15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z′ value of 0.79 and 0.4 in a 96-and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme-and cell-based assays for the identification of hits and to determine potency by IC 50 determinations. (Journal of Biomolecular Screening 2010:671-679)
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