T E C H N I C A L N O T E S

CHROMATOGRAPHIC EVIDENCE FOR HEAT-INDUCED INTERACTION O F a-LACTALBUMIN AND

P-LACTOGLOBULIN

The heat-induced interaction of p-lacto- globulin (P-Lg) and K-casein has received much study, but comparatively little work has been done on heat-induced interactions among the whey proteins. I t has been suggested that an interaction may occur between P-Lg and a-lac- talbuniin (a-La) when these proteins mixed in solution are heated (3, 4, 8 ) . Yanlauchi (S) , af ter heating whey proteins f o r 10 rnin a t 70 C in dilute sodiu~n chloride solution, found a new electrophoretic peak between a-La and P-Lg. Hostettler and Steiu (3) reported that the proteiu in the sediment from stored evapo- rated milk coutained a n a-La-&Lg complex whose electrophoretic mobility was intermediate between those of the proteins singly. This study presents chromatographic evidence f o r the heat-induced interaction between a-La and 8-Lg .

M..ZTERIALS AND METHODS

The ji-Lg used was obtained froin the Nu- tritional Biochelnical Company; the a-La was prepared by chromatography of the whey pro- teins. Peal; d (Figure 1 ) is considered to be

urnn was ca l ru la t~d from its absorptivity a t 280 nlp (9) .

Heating was performed a t 75 C for 30 min in 0.02 M sodium phosphate buffer of p H 6.7. After being heated, the samples were imme- diately cooled in an ice bath and equilibrated with 0.02 M sodium phosphate buffer of p H 7.0 by dialysis.

RESULTS AND DISCUSSION

Results were calculated from the following equations :

( I - A,/A,)100 (1) ( 1 - a4 6 6 / 6 8 AB) (100) (a ] ( 1 - d,/Al)lOO (3)

where A is total absorbance of one or more peaks of the various samples a s follows: Al, peak cl. unheated sample of a-La (Figure 1 ) ; A,, peak d, heated sample of a-La (Figure 2) ; A,, peak d, unheated whey proteins (Figure 3) ; A,, all pealis of the chroruatogram of un- heated whey proteins; A , peak d , heated whey

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FIG. 1. Chromatogram of 73 mg of a-laetalbumin.

almost entirely a-La. The whey was prepared by filtering the casein from skiinnlilk acidified with 1 N HCl to p H 4.6. This whey was al- lowed to staud a t that pH about 8 hr, and then filtered to remove any residual casein.

All chromatography was performed a t room temperature on DEAE cellulose by the step- wise method of Yaguchi et al. (7 ) . The effluent was collected by a fraction collector with a volu~uetric siphon of 20-ml capacity. Absorb- ance ( A ) a t a wavelength of 280 nip was de- termined with a Beckman DTJ spectrophotom- eter. The amount of a-La applied to the col-

'Data from a thesis by H. G. Hunziker, in partial fulfillment of the requirement for the M.S. degree, University of California.

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FIG. 2. Chroniatogram of 7 3 mg of alpha-lac- talhun?in heated a t 75 C for 30 nlin in phosphate buffer a t pH 6.7.

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FIG. 3. C!hronlatogram of unheated whey pro- tpins (control).

734 JOURNAL OF DAIRY SCIENCE

proteins (Figure 4) ; A., all peaks of the chro- matogram of heated whey proteins; A, peak G heated mixture of a-La and fl-Lg (Figure 5) .

When a-La was heated alone, the decrease iu this protein was calculated from Equation (1) to be 14%. However, when the whey proteins were heated the decrease in a-La [calculated from Equation (2)] was 36%, and when a mixture of a-La and fl-Lg was heated this de- creased [calculated from Equation (3)] was 84%.

The amount of a-La in peak d decreased only 14% when heated alone, but decreased 84% when heated in the presence of fl-Lg. These results indicate that fi-Lg changes the chromatographic behavior o£ a-La when these two proteins are heated together. A compari- son of Figures 3 and 4 indicates that fl-Lg also affects the chromatographic 'behavior of a-La when whey proteins are heated, though not so much as when only fi-Lg and a-La are heated together. The ratio of fl-Lg to a-La was nearly

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FIG. 4. C h r o m a t o g r m n of whey p ro te ins hea ted a t 75 C fox" 30 mln in phospha t e buffer a t p H 6.7.

