Lucia Becucci

Kinetics of electron and proton transfer to ubiquinone-10 and from ubiquinol-10 in a self-assembled phosphatidylcholine monolayer

Upon incorporating from 0.5 to 2 mol% ubiquinone-10 (UQ) in a self-assembled monolayer of dioleoylphosphatidylcholine (DOPC) supported by mercury, the kinetics of UQ reduction to ubiquinol-10 (UQH2) as well as that of UQH2 oxidation to UQ were investigated in borate buffers over the pH range from 8 to 9.5 by cyclic voltammetry. A general kinetic approach was adopted to interpret the dependence of the applied potential upon the scan rate at constant pH and upon pH at constant scan rate, while keeping the initial reactant concentration and the faradaic charge constant. The oxidation of UQH2 to UQ in DOPC monolayers occurs via the reversible release of one electron with formation of the semiubiquinone radical cation UQH2.+, followed by its rate-determining deprotonation by hydroxyl ions with formation of the UQH. neutral radical; the latter is then instantaneously oxidized to UQ. Analogously, the rate-determining step in UQ reduction to UQH2 consists in the protonation by hydrogen ions of the semiubiquinone radical anion UQ.- resulting from the reversible uptake of one electron by UQ. However, a non-negligible fraction of UQ.- uptakes protons very slowly, and hence, retains its intermediate oxidation state during the recording of the cyclic voltammetric peak for UQ reduction.

A novel model of the hanging mercury drop electrode

A novel version of a home-made hanging mercury drop electrode (HME) prepared in our laboratory is presented. Its main technical features are described, as well as the advantages it offers over the commercial ones. With this version the area of the electrode surface can be measured with great accuracy, and the reproducibility of the area from one mercury drop to another is better than 1 × 10−1 cm2, corresponding to less than 1% of the total amount of the surface area of an average mercury drop. The constancy of the area, for time periods of up to 90 min, and the reproducibility from one drop to another make this version of HME suitable for use in the analyical field in the study of adsorption or electron transfer phenomena when it is necessary to know the exact electrode surface area in order to determine surface density charge or differential capacitance values.

Surface charge density measurements on mercury electrodes covered by phospholipid monolayers

Abstract A novel method to measure the surface charge density at a hanging mercury drop electrode (HMDE) coated with a selfassembled phospholipid monolayer mimicking a biological membrane is described. The charge density is obtained by analogical integration of the capacitive current which flows at constant applied potential as a consequence of a slight contraction of the mercury drop. The contraction must be carried out while keeping the neck of the lipidcoated mercury drop in contact with the lipid film spread on the surface of the electrolytic solution. The validity of this procedure is tested by comparison with differential capacity measurements carried out at a lipidcoated HMDE, fully immersed into the solution, under otherwise identical conditions. The potential of this method in the investigation of the properties of selfassembled lipid monolayers, either pure or incorporating lipophilic species, is briefly outlined. The method is applied to the determination of the charge density of tetraphenylphosphonium cations adsorbed in the polar head region of a phosphatidylserine monolayer supported by the HMDE.

On the Function of Pentameric Phospholamban: Ion Channel or Storage Form?

Phospholamban (PLN) is an integral membrane protein that inhibits the sarcoplasmic reticulum Ca(2+)-ATPase, thereby regulating muscle contractility. We report a combined electrochemical and theoretical study demonstrating that the pentameric PLN does not possess channel activity for conducting chloride or calcium ions across the lipid membrane. This suggests that the pentameric configuration of PLN primarily serves as a storage form for the regulatory function of muscle relaxation by the PLN monomer.

Total and free charge densities on mercury coated with self-assembled phosphatidylcholine and octadecanethiol monolayers and octadecanethiol/phosphatidylcholine bilayers

Abstract The thermodynamic ‘total’ charge density is the charge to be supplied to the electrode to keep the applied potential constant when the electrode surface is increased by unity, while the extrathermodynamic ‘free’ charge density is the charge actually experienced by the diffuse layer ions. The total charge density at dioleoylphosphatidylcholine (DOPC) and octadecanethiol (ODT) monolayers and mixed ODT/DOPC bilayers self-assembled on mercury from aqueous solutions was determined from chronocoulometric single potential steps to a final potential negative enough to cause complete desorption of the film. The effect of different alkali metal ions and of tetramethylammonium on DOPC desorption was examined. The total charge for ODT monolayers and ODT/DOPC bilayers, +56±3 μC cm −2 , agrees with the value obtained by integration of the current under the reductive desorption voltammetric peaks, only provided the scan rate is higher than 100 mV s −1 . An approximate model of the interface of the ODT-coated electrode, which accounts for partial charge transfer from sulfur to mercury and for the degree of dissociation of the sulfhydryl group upon self-assembly, was employed to estimate the free charge density.

Equilibrium distribution of K+ ions in the hydrophilic spacer of tethered bilayer lipid membranes

Tethered bilayer lipid membranes (tBLMs) have been extensively employed to investigate the function of channel-forming peptides and proteins. They consist of a lipid bilayer tethered to the surface of an Au or Hg electrode through an oligopeptide or polyethyleneoxy “hydrophilic spacer”. Upon incorporating an ion-selective channel in the tBLM, an equilibrium distribution of the permeating ion along the hydrophilic spacer is attained at each applied potential. An electrochemical model of tBLMs is developed to calculate the equilibrium concentration profile of the permeating ion along the hydrophilic spacer as a function of the applied potential. The limited spaciousness of the hydrophilic spacer is accounted for by imposing a maximum local volume concentration to the ion. The present approach is general. In this work, it is applied to a particular mercury-supported (tetraethyleneoxy spacer)|(phospholipid bilayer) tBLM bathed by an aqueous solution of 0.1 M KCl. Penetration of K+ ions into the hydrophilic spacer was realized by incorporating in this tBLM either the ion carrier valinomycin or the ion channel gramicidin. The equilibrium charge of the cation in the hydrophilic spacer at each applied potential was determined by jumping to the given potential from a fixed potential positive enough to exclude the presence of K+ ions in the spacer.

