A fluorimetric assay for human reticulocyte 15-lipoxygenase-1 activity

A rapid and sensitive fluorescence-based assay for the determination of human 15-lipoxygenase-1 (15-LOX-1) activity is described in this article. The assay utilizes the ability of 15-LOX-1-generated lipid hydroperoxides to oxidize nonfluorescent dihydrorhodamine 123, producing the highly fluorescent dye rhodamine 123. Formation of rhodamine 123 can be monitored through fluorescence spectroscopy using Ex/Em of 500 nm/536 nm. The IC₅₀ values of three well-known 15-LOX-1 inhibitors, nordihydroguaiaretic acid, quercetin, and fisetin, were evaluated in 96- and 384-well formats, and they conform to previously reported data. We believe this assay can be broadly used for the discovery of novel lipoxygenase inhibitors.