INACTIVATION OF HORSERADISH-PEROXIDASE BY PHENOL AND HYDROGEN-PEROXIDE - A KINETIC INVESTIGATION

Inactivation of horseradish peroxidase (HRP) was examined in the prese nce of hydrogen peroxide alone and in the presence of hydrogen peroxid e plus phenol. HRP is inactivated upon exposure to hydrogen peroxide ( H2O2) by the combination of two possible pathways, dependent upon hydr ogen peroxide concentration. At low H2O2 concentrations (below 1.0 mM in the absence of phenol), inactivation is predominantly reversible, r esulting from the formation and accumulation of catalytically inert in termediate compound III. As H2O2 concentrations increase, an irreversi ble mechanism-based inactivation process becomes predominant. The over all inactivation comprised of both processes exhibits a second-order i nactivation rate constant (k(app)) of 0.023 +/- 0.005 M(-1)s(-1) at pH 7.4 and 25 degrees C. In the presence of both hydrogen peroxide fixed at 0.5 mM and phenol, HRP was inactivated in an irreversible, time- a nd phenol concentration-dependent process, also mechanism-based, with a k(app) Of 0.019 +/- 0.004 M(-1)s(-1).