The enthalpy titration of troponin C with calcium

Microcalorimetric titrations have been used to study the binding of Ca2+ to troponin C, the Ca-binding component of troponin. Troponin C was extracted from rabbit skeletal muscle and Ca2+ was added to Ca-free troponin C in the presence of 1 mM Mg2+ at pH 8.83 at 10 degrees C. In these conditions proton exchanges of troponin C on Ca-binding are negligible. In case of troponin C there is neither aggregation nor dissociation when Ca2+ is added, a phenomenon controlled by bound Ca, which caused some difficulty in the analysis of results for troponin. Using an iterative curve fitting procedure the following parameters of two classes of Ca-binding sites have been determined from the calorimetric results: number of binding sites in 1st class, 1.81; in 2nd class, 0.69; log of binding constant in 1st class, 7; in 2nd class, 5.0; standard enthalpy change for 1st class, -10.00 kJ . mol-1; that in 2nd class, -25.64. The standard free energy and entropy changes have been calculated from these values. The results indicate that the standard entropy change for Ca2+-troponin C interaction is positive and strikingly resembles the values for the interaction of organic sequestering agents such as glycol-EDTA with Ca2+.