Abstract
Differential scanning calorimetry (DSC) measurements were performed on the thermal denaturation of ribonuclease a and ribonuclease a complexed with an inhibitor, cytidine or uridine 3''-monophosphate, in sodium acetate buffered solutions. Thermal denaturation of the complex results in dissociation of the complex into denatured ribonuclease a and free inhibitor. Binding contrasts of the inhibitor to ribonuclease a were determined from the increase in the denaturation temperature of ribonuclease a in the complexed form and from the denaturation enthalpy of the complex. Binding enthalpies of the inhibitor to ribonuclease a were determined from the increase in the denaturation enthalpy of ribonuclease a complexed with the inhibitor. For the cytidine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 87 .+-. 8 M-1 (pH 7.0) to 1410 .+-. 54 M-1 (pH 5.0), while the binding enthalpies increased from 17 .+-. 13 kJ mol-1 (pH 4.7) to 79 .+-. 15 kJ mol-1 (pH 5.5). For the uridine inhibitor in 0.2 M sodium acetate buffered solutions, the binding constants increase from 104 .+-. 1 M-1 (pH 7.0) to 402 .+-. 7 M-1 (pH 5.5), while the binding enthalpies increase from 16 .+-. 5 kJ mol-1 (pH 6.0) to 37 .+-. 4 kJ mol-1 (pH 7.0). The binding constants and enthalpies of the cytidine inhibitor in 0.05 M sodium acetate buffered solutions increase respectively from 328 .+-. 37 M-1 (pH 6.5) to 2200 .+-. 364 M-1 (pH 5.5) and from 22 kJ mol-1 (pH 5.5) to 45 .+-. 7 kJ mol-1 (pH 6.5). The denaturation transition cooperatives of the uncomplexed and complexed ribonuclease a were close to unity, indicating that the transition is two state with a stoichiometry of 1.