The modification of arginine-36 in elapid venom cardiotoxin using 1,2-cyclohexanedione

Abstract

The importance of the invariant arginine-36 in cardiotoxin was investigated by chemical modification. Reaction of the single arginine residue in Naja haje annulifera venom cardiotoxin V 111 with 1,2-cyclohexanedione yielded derivative 1 (cardiotoxin V 111 modified at Arg-36). Purification was achieved using cation-exchange chromatography in the presence of borate ions. The reaction was reversible by incubation with hydroxylamine at 37°C. Derivative 1 had an intravenous LD 50 of 17 μg·g −1 in mice, the borate complex of the derivative an LD 50 of 14 gmg·g −1, and the maternal resulting from the reversal of the reaction, 5.9 μg·g −1, as compared to an LD 50 of 4.5 μg·g −1 for the native toxin. Circular dichroism spectra in borate buffer (pH 8.0) and glycine-HCl (pH 3.5) showed that the derivative had not undergone severe conformational changes. Because some lethality was retained after making the derivative, it is doubtful whether Arg-36 can be considered to be of crucial importance for the biological action of cardiotoxin and it probably fulfils a structural role.