identification of the k-casein among the components of whole goat casein is 0022030266879468

8/13/2019 Identification of the K-Casein Among the Components of Whole Goat Casein is 0022030266879468

1/4

Identification of the K C asein A m on g the Com ponentsof W hole G oat Casein

C A Z I T T L E an d J H C U S T E REastern Re gion al Research Laboratory

Eastern Utiliz atio n Research and Development DivisionUSDA Philadelphia Pennsylvania

Abs t rac t

Goat ,-casein in the reduced state, prob-ably the nat ura l state, has a mobility onacrylamide gel electrophoresis at pH 9,very close to tha t of the fi-easeins. The goat

K-casein was identified by clotting withrennin, by specific changes in the electro-phoretic pattern after rennin action, andby nonpreeip itation with calcium ions. Itstabilized goat a,-casein so that a~-caseinwas not precipitated with calcium ions.PolyaczTlamide gel electrophoresis of goatK-casein in some cases gave a single band(together with identifiable contaminants),and, in ot her cases, gave a mul tipl ici ty ofslower-moving bands. In all cases only asingle major band was observed when the

~-casein was reduced with mereaptoethanol.Components with the mobilities of para-K-casein were pre sent in considerable amount sin some preparations of ~-casein.

Goat casein is characterized by two strongbands in the fl-casein region ~ on gel electro-phoresis at pH 9. On trea ting whole goat caseinwith urea-sulfuric acid for the isolation ofu-casein (11), preparations were obtained thaton eleetrophoresis gave a principal band movingin the fi-casein region. Other prepara tion s on

electrophoresis gave multiple slower-movingbands, but these preparations, too, gave a prin-cipal band moving in the fl-easein region whentreated with mer eaptoetha nol for electrophoresis.This paper will show that the preparations ob-tained by the urea-sulfuric acid method areindeed K-casein and will report some interestingproperties of goat u-casein.

M a t e r i a l s a n d M e t h o d s

Go at mi lk . Fresh goat milk, both pooledand f rom two indivi dual goats, was used in thefollowing studies. No majo r differences wereobserved in the pooled and in these individualsamples. The milk was warmed to 30 to 35 Cand skimmed in a separator. Prep arat ion of

Received for publ ication March 9, 1966.

the caseins from this milk is described in thenext section.

Po ly acry l amid e g e l e l ee t ro p h o res i s .Electro-phoresis was performed in a vertical cell atpH 9.0 in a 4.5 ~ urea with an aemTlamide con-eentration of 7.0 (7). In some experimentsmercaptoetha nol was added (10).

Exper imental P rocedures and Resul t s

Wh o le case in was prepared from skimmedgoat milk by acidification with HCI . The mosteffective floeeulation and rapid filtration wasobtained when the p H was 4.2. The casein waswashed twice with water main tain ed at the samepH. The casein was subsequent ly washed withethanol, acetone, and ethyl ether, a processwhich extracted lipids and dried the casein.

~ Casein was prepared from whole goatcasein by the sulfuric acid-urea procedure (11).Goat casein behaved in this procedure as didbovine casein. The lyophile- dried K-casein ob-tained was about 13 of' the whole casein used.These impure (see electrophoretie patterns)K-caseins had t he following composition: 8volatile mat ter (mo istu re) , 5 fixed ash,15.5 N, 0.6 P, and 0.8 S, all on amoistur e-fre e basis. The same values for thewhole casein were 8, 2, 15.6, 0.8, and 0.8.

The sialic acid content of these preparations

was determined by the method of Warren (9).The whole caseins contained 0.13 , the u-caseins 0.3 sialie acid. Alt hough these goat~-caseins still contain other components, it iseviden~ that they contain considerably less sialieacid than bovine K-casein (2.0 ). Sialic acidcontent of goat and cow whole casein, is respee-tively, 0.13 and 0.36 . AlMs and Joll~s (1)have foun d goat casein to cont ain 0.13 sialieacid, the glyeopeptide 3.0 .

The precipit ate obtained in the ~-casein isola-tion, termed a ~ f l casein, was suspended in

water, dissolved with NaOH, dialyzed for 18 hragai nst H~O, pre cip ita ted with 0.1 N HC1, anddried with organic solvents, as was the wholecasein. Pol yacr ylam ide gel eleetrophoresis of

M. P. Thompson, unpublished studies ; R.Aschaffenburg, personal word to M. P. Thompson.

788


2/4

I D E N T I F I C A T I O N O F K C A S E I N 7 8 9

~IG. i. Polyacrylamide gel electrophoresis ofwhole goat casein, K-casein, and a~ fl casein. 1, 2Whole case in; 3, ~ K-caseln; 5, 6 a~-fl casein. Pat-terns 2, 4, and 6 were obtained with mereapto-ethanol present. The doublet of intermed iate mo-bility, very evident in Patterns I, 2, 5, and 6, isfl-casein. The components of grea tes t mobili ty area~-casein. The majo r band of the mercoptoethanol-trea ted K-casein, presumably reduced K-casein,moves jus t ahead of the fl-casein. This prepara tionwithout mercaptoethaaol gives the multiple bands,presumably corresponding to oxidized K-casein,showing between the fl-caseins and the origin.

