Binding of Mg 2+ and Ca 2+ to β -casein A 1 : a multi-nuclear magnetic resonance study

Summary 25 Mg, 43 Ca and 31 P NMR have been used to study the binding of Mg 2+ and Ca 2+ ions to β-casein A 1 . The concentration dependence of the line width of the 25 Mg NMR signal shows that β-casein contains at least two different types of binding sites for Mg 2+ ions, one with strongly bound, slowly exchanging ions and one with more weakly bound ions which undergo fast exchange. The strong Mg 2+ binding site has an unexpectedly high binding constant, K b strong 10 4 M –1 , which has not been reported earlier. Mg 2+ and Ca 2+ compete for the Ca 2+ binding sites of β-casein, while Na + does not compete for these binding sites under physiological conditions. The dependence of the 43 Ca NMR chemical shifts on total concentration of Mg 2+ and Ca 2+ , in the presence of β-casein, could be equally well fitted with a model assuming up to five identical and independent sites as with a model assuming five or more sites with negative cooperativity. The proton dissociation constant, p k a , for the strongest Ca 2+ binding site was found to be 7·1.