2.5. IC50 and Ki Determination

Kinetic inhibition assays were conducted to determine the IC₅₀ values for flavonoid inhibitors and the experiments were conducted on a Cary Eclipse spectrophotometer with an excitation of 305 nm, emission wavelength of 405 nm and bandpasses of 5 nm. Plx2A (5 μmol/L) was mixed with 250 μmol/L ε-NAD⁺, at various inhibitor concentrations in GH buffer. Triplicate reactions at 25 °C were monitored for 10 min intervals and then the initial slope was calculated for each trace. The kinetic data were fit to a Michaelis–Menten function generated in OriginLab Pro ver8 software to determine the IC50 value. The mechanism of flavonoid inhibition was determined for Quercetin as the model compound and entailed collection of full Michaelis–Menten datasets in the presence of 0, 10, 20, 30, and 40 μmol/L of inhibitor in a final DMSO concentration of 15% (v/v). Plx2A GH activity is not affected by DMSO in the reaction buffer until 20% (v/v). The inhibition data were transformed to the Lineweaver–Burk function and plotted to determine the inhibition pattern. Secondary plots were then generated to calculate the K_i value, which represents the Quercetin binding constant for Plx2A.