Electrophorus electricus ache

We purchase lyophilized Electrophorus electricus AChE, demeton-S-methyl, and diisopropylfluorophosphate from Sigma Aldrich. We run a modified protocol from Cherny et al.19 (link), where standard Ellman assays recover kinetic parameters26 (link) and determine the ability of Smu. 1393c to protect AChE from the effects of OP simulants. We incubate equal amounts of Smu. 1393c and OP simulant (final concentration 500 nM) at 20 °C for 30 minutes. We add AChE to each sample and incubate at 20 °C for 60 minutes. As a detectable marker we add 500 μM of the Ellman reagent 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) to the samples. We vary the amount of acetylthiolcholine (0–20 mM) and bring each sample to a volume of 50 μL using buffer (200 mM NaCl, 20 mM TRIS at pH 7.4). Each assay is performed in triplicate. We plot the kinetic parameters, obtained using SpectraMax M3 plate reader, and fit them to the Michaelis-Menton kinetic equation.

AChE activity was assayed as described by Ellman et al. [64 (link)], with some modifications [65 (link)]. Fifteen μL of Electrophorus electricus AChE (Sigma-Aldrich, Darmstadt, Germany) in buffer phosphate (pH 7.6) and 15 μL of the tested compounds (1.4–4350 μM in methanol) were dissolved in 200 μL in the same buffer, and plated in 96-well plates. The mixtures were incubated for 30 min at room temperature before the addition of 30 μL of the substrate solution (15 μL 0.5 M DTNB, 15 μL 0.6 mM ATCI in buffer, pH 7.6). The absorbance was read on a Microplate Reader Biochrom EZ 800 at 403 nm, after three minutes. Enzyme activity was calculated as a percentage compared to an assay using a buffer without any inhibitor, according to the following equation:
where Abs_control is the absorbance of the control, containing 15 μL enzyme in buffer, 200 μL buffer and 15 μL solvent (methanol/water for GAL); Abs_sample is the absorbance of the sample, containing 15 μL enzyme in buffer, 200 μL buffer and 15 μL solution of the tested compound (in methanol/water for GAL).