Horseradish Peroxidase / Hydrogen Peroxide - catalyzed Binding of Aromatic Hydrocarbons to DNA

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.