Suicide inactivation of horseradish peroxidase by excess

Abstract

The inactivation of peroxidases by its oxidant substrate H2O2 limits the usefulness of these versatile enzymes. Here,we investigated the effect of reaction conditions on inactivation of horseradish peroxidase by excess H2O2. Inactivation was morepronounced at pH extremes, indicating that reactions in which the oxidation products induce significant changes in reaction pHcould accentuate the loss of peroxidase activity. In reactions carried out in sodium acetate buffer, higher inactivation rates wereobserved when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicalsfrom the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher concentrationsof the redox mediator caused a slight increase in inactivation, likely due to the formation of reactive promethazine radicals, whichin turn attack the protein via a mechanism different from that caused by excess H2O2. These findings will help in defining theoptimal reaction conditions that preserve the activity of the peroxidase molecules.