A spectrophotometric method to assay epoxide hydrolase activity.
The Aspergillus niger epoxide hydrolase activity was assayed by spectrophotometric using (rac) p-nitrostryrene oxide (pNSO) as substrate. Both the substrate (pNSO) and the reaction product, p-nitrostryrene diol (pNSD), had a strong absorbance in UV at 280 nm. The assay was based on the measure of the pNSD absorbance of the water phase after extraction of the non-reacted pNSO with a solvent. Among the five solvents tested, chloroform was selected since it extracted more than 99% of the epoxide and only 32% of the produced diol. This extraction yield was independent of the diol and epoxide concentrations and it was fairly reproducible. Using different enzyme amounts, the reaction kinetics were linear for the first 10 min corresponding to degrees of conversion less than 5% for the epoxide. Two controls were run simultaneously, one with the substrate alone (epoxide hydrolysis and non-complete extraction) and one with the enzyme alone (enzyme absorbance at 280 nm). The resulting DeltaOD/min was linear with the amount of enzyme added within a large range from 2 to 80 microg of the EH preparation. The new spectrophotometric assay correlates well with the previous HPLC assay and could be used routinely for an easy and fast evaluation of EH activity. The kinetic parameters of (rac) pNSO hydrolysis by A. niger epoxide hydrolase could be easily determined and K(M) (1.1 mM) compared well with that previously reported (1.0 mM).
Bhatnagar
T
T
Biocatalyse et Chimie Fine, Université de la Méditerranée, Faculté des Sciences de Luminy, case 901, 13288 cedex 9, Marseille, France.
Manoj
K M
KM
Baratti
J C
JC
D001234:Aspergillus niger / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004126:Dimethylformamide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004851:Epoxide Hydrolases / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D013053:Spectrophotometry / Q000379:methods*; D014466:Ultraviolet Rays
D004852:Epoxy Compounds; C033332:4-nitrostyrene oxide; D004126:Dimethylformamide; D004851:Epoxide Hydrolases