Development of a Fluorescent Intensity Assay Amenable for High-Throughput Screening for Determining 15-Lipoxygenase Activity

作者：Dahlstrom Marta^*; Forsstrom Daniel; Johannesson Malin; Huque Andersson Yasmin; Bjork Marie; Silfverplatz Erik; Sanin Andrei; Schaal Wesley; Pelcman Benjamin; Forsell Pontus K A

来源：Journal of Biomolecular Screening, 2010, 15(6): 671-679.

DOI：10.1177/1087057110373383

• 15-lipoxygenase
• cell-based assay
• arachidonic acid
• lipid hydroperoxides
• screening
• cell-based assays

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)

• 出版日期2010-7