The Non-Steady-State Membrane Potential of Ion Exchangers with Fixed Sites

A system of equations, based upon the assumption that the only force acting on each ionic species is due to the gradient of its electrochemical potential, is used to deduce, in the non-steady state for zero net current, the expression of the difference of electric potential between two solutions separated by an ion exchange membrane with fixed monovalent sites. The membrane is assumed to be solely permeable to cations or anions, depending on whether the charge of the sites is -1 or +1, and not to permit any flow of solvent. Under the assumptions that the difference of standard chemical potentials of any pair of permeant monovalent species and the ratio of their mobilities are constant throughout the membrane, even when the spacing of sites is variable, explicit expressions are derived for the diffusion potential and total membrane potential as functions of time and of solution activities. The expressions are valid for any number of permeant monovalent species having ideal behavior and for two permeant monovalent species having “n-type” non-ideal behavior. The results show that for a step change in solution composition the observable potential across a membrane having fixed, but not necessarily uniformly spaced, sites becomes independent of time once equilibria are established at the boundaries of the membrane and attains its steady-state value even while the ionic concentration profiles and the electric potential profile within the membrane are changing with time.     

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.     

Amyloid-β Membrane Binding and Permeabilization are Distinct Processes Influenced Separately by Membrane Charge and Fluidity

The 40 and 42 residue amyloid-β (Aβ) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Aβ becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Aβ40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Aβ into these toxic species. Aβ40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Aβ40 [Y10W]). We extend previous observations that Aβ40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Aβ40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Aβ behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.     

The influence of pH on cystine and dibasic amino acid transport by rat renal brushborder membrane vesicles

The uptake of cystine and lysine by rat renal brushborder membrane vesicles was examined at various intravesicular and extravesicular hydrogen ion concentrations to discern whether ionic species are determinants of specificity for the shared transport system and whether hydrogen ion gradients play a role in determining uptake values. When intravesicular and extravesicular pH are identical, the highest uptake of cystine occurred at pH 7.4, with lesser uptake at pH 6.0 and 8.3. Since cystine is electroneutral at pH 6.0 and 90% anionic at pH 8.3, it appears that neither form of the amino acid is a preferred species for transport. A similar relationship between pH and uptake occurs for lysine, which is cationic at pH below 8.5. This suggests that pH affects the functioning of the membrane carrier system independent of ionic species of the substrate. There is no apparent relationship of cystine uptake to hydrogen ion gradients across the membrane. Over the range of extravesicular pH studied, optimal cystine uptake occurred whenever the intravesicular pH was 7.4. Competitive interactions between unlabeled amino acids and labeled cystine were not affected by the extravesicular pH and, therefore, did not seem determined by the ionic species of cystine.     

Poisson-Boltzmann Theory for Membranes with Mobile Charged Lipids and the pH-Dependent Interaction of a DNA Molecule with a Membrane

We consider a planar stiff model membrane consisting of mobile surface groups whose state of charge depends on the pH and the ionic composition of the adjacent electrolyte solution. To calculate the mean-field interaction potential between a charged object and such a model membrane, one needs to solve a Poisson-Boltzmann boundary value problem. We here derive and discuss the boundary condition at the membrane surface, a condition that is generally appropriate for biological membranes where two charge-regulating mechanisms are present at the same time: the pH-dependent chemical charge regulation and a regulation through the in-plane mobility of the surface groups. As an application of this general formalism, we consider the specific example of a single DNA molecule, approximated by a cylinder with smeared-out surface charges, interacting with such a model membrane. We study the effect that the two competing charge-regulating mechanisms have on the DNA/membrane interaction and the distribution of surface ions in the plane of the membrane. We find that, at short DNA-membrane distances, membrane fluidity can have a considerable impact on the DNA adsorption behavior and can lead to such counterintuitive phenomena as the adsorption of a negatively charged DNA onto a (on average) negatively charged membrane.     

