Inactivation and reactivation kinetics of horseradish peroxidase in phosphate buffer and buffer–dimethylformamide solutions

The influence of the regeneration of horseradish peroxidase activity at 4 °C on both the thermal inactivation and the reactivation kinetics of the enzyme was studied in phosphate buffer and a mixture of this buffer with 10% (v/v) N,N-dimethylformamide, at temperatures ranging from 70 to 85 °C. A series-type model was fitted to the experimental data that exhibited typical biphasic patterns, which became less pronounced as enzyme activity was regenerated. The kinetic parameters were found to be, in general, significantly different in both inactivation media. The magnitude of the activation energies for both the reactions of formation and inactivation of the intermediate enzyme indicated that conformational changes might play a major role in both processes when the solvent is added. A logarithmic model, although lacking a theoretical background, was able to predict the enzyme activity regeneration upon storage of the inactivated samples at 4 °C. There was evidence that the rate of reactivation may be dependent on the amount of intermediate formed in the first reaction or remaining after inactivation, as suggested by both the inactivation temperatures and extent of heating time relationships verified in this work.