FIGURE 1.
HPLC-RAM identification of the O-methylated metabolites (M1 and M2) of the representative PAH-catechols produced by human recombinant COMT. The reactions were conducted under anaerobic conditions in 10 mm KPO4 buffer, pH 7.8, 1.0 mm dithiothreitol, 1.0 mm MgCl2, 50 μm [3H]AdoMet, 20 μm PAH o-quinone, and 0.225 or 0.45 μg of human recombinant COMT at 25 °C. The reactions were quenched by 1% formic acid and extracted with toluene. The organic phase was then dried and dissolved in MeOH for HPLC-UV-RAM analysis. A and B, HPLC-UV-RAM chromatogram of O-methylated B[a]P-7,8-catechols; C and D, HPLC-UV-RAM chromatogram of O-methylated 5MC-1,2-catechols; E and F, HPLC-UV-RAM chromatogram of O-methylated B[g]C-11,12-catechols. The red triangles indicate the beginning and end of peak integration in the chromatograms. AU, absorbance units.