Spectrophotometric Assay based on Horseradish Peroxidase- Catalysed Hydrogen Peroxide using Amino Antipyrine and Resorcinol as Chromogenic Reagents for Sensitive Detection of Peroxidase in Plant Extracts

We reported a unique enzyme- catalyzed hydrogen peroxide system for the detection of Horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The catalytic system involving 4-Aminoantipyrine (4-AAP) and resorcinol (RN) as chromogens. In this study, the red-colored quinone-imine radical generated by the oxidation of RN with peroxidase catalyzed H2O2 oxidation, coupling with the aminyl radical of 4-AAP that has absorption peak at 500 nm. The absorption intensity of the quinone-imine radical scaled linearly with H2O2 concentration in the ranges of 1.1522μM-294.98 μM and 0.5761- μM- 82.96 μM with a detection limit of 0.12 μM and quantification limit of 0.225 μM and peroxidase concentration 0.0425-5.8 nM and 0.02-2.99 nM for kinetic and fixed time methods could be achieved. The kinetic parameters Km and Vmax were found to be 172.41 μM and 0.1136 EU min-1. Moreover, compared with standard guaiacol system, HRP/ H2O2/4-AAP/RN system had wide linear ranges, low detection limit, Km and Vmax value. Noteworthy, the present study was successful applied to monitor peroxidase concentration in different plants extracts with satisfied results. Overall, the proposed system can be adopted as a candidate system for the determination of H2O2.