A colorimetric method for the determination of lipoxygenase activity suitable for use in a high throughput assay format.
A rapid and sensitive method for measuring the activity of lipoxygenase is described. The assay is based on the principle that under acidic conditions a lipid hydroperoxide can oxidize Fe2+ to Fe3+ which then oxidizes xylenol orange to form a product that absorbs strongly in the visible region (see Z-Y. Jiang, A. C. S. Woollard, and S. P. Wolff, Lipids 26, 853-856, 1991). This methodology was modified to measure lipoxygenase activity and the system was optimized using platelet 12-lipoxygenase. This assay is suitable for use in a 96-well microtiter plate and may be utilized as a high throughput screen for the identification of novel lipoxygenase inhibitors.
Waslidge
N B
NB
Enzymology Group, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.
Hayes
D J
DJ
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase / Q000097:blood*; D001792:Blood Platelets / Q000201:enzymology*; D003124:Colorimetry; D005290:Ferric Compounds / Q000737:chemistry; D005456:Fluorescent Dyes; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood; D015289:Leukotrienes / Q000097:blood; D016014:Linear Models; D010084:Oxidation-Reduction; D010636:Phenols; D010975:Platelet Aggregation Inhibitors / Q000097:blood; D012680:Sensitivity and Specificity; D012995:Solubility; D013053:Spectrophotometry; D013454:Sulfoxides; D013997:Time Factors; D014992:Xylenes
D005290:Ferric Compounds; D005456:Fluorescent Dyes; D006893:Hydroxyeicosatetraenoic Acids; D015289:Leukotrienes; D010636:Phenols; D010975:Platelet Aggregation Inhibitors; D013454:Sulfoxides; D014992:Xylenes; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C021201:12-HPETE; D001092:Arachidonate 12-Lipoxygenase; C016833:xylenol orange