Optimizing the o-Phenylenediamine Assay for Horseradish Peroxidase: Effects of Phosphate and pH, Substrate and Enzyme Concentrations, and Stopping Reagents

Horseradish peroxidase, assayed with o-phenylenedi-amine, is irreversibly inactivated when incubated in phosphate buffer, 100 mmol/L, at pH 5. The inactivation depends on both duration of incubation and phosphate concentration. Phosphate was the most potent inactivator and citrate the least potent of a series of buffers tested. The inactivation is not attributable to ionic strength per se or to Na+ or K+. The observed inactivation did not occur at high concentrations (2500 nmol/L, 0.1 g/L) of enzyme; however, this “protective ” effect could not be reproduced by adding bovine serum albumin or a surfactant (Tween 20) to lower concentrations of enzyme. The inactivation was independent of commercial source of the enzyme or the kind of chromogenic assay used. On the basis of this information, we optimized the assay so that it gave eightfold greater absorbance values than those reported by others. The improved assay was sensitive to as little as 0.4 pmol/L (16 ng/L) of peroxidase, and was linear over the range of 0.4 to 5 pmol/L (16-200 ng/L). Additional Keyphrases: enzyme activity. variation,source of Although horseradish peroxidase (HRP; EC 1.11.1.7)2 is widely used as an auxiliary enzyme for oxidases and as a marker in enzyme-labeled immunoassays, its stability at different pH values and in different buffers has not been systematically investigated. We find that HRP activity di-minishes rapidly when HRP is stored at 25 #{176}Cin 100 mmol/L phosphate buffer, pH 5. We describe here the effects of pH and phosphate buffer concentration on the stability of HRP, and the conditions for optimizing the assay of HRP with o-phenylenediamine (OPD)