A thin layer chromatographic method for determining the enzymatic activity of peroxidases catalyzing the two-electron reduction of lipid hydroperoxides.
Thiol-dependent peroxidases catalyzing the reductive detoxification of lipid hydroperoxides (LOOHs) are crucial antioxidant components of mammalian cells. There is a growing interest in manipulating expression of such enzymes to better understand their biological roles. A new approach for determining their cellular activity is described, whereby LOOH reduction kinetics are tracked by high performance thin layer chromatography with peroxide-sensitive tetramethyl-p-phenylenediamine detection (HPTLC-TPD). The approach was tested on a tumor cell transfectant clone (7G4) over-expressing selenoperoxidase GP x 4. Timed incubation of Triton-solubilized 7G4 cells with GSH and peroxidized phosphatidylcholine (PCOOH), followed by lipid extraction, HPTLC-TPD and densitometry revealed an exponential decay of PCOOH at a rate approximately 80-times greater than that for GP x 4-deficient controls (VC). A TPD-detectable cholesterol hydroperoxide (7alpha-OOH) was also reduced much faster by 7G4 than VC extracts. Spraying with H(2)SO(4) after TPD revealed both 7alpha-OOH loss and resolved diol product (7alpha-OH) accumulation, the kinetics of which were identical. The approach described is relatively convenient, highly specific, and much more sensitive than conventional assays for cellular LOOH reducing enzymes.
Kriska
Tamas
T
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Girotti
Albert W
AW
D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods*; D005979:Glutathione Peroxidase / Q000032:analysis; D006801:Humans; D008054:Lipid Peroxides / Q000378:metabolism*; D010544:Peroxidases / Q000032:analysis*; D010713:Phosphatidylcholines / Q000378:metabolism; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D008054:Lipid Peroxides; D010713:Phosphatidylcholines; C057590:phosphatidylcholine hydroperoxide; C002843:cholesterol hydroperoxide; D002784:Cholesterol; D010544:Peroxidases; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase