Purification and partial characterization of peroxidase from human term placenta of non-smokers: Metabolism of benzo(a)pyrene-7,8-dihydrodiol

Peroxidase (Donor: H2O2 oxidoreductase EC 1.11.1.7) from human term placent ae of non-smokers was purified to homogeneity by a combination of NH4Cl ext raction, affinity chromatography, (NH4)(2)SO4 precipitation, ion-exchange a nd gel filtration chromatography. The homogeneity of purified human placent al peroxidase (HTPP) was confirmed by gel filtration, reverse phase high pe rformance liquid chromatography (HPLC) and SDS-PAGE. Peroxidase was found t o be a membrane bound enzyme. A high concentration of NH4Cl (1.2 M) was nee ded to extract and solublize the enzyme. Removal of the salt resulted in ir reversible precipitation of the enzyme. The protein exhibited a molecular m ass of 126 000 kDa according to gel filtration and approximately 60 000 kDa by SDS-PAGE, indicating that the peroxidase is a homodimer. The purified p eroxidase showed an optimum pH range of 7 to 8.5 and the K-m for H2O2 and g uaiacol were found to be 0.08 mM and 10.0 mM, respectively. The purified pe roxidase oxidized several substrates, namely potassium iodide, tetramethyl benzidine, guaiacol, ortho dianisidne and tyrosine. The enzyme was resistan t to thermal denaturation up to 70 degrees C and also to chaotropic agents, guanidinium chloride and urea. Spectral properties indicated the presence of Soret band at 433 which shifted to 451 nm on complexation with cyanide. The circular dichroism studies showed that HTPP has a predominantly helical secondary structure. The enzyme showed similarities to the myeloperoxidase with regard to spectral and catalytical properties but differed significan tly in amino acid composition, the R-z value and molecular mass. Purified H TPP differed from eosinophil peroxidase in all physico-chemical properties indicating that it is not of eosinophil origin, but mall represent a distin ct, constitutive peroxidase in human placenta. Further, purified peroxidase catalyzed oxidation of benzo(a)pyrene-7,8-dihydrodiol in presence of tyros ine and hydrogen peroxide to BP-tetrols, the hydrolytic products of BP-diol -epoxides, demonstrating the ability of peroxidase in bioactivation of benz o(a)pyrene in human placenta. (C) 2000 Harcourt Publishers Ltd.