Examination of Casein Micelle Structure by a Method for Reversible Covalent Immobilization

Proteins on the surface of native casein micelles were examined by a reversible method for covalent attachment. Accordingly, native micelles and various sized fractions were immobilized covalently by exposure to thioester-derivatized glass beads. Following dissociation of the micelle in 4M urea and removal of unbound monomers from the glass, the covalently bound monomers were released with hydroxylamine, identified, and quantitated by a micro method for ion exchange chromatography. Each of the major caseins (as-, ~-, and g-caseins) was accessible for covalent reaction with the glass surface; however, the percent of K-casein was increased substantially relative to composition of whole casein. Moreover, the relative amount of K-casein that became attached covalently appeared to be greater when smaller micelles were immobilized. Consequently, these results argue against those miceUar models that require a) an inside location for g-casein, b) exposure of only K-casein on the surface, c) a uniform distribution of casein monomers in the micelle, or d) a uniform composition for all casein submicelles. Likewise, results are consistent with models that require a preference for K-casein on the micellar surface but allow for access to a s -and j3-caseins and which require a variable composition of the submicelles.