HEMOLYTIC-ACTIVITY OF STONUSTOXIN FROM STONEFISH (SYNANCEJA-HORRIDA) VENOM - PORE FORMATION AND THE ROLE OF CATIONIC AMINO-ACID-RESIDUES

Stonustoxin (SNTX) is a two-subunit protein toxin purified from the ve nom of the stonefish (Synanceja horrida), which induces potent haemoly tic activity. We examined the pore-forming property of this non-enzymi c protein by an osmotic protection assay. SNTX-induced haemolysis was completely prevented by osmotic protectants of adequate size [poly(eth ylene) glycol 3000; molecular diameter approx. 3.2 nm]. Uncharged mole cules of smaller size, such as raffinose and poly(ethylene) glycol 100 0- 2000, failed to protect against cell lysis. These findings indicate that SNTX induces the formation of hydrophilic pores in the cell memb rane, which results in the lysis of erythrocytes. Since cationic resid ues contribute significantly to the cytolytic activity of several othe r pore-forming toxins, we examined the role of positively charged lysi ne and arginine residues in the haemolytic activity of SNTX. SNTX lost its haemolytic activity when the positively charged side chains of ly sine residues were neutralized or converted into negatively charged si de chains upon carbamylation or succinylation respectively. The haemol ytic activity of SNTX was also inhibited by the modification of positi vely charged arginine residues using 2,3-butanedione. The loss of haem olysis showed strong correlation with the number of Lys or Arg residue s modified. CD analyses, however, showed that the conformation of SNTX was not significantly affected by these chemical modifications. Furth er, the haemolytic activity of SNTX was competitively inhibited by var ious negatively charged lipids, such as phosphatidylserine, cardiolipi n and monosialogangliosides. These results indicate that SNTX induces potent haemolytic activity through the formation of pores in the cell membrane; and that cationic residues play a crucial role in its cytoly tic mechanism.