Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of su bstance P (DNC-SP) with negatively charged small unilamellar vesicles compo sed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycero l (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniqu es. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic stre ngths. The experimental association isotherms were analyzed using two bindi ng approaches: (i) the Langmuir adsorption isotherm and the partition equil ibrium model, that neglect the activity coefficients; and (ii) the partitio n equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP b inding curve at different lipid composition was achieved by means of the Go uy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M -1 (9/1, mol/mol) and 6.9 x 10(3) M-1 (7/3, mol/mol), whereas for PC/PS mem branes an average value of 6.8 x 10(3) M-1 was estimated. These partition e quilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M-1). as theoretically expect ed. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge d ensity of the vesicles. Also, the potential of fluorescent dansylated SP an alogue in studies involving interactions with cell membranes is discussed. (C) 2000 Elsevier Science B.V. All rights reserved.