Kinetic Study of Peroxidase‐Catalyzed Coupling of Benzene‐1,4‐diamine and N‐(2‐Aminoethyl)naphthalen‐1‐amine: Development of Micromolar Hydrogen Peroxide Reaction System

Abstract

A novel chromogenic method to measure the peroxidase activity using para-phenylenediamine dihydrochloride (¼ benzene-1,4-diamine hydrochloride; PPDD) and N-(1-naphthyl)ethylenediamine dihydrochloride (¼ N-(2-aminoethyl)naphthalen-1-amine; NEDA) is presented. The PPDD entraps the free radical and gets oxidized to electrophilic diimine, which couples with NEDA to give an intense redcolored chromogenic species with maximum absorbance at 490 nm. This assay was adopted for the quantification of H 2 O 2 between 20 and 160 mm. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 4.47 Â 10 4 m À1 min À1 and 3.38 Â 10 À4 min À1, respectively. The catalytic constant (k cat ) and specificity constant (k cat /K m ) at saturated concentration of the co-substrates were 0.0245 Â 10 3 min À1 and 0.0445 mm À1 min À1, respectively. The chromogenic coupling reaction has a minimum interference from the reducing substances such as ascorbic acid, l-cystein, citric acid, and oxalic acid. The method being simple, rapid, precise, and sensitive, its applicability has been tested in the crude vegetable extracts that showed peroxidase activity.