The pH-dependent Dissociation of β-Casein from Human Milk Micelles: Role of Electrostatic Interactions

Abstract

Evidence has been reported that supports the role of hydrophobic interactions in the association of the monomers and in the dissociation of different phosphorylation levels of β-casein (CN) from human casein micelles. Here, the role of electrostatic interactions in the formation and structure of human casein micelles was examined by determining the β-CN composition of micelle pellets from milk samples adjusted in the range from pH 5 to 10. As the pH was decreased from normal (∼pH 7.5) at 37°C, the proteins remained associated with the micelle, and the relative amounts of all of the phosphorylated forms remained essentially constant. As the pH was increased from normal, protein was lost from the micelles to the supernate. When the relative micelle compositions were corrected for the loss, all of the phosphorylated forms decreased in total amount: the largest decrease for both the nonphosphorylated and singly phosphorylated forms (∼95%), a sizeable decrease for the doubly phosphorylated form (∼70%), and only about a 25% decrease for the triply, quadruply, and fully phosphorylated forms. Laser light scattering measurements on micelle pellets resuspended in simulated milk ultrafiltrate at 37°C yielded mean particle radii of ∼63nm at pH 7.5 and ∼35nm near pH 6. These results suggest that micelle stabilization depends not only on the net charge on the negatively charged organic phosphate esters and the inorganic orthophosphate, which vary with pH and calcium binding, but also on the positively charged amino groups of basic amino acid residues in the proteins.