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The effects of Mg2+ ion, pH, and KCl concentration on Ca2+ binding to calmodulin were studied by using a Ca2+ ion-sensitive electrode. The Ca2+ ion affinity of calmodulin increased with increasing pH or decreasing KCl concentration. Cooperativity between the Ca2+-binding sites was observed, and increased with decreasing pH or increasing KCl concentration. Free Ca2+ ion concentration was decreased by adding MgCl2 ion at low Mg2+ concentration and increased at higher concentrations in the presence of small amounts of Ca2+ ion. The decrease of free Ca2+ ion concentration by Mg2+ ion strongly suggests cooperativity between the Ca2+-binding sites, and it is difficult to explain the decrease in terms of the ordered binding models previously proposed. These results can be explained by a simple model which has four equivalent binding sites that bind Ca2+ and Mg2+ competitively, and showing cooperativity when either Ca2+ or Mg2+ is bound. Mg2+ ion binding to calmodulin was measured in the presence or absence of Ca2+ to confirm the validity of this model, and no Mg2+-specific site was observed.

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The effects of pH,Mg2+, and ionic strength on Ca2+ binding to rabbit skeletal troponin C were studied by using a Ca2+ sensitive electrode. Troponin C has two high affinity and two low affinity sites and the Ca2+ affinity of both sites was increased by increasing pH in a pH range from pH 5.6 to 10.4. The affinity was decreased by increasing ionic strength. The change of the Ca2+ affinity can be explained by the electrostatic interaction between Ca2+ and the protein. At alkaline pH, the four Ca2+ binding sites bind Ca2+ with the same affinity and the distinction between the high and the low affinity sites vanished. This result shows that the difference of the Ca2+ affinity is owing to differences of the secondary or the tertiary structure of the Ca2+ binding sites, not owing to a difference of the primary structures of the Ca2+ binding sites. The two high affinity sites bound two Ca2+ ions cooperatively in neutral pH. The cooperativity was diminished at both acidic and alkaline pH. Mg2+ ion decreased the affinity of the low affinity sites.