Thermally induced conformational changes in horseradish peroxidase

Detailed differential scanning calorimetry (DSC), steady-state tryptophan f luorescence and far-UV and visible CD studies, together with enzymatic assa ys, were carried out to monitor the thermal denaturation of horseradish per oxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were compleme ntary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tr yptophan fluorescence emission, corresponded to changes in the tertiary str ucture of the enzyme. The results, supported by data about changes in enzym atic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, sug gesting that denaturation is under kinetic control. It is shown that the pr ocess of HRPc denaturation can be interpreted with sufficient accuracy in t erms of the simple kinetic scheme N (k) under right arrow D where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denat ured state. On the basis of this model, the parameters of the Arrhenius equ ation were calculated.