反应详情

The assay buffer “AB” contains 50 mM Hepes pH7.5, 100 μM Dithiothreitol, 0.006% Triton-X100. The following components are added in a black polystyrene Costar plate up to a final volume of 55 μL: 1.5 μL DMSO, or inhibitor dissolved in DMSO and 53.5 μL of a FabI/NADH/NAD+ mixture in AB. After 60 min of pre-incubation at room temperature, the reaction is started by addition of 5 μL of Crotonoyl-CoA to a final volume of 60 μL. This reaction mixture is then composed of 40 nM FabI (produced in house from E. coli, C-terminal 6-His tagged), 20 μM NADH (Biochemika), 10 μM NAD+ (Biochemika), 50 μM Crotonoyl-CoA (Biochemika) and compound at defined concentration. Fluorescence intensity of NADH (lex=360 nm, lem=520 nm) is measured immediately after Crotonoyl-CoA addition, and 2 hours later by a Fluostar Optima (BMG). Enzyme activity is proportional to the signal decrease from which inhibition percentages are derived. For IC50 determinations, the inhibitor is tested at 6 to 10 different concentrations, and the related inhibitions are fitted to a classical langmuir equilibrium model using XLFIT (IDBS).