the goat whole casein, K-casein, and the a~-flcasein preparation are shown in Figure 1, bothwithout and with mercaptoethanol. Some of the

K-casein preparations gave a pattern almostidentical to no. 4 without the use of mercapto-ethanol. The urea- sulf uric acid method ex-cluded most of the fl-easein, but considerablea~-casein is still present in this K-casein prepa-raion. Other K-casein pre par ati ons have beenconsiderably freer of a~-casein (see Figure 2),with most of this component in the a~ fl caseinprepara tion. An electrophoretic comparison ofgoat whole casein with the bovine casein isshown in Fi gu re 2. Alth ough the fl-easeins have

about the same mobility, the goat a~-casein hasa lower mobility than the bovine as-casein. Alsoshown is another K-casein preparation, withoutand with mercaptoethanol. This prep arati onshows the presence of some spontaneouslyformed para-K-easein (see late r). The influenceof mereaptoethanol on another, more typicalprepa ratio n (sharp bands, little of the diffusestaining, only trace of para-K-easein) of K-casein is shown in Figure 3.

Since the K-casein preparation gave thestrong band in the fl-casein region, tests wereperformed to determine whether the prepara-tion had the character istics o f K-casein. Neut ral1 solut ions of goa t whole, K-, and as-caseinswere mixed with 0.1 M CaCl~, final concentra-tion 0.01 M. The solution of whole casein be-came white, the K-casein solution remainedclear, and the a~-casein preci pita ted. The samecaseins at pH 6.5 with 0.05 M NaC1 (no CaCl~)present were treated with rennin (1:100) at37 C. The whole casein and the as-casein re-maine d clear ; the K-casein became white. Thegoat as-casein, 80 pre cipi tate d by 0.020 MCaCl~, was stabilized by the K-casein at a weightratio of 1:7, so that only 20 of the a,-caseinwas precipitated.

All these properties are characteristic of K-casein, based on what is known about bovineK-casein. The most striking demo nstra tion tha tthe sulfuric acid-urea preparation is indeedmainly K-casein was obtained by following theaction of rennin with polyacrylamide gel elec-trophoresis. The casein was treated with ren-nin, as described above, but in this experiment

FIG. 2. Eleetrophoretic comparison of goa t andbovine whole caseins and goat K-casein withoutand with mercaptoethanol present. 1. Whole goatcasein. 2. Goa t K-casein. 3. Goat ~-casein wi th mer-captocthanol present. 4. Whole bovine casein. Thetwo slow-moving bands in no. 3 correspond topara-K-casein.

FIG. 3. Influence of mercaptoethanol on po]y-acrylamide gel electrophoretic pattern of goat K-casein. 1. K-Casein without mercaptoethanol. 2. K-Casein with mercaptoethano].


3/4

790 C . A . Z I T T L E A N D f t. I -I ~ 3 U S T E t ~

no NaC1 was added. Small samples were re-moved at i ntervals and NaC1 added to de terminethe course of ren nin action. The action ap-peared to be complete at 30 rain treat ment for70 rain gave the same electrophoretic results)an d the sam ple wa s f rozen an d f reeze-dr ied.Th e electrophoret ic resu lts are sh ow n in Figure4 . T he s trong ba nd in the B-casein reg ion then-casein band is to tally trans forme d by renninto tw o b an d s o f mu ch less mo bi l ity. Th ese r e-sults are reminiscent of the action of rennin onbovine K-casein 4, 5), a lthough both goa t K-and para-K-caseins have greater positive mo-bilities than the corresponding bovine compo-nents. Ident ical patt erns were obtained whetherthe ren ni n acted for 30 or 70 rain. The complexpat ter n of the conta nfinatin g as-casein remainedunchanged, suggesting that during the periodthe rennin acted no nonspecific proteolysisoccurred.

One preparation of K-casein, obtained fromthe whole casein of a second lot of pooled goatnfilk, showed some differences from the otherprepa ratio ns. These differences are shown inthe electrophoretie patt erns in Fig ure 5. Thepa tt er n no. 1) is more diffuse and, whentreated with mereaptoe thanol no. 2), two slow-moving bands appear near the origin, togetherwith the fa ste r-mo ving K-casein band. The ma-jor of these two bands appears in other prepa-rations, but not to the same extent. Resultsobtained with ren nin Figur e 4) suggested thatthese bands corresponded to para-K-casein.Treatment of this preparation with rennin, fol-lowed by mercaptoethanol reduction, trans-for med al l of the K-casein to components no. zl)whose mobilities coincided with the slow-movingbands, strongly suggesting that these bands cor-responded to para-K-casein that had been spon-taneously formed.

