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 GPx4. 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 ∼80-times greater than that for GPx4-deficient controls (VC). A TPD-detectable cholesterol hydroperoxide (7α-OOH) was also reduced much faster by 7G4 than VC extracts. Spraying with H2SO4 after TPD revealed both 7α-OOH loss and resolved diol product (7α-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.