| Assay Method Information | |
| | Determination of in vitro ChE inhibition |
| Description: | The inhibitory potencies against huBChE and murine AChE (mAChE) were determined for all of these synthesized compounds using the method of Ellman (Ellman G. L. et al. Biochem. Pharmacol. 1961, 7, 88-95). 5,5-Dithiobis (2-nitrobenzoic acid) (Ellman's reagent; DTNB), and the butyrylthiocholine and acetylthiocholine iodides were purchased from Sigma-Aldrich (Steinheim, Germany). mAChE and recombinant huBChE at the stock concentration of 4.6 mg/mL in 10 mM MES buffer (pH 6.5) were used. The enzyme solutions were prepared by dilution of the concentrated stocks in phosphate-buffered solution (0.1 M, pH 8.0). The reactions were carried out in a final volume of 300 μL of 0.1 M phosphate-buffered solution, pH 8.0, containing 333 μM DTNB, 5 10-4 M butyrylthiocholine/acetylthiocholine and 1 10−9 M or 5 10−11 M huBChE or mAChE, respectively. The reactions were started by addition of the substrate, at room temperature. The final content of the organic solvent (DMSO) was always 1%. The formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with the thiocholines was monitored for 1 min as the change in absorbance at 412 nm, using a 96-well microplate reader (Synergy H4; BioTek Instruments, Inc., USA). To determine the blank value (b), phosphate-buffered solution replaced the enzyme solution. The initial velocity (v0) was calculated from the slope of the linear trend obtained, with each measurement carried out in triplicate. For the first inhibitory screening, stock solutions of the test compounds (1 mM) were prepared in DMSO. The compounds were added to each well at a final concentration of 10 μM. |
| Affinity data for this assay | |
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