TY - JOUR
T1 - Spectrophotometric Substrates for Cytosolic Epoxide Hydrolase
AU - Dietze, E. C.
AU - Kuwano, E.
AU - Hammock, B. D.
PY - 1994/1
Y1 - 1994/1
N2 - 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 Vmax of 22 μmol min-1 mg-1 and a Km of 16 μM when assayed with a conventional spectrophotometer. When assayed under the same conditions with a 96-well plate reader, the measured Vmax was 15 μmol min-1 mg-1 and the Km 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.
AB - 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 Vmax of 22 μmol min-1 mg-1 and a Km of 16 μM when assayed with a conventional spectrophotometer. When assayed under the same conditions with a 96-well plate reader, the measured Vmax was 15 μmol min-1 mg-1 and the Km 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.
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U2 - 10.1006/abio.1994.1023
DO - 10.1006/abio.1994.1023
M3 - Article
C2 - 8135350
AN - SCOPUS:0028013506
VL - 216
SP - 176
EP - 187
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -