DELINEATION OF THE FUNCTIONAL SITE OF A SNAKE-VENOM CARDIOTOXIN - PREPARATION, STRUCTURE, AND FUNCTION OF MONOACETYLATED DERIVATIVES

Toxin γ, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with Acctic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoAcctylated derivatives were obtained, and those modified at positions 1,2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin -γ, composed of three adjacent loops (I, II, and III) rich in β-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin γ to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin γ. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins. © 1990, American Chemical Society. All rights reserved.