IMPROVED RADIOLABELED SUBSTRATES FOR SOLUBLE EPOXIDE HYDROLASE

Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [C-14]-cis-9,10-epoxystearic acid and its corresponding diol ([C-14]-9,10-dihydroxystearic acid) is described. The cis fatty acid oxide exhibits higher specific activity of hydration with sEH from mouse, rat, human, and potato compared to trans-stilbene oxide (TSO). The K-m and V-max obtained for [C-14]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 mu M and 3460 nmol/min/mg protein, respectively. [C-14]-cis-9,10-Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-H-3]-trans-1,3-diphenylpropene oxide ([H-3]-tDPPO) and [2-H-3]-cis-1,3-diphenylpropene oxide ([H-3]-cDPPO) were synthesized and rapid radiometric assays for epoxide hydrolases (EHs) were developed by differential partitioning of the epoxide into iso-octane and its corresponding diol into aqueous phase containing methanol. It was shown that sEHs from mouse, rat, human, and potato rapidly hydrolyze [H-3]-tDPPO and in comparison to TSO have 20-, 49-, 28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [H-3]-tDPPO at 26,200 nmol/min/mg protein, and a K-m of 2.80 mu M is observed. (C) 1995 Academic Press, Inc.