Modification of sticholysin II hemolytic activity by free radicals

Sticholysin II is a highly hemolytic toxin present in the caribbean sea anemone Stichodactyla helianthus. Pre-incubation of St II with 2,2'-azobis(2-amidinopropane), a source of peroxyl radicals in air saturated solution, readily reduces its hemolytic activity. Analysis of the amino acids present in the protein after its modification shows that only tryptophan groups are significantly modified by the free radicals. According to this, the loss of hemolytic activity correlates with the loss of the protein intrinsic fluorescence. The results indicate that, at high toxin concentrations, nearly a tryptophan residue and 0.2 toxin molecules are inactivated by each radical introduced into the system. Association of St II to multilamellar liposomes (egg yolk phosphatidyl choline:sphingomyelin 1:1) increases the toxin intrinsic fluorescence, indicating a more hydrophobic average environment of the five tryptophan groups of the protein. In agreement with this, incorporation of St II to the liposomes reduces the rate of fluorescence loss during its modification by free radicals, particularly at long incubation times. These results are explained in terms of two populations of tryptophans that are quenched at different rates by acrylamide and whose rates of inactivation by free radicals are also different.