| Assay Method Information | |
| | Inhibition Assay |
| Description: | AChE inhibitory activity of compounds (Ia)-(Ie) and (-)-(Ib) and (+)-(Ib) was evaluated spectrophotometrically at 25° C. by the method of Ellman et al., using recombinant human AChE (hAChE) and Electrophorus electricus (ee) AChE and acetylthiocholine iodide as substrate (cf. G. L. Ellman et al., New and Rapid Colorimetric Determination of Acetylcholinesterase Activity Biochem. Pharmacol. 1961, vol. 7, pp. 88-95). For eeAChE inhibitory activity determination, the reaction took place in a final volume of 300 uL of 0.1 M phosphate-buffered solution pH 8.0, containing 0.03 unit/mL of ee AChE and 333 uM 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) solution used to produce the yellow anion of 5-thio-2-nitrobenzoic acid. Inhibition curves were performed in duplicates using at least 10 increasing concentrations of inhibitor and preincubating at 37° C. for 20 min. One duplicate sample without inhibitor was always present to yield 100% of AChE activity. Then, substrate acetylthiocholine (450 uM) was added and the reaction was developed at 37° C. for 5 min. The color production was measured at 414 nm. |
| Affinity data for this assay | |
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