We have used fluorescence microscopy, fluorescence photobleaching recovery (FPR), and atomic force microscopy (AFM) to investigate the formation of tethered lipid bilayers on plane aluminum oxide or glass surfaces. The bilayers were assembled with the help of a two-step methodology recently proposed for microporous templates (Proux-Delrouyre et al. J. Am. Chem. Soc. 2001, 123, 8313). The first step consists of the accumulation of intact biotinylated vesicles (PC + DOPE) on a streptavidin sublayer itself immobilized on the substrate. The second step, clearly time separated, is the deliberate triggering of bilayer formation with the help of poly(ethylene glycol) (PEG), a fusion agent of lipidic vesicles. AFM and FPR measurements confirm that the vesicles do not spontaneously fuse during the first step provided that the streptavidin sublayer is present on the substrate. On the contrary, the treatment with PEG provokes the fast formation of a continuous lipid bilayer, as attested at the hundred nanometer scale by the AFM images and at the hundred micrometer scale by the lateral diffusion of a fluorescent probe (D ) 2.2 × 10 -8 cm 2 s -1 for NBD-DMPE at 22 °C). † Part of the Langmuir special issue entitled The Biomolecular Interface.
The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.
Physiological mole fractions of long isoprenic chain ubiquinone (UQ[10]) and plastoquinone (PQ9) were incorporated inside a supported bilayer by vesicle fusion. The template of the bilayer was an especially designed microporous electrode that allows the direct electrochemistry of water insoluble molecules in a water environment. The artificial structure, made by self-assembly procedures, consisted of a bilayer laterally in contact with a built-in gold electrode at which direct electron transfers between the redox heads of the quinones molecules and the electrode can proceed. The mass balances of quinone and lipid in the structure were determined by radiolabeling and spectrophotometry. A dimyristoyl phosphatdylcholine stable surface concentration of 250 +/- 50 pmol x cm(-2), unaffected by the presence of the quinone, was measured in the fluid monolayer. The mole fraction of quinone was between 1 and 3 mol%, remaining unchanged when going from the vesicles to the supported layers. The lipid molecules and the quinone pool were both laterally mobile, and cyclic voltammetry was used to investigate the redox properties of UQ10 and PQ9 over a wide pH range. Below pH 12, the two electrons-two protons electrochemical process at the gold electrode appeared under kinetic control. Thus all thermodynamic deductions must be anchored in the observed reversibility of the quinone/hydroquinol anion transformation at pH > 13. Within the experimental uncertainty, the standard potentials and the pK(a)'s of the pertinent redox forms of UQ10 and PQ9 were found to be essentially identical. This differs slightly from the literature in which the constants were deduced from the studies of model quinones in mixed solvents or of isoprenic quinones without a lipidic environment.
The functionality of the membrane-bound, ubiquinone-dependent pyruvate oxidase from the respiratory chain of Escherichia coli was reconstituted with a supported lipidic structure. The artificial structure was especially designed to allow the electrochemical control of the quinone pool through the lateral mobility of the ubiquinone (Q(8)) molecules. The kinetic coupling of the enzyme bound to the lipid structure with the quinone pool was ensured by the regeneration of the oxidized form of ubiquinone at the electrochemical interface. Such an experimental approach enabled us to carry out an unprecedented determination of the kinetic parameters controlling the reaction between the enzyme bound and the electron carrier under conditions taking rigorously into account the fact that the freedom of motion is restricted to two dimensions. The kinetic constants we found show that the activated enzyme can be efficiently regulated by the oxidation level of the quinone pool in natural membranes.
A plane gold-supported bilayer was prepared on an electrode by
fusion of phospholipid (dimyristoylphosphatidylcholine (DMPC)) vesicles onto an alkanethiol
(octadecylmercaptan (OM)) self-assembled
monolayer (SAM). Escherichia coli pyruvate oxidase
(Pox), a peripheral membrane enzyme, was incorporated
into the supported bilayer. This supramolecular assembly was
characterized by contact angle goniometry,
electrochemical blocking studies, double-layer capacitance, and BIAlite
(surface plasmon resonance)
measurements. Electrochemistry of ferrocenemethanol at the gold
surface was blocked by the well-ordered
alkane chains of the OM monolayer. In order to prevent this
blocking effect, dibenzyl disulfide (DBDS)
was used to produce defect sites in the OM monolayer and to allow the
reversibility of ferrocene
electrochemistry. BIAlite measurements showed that fusion of DMPC
on the OM + DBDS monolayer was
not significantly different from the fusion of DMPC on the OM
monolayer. Pox incorporation into the
(OM + DBDS)/DMPC gold-supported bilayer was detected by BIAlite
measurements. The activity of
incorporated Pox was detected by the electrocatalytic current produced
when substrate and the electron
acceptor, ferricinium methanol, were present in solution.
A new allergen from horse dander, Equ c 5 has been purified. Its biochemical and biophysical properties have been characterized and compared with those of Equ c 1, Equ c 2 and Equ c 4. Their molecular masses, determined by mass spectrometry, were 22 kDa for Equ c 1, 16 kDa for Equ c 2, 18.7 kDa for Equ c 4 and 16.7 kDa for Equ c 5. Their pI values were between 3.8 and 5.25. Equ c 2 and Equ c 5 are not glycosylated, while Equ c 4 contains a tri‐antennary tri‐sialylated N‐linked glycan. Linkages of terminal N‐acetylneuraminic acid to galactose were: α‐(2→6) in Equ c 4, and both α‐(2→3) and α‐(2→6) in Equ c 1. Oligosaccharide portions of Equ c 1 or Equ c 4 were barely involved in IgE‐immunoreactivity. Partial N‐terminal sequence of Equ c 4 shares a significant sequence homology with the rat submandibular gland protein A. No matching was found for two internal peptides of Equ c 5. Surfactant properties of horse allergens as well as other proteins were investigated. In contrast to Equ c 2 and Equ c 3, solutions of Equ c 1, Equ c 4 and Equ c 5 significantly lowered the surface tension. Relationship between a property such as this, involving oriented hydrophobic patches of a molecule and allergenicity, is addressed.
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