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

Authors: Wixtrom, R N; Silva, M H; Hammock, B D

Published In Anal Biochem, (1988 Feb 15)

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.

PubMed ID: 3369689

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