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

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 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed 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 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X 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 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 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.

Peptide substrates for chymosin (rennin): conformational studies of kappa-casein and some kappa-casein-related oligopeptides by circular dichroism and secondary structure prediction.

Circular dichroism spectra of a series of synthetic, kappa-casein-related oligopeptide substrates for chymosin in water and in surfactant solution were determined. The results show that there is a good correlation between the beta-structure forming potential of these peptides as found by using structure-predictive methods and the conformation in dilute sodium dodecyl sulfate solutions. The results support earlier suggestions concerning enzyme-substrate interaction which were made on the basis of X-ray analysis of acid proteinases. A predicted secondary structure of the whole kappa-casein molecule obtained by using a combination of three methods is also presented.

Some association properties of bovine SH-kappa-casein.

(1) It is shown that kappa-casein association is characterized by a critical micelle concentration which decreases as the ionic strength is increased. (2) The kappa-casein polymer molecular weight was calculated from the weight-average apparent molecular weight by taking into consideration the monomer concentration and the excluded volume. The degree of polymerization is 30 and does not depend on ionic strength between 0.1 and 1 M. (3) The non-electrical contribution to the standard free energy of association is -38 kJ/mol monomer. The electrical part is small: 1-2 kJ/mol monomer depending on the ionic strength and kappa-casein genetic variant. (4) The limitation of size and the size itself of the kappa-casein polymer can be explained by the theory of self-assembly of virus particles by Caspar and Klug (D.L.D. Caspar and A. Klug, Cold Spring Harb. Symp. Quant. Biol. 27 (1962)1). (5) Extending this theory to casein micelle assembly, it is predicted that micelles are distributed preferentially over a restricted number of sizes.

The association of bovine SH-kappa-casein at pH 7.0.

The self-association of SH-kappa-casein at pH 7.0 in 0.01 M-EDTA, 0.001 M-dithiothreitol buffer, containing 0.1, 0.2, 0.5 or 1.0 M-NaCl is of a monomer-polymer type. The polymer is a spherical particle, diam. 23 nm and mol. wt 570000 (30 monomers). These parameters are not greatly influenced by variations in ionic strength above 0.1. The critical micelle concentration, which is a property of a monomer--polymer equilibrium, decreases with increasing ionic strength. The standard free energy of association is about -36 kJ/mol. monomer at 20 degrees C.

Purification and some physicochemical properties of bovine kappa-casein.

1. A description is given of the fractionation of kappa-casein on DEAE-cellulose using a pH gradient. With this method an improved separation of the kappa-casein components with a higher negative charge is obtained. 2. It is shown that at least one of the kappa-casein fractions has a second phosphate ester group. The heterogeneity of kappa-casein therefore is not exclusively caused by a varying N-acetylneuraminic acid content. 3. Ultracentrifuge experiments and exclusion gel chromatography show that the purified kappa-casein fraction having the lowest electrophoretic mobility exhibits a monomer-polymer association equilibrium. The free energy of association per mol monomer in 0.2 M NaCl is approximately --36 kJ-mol-1.