Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes.
We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-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 membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium 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 expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.
Gómez
C M
CM
Departament de Bioquímica i Biologia Molecular, Universitat de València, Spain.
Codoñer
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Campos
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Abad
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D002103:Cadaverine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D008956:Models, Chemical; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D013373:Substance P / Q000031:analogs & derivatives* / Q000378:metabolism*
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010743:Phospholipids; D013373:Substance P; C008542:monodansylcadaverine; D002103:Cadaverine