Dithionitrobenzoic Acid

The time course of ethyl-paraoxon hydrolysis by SisLac at 70°C was monitored following the p-nitrophenolate production at 405 nm (ε_405nm = 17 000 M⁻¹cm⁻¹) in 1-cm path length cell with a Cary WinUV spectrophotometer (Varian, Australia) and using the Cary WinUV software. Standard assays (500 µL) were performed in paraoxonase buffer CHES 50 mM pH 9, NaCl 150 mM, CoCl₂ 0.2 mM, EtOH 6% (v/v), with pH adjusted with NaOH at 70°C.
At 25°C, the phosphotriesterase, esterase and lactonase activities were analyzed monitoring absorbance variations in 200 µL reaction volumes using 96-well plates (6.2-mm path length cell) and a microplate reader (Synergy HT) using the Gen5.1 software at 25°C. For each substrate, assays were performed using organic solvent concentrations below 1%. The monitoring wavelength, the solvent used, the molar extinction coefficient and the concentration range for each substrate (Fig. 1, S1 & S2) are summarized in Table S2. Phosphotriesterase and esterase activities were performed in activity buffer. When required, DTNB at 2 mM was added to the buffer to follow hydrolysis of substrate releasing thiolate group (malathion (Fig. S1V)). Catalytic parameters for some phosphotriesters were also recorded using SDS at concentrations 0.01 and 0.1% (w/v). Lactone hydrolysis assays were performed in lactonase buffer (Bicine 2.5 mM pH 8.3, NaCl 150 mM, CoCl₂ 0.2 mM, Cresol purple 0.25 mM and 0.5% DMSO) using cresol purple (pK_a 8.3 at 25°C) as pH indicator to follow the acidification related to the lactone ring hydrolysis. Molar coefficient extinction was measured by recording absorbance of the buffer over a range of acetic acid concentrations (0–0.35 mM). The absorbance values versus acetic acid concentration were fitted to a linear regression (GraphPad Prism 5 software) with a slope corresponding to molar extinction coefficient (see Table S2). For all experiments, each point was made in triplicate and the Gen5.1 software was used to evaluate the initial velocity at each substrate concentration. Mean values were fitted to the Michaelis-Menten equation using Graph-Pad Prism 5 software to obtain the catalytic parameters. In the case of C4 AHL hydrolysis for which the substrate concentration that enable to determine the enzyme V_max could not be reached, the catalytic efficiency has been determined by fitting the linear part of the Michaelis-Menten plot to a linear regression.

For the estimation of reduced glutathione, the 1 mL of tissue homogenate was precipitated with 1 mL of 10% TCA. To an aliquot of the supernatant, 4 mL of phosphate solution and 0.5 mL of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent were added and absorbance was taken at 412 nm [28 (link)]. The values were expressed as nM of reduced glutathione per mg of protein: $\begin{array}{c}\text{GSH level}=\frac{Y-0.00314}{0.0314}\times \frac{D_F}{B_T\times V_U},\end{array}$ where Y is Abs₄₁₂ of tissue homogenate, D_F is dilution factor (1), B_T is brain tissue homogenate (1 mL), and V_U is aliquot volume (1 mL).

Acetylcholinesterase from electric eel type VI-S, butyrylcholinesterase from equine serum, acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB), bovine serum albumin (BSA), tris buffer, and galantamine were purchased from Sigma–Aldrich. The organic solvents (methanol and ethanol) and reagents used in the analysis were of analytical grades.

The assay was carried out according to the modified Ellman's method, as described in the previous publications [22 , 24 (link)]. The extracts were dissolved in methanol to make 10 mg/mL and diluted with water to obtain 1 mg/mL. These samples were serially diluted to get a range of sample concentrations of 0.01–300 μg/mL. Sample solutions were added to a 96-well microplate (25 μL), followed by the addition of 1.5 mM ATCI (25 μL) as a substrate for the AChE enzyme, and BTCI (25 μL) as a substrate for the BChE enzyme. The substrate was then hydrolyzed using 3 mM DTNB (125 μL), tris-buffer (50 μL), and AChE or BChE enzymes (25 μL) 0.22 μ/mL. Before measurement, the solutions were shaken for 30 s in a microplate reader (Thermo Scientific Multiskan FC). The yellow color from the product, 5-thio-2-nitrobenzoate, was measured at 405 nm every 5 s for 2 min. Every experiment was carried out in triplicates. Methanol 10% was used as a control. The enzyme activity was calculated as a percentage of the velocity of the sample compared with the negative control. The inhibitory activity was calculated based on the equation. $\begin{array}{c}\%\mathbf{I}\mathbf{n}\mathbf{h}\mathbf{i}\mathbf{b}\mathbf{i}\mathbf{t}\mathbf{i}\mathbf{o}\mathbf{n}=\frac{\left(\mathbf{V}\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}-\mathbf{V}\mathbf{s}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}\right)}{\mathbf{V}\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}}\times \mathbf{100}\text{,}\end{array}$ where V: mean velocity.