Lipoxygenase enzyme inhibition assay

Enzyme assays (100 μL final volume) were conducted in Corning 96 half-well black, flat bottom assay plates and contained final concentrations of 50 μM arachidonic acid as a substrate, 1:10 (v:v) cholate mix (2% (w:v) sodium cholate and 2% (v:v) DMSO), 40 μM dihydrorhodamine 123 and 3 U/mL rLOX15 in buffer A (100 mM Tris-HCl, pH 7.5). Stock solutions of test compounds were prepared in DMSO and then diluted to final assay concentrations in cholate mix such that final cholate and DMSO concentrations were maintained at 0.2%. Reactions were initiated by addition of 50 μL of enzyme mix (80 μM dihydrorhodamine 123 and 6 U/mL rLOX15 in buffer A) to wells containing 50 μL substrate and cholate mix in Buffer A and linear rates were assessed via fluorescence, using excitation/emission of 500/536 nm, on a Molecular Devices Spectramax M2 every 10 seconds for 5 minutes at room temperature.