Figure 1.

Purification of phrixotoxins from Phrixotricus auratus venom. (A) Reverse phase HPLC chromatogram monitored at 220 nm illustrating the separation of 10 fractions – A–J – after loading 500 μl of diluted venom in a Beckman ODS C18 column. The stepwise solvent B (0.1% TFA in acetonitrile) gradient (from 15–30% over 60 min, from 30–35% over 10 min, then from 35–60% over 10 min) is indicated. Collected fractions E and F, active on Kv4.3 channels, are shaded. (B,C) Ion exchange chromatograms illustrating the second purification step for fractions PaE and PaF as described in ‘Methods'. A stepwise gradient for fraction PaE, from 50–70% over 15 min, then from 70–100% over 10 min of solvent D (ammonium acetate 1 M) was used and allowed the separation of five fractions. Activity against Kv4.3 channels was recovered in PaE5. The other active fraction PaF4 was purified from the fraction PaF with a linear gradient from 50–100% solvent D over 25 min. The effluent was monitored at 280 nm. (D, E) The fractions PaE5 and PaF4 were further purified on C18 reverse phase column with a linear solvent B gradient from 10–50% over 40 min.