A 2, a nd A 3. TheT S -c a se in s A a n d B in u n f r a c t io n a t e d c a -se ins can be iden t i f ied by e lec t rophores is a ta c id p t t . T h e T S -A in s a m p le s t y p e d f l -c a se inA s a p p e a r s t o d i f f e r f r o m th e o th e r A ty p e s ,s in c e i t sh o w s tw o b a n d s o f d i f f e r e n t m o b i l i t yf r o m t h a t o f T S - A a n d T S- B . P r e l i m i n a r ya m in o a c id a n a ly s i s a n d th e d i s c g e l e l e c t ro -p h o r e t i c d a t a i n d i c a t e t h a t t h e T S - c a s e i n sf rom samples typed A and B , l ike the ~ / -ea -s e ins , a r e p o ly m o rp h ic (1 0 ) a n d , f u r th e rm o re ,t h a t t h e y c o n t a i n m o r e p r o l i n e t h a n a n y o t h e rc a se in s s tu d i e d . B a se d o n d i s c g e l e l e c tro -

p h o re s i s , t h e R - a n d S -c a se in s c o u ld a l so b ep o l y m o r p h s o f t h e s a m e p r o t e i n . F u r t h e ra n a ly se s a r e i n p ro g re s s t o d e t e rm in e i f t h i sis so.N o a t t e m p t h a s b e e n m a d e t o i d e n t i f y t h em in o r s lo w -m o v in g c a se in c o m p o n e n t s b y h u m -

F~G. 5. Disc gel electro phoretic patte rns, at p:H9.6 and 4 ~ u rea of puri fied r- and fl-caseins iso-la te d fro m caseins ty pe d fl-, -~-easein A 2, and fl-,r-casein B.J. DAIRY SCIEI~CE ~rOL. 52 NO. 8

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1 1 6 0 a R O V E ST AB LE 1 . C o m p a r i s o n o f a m i n o a c i d r e s i d u e s o f ~ - a n d B - c a s e in s t y p e d A 2 a n d B ( 9 ) .

7 - A 2 ~ -B f l - A 2 f l - BR e s i d u e s p e r m o l e c u l e

L y s i n e 1 2 1 2 1 1 1 1H i s t i d i n e 6 7 5 6A r g i n i n e 3 4 4 5A s p a r t i c A c i d 9 9 9 9T h r e o n i n e 1 0 1 0 9 9S e r i n e 1 3 1 2 1 5 1 4G l u t a m i e A c i d 3 9 3 9 3 9 3 9P r o l i n e 4 1 4 0 3 4 3 3G l y e i n e 5 5 5 5A l a n i n e 6 6 5 5V a l i n e 2 0 2 0 1 8 1 8M e t h i o n i n e 7 7 6 6I s o l e u c i n e 8 8 1 0 1 0L e u c in e 2 3 2 3 2 1 2 1T y r o s i n e 5 5 4 4P h e n y l a l a n i n e 1 1 1 ] 9 9T r y p t o p h a n 1 1 1 1M o l w t a 2 5 ,0 2 0 2 5 ,1 30 2 3 ,5 9 0 2 3 ,70 0

a C a l c u l a t e d f r o m c o m p o s i t i o n s h o w n , i n c l u d i n g p h o s p h o r u s a n d , i n t h e c a s e o f y - c a s e i n ,o n e r e s i d u e o f h e x o s e .b e r i n g t h e b a n d s o b t a i n e d b y s t a r c h g e l e le c -t r o p h o r e s i s a c c o r d i n g t o t h e s y s t e m d e v e l o p e db y W a k e a n d B a l d w i n ( 4 0 ), s i n ce th e m i n o rc a s e i n s d e s c r i b e d h e r e w e r e i d e n t i fi e d u s i n gt h e d i s c g e l e l e c t r o p h o r e t i c t e c h n i q u e . I t i sp r o b a b l e t h a t s o m e o f th e s e c a s e i n s c o r r e s p o n dt o B a n d s 0 . 1 8 , 0 . 3 4 , a n d 0 . 4 1 f o u n d , a s m e n -t i o n e d e a r l i e r , i n t h e ) , - f r a c t i o n o f M a c k i n l a ya n d W a k e ( 2 6 ), s i nc e t h i s f r a c t i o n w a s e l u t e df i rs t f r o m a D E A E - c e l l u l o s e co l um n . I t a p -p e a r s t h a t m o s t o f t h e r e c e n t s t u d i e s o n c a s e i nh a v e m a d e u s e o f v e r t i c a l a c r y l a m i d e g e l e l e c -t r o p h o r e s i s . F o r t h i s r e a s o n , a n d f o r c o m p a r i -s o n w i t h t h e d i s c g e l t e c h n i q u e , t h e p a t t e r n so b t a i n e d b y b o t h m e t h o d s h a v e b e e n s h o w nw h e r e p o s s i b l e .

