2-amino-4-nitrophenol and 3-3--5-5--tetramethylbenzidine

The steady-state kinetics of the horseradish peroxidase (HRP)-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) has been studied in the presence of 2-amino-4-nitrophenol (ANP), gallic acid (GA) or 4,4'-dihydroxydiphenylsulfone (DDS) and their polydisulfides poly(ADSNP), poly(DSGA), poly(DSDDS) at 20 degrees C in 10 mM phosphate buffer, pH 6.4, supplemented with 5-10% dimethylformamide. The second-order rate constants for the reactions of ANP, GA, poly(DSGA) and poly(DSDDS) with HRP-Compound I (k2) and Compound II (k3) have been determined at 25 degrees C in 10 mM phosphate buffer, pH 6.0 by stopped-flow spectrophotometry. ANP, GA and their polydisulfides strongly inhibited HRP-catalyzed TMB oxidation. Inhibition constants (Ki) and stoichiometric coefficients of inhibition (f) have been determined for these reactions. The most effective inhibitor was poly(DSGA) (Ki=1.3 microM, f=35.6). The oxidation of substrate pairs by HRP, i.e., TMB-DDS and TMB-poly(DSDDS) at pH 7.2 resulted in a approximately 8- and approximately 12-fold stimulation of TMB oxidation rates, respectively. The mechanisms of the HRP-catalyzed co-oxidation of TMB-phenol pairs are discussed.

Topics: Antioxidants; Benzidines; Catalysis; Disulfides; Enzyme Inhibitors; Gallic Acid; Horseradish Peroxidase; Hydrogen-Ion Concentration; Kinetics; Nitrophenols; Oxidation-Reduction

The effects of different concentrations of 2-amino-4-nitrophenol (ANP) and of its polydisulfide (poly(ADSNP)) on peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) were studied at 20 degrees C in reversed micelles of AOT (0.2 M) in heptane and in mixed reversed micelles of AOT (0.1 M)-Triton X-100 (0.1 M) in isooctane supplemented with 15% hexanol. The oxidation of TMB was activated nearly twofold in the presence of ANP and nearly fourfold in the presence of poly(ADSNP) in reversed micelles of AOT, whereas in the mixed micelles oxidation of the TMB-ANP pair was associated with inhibition of TMB conversion and poly(ADSNP) activated oxidation of TMB. The co-oxidation of TMB with 4,4'-dihydroxydiphenylsulfone (DDS) and with its polydisulfide (poly(DSDDS)) at different concentrations of phenol components was accompanied by activation of TMB conversion in 0.01 M phosphate buffer (pH 6.4) supplemented with 5% DMF and in reversed micelles of AOT in heptane. The effect of pH of the aqueous solution on the initial oxidation rate of the TMB-DDS and TMB-poly(DSDDS) pairs and also the effect of hydration degree of reversed micelles of AOT on conversion of the same pairs by peroxidase were studied. A scheme of peroxidase-dependent co-oxidation of "aromatic amine-phenol" pairs is proposed and discussed. A significant part of this scheme is a nonenzymatic exchange of phenoxyl radicals with amines and of aminyl radicals with phenols.

Topics: Benzidines; Chromogenic Compounds; Disulfides; Kinetics; Micelles; Nitrophenols; Oxidation-Reduction; Peroxidase