Oxidation of Benzidine and Its Derivatives by Thyroid Peroxidase

V. V. Sentchouk^1* and E. E. Grintsevich²

¹Department of Biochemistry, Faculty of Biology, Belarus State University, ul. Skoriny 4, Minsk 220080, Belarus; fax: (375-172) 09-5851; E-mail: sentchouk@bsu.by; biochem@bsu.by

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

^* To whom correspondence should be addressed.

Received February 28, 2003; Revision received April 17, 2003

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Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3´,5,5´-tetramethylbenzidine (TMB). The kinetic characteristics (k_cat and K_m) of benzidine (BD), 3,3´-dimethylbenzidine (o-tolidine), 3,3´-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25°C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K_m values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3´, 5, and 5´ positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k_cat/K_m was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.

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KEY WORDS: human thyroid peroxidase, horseradish peroxidase, tetramethylbenzidine, o-tolidine, o-dianisidine, benzidine, peroxidase oxidation, kinetic characteristics, DNA damage

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