Development of a new colorimetric assay for lipoxygenase activity

Abstract

Lipoxygenases (LOXs) are a family of non-heme iron-containing dioxygenases that catalyze the hydroperoxidation of lipids, containing a cis,cis-1,4-pentadiene structure. A rapid and reliable colorimetric assay for determination of the activity of three human functional lipoxygenase isoforms (5-lipoxygenase, platelet 12-lipoxygenase, and 15-lipoxygenase-1) is developed in this article. In the new assay, LOX-derived lipid hydroperoxides oxidize the ferrous ion (Fe2+) to the ferric ion (Fe3+), the latter of which binds with thiocyanate (SCN–) to generate a red ferrithiocyanate (FTC) complex. The absorbance of the FTC complex can be easily measured at 480nm. Because 5-LOX can be stimulated by many cofactors, the effects of its cofactors (Ca2+, ATP, dithiothreitol, glutathione, l-α-phosphatidylcholine, and ethylenediaminetetraacetic acid) on the color development of the FTC complex are also determined. The assay is adaptive for purified LOXs and cell lysates containing active LOXs. We use the new colorimetric assay in a 96-well format to evaluate several well-known LOX inhibitors, the IC50 values of which are in good agreement with previously reported data. The reliability and reproducibility of the assay make it useful for in vitro screening for inhibitors of LOXs and, therefore, should accelerate drug discovery for clinical application.