2.4. sEH Enzymatic Assay

The sEH assay was performed with minor modifications in accordance with instructions present with the Cayman sEH kit. The sEH assay buffer was diluted ten-fold in HPLC water. For determining inhibitory activity, 90 μL of the diluted buffer was added to either 2.5 μL sEH (diluted fifty-fold in kit buffer) and 5 μL MeOH, or a solution of inhibitor dissolved in MeOH. Next, the substrate (PHOME) (2.5 μL) was added to each mixture, and sEH hydrolysis was allowed to proceed at 37 °C. The products were monitored at 330 nm excitation and 465 nm emission for approximately 40 min.

Inhibitory activity was calculated according to the following equations:

Where ΔC and ΔI are the intensity of control and inhibitor, respectively, after 40 min.

Where y₀ is minimum value of the y-axis, a is the difference between maximum and minimum values and b is the x value at 50% of the a value.