f l - C a s e i n c o n t a i n s f o u r o r f i v e m o l e s o f p h o s -p h o r u s p e r m o l e o f c a s e i n , d e p e n d i n g o n t h et y p e ( 3 7 ) , w h i l e t h e v - c a s e i n s A 2 a n d B c o n -r a i n o n l y o n e m o l e o f p h o s p h o r u s ( 9 ) . T h i sr e l a t i v e l y s m a l l a m o u n t o f p h o s p h o r u s i nv - c a s e i n i s p r o b a b l y r e f l e c t e d i n i t s s l o w e r e l e c -t r o p ho r e g ie m o b i l i t y a t a n a l k a l i n e p H , a s c o m -p a r e d w i t h t h a t o f fl - ca s e in . I t i s n o t i n c o n -c e i va b l e , t h e n , t h a t o t h e r m i n o r p r o t e i n s s u c ha s t h e S - a n d T S - c a s e i n s , w i t h m o b i l i t ie s l e s st h a n t h a t o f ~ ,- c as e in , m i g h t c o n t a i n n o p h o s -p h o r u s . T h i s w o u l d b e i n c o n s i s t e n t w i t h t h ed e f i n i t i o n o f c a s e i n a s a p h o s p h o p r o t e i n , e v e nt h o u g h p r e l i m i n a r y a m i n o a c i d a n a l y s e s i n d i -J. DAIRY SCIENCE VOL. 52 NO. 8

c a t e t h a t t h e s e m i n o r p r o t e i n s b e l o n g t o t h ef l- a n d v - c a s e i n f a m i l y , s i n ce t h e y c o n t a i n ,l i k e f l- a n d 7 - c a s e i n , n o c y s t i n e b u t r e l a t i v e l yl a r g e a m o u n t s o f g l u t a m i c a c i d , p r o l i n e , v a li n e ,a n d l e u c i n e .

a s2 - , a s 3 -C a s e i ~s a n d 0 .8 6 ca s e i n . A c a s e i nc o r r e s p o n d i n g t o B a n d 0 .8 6, a s a l r e a d y m e n -t io n e d, w a s f o u n d b y M a c k i n l a y a n d W a k e2 6 ) t o d i s a p p e a r w h e n c a s e i n w a s r e d u c e d a n da l k y l a t e d p r i o r t o z o n e e le c t r o p h o r e s i s . T h i sp r o t e i n h a s n o w b e e n i s o la t e d b y H o a g l a n da n d K a l a n ( 16 ) . I t w a s p a r t i a l l y p u r i fi e d b yc h r o m a t o g r a p h y o n D E A E - c e l l u l o s e . T h e f r a c -

t i o n e n r i c h e d in p r o t e i n c o r r e s p o n d i n g t o B a n d0 . 8 6 w a s t h e n f r a c t i o n a t e d b y p r e p a r a t i v ep o l y a c r y l a m i d e e l e c t r o p h o re s i s . T h e p u r i f ie dp r o t e i n o n r e d u c ti o n w i t h m e r c a p t o e t h a n o l h a sa g r e a t e r m o b i l i t y th a n t h e u n r e d u c e d p r o t e i nb y z o n e e l e c t r o p h o r e s i s a t p H 9 .2 a n d , a s m i g h tb e e x p e c t ed , c o n t a i n s c y s t in e . T h i s p r o t e i nd i f f e r s f r o m t h e a s 1 - , f l - , a n d v - c a s e i n s , w h i c hc o n t a i n n o c y s t i n e . A l s o , i t is r e l a t i v e l y lo wi n p r o l i n e c o n t e n t b u t l i k e o t h e r c a s e i n s h ig hi n g l u t a m i c a c i d . T h e c a s e i n c o r r e s p o n d i n g toB a n d 0 . 86 is a p h o s p h o r u s - c o n t a i n i n g p r o t e i n .C a s e i n c o n t a i n s tw o m i n o r c o m p o n e n t s w h i c hs h o w m o b i l i t ie s s l i g h t l y s l o w e r t h a n t h a t o fa s l - c a s e i n b y g e l e l e c t r o p h o r e s i s a t a l k a l i n ep g . T h o m p s o n ( 34 ) i s o la t e d t h e s e p r o t e i n s b yu r e a f r a c t i o n a t i o n o f c a s e in , th e n b y a l c o h ol -

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CASEIN COMPONENTS 6

l~m. 6 . Disc gel e lectrophoret ie pat terns , a t pH 4.3 and 8 x urea, of par t ia l ly pur if ied S- ,TS-A, TS-B, and R-caseins, toget her with the uufra ct ion ate~ caseins , a , S-casein; b , TS-caseinA; e , TS-caseln B; d , R-cas eia; e , a mixture of S- , TS-A, TS-B, and R-c asein; f , casein typed- , 7-casein B; g , casein typed f l - 7-casein A 2.

FIG. 7 . V er t ical gel e leetrophoret i pat te rns a t p i t 3 .0 and 4 .5 ~ ure a, of par t ia l ly pur if iedm inor case ins, toge ther w i th an unf rac t iona ted casein s ample , a , Unf rae t iona ted case in typedB-, 7-casein A2; b , 7-casein A2; e , 7-casein B; d , R-casein; e , TS-casein B ; f , T S-casein A ;g S-casein.J. DAn~Y SC ON CE VOL. 52, NO.

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1162 GROVEST AB LE 2 . T y p e s o f f i- , ~ , -, T S - , R - , a n d S - c a s e i n s f o u n d i n c a s e i n s a m p l e s o b t a i n e d f r o m i n d i -v i d u a l m i l k s .

R - o r S -B - C a s e i n ~ - C a s e i n T S - C a s e i n C a s e i nA 1 A 1 A RA ~ A : A RA A A a RB B B SC - - A R

a P r e l i m i n a r y e v i d e n c e s u g g e s t s t h a t t h i s s a m p l e s h o w s a m o b i l i t y s l i g h t l y d i f fe r e n t f r o mo t h e r t y p e s d e s i g n a t e d A a n d t h a t i t c o n t a i n s a n o t h e r p o l y m o r p h o f m o b i l i t y s lo w e r t h a nt h a t o f T S - B . T h e T S - A 3 d e s i g n a t i o n i s t e m p o r a r y , u n t i l f u r t h e r s t u d i e s c a n b e m a d e o nt h i s p r o t e i n .a m m o n i m n a c e t a t e f r a c t i o n a t i o n a n d f in a l l yb y D E A E - c e l l u l o s e co l um n c h r o m a t o g r a p h y .L i k e l - c a s e i n , t h e y a r e c a l c i u m - s e n s i t i v e a n da r e s t a b i l i z e d a g a i n s t c a l c i m n p r e c i p i t a t i o n b yK - c as e in . T h e y w e r e n a m e d as2- a n d a s 3 - c a s e i ni n o r d e r o f t h e i r d e c r e a s i n g m o b i l i t y . T h e yc o r r e s p o n d i n m o b i l i t y t o t h e z o n e n u m b e r s1 .0 4 a n d 1 .0 0 o f W a k e a n d B a l d w i n ' s p a t t e r n s( 4 0 ) . A c o m p a r i s o n o f t h e a m i n o a c i d c o m -p o s i t i o n o f t h e a s 2 - a n d u s a - c a s e i n s i n d i c a t e st h a t t h e y b o t h c o n t a i n c y s t e i n e o r e y s t i n e , b u ts h o w s m a l l d i f f e r e n c e s i n ] y s in e , a s p a r t i c a c i d ,g l u t a m i c a c i d , p r o l i n e , a n d i s o l e u e i n e . T h e yd i f fe r s i g n i f i c a n t l y f r o m a s l - c a s e in i n c o m p o s i -t i o n , a n d m o r e c l o s e l y r e s e m b l e t h e c a s e i n o fZ o n e 0 .8 6 w i t h r e s p e c t t o t h e p r e s e n c e o f c y s -f i n e a n d a r e l a t i v e l y lo w a m o u n t o f p r o l i n e .T h e r e i s s o m e e v i d e nc e t h a t t h e p r o t e i n o fZ o n e 0.8 6 r e p r e s e n t s t h e o x i d i z e d f o r m o fa s 2 - ( Z o n e 1 . 0 4 ) a n d U sa ( Z o n e 1 . 0 0 ) c a s e i n s . 1I f t h i s i s t r u e , i t w o u l d b e o f i n t e r e s t t o k n o ww h e t h e r t h e s e p r o t e i n s c o u ld b e s u b s t r a t e s f o rt h e e n z y m e , s u l f h y d r y l o x id a s e , r e c e n t l y f o u n di n m i l k b y K i e r m e i e r e t a l . ( 20 , 2 1 ) .Proteose-peptone fraction. W h e n s k i m m i l ki s h e a t e d a t 9 5 t o 1 00 C f o r 3 0 r a i n , t h e n a d -j u s t e d t o p H 4 . 6 b y a d d i t i o n o f a c i d , m o s t o ft h e w h e y p r o t e i n s a r e d e n a t u r e d b y t h e h e a tt r e a t m e n t a n d p r e c i p i t a t e w i t h t h e c a s e in ,l e a v i n g a p r o t e o s e - p e p t o n e f r a c t i o n i n s o l u t io n( 3 8 ) . A r e c e n t r e v i e w o f t h i s s u b j e c t h a s b e e nm a d e b y M c K e n z i e ( 27 ) . L a r s o n a n d R o l l e r i( 2 4) d e t e r m i n e d b y m o v i n g - b o u n d a r y e l e c t ro -p h o r e s i s t h a t t h e p r o t e o s e - p e p t o n e f r a c t i o nc o n s i s t s o f t h r e e c o m p o n e n t s d e s i g n a t e d 3 , 5,a n d 8, o f i n c r e a s i n g m o b i l i ty . C o m p o n e n t 8s h o w e d a m o b i l i t y a t p t I 8 .6 f a s t e r t h a n t h a t o fs e r u m a l b u m i n ; C o m p o n e n t 5 w a s b e t w e e n~ - l a e t a l b u m i u a n d f l - l a c t o g l o b u l i n ; a n d C o m p o -

1 1~ . p . T h o m p so n an d P . D . t t o ag ] an d , p e r so n a lco m m u n ica t io n .J . D A r a Y S CI EN C E V O L . 5 2 N O . 8

n e n t 3 h a d a m o b i l i t y s l i g h t l y s l o w e r t h a nt h a t o f a - la c t a lb u m i n . I t w a s n o t k n o w n w h e t h e rt h e s e p r o t e i n s w e r e p r e s e n t i n n a t i v e m i l k o rw h e t h e r t h e y w e r e a r t i f a c t s r e s u l t i n g f r o m t h eh e a t t r e a t m e n t , a s t h o u g h t b y e a r l y w o r k e r sin t h e f i e ld .

