Electrochemical and PM-IRRAS studies of the effect of cholesterol on the structure of a DMPC bilayer supported at an Au (111) electrode surface, part 1: properties of the acyl chains

Abstract

Charge density measurements and polarization modulation infrared reflection absorption spectroscopy were employed to investigate the spreading of small unilamellar vesicles of a dimyristoylphosphatidylcholine (DMPC)/cholesterol (7:3 molar ratio) mixture onto an Au (111) electrode surface. The electrochemical experiments demonstrated that vesicles fuse and spread onto the Au (111) electrode surface, forming a bilayer, at rational potentials -0.4 V < (E - Epzc) < 0.4 V or field strength <6 x 10(7) V m(-1). Polarization modulation infrared reflection absorption spectroscopy experiments provided information concerning the conformation and orientation of the acyl chains of DMPC molecules. Deuterated DMPC was used to subtract the contribution of C-H stretching bands of cholesterol and of the polar head region of DMPC from spectra in the C-H stretching region. The absorption spectra of the C-H stretch bands in the acyl chains were determined in this way. The properties of the DMPC/cholesterol bilayer have been compared with the properties of a pure DMPC bilayer. The presence of 30% cholesterol gives a thicker and more fluid bilayer characterized by a lower capacity and lower tilt angle of the acyl chains.

FIGURE 1

(a) Surface charge density on the gold electrode plotted versus electrode potential for (□) 0.1 M NaF supporting electrolyte, (▪) pure DMPC bilayer, and (•)mixed 7:3 DMPC/cholesterol bilayer spread from a vesicle solution. (b) Surface pressure π versus potential plot calculated by integration of the charge density curves: (▪) pure DMPC bilayer and (•) mixed 7:3 DMPC/cholesterol bilayer spread from the vesicle solution.

FIGURE 2

The σ_M/C component of the potential drop across the membrane (Δφ_M−S) and (E − E_pzc) as a function of applied potential E. (▪)σ_M/C component for the pure DMPC bilayer and (•) for the mixed 7:3 DMPC/cholesterol bilayer spread from the vesicle solution. (-□-) (E − E_pzc) for the pure DMPC bilayer and (-○-) for the mixed 7:3 DMPC/cholesterol bilayer spread from the vesicle solution

FIGURE 3

(a) PM-IRRAS spectra in the CH stretching region of the h-DMPC bilayer (dashed line) and the mixed 7:3 h-DMPC/cholesterol bilayer (solid line) on an Au (111) electrode surface in 0.1 M NaF/D₂O solution at potentials indicated on the figure. The top two traces plot spectra calculated for a 5.5-nm-thick film of randomly oriented molecules, using the optical constants for h-DMPC or h-DMPC/cholesterol (7:3) mixtures for solutions in CCl₄ (top line) and vesicle dispersions in D₂O (second line). (b) PM-IRRAS spectra in the CH stretching region of the mixed 7:3 DMPC-d₅₄/h-cholesterol on an Au (111) electrode surface in 0.1 M NaF/D₂O solution at potentials indicated on the figure. The top two traces plot spectra calculated for a 5.5-nm-thick film of randomly oriented molecules, using the optical constants for the DMPC-d₅₄/h-cholesterol (7:3) mixture determined from transmission spectra of a solution in CCl₄ (top line) and a vesicle dispersion in D₂O (second line). Abbreviation a.u. denotes absorbance units.

FIGURE 4

(a) IR bands of the acyl chains of DMPC/cholesterol mixed bilayers on an Au (111) electrode surface in 0.1 M NaF/D₂O solution at the indicated potentials. The top two thicker traces plot spectra for a 5.5-nm-thick film of randomly oriented molecules, calculated from optical constants determined from transmission spectra of a solution in CCl₄ (top line) and a vesicle dispersion in D₂O (second line). (b) Example of a deconvolution of the overlapping ν_as (CH₃), ν_as(CH₂), ν_s (CH₃), ν_s (CH₂) bands and two Fermi resonances between the overtones of the symmetric bending mode and symmetric methyl and methylene stretching modes for the DMPC/cholesterol mixed bilayer at E = −0.2 V. Abbreviation a.u. denotes absorbance units.

FIGURE 5

(a) ν_s (CH₂) band center positions and (b) the FWHM as a function of electrode potential. (▵, potential changed in negative direction) and (□, potential changed in positive direction) for the pure h-DMPC bilayer; (▴, potential changed in the negative direction) and (▪, potential changed in the positive direction) for the mixed 7:3 h-DMPC/cholesterol bilayer. (♦, potential moved in the negative direction) and (★, potential moved in the positive direction) show ν_s(CD₂) band center (a) and FWHM (b) for the DMPC-d₅₄/h-cholesterol bilayer.

FIGURE 6

(a) The potential dependence of the angle (θ) between the directions of the transition dipole moment and the electric field of the photon (normal to the surface) for the mixed DMPC/cholesterol bilayer on the Au (111) electrode in 0.1 M NaF/D₂O solution for: (a) ν_s(CH₂) (▪) potential changed in the positive direction, (□) potential changed in the negative direction; (b)ν_as(CH₂) (▴) potential changed in the positive direction, (▵) potential changed in the negative direction; (c) δ(CH₂) (•) potential changed in the positive direction, (○) potential changed in negative direction.

FIGURE 7

Dependence of the tilt angle of the acyl chains on the electrode potential for the pure DMPC bilayer; dashed lines, (○) potential changed in the positive and (▵) potential changed in the negative direction. Solid lines, (•) and (▴) denote corresponding changes of the tilt angle for the mixed h-DMPC/h-cholesterol bilayer.

FIGURE 8

(a) PM-IRRAS spectra for the CD stretching region of a mixed DMPC-d₅₄/h-cholesterol bilayer on an Au (111) electrode in 0.1 M NaF/H₂O solution at the indicated potentials. The top two traces plot spectra calculated for a 5.5-nm-thick film of randomly oriented molecules, using optical constants for DMPC-d₅₄/h-cholesterol (7:3) determined from transmission spectra of the solution in CCl₄ (top line) and the dispersion of vesicles in H₂O (second line). (b) Example of a deconvolution of the overlapping ν_as (CD₃), ν_as(CD₂), ν_s (CD₃), ν_s (CD₂) bands and two Fermi resonances between overtones and symmetric methyl (ν_s(CD₃)) modes for the mixed DMPC-d₅₄/h-cholesterol bilayer at E = −0.2 V. Abbreviation a.u. denotes absorbance units.

FIGURE 9

Dependence on the electrode potential of the angle (θ) between the direction of the transition dipole moment and the electric field of the photon (normal to the surface) for the mixed DMPC-d₅₄/h-cholesterol bilayer at an Au (111) electrode in 0.1 M NaF/H₂O solution; (a) ν_s (CD₂) and (b) ν_as(CD₂) band, (•) potential changed in the positive and (▴) potential changed in the negative direction.