Determination of membrane potential with lipophilic cations. Comparison of estimated values with various phosphonium ions

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–5) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the binding of probes to the membrane was measured. For the probes of n = 0 and n = 1, and for TPP⁺, binding followed the Langmuir adsorption isotherm. For other probes, analysis revealed the presence of two, high- and low-affinity, binding sites. Upon illumination, which generated the membrane potential, the probe molecules were accumulated into the vesicles. If we ignore the membrane-potential-dependent binding of the probe molecules, the estimated values are larger when the probe used is more hydrophobic. We have tested some models describing the amount of probe bound on membranes in terms of concentration of free probe inside and outside the vesicles. No model has fulfilled the criterion of valid estimation that the membrane potentials estimated are independent of probes used. An experimental method for the estimation of true membrane potential is proposed. Effects of tetraphenylboron on the estimation of membrane potential and on the transport rate of phosphonium cations were examined.     

Partitioning of triphenylalkylphosphonium homologues in gel bead-immobilized liposomes: chromatographic measurement of their membrane partition coefficients

Unilamellar liposomes of small or large size, SUVs and LUVs, respectively, were stably immobilized in the highly hydrophilic Sepharose 4B or Sephacryl S-1000 gel beads as a membrane stationary phase for immobilized liposome chromatography (ILC). Lipophilic cations of triphenylmethylphosphonium and tetraphenylphosphonium (TPP+) have been used as probes of the membrane potential of cells. Interaction of TPP+ and triphenylalkylphosphonium homologues with the immobilized liposomal membranes was shown by their elution profiles on both zonal and frontal ILC. Retardation of the lipophilic cations on the liposome gel bed was increased as the hydrophobicity of the cations increased, indicating the partitioning of lipophilic cations into the hydrocarbon region of the membranes. The cations did not retard on the Sepharose or Sephacryl gel bed without liposomes, confirming that the cations only interact with the immobilized liposomes. Effects of the solute concentration, flow rate, and gel-matrix substance on the ILC were studied. The stationary phase volume of the liposomal membranes was calculated from the volume of a phospholipid molecule and the amount of the immobilized phospholipid, which allowed us to determine the membrane partition coefficient (KLM) for the lipophilic cations distributed between the aqueous mobile and membrane stationary phases. The values of KLM were generally increased with the hydrophobicity of the solutes increased, and were higher for the SUVs than for the LUVs. The ILC method described here can be applied to measure membrane partition coefficients for other lipophilic solutes (e.g., drugs).     

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.     

Interactions between liposomes and cations in aqueous solution

An investigation on the dependence of electrophoretic mobilities of unilamellar vesicles of phosphatidylcholine-cholesterol-phosphatidylinositol (PC-Chol-PI) on the concentration of several cations with variations in the relation charge/radius in the range Na+, K+, Cs+, Mg2+, Ca2+, Ba2+, Al3+, and La3+ has been realized. Plots of zeta potential against ion concentration exhibit a maximum for all the cations under study, the position of the maximum is greatly affected by the charge of the ion. From the feature of these plots two phenomenon were observed: an initial binding of cations into the slipping plane for ion concentration below the maximum and a phenomenon of vesicle association for concentration above the maximum. To confirm these observations measurements on dynamic light scattering were performed to obtain the corresponding size distribution of the liposomes at different ion concentrations. Finally the ability of the Stern isotherm to describe the adsorption of the cations to vesicles was tested by two methods. The two main parameters of the theory: the total number of adsorption sites per unit area, N1, and the equilibrium constant, K; (and consequently the free energy of adsorption, deltaG0ads) were calculated for the different ions, showing good agreement. The equilibrium constants of adsorption have been found to obey a linear relationship with ion radius the slope of which decreases with the ion charge.     

Membrane potential difference of isolated plant vacuoles: positive or negative? I. Evidence for membrane binding of cationic probes

The binding of membrane potential cationic probes was studied on phospholipidic liposomes by equilibrium dialysis and microelectrophoresis. Surface binding of lipophilic cations (benzyltributylammonium or tetraphenylphosphonium) appears to be the major accumulation mechanism in liposomes and simulates the existence of a negative transmembrane potential (E_m), in absence of any transmembrane ionic gradient. Furthermore, this apparent negative potential has a classical response with regard to common E_m effectors, namely a depolarization induced by KCl or FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone). The relevance of these results to the study of transtonoplast potential difference on isolated vacuoles was investigated. Tetraphenylphosphonium was shown to bind to the tonoplast, the essential features of binding and interaction with E_m effectors being similar in vacuoles and liposomes. Therefore the assumption of negligible binding of cationic probe to vacuoles, classically admitted in determinations of vacuolar E_m using lipophilic cations, is untenable.     