Ionic interactions in nanofiltration of beta casein peptides

Nanofiltration (NF) membrane technology shows interesting potentials for separating organic components on the basis of solute charge and size in the range of 300-1000 g mol-1. Separation properties of two inorganic NF membranes were studied with a set of 10 small peptides (molecular mass range: 300-900 g mol-1; 3 < pI < 10) contained in a well-characterized tryptic beta casein hydrolysate. Peptides transmission strongly depended on ionic interactions in the system. Physicochemical conditions such as ionic strength and especially pH were crucial to the separation, because the membrane and peptides showed amphoteric properties. Thus, the three categories of peptides (acid, basic, neutral) were separated according to their pI because of presumed concentration gradients of charged peptides at the membrane: positive for basic peptides and negative for acid peptides. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a membrane coion and a complete reversion of selectivity when it was a membrane counterion. Copyright 1998 John Wiley & Sons, Inc.     

Liquid membrane potential in nonisothermal systems.

Electrical membrane potential equations for liquid ion exchange membranes, characterized by the presence of uncharged associated species and by exclusion of co-ions (no electrolyte uptake) have been derived. The irreversible thermodynamic theories already developed for solid membranes with fixed charged site density have been extended to include the different physicochemical aspects of the liquid membranes. To this purpose the dissipation function has been written with reference to the fluxes of all the species present in the membrane. It has been found that the mobile charged site, the counterions, and the uncharged associated species contribute to the electrical membrane potential through their phenomenological coefficients. The electrical membrane potential equations have been integrated in isothermal and nonisothermal conditions for monoionic and biionic systems. The theoretical predictions have been experimentally tested by studying the electrical potential of liquid membranes formed with solutions of tetraheptylammonium salts in omicron-dichlorobenzene.     

A nonlinear electrostatic potential change in the T-system of skeletal muscle detected under passive recording conditions using potentiometric dyes

Voltage-sensing dyes were used to examine the electrical behavior of the T-system under passive recording conditions similar to those commonly used to detect charge movement. These conditions are designed to eliminate all ionic currents and render the T-system potential linear with respect to the command potential applied at the surface membrane. However, we found an unexpected nonlinearity in the relationship between the dye signal from the T-system and the applied clamp potential. An additional voltage- and time-dependent optical signal appears over the same depolarizing range of potentials where change movement and mechanical activation occur. This nonlinearity is not associated with unblocked ionic currents and cannot be attributed to lack of voltage clamp control of the T-system, which appears to be good under these conditions. We propose that a local electrostatic potential change occurs in the T-system upon depolarization. An electrostatic potential would not be expected to extend beyond molecular distances of the membrane and therefore would be sensed by a charged dye in the membrane but not by the voltage clamp, which responds solely to the potential of the bulk solution. Results obtained with different dyes suggest that the location of the phenomena giving rise to the extra absorbance change is either intramembrane or at the inner surface of the T-system membrane.     

Purification of singly PEGylated α-lactalbumin using charged ultrafiltration membranes

One of the challenges in producing a PEGylated therapeutic protein is that the PEGylation reaction typically generates a mixture of both singly and multiply PEGylated species. The objective of this study was to examine the feasibility of using ultrafiltration for the purification of a singly PEGylated protein from the multiply PEGylated conjugates. Data were obtained with α‐lactalbumin that was PEGylated with a 20 kDa activated PEG, with the ultrafiltration performed over a range of pH and ionic strength using both unmodified and negatively charged composite regenerated cellulose membranes. Purification of the singly PEGylated α‐lactalbumin from the multiply PEGylated species was accomplished using a diafiltration process with a negatively charged membrane at pH 5 and an ionic strength of 0.4 mM, conditions that maximized the electrostatic exclusion of the multiply PEGylated species from the charged membrane. The diafiltration process provided more than 97% yield with greater than 20‐fold purification between the singly and doubly PEGylated proteins and nearly complete removal of the more heavily PEGylated species. The singly PEGylated α‐lactalbumin was recovered as a dilute filtrate solution, although this dilution could be eliminated using a cascade filtration or the final product could be re‐concentrated in a second ultrafiltration as part of the final formulation. These results demonstrate the feasibility of using ultrafiltration for the purification of singly PEGylated protein therapeutics. Biotechnol. Bioeng. 2011; 108:822–829. © 2010 Wiley Periodicals, Inc.     

