Chemical modification of equinatoxin II, a lethal and cytolytic toxin from the sea anemone Actinia equina L.

The role of arginine and tyrosine in cytolytic properties of equinatoxin II, isolated from the sea anemone Actinia equina L., was studied by means of chemical modifications. The toxin was modified with 2,3 butanedione and tetranitromethane, respectively. The extent of modification and physico-chemical properties of the modified proteins were checked with amino acid analysis, isoelectric focusing and circular dichroic spectra. Extensive treatment of the toxin with 2,3 butanedione modified seven arginines and also two tyrosines, with resulting loss of hemolytic activity. Modification of two out of nine arginine residues resulted in a 25% loss of hemolytic activity, whereas nitration of three out of ten tyrosines decreased hemolytic activity by 95%. The nitrated toxin had at least a 30-fold higher i.v. LD50 than the native toxin. None of the modifications significantly affected the secondary structure of the toxin as revealed by the CD spectra. It is concluded that tyrosine residues are involved in both lethal and cytolytic activity, while the role of arginine residues is not evident because of the non-specific alteration of tyrosine residues with 2,3 butanedione.