Assay Method Information

Assay Name:  Lipoxygenase UV-Vis Assay
Description:  The inhibitor compounds were screened initially using one concentration point on a Perkin-Elmer Lambda 40 UV-Vis spectrometer. The percent inhibition was determined by comparing the enzyme rates of the control (DMSO solvent) and the inhibitor sample by following the formation of the conjugated diene product at 234 nm (ε=25,000 M−1 cm−1). The reactions were initiated by adding either of 40 nM 12-LOX, 40 nM 12/15-LOX, 0.5 μM 15-LOX-2 or 200 nM 5-LOX (ammonium sulfate suspension) to a cuvette with a 2 mL reaction buffer constantly stirred using a magnetic stir bar at room temperature (22° C.). It should be noted that LOX isozymes are often expressed in the inactive demetallated form, so it is best to utilize activity to determine the optimal LOX concentration for the assay (optimal rate of approximately 0.001 abs/sec at 10 μM AA). Reaction buffers used for various lipoxygenase were as follows-25 mM HEPES (pH 7.3), 0.3 mM CaCl2, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, 10 μM AA for the crude, ammonium sulfate precipitated 5-LOX; 25 mM Hepes (pH 8), 0.01% Triton X-100, 10 μM AA for 12-LOX and 25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, 10 μM AA for 12/15-LOX and 15-LOX-2. The substrate concentration was quantitatively determined by allowing the enzymatic reaction to go to completion in the presence of 15-LOX-2. For the inhibitors that showed more than 50% inhibition at the one point screens (25 μM inhibitor), IC50 values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration (Approximate range: 0.1 to 25 μM inhibitor), followed by a hyperbolic saturation curve fit (assuming total enzyme concentration [E]<<Ki app, so IC50 Ki app). It should be noted that all of the potent inhibitors displayed greater than 80% maximal inhibition, unless stated in the tables. Inhibitors were stored at −20° C. in DMSO.
Affinity data for this assay
 

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