Development and evaluation of kinetic spectrophotometric assays for horseradish peroxidase by catalytic coupling of paraphenylenediamine and mequinol.

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 x 10(-6) to 80 x 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 x 10(6) M(-1) min(-1) and 2.86 x 10(-4) min(-1), respectively. The catalytic constant (k(cat)) of the proposed method was 0.2080 x 10(3) 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.