A fluorescent micro‐assay utilizing purified microsomal epoxide hydrolase

A low volume (10 μL) kinetic fluorescent assay to evaluate human microsomal epoxide hydrolase (mEH) activity with the α‐cyano‐methyl ester substrate, PHOME (Analyt. Biochem. (2006) 355, 71–80), has been optimized. mEH was successfully cloned into a mammalian expression vector, expressed transiently in FreeStyle 293F cells, and purified by affinity chromatography (purity > 80%). In the presence of 5 μM PHOME, reaction rates were linearly proportional to added enzyme to 0.1 μM and the reactions were linear for up to 60 minutes with respect to product formation. The lowest discernable reaction rate of 5.5 nM/min was obtained with 10 nM mEH; with an assay signal:noise of 3:1. Optimized assay buffer contained 25 mM HEPES pH 7.0, 0.01% CHAPS, and 0.005% casein. Purified mEH activity was selectively inhibited with the addition of 50 mM ZnSO4 or with the known mEH inhibitor, Valpromide (IC50 = 1 mM and 5 μM, respectively), indicating that product formation was due solely to mEH. The potency of Valpromide was shown to give similar values in an alternative assay format using mEH enriched microsomes (IC50 = 8.0 μM). AUDA, a potent sEH inhibitor, was not an inhibitor of purified mEH. Thus, purified mEH provides a means to develop a low volume, high throughput assay to further our efforts towards developing inhibitors that can selectively discriminate sEH versus mEH activity.