Spectrophotometric Substrates for Cytosolic Epoxide Hydrolase

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°C, had a V_max of 22 μmol min^-1 mg^-1 and a K_m of 16 μM when assayed with a conventional spectrophotometer. When assayed under the same conditions with a 96-well plate reader, the measured V_max was 15 μmol min^-1 mg^-1 and the K_m was 6.6 μM. 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.