Elsevier

Analytical Biochemistry

Volume 231, Issue 1, October 1995, Pages 188-200
Analytical Biochemistry

Regular Article
Improved Radiolabeled Substrates for Soluble Epoxide Hydrolase

https://doi.org/10.1006/abio.1995.1520Get rights and content

Abstract

Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [14C]-cis-9,10-epoxystearic acid and its corresponding diol ([14C]-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 Km and Vmax obtained for [14C]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 μM and 3460 nmol/min/mg protein, respectively. [14C]-cis-9,10-Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-3H]-trans-1,3-diphenylpropene oxide ([3H]-tDPPO) and [2-3H]-cis-1,3-diphenylpropene oxide ([3H]-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 [3H]-tDPPO and in comparison to TSO have 20-, 49-, 28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [3H]-tDPPO at 26,200 nmol/min/mg protein, and a Km of 2.80 μM is observed.

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