Role of cationic residues in cytolytic activity: modification of lysine residues in the cardiotoxin from Naja nigricollis venom and correlation between cytolytic and antiplatelet activity.

Cardiotoxins and postsynaptic neurotoxins from snake venoms have similar primary, secondary, and tertiary structures. Cardiotoxins, however, in contrast to neurotoxins, exhibit general cytotoxicity. Comparison of the distribution of hydrophobic and charged amino acid residues in the three-dimensional structures of lytic cardiotoxins and nonlytic neurotoxins indicates the presence of a cationic site associated with a hydrophobic surface in cardiotoxins, but not in neurotoxins. A cationic site flanked by a hydrophobic site is a common structural feature shared by a wide variety of unrelated cytolysins and is predicted to determine the lytic activity of a large group of cytolysins. To determine the essential nature of the cationic site in cardiotoxin CTX-1 from Naja nigricollis crawshawii venom, we modified the positive charges of nine Lys residues to negative, neutral, or positive charges by succinylation, carbamylation, or guanidination, respectively. Circular dichroism studies indicated that these modifications did not affect the conformation of the cardiotoxin. Binding of the modified cardiotoxins to phospholipids was demonstrated by changes in the intrinsic fluorescence of native and modified CTX-1 after binding to phospholipid vesicles, and by resonance energy transfer with anthracene-phospholipid vesicles. Phospholipid binding was not affected by these modifications, but their binding preference was determined by the electrostatic interactions between the polypeptide and phospholipid. Both positively charged native and guanidinated CTX-1 showed direct lytic activity on human erythrocytes and platelets, whereas the succinylated or carbamylated derivatives did not show lytic activity. The loss of lytic activity cannot be related to conformational changes or phospholipid binding abilities of the modified cardiotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)