Kinetics of rennin action on casein prepared by ultracentrifugation

Abstract

The kinetics of the primary phase of rennin action on casein micelles and on total casein prepared by ultracentrifugation have been studied. It was observed that the values of K m and V varied for the different casein samples. Addition of whey proteins to the reaction mixture or variation of the concentrations of Na + and K+ had no effect on K m and V, but there was a consistent increase in the values of both these parameters when /c-casein was added. It was concluded that these changes were due to an increase in the accessibility of the /c-casein resulting from the rearrangement of the casein micelles. These results are in agreement with our earlier conclusion that the K m for rennin action on casein is dependent on the carbohydrate composition of the molecule.In a previous paper (Castle & Wheelock, 1972) we showed that there was a wide variation between cows in the values of K m and F obtained for the primary phase of rennin action on whole milk. It was also observed that for an individual milk sample the value of K m but not of V varied with rennin concentration. It was suggested that this variation was partly due to differences between milks in the amount and composition of the /c-casein and possibly also in the composition of the non-casein fraction of the milk.The present experiments were designed to study the kinetics of rennin action on casein in order to obtain further information on the factors which contribute to the differences observed for K m and V between milks of different animals. MATERIALS AND METHODSMilk samples were obtained from individual cows which were known to be free from bacterial infections of the udder.Casein micelles and total casein (casein micelles + soluble casein) were prepared by an adaptation of the method of Bohren & Wenner (1961). The micelles were sedimented by centrifuging skim-milk in a Superspeed 40 (Measuring and Scientific Equipment Ltd, Crawley, Sussex) preparative ultracentrifuge at 100000 g for 1 h at 20 °C. The total casein was prepared in exactly the same way except that 2-0 ml of 3-0 M-CaCl 2 /100 ml milk was added prior to centrifugation in order that the soluble casein would form a sediment with the micelles. After the addition of the CaCl 2 , the pH of the milk was adjusted to its original value with 1-0 M-KOH.

Influence of milk protein concentration on the gelling activity of chymosin and bovine pepsin

“…Other investigations of milk, whole casein and /c-casein have studied further the effect of factors such as pH, temperature, ionic strength, enzyme concentration on the enzymic stage and on coagulation (Mackinlay & Wake, 1971;Beeby, Hill & Snow, 1971). Castle & Wheelock (1973), attempting to explain the differences in the clotting times obtained with different concentrations of milk substrates from various sources, concentrated the native phosphocaseinate by ultracentrifugation. By diluting this concentrate, they obtained substrates with different casein contents on which they studied the enzymic reaction.…”