Wixtrom R N, Hammock B D
Department of Entomology, University of California, Davis 95616.
Anal Biochem. 1988 Oct;174(1):291-9. doi: 10.1016/0003-2697(88)90548-9.
Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.
本文描述了两种用于检测胞质环氧化物水解酶的便捷灵敏的连续分光光度法。这些方法基于环氧化物底物及其二醇产物在紫外光谱上的差异。1,2 - 环氧 - 1 -（对硝基苯基）戊烷（ENP5）的水解伴随着302nm处吸光度的降低，而1,2 - 环氧 - 1 -（2 - 喹啉基）戊烷（EQU5）的水化作用则使315.5nm处的吸光度增加。纯化的小鼠肝脏胞质环氧化物水解酶对ENP5和EQU5的Km、Vmax值分别为1.7μM、11,700 nmol/min/mg和25μM、8300 nmol/min/mg。这两种底物的水解速度均明显快于反式氧化茋，反式氧化茋是目前用于测量胞质环氧化物水解酶活性最常用的底物。在正常测定条件下，未检测到底物的自发水解。这些方法适用于全组织匀浆以及纯化的酶制剂。对硝基苯乙烯氧化物和对硝基苯基缩水甘油醚也进行了检测，发现它们是小鼠肝脏胞质环氧化物水解酶非常差的底物。
