Dynamic excitations in membranes induced by optical tweezers

We present the phenomenology of transformations in lipid bilayers that are excited by laser tweezers. A variety of dynamic instabilities and shape transformations are observed, including the pearling instability, expulsion of vesicles, and more exotic ones, such as the formation of passages. Our physical picture of the laser-membrane interaction is based on the generation of tension in the bilayer and loss of surface area. Although tension is the origin of the pearling instability, it does not suffice to explain expulsion of vesicles, where we observe opening of giant pores and creeping motion of bilayers. We present a quantitative theoretical framework to understand most of the observed phenomenology. The main hypothesis is that lipid is pulled into the optical trap by the familiar dielectric effect, is disrupted, and finally is repackaged into an optically unresolvable suspension of colloidal particles. This suspension, in turn, can produce osmotic pressure and depletion forces, driving the observed transformations.     

Transport of long-chain native fatty acids across lipid bilayer membranes indicates that transbilayer flip-flop is rate limiting

Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, studies using lipid membranes indicate that FA are capable of spontaneous flip-flip, raising the possibility that rapid transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA across lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid membranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previously. In this study, we have used the fluorescent probe ADIFAB to monitor the time course of FA movement from the outer to the inner aqueous compartments and from the lipid membrane to the outer aqueous compartment of lipid vesicles. These two measurements, together with measurements of the lipid:aqueous partition coefficients, allowed the determination of the rate constants for binding (kon), flip-flop (kff), and dissociation (koff) for the transport of long-chain natural FA across lipid vesicles. These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 A diameter, prepared by extrusion and giant unilamellar vesicles (GUV), prepared by detergent dialysis, that are >/=2000 A diameter. The results of these studies for vesicles composed of egg phosphatidylcholine (EPC) and cholesterol