B r u n n e r a n d T h o m p s o n ( 5 ) c o m p a r e d s e v e ra lm e t h o d s o f i s o l a t i n g t h e p r o t e o s e - p e p t o n e - l i k ef r a c t i o n s fr o m t h e s a m e m i l k . A l l p r e p a r a -t i o n s w e r e h e t e r o g e n e o u s b y m o v i n g - b o u n d a r ye l e c t r o p h o r e s i s , a l t h o u g h t h e y a p p e a r e d t o c o n -t a i n a c o m m o n m a j o r c o m p o n e n t , a n d th e y a l lc o n t a i n e d c a r b o h y d r a t e a n d a r e l a t i v e l y h i g ha m o u n t o f p h os p h o r u s . O n u l t r a c e n t r i f u g a t i o n ,t w o b o u n d a r i e s w e r e o b s e r v e d , w i t h s e d i m e n t a -t i o n co e f f ic i en t s o f a b o u t 0 .8 S an d 2 .8 S .I n d i c a t i o n s a r e t h a t e l e c t r o p h o r e t i c C o m p o -n e n t 5 i s a n a t i v e p r o t e i n i n m i l k , s i n c e J e n n e s s( 1 7 ) h a s i s o l a t e d a p r o t e i n r e s e m b l i n g t h i sc o m p o n e n t u s i n g m i l d f r a c t i o n a t i o n p r o c e d u r e s .F r a c t i o n a t i o n w a s a c c o m p l i s he d b y s a l t in g o u tt h e c a s ei n , l a c t o p e r o x i d a s e , a n d o t h e r p r o t e i n sb y s a t u r a t i o n o f r a w s k i m m i l k w i t h s o d i u mc h l o ri d e . T h e p r e c i p i t a t e w a s r e d i s p e r s e d a n d

t h e c a s e i n p r e c i p i t a t e d b y a d d i t i o n o f a c i d top i t 4 .6 . A n i o n e x c h a n g e r e s i n w a s t h e n u s e dt o a d s o r b l a e t o p e ro x i d a s e f r o m t h e p r o t e i n t h a tr e m a i n e d i n s o l u t io n , t h e n C o m p o n e n t 5 w a ss e l ec t iv e l y p r e c i p i t a t e d b y c a r e f u l a d j u s t m e n to f th e p t I t o a b o u t 4. 5. C o m p o n e n t 5 w a s 9 0p u r e , b a s e d o n a r e a m e a s u r e m e n t s o f i t s e l e c -t r o p h o r e t i c p a t t e r n o b t a i n e d a t p H 8 .6, b u ts o m e w h a t m o r e h e t e r o g e n e o u s a t p i t 2 .3 . T h ee l e c t ro p h o r e t ic m o b i l it y o f C o m p o n e n t 5 w a s- - 4 . 5 X 1 0 - 5 / c m 2 / V - I / s e c - 1 a t 0 . 1 i o n i c s t r e n g t ha n d p Y I 8 .6 i n v e r o n a l b u f fe r . I t c o n t a i n s a b o u t1 . 2 p h o s p h o r u s , i s s o l u b l e i n c a l c iu m c h l o r i d ea t 0 . 02 a n d 0 . 2 ~ c o n c e n t r a t i o n s , a n d i s n o tc l o t t e d b y r e n n i n , e i t h e r i n t h e p r e s e n c e o r i nt h e a b s e n c e o f c a l c i u m .B r u n n e r a n d c o - w o r k e r s ( 2 2 , 2 3, 29 ) m o r e r e -c e n t l y h a v e i s o l a t e d a n d f u r t h e r c h a r a c t e r i z e d

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C SEIN COMPONENTS 1 1 6 3C o m p o n e n t s 3 , 5 , a n d 8 . C o m p o n e n t 3 , aw h e y p r o t e i n b y t h e i r d e s ig n a t io n , w a s i s o -l a te d f r o m b o t h r a w a n d h e a t e d s k i m m i l k b y ac o m b i n a t i o n o f s a l t i n g - o u t w i t h a m m o n i u m s u l -f a t e a n d p r e p a r a t i v e g e l e l e c t r o p h o r e s i s ( 2 9 ) .T h e p r o t e i n g a v e a s i n g l e z o n e b y p o l y a c r y l a -m i d e g e l e l e c t r o p h o r e s i s a n d ~ sh ow e d, b y m o v i n go u t b o u n d a r y e l e c t r o p h o r e t i c d a t a , a n i s o e le c t ri cp o i n t o f p H 3 .7 . I t h a d a s e d i m e n t a t i o n c o ef fi-c i e n t o f 3 . 4 S a n d a m o l e c u l a r w e i g h t o f 1 2 5 , -0 0 0 a s d e t e r m i n e d b y s e d i m e n t a t i o n - e q u i l i b r i u m .I t c o n t a i n e d 1 7 . 2 c a r b o h y d r a t e , c o n s i s t in g o f7 . 2 h e x o s e ( g a l a c t o s e a n d m a n n o s e ) , 6 . 0h e x o s a m in e ( g l u c o sa m i n e a n d g a l a c to s a m i n e ) ,1 . 0 f u c o s e , 3 . 0 s i at ic a c i d ; i t a l s o c o n t a i n e d0 . 5 p h o s p h o r u s . C o m p o n e n t 3 c o n t a in e ds m a l l a m o u n t s o f a r o m a t i c a m i n o a c i d s a n dm e t h i o n i n e , b u t l a r g e a m o u n t s o f a s p a r t i c a n dg l u t a m i c a c i d s a n d n o c y s t i n e o r c y s t e i n e .

C o m p o n e n t 8 w a s i s o l a t e d f r o m t h e p r o t e o s e -p e p t o n e f r a c t i o n o f ac i d w h e y a n d f r o m b o t hi soe l ec t r i c and mi ce l l a r c a se i n compl exes (22) .T h i s c o m p o n e n t i s f o u n d a s s o c i a t e d w i t h K - c a -s e in a n d m a y b e i d e n t i c a l t o X -c as ein ( 2 5 ) . I tc o n t a in s c a r b o h y d r a t e a n d 1 . 2 p h o s p h o r u s .I n s u b s e q u e n t w o r k C o m p o n e n t 8 w a s r e s o l v e db y g e l f i l t r a t i o n i n t o t w o h e t e r o g e n e o u s z o n e s ,d e s i g n a t e d 8 - f a s t a n d 8 -s l o w ( 2 3 ) . B o t h a r ep h o s p h o g l y c o p r o t e i n s , s i n c e t h e y c o n t a i n 0 . 9 7a n d 3 . 2 9 p h o s p h o r u s , r e s p ec t iv e l y , a n d v a r y -i n g a m o u n t s o f h e x o s e , h e x o s a m i n e , a n d s i a l i ca c i d . C o m p o n e n t 5 w a s a l s o i s o l a t e d , a l t h o u g ht h e f ra c t i o n a t i o n p r o c e d u r e w a s n o t g i v en . C o m -p o n e n t 5 s h o w e d u d o u b l e t o n p o l y a c r y l a m i d eg e l a t p H 8 .6 . T h e C o m p o n e n t 8 f r a c t i o n s c o n -t a i n h i g h c o n c e n t r a t i o n s o f g l u t a m i c a c i d , a s -p a r t i c a c i d , s e r i n e , a n d t h r e o n i n e , b u t n o c y s -t e i ne or cys t i ne . Sed i me nt a t i on coe f f i ci en tsS 2o .w f o r C o m p o n e n t s 5, 8 - f a s t , a n d 8 - s lo ware 1 .22 S , 0 . 78 S , and 1 .35 S . Th e cor re spo nd -i n g m o l e c u l a r w e i g h t s a s d e t e r m i n e d b y s e d i -m e n t a t i o n - e q u i l i b r i u m a r e 1 4 , 3 0 0 , 4 , 1 0 0 , a n d9 ,9 0 0. S i n c e C o m p o n e n t s 5 , 8 - f a s t , a n d 8 - s lo wa r e f o u n d i n m i c e l l a r c a s e i n a s w e l l a s t h es e r u m , B r u n n e r a n d c o - w o r k e r s ( 5 ) s u g g e s t t h a tt he se pro t e i ns a re ca se i ns .G a n g u l i e t al . ( 7 ) f o u n d t h a t t h e p r o t e o s e -p e p t o n e f r a c t i o n a m o u n t s t o a b o u t 1 7 3 m g p e r1 0 0 m l o f m i l k a n d t h a t t h i s v a l u e i s g r e a t e r i nc o l o s t ru m . T h e y d e t e rm i n e d t h e a m i n o a c i dc o m p o s i t i o n o f t h e p r o t e o s e - p e p t o n e f r a c t i o na n d a l s o s h o w e d t h a t c y s t i n e a n d s e r i n e w e r e

t h e o n l y N - t e r m i n a l a m i n o a c i d s p r e s e n t .Acid glycoproteins B e z k o r o v a i n y ( 3 , 4 )f o u n d t h a t t h e a c i d g l y c o p r o t e i n s f r o m b o v i n eb l o o d s e r u m , e o l o s t r u m a c i d w h e y , a n d m i l ka c i d w h e y w e r e a d s o r b e d b y D E A E - c e l l u l o s ea t p H 4 .3 a n d l o w i o n i c s t r e n g t h . T h e a c i d