A radiolabel and electron microscopy study of the interaction of liposomes with synaptosomal membranes

The quantitative and qualitative interaction of liposomes with synaptosomes isolated from rat brain was examined using radiolabeled phospholipids and electron microscopy. Liposomes were prepared by sonication and detergent dialysis. Binding (adsorption) of radiolabeled phospholipid to synaptosomes was saturable when liposomes were in the liquid-crystalline state, were electrically neutral (egg-phosphatidylcholine), or carried increasing fractions (10:2 and 10:4 molar ratio) of negatively charged phosphatidic acid. Analysis using the Langmuir isotherm equation indicated a biphasic adsorption behavior. Adsorption increased with increasing temperature (4 degrees C and 37 degrees C). Binding was nonsaturable when liposomes were positively charged with stearylamine or composed of dimyristoylphosphatidylcholine and phosphatidylinositol (10:2 molar ratio). Due to the latter composition's solid state at 4 degrees C, temperature dependency was inverse. Electron micrographs revealed disc-shaped areas of adsorption that were free of integral membrane particles which appeared to form a condensed layer surrounding the areas of liposome adsorption. Following interaction with stearylamine-containing liposomes the vesicular structure of synaptosomes appeared largely destroyed. It is concluded that both liposome surface charge and membrane fluidity determine the extent of interaction with biological membranes.     

Domains and anomalous adsorption isotherms of dipalmitoylphosphatidylcholine membranes and lipophilic ions: pentachlorophenolate, tetraphenylborate, and dipicrylamine.

Dipalmitoylphosphatidylcholine (DPPC) vesicles acquire negative surface charge on adsorption of negatively charged pentachlorophenolate (PCP-), and lipophilic ions tetraphenylborate (TPhB-), and dipicrylamine (DPA-). We have obtained (a) zeta-potential isotherms from the measurements of electrophoretic mobility of DPPC vesicles as a function of concentration of the adsorbing ions at different temperatures (25-42 degrees C), and (b) studied the effect of PCP- on gel-to-fluid phase transition by measuring the temperature dependence of zeta-potential at different PCP- concentrations. The zeta-potential isotherms of PCP- at 25, 32, and 34 degrees C correspond to adsorption to membrane in its gel phase. At 42 degrees C the zeta-potential isotherm corresponds to membrane in its fluid phase. These isotherms are well described by a Langmuir-Stern-Grahame adsorption model proposed by McLaughlin and Harary (1977. Biochemistry. 15:1941-1948). The zeta-potential isotherm at 37 degrees C does not follow the single-phase adsorption model. We have also observed anomalous adsorption isotherms for lipophilic ions TPhB- and DPA- at temperatures as low as 25 degrees C. These isotherms demonstrate a gel-to-fluid phase transition driven by ion adsorption to DPPC membrane during which the membrane changes from weakly to a strongly adsorbing state. The anomalous isotherm of PCP- and the temperature dependence of zeta-potential can be described by a two-phase model based on the combination of (a) Langmuir-Stern-Grahame model for each phase, (b) the coexistence of gel and fluid domains, and (c) depression of gel-to-fluid phase transition temperature by PCP-. Within the anomalous region the magnitude of zeta-potential rapidly increases concentration of adsorbing species, which was characterized in terms of a Esin-Markov coefficient. This effect can be exploited in membrane-based devices. Comments are also made on the possible effect of PCP, as an uncoupler, in energy transducing membranes.     

