Monomer Characterization and Studies of Self-Association of the Major ß-Casein of Human Milk

¹ Departments of Biochemistry and Pediatrics, Loma Linda University School of Medicine, Loma Linda, CA 92350

The casein form that has four organic phosphoryl groups, ß-casein (CN)-4P, is the major constituent (35%) of the ß-CN fraction of human milk and should play an important role in micelle structure and formation. In 3.3 M urea, the monomer is present with a molecular mass of 24,500 Da and a sedimentation coefficient of 1.3 S (Svedberg units, 10^–13s). In 0.02 M NaCl and 0.01 M imidazole (low salt buffer) at pH 7, the sedimentation coefficient was 1.5 S, which increased to 14 S at 37°C. Laser light scattering in low salt buffer and 9 mg/ml of protein indicated monomers with a radius of about 4 nm at 4°C. The size of the radius increased as temperature increased, and, at 37°C, the radius was about 12 nm. The molecular mass suggested the presence of about 47 monomers per polymer. In 0.25 M NaCl and with 10 mM Ca²⁺ prior to precipitation, the polymer attained a maximum radius of about 15 nm, which perhaps is the size of the smallest human milk micelles. The low value for reduced viscosity of 8.2 ml/g for the calcium-induced polymer was independent of protein concentration, suggesting a spherical shape and fixed size. Calcium apparently binds strongly to the phosphates; the dissociation constant was 8.1 x 10^–4 M. Other constituents of milk, such as inorganic orthophosphate, may contribute to differences in the manner by which ß-CN, with various phosphorylation levels, participate in micelle formation.

Key Words: human milk • human ßbeta;-casein • protein self-association • protein-ion interactions

Submitted on May 7, 1996
Accepted on January 17, 1997

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