Kinetics and mechanism of St I modification by peroxyl radicals

St I is a toxin present in the Caribbean Sea anemone Stichodactyla helianth us which is highly hemolytic in the nanomolar concentration range. Exposure of the toxin to free radicals produced in the pyrolysis of 2,2'-azobis(2-a midinopropane) hydrochloride leads to a progressive loss of hemolytic activ ity. This loss of hemolytic activity is accompanied by extensive modificati on of tryptophan residues. On the average, three tryptophan residues are mo dified by each inactivated toxin. The loss of hemolytic activity of St I ta kes place without significant changes in the protein structure, as evidence d by the similarity of the fluorescence and CD spectra of native and modifi ed proteins. Also, the native and modified ensembles present a similar resi stance to their denaturation by guanidinium chloride. The hemolytic behavio r and the performance of the toxin at the single-channel level when incorpo rated to black lipid membranes suggest that the modified ensemble can be co nsidered as composed of inactive toxins and active toxins whose behavior is similar to that of the native proteins. These results, together with the l ack of induction time in the activity loss, suggest that the fall of hemoly tic activity takes place by an all-or-nothing inactivation mechanism in whi ch the molecules become inactive when a critical amino acid residue is modi fied.