Differential scanning calorimetry of the irreversible thermal unfolding of bovine superoxide dismutase

The thermal denaturation of bovine superoxide dismutase has been studied by means of differential scanning calorimetry (DSC). Analysis of the calorimetric profile by means of Freire and Biltonen's algorithm to determine the accessible states in the transition path did not succeed. At least three factors influenced this analysis negatively: (a) under the experimental conditions used, the denaturation of the enzyme was completely irreversible, as judged by the absence of any endotherm on re-scanning; (b) the denaturation process was followed by a change in molecularity, as the dimeric nature of the enzyme suggested; and (c) there was distortion at the high temperature side of the DSC profile, caused by the existence of an exothermic aggregation process. Moreover, the difference between the van't Hoff and the calorimetric enthalpy on one hand, and the dependence of the specific heat C-p on scan rate on the other, allowed us to exclude a simple two-state reversible or irreversible transition for the denaturation of the enzyme. In the light of these results, we attempted to fit the experimental C-pexc curve using the SIMPLEX minimization algorithm by taking in account only three processes: two endothermic and one exothermic. The deconvolution of the calorimetric profile agreed very well with the experimental data and allowed us to suggest a tentative mechanism for the thermal denaturation of this dimeric enzyme.