Kinetics and thermodynamics of association of a phospholipid derivative with lipid bilayers in liquid-disordered and liquid-ordered phases

Abstract

We have measured the rates of insertion into, desorption from, and spontaneous interlayer translocation (flip-flop) in liquid-disordered and liquid-ordered phase lipid bilayer membranes, of the fluorescent phospholipid derivative NBD-dimyristoylphosphatidyl ethanolamine. This study made use of a recently described method that exploits a detailed knowledge of the binding kinetics of an amphiphile to bovine serum albumin, to recover the insertion and desorption rate constants when the albumin-bound amphiphile is transferred through the aqueous phase to the membrane and vice versa. The lipid bilayers, studied as large unilamellar vesicles, were prepared from pure 1-palmitoyl-2-oleoylphosphatidylcholine in the liquid-disordered phase; and from two cholesterol-containing binary lipid mixtures, 1-palmitoyl-2-oleoylphosphatidylcholine and cholesterol (molar ratio of 1:1), and egg sphingomyelin and cholesterol (molar ratio of 6:4), both in the liquid-ordered phase. Insertion, desorption, and translocation rate constants and equilibrium constants for association of the amphiphile monomer with the lipid bilayers were directly measured between 15 degrees and 35 degrees C, and the standard free energies, enthalpies, and entropies, as well as the activation energies for these processes, were derived from this data. The equilibrium partition coefficients for partitioning of the amphiphile between the aqueous phase and the different membrane phases were also derived, and permitted the estimation 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.

FIGURE 1

Determination of the critical aggregation concentration of NBD-DMPE in aqueous solution. The change in slope of a plot of the concentration-normalized fluorescence intensity of NBD-DMPE in buffered aqueous solution versus log([NBD-DMPE]) was considered to be the critical aggregation concentration for the amphiphile.

FIGURE 2

(A) Titration curve used to determine K_B for the binding of NBD-DMPE to BSA at 35°C. The insert compares the fluorescence spectra of NBD-DMPE in aqueous solution and bound to BSA. (B) Time course of the binding of NBD-DMPE, dispersed in aqueous solution at a concentration above its CAC, to BSA at 25°C.

FIGURE 3

Comparison of the fluorescence emission spectra of NBD-DMPE in aqueous solution (a), bound to BSA (b), and associated with LUVs prepared from POPC (c).

FIGURE 4

(A) Experimentally observed kinetics, at 35°C, of the transfer of NBD-DMPE between BSA and LUVs in the l_d phase prepared from POPC, ♦, and in the l_o phase prepared from an equimolar mixture of POPC and cholesterol, ▪, and from a 6:4 molar mixture of SpM and cholesterol, •. Each curve was fitted by numerical integration of Eq. 2. The respective residuals, as Δ[AL_v]/[A]_T (%), are shown in B–D for POPC, POPC-Chol, and SpM-Chol, respectively. The recovered rate constants are presented in Table 1.

FIGURE 5

Time course of the bleaching of NBD-DMPE associated with LUV membranes by sodium dithionite at 35°C. The curves are for LUVs prepared from ♦, POPC (l_d phase); ▪ an equimolar mixture of POPC and cholesterol (l_o phase); and •, a 6:4 (molar ratio) mixture of SpM and cholesterol (l_o phase). The rates of translocation were obtained from the initial rates (also shown). The recovered rate constants are presented in Table 1.

FIGURE 6

Arrhenius plots for the translocation of NBD-DMPE between the monolayers of LUVs prepared from ♦, POPC (l_d phase); ▪, an equimolar mixture of POPC and cholesterol (l_o phase); and •, a 6:4 (molar ratio) mixture of SpM and cholesterol (l_o phase). The lines are the linear fits to the two independent experiments shown and the recovered parameters are presented in Table 1.

FIGURE 7

Arrhenius plots for (A) insertion, and (B) desorption of NBD-DMPE into/from lipid bilayers. Data are the average and standard deviation of five independent experiments for ♦, POPC (l_d phase); ▪, an equimolar mixture of POPC and cholesterol (l_o phase); and •, a 6:4 (molar ratio) mixture of SpM and cholesterol (l_o phase), in each panel. The lines are weighted linear fits to the data and the recovered parameters are presented in Table 1.

FIGURE 8

The van't Hoff plots for the association of NBD-DMPE monomers with lipid bilayer membranes. Data are for ♦, POPC (l_d phase); ▪, an equimolar mixture of POPC and cholesterol (l_o phase); and •, a 6:4 (molar ratio) mixture of SpM and cholesterol (l_o phase). The lines are weighted linear fits to the data and the recovered parameters are presented in Table 1.

FIGURE 9

Model for the passage through the transition state during the transfer of NBD-DMPE between the aqueous and membrane phases, and vice versa.