Effect of N-bromosuccinimide-modification of tyrosine side chains of cardiotoxin II of the Indian cobra on biological activity.

The essential role of tyrosine residue(s) of cardiotoxin II in the biological activity of the toxin was evaluated using N-bromosuccinimide. N-bromosuccinimide effected oxidation of the tyrosine residues in cardiotoxin II with enhancement in absorbance at 260 nm. The influence of various solvent media such as acetate-formate buffer (pH 4·0),0·01 Ν H2SO4 (pH 2·0) and Tris-HCl buffer (pH 8·5) on oxidation of tyrosine residues was exa mined. In comparison with 0·01 Ν H2S O4, acetate-formate buffer could prevent secondary oxidations as revealed by lower consumption of oxidant, N-bromosuccinimide, to achieve oxidation. In Tris-HCl buffer oxidation of tyrosine did not take place effectively. N-iodosuccinimide caused only limited oxidation as evident from minor increase in absorbance at 260 nm. N-chlorosuccinimide was completely ineffective. Oxidation of cardiotoxin II with 3·75 equivalents of N-bromosuccinimide tyrosine residue led to complete loss of lethal activity. However, the derivative retained the ability to protect bacterial protoplasts from lysis in solutions of low tonicity. Unlike cardiotoxin II oxidized with N-chlorosuccinimide (50 equivalents/mol of toxin) which retained lethal activity as well as the ability to protect protoplasts from lysis, performic acid-oxidized toxin had lost both the activities.

Non-identity of reaction centres for pyrophosphatase and toxic actions of cardiotoxin II: The status of cardiotoxin II as a metalloprotein.

Cardiotoxin II of the Indian cobra (Naja naja) contains approximately four Mg2+ per mol. Complete demetallation of the toxin is achieved by three cycles of treatment with ethylenediamine tetraacetate and gel filtration. Reconstitution of toxin by treatment of the apo-protein with Mg2+ restores metal content and inorganic pyrophosphatase activity only to the extent of two atoms/mol and 65%, respectively. Use of Mg (II)-EDTA in the reconstitution experiment yields restoration of half the original enzyme activity. Mg2+ is required for the inorganic pyrophosphatase action of the toxin. A definitive statement on the non-essentiality of Mg2+ for the lethal toxicity of the toxin is not possible at present, although experimental observations indicate that demetallated toxin is as toxic as the native toxin. Based on this and the differing sensitivities of the enzyme and toxic activities of the toxin to heat, it is suggested that the reaction centres in the toxin for the two activities are different and that the pyrophosphatase activity is not causally connected with the lethal toxicity of the toxin.