FIGURE 1.

Purification of APHC1. The first separation stage of a dried ethanol extract of sea anemone H. crispa nematocysts was done on a water-equilibrated hydrophobic column Polychrom-1 (7 × 30 cm). Fractions were eluted by stepwise ethanol gradient with a flow rate of 1.2 liters/h. Active fraction (marked as a gray box on overall separation steps) has been separated on the second stage by ion exchange chromatography on Bio-Rex 70 column (2.5 × 60 cm). The separation was done in 5 mm ammonium acetate buffer (pH 4.5) by flow rate 22 ml/h in a linear gradient of NaCl concentration. The third stage of purification was performed with a flow rate 70 ml/h on the ion exchange column SP-Sephadex C-25 (2.5 × 40 cm), with the same 5 mm ammonium acetate buffer (start buffer, pH 4.5) in combined gradient of NaCl concentration and pH value. Final purification (stage 4) was achieved on a reverse-phase column Jupiter C₅ (4.6 × 150 mm) in 0.1% trifluoroacetic acid with a flow rate of 1 ml/min using a linear gradient of acetonitrile concentration.