| Assay Method Information | |
| | Test of the Ability to Inhibit Enzymes: Horse Plasma Butyrylcholinesterase (eqBuChE) and Human Plasma Butyrylcholinesterase (hBuChE) |
| Description: | The spectrophotometric Ellman's test [Ellman et al.] modified for assays in 96-well plates was used to test the activity. Reagents: 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), eqBuChE and butyrylthiocholine iodide (BTC) were purchased from Sigma-Aldrich (Steinheim, Germany). hBuChE was from Vivonics (Bedford, MA, USA). The enzymes were prepared by dissolving in demineralized water to a concentration of 0.384 U/ml. Stock solutions of test compounds were prepared by dissolving in DMSO to a concentration of 0.0034 M. They were then diluted with water to obtain the appropriate concentrations in the wells. In the first step of the test, 25 μl of test compound (or DMSO/water mixture at the appropriate ratio for blank samples) was incubated in 0.1 M phosphate buffer (200 μl, pH=8.0) with DTNB (20 μl; 0.0025M) and with the appropriate enzyme (20 μl; eqBuChE or hBuChE) at 25° C. (animal enzyme) or 36° C. (human enzyme). After a 5 minutes incubation period, 20 μl of BTC solution (0.00375 M) was added. After another 5 minutes, the changes in absorbance of the solutions were measured at 412 nm using a multifunctional microplate reader (EnSpire Multimode; PerkinElmer; Waltham, MA, USA). All compounds were tested at a screening concentration of 10 μM (eqBuChE) or 1 μM (hBuChE). The percentage of enzyme inhibition was calculated from the equation 100-(S/B)×100, where S and B were the enzymatic activity in the test sample well and in the well without sample, respectively. For compounds with an enzyme inhibitory ability at a screening concentration greater than 50% (eqBuChE) or 80% (hBuChE), IC50 values were determined. To determine the IC50 value, seven different concentrations of each compound were used to obtain an enzyme activity from 5% to 95%. All reactions were performed in triplicate. IC50 values were calculated using non-linear regression (GraphPad Prism 5 [GraphPad Software, San Diego, CA, USA 5.00]) for the percentage of enzyme inhibition against the inhibitor concentration used. Tacrine and donepezil were used as reference compounds. |
| Affinity data for this assay | |
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