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Horseradish peroxidase/hydrogen peroxide-catalyzed binding of aromatic hydrocarbons to DNA.

E. Rogan,P. Katomski,R. Roth,E. Cavalieri

Published 1979 in Journal of Biological Chemistry

ABSTRACT

Covalent binding of aromatic hydrocarbons to DNA is catalyzed by horseradish peroxidase with HzOz. Under optimal reaction conditions (30 PM hydrocarbon, 5 x lo-“ M HzOz, and 0.2 mg/ml of horseradish peroxidase at pH 7.0), binding was linear for 30 min. Binding of benzo[a]pyrene, 6-methylbenzo[a]pyrene, and 3-methylcholanthrene ranged from 40 to 100 pmol/mol of DNA. Fluorescence spectra of 6-methylbenzo[u]pyrene enzymatically bound to DNA were almost identical to the spectra of 6-bromomethylbenzo[a]pyrene bound to DNA and 6-methylbenzo[u]pyrene alone, indicating that 6-methylbenzo[a]pyrene was bound to DNA at the 6-methyl group. The mechanism of activation of benzo[u]pyrene was studied using 16; 1,3,6; 7 and 8-3H, %]benzo[u]pyrenes. Loss of 94% of the tritium from [6-3H, “C]benzo[u]pyrene showed that binding occurred at C-6. No tritium was lost from [S3H, 14C]benzo[u]pyrene, demonstrating that tritium was not randomly lost from benzo[u]pyrene. Benzo[a]pyrene is most probably activated by HRP/ HzOz by 1) l-electron oxidation with formation of a radical cation having charge localized at C-6 or 2) formation of the 6-oxybenzo[u]pyrene radical from the possible metabolite 6-hydroxybenzo[u]pyrene. To distinguish between these, reaction mixtures were analyzed by high pressure liquid chromatography. Approximately 2% of the substrate was bound to DNA, but no metabolites were detected. These results provide strong, but not conclusive, evidence that HRP/H202catalyzed binding of hydrocarbons to DNA occurs by lelectron oxidation.

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