Fractionation of Skimmilk Casein Micelles by Rate-Zone and Isopycnic-Zone Ultracentrifugation in Sucrose Gradients

Abstract

Rate-zone and isopycnic-zone ultracentrifugation techniques with sucrose gradients in a swinging-bucket rotor were utilized for fractionating skimmilk proteins with emphasis upon characterizing the different size casein micelles. Rate-zone ultracentrifugation separated skimmilk casein micelles from soluble casein and whey proteins, and further fractionated the different size micelles. Up to ,-92% of skimmilk casein mieelles was sedimented to the bottom of a 17.2 to 27.5% w/v sucrose gradient by centrifuging at 206,000 g for two hours at 0 to 5 C. Zonal electrophoresis data, uncorrected for differences in dyebinding coefficients, revealed that the smallest casein micelles and soluble casein contained ~8.8% as-casein , 89% fl-casein and 2.2% ~-easein and the largest micelles recovered in the pellet fraction contained ~80% as-casein , 19% fl-casein, and 0.8% ~-casein. Isopycnic-zone ultracentrifugatiou handed whey proteins and soluble casein in ~8 to 10% w/v sucrose (p = 1.02 to 1.04 g/ml), but failed to band casein micelles even in =~70% w/v sucrose gradients. It was concluded that the high concentrations of sucrose increased buoyant density of the casein mieelles by removing their solvation and hydration layers, and thus, the isopyenie-zone technique is not applicable for studying casein mieelles in their native state.