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Kinetics and Thermodynamics of Association of a Fluorescent Lysophospholipid Derivative with Lipid Bilayers in Liquid-Ordered and Liquid-Disordered Phases

Departamento de Química, Universidade de Coimbra, 3004-535 Coimbra, Portugal

Correspondence: Address reprint requests to Winchil L. C. Vaz, Depto. de Química, Universidade de Coimbra, 3004-535 Coimbra, Portugal. Tel.: 351-239-824861; Fax: 351-239-827703; E-mail: wvaz{at}ci.uc.pt.

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) of the fluorescent lysophospholipid derivative NBD-lyso-1-myristoylphosphatidylethanolamine in l_d and l_o phase lipid bilayer membranes. The lipid bilayers, studied as LUV, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine, in the l_d phase; and from two Chol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and Chol (molar ratio of 1:1) and SpM and Chol (molar ratio of 6:4), both in the l_o phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were measured between 15°C and 35°C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes were derived from these data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and an estimation was made of hypothetical partition coefficients and the respective energetic parameters for partitioning between the different lipid phases if these were to coexist in the same membrane. We show that, contrary to general belief, the association of NBD-lysoMPE with lipid bilayers is not a diffusion-controlled process, the rate-limiting step in insertion being the formation of a free area in the membrane surface of an adequate size for insertion to occur.

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