Fig. 1

Fractionation of Rhinella schneideri poison (RsP). a Chromatographic profile of RsP on CM-cellulose-52. The column was equilibrated with 0.05 M ammonium bicarbonate, pH 7.8. The sample (extract from 500 mg) was applied at a flow rate of 20 drops/min; and adsorbed /components were eluted using a convex concentration gradient of NH₄HCO₃ (0.05 to 1.0 M, pH 7.8). Fractions (3.0 mL/tube) were collected at 4 °C. b SDS-PAGE using 13.5 % separation gel. Lanes 1, 2 and 3 – ultra- mass markers, respectively. Lane 4 – fraction C1; lanes 5 and 6 – RsP. c Chromatographic low (Sigma-Aldrich, USA), low-high (GE Healthcare, Sweden) and high (GE Healthcare, Sweden)molecular profile of fraction C1 on DEAE-Sepharose. The column was equilibrated with 0.05 M Tris–HCl, pH 7.8 (buffer A). The sample (56.6 mg of C1) was applied at a flow rate of 0.5 mL/min; and adsorbed components were eluted using a linear gradient from 0–1 M NaCl in equilibrating buffer (buffer B). Elution with 100 % buffer B was achieved after 150 mL. d SDS-PAGE using 13.5 % separation gel. Lane 1 – RsP; lane 2 – fraction C1; lane 3 – fraction D3; lane 4 – ultra-low molecular mass markers (Sigma-Aldrich, USA). e Chromatographic profile of fraction D3 on Sephacryl S-200. The column, equilibrated with PBS, pH 7.4, was eluted with this same buffer (flow rate: 0.4 mL/min) and fractions of 1 mL were collected. In (a) and (c), the elution profiles were monitored at 280 nm, while in (e) the profile was monitored at 254 nm. f SDS-PAGE using 13.5 % separation gel. Lanes 1 and 2 – fraction S5; lanes 3 and 5 – ultra-low-molecular-mass markers (Sigma-Aldrich, USA); lanes 4 and 6 – low-molecular-mass markers (GE Healthcare, Sweden); lane 7 – fraction S2