4.2. Assay methods

AChE concentrations were determined spectrophotometrically, using a value of ε _{280 nm} (1 mg/ml) = 17.4. 59 The TcAChE concentration is expressed as the concentration of the dimer (molecular weight 130,000), assuming a subunit molecular weight of 65,000. 35 AChE activity was monitored by the Ellman procedure, 60 using ATC as the substrate. Measurements were performed in 0.01% sodium azide/0.1% Tergitol/200 mM NaCl/50 mM Tris, pH 8.0, at 25°C. The ATC concentration was determined by measurement of the absorption of the Ellman reagent after complete hydrolysis of the substrate by TcAChE. It was about 80% of the nominal concentration based on weight.

Thermal inactivation experiments were performed as follows: TcAChE samples, at 2×10⁻⁹ M, were incubated in 0.01% sodium azide/0.1% Tergitol/200 mM NaCl/50 mM Tris, pH 8.0, containing the appropriate concentration of the divalent metal ion. Loss of enzyme activity was monitored for greater than two half‐lives of inactivation, by 50‐ to 100‐fold dilution of aliquots into the Ellman reaction mixture, and assaying at 25°C, as above. The data points for the time‐course of residual activity were fitted to a mono‐exponential decay function (not shown), and the increases in t _1/2 values were used to calculate the degree of enhancement of stability. Thermal inactivation of the other three AChEs was similarly monitored by dilution of suitable aliquots into the incubation buffer, with or without the divalent metal ion, which had been pre‐equilibrated at the appropriate reaction temperature. Thermal inactivation of TcAChE was monitored at 39°C, of HuAChE and EeAChE at 49°C, and of DrAChE at 47°C.