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 monitor 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 intensity reversibly decreased by increasing the pH, whereas lipid vesicles, at every pH, caused an increase and a blue shift. The amount of toxin binding to the lipid vesicle was increased by the presence of sphingomyelin. With sphingomyelin-containing vesicles half-saturation occurred at a lipid/toxin molar ratio of about 40, whereas with phosphatidylcholine no saturation appeared up to a ratio of 300. One hydrophilic neutral quencher (acrylamide) and two lipid-confined phosphatidyltype quenchers [bis(9,10-dibromostearoyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine] were used to assess the exposure of the emitting centres to the solvent and/or to the lipid. Most of the indolyl residues were found to be solvent-exposed in the water-soluble form of the toxin, as inferred from acrylamide quenching. Upon association with lipid vesicles, the fraction accessible to acrylamide dropped considerably, meanwhile the toxin became sensitive to lipid-soluble quenchers. Taken together these results suggest that insertion of equinatoxin II into sphingomyelin-containing bilayers is facilitated by high pH and results in the transfer of one or more exposed tryptophanyl residues into the liquid 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 phase. We found that the rate constants for vesicles permeabilization and for changes of intrinsic tryptophanyl fluorescence had a different dependence on the lipid/toxin ratio suggesting they correspond to separate steps in the toxin lipid interaction.