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Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol

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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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Authors and Affiliations

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

    Padmarajaiah Nagaraja, Anantharaman Shivakumar & Ashwinee Kumar Shrestha

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  1. Padmarajaiah Nagaraja
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  2. Anantharaman Shivakumar
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  3. Ashwinee Kumar Shrestha
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Correspondence to Padmarajaiah Nagaraja.

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Nagaraja, P., Shivakumar, A. & Kumar Shrestha, A. Development and evaluation of kinetic spectrophotometric assays for horseradish Peroxidase by Catalytic Coupling of Paraphenylenediamine and mequinol. ANAL. SCI. 25, 1243–1248 (2009). https://doi.org/10.2116/analsci.25.1243

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  • DOI: https://doi.org/10.2116/analsci.25.1243

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