Anion binding to lipid bilayers: a study using fluorescent lipid probes

Anion-induced fluorescence quenching of lipid probes incorporated into the liposomal membrane was used to study the binding of anions to the lipid membrane. Lipid derivatives bearing nonpolar fluorophore located either in the proximity of the polar headgroups (anthrylvinyl-labelled phosphatidylcholine, ApPC; methyl 4-pyrenylbutyrate, MPB) or in the polar region (rhodamine 19 oleyl ester, OR19) of the bilayer were used as probes. The binding of iodide to the bilayers of different compositions was studied. Based on the anion-induced quenching of the fluorescence, the isotherm of adsorption of the quencher (iodide) to the membrane was plotted. For anions, which are non-quenchers or weak quenchers (thiocyanate, perchlorate or trichloroacetate), the binding parameters were obtained from the data of the competitive displacement of iodide by these anions. The association constants of the anion binding to the bilayer (Ka) were determined for the stoichiometry of 1 ion/1 lipid and also for the case of independent anion binding. At the physiological concentration of the salt, which does not bind noticeably to the membrane (150 mM NaCl), anion binding could be satisfactorily described by the Langmuir isotherm. The approach applied here offers new possibilities for the studies of ion-membrane interactions using fluorescent probes.     

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations,rubidium ion,and a cyanine dye as probes

Summary Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C₃(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [³H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C₃(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by⁸⁶Rb⁺ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 m for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas⁸⁶Rb⁺ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.     

Mitochondrial membrane potential estimated with the correction of probe binding

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and / or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207–215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0−4) and tetraphenylphosphonium (TPP⁺). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50–800 μM) and low (under 20 μM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the ‘state of no binding’, the membrane potential of intact mitochondria was estimated to be −147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl₂, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25°C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the Langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 μM.     

Thermodynamics of ouabain binding to human erythrocytes

A previous study reported that the uptake and release kinetics of ouabain by human erythrocytes in suspension could well be explained by a physical model which involves the slow Langmuir binding of the drug to the erythrocyte membrane. The purpose of the present investigation was to assess quantitatively the thermodynamics of this drug-membrane receptor interaction in order to evaluate the consistency of these parameters with the proposed kinetics model.Cellular drug uptake and release experiments were conducted at 20, 30 and 40°C, and the Langmuir adsorption and desorption rate constants as well as the Langmuir adsorption isotherms determined from the rate data. With the knowledge of these Langmuir parameters, it was possible to estimate the magnitude of all relevant thermodynamic properties by the use of established physicochemical theories.The activation energies and entropies for the ouabain adsorption and desorption processes were computed as 105 kJ/mol, 231 J/K per mol, 180 kJ/mol and 245 J/K per mol, respectively. The kinetic and isosteric heats of adsorption were found to be ?75.0 and ?72.4 kJ/mol, respectively. These findings suggest that the ouabain-erythrocyte membrane interaction represents a case of activated chemisorption which follows the Langmuir isotherm, thus, further underscoring the appropriateness of the Langmuir binding kinetics model.     

Mitochondrial membrane potential estimated with the correction of probe binding

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and/or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207-215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50-800 microM) and low (under 20 microM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the 'state of no binding', the membrane potential of intact mitochondria was estimated to be -147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl2, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25 degrees C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 microM.     

Adsorption of cellulase fromTrichoderma viride on microcrystalline cellulose

Summary The adsorption behaviour of cellulase fromTrichoderma viride on microcrystalline celluloses with different specific surface areas was studied. The adsorption was found to fit a Langmuir isotherm. There was an increase in the maximum adsorption amount (A_max) as the specific surface area of microcrystalline cellulose increased. The values of A_max and adsorption equilibrium constant (K) decreased with increasing temperature. Thermodynamic parameters in adsorption were calculated from K. It was found from the enthalpy of adsorption, that van der Waals-Type interaction was responsible for adsorption of cellulase on microcrystalline cellulose. The adsorption process was exothermic and an adsorption enthalpy-controlled reaction.     

The effect of lateral mobility on the binding and reaction rate at membrane sites

The rates of membrane processes like transport, hormone action and enzyme reaction depend on the prevailing temperature. Arrhenius plots of such rates show a break at the phase transition temperature. We have very little insight into the molecular mechanism of the effect of phase transition on the membrane phenomena. In general above this temperature there is an onset of two dimensional mobility of the surface sites. Here, we point out that the binding of ligands to these sites should then be described by an adsorption isotherm appropriate for mobile rather than fixed sites. This introduces an additional factor in the rate equation, which correlates well with the observed changes in the rates at membrane sites on gel to liquid crystalline transition.     

