Dietze E C, Kuwano E, Hammock B D
Department of Entomology, University of California, Davis 95616.
Anal Biochem. 1994 Jan;216(1):176-87. doi: 10.1006/abio.1994.1023.
In this study, we demonstrate the utility of a broad class of spectrophotometric substrates for the assay of cytosolic epoxide hydrolase purified from murine liver. These substrates, epoxy esters or carbonates, cyclize spontaneously upon or during hydrolysis of the epoxide functionality. The alcohol released by cyclization may then be assayed directly or by coupling to a second reaction. The alcohol produced, or its secondary reaction products, can be selected to give an absorption in the visible or near-uv range of the spectrum. This allows the synthesis of a wide variety of useful spectrophotometric substrates. 4-Nitrophenyl (2S,3S)-2,3-epoxy-3-phenylpropyl carbonate, at pH 6.4 and 25 degrees C, had a Vmax of 22 mumol min-1 mg-1 and a Km of 16 microM when assayed with a conventional spectrophotometer. When assayed under the same conditions with a 96-well plate reader, the measured Vmax was 15 mumol min-1 mg-1 and the Km was 6.6 microM. Some of these compounds were also found to be substrates for glutathione S-transferase, microsomal epoxide hydrolase, and porcine liver carboxylesterase. Indeed, 4-nitrophenyl 3,4-epoxy-3-phenylbutanoate was a 3.4-fold better substrate for porcine liver carboxylesterase than 4-nitrophenyl acetate when initial rates of hydrolysis were measured under the same conditions.
在本研究中，我们展示了一大类分光光度法底物在测定从小鼠肝脏中纯化的胞质环氧化物水解酶时的实用性。这些底物，环氧酯或碳酸酯，在环氧化物官能团水解时或水解过程中会自发环化。然后可以直接测定环化释放的醇，或者通过与第二个反应偶联来测定。所产生的醇或其二级反应产物，可以选择使其在光谱的可见光或近紫外范围内产生吸收。这使得能够合成各种各样有用的分光光度法底物。在pH 6.4和25℃条件下，用传统分光光度计测定时，4-硝基苯基(2S,3S)-2,3-环氧-3-苯基丙基碳酸酯的Vmax为22 μmol min-1 mg-1，Km为16 μM。在相同条件下用96孔板读数器测定时，测得的Vmax为15 μmol min-1 mg-1，Km为6.6 μM。还发现其中一些化合物是谷胱甘肽S-转移酶、微粒体环氧化物水解酶和猪肝羧酸酯酶的底物。实际上，在相同条件下测量水解初始速率时，4-硝基苯基3,4-环氧-3-苯基丁酸酯作为猪肝羧酸酯酶的底物比4-硝基苯基乙酸酯好3.4倍。
