A phosphate-induced sub-micelle-micelle equilibrium in reconstituted casein micelle systems

Summary

In reconstituted casein systems, complete sub-micelles were previously observed only under extreme conditions of ionic strength, namely a NaCl concentration of 2 M or greater. However, studies with the analytical ultracentrifuge on phosphate-containing casein systems revealed that under certain conditions, a stable sub-micelle-micelle equilibrium was established. Conditions which were standard throughout were 7.5 mg/ml protein, 0.04 M-NaCl, pH 6.6 and 37 °C. The concentrations of added CaCl² (Ca²⁺) and inorganic phosphate (P₁) were variable. With no P₁ present, Ca-sensitive caseins precipitated or formed micelles when k-casein was present, between 6 and 7 mM-Ca²⁺. With 10–20 mM-P₁ precipitation or micelle formation began at about 4 mM-Ca²⁺ and was complete at about 5 mM-Ca²⁺. Over this interval, during micelle formation, a sub-micelle peak with s_{20, w} of about 13 S was observed in equilibrium with the broad micelle peak. In a system containing a 2:2:1 weight ratio of ±_sI-:²-:k-casein, this sub-micelle was isolated at 4 mM-Ca²⁺ and 2.5 mM-P₁. The mol. wt was 760000 and it thus contained approximately 33 monomer caseins. The reconstituted micelle system formed in the absence of P₁ was quite temperature sensitive, forming at 37 °C but disappearing upon cooling. In the presence of P₁ the micelles formed at 37 °C but were stable to cooling as are natural micelles. Evidently, a combination of hydrophobic and electrostatic interactions are involved in natural micelle formation, probably with the production of salt bridges of Ca and phosphate ions between sub-micelles.