STABILIZATION OF CALCIUM-SENSITIV E (a,) CASEIN BY KAPPA-CASEIN: EFFECT OF CHYMOTRYPSIN AND HEAT ON KAPPA-CASEIN

It has been found helpful in studies of the caseins to characterize the calcium-sensitive casein by its quantitative precipitation with calcium chloride (6), and the kappa-casein by its ability to quantitatively stabilize the calcium- sensitive casein so that it does not precipitate with calcium chloride (5). The latter test, which has not been reported in detail, is per- formed with calcium-sensitive (as) casein (0.3%), at pH 6.7, with varying amounts of kappa-casein, with a 0.020 M concentration of calcium chloride. The test is set up in 15-ml. centrifuge tubes; total volume of the test mix- ture is 10.0 ml. A 2% solution of the a,-easein at pH 7.8 is used; subsequent addition of cal- cium chloride brings the pH of the mixture to the desired pit of 6.7. The solution of as-casein (30 to 34 rag.) is added to the tubes, followed by the required volumes of water, and amounts of kappa-casein such that the kappa/as ratios vary from about 0.03 to 0.12. Finally, 2.0 mh of 0.1 M calcium chloride is added and the mixture stirred with a spatula. The test mix- tures are kept at 30 ° C. for 15 rain., then cen- trifuged at about 3,000 X G for 5 rain. Sam- ples of the supernatant solutions are withdrawn, appropriate dilutions made, and 1 drop of 0.5 M NaOI-I added to clarify the dilutions. The casein in solution is determined from the light absorption at 280 mix. A factor of 1.0 is used for convenience for converting light absorption (optical density) to milligrams of protein per milliliter. The amount of kappa-casein used in the test is deducted from the casein in solu- tion and the results expressed as per cent of as-casein in solution. Solutions of a~-casein for the test are stored at 7 ° C. and are not kept more than three days, since the soluble portion at zero kappa concentration tends to increase. A stabilization test with an aged as-casein solution parallels that obtained with a fresh solution, but is at a higher level. A typical curve for kappa-casein by this test procedure