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Related Experiment Videos

Improved radiolabeled substrates for soluble epoxide hydrolase

B Borhan1, T Mebrahtu, S Nazarian

  • 1Department of Chemistry, University of California, Davis 95616, USA.

Analytical Biochemistry
|October 10, 1995
PubMed
Summary

Two new radiometric assays were developed for soluble epoxide hydrolase (sEH) using novel substrates. These assays offer sensitive and rapid methods for studying sEH activity in various species, including humans.

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Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Soluble epoxide hydrolase (sEH) plays a crucial role in regulating bioactive lipid mediators.
  • Existing assays for sEH activity can be time-consuming or lack sensitivity.
  • Development of rapid and sensitive assays is essential for comprehensive sEH research.

Purpose of the Study:

  • To develop and validate two novel, rapid radiometric assays for quantifying soluble epoxide hydrolase (sEH) activity.
  • To evaluate the efficacy of these assays using various sEH sources, including mouse, rat, human, and potato.
  • To characterize the kinetic parameters of sEH with the newly developed substrates.

Main Methods:

  • Radiometric assay utilizing [(14)C]-cis-9,10-epoxystearic acid and thin-layer chromatography.
  • Synthesis of radiolabeled [2-(3)H]-trans-1,3-diphenyl-propene oxide ([(3)H]-tDPPO) and [2-(3)H]-cis-1,3-diphenylpropene oxide ([(3)H]-cDPPO).

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  • Development of a differential partitioning assay for epoxide hydrolases (EHs) using [(3)H]-tDPPO and [(3)H]-cDPPO.

    • Main Results:

    • The [(14)C]-cis-9,10-epoxystearic acid assay demonstrated high sensitivity and specific activity with sEH from multiple species.
    • Kinetic analysis of mouse sEH with [(14)C]-cis-9,10-epoxystearic acid yielded K(m) of 11.0 microM and V(max) of 3460 nmol/min/mg protein.
    • The [(3)H]-tDPPO assay showed significantly higher hydrolysis rates compared to trans-stilbene oxide (TSO) across different species, with mouse sEH exhibiting a V(max) of 26,200 nmol/min/mg protein and a K(m) of 2.80 microM.

    Conclusions:

    • The developed radiometric assays provide sensitive and rapid methods for assessing sEH activity.
    • [(14)C]-cis-9,10-epoxystearic acid may represent a more biologically relevant substrate for sEH.
    • [(3)H]-tDPPO serves as an effective substrate for rapid radiometric assays of sEH in diverse biological systems.