Estimation of transmembrane potentials from phase equilibria of hydrophobic paramagnetic ions.

Positively charged hydrophobic spin labels have been synthesized which respond to transmembrane potentials in sonicated liposomes. Electron paramagnetic resonance spectroscopy is used to show that the distribution of these probes between aqueous and membrane phases changes as a function of transmembrane potential. When liposomes are made more inside-negative, the fraction of membrane associated probe increases while the fraction of probe in the aqueous phase decreases. The results are in quantitative agreement with a simple equilibrium thermodynamic theory which allows estimation of absolute transmembrane potentials in phospholipid vesicles.     

Study of intervesicular phospholipid exchange by NMR

NMR spectroscopy with the use of non-penetrating paramagnetic probes permits in situ determination of the composition of the outer surface of phospholipid vesicles. The method was employed to follow the phospholipid exchange between phosphatidylinositol and phosphatidylcholine vesicles induced by a postmicrosomal protein fraction from rat liver. The effects of these proteins on the lipid bilayer and the structure of the vesicles produced by exchange were studied.     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

Effect of lysophosphatidylcholine on the surface hydration of phospholipid vesicles

The interfacial properties of the negatively charged dimyristoyl-phosphatidylglycerol (DMPG) and the zwitterionic dimyristoyl-phosphatidylcholine (DMPC) vesicles mixed with the fusion inhibitor lysomyristoylphosphatidylcholine (LMPC) are investigated by electron paramagnetic resonance (EPR). At 35 degrees C, addition of 20 mol% of LMPC to the DMPG vesicles increases the effective concentration of water in the interfacial layer of DMPG vesicles from 19.3 M to 27.7 M, whereas in the case of mixed DMPC-LMPC vesicle the effective water concentration in the interfacial layer of DMPC vesicles only changes from 15.1 M to 18.4 M. The hydrogen bonding structure in both mixed DMPG-LMPC and mixed DMPC-LMPC vesicles becomes stronger with an increasing fraction of LMPC in the vesicles. The average area per phospholipid decreases in mixed DMPC-LMPC vesicles, while it increases in mixed DMPG-LMPC vesicles as the proportion of LMPC in the vesicle increases. The inhibitory nature of LMPC in both vesicle and biological fusion comes from the increase in surface hydration, as well as from the dynamic cone shape of LMPC in the phospholipid bilayer.     

The association of D-glucose with unilamellar phospholipid vesicles

The equilibrium uptake of hydrophilic solutes, D-glucose and L-carnitine, by large unilamellar phospholipid vesicles composed of egg lecithin (PC), phosphatidic acid (PA), and various concentrations of cholesterol (Chol) has been measured. Calculation of the encapsulated volume of PC-PA and PC-PA-Chol vesicles, based on electron-microscopy data, agreed with the values directly measured by fluorescence techniques. Likewise, vesicle surface areas determined directly and from electron microscopy were in good agreement. Equilibrium uptake experiments by these well-characterized vesicles showed that glucose was taken up in excess of that amount predicted on the basis of the encapsulated aqueous volume. In contrast, the equilibrium uptake of carnitine can be predicted solely on the basis of the vesicle encapsulated volume. Each excess glucose molecule was found to be associated with from 7 to 5200 phospholipid molecules for 100 and 0.1 mM glucose, respectively. Uptake of glucose by PC-PA-Chol vesicles is independent of the cholesterol concentration and is similar to that observed in PC-PA vesicles. The cholesterol concentration independence and oil/buffer partitioning studies with octane and octanol, coupled with previous studies, strongly suggest that excess glucose is located in the vicinity of the phospholipid head group. A probable mechanism would have phospholipid, water and glucose all involved in the interaction rather than a competition between water and glucose for the phospholipid surface, as has been suggested in the literature.     

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.     

Interaction of the membrane-bound D-lactate dehydrogenase of Escherichia coli with phospholipid vesicles and reconstitution of activity using a spin-labeled fatty acid as an electron acceptor: a magnetic resonance and biochemical study.

