C8 column

Venom was collected from Macrovipera lebetina transmeditarannea snakes at the serpentarium of Tunis Pasteur Institute and subjected to a purification process. The venom was loaded onto a Sephadex G-75 column (Pharmacia, Uppsala, Sweden). Subsequently, Leb-C was isolated following a previously described procedure [26 (link)]. In brief, Fraction I, obtained from the Sephadex G-75 column, was applied to reverse-phase high-performance liquid chromatography using a C8 column (250 × 4.6 mm, 5 mm; Beckman coulter, Marseille, France) that was pre-equilibrated with a solution of 0.1% trifluoroacetic acid in 20% acetonitrile. The elution was performed at a flow rate of 0.8 mL/min, employing an acetonitrile linear gradient of 20–65% over a period of 40 min.

Cerastes cerastes snake venom was collected from the Pasteur Institute’s Serpentarium (Tunis, Tunisia) and stored at –20 °C. Crude venom (300 mg) was dissolved in 0.2 M ammonium acetate, pH 6.8, and fractionated by a Sephadex G-75 (Pharmacia, Uppsala Sweden) column. Fraction II, which contains medium-molecular-weight proteins (<30 kDa), was lyophilized subjected to a reverse phase C8 column (250 × 4.6 mm², 5 μm; Beckman; Fullerton, CA, USA) eluted with a linear acetonitrile gradient 10–65% over 45 min at a flow rate of 0.8 mL/min. The homogeneity of CC5 and CC8 were assessed by a second step of high-performance liquid chromatography (HPLC) on a C18 column (250 × 4.6 mm², 5 μm; Beckman) as previously described [21 (link)].

Aliquots of the protein fraction collected from the size exclusion chromatography were subjected to RP-HPLC separation. The protein was loaded on a Vydac (Deerfield, IL, USA) C8 column (4.6 × 250 mm), using a Beckman System Gold apparatus (Fullerton, CA, USA). Elution was performed by a multistep linear gradient of eluent B (0.08% TFA in acetonitrile) in eluent A (0.1% TFA) at a flow rate of 1 ml/min. The eluate was monitored at 220 and 280 nm.