Leucine standard

The DH was measured using the 2,4,6 trinitrobenzene sulfonic acid (TNBS) method [8 (link)]. A 50 mL sample of a 0.02% protein solution was prepared by dissolving 1 g of WPH powder in distilled water. Protein solutions were stirred for 30 min to ensure that the powder was fully dissolved. Then, 1 g of the protein solution was added to 9 g of 1% (w/w) sodium dodecyl sulfate (SDS) solution (Sigma Aldrich, St. Louis, MO, USA). In test tubes, a 0.1 mL aliquot of protein solution, 2 mL of phosphate buffer (pH 8.2), and 2 mL of 0.1% (w/w) TNBS solution (Sigma Aldrich, St. Louis, MO, USA) were added. A leucine standard linear curve was obtained by diluting 530 mg/L leucine standard (Sigma Aldrich, St. Louis, MO, USA) in 1% SDS (w/w) to obtain 42.4, 84.8, 127.2, 169.6, and 212 mg/L amino nitrogen leucine standards. All tubes were vortexed and placed in the water bath at 50 °C for 60 min. The reaction was stopped by adding 4 mL of 0.1 N HCL (Chemicals, Gibbstown, NJ, USA) to each tube. Standards and samples were measured at 340 nm using a UV/VIS spectrophotometer (Metash Instruments Inc., Shanghai, China) against a 1% SDS blank solution. The leucine standard curve was plotted, then calculations were carried out accordingly.

Naja atra venom lyophilized powder was obtained from the Snake Venom Research Institute of Guangxi Medical University and stored at -80°C. Ultrapure water was filtered by Milli-Q (USA) and then used. Formic acid (HPLC purity) and acetonitrile (HPLC purity) were purchased from Merck (Germany). ammonium acetate (HPLC purity), inosine standard, horse uric acid standard, goose deoxycholic acid standard, thymidine standard, glycocholic acid, glutamine standard, leucine standard, phenylalanine standard, proline standard and arginine standard were purchased from Sigma (USA). Serum samples from patients bitten by Naja atra were provided by the Emergency Department of the First Clinical Medical College of Guangxi Medical University and the Emergency Department of the Second Clinical Medical College of Guangxi Medical University.