琼脂糖
亲和层析
化学
环氧化物水解酶
色谱法
配体(生物化学)
胞浆
烷基
酶
生物化学
有机化学
微粒体
受体
作者
Roger N. Wixtrom,Marilyn H. Silva,Bruce D. Hammock
标识
DOI:10.1016/0003-2697(88)90256-4
摘要
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.
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