2.5. Enzyme Inhibition Assays
Solution-based enzyme assays were performed on SpectraMax M5 96 well plate readers (Molecular Device, Sunnyvale, CA, USA) as described previously [17]. Simply explained, peptides were first incubated with enzyme for 20 min, and then the substrate solution was added into the wells to measure the enzyme activity. At least three replicates per peptide were included. The β-gal-catalyzed hydrolysis of RBG was fluorescently monitored at 590 nm (resorufin) with the excitation at 540 nm. For a typical assay, ~0.3 nM or 1 nM β-Gal was incubated with peptides and 100 μM RBG substrate in pH 7.4, 10 mM potassium phosphate buffer with 100 μM MgCl2 at 25 °C. The reaction rate was determined by the initial velocity of the linear reaction. The percentage of reduced/inhibited enzyme activity was calculated as below:Percentage of reduced activity=(1−Inhibited activityNoninhibited activity)×100%
For recovery of PEP-1 inhibited enzyme activity, 1 nM β-Gal was first incubated with PEP-1 for 20 min for inhibiting activity, and then NEG peptide was added to the solution and incubated for another 20 min before the activity assay.