Partial purification, characterization, and binding study of a peroxidase from human placenta.

Peroxidases can metabolize a variety of xenobiotics to reactive intermediates capable of binding to protein or DNA. The potential role of these enzymes in fetotoxicity has been unexplored. In this study, a protein with peroxidase activity from human term placenta (HTPP) was partially purified by Con-A-affinity chromatography from CaCl$\sb2$ extracts of the particulate fraction. HTPP appears to be a membrane-bound glycoprotein and metabolizes several model peroxidase substrates. HTPP activity was found in all subcellular membranes but not in cytosol. Arachidonic acid-dependent oxidation of guaiacol was not observed, suggesting that the peroxidase activity was not due to prostaglandin synthase. Moreover, HTPP preparations were devoid of catalase and spectrally dissimilar to human hemoglobin, cytochrome P-450, eosinophil peroxidase, and myeloperoxidase, suggesting an endogenous origin. A molecular weight of approximately 119,000 daltons was determined for HTPP by gel filtration. Cathodic slab-PAGE of cetyltrialkylammonium bromide-solubilized HTPP yielded two peroxidase-staining bands. No peroxidase-staining bands were visible on anodic slab-PAGE. SDS-PAGE of Con-A-purified HTPP revealed numerous bands, two of which co-migrated with lactoperoxidase and had estimated molecular weights of 80,000 and 87,000 daltons. Con-A-purified HTPP catalyzed the in vitro binding of 2-aminofluorene (2-AF) to protein and DNA, which was significantly inhibited by azide and BHA. 7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) was metabolized to protein-binding intermediates, but was not oxidized to derivatives which bound DNA in the presence of Con-A-purified HTPP and H$\sb2$O$\sb2$. Con-A-purified HTPP failed to catalyze the in vitro binding of benzo(a)pyrene to either protein or DNA under the same conditions. Measurable H$\sb2$O$\sb2$- but not NADPH-dependent in vitro binding of 2-AF to protein was observed in washed term placental membranes. The results indicate that a human placental peroxidase catalyzes the oxidation of certain carcinogens to reactive intermediates capable of binding to biological macromolecules, suggesting a potential role in the bioactivation of fetotoxicants.PhDBiochemistryEnvironmental scienceHealth and Environmental SciencesPublic healthPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128278/2/8907116.pd