Batch and continuous adsorption of strontium by plant root tissues

Strontium (Sr) ions in aqueous solutions could be adsorbed by root tissue powders of Amaranthus spinosus, a common weed found in the fields. The adsorption isotherm could be fitted by either the Langmuir or the Freundlich model with the maximum adsorption capacity being 12.89 mg/g from the Langmuir isotherm. The maximum adsorption capacity of the biosorbent decreased with increasing temperature, whereas alkaline pretreatment enhanced the adsorption capacity 1.9 fold. Alginate gel beads (1 mm diameter) containing the root tissue powders were prepared and packed in a column for continuous adsorption/desorption of Sr in solution. Efficient desorption of Sr could be carried out with 0.1 CaCl₂ to give a concentrated Sr solution with 94% recovery.     

Hydrophobic interaction adsorption of whey proteins: effect of temperature and salt concentration and thermodynamic analysis

The adsorptive behavior of bovine serum albumin (BSA) and beta-lactoglobulin (beta-lg) on hydrophobic adsorbent was studied at four temperatures and different salt concentrations. The Langmuir model was fitted by experimental equilibrium data showing that an increase in temperature and salt concentration results in an increase on the capacity factor of both proteins. A thermodynamic analysis coupled with isotherm measurements showed that salt concentration and temperature affected the enthalpic and entropic behavior of the adsorption process of both proteins, mainly to the beta-lg. The fast variation in the Z value for temperature over than 303.1K suggest a great conformational change occurring in the beta-lg structure, which almost duplicated the maximum adsorption capacity of this protein.     

Purification and characterisation of a cellulose binding endoxylanase fromCellulomonas flavigena

Summary An extracellular endoxylanase capable of binding to avicel was isolated and purified fromCellulomonas flavigena. The purified xylanase, posessing no cellulose hydrolysing activity, had a molecular weight of 53K as determined electrophoretically. Steady state adsorption to avicel was reached within 5 minutes at 20°C. The adsorption followed the Langmuir isotherm with a dissociation constant at 0°C of 7.9E-8M and a saturation coefficient of 12 mg per gram with binding being stable for a wide range of ionic strengths down to 0.1M.     

The characteristics of Escherichia coli adsorption of arsenic(III) from aqueous solution

The potential of using Escherichia coli (E. coli) as adsorbent for the adsorption of As(III) from aqueous solution was assessed. Various parameters like pH, initial concentration and temperature have been investigated. It is found that the adsorption of As(III) is pH dependent and the optimum value was 2.0. Kinetics studies revealed that the equilibrium of the adsorption of As(III) on to E. coli was obtained within 30 min and the process was followed with the pseudo-2nd-order kinetic model. The equilibrium adsorption data obtained at different temperatures were fitted better with Langmuir than Freundich isotherm. The adsorption capacity of E. coli for As(III) is increased with the increasing of temperature and the maximum adsorption capacity is 1.111 mg/g which obtained at 40°C. Potentiometric titration and Fourier transform infra-red spectroscopy were applied to reveal the mechanisms of the As(III) binding. Amino, amide, amine group and C–H of the alkane are determined to be involved in As(III) binding based on the results.     

A critical assessment of the use of lipophilic cations as membrane potential probes

A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP⁺) as membrane potential probes in prokaryotes, uekaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials of lipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand in valid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.     

Effects of lipophilic cations on motility and other physiological properties of Bacillus subtilis.

Lipophilic cations (tetraphenylarsonium, tetraphenylphosphonium, and triphenylmethylphosphonium) caused a number of major changes in the physiology of Bacillus subtilis. Macromolecular synthesis was inhibited, adenosine 5'-triphosphate concentration increased, swimming speed was reduced, tumbling was suppressed, and the capacity to take up the cations was greatly enhanced; respiration was not significantly altered. The effects occurred at lipophilic cation concentrations in the range commonly employed for measurement of membrane potential. Neither the enhancement of cation uptake nor the motility inhibition was a consequence of alteration of membrane potential, since both effects were still seen in the presence of valinomycin, with the extent of 86Rb+ uptake indicating a constant potential. Because suppression of tumbling accompanied speed reduction, as has also been found when protonmotive force is reduced, it is likely that lipophilic cations are perturbing the process of conversion of proton energy into work, rather than simply causing structural damage.