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

Roger N. Wixtrom, Marilyn H. Silva, Bruce D. Hammock

Research output: Contribution to journalArticlepeer-review

82 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)71-80
Number of pages10
JournalAnalytical Biochemistry
Issue number1
StatePublished - Feb 15 1988


  • affinity chromatography
  • enzyme purification
  • epoxide hydrolase
  • epoxy-activated Sepharose

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology


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