ABSTRACT:It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion-and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97%) with the already reported cytolysins from other sea anemone species.

ABSTRACT:The unifying characteristic of cnidarians is the production of protein and polypeptide toxins. The present study describes the identification of a hemolytic toxin from the soft coral Sarcophyton trocheliophorum. The crude extract was highly cytotoxic (EC 50 = 50 ng/mL) against human erythrocytes. It was also tested for hemolytic activity by the blood agar plate method, resulting in a hemolytic halo of 12 mm with 50 µg of protein. The stability of the venom under different physiological conditions was analyzed. The venom hemolytic activity was augmented by alkaline and neutral pH whereas it was reduced in acidic pH. The activity was stable up to 60°C. The hemolytic activity was completely abolished by the addition of serum and reduced significantly during frequent freezing-thawing cycles. Toxin purification was performed by ammonium sulfate precipitation and subsequently desalted by dialysis against 10 mM sodium phosphate buffer (pH 7.2), followed by anion exchange chromatography on DEAE cellulose column and gel filtration chromatography using Sephadex G-50 matrix. The purified active fractions possessed a prominent protein of approximately 45 kDa, as revealed by SDS-PAGE.

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAESepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE. Key words:Heteractis magnifica, cytolysin, hemolysin, pore-forming toxin. Dr. Daphne Fautin (17)] was collected from Andaman Islands, India, at a depth of 5 m by scuba diving. Samples were deposited in the National Marine Repository at the National Institute of Oceanography, Goa, India. In the laboratory, the live animal was induced by osmotic thermal stress to eject the epithelial mucus that contains the toxins (6). Cnidarian toxins are stored in the cnidocytes, which are intracellular organelles that fire under pressure or osmotic variations and deeply injects the venom. Approximately 2 kg of anemone was placed in 1 L of warm distilled water (40 to 45°C) for 15 minutes with intermittent stirring to minimize tissue damage. After 15 minutes, the anemone was removed and the solution was filtered. The filtrate was stored as 100 mL aliquots in liquid nitrogen during transportation. The extract was removed from liquid nitrogen after transportation and stored at -70°C until analysis. When required, the aliquots were thawed and concentrated by lyophilization and reconstituted in phosphate buffered saline at pH 7.4. Short CommuniCationConcentration of the protein was determined by the method described by Lowry et al. (18) using bovine serum albumin (BSA) as the standard.Hemolytic activity of the toxin was measured quantitatively in terms of attenuance on human red blood cells at room temperature using Spectramax Microtiter® plate reader (Molecular devices, USA). Freshly collected human blood with heparin was centrifuged to remove the buffy coat, and the erythrocytes (RBC) obtained were washed three times in 0.85% saline and stored at 4°C. Toxins at desired concentrations were added in the first well to erythrocyte buffer (140 mM NaCl, 10 mM Tris-HCl, pH 7.4), and then serially diluted two-fold. RBCs (100 μL; D 630 = 0.5) in erythrocyte buffer were added to the toxins, and hemolysis was monitored by measuring attenuance at 630 nm for 20 minutes at room temperature. The final volume was 200 μL per well. The percentage of hemolysis was determined at the end of the assay using the following equation of Malovrh et al. (19):In which D obs was the measured attenuance in the well after 20 minutes and D max is the maximal attenuance by distilled water and D min is the minimal a...