g l y c o p r o t e i u s c o u l d t h e n b e e lu t e d a t p i t 5 .0w i t h b u f f e r s o f i n c r e a s i n g i o n i c s t r e n g t h . C I ~ -c e l l u l o s e w a s a l s o u s e d f o r f u r t h e r f r a e t i o n a -t io n . T w o m a j o r f r a c t i o n s , d e s i g n a t e d M - 1a n d M - 2 , w e r e i s o l a te d a n d s t u d i e d . T h e b lo o ds e r u m w a s s h o w n t o c o n t a i n m o s t l y o r o s o m u c o i di n t h e M - 1 f r a c t i o n a n d a n M - 2 g l y c o p r o t e i n .A f r a c t i o n c o n t a i n in g o r o s o m u c o i d a n d M - 2 g l y -c o p r o t e i n w a s a l s o i s o l a t e d f r o m b o v i n e c o l o s -t r u m , w h i l e m i l k c o n t a i n e d n o o r o s o m u c o i d a n do n l y t r a c e a m o u n t s o f t h e M - 2 g l y c o p r o t e i n .H o w e v e r , b o t h m i l k a n d c o l o s t r u m s h o w e d i nt h e 1V [-1 f r a c t i o n a p h o s p h o g ~ y c o p r o t e i n o f l o wa b s o r p t i o n a t 28 0 m ~ . I t s c a r b o h y d r a t e a n dp h o s p h o r u s c o n t e n t a n d i ts e l e c tr o p h o r e t ic m o -b i l i t y a t pH 4 . 5 a n d 8 . 6 w a s s h n i l a r t o t h a t o ft h e p r o t e o s e - p e p t o n e f r a c t i o n a n d ~- ca se in . T h ep h o s p h o g l y c o p r o t e i n d i f f e r e d f r o m ~ - c a s e i n , a si n d i c a t e d b y d i f f e r e n c e s i n i t s s e d i m e n t a t i o n b e -h a v i o r a n d a m i n o a c i d c o m p o s i t i o n . T h e p h o s -p h o g l y c o p r o t e i n c o n t a i n e d 3 . 1 h e x o s e , 2 . 4N - a c e t y l h e x o s a m i n e , a n d 4 . 0 s i al ic a c id . I t sp h o s p h o r u s c o n t e n t w a s 0 . 4 4 .T h e M - 1 a c i d g l y c o p r o t e i n f r a c t i o n s o b -t a i n e d f r o m t h e m i l k a n d c o l o s t r u m o f i n d i -v i d u a l c o w s w e r e f u r t h e r r a c ti o n a t e d b y g e lf i l t r a t i o n a n d c h r o m a t o g r a p h y o n D E A E -S e p h a d e x , t o g i v e s a m p l e s t h a t w e r e h o m o g e n e -o u s b y n l t r a c e n t r i f u g a t i o n a n d m o v i n g - b o u n -d a r y e le c t r o p h o r e s is . T h e s e s a m p l e s sh o w e de l e c t r o p h o r e t i c m o b i l i t i e s a t p H 8 . 6 o f - - 5 . 7t o - - 5 . 8 5 1 0 - S /c m 2 / ~ T - 1 /s e c 1 a n d s e d i m e n t a -t i on coe f f i c i en t s o f ] . 03-1 .30 S , wi t h mol ecu l a rw e i g h t s o f f r o m 9 , 50 0 to 1 2,8 0 0. S i n c e t h e yc o n t a i n e d s e v e r a l N - t e r m i n a l a m i n o a c id s a n di n s o m e s a m p l e s s h o w e d s e v e r a l b a n d s o n g e le l e c t r o p h o r e s i s , t h e y m a y c o n s i s t o f a s e r i e s o fr e l a t e d m o l e c u l a r sp e c ie s . T h e a m i n o a c i d a n dc a r b o h y d r a t e c o m p o s i t i o n o f M - 1 g l y c o p r o t e i n sf r o m i n d i v i d u a l m i l k s w a s a l s o v a r i a b I e . T h ea c i d g l y c o p r o t e i n s c o n t a i n e d r e l a t i v e l y l a r g ea m o u n t s o f g l u t a m i c ac i d , p r o l in e , t h r e o n i n e ,and i so l euc i ne , whi l e t he ba s i c ami no ac i dsw e r e l o w . V a l u e s f o r c y s t i n e w e r e n o t r e p o r t e d .L i k e t h e c a s e i n s , t h e y s h o w e d h i g h n e g a t i v es p e c if ic r o t a t i o n s . T h e c o l o s t r u m M - 1 g l y c o -p r o t e i n f r a c t i o n c o n t a i n e d g a l a c t o s e , g l u c o s a -mi ne , ga l ac t osa mi n e , and s i a li c a c i d . S i m i l a rc a r b o h y d r a t e s w e r e f o u n d i n t h e m i l k g l y c o -p r o t e i n f r a c t i o n , w i t h t h e e x c e p t i o n t h a t t h et y p e o f h e x os a m i n es w a s n o t d e t er m i n e d . T h es i a l i c a c i d w a s e a s i l y r e m o v e d b y v i r a l n e u r a -m i n id a s e. P h o s p h o r u s v a l u es w e r e n o t re p o r t e df o r t h e s e p r e p a r a t i o n s .A c k n o w l e d g m e n t s

T h e a u t h o r i s i n d e b t e d t o J IT C u s t e r f o r t h ev e r t i c a l g e l e l e c t r o p h o r e t i c p a t t e r n s a n d t h a n k sJ . D A IR Y SC IEN C E V O L . 5 2 N O . 8

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1 1 6 4 G R O V E S

D r . M . P . T h o m p s o n f o r p r o v i d i n g u n p u b l is h e dd a t a o n t h e a s 2 - a n d a s 3 - e a s e i n s .eferences( 1 ) A s c h a f f e n b u r g , R . 1 9 61 . I n h e r i t e d c a s e in

v a r i a n t s in c o w 's m i l k . N a t u r e , 1 9 2 : 4 3 1 .( 2 ) A s c h a f f e n b u r g , R . 1 9 63 . I n h e r i t e d c a s e i nv a r i a n t s i n c o w ' s m i l k . I I . ] 3 r e e d d i f f e r e n c e si n t h e o c c u r re n c e o f f i - c a s e in v a r i a n t s . J .D a i r y R e s ., 3 0 : 2 5 1 .( 3 ) t ~ e z k o r o v a i n y , A . 1 9 6 5 . C o m p a r a t i v e s t u d yo f t h e a c i d g l y e o p r o t e i n s i s o l at e d f r o mb l o o d s e r u m , c o l o s t r u m a n d m i l k w h e y .A r c h . ] 3 i o c h e m . B i o p h y s . , 1 1 0 : 5 5 8 .( 4 ) ] 3 e z k o r o v a in y , A . 1 9 6 7 . P h y s i c a l a n d c h e m i -c a l p r o p e r t i e s o f b o v i n e m i l k a n d c o l o s t ru mw h e y M - 1 g l y c o p r o t e i n s . J . D a i r y S c i. ,50 : 1368.( 5 ) ] 3 r u n n e r , J . 1 % ., a n d M . P . T h o m p s o n . 1 9 6 1 .C h a r a c t e r i s t i c s o f s e v e r a l m i n o r - p r o t e i nf r a c t i o n s i s o la t e d f r o m b o v i n e m i lk . J .D a i r y S c i . , 4 4 : 1 2 2 4 .( 6 ) E i - N e g o u m y , A . M . 1 9 67 . P o l y m o r p h i s mi n 5 , - c a s e i n f r a c t i o n s f r o m t h e m i l k o f i n d i -v i d u a l c o w s . ] 3 i o e h im . e t ] 3 io p h y s . A c t a ,140 : 503 .( 7 ) G a n g u l i , N . C . , B . S . G u p t a , O . N . A g a r -w a l a , a n d V . R . ] 3 h a l e r a o . 1 9 6 5 . S t u d i e so n t h e p r o t e o s e - p e p t e n e f r a c t i o n o f m i l k :I s o l a t i o n a n d p r o p e r t i e s o f p r o t e o s e - p e p t o n ea n d p r o t e o s e f r o m c o w a n d b u f f a l o m i l k .

I n d i a n 5 . ] 3 i o c h e m . , 2 : 1 8 9 .( 8 ) G o r d o n , W . G . , W . F . S e m m e t t , a n d M . B e n -d e r . 1 9 5 3. A m i n o a c i d c o m p o s i t i o n o f 7 -c a -s e i n . 5 . A m e r . C h e m . S o e ., 7 5 : 1 6 7 8 .( 9 ) G r o v e s , M . L . , a n d W . G . G o r d o n . 1 9 6 9. I s o -a t i o n a n d c o m p o s i t i o n o f p o ~ y m o r p h s o f~- a n d E - c a s e in . ( U n p u b l i s h e d d a t a . )( 1 0 ) G r o v e s , M . L . , W . G . G o r d o n , a n d C . A .K i d d y . 1 9 6 8. P o l y m o r p h i s m o f e l e c tr o -p h o r e t i c a l l y s l o w - m o v in g c a s ei n s a n d t h e i rr e l a t i o n s h i p t o t h e 7 - c a s e i n a n d f l - e a se i nv a r i a n t s . J . D a i r y S e i. , 5 1 : 9 4 6 .( 1 1 ) G r o v e s , M . L ., C . A . K i d d y , a n d W . G .G o r d o n . 1 9 6 8. P o l y m o r p h i s m o f t e m p e r a -t u r e s e n s it i v e (T S ) - c a s e i n s . 1 5 6 t h N a t i o n a lM e e t i n g A m e r . C h e m . S o e . , A t l a n t i c C i t y ,N . J . , S e p t e m b e r 8 t o 13 . A b s t r . B i o l ., 2 5 5 .( 1 2 ) G r o v e s , M . L . , a n d C . A . K i d d y . 1 9 6 8. P o l y -m o r p h i s m o f y - ca s ei n i n c o w ' s m i l k . A r c h .]31ochem. B iop hy s . , 12 6 : 188 .( 1 3 ) G r o v e s , M . L ., T . L . M e M e e k i n , N . J . H i p p ,a n d W . G . G o r d o n . 1 9 62 . P r e p a r a t i o n o ff l- a n d 5 , - c a se i n s b y c o l u m n c h r o m a t o g r a p h y .] 3 io c h im . e t B i o p h y s . A c t a , 5 7 : 1 97 .( 1 4 ) t t i p p , N . J . , M . L . G r o v e s , 5 . I t . C u s t e r , a n dT . L . M c M e e k l n . 1 9 5 0 . S e p a r a t i o n o f 7 - c a -s e in . 5 . A m e r . C h e m . S o e. , 7 2 : 4 9 2 8 .( 1 5 ) H i p p , N . J . , M . L . G r o v e s, J H . C u s t e r , a n dT . L . M c M e e k in . 1 9 52 . S e p a r a t i o n o f a ' ,f l- , a n d - / - c a s ei n . J . D a i r y S c i . , 3 5 : 2 7 2 .( 1 6 ) H o a g l a n d , P . D . , a n d E . ]3 . K a l a n . 1 9 6 5 .A m i n o a c i d c o m p o s i t i o n o f a m i n o r p r o t e i no f c a s ei n . 1 5 0 t h N a t i o n a l M e e t i n g A m e r .J . D A I R Y S CI E I~ C ]~ V O L . 5 2 N O . 8

