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 journalArticle

79 Citations (Scopus)

Abstract

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
Volume169
Issue number1
DOIs
StatePublished - Feb 15 1988

Fingerprint

Epoxide Hydrolases
Sepharose
Purification
Gels
Sulfhydryl Compounds
Impurities
Ligands
Affinity chromatography
Enzyme activity
Enzymes
Electrophoresis
Affinity Chromatography
Sodium Dodecyl Sulfate
Liver
Polyacrylamide Gel Electrophoresis
Tissue
Recovery

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels. / Wixtrom, Roger N.; Silva, Marilyn H.; Hammock, Bruce D.

In: Analytical Biochemistry, Vol. 169, No. 1, 15.02.1988, p. 71-80.

Research output: Contribution to journalArticle

Wixtrom, Roger N. ; Silva, Marilyn H. ; Hammock, Bruce D. / Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels. In: Analytical Biochemistry. 1988 ; Vol. 169, No. 1. pp. 71-80.
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