Development and kinetic validation of an assay for the quantitative determination of peroxidase: Application in the detection of activity in crude plant tissues

A simple, rapid, and sensitive direct spectrophotometric assay of peroxidase activity based on its reaction with a chromogenic reagent 2,4-dimethoxyaniline (DMA) in aqueous media of pH 3.7-7.8 is reported. This method is based on the intermolecular coupling of activated DMA in the presence of peroxidase and H2O2 to produce a violet-colored species having λmax 540 nm. This method has been used in the assay of H2O2 in the range of 40-330 μM. At a H2O2 concentration of 660 μM, the peroxidase linearity is established in the range of 0.2022-1.13 and 0.0079-0.756 nM by rate and one-time detection method, respectively. Two substrate kinetic ping-pong mechanism is established. A Hanes-Woolf plot is used in the evaluation of Michaelis-Menten constants of H2O2 and dimethoxyaniline. The catalytic efficiency and catalytic power of the proposed method are 2.67 × 105 min−1 M−1 and 2.05 × 10−4 min−1, respectively. The apparent k3 at H2O2 concentration ranging between 40 and 1320 μM is found to be 469 min−1. This method has been applied in the crude plant extracts with medicinal value.