INTRINSIC TRYPTOPHAN FLUORESCENCE OF EQUINATOXIN-II, A PORE-FORMING POLYPEPTIDE FROM THE SEA-ANEMONE ACTINIA-EQUINA L, MONITORS ITS INTERACTION WITH LIPID-MEMBRANES

Equinatoxin II is a cytolytic polypeptide from the sea anemone Actinia equina L. which forms pores in natural and artificial membranes. The intrinsic fluorescence of its five tryptophanyl residues was used to m onitor the conformational changes induced by denaturing agents, pH and lipids. In the presence of denaturants, the emitted fluorescence peak , normally occurring at 335 nm, was reduced in height by about 65% and red-shifted to 354 nm indicating unfolding. The toxin fluorescence in tensity reversibly decreased by increasing the pH, whereas lipid vesic les, at every pH, caused an increase and a blue shift. The amount of t oxin binding to the lipid vesicle was increased by the presence of sph ingomyelin. With sphingomyelin-containing vesicles half-saturation occ urred at a lipid/toxin molar ratio of about 40, whereas with phospha t idylcholine no saturation appeared up to a ratio of 300. One hydrophil ic neutral quencher (acrylamide) and two lipid-confined phosphatidylty pe quenchers [bis(9,10-dibromostearoyl)-sn-glycero-3- line and -2-(1-p yrenedecanoyl)-sn-glycero-3-phosphocholine] were used to assess the ex posure of the emitting centres to the solvent and/or to the lipid. Mos t of the indolyl residues were found to be solvent-exposed in the wate r-soluble form of the toxin, as inferred from acrylamide quenching. Up on association with lipid vesicles, the fraction accessible to acrylam ide dropped considerably, meanwhile the toxin became sensitive to lipi d-soluble quenchers. Taken together these results suggest that inserti on of equinatoxin II into sphingomyelin-containing bilayers is facilit ated by high pH and results in the transfer of one or more exposed try ptophanyl residues into the lipid phase. Calcein-loaded vesicles, with or without a lipid quencher, were used to monitor simultaneously the formation of pores and the transfer of the tryptophans to the lipid ph ase. We found that the rate constants for vesicles permeabilization an d for changes of intrinsic tryptophanyl fluorescence had a different d ependence on the lipid/toxin ratio suggesting they correspond to separ ate steps in the toxin lipid interaction.