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

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Abstract
Rate-zone and isopycnic-zone ultracentri- fugation techniques with sucrose gradients in a swinging-bucket rotor were utilized for fractionating skimmilk proteins with em- phasis upon characterizing the different size casein micelles. Rate-zone ultracen- trifugation separated skimmilk casein micelles from soluble casein and whey pro- teins, and further fractionated the different size micelles. Up to ,-92% of skimmilk casein mieelles was sedimented to the bot- tom of a 17.2 to 27.5% w/v sucrose gradi- ent by centrifuging at 206,000 × g for two hours at 0 to 5 C. Zonal electrophoresis data, uncorrected for differences in dye- binding coefficients, revealed that the small- est casein micelles and soluble casein con- tained ~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.