Water Interactions with Bovine Caseins by Hydrogen-2 Nuclear Magnetic Resonance Relaxation Studies: Structural Implications

A method was developed for determining hydration from the protein concentration dependence of deuteron magnetic resonance relaxation rates. Measurements were made in D 2 0 on both casein micelles and submicelles. From the protein concentration-dependent relaxation rates, the second virial coefficients of the proteins were obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, hydration and correlation times were calculated for the proteins; from the latter parameter the Stokes radius was obtained. Molecular weights, calculated from the Stokes radius using the Stokes-Einstein relationship and the partial specific volume, were in the range of those published for caseins in the absence of Ca 2+ (submicelles). For casein submicelles variations of Stokes radius and hydration with temperature were in agreement with hydrophobically driven aggregations and in accord with known changes in molecular state. Similar temperature variations of Stokes radius and hydration were observed for casein micelles; however, their absolute values, although greater than those of submicelles, were less than expected. These data are interpreted with respect to a model in which trapped water in the micelles is associated with discrete submicellar structures.