an improved method for the isolation of κ-casein

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24 ° B1OCHIMICA ET BIOPHYSICA ACTA AN IMPROVED METHOD FOR THE ISOLATION OF ~-CASEIN* H. A. McKENZIE AND R. G. WAKE Department o] Physical Biochemistry, Australian National University, Canberra, A. C. T. and Department o[ Biochemistry, Stan]ord University, Palo Alto, Cali]. (U.S.A.) (Received July 26th, 196o) SUMMARY An improved method for the isolation of K-casein, the rennin sensitive material of whole casein, is described. Acid casein is used as starting material and a comparatively high yield of product is obtained. The K-casein has been characterized by sedimentation velocity, sensitivity to calcium and rennin, and by zone electrophoresis in con- centrated urea. INTRODUCTION WAUGH AND VON HIPPEL1 first demonstrated the existence of a fraction in bovine casein which is responsible for the stabilization of the micelles formed with the other casein components in the presence of calcium ions. This stabilizing fraction, or K- casein, is specifically attacked by the milk clotting enzyme, rennin 1, 2. A method for the isolation of K-casein in low yield but relatively pure form, as judged by the absence of appreciable quantities of more slowly sedimenting material at low temperature and neutral pH, has recently been described a. In the present paper an improved method for its isolation is given. It is simple, uses acid casein as starting material, and gives a comparatively high yield of product which is free from slowly sedimenting components. In addition, the fractions have been characterized by the technique of zone electrophoresis in concentrated urea 4 in order to follow the fractionation procedure and to detect minor impurities. 20 g acid casein, prepared by the method of HIPP et al. 5, are dissolved at pH 7.0- 7.5 with I N NaOH and the final volume is made up to 300 ml. After cooling to 2 °, 30 ml 4 M CaC12 are added at pH 6.5-7.0 as described by WAUGH AND YON HIPPELL Stirring is continued for I h and then the calcium-sensitive casein is precipitated by warming to 35 °. The precipitate (fraction I) is removed by centrifuging at room temperature, first for 3o rain at 9o0 × g and then for 3o rain at 4o,ooo × g. To the clear supernatant are added IOO ml 1.5 M potassium oxalate at pH 6.5-7.0 as de- scribed by WAUGH AND VON HIPPEL1. The calcium oxalate is removed by centrifuging for 30 rain at 40,000 × g and 5 °. The supernatant is warmed to 25 °, and anhydrous Na,SO4 (25 g/Ioo ml) added with stirring. After I h the precipitate (fraction II) is obtained by centrifugation at 9o0 × g for 30 rain. It is dissolved in 60 ml water and * Preliminary work was carried out in the Physico-Chemical Unit, C.S.I.lZ.O. Division of Food Preservation and the Biochemistry Department, University of Sydney (Australia). Biochim. Biophys. Acta, 47 (1961) 240-242

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Page 1: An improved method for the isolation of κ-casein

24 ° B1OCHIMICA ET BIOPHYSICA ACTA

AN I M P R O V E D M E T H O D F O R T H E I S O L A T I O N OF ~-CASEIN*

H. A. M c K E N Z I E AND R. G. W A K E

Department o] Physical Biochemistry, Australian National University, Canberra, A. C. T. and Department o[ Biochemistry, Stan]ord University, Palo Alto, Cali]. (U.S.A.)

(Received July 26th, 196o)

SUMMARY

An improved method for the isolation of K-casein, the rennin sensitive material of whole casein, is described. Acid casein is used as starting material and a comparatively high yield of product is obtained. The K-casein has been characterized by sedimentation velocity, sensitivity to calcium and rennin, and by zone electrophoresis in con- centrated urea.

INTRODUCTION

WAUGH AND VON HIPPEL 1 first demonstrated the existence of a fraction in bovine casein which is responsible for the stabilization of the micelles formed with the other casein components in the presence of calcium ions. This stabilizing fraction, or K- casein, is specifically at tacked by the milk clotting enzyme, rennin 1, 2.

A method for the isolation of K-casein in low yield but relatively pure form, as judged by the absence of appreciable quantities of more slowly sedimenting material at low temperature and neutral pH, has recently been described a. In the present paper an improved method for its isolation is given. I t is simple, uses acid casein as start ing material, and gives a comparat ively high yield of product which is free from slowly sedimenting components. In addition, the fractions have been characterized by the technique of zone electrophoresis in concentrated urea 4 in order to follow the fractionation procedure and to detect minor impurities.

20 g acid casein, prepared by the method of HIPP et al. 5, are dissolved at p H 7.0- 7.5 with I N NaOH and the final volume is made up to 300 ml. After cooling to 2 °, 30 ml 4 M CaC12 are added at p H 6.5-7.0 as described by WAUGH AND YON HIPPELL Stirring is continued for I h and then the calcium-sensitive casein is precipitated by warming to 35 °. The precipitate (fraction I) is removed by centrifuging at room temperature, first for 3o rain at 9o0 × g and then for 3o rain at 4o,ooo × g. To the clear supernatant are added IOO ml 1.5 M potassium oxalate at pH 6.5-7.0 as de- scribed by WAUGH AND VON HIPPEL 1. The calcium oxalate is removed by centrifuging for 30 rain at 40,000 × g and 5 °. The supernatant is warmed to 25 °, and anhydrous Na,SO4 (25 g/Ioo ml) added with stirring. After I h the precipitate (fraction II) is obtained by centrifugation at 9o0 × g for 30 rain. I t is dissolved in 60 ml water and

* Preliminary work was carried out in the Physico-Chemical Unit, C.S.I.lZ.O. Division of Food Preservation and the Biochemistry Depar tment , Universi ty of Sydney (Australia).