Controlling protein transport in ultrafiltration using small charged ligands

Previous studies have demonstrated that protein transport during ultrafiltration can be strongly influenced by solution pH and ionic strength. The objective of this study was to examine the possibility of controlling protein transmission using a small, highly charged ligand that selectively binds to the protein of interest. Experiments were performed using bovine serum albumin and the dye Cibacron Blue. Protein sieving data were obtained with essentially neutral and negatively charged versions of a composite regenerated cellulose membrane to examine the effects of electrostatic interactions. The addition of only 1 g/L of Cibacron Blue to an 8 g/L BSA solution reduced the BSA sieving coefficient through the negatively-charged membrane by more than two orders of magnitude, with this effect being largely eliminated at high salt and with the neutral membrane. Protein sieving data were in good agreement with model calculations based on the partitioning of a charged sphere in a charged pore accounting for the change in net protein charge due to ligand binding and the increase in solution ionic strength due to the free ligand in solution.     

Ion concentration and charge adjacent to electrically charged cell membrane

Equations describing ion concentration profiles and electric charge in electrolyte solutions adjacent to an electrically charged cell membrane model in the electrochemical equilibrium state are developed and completely solved. The membrane system model consists of an infinitely large planar sheet of finite thickness separating two electrolyte solutions. Electric charges in the membrane model consist of planes of charge parallel to the surfaces of the planar sheet. The charge in solution adjacent to each surface of the membrane is due to differences in the total anion and cation concentrations in each solution.Expressions of concentration and charge are functions of the quantity and location of charge in the membrane, the various permittivities and thickness of the membrane, and the ionic compositions, permittivities, and temperature of the electrolyte solutions.The validity and relation of the model to real membranes are discussed.     

DNA Strands Attached Inside Single Conical Nanopores: Ionic Pore Characteristics and Insight into DNA Biophysics

Single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume. Tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution. Here, we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single-stranded DNA molecules. We find that the DNA occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte. The results are explained by the ionic strength dependence of the persistence length of DNA. At low KCl concentrations (10 mM) the molecules assume an extended and rigid conformation, thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted DNA at high salt concentrations. Attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species.     

Comparison of p25 presequence peptide and melittin. Red blood cell haemolysis and band 3 aggregation.

The 25 residue presequence (p25) for subunit IV of yeast cytochrome oxidase had previously been shown to possess structural and behavioural characteristics in common with the bee venom polypeptide, melittin. The present study extends the results of leakage experiments on model-membrane systems to the haemolysis of human erythrocytes, which both peptides are shown to accomplish in a manner sensitive to membrane potential. In addition, the laser flash-induced transient dichroism technique for measuring protein rotational diffusion has been used to show that both peptides aggregate band 3, the major integral membrane protein of the erythrocyte. Aggregation cannot be reversed by high ionic strength; this serves to differentiate these peptides from other positively charged species such as polylysine that aggregate band 3 at low ionic strength. These results suggest that aggregation of membrane proteins may possibly prove to be a feature of the interaction of p25 signal peptide with mitochondrial membranes.     

Alterations in the structure of the electrical double layer adjacent to a charged membrane arising from electromagnetically induced changes in the activity of membrane bound enzymes

Electromagnetic fields of very low amplitude have been reported to influence a number of cellular functions. Many of these effects have a high degree of frequency specificity. Herein it is suggested that some of these reported results could be explained by a fieldinduced alteration in the enzymic activity of integral membrane proteins. It is shown that such a field-induced transition from an initial nonequilibrium steady-state to a final nonequilibrium steady-state can lead to an alteration in the concentration profiles of those charged species in the cell's ambient electrolyte that comprise the so-called electrical double layer. Examples of variations in the concentration profiles of those ions that react with a membrane-bound enzyme, as well as nonreacting ionic species, are given. The modulation of such effects by systematic variations in extracellular pH and ionic strength is discussed.     

Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Interactions of voltage-sensing dyes with membranes. III. Electrical properties induced by merocyanine 540

The effects of merocyanine 540 on the electrical properties of lipid bilayer membranes have been investigated. The alterations this dye was found to produce in the intrinsic conductances of these membranes were minimal, but it profoundly altered the conductances produced by extrinsic permeant species. These alterations were much larger for neutral membranes than for negatively charged ones. The dye increased the conductances mediated by positively charged permeant species and decreased those by negatively charged permeant species, suggesting that it produces a negative electrostatic potential on the membrane; it also altered the kinetics and the voltage dependencies of permeation by these charge carriers. The magnitudes of dye-mediated conductance changes were much larger for positively charged permeants than for negatively charged ones; also, changes in ionic strength altered these dye effects in opposite directions from those predicted by the Stern equation, and the dependence of the conductance alteration on dye concentration was steeper than that predicted by this equation. Finally, only very small changes in liposome zeta potentials were induced by the dye. Calculations show that a large fraction of these effects can be accounted for by the dipole potential produced by merocyanine at the membrane surface, but that additional effects of the dye must be postulated as well.     

Alterations in the structure of the electrical double layer adjacent to a charged membrane arising from electromagnetically induced changes in the activity of membrane bound enzymes

Electromagnetic fields of very low amplitude have been reported to influence a number of cellular functions. Many of these effects have a high degree of frequency specificity. Herein it is suggested that some of these reported results could be explained by a field-induced alteration in the enzymic activity of integral membrane proteins. It is shown that such a field-induced transition from an initial nonequilibrium steady-state to a final nonequilibrium steady-state can lead to an alteration in the concentration profiles of those charged species in the cell's ambient electrolyte that comprise the so-called electrical double layer. Examples of variations in the concentration profiles of those ions that react with a membrane-bound enzyme, as well as nonreacting ionic species, are given. The modulation of such effects by systematic variations in extracellular pH and ionic strength is discussed.     

Triethanolammonium acetate as a multifunctional ionic liquid in the palladium-catalyzed green Heck reaction

An efficient green Heck reaction protocol was performed using a triethanolammonium acetate ionic liquid–palladium(II) catalytic system. The ionic liquid used acts as a reaction medium, base, precatalyst-precursor, and mobile support for the active Pd species. Our experimental investigation indicates that performing the Heck reaction in ionic liquid is superior to the same procedure carried out in triethanolamine. The mechanism of the reaction of triethanolammonium acetate with PdCl₂ was examined using density functional theory (M06 method). It was found that two Pd(II) complexes are formed, one of which acts further as a precatalyst yielding catalytically active Pd(0) complex. The calculated activation energies are in agreement with our experimental findings.     

Association of Cytochrome c with Membrane-Bound Cytochrome c Oxidase Proceeds Parallel to the Membrane Rather Than in Bulk Solution

Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c₅₅₂, and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions.     

Compositional domain immiscibility in whole myelin monolayers at the air-water interface and Langmuir-Blodgett films

Monomolecular layers of whole myelin membrane can be formed at the air-water interface from vesicles or from solvent solution of myelin. The films appear microheterogeneous as seen by epifluorescence and Brewster angle microscopy. The pattern consists mainly of two coexisting liquid phases over the whole compression isotherm. The liquid nature of the phases is apparent from the fluorescent probe behavior, domain mobility, deformability and boundary relaxation due to the line tension of the surface domains. The monolayers were transferred to alkylated glass and fluorescently labeled against myelin components. The immunolabeling of two major proteins of myelin (myelin basic protein, proteolipid-DM20) and of 2',3'-cyclic nucleotide 3'-phosphodiesterase shows colocalization with probes partitioning preferentially in liquid-expanded lipid domains also containing ganglioside G(M1). A different phase showing an enrichment in cholesterol, galactocerebroside and phosphatidylserine markers is also found. The distribution of components is qualitatively independent of the lateral surface pressure and is generally constituted by one phase enriched in charged components in an expanded state coexisting with another phase enriched in non-charged constituents of lower compressibility. The domain immiscibility provides a physical basis for the microheterogeneity found in this membrane model system.