3.10. Carbonic Anhydrase II Inhibition

The total reaction volume of 200 µL included 20 µL of test compounds prepared in DMSO, followed by the addition of 140 µL of the HEPES–tris buffer, 20 µL of purified bovine erythrocyte CA-II (0.1 mg/mL) prepared in buffer, and 20 µL of a solution of 4-nitrophenyl acetate [70]. A 20 µL amount of test compound was incubated with the enzyme (EC 4.2.1.1, Sigma-Aldrich, St. Louis, MO, USA) for 15 min in a 96-well flat-bottom plate. The rate of product formation was monitored with the addition of 20 µL of 4-NPA as substrate, prepared in methanol at the final concentration of 0.7 mM, at 25 °C for 30 min with regular intervals of 1 min, by using microplate readers (Bio-Rad, Molecular Devices, CA, USA). HEPES-tris was used as a buffer for the reaction at the final concentration of 20 mM at pH 7.4. The percent inhibition was calculated by using the following formula: