Calorimetric Studies on Calcium and Magnesium Binding by Troponin C from Bullfrog Skeletal Musele

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Microcalorimetic titrations were carried out to measure the thermodynamic functions of bullfrog skeletal muscle troponin C (TnC) in the interaction with Ca²⁺ and Mg²⁺ at 25°C and at pH 7.0. Enthalpy titration curves with Ca²⁺ were composed of three stages both in the presence and in the absence of Mg²⁺. The first (0-2 mol Ca²⁺/mol TnC) and the third (>3mol Ca²⁺/mol TnC) stages were exothermic and the second stage (2-3mol Ca²⁺/mol TnC) was endothermic. Mg²⁺ affected the first stage to decrease the amount of heat produced but not the second and third stages. The enthalpy titration with Mg²⁺, in the absence of Ca²⁺, was slightly exothermic initially and then became endothermic beyond 2-3 mol Mg²⁺/mol TnC. Absorption of heat was observed throughout the additions of Mg²⁺ in the presence of 1mM Ca²⁺. The results indicate that bullfrog TnC has two high-affinity Ca²⁺-Mg²⁺ sites, two low-affinity Ca²⁺ -specific sites, and two or around two Mg²⁺-specific sites. Based on the enthalpy and entropy changes, the Ca²⁺ binding reactions of TnC were classified into three types, indicating thermodynamic variety in the binding sites of the molecule.