在取代酚及其多硫化物存在下，过氧化物酶催化3,3',5,5'-四甲基联苯胺的共氧化反应。

Peroxidase-catalyzed co-oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of substituted phenols and their polydisulfides.

作者信息

Metelitza D I, Karasyova E I, Grintsevich E E, Thorneley R N F

机构信息

Institute of Bioorganic Chemistry, National Academy of Sciences, Kuprevich str., 5/2, Minsk 220141, Belarus.

出版信息

J Inorg Biochem. 2004 Jan;98(1):1-9. doi: 10.1016/j.jinorgbio.2003.10.007.

DOI:10.1016/j.jinorgbio.2003.10.007

PMID:14659626

Abstract

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.

摘要

在20℃下，于pH 6.4的10 mM磷酸盐缓冲液中，添加5 - 10%二甲基甲酰胺，研究了辣根过氧化物酶（HRP）催化3,3',5,5'-四甲基联苯胺（TMB）氧化反应的稳态动力学，反应体系中存在2-氨基-4-硝基苯酚（ANP）、没食子酸（GA）或4,4'-二羟基二苯砜（DDS）及其多硫化物聚（ADSNP）、聚（DSGA）、聚（DSDDS）。通过停流分光光度法，在25℃下于pH 6.0的10 mM磷酸盐缓冲液中测定了ANP、GA、聚（DSGA）和聚（DSDDS）与HRP-化合物I（k2）和化合物II（k3）反应的二级速率常数。ANP、GA及其多硫化物强烈抑制HRP催化的TMB氧化。已测定了这些反应的抑制常数（Ki）和抑制化学计量系数（f）。最有效的抑制剂是聚（DSGA）（Ki = 1.3 μM，f = 35.6）。在pH 7.2时，HRP对底物对即TMB-DDS和TMB-聚（DSDDS）的氧化分别使TMB氧化速率提高了约8倍和约12倍。讨论了HRP催化TMB-苯酚对共氧化的机制。