The interaction with phospholipid vesicles of the membrane-bound respiratory enzyme D-lactate dehydrogenase of Escherichia coli has been studied. Proteolytic digestion studies show that D-lactate dehydrogenase is protected from trypsin digestion to a larger extent when it interacts with phosphatidylglycerol than with phosphatidylcholine vesicles. Wild-type D-lactate dehydrogenase and mutants in which an additional tryptophan is substituted in selected areas by site-specific oligonucleotide-directed mutagenesis have been labeled with 5-fluorotryptophan. 19F nuclear magnetic resonance studies of the interaction of these labeled enzymes with small unilamellar phospholipid vesicles show that Trp 243, 340, and 361 are exposed to the lipid phase, while Trp 384, 407, and 567 are accessible to the external aqueous phase. Reconstitution of enzymatic activity in phospholipid vesicles has been studied by adding enzyme and substrate to phospholipid vesicles containing a spin-labeled fatty acid as an electron acceptor. The reduction of the doxyl group of the spin-labeled fatty acid has been monitored indirectly by nuclear magnetic resonance and directly by electron paramagnetic resonance. These results indicate that an artificial electron-transfer system can be created by mixing D-lactate dehydrogenase and D-lactate together with phospholipid vesicles containing spin-labeled fatty acids.     

Effect of lysophosphatidylcholine on the surface hydration of phospholipid vesicles

The interfacial properties of the negatively charged dimyristoyl-phosphatidylglycerol (DMPG) and the zwitterionic dimyristoyl-phosphatidylcholine (DMPC) vesicles mixed with the fusion inhibitor lysomyristoylphosphatidylcholine (LMPC) are investigated by electron paramagnetic resonance (EPR). At 35 °C, addition of 20 mol% of LMPC to the DMPG vesicles increases the effective concentration of water in the interfacial layer of DMPG vesicles from 19.3 M to 27.7 M, whereas in the case of mixed DMPC-LMPC vesicle the effective water concentration in the interfacial layer of DMPC vesicles only changes from 15.1 M to 18.4 M. The hydrogen bonding structure in both mixed DMPG-LMPC and mixed DMPC-LMPC vesicles becomes stronger with an increasing fraction of LMPC in the vesicles. The average area per phospholipid decreases in mixed DMPC-LMPC vesicles, while it increases in mixed DMPG-LMPC vesicles as the proportion of LMPC in the vesicle increases. The inhibitory nature of LMPC in both vesicle and biological fusion comes from the increase in surface hydration, as well as from the dynamic cone shape of LMPC in the phospholipid bilayer.     

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.     

The distribution of oleic acid between chylomicron-like emulsions, phospholipid bilayers, and serum albumin. A model for fatty acid distribution between lipoproteins, membranes, and albumin

In the process of lipoprotein lipolysis, masses of fatty acid are generated at the surface of the lipoprotein. The newly generated fatty acid may at least partly redistribute from the site of lipolysis to phospholipid-rich membranes and to albumin. We have studied the distribution of [1-13C]oleic acid in model systems consisting of chylomicron-like triacylglycerol-rich emulsions, unilamellar phosphatidylcholine vesicles, and bovine serum albumin. By using high resolution 13C NMR spectroscopy it was possible to distinguish fatty acid in each compartment (emulsion, vesicle, albumin) and quantitate the fatty acid distribution under various conditions of lipid compartment concentration and aqueous pH. When emulsions and vesicles were present in equivalent mass amounts, fatty acid exhibited a profound preference for the lipid bilayers. The release of oleic acid to phospholipid bilayers was presumably also a function of its high molar stoichiometry (5:1) with the albumin present. More equitable distributions of fatty acid between vesicles and emulsions were seen when higher concentrations of emulsion were used. The distribution of fatty acid between compartments was in good agreement with predictions made using the apparent ionization constant, expressed as pKapp, of 7.5 and the surface to core (phospholipid to triacylglycerol) distribution coefficient of 7.0, measured for unionized oleic acid in chylomicron particles (Spooner, P. J. R., Bennett Clark, S., Gantz, D. L., Hamilton, J. A., and Small, D. M. (1988) J. Biol. Chem. 263, 1444-1455). These results indicate that the affinities of fatty acid for phospholipid bilayer and chylomicron-like emulsion surfaces are equivalent. Redistribution of lipolytically generated fatty acid from chylomicron surface to cell membrane may simply be driven by the predominant quantity of the cell membrane surfaces.     

