Association of mixtures of the two major forms of β-casein from human milk

Human milk β-casein (CN) is unique in that it may be phosphorylated at any level from zero (β-CN-0P) to five (β-CN-5P) organic phosphates per molecule. The 2P and 4P forms are the major components, with about 30 to 35% each. Here, we present the association properties of mixtures of these two moieties of human β-CN. The aggregation patterns, as functions of temperature and ionic strength of these mixtures, generally follow those for the individual components. However, the mixtures yielded polymers with slightly different properties, which indicates extensive interaction between the two. Some properties of the mixtures were more like those for the 2P form, such as association in low salt buffer to give a peak with a sedimentation coefficient, S_20,w, of ≈11 S, in contrast to the 2P form alone with a peak of ≈13 S and 4P alone with only a small amount of material with S_20,w greater than 2 S at 27°C. The solubility and interactions in the presence of Ca²⁺ ions were intermediate but more like the 4P form. A protein-concentration dependence for s_20,w was seen, and laser light scattering indicated that there was an increase in size and/or a change in shape as the protein concentration increased. From the results, it is apparent that submicellar oligomers are probably formed by rapidly established equilibrium association reactions. The presence of an equal amount of the 2P form along with the 4P form does not appear to be a disadvantage in casein micelle formation and function.