V 530 double beam spectrophotometer

The inhibitory activity of the novel compounds on human recombinant AChE and on human serum BChE (Sigma, Milan, Italy) was evaluated spectrophotometrically by the method of Ellman et al. [34 (link)]. The AChE stock solution was prepared by dissolving human recombinant AChE lyophilized powder in 0.1% Triton X-100/0.1 M potassium phosphate, pH 8.0. The stock solution of human serum BChE was prepared by dissolving the lyophilized powder in aqueous 0.1% gelatine. The stock solutions of the novel compounds (1 mM) were prepared in MeOH. The assay solution contained 340 μM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 0.02 unit/mL hAChE or hBChE, and 550 μM substrate (acetylthiocholine iodide or butyrylthiocholine iodide, for AChE and BChE, respectively), in 0.1 M potassium phosphate, pH 8.0. Assay solutions with and without inhibitor were preincubated at 37 °C for 20 min, and then the substrate was added. Blank solutions containing all components except the enzymes were prepared in parallel to correct for non-enzymatic hydrolysis of the substrate. Initial rate assays were performed at 37 °C with a Jasco V-530 double beam spectrophotometer. At least five increasing concentrations of the inhibitors, which produced 20–80% inhibition of the enzymatic activity, were assayed. IC₅₀ values were calculated using Microcal Origin 3.5 software (Microcal Software, Inc., Darmstadt, Germany).