Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels.
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.
Wixtrom
R N
RN
Department of Entomology, University of California, Davis 95616.
Silva
M H
MH
Hammock
B D
BD
D000328:Adult; D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology*; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004851:Epoxide Hydrolases / Q000302:isolation & purification*; D004852:Epoxy Compounds; D005260:Female; D006801:Humans; D008099:Liver / Q000201:enzymology; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D012685:Sepharose
D004852:Epoxy Compounds; D012685:Sepharose; D004851:Epoxide Hydrolases