Peroxidase-catalyzed co-oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of substituted phenols and their polydisulfides.
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.
Metelitza
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DI
Institute of Bioorganic Chemistry, National Academy of Sciences, Kuprevich str., 5/2, Minsk 220141, Belarus. metelitza@iboch.bas-net.by
Karasyova
E I
EI
Grintsevich
E E
EE
Thorneley
R N F
RN
D000975:Antioxidants / Q000737:chemistry; D001560:Benzidines / Q000737:chemistry*; D002384:Catalysis; D004220:Disulfides / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005707:Gallic Acid / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009596:Nitrophenols / Q000737:chemistry*; D010084:Oxidation-Reduction
D000975:Antioxidants; D001560:Benzidines; D004220:Disulfides; D004791:Enzyme Inhibitors; D009596:Nitrophenols; C021758:3,3',5,5'-tetramethylbenzidine; D005707:Gallic Acid; D006735:Horseradish Peroxidase; C036645:2-amino-4-nitrophenol