Formation of reconstituted casein micelles with human β-caseins and bovine κ-casein

Human β-casein (CN) is the major protein of the human milk casein fraction (∼80%) and exists in six calcium-sensitive forms, having zero to five organic phosphates per molecule. The major forms are the doubly-phosphorylated (β-CN-2P; ∼30%) and the quadruply phosphorylated (β-CN-4P; ∼35%) forms. Although calcium-insensitive, κ-CN is known for its role in preventing the precipitation of β-CN in the presence of Ca⁺², but it is not known how the different levels of phosphorylation may affect this. In the present investigation, turbidity, measured at 400 nm, was determined at increasing temperatures (4 up to 37°C) for solutions of β-CN-2P and β-CN-4P (3 mg/ml in 0.02 M NaCl, 0.01 M imidazole, pH 7) individually and also mixed with bovine κ-CN in 6/1 and 3/1 weight ratios of β/κ and containing 0, 5, and 10 mM Ca ⁺². The results indicate that the first step of micelle formation probably leads to polymers of limited size, the only complexes available to β-CN-2P under most conditions. With β-CN-4P, these polymers aggregate further to give reconstituted micelles, probably because of the ability to form crosslinks at this phosphorylation level. The formation of reconstituted micelles under various conditions of pH, Ca⁺² concentration and κ-CN content indicates that both hydrophobic interactions and Ca ⁺² bridges or crosslinks may contribute to protein aggregation and micelle building.