Binding of Mg²⁺ and Ca²⁺ to β-casein A¹: A multinuclear magnetic resonance study

²⁵Mg, ⁴³Ca and ³¹P NMR have been used to study the binding of Mg²⁺ and Ca²⁺ ions to β-casein A¹. The concentration dependence of the line width of the 25Mg NMR signal shows that β-casein contains at least two different types of binding sites for Mg²⁺ ions, one with strongly bound, slowly exchanging ions and one with more weakly bound ions which undergo fast exchange.
The strong Mg²⁺ binding site has an unexpectedly high binding constant, K_b^strong 10⁴ M^–1, which has not been reported earlier. Mg²⁺ and Ca²⁺ compete for the Ca²⁺ binding sites of β-casein, while Na⁺ does not compete for these binding sites under physiological conditions. The dependence of the ⁴³Ca NMR chemical shifts on total concentration of Mg²⁺ and Ca²⁺, 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, pk_a, for the strongest Ca²⁺ binding site was found to be 7·1.