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.
Joseph
P
P
Toxicology Program, College of Public Health, University of South Florida, Tampa 33612.
Srinivasan
N S
NS
Kulkarni
A P
AP
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