Suicide inactivation of horseradish peroxidase by excess hydrogen peroxide: The effects of reaction pH, buffer ion concentration, and redox mediation

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 more pronounced at pH extremes, indicating that reactions in which the oxidation products induce significant changes in reaction pH could accentuate the loss of peroxidase activity. In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher concentrations of the redox mediator caused a slight increase in inactivation, likely due to the formation of reactive promethazine radicals, which in turn attack the protein via a mechanism different from that caused by excess H₂O₂. These findings will help in defining the optimal reaction conditions that preserve the activity of the peroxidase molecules.