¢q

i i i i i ; i i i° i i"i i ' i'i' zo

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Fro. 5. C h r o m a t o g r a m of a m i x t u r e of 250 m g of beta-lactog]obt~lin and 73 m g of a]pha-]acta tbu- rain h e a t e d a t 75 C fox' 30 rain in phospha t e buffer a t p H 6.7.

the same in both cases, considering that the ratio of fi-Lg to a-La in whey is about 3:1.

Zittle et al. (10) showed that fl-Lg com- plexes with ~-casein when these proteins mixed in solution are heated. Sawyer et al. (6) sug- gested that this complexing occurs via the sulfhydryl-disulfide reaction. Since fl-Lg con- tains sulfhydryl groups (5) and a-La contains disulfide groups (1, 2), it is evident the similar sulfhydryl-disulfide reactions may occur be- tween these two proteins.

H. G. HUNZIKER AND

N. P. TARASSUK Departnient of Food Science

and Technology University of California, Davis

t~EFERE~CES

(1) GOgDON, W. G., SE~SIE'rT, W. F., ANI) ZIEG- LER, J. 1954. Crys ta l l iue a -Lac t a lbumin : A n h n p r o v e d Method for I t s Isolat ion. Sul- f u r Dis t r ibut ion . J . Am. Chem. See., 76: 287.

(2) GOI%DON, W. G., AND ZIEGLER, ,]-. 1955. Amino Acid Composi t ion of Crystal l i~e a- Lac t a lbumin . Arch. ]3iochem. Biophys. , 57: 80.

(3) HOSTETTLER, H., AND STEIN, J . ]958. ~2ber d a s Elekt rophore t i sche Verha l ten der Milch- pro te ine bei verschlcdenen Erhe lzungsver - f ah ren . E lek t rophore t i sehe U n t e r s u c h u n g e n an evapor ier ter Milch. Londw. J a h r b . Schweiz, 72 : 163.

(4) JENNESS, R. 1858. H e a t Dena tu r a t i on of Milk Prote ins . Am. Chem. Soc. Abs t r . o f Papers , 133: 58c.

(5) PIEZ, K. A., DAVlE, E. W., FOLG ,I. E., AND GLADNER, J . A. 1961. f l -Lactoglobul ins A a n d B. I. Chromatograph ic Separa t ion a n d Amino Acid Composi t ion. J . ]~iol. Chem., 236 : 2912.

(6) SAWYEg, W. tI. , COULTER, S. T., AND JEN- ~¢ESS, R. 1963. Role of Su l fhydry l Groups in %he In terac t ion o£ K-Casein and fl-Lac- tog]obu]in. J . Dairy Sei., 46: 564.

(7) YAGUCHI, M., TARASSUK, N. P., AND HUI'.I- Z.IKEa, H. G. 1961. C h r o m a t o g r a p h y of Milk P ro t e ins on An lon-Exchange Cellu- lose, J . Da i ry Sci., 44: 589.

(8) YA~AUCHI, K. 1961. Inf luence of S u g a r s on the E]eet rophoret ic Change of Whole W h e y Pro te ins Caused by Hea t ing . J a p a - nese J . Zootech. Sci., 31: 304.

(9) /ITTLE, C. A., AND DELLAMONICA, E. S. ]955. Separa t ion of P ro t e ins by Gradient Solvent Ex t r ac t i on of a P ro t e in Precipita±e. Arch. ]=/iochem. Biophys. , 58: 31.

(10) ZITTLE, C. A., TI~[O~IPSON, M. P., CUSTEI%, J . H., AND CE~B[mZS, J . 1962. ~-Cascin f l -Lactoglobul in In t e rac t ion in Solut ion when Heated . J . Da i ry Sci., 45: 807.