Characterization of bovine κ-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography

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Abstract
Bovine .kappa.-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30.degree. C. To this end the trichloroacetic acid-soluble reaction products were analyzed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 .mu.M; the overall substrate properties of these components as reflected by the kinetic parameter kcat/Km ranged from 5 to 8 .mu.M-1 .cntdot. s-1. Irreversible polymerization of the carbohydrate-free fraction brought about a-more-than-2-fold increase in Km, the kcat value remaining virtually constant. The kcat/Km found for the cleavage of whole of .kappa.-casein at pH 6.6 was of the same magnitude as the kcat/Km found for the polymerized carbohydrate-free fraction (i.e. about 3 .mu.M-1 .cntdot. s-1). No indication of substrate inhibition was found for the carbohyrate-free fraction.