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.     

II. Phospholipid exchange and size enlargement in sonicated vesicles induced by a ‘critical’ fatty acid concentration

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level (‘critical’ concentration). This ‘critical’ concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement.Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidylcholine vesicles increased abruptly above a ‘critical’ fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the ‘critical’ concentration. The phospholipid exchange occurred both in fluid- and in solid-state vesicles. The ‘critical’ fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process.The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.     

Interaction of lipid vesicles with monomolecular layers containing lung surfactant proteins SP-B or SP-C

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air-fluid interface of the lung. The Wilhelmy plate method was used to study the involvement of SP-B and SP-C in the formation of phospholipid monolayers. The proteins were either present in the phospholipid vesicles which were injected into the subphase or included in a preformed phospholipid monolayer. In agreement with earlier investigators, we found that SP-B and SP-C, present in phospholipid vesicles, were able to induce the formation of a monolayer, as became apparent by an increase in surface pressure. However, when the proteins were present in a preformed phospholipid monolayer (20 mN/m) at similar lipid to protein ratios, the rate of surface pressure increase after injection of pure phospholipid vesicles into the subphase at similar vesicle concentrations was 10 times higher. The process of phospholipid insertion from phospholipid vesicles into the protein-containing monolayers was dependent on (1) the presence of (divalent) cations, (2) the phospholipid concentration in the subphase, (3) the size of the phospholipid vesicles, (4) the protein concentration in the preformed monolayer, and (5) the initial surface pressure at which the monolayers were formed. Both in vesicles and in preformed monolayers, SP-C was less active than SP-B in promoting the formation of a phospholipid monolayer. The use of preformed monolayers containing controlled protein concentrations may allow more detailed studies on the mechanism by which the proteins enhance phospholipid monolayer formation from vesicles.     

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles

The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.     

Synthesis and characterization of a new fluorescent phospholipid

A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2′-thio-1′-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and β-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.     

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.     

Precision parameters from spin-probe studies of membranes using a partitioning technique. Application to two model membrane vesicles

A new version of the ESR spin probe partitioning method is developed and applied to the study of hydration properties of dimyristoyl-phosphatidylglycerol (DMPG) and dimyristoyl-phosphatidylcholine (DMPC) vesicles as functions of salt concentration and temperature above the lipid phase transition. The small spin probe di-tert-butyl nitroxide (DTBN) is used in order to achieve motionally narrowed Electron Spin Resonance (ESR) spectra which may be analyzed with high precision. The new method relies on the use of the second harmonic display of the ESR spectrum followed by spectral line fitting. Spectral fitting yields precise ESR parameters giving detailed information on the surroundings of the spin probe in both phospholipid and aqueous phases. The nitrogen hyperfine coupling constant of DTBN arising from those probes occupying the vesicles is used to study the hydration of the vesicle surface. The hydration properties of the negatively charged vesicle surface of DMPG vesicles are affected by the addition of salt at all temperatures. In contrast, the hydration of DMPC vesicles does not change with salt concentration at the low temperatures. However, at higher temperatures the hydration properties of DMPC vesicle are affected by salt which is interpreted to be due to the faster motion of the phospholipid molecules. The partitioning of the spin probe increases with salt concentration for both DMPG and DMPC vesicles, while water penetration decreases simultaneously. The spin probe in the phospholipid bilayer exhibits anisotropic motion and the extent of the anisotropy is increased at the higher salt concentrations.     

Effect of succinylphosphatidylcholine on phosphatidylcholine vesicles. Structural studies by gel chromatography,electron microscopy and nuclear magnetic resonance

The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn²⁺ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10–20 mM.Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine.Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical.Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215–240 Å, and an almost constant internal volume.Between 30 and 60% of succinylphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.