Epomes

Epoxy fluor 7 (EF7), EETs, and EpOMEs were from Cayman Chemicals. Fluorescence/absorbance was monitored using an EnVision Multilabel plate reader (PerkinElmer). EH activity was measured using the EF7 substrate (47 (link)) in a 200-µl reaction mixture containing 5 µM EF7 and 10-µg recombinant enzyme in phosphate-buffered saline (PBS) (expressed in E. coli as previously described [85 (link)]). Diol formation (measuring 6-methoxy-2-naphthaldehyde production) was quantified by measuring fluorescence (excitation at 340 nm and emission at 450 nm). Calculation of the initial (linear) rates of substrate hydrolysis was determined using GraphPad Prism software (v5). EH activity against the 11(12)-EET and 14(15)-EET regioisomers was measured using the 11(12)- or 14(15)-DHET ELISA kit, according to the manufacturer’s protocol (Detroit R&D), with some modifications. Reaction mixtures (100 µl) contained 6 or 12 µM EET and 100 µg EH recombinant enzyme in PBS and were incubated at 37°C for 1.5 h. Sample buffer (900 µl) was added to the reaction mixture, and 100 µl was loaded onto the ELISA wells in duplicate. Absorbance was measured at 450 nm.