C h e m . S oc ., A t l a n t i c C i t y , N . J . , S e p t e m b e r1 2 t o 1 7 . A b s t r . , 7 8 C .( 1 7 ) J e n n e s s , R . 1 9 5 9 . C h a r a c t e r i z a t i o n o f m i l ks e r u m p r o t e i n c o m p o n e n t 5 . J . D a i r y S e i .,42 : 895.( 1 8 ) J e n n e s s , R ., ]3 . L . L a r s o n , T . L . M c M e e k i n ,A . M . S w a n s o n , G . H . W h i t n a h , a n d 1%. M e L .W h i t n e y 1 9 56 . N o m e n c l a t u r e o f t h e p r o -t e i n s o f b o v i n e m i l k . J . D a i r y S o l. , 3 9 : 5 3 6.( 1 9 ) K i d d y , C . A . , 1 . F . P e t e r s o n , a n d ~ . C .K o p f l e r . 1 9 6 6 . G e n e t i c c o n t r o l o f t h e v a r i -a n t s o f f l -c a s e in A . J . D a i r y S c i. , 4 9 : 7 4 2 .( 2 0 ) K i e r m e i e r , F . , a n d E . P e t z . 1 9 6 7 . A n en -z y m e i n m i l k w h i c h o x i d i z e s s u l f h y d r y ]g r o u p s . I . I s o l a t i o n a n d c h a r a c t e r i z a t i o n o ft h e e n zy m e . Z . L e b e n s m . - U n t e r s u c h . u . -F o r s c h . , 1 3 2 : 3 4 2 .( 2 1 ) K i e r m e i e r , F . , a n d K . R a n f f t . 1 9 68 . A ne n z y m e i n m i l k w h i c h o x i d i z e s s u l f h y d r y lg r o u p s . I V . E f f e c t o f s t o r a g e a n d h e a t i n gt e m p e r a t u r e . Z . L e b e n s m . - U n t e r s u c h . u .-F o r s c h . , 1 3 8 : 8 0 .( 2 2 ) K o l a r , C . W . , J r . , a n d f t . 1%. ] 3 r u n n e r . 1 9 6 5 .I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f m i l k p r o -t e i n C o m p o n e n t '8 . J . D a i r y S c i ., 4 8 : 7 72 .( 2 3 ) K o l a r , C . W . , J r . , a n d J . 1%. ] 3 r u n n e r . 1 9 6 8 .C h a r a c t e r i s t i c s o f p r o t e o s e - p e p t o n e c o m p o -n e n t s f i v e a n d e i g h t . J . D a i r y S c i ., 5 1 : 9 4 5 .( 2 4 ) L a r s o n , B . L . , a n d G . D . R o l l e r i . 1 9 5 5 . H e a td e n a t u r a t i o n o f t h e s p e ci fi c s e r u m p r o t e i n si n m i l k . J . D a i r y S c i ., 3 8 : 3 5 1 .( 2 5 ) L o n g , J . , Q . V a n W i n k l e , a n d I . A . G o u l d .1 9 5 8. I s o l a t i o n a n d i d e n t i f i c a t i o n o f k - ca -s e in . J . D a i r y S c i. , 4 1 : 3 1 7 .( 2 6 ) M a c k i n l a y , A . G . , a n d R . G . W m k e . 1 9 6 5 .F r a c t l o n a t l o n o f S - c a r b o x y - m e t b y l - K - c a s e i na n d c h a r a c t e r i z a t i o n o f t h e c o m p o n e n t s .B i o c h i m . e t B i o p h y s . A c t a , 1 0 4 : 1 6 7.

( 2 7 ) M c K e n z i e , H . A . 1 9 6 7. M i l k p r o t e i n s . A d -v a n c e s i n P r o t e i n C h e m ., 2 2 : 5 5 .( 2 8 ) M e l l a n d e r , O . 1 9 3 9 . E l e c t r o p h o r e t i c s tu d i e so n c a s e i n . ] 3 i o c h e m . Z . , 3 0 0 : 2 4 0 .( 2 9 ) N g , W e S u , a n d J . 1%. B r u n n e r . 1 9 6 7 . W h e yp r o t e i n c o m p o n e n t t h r e e . J . D a i r y S c i. ,50 : 950 .( 3 0 ) P e t e r s o n , 1%. F . 1 9 6 3. H i g h r e s o l u t i o n o fm i l k p r o t e i n s o b t a i n e d b y g e l e l e c t r o p h o r e s i s .J . D a i r y S c i . , 4 6 : 1 1 3 6 .( 3 1 ) P e t e r s o n , R . F . , a n d F . C . K o p f l e r . 1 9 6 6 .D e t e c t i o n o f n e w t y p e s o f f l - c as e i n b yp o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s a t a c i dp H : a p r o p o s e d n o m e n c l a t u r e . B i o c h e m .] 3 i o p h y s . R e s . C o m m u n . , 2 2 : 3 8 8 .( 3 2 ) P e t e r s o n , 1%. F . , L . W . N a u m a a , a n d D . F .H a m i l t o n . 1 9 66 . A m i n o a c i d c o m p o s i t i o no f s i x d i s t i n c t t y p e s o f fl - ca s e in . J . D a i r ySc i . , 49 : 601 .( 3 3 ) 1% o se , D . 1 9 6 8 . R e l a t i o n b e t w e e n m i c e l l a r

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J. DAI~Y SCIenCE VOL 52, No. 8