F I G 4. Eleetrophoretic patterns il lustrating theaction of rennin on K-casein. :1. 70-nfin treatment.2. 30-min treatment. 3. Untreated K-casein.

t s

K

I 3 4

Fro. 5. Polyacrylamide gel electrophoretic pat-terns of goat K-casein containing considerablepara--casein-like material initia lly, without and

with mercaptoethanol, before and after treatmentwith rennin. 1. K-Casein. 2. K-Casein treated withmercaptoethanol. 3. K-Casein treated with rennin.4. K-Casein treated with renni n a~ld reduced withmercaptoethanol.

iscussion

In goat whole casein two strong bands ap-pear in the B-casein region on ac~Tlamide gelelectrophoresis. In none of many samples ex-amined has a single band been observed in thisregion. 1 Present results indicate that thesebands obscure the reduced K-casein band or thatone of these bands is K-casein. The positi on ofthe K-casein band in the purified preparationrelative to the position of the two strong bandsin the whole casein, as well as the position ofthe two strong bands remaining in the as- 8casein preparation, suggest that the K-casein isobscured by these B-casein components. Theidentity of the component separated by urea-sulfuric acid as K-casein was established by anum ber of properties. The most striki ng is thechange in electrophoretic mobility broughtabout by the action of rennin. Major and minorbands of much less mobility are obtained, remi-nisce nt of bovine K-casein 4, 5). Studies withpure goat K-casein are needed to show whetherthese para-K-easein bands arise from one ortwo K-casein component s [see Reference 5) ].

Appa ren tly, goat K-casein can change to par a-K-casein without t rea tme nt with remfin. Thischange was very marked in one preparation ofgoat K-casein studied. This tra nsf orm ati on hasbeen observed with bovine K-casein, but only toa small extent. This change has been attr ibuted

3) to spontaneous breakdown of the rennin-sensitive bond or to the action of microorgan-isms. The nat ura lly occurr ing milk proteasewould appear to be a possible cause also, al-though assay 12) o f the goat whole casein giv-ing the K-casein just mentioned showed rela-tively little protease.


4/4

IDENTIFICATION OF ~-CASEIN 79

T h e tw o t y p e s o f e l e c t r o p h o r e t i c p a t t e r n s o b -t a i n e d w i t h K - c a se in , a s i n g l e b a n d a n d a m u l t i -p l i c i t y o f b a n d s, h a v e i n t e r e s t i n g i m p l i c a t i o n s .I n v i e w o f t h e si n g l e b a n d o b t a i n e d w h e n r e -d u c t io n is a c c o m p l i s h e d w i t h m e r c a p t o e t h a n o la n d f r o m a n a l o g y w i t h b o v i n e K - ca se in , t h es i n g l e b a n d i s i n t e r p r e t e d t o r e p r e s e n t t h er e d u c e d K - c as ei n. P r e s u m a b l y , t h i s i s t h e f o r ms y n t h e s iz e d b y t h e g o a t , s in c e s o m e p r e p a r a -t i o n s s h o w t h i s e l e c t r o p h o r e t i c p a t t e r n e v e nw i t h o u t r e d u c t io n . W h e n o x i d a t i o n o c c u rs , a n dt h i s a p p a r e n t l y c a n o c c u r s p o n t a n e o u s l y , am u l t i p l i c i t y o f c o m p o n e n t s r e s u l ts , a s s h o w n i nt h e e l e c t r o p h o r e t i c p a t t e r n s . T h e r e s u l t o f o x i -d a t i o n o f g o a t K - c a se in d if f e rs f r o m t h a t o f t h ec o w. I n c o w K - c a se i n, a s i z e h e t e r o g e n e i t y a p -p a r e n t l y r e s u lt s t h a t is a p p a r e n t a s a s m u d g eo n g e l e l e c t r o p h o r e s i s , w h e r e a s i n t h e g o a t K -c a s ei n , a p p a r e n t l y p o l y m e r s o f d i s c r e te s iz e sr e s u l t p r e f e r e n t i a l l y .

T h e n a t u r e o f t h e i n t e r a c t i o n s t h a t l e a d t ot h e a p p a r e n t g r e a t h e t e r o g e n e i t y in b o v i n e K -c a s e i n i s n o t c l e a r , a l th o u g h f o r m a t i o n o f d i -s u lf ie d b o n d s m u s t h a v e a m a j o r r o l e . T h e f o r -m a t i o n o f p o l y m e r s o f d i s c r e t e s i z e s p o s t u l a t e df o r g o a t K - c a se in h a s a c o u n t e r p a r t i n th eh u m a n s e r u m h a p t o g l o b u l i n s ( 2 ) . T h e h a p t o -g l o b u l in p o l y m e r s a r e tr a n s f o r m e d t o m o n o m e r sb y t r e a t m e n t w i t h m e r c a p t o e t h a n o l ( 8 ) .

