| Assay Method Information | |
| | Cholinesterase Inhibition Assay |
| Description: | Cholinesterase inhibition was assayed spectrophotometrically at 412 nm at 25°C [Pietsch et al., J. Med. Chem., 48:8270-8288; Ellman et al., Biochem. Pharmacol., 7:88-95]. Product formation was monitored over 5 min. Assay buffer was 100 mM sodium phosphate, 100 mM NaCl, pH 7.3. Enzyme stock solutions were prepared with assay buffer in the following concentrations ∼ 100 U/mL (EeAChE),∼ 3 U/mL (hAChE), ∼ 10 U/mL (hBChE), and were kept at 0°C. Appropriate dilutions of the EeAChE (1:30) and hBChE (1:10) solutions were done immediatelybefore starting the measurements. Solutions of ATCh (5, 10, 15, or 20 mM), BTCh (10 mM), and DTNB (7 mM) were prepared in assay buffer and kept at 0°C. Stock solutions of the inhibitors were prepared in acetonitrile. EeAChE was assayed as follows. Into a cuvette containing 830 μL assay buffer, 50 μL of the DTNB solution, 50 μL acetonitrile, 10 μL of an inhibitor solution, and 10 μL of an enzyme solution were added and thoroughly mixed. After incubation for 15 minat 25°C, the reaction was initiated by adding 50 μL of the ATCh solution. The following final concentration were used, ∼ 0.033 U/mL of EeAChE, 250, 500, 750, or 1000 μM of ATCh, 350 μM of DTNB, 6% acetonitrile. Similarly, hAChE (final concentration ∼ 0.03 U/mL) was assayed with 500 μM ATCh, and hBChE (finalconcentration ∼ 0.01 U/mL) with 500 μM BTCh. The rates of enzyme-catalyzed substrate hydrolysis were corrected by those of the non-enzymatic hydrolysis ofATCh or BTCh, respectively, as determined by using 10 μL of assay buffer, instead of the enzyme solution. A Km value of 550 μM19 for the substrate ATCh used in the EeAChE assay was taken for kinetic analyses. |
| Affinity data for this assay | |
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