Analytical Sciences

Development and Evaluation of Kinetic Spectrophotometric Assays for Horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and Mequinol

Padmarajaiah NAGARAJA, Anantharaman SHIVAKUMAR, and Ashwinee KUMAR SHRESTHA

Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India

This paper presents a novel spectrophotometric method to measure peroxidase activity using paraphenylenediamine dihydrochloride (PPDD) and Mequinol (MQ). The PPDD traps the free radical, and gets oxidized to electrophilic 1,4-diimine; this couples with MQ to an give intense violet-colored chromogenic species with the maximum absorbance at 560 nm. This assay was adopted for the quantification of hydrogen peroxide between 10 × 10⁻⁶ to 80 × 10⁻⁶ M. From the kinetic data, a two-substrate ping-pong mechanism of peroxidase was established. Catalytic efficiency and catalytic power of commercial peroxidase were 0.204 × 10⁶ M⁻¹ min⁻¹ and 2.86 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) of the proposed method was 0.2080 × 10³ min⁻¹. As a simple, rapid, precise and sensitive technique, PPDD-MQ stands as a potential replacement for the traditional guaiacol method. Applications to the plant extracts increase its relevance in the field of biochemical analysis.

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