T h e s e o b s e r v a t i o n s w i t h g o a t d - c a s e i n , a n dt h e r e c e n t r e p o r t o n t h e h e a t s t a b i l i t y o f g o a tm i l k ( 6 ) , i n d i c a t e t h a t g o a t d - c a s e in is a n in t e r -e s ti n g c a se i n f o r f u r t h e r s t ud y , p a r t i c u l a r l yc o m p a r a t i v e s t u d i es w i t h t h e b o v i n e ~ -e a se i n.

cknowledgmentsT h e a u t h o r s a r e i n d e b t e d t o Wi l l i a m C . Wa g n e r ,

B o y e r t o w n , P e n n s y l v a n i a , f o r t h e g o a t m i l k a n dt o D r. M a r v i n P. T h o m p s o n f o r h e l p f u l d i s c u s -s i o n s o f t h e e l e c t r o p h o r e t i e d a t a . D r. T h o m p s o n

h a s m a d e m a n y e l e c t r o p h o r e t i e e x a m i n a t i o n s o ft h e p r o t e i n s i n g o a t m i l k i n t h e c o u r s e o f t y p i n gsu rveys .

References

(1 ) A la i s , C ., and Jo l l~s , P. 1961 . Co m para t iveS t u d y o f t h e C a s e i n - g l y c o p e p t i d e s O b t a i n e dA f t e r l n n l n D i g e s t i o n o f t h e C a se i ns o ft h e M i l k o f C o w, S h e e p a n d G o a t. I I . S t u d yo f th e ~ o n - p e p t i d i c P a r t . B i o c hi m . e t B i o -phys . Ac ta , 51 : 315 .

( 2 ) A l l i s o n , A . C . G e n e t i c C o n t r o l o f H u m a nH a p t o g l o b i n S y n t h e s i s . 1 95 9. N a t u r e , ] 8 3 :1312.

( 3 ) M a c k i n l a y, A . G . , H i l l , R . J . , a n d Wa k e ,R . G . 1966 . The Ac t io n o f Re nn in on K-C a s ei n . T h e H e t e r o g e n e i t y a n d O r i g i n o ft h e I n s o l u b l e P r o d u c t s . B i o c h i m . e t B i o -phys . Ac ta , 115 : 103 .

( 4 ) M a c k i n ] a y, A . G . , a n d W a k e , R . G . ] 9 6 4 .

T h e H e t e r o g e n e i t y o f K -C a se in . B i o c h i m . e tBiophys . Ac ta , 93 : 378 .( 5 ) M a e k i n l a y, A . G . , a n d W a k e , R . G . ] 9 6 5 .

F r a c t i o n a t i o n o f S - C a r b o x y m e t h y l - K - C a s e l 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 ioch im. e t B iophys . Ac ta , 104 : 167 .

( 6 ) O r m i s t o n , E . E . , a n d H e r r e i d , E . O . ] 9 6 5 .E f f e c t o f S t e r i l i z a t i o n a n d S t o r a g e o n t h eF l a v o r o f G o a t M i l k . J . D a i r y S c i ., 48 : 50 1.

( 7 ) P e t e r s o n , R . F. 1 96 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 -pho res i s . J . Da i ry Sc i . , 46 : 1136 .

( 8 ) S m i t h i e s , O . ] 9 6 5 . D i s u l f i d e - B o n d C l e a v a g ea n d F o r m a t i o n i n P r o t e i n s . S c i e n c e , 1 5 0 :1595.

( 9 ) W a r r e n , L . 1 9 59 . T h e T h i o b a r b i t u r i c A c i dA s s a y o f S i a l i e A c i d s . J . B i o l . C h e m . , 2 3 4 :1971.

( 1 0 ) Wo y c h i k , J . H . 1965 P r e p a r a t i o n a n d P r op -e r t i e s o f Re duce d K-Case in . Arc h . B ioehem.Biophys . , 109 : 542 .

( 1 ] ) Z i t t l e , C . A . , a n d C u s t e r, J . H . ] 9 6 3 . P u r i -f i c a t io n a n d S o m e o f t h e P r o p e r t i e s o f a s -Case in and K-Casein. J . D a i r y Sc i . , 46 :

1183.( 1 2 ) Z i t t l e , C . A . 1 96 5. P u r i f i c a t i o n o f P r o t e a s ein Cow's Mi lk . J . Da i ry Sc i . , 48 : 771.

identification of the k-casein among the components of whole goat casein is 0022030266879468

Documents