Butyrylthiocholine buc iodide

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)].

The AChE and BChE inhibitory activities of compounds were determined by using a slightly modified Ellman’s method [30 (link),31 (link)]. Electric eel AChE, equine serum BChE, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), phosphate buffer solution (PBS, pH 8.0), acetylthiocholine (ATC) iodide, and butyrylthiocholine (BUC) iodide were purchased from Sigma-Aldrich (Steinheim, Germany). Tacrine was used as positive control. Enzyme solutions were prepared at 2.0 U/mL in 2-mL aliquots. The assay medium consisted of 10 μL of enzyme, 40 μL of PBS, 20 μL of 0.01 M DTNB, and 10 μL of tested compound. Assayed solutions of tested compounds were pre-incubated with corresponding ChE for 5 min. The reaction was initiated by the addition of 20 μL of 0.01 M substrate (ATC or BUC). 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 from the measured data as follows: I = (A_c − A_i)/A_c × 100%, where A_i and A_c represent the change in the absorbance in the presence of an inhibitor and without an inhibitor, respectively.