Human acetylcholinesterase (hAChE), bovine serum albumin (BSA), acetylthiocholine (ATCh), 5, 5’-dithiodis-2-nitrobenzoic acid
(DTNB) and 2-PAM were purchased from Sigma-Aldrich. Sarin, VX, tabun and soman were from Anti chemical command and
Engineering Institute of the Chinese people's Liberation Army. (Caution! Nerve agents used in our research are highly toxic and must
be handled with extreme care by well-trained personnel. Use of these materials has been approved by Anti chemical command and
Engineering Institute of the Chinese people's Liberation Army. Paraoxon, parathion, phorate and dichlorvos were from commercial
sources. After reactivation studies, biochemical samples were neutralized by stirring with 2 M NaOH for 12 h, the remaining solutions
were brought back to pH ∼7 and disposed in chemical waste.) Centrifugation was conducted at 4 °C in a 3-18K instrument from Sigma,
the absorption was measured on a Bio Rad Microplate Reader Model 550 (Parts).
At the beginning of each experiments, the stock solution of hAChE (dissolved in 20 mM HEPES, pH 8.0, contain 0.1% TRITON
X-100) were diluted by PBS (0.1 M, pH=7.4, 0.1% BSA) and stored at 0-4°C. A solution of oxime (10 mM) were prepared in water
containing 2.5% acetic acid and 10% PEG-400 and it was further diluted by PBS (0.1 M, pH=7.4) to the required concentrations. It was
found that there was no effect of CH3COOH and PEG-400 on hAChE by a control experiment. All the biological evaluation experiment
were conducted triplicate in 96-well plate, the enzyme activity was measured by the time-dependent hydrolysis of ATCh in which the
product thiocholine was detected by reaction with the Ellman’s reagent DTNB and absorbance at 412 nm [36].
2.3. hAChE inhibition experiments
Initially a stock solution of hAChE (20 U/mL, from sigma) was diluted 2000-fold with PBS (0.1 M, pH 7.4, 0.1% BSA). 20 μL of
diluted hAChE was incubated with 10 μL of each oxime (final concentrations: 10, 50, 200, 500 and 1000 µM) for 15min at 25 °C. A
blank experiment (positive control) was run in parallel in which oxime was replaced by PBS. For each sample above in the 96-well
plate, 30 µl of ATCh (3.0 mM in 0.1 M PBS, pH 8.0, 0.1% BSA) along with 150 µl DTNB (0.75 mM in 0.1 M PBS, pH 7.0) and 10 µl
HCl (0.1 M) was added in each well. Then the resulting mixture was centrifuged at 4 °C for 1 min to remove bubbles, the reaction
product was monitored immediately by testing the absorption value at 412 nm (0<abs<2). Enzyme activity was calculated by using the
formula: %Activity= 100*S/P. (S=absorption value of test substance; P = absorption value of positive control (100% activity)). IC50 was
determined by non-linear fitting using the standard IC50 equation: %Activity = 100*IC50/(IC50+[Ox]).
2.4. hAChE reactivation experiments
A stock solution of hAChE (20 U/mL, from sigma) was diluted 2000-fold with PBS (0.1 M, pH 7.4, 0.1% BSA). The concentrations
of different nerve agents and pesticides were determined by a pre-experiment similar to the inhibition experiment to attain an inhibition
plateau between 80% to 95%, the final concentration were as followings: VX, 3*10-8M; sarin, 6* 10-7M; tabun, 6*10-8M; soman,
3*10-8M, paraoxon, 4.5*10-8M, parathion, 6*10-5M, phorate, 8*10-3M, dichlorvos, 6*10-6M. The diluted hAChE (20 μL) was incubated
with different nerve agents and pesticides (10 μL) at 25 °C for 15 min (at 4 °C for soman to delay rapid aging). Then the inhibited
enzyme was incubated with oximes (15 μL, 0.3 mM) at 25 °C for 30 min (final concentration of oximes was 0.1 mM). At last, activity
of the enzyme in the incubation mixture was measured by using same method described in the section of inhibition experiment. Blank
samples were run in parallel and consisted of: (a) A positive control (P): uninhibited enzyme (20 μL) was used instead of the inhibited
enzyme; (b) a negative control (N): PBS (25 μL, 0.1 M, pH 7.4, 0.1% BSA) was used instead of oximes. Reactivation was calculated
using the formula: %Reactivation 100*(S-N)/(P-N) [37].
2.5. Determination of reactivation kinetics
In order to determine the reactivation rate constant (Kr), dissociation constant (KD) and second order reactivation rate constant (Kr2)
of the selected reactivators, the reactivation rate at different time intervals and at different concentrations were measured. Initially the
diluted hAChE (20 μL) was incubated with different nerve agents and pesticides (10 μL) at 25 °C for 15 min. Then the inhibited
enzyme was incubated with oximes in various concentrations, and 30 µl of ATCh (3.0 mM in 0.1 M PBS, pH 8.0, 0.1% BSA) along
with 150 µl DTNB (0.75 mM in 0.1 M PBS, pH 7.0) and 10 µl HCl (0.1 M) was added immediately. The reaction product was
monitored every 5 minutes, up to 2 hours by testing the absorption value at 412 nm (0<abs<2). Same blank samples were run in
parallel as describing in the section of reactivation section. The observed first-order rate constant Kobs for each oxime concentration, the
dissociation constant KD of inhibited enzyme-oxime complex (EP-OX) and the reactivation rate constant Kr were calculated by
non-linear fitting using the standard oxime concentration dependent reactivation equation derived from the following scheme [38, 39]:
In this scheme, EP is the phosphylated enzyme, [EP-OX] is the reversible Michaelis-type complex between EP and the oxime [OX],
E is the active enzyme and P-OX the phosphylated oxime. KD is equal to the ratio (K-1+Kr)/K1, and it typically approximates the
dissociation constant of the [EP-OX] complex, where from it follows that: Kr2 = Kr/KD.
Experimental details to determine the concentration dependence of the apparent reactivation rate Kobs for the reactivation of VX-,
sarin- and tabun-inhibited hAChE are described in the supporting information.