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Detection of mammalian 5‐lipoxygenase activity using the fluorescent probe dihydrorhodamine 123
Author(s) -
Zhao Xue,
Lu Weiqiang,
Song Cheng,
Huang Jin
Publication year - 2014
Publication title -
european journal of lipid science and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.614
H-Index - 94
eISSN - 1438-9312
pISSN - 1438-7697
DOI - 10.1002/ejlt.201300104
Subject(s) - fluorescence , chemistry , plate reader , lipoxygenase , arachidonate 5 lipoxygenase , biochemistry , rhodamine 123 , cofactor , chromatography , enzyme , multiple drug resistance , arachidonic acid , physics , quantum mechanics , antibiotics
In this article, we describe a new, fast and reliable method for determining 5‐LOX activity using the fluorescent probe dihydrorhodamine 123 (DHR). In the new assay, the products of 5‐LOX, 5S‐hydroperoxy‐6E,8Z,11Z,14Z‐eicosatetraenoic acid, can directly oxidize non‐fluorescent DHR to highly fluorescent rhodamine 123. The fluorescence intensity can be measured using fluorescence spectroscopy at Ex/Em = 500/536 nm. As 5‐LOX requires some cofactors to achieve its optimum activity, the effects of its cofactors on the fluorescence assay are also investigated. Using this new assay, we evaluated the inhibitory activity of two well‐known 5‐LOX inhibitors (NDGA and AA861) in a 384‐well format and the IC 50 values of them are consistent with previously references. The fluorescence assay is amenable to both purified 5‐LOX and 5‐LOX in cell lysates and will be widely used for the discovery of novel 5‐LOX inhibitors for clinical application.