Analytical Sciences
Online ISSN : 1348-2246
Print ISSN : 0910-6340
ISSN-L : 0910-6340
Original Papers
Development and Evaluation of Kinetic Spectrophotometric Assays for Horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and Mequinol
Padmarajaiah NAGARAJAAnantharaman SHIVAKUMARAshwinee KUMAR SHRESTHA
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2009 Volume 25 Issue 10 Pages 1243-1248

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Abstract

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 (kcat) 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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© 2009 by The Japan Society for Analytical Chemistry
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