Peroxidase-catalyzed Oxidation of 3,3",5,5"-Tetramethylbenzidine in the Presence of 2,4-Dinitrosoresorcinol and Polydisulfide Derivatives of Resorcinol and 2,4-Dinitrosoresorcinol

A comparative study of the kinetics of peroxidase-catalyzed oxidation of 3,3",5,5"-tetramethylbenzidine (TMB) in the presence of 2,4-dinitrosoresorcinol (DNR), its polydisulfide derivative [poly(DNRDS)], and resorcinol polydisulfide [poly(RDS)], substances that competitively inhibit the formation of TMB conversion product, was carried out. The inhibition constants Ki for DNR, poly(DNRDS), and poly(RSD) were determined at 20°C and pH 6.4 to be 110, 13.5, and 0.78 μM, respectively. The stoichiometric coefficients of inhibition were calculated to be 0.38 and 76 for poly(DNRDS) and poly(RDS), respectively. In the pH range 6.4–7.0, the initial rates of the peroxidative oxidation of TMB, and its mixtures with DNR and poly(DNRDS) and the Ki value for poly(RDS) substantially decreased with increasing pH. The kinetic parameters of poly(RDS) (Ki 0.22–0.78 μM and f 76) suggest that it is the most efficient inhibitor of peroxidase oxidation of TMB: in micromolar concentrations, it completely stops this process and can be used in EIA.