MODIFICATION OF STICHOLYSIN-II HEMOLYTIC-ACTIVITY BY FREE-RADICALS

Sticholysin II is a highly hemolytic toxin present in the caribbean se a anemone Stichodactyla helianthus. Pre-incubation of St II with 2,2'- azobis(2-amidinopropane), a source of peroxyl radicals in air saturate d solution, readily reduces its hemolytic activity. Analysis of the am ino acids present in the protein after its modification shows that onl y tryptophan groups are significantly modified by the free radicals. A ccording to this, the loss of hemolytic activity correlates with the l oss of the protein intrinsic fluorescence. The results indicate that, at high toxin concentrations, nearly a tryptophan residue and 0.2 toxi n molecules are inactivated by each radical introduced into the system . Association of St II to multilamellar liposomes (egg yolk phosphatid yl choline:sphingomyelin 1:1) increases the toxin intrinsic fluorescen ce, indicating a more hydrophobic average environment of the five tryp tophan groups of the protein. In agreement with this, incorporation of St II to the liposomes reduces the rate of fluorescence loss during i ts modification by free radicals, particularly at long incubation time s. These results are explained in terms of two populations of tryptoph ans that are quenched at different rates by acrylamide and whose rates of inactivation by free radicals are also different, (C) 1998 Elsevie r Science Ltd. All rights reserved.