Association of the quadruply phosphorylated β-casein from human milk with the nonphosphorylated form

Abstract

Human β-casein (β-CN) is phosphorylated at levels from zero (β-CN-0P) to five (β-CN-5P). The major constituent is the 4P form (∼35%), whereas the 0P form (∼5%) has been implicated in the formation of a framework upon which the forms with higher levels of phosphorylation may aggregate. At 4°C in 0.01 M imidazole and 0.02 M NaCl, pH 7, with a 1:1 (wt:wt) 0P:4P ratio and a total protein concentration of 3 mg/ml, the s20,w was 1.4 S (monomer). Laser light scattering gave a radius of ∼4.5 nm. As the temperature, T, increased, s20,w increased to 2 S. At 25°C, peaks of 9.5 S and 2 S were observed. This transition T was different from that of either form. At 37°C, a single peak was again observed with s20,w of 17.5 S, compared with 42 S for the 0P and 14 S for the 4P form. Laser light scattering at 37°C revealed a polymer of ∼16 nm radius and D20,w of 1.55 cm2/s. A combination of D20,w and s20,w gave a relative molecular mass suggesting about 45 monomers per polymer. An incubation of 3 h or more at 37°C caused further aggregation, characteristic of the 0P form, and supported the concept of framework formation. At pH 6.6, s20,w was 38 S compared with 1.4 S at pH 10.4. Hydrostatic pressure did not have a large effect but supported a soap micelle-like structure for the polymer. The turbidity of the mixture increased with the amount of CaCl2 and T until the protein precipitated. The properties of the 1:1 mixture of these human β-CN are intermediate but probably more biased toward those for the 4P form.