Effect of Zn2+ on the thermal denaturation of carboxypeptidase B

A differential scanning calorimetry study on the thermal denaturation of porcine pancreas carboxypeptidase B (in 20 mM pyrophosphate buffer, pH 9.0) has been carried out. The calorimetric transitions have been found to be calorimetrically irreversible and to depend on the Zn2+ concentration in the buffer. The effect of the Zn2+ concentration on the temperatures corresponding to maximum heat capacity appears to conform the dictates of the van't Hoff equation. In spite of this, analysis of the scanning rate effect on the transitions, together with studies on the thermal inactivation kinetics, show that the heat absorption is entirely determined by the rate of formation of the final (irreversibly denatured) state of the protein; therefore, analysis of the calorimetric transitions according to equilibrium thermodynamics models is not permissible. The effect of Zn2+ on the calorimetric transitions can be explained on the basis of a simple kinetic model that does not assume chemical equilibrium to be established between the significantly populated states of the protein.