Placental peroxidase--further purification of the enzyme and oxidation of thiobenzamide.

Human term placental peroxidase [donor: hydrogen peroxide (H2O2) oxidoreductase] from non-smoking women was purified by extraction of the membrane fraction with 0.5 M Ca2+ followed by affinity chromatography on concanavalin A, hydrophobic chromatography on phenyl sepharose CL-4B and gel filtration chromatography on sephacryl S-200 columns. The final enzyme preparation was 110-fold pure when compared to the Ca2+ extract and the overall recovery of the procedure was 55 per cent. SDS-PAGE of the final product showed the presence of four protein staining bands of molecular weights 30, 32.5, 35 and 55 KDa. The purified peroxidase eluted as a single peak from the sephacryl S-200 column suggesting apparent homogeneity of the protein. The purified placental peroxidase oxidized thiobenzamide to its sulfoxide. The highest rate of TB S-oxidation under optimum conditions was 18 +/- 2.15 mumoles/min/mg protein. The H2O2-dependent thiobenzamide S-oxidation catalysed by the placental peroxidase was inhibited by KCN and NaN3 with IC50 values of 16.9 and 34 microM respectively.