A 1H-NMR study of bovine casein micelles; influence of pH, temperature and calcium ions on micellar structure.

The influence of pH, temperature and Ca depletion on bovine casein micelle suspensions in D2O containing simulated milk ultrafiltrate was studied by 1H-NMR spectroscopy. In the pH range of 5.8-7.5 the spectrum of the micelles showed very little pH dependence, indicating that no changes occurred in the dynamic behaviour of the proteins constituting the micelle. The NMR spectrum of casein micelles was strongly temperature dependent, particularly in the temperature range of 60-98 degrees C. Increase in temperature resulted in a strong increase in spectral intensity concomitant with changes in the spectral characteristics. In micelle suspensions these changes were reversible, and indicated that at elevated temperatures the rigid structure of the casein micelle started to melt, leading to an increased mobility of appreciable parts of the proteins in the micelle. Ca depletion of the casein micelles by addition of EDTA resulted in an increase in spectral intensity, which arose from the presence of casein components in the serum phase. The spectrum of these serum phase particles resembled closely the spectrum of a solution of total casein in simulated milk ultrafiltrate and was quite different from the spectrum of casein micelles. The implications of these results with respect to models of the structure of bovine casein micelles are discussed.

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.

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.