BChE (EC 3.1.1.8, from human serum), AChE (EC 3.1.1.8, from human erythrocyte), butyrylthiocholine (BUC) iodide, and acetylthiocholine (ATC) iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme was dissolved and pre-prepared at 2.0 U/mL. 10 μL of enzyme, 20 μL of 0.01 M 5,5′-dithiobis(2-nitrobenzoic acid), 10 μL of tested compound, and 40 μL of PBS were pre-incubated together for 5 min. Io initiate the reaction, 20 μL of 0.01 M BUC or ATC was added. The activity was determined by measuring the increase in absorbance at 410 nm at 37 °C in 2 min intervals using Tecan Spark multimode microplate reader (Mannedorf, Switzerland). The percentage of inhibition (I) was calculated according to the formula: I = (Ac − Ai)/Ac × 100%, with Ai and Ac representing the change in the absorbance in the presence of an inhibitor and without an inhibitor, respectively [28 (link),29 (link)].
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