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.
Toxicology Program, College of Public Health, University of South Florida, Tampa 33612.
P
P
Joseph
N S
NS
Srinivasan
A P
AP
Kulkarni
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009584:Nitrogen / Q000494:pharmacology; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011190:Potassium Cyanide / Q000494:pharmacology; D011247:Pregnancy; D013854:Thioamides / Q000378:metabolism*
D013854:Thioamides; C003320:sodium nitride; C015539:thiobenzamide; D006861:Hydrogen Peroxide; D009195:Peroxidase; D011190:Potassium Cyanide; D009584:Nitrogen