Surface Dipole Potential at the Interface between Water and Self-Assembled Monolayers of Phosphatidylserine and Phosphatidic Acid

The nature and magnitude of the surface dipole potential chi at a membrane/water interface still remain open to discussion. By combining measurements of differential capacity C and charge density sigma at the interface between self-assembled monolayers of phosphatidylserine and phosphatidic acid supported by mercury and aqueous electrolytes of different concentration and pH, a sigmoidal dependence of chi upon sigma is revealed, with the inflection at sigma = 0. This behavior is strongly reminiscent of the surface dipole potential due to reorientation of adsorbed water molecules at electrified interfaces. The small increase in C with a decrease in the frequency of the AC signal below approximately 80 Hz, as observed with phospholipid monolayers with partially protonated polar groups, is explained either by a sluggish collective reorientation of some polar groups of the lipid or by a sluggish movement of protons across the polar head region.

The intrinsic pKa values for phosphatidic acid in monolayers deposited on mercury electrodes

The intrinsic Ka values of the phosphate group of phosphatidic acid (PA) in self-organized monolayers deposited on a hanging mercury drop electrode were determined by a novel procedure based on measurements of the differential capacity C of this lipid-coated electrode. In line with the Gouy-Chapman theory, plots of 1C at constant bulk pH and variable KCl concentration against the reciprocal of the calculated diffuse-layer capacity Cd,0 at zero charge exibit slopes that decrease from an almost unit value to zero as the absolute value of the charge density on the lipid increases from zero to ≈ 2 μC cm−2. The values so determined are K1 = 108 M−1 and K2 = 104 M−1. The plots of 1C against 1Cd,0 for PA exhibit slopes that pass from zero to a maximum value and then again to zero as pH is varied from 7.5 to 1.5, indicating that the charge density of the lipid film passes from slightly negative to slightly positive values over this pH range. An explanation for this anomalous behavior is provided.

Thallous ion movements through gramicidin channels incorporated in lipid monolayers supported by mercury

The potential independent limiting flux of hydrated Tl(+) ions through gramicidin (GR) channels incorporated in phospholipid monolayers self assembled on a hanging mercury-drop electrode is shown to be controlled both by diffusion and by a dehydration step. Conversely, the potential independent limiting flux of dehydrated Tl(+) ions stemming from Tl amalgam electro-oxidation is exclusively controlled by diffusion of thallium atoms within the amalgam. Modulating the charge on the polar heads of dioleoylphosphatidylserine (DOPS) by changing pH affects the limiting flux of hydrated Tl(+) ions to a notable extent, primarily by electrostatic interactions. The dipole potential of DOPS and dioleoylphosphatidylcholine (DOPC), positive toward the hydrocarbon tails, does not hinder the translocation step of Tl(+) ions to such an extent as to make it rate limiting. Consequently, incorporation in the lipid monolayer of phloretin, which decreases such a positive dipole potential, does not affect the kinetics of Tl(+) flux through GR channels. In contrast, the increase in the positive dipole potential produced by the incorporation of ketocholestanol causes the translocation step to contribute to the rate of the overall process. A model providing a quantitative interpretation of the kinetics of diffusion, dehydration-hydration, translocation, and charge transfer of the Tl(+)/Tl(0)(Hg) couple through GC channels incorporated in mercury-supported phospholipid monolayers is provided. A cut-off disk model yielding the profile of the local electrostatic potential created by an array of oriented dipoles located in the lipid monolayer along the axis of a cylindrical ion channel is developed.

Estimate of the potential difference across metal/water interfaces and across the lipid bilayer moiety of biomimetic membranes: an approach

Mercury is a particularly advantageous support of lipid, thiol and disulfide self-assembled monolayers (SAMs), because it provides them with a perfectly smooth and fluid surface and allows their gradual expansion in aqueous solution with progressive tilt of the adsorbed molecules, without water incorporation. These unique advantageous features permit an estimate of the extrathermodynamic absolute potential difference, ϕ, across the Hg|water interface and of the surface dipole potential of mercury-supported SAMs. Two different models of metal-supported tethered bilayer lipid membranes (tBLMs) incorporating a cation-selective channel predict that the ϕ value at the inflection point of a plot of the in-phase component, Y′, of the electrochemical admittance against the applied potential E is almost coincident with the surface dipole potential, χs, of the hydrophilic spacer moiety of the tBLM. This prediction allows an estimate of the absolute potential difference, ϕ, across the interface between any metal capable of supporting tBLMs and the bulk aqueous phase, provided the χs value of the tBLM is known. Moreover, the potential difference across the lipid bilayer moiety of the tBLM (i.e., the transmembrane potential) is shown to be practically equal to χs at the inflection point of the corresponding Y′ vs.E plot. This approach is applied to polycrystalline Au and Ag(111).