Peroxidase-catalyzed oxidative coupling of paraphenylenediamine with 3-dimethylaminobenzoic acid: application in crude plant extracts.

This paper presents a novel spectrophotometric method to measure peroxidase activity using paraphenylenediamine dihydrochloride (PPDD) and 3-dimethylaminobenzoic acid (DMAB). The PPDD traps free radicals and becomes oxidized to electrophillic 1,4-diimine, which couples with DMAB to give an intense green-colored chromogenic species with maximum absorbance at 710 nm. This assay was adopted for the quantification of hydrogen peroxide between 5 and 45 microM. From the kinetic data, a two-substrate ping-pong mechanism of peroxidase was established. The catalytic efficiency and catalytic constant (k(cat)) of the proposed assay were 0.54 x 10(6) M(-1) min(-1) and 0.0436 x 10(3) min(-1), respectively. As a simple, rapid, precise, and sensitive technique, PPDD-DMAB stands as a potential replacement for the traditional guaiacol method. Application of this method in plant extracts opens its relevance in the field of biochemical analysis.