Biochim. Biophys. Acta, 47 (1961) 240-242

Page 2: An improved method for the isolation of κ-casein

ISOLATION OF KAPPA-CASEIN 241

dialyzed with stirring against 4 1 0.005 M NaC1 at 2 ° for 4 h, and against a further 4 1 0.005 M NaC1 overnight. The solution (approx. IOO ml) is taken from the dialysis bag, warmed to room temperature, and the p H adjusted to 7.2 with 0.2 N NaOH. An equal volume of absolute ethanol is added with stirring, followed by 2 M ammonium acetate in 50 ~ ethanol until a definite mucilaginous precipitate forms (approx. 3.5 ml are needed) : care should be taken so that excess ammonium acetate is not added, as the precipitate will then redissolve. After stirring for 3o min the supernatant (fraction III) is poured off, and the precipitate (fraction IV) is washed with 20o ml of 50 ~/o ethanol containing 3 ml 2 M ammonium acetate and then dissolved in 3o ml 6 M urea. The urea is removed by dialysis, with stirring, against 4 1 0.0o5 M NaC1 for 4 h at 2 ° and then against a further 4 1 0.005 M NaC1 overnight. The urea should not be removed by dialysis against water, nor should the dialysis be continued further than recommended. If this is done the pH of the solution will fall and the K-casein will precipitate. I t is then only soluble with difficulty. The solution is taken from the dialysis bag, adjusted to p H 7.2 with a few drops of 0.2 M NaOH, made up to 5 ° ml and the K-casein reprecipitated in 50 ~o ethanol as before. After dissolving in a mini- mum quant i ty of 6 31" urea, the latter is removed by exhaustive dialysis against o.x M NaC1 at 2 °. The solution is generally cloudy owing to the presence of fat. This can be removed by centrifugation at 90,000 × g and 2 ° for 30 min. The clear solution of K-casein is withdrawn from the bot tom of the centrifuge tube and the thin layer of fat discarded. After freeze-drying 0.5-0. 7 g K-casein is obtained, or a yield of about 20 % assuming 15 ~o K-casein in whole casein. The procedure has also been carried out successfully, using 200 g acid casein as starting material.

Starch-gel electrophoresis patterns 4 of the various fractions obtained during the preparation of K-casein are shown in Fig. I. The K-casein is the slowly moving material which spreads out and tends to form bands between the o.41 and o.65 positions: its behaviour on starch-gel electrophoresis is discussed by WAKE AND BALDWIN 4. The ~-casein isolated by the present procedure moves in the same manner as the rennin- sensitive fraction of whole casein, when both are analyzed under identical conditions. The K-casein overlaps with material in the o.18, o.34 and o.41 positions; these bands are not well defined in the photograph shown here. To check for the absence of these

0.18 0.340.41 0.65 0.80 1.00

' I Innlll I l n l l t

Fig. i. Starch-gel electrophoresis patterns of fractions obtained during the preparation of K-casein. a, whole casein; b, fraction I; c, fraction II; d, fraction I I I ; e, K-casein. See ref. 3 for details of

conditions.

Biochim. Biophys. Acta, 47 (1961) 24o-242

Page 3: An improved method for the isolation of κ-casein

2 4 2 H . A . MCKENZIE, R. G. WAKE

bands the K-casein was analyzed by an agar-gel electrophoresis procedure 4 using relatively high protein concentrations in the starting slots. No trace of these bands could be detected.

The sedimentation behaviour of a 0. 7 % solution of K-casein at pH 6. 5 and 4 ° in o.I M NaC1 is shown in Fig. 2. There is essentially no material sedimenting more slowly than the fast-moving K-casein peak which has an s2o,w of 13. 9 S. Sedimentation of K-casein (0.3 %) in sodium phosphate buffer at pH 6. 9 (1"/2 o.Io, 0.o8 M NaC1) and 2o ° shows essentially the same behaviour, with s2o,w -~ 13.6 S.

Fig. 2. Sed imen ta t ion -ve loc i ty of u-case in a t neu t r a l p H a n d low t e m p e r a t u r e . The Spinco Model E u l t r acen t r i fuge was opera ted a t 59,780 rev . /min , t he p h o t o g r a p h was t a k e n 22 m i n af te r r e a c h i n g

full speed, a n d t he angle of t he schlieren phase p la te was 75 °. See t e x t for o the r details .

The materialisolated by the present method gives no precipitate with 0.4 M CaCI~ at room temperature, and forms stable micelles with second cycle soluble casein in the presence of calcium at 3 o°. These micelles were clotted by rennin. At pH 6.5 in o.I M NaC1 and 3 °0 (calcium absent) rennin converts it rapidly into insoluble para-~-casein.

The possible heterogeneity of K-casein has been discussed previously 4.

ACKNOWLEDGEMENT

We a reg ra t e fu l to Dr. R . L . BALDWlN for hishelpfulsuggestions.

REFERENCES

1 D. F. WAUOH AND I O, H. VON HIPPEL, J. Am. Ckem. Soc., 78 (1956) 4576. R. G. WAKE, Australia** J. Biol. Sci., 12 (1959) 479.

a R. G. WAKE, Australian J. Biol. Sci., 12 (1959) 538. 4 R. (2-. WAKE AND R. L. BALDWlN, Biochim. Biophys. Acta, 47 (1961) 225. 5 iN. J. HIPP, M. L. GROVES, J. H . CUSTER AND T. L. MCMEEKIN, J. Dairy Sci., 35 (1952) 272-

Biochim. Biophys. Acla, 47 (1961) 24o-24z