Streptococcus equi hyaluronic acid

Venom protein concentrations ranging from 20 to 200 μg were mixed with 100 μL of 0.2 M sodium acetate (pH 6), 0.15 M NaCl buffer, and 100 μL of 1 mg/mL solution of Streptococcus equi hyaluronic acid (Sigma-Aldrich) as a substrate for a final volume of 250 μL. After 15 min incubation at 37 °C, the reaction was stopped by adding 1 mL of 2.5% hexadecyl trimethyl ammonium bromide in 2% NaOH for 30 min at room temperature. Turbidity was determined at 400 nm in a plate reader using a spectrophotometer (Benchmark Plus, Bio-Rad, USA). Bovine testes type IV-S hyaluronidase (Sigma-Aldrich) was used as control [71 (link)].

Dulbecco’s Modified Eagle’s Medium F12 HAM (DMEM/F12), Dulbecco’s Modified Eagle’s Medium with 4500 mg/L glucose (DMEM high glucose), foetal bovine serum (FBS), phosphate-buffered saline (PBS), crystal violet, formaldehyde, lipopolysaccharide (LPS), DMSO, albumin from bovine serum (BSA): fraction V ≥ 98% (A3294), hyaluronidase from bovine testes type I-S, Streptococcus equi hyaluronic acid (HA), cetyltrimethylammonium bromide (CTAB), quercetin dihydrate, ursolic acid (other terpenoids were obtained by isolation), diclofenac sodium, dutasteride, and testosterone propionate were obtained from Sigma-Aldrich (Seelze, Germany). Acetate buffer pH 4.5 was purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA).