Detection of mammalian 5-lipoxygenase activity using the fluorescent probe dihydrorhodamine 123

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=500/536nm. 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 IC₅₀ 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.