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A fluorimetric assay for human reticulocyte 15-lipoxygenase-1 activity
- Lu, Weiqiang, Zhao, Xue, Zou, Shien, Huang, Jin
- Analytical biochemistry 2012 v.426 no.1 pp. 66-68
- fluorescence emission spectroscopy, fluorescent dyes, fluorometry, humans, hydroperoxides, inhibitory concentration 50, lipoxygenase, nordihydroguaiaretic acid, quercetin
- 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 500nm/536nm. 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.