Choline oxidase

Chemicals that were used
in the study were purchased from different sources: CuCl₂·2H₂O, glutaraldehyde (GLA, 25 wt % in water), tri-sodium
citrate 5,5-hydrate, FeCl₃·6H₂O, FeSO₄·7H₂O, d-(+)-glucose (Glu), phenol,
Na₂HPO₄·2H₂O, and NaH₂PO₄·2H₂O were purchased from Merck; uric
acid (UA), (+)-catechin hydrate (CAT), bilirubin (Bil), caffeic acid
(CFA), fetal bovine serum (FBS), quercetin (QR), catalase from bovine
liver, uricase (UOx) from Candida sp., glucose oxidase
(GOx) from Aspergillus niger, choline
oxidase (ChOx) from Alcaligenes sp., peroxidase from
horseradish (HRP), choline chloride (ChCl), glycine, neocuproine (Nc)
hydrochloride hydrate, and trizma base were purchased from Sigma;
(3-aminopropyl)triethoxysilane (APTS), N-acetyl-l-cysteine (NAC), tetraethyl orthosilicate (TEOS), urea, 4-aminoantipyrine
(4-AAP), ascorbic acid (AA), gallic acid monohydrate (GA), l-glutathione reduced (GSH) were from Sigma-Aldrich; and trans-ferulic acid (FA), (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (trolox) (TR), and l-cysteine (CYS) were from Aldrich.

Inhibition of human ATX was determined using the fluorogenic Amplex-Red in vitro assay, which was adapted from Ferry et al. (Ferry et al., 2008 (link)). DMSO-containing compound solutions were transferred to a 384-well assay plate (Greiner #781096, Monroe, NC, United States) and 6.6 nM in-house generated ATX enzyme in assay buffer (50 mM Tris-HCl, 120 mM NaCl, 20 mM CaCl₂, 5 mM KCl, 0.01% Triton-X100, pH 8.0) was added. After a 10-min incubation at room temperature, choline oxidase (7.3 U/ml; Sigma-Aldrich #C5896-1KU, St. Louis, MO, United States) and horseradish peroxidase (14.7 U/ml; F. Hoffmann-La Roche Ltd. #11378783, 1MU/4.311 g lyophilizate, Basel, Switzerland) were added and mixed. Thereafter, 110 μM LPC 18:1 (Avanti Polar Lipids #845875SP, Alabaster, AL, United States) and 183.3 μM Amplex Red (Chemodex #A-022, St. Gallen, Switzerland) were added. Plates were sealed and incubated for 5 min. The final assay concentrations were ATX 3 nM, choline oxidase 2 U/ml, horseradish peroxidase 4 U/ml, LPC 18:1 30 μM, Amplex Red 50 μM, and DMSO 2%. Background values were measured within 15 min and activity values after 90 min of incubation (Perkin Elmer Envision; Ex 535 nm/Em 587 nm; Perkin Elmer AG, Schwerzenbach, Switzerland).

All chemicals from commercial sources were of analytical grade. Choline oxidase (EC 1.1.3.17) from Alcaligenes sp. and its substrate choline, potassium hydrogen phosphate, hydrogen peroxide, the nitrogen mustard simulant bis(2-chloroethyl) amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Potassium chloride, potassium ferricyanide and iron chloride were obtained from Carlo Erba (Milan, Italy). A PalmSens3 (Houten, The Netherlands) Potentiostat was used for amperometric/voltammetric measurements.

PLD hydrolytic activity was typically measured by spectrophotometric assay using PC as the substrate.²⁵ (link) The reaction mixture (total volume, 100 µL) consisted of 0.5% (w/v) soybean lecithin, 0.1% (v/v) Triton X-100, 40 mM Tris–HCl (pH 7.5), 10 mM CaCl₂ and 40 µL of an enzyme sample. After incubation at 55°C for 20 min, the reaction was terminated by addition of 50 µL solution containing 50 mM EDTA and 100 mM Tris-HCl (pH 7.5), and the PLD enzyme was immediately denatured by heating at 100°C for 5 min. After cooling the reaction mixture to room temperature for 5 min, 500 µL of 50 mM Tris–HCl (pH 7.5) containing 1 mg phenol, 0.3 mg 4-aminoantipyrine, 3 U/mL of choline oxidase (Sigma, China), and 2 U/mL of horseradish peroxidase (Sigma, China) was added. After incubation at 37°C for 20 min, the absorbance of the reaction mixture was measured at 505 nm (Multiskan Ascent, ThermoFisher, USA). The calibration curve was obtained by using a standard solution of choline chloride instead of the enzyme solution. One unit (U) of hydrolytic activity of PLD was defined as the amount of enzyme that produced 1 µmol choline per min. All assays were performed in triplicate.

Acetylcholinesterase (Type VI-S, EC 3.1.1.7, from Electrophorus electricus, lyophilized powder, 217 U/mg) and choline oxidase (EC 1.1.3.17, from Alcaligenes sp., lyophilized powder, 15 U/mg), acetylcholine chloride (ACh), bovine serum albumin (BSA) and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich. Certified reference pesticide standards were all purchased from Dr. Ehrensdorfer (Augsburg, Germany). Water-soluble CdSe/ZnS core/shell quantum dots (QDs) were obtained from Ocean Nanotech (Springdale, AR, USA). Phosphate buffer solution (PBS) (pH 8.0, 10 mM) and Milli-Q ultrapure water (Millipore, ≥18 MΩ·cm) were used throughout. All chemicals were used without further purification. Individual stock solutions of the pesticides were prepared in acetonitrile and stored at 4 °C. A series of working solutions were prepared daily by an appropriate dilution by PBS (pH 8.0, 10 mM) which contains 0.5 mg/mL of BSA.