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Inhibition of Peroxidase-Catalyzed Oxidation of 3,3´,5,5´-Tetramethylbenzidine by Aminophenols

I. V. Naumchik1, E. I. Karasyova1, D. I. Metelitza1*, I. P. Edimecheva2, V. L. Sorokin2, and O. I. Shadyro2

1Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Akademika Kuprevicha 5/2, 220141 Minsk, Belarus; fax: (375)-(172) 63-7274; E-mail: metelitza@iboch.bas-net.by

2Faculty of Chemistry, Belorussian State University, ul. Leningradskaya 14, 220050 Minsk, Belarus; fax: (375)-(172) 09-5464; E-mail: shadyro@open.by

* To whom correspondence should be addressed.

Received April 7, 2004; Revision received August 23, 2004
Peroxidase-catalyzed oxidation of 3,3´,5,5´-tetramethylbenzidine (TMB) was inhibited by o-aminophenol (AP), 2-amino-4-tert-butylphenol (ATBP), 2-amino-4,6-di-tert-butylphenol (ADTBP), and 4-tert-butylpyrocatechol (TBP). Inhibitors were characterized by inhibition constant Ki and stoichiometric coefficient f, the number of radicals terminated by one inhibitor molecule. The most efficient inhibitor is ADTBP characterized by Ki = 36 µM in 0.015 M phosphate citrate buffer, pH 6.0, at 20°C. According to their antiradical efficiency, the studied inhibitors can be arranged as follows: ADTBP > ATBP > AP > TBP. The role of the NH2 group in the inhibitory capacity of aminophenols is discussed. Using gas-liquid chromatography, kinetics of consumption of the initial components and accumulation of the reaction products on peroxidase-catalyzed oxidation of the TMB-TBP pair was studied; the data clarify the stages of a complex process of co-oxidation of amines and phenols.
KEY WORDS: horseradish peroxidase, tetramethylbenzidine, aminophenols, inhibition, inhibition constants, stoichiometric inhibition coefficients