Calcium-induced associations of the caseins: A thermodynamic linkage approach to precipitation and resolubilization

Abstract

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wyman's thermodynamic linkage. Salting-out has been described by a salt-binding constant, k 1, the number of moles of salt bound per mole of protein, n, and S 1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k 2, m, and S 2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of α s1 -casein ( α s1 -A), ( α s1 -B), and β-casein C (β-C) at 37 °C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k 1 values of 157, 186, and 156 liters · mol −1 and n values of 8, 8, and 4, respectively. The values of k 1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 °C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here β-C is totally soluble at all calcium concentrations and α s1 -B and α s1 -A were now found to have salting-in parameters, K 2 and m, of 2.5 liters · mol −1 and 4, and 11 liters · mol −1 and 8, respectively. α s1 -A is more readily salted-in and studies on the variation of S 1 with added KCl for this protein at 1 °C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K + and Ca 2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k 1 and k 2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in α s1 -B which is deleted in α s1 -A; it is speculated that such bonds may play a role in other calcium-induced changes in protein Solubility.