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

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I. V. Naumchik¹, E. I. Karasyova¹, D. I. Metelitza^1*, 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

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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 K_i and stoichiometric coefficient f, the number of radicals terminated by one inhibitor molecule. The most efficient inhibitor is ADTBP characterized by K_i = 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.

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KEY WORDS: horseradish peroxidase, tetramethylbenzidine, aminophenols, inhibition, inhibition constants, stoichiometric inhibition coefficients

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