Binding of Cations to Caseins. Site Binding, Donnan Binding, and System Characteristics

Ion-protein interactions in the formation of core polymers and cores of casein micelles are determined by examining α_s-casein, β-casein, and their unit weight ratio mixture at final pH 6.6 and 37°. Increasing protein association is correlated with monomer net charge, protein solvation, and site binding of calcium and hydrogen ions. To obtain these data, isoionic proteins are used and proton releases are obtained (a) as monovalent cation concentration, I, is brought to 0.04, 0.08, and 0.16 M, and (b) at each I, as calcium concentration is brought progressively to 0.08 M. Protein solvations are measured for precipitates, and calculated from apparent monomer net charge for protein in solution. Apparent molar calcium binding, ν_Ca,A, is measured after equilibrium dialysis. Since net fixed charge occuss on short, solvent-accessible acidic peptides, Donnan binding of calcium ion is examined. A model consistent with the theory of Overbeek is used to obtain monomrr net charge, and Donnan binding corrections, ν_Ca,D. Site-bound calcium is obtained by difference. Results and conclusions are: ν_Ca,D are small at low ν_Ca,A (~15%) and decrease as ionic strength and/or ν_Ca,S increase. For α_S-casein, β-casein, and the mixture; and independent of I, (a) precipitation is initiated at the same ν_Ca,S and Z (respectively, 9.3, 5.4, and 10.4, and -9, -6, and -1.5), and (b) at 0.08 M calcium, proton release and ν_Ca,S are essentially constant (6.6, 4.8, and 6.3, and 20, 11.3, and 16.3). At these ν_Ca,S there is apparently a reversal of Z in precipitate (to approximately +10.4, +4.4, and +8.9). Since solubility and solvation do not reverse significantly, core polymer cross-linking by calcium is proposed as a source of interaction energy. The mixture is not intermediate in precipitate characteristics of ν_Ca,S, Z, and solvation. In the mixture, cominghng of monomers in core polymers produces interactions which convert potential interpolymer cross-linking sites into intrapolymer cross-linking sites.