| Assay Method Information | |
| | AChE Assay |
| Description: | The inactivation of AChEs was determined using a colorimetric assay [Ellman et al., Biochem. Pharmacol., 7:88-95]. Carrier solvents (ACN, methanol, ethanol, or acetone) showed that 1% (v/v) organic solvent caused a significant decrease (>5%) in rMAChE enzyme activity. ACN was selected as a carrier because it showed no effect on enzyme activity at 0.5% (v/v). All the fluorophore amides showed interference at 412 nm at ≥100 μM, and therefore inhibition studies were conducted at ≤100 μM. The inhibition of rMAChE and EEAChE by compounds 1-7 was determined as follows. DTNB solution and a solution of AChE yielding a 0.1 Abs unit/min rate in PBS (2.80 mL; pH 7.6) were placed in a cuvette at 20°C. To this solution was added either: (a) 10 μL of ACN as the control, or (b) 10 μL of ligand solution (10 mM in ACN). After 6 min incubation, the remaining enzyme activity was determined by adding 20 μL aliquots of the ATCh-I solutions and the hydrolysis rate was monitored at 412 nm over a period of 10 min ( 15 s intervals). The final concentrations of the reactants during enzyme assay were: 0.33 mM DTNB, 0.59 mM ATCh-I, 0.58 mM NaHCO3, and 0.05 mM inhibitor. |
| Affinity data for this assay | |
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