Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels

Anal Biochem. 1988 Feb 15;169(1):71-80. doi: 10.1016/0003-2697(88)90256-4.


Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Animals
  • Chromatography, Affinity
  • Cytosol / enzymology*
  • Dialysis
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Epoxide Hydrolases / isolation & purification*
  • Epoxy Compounds
  • Female
  • Humans
  • Liver / enzymology
  • Macaca mulatta
  • Male
  • Mice
  • Sepharose


  • Epoxy Compounds
  • Sepharose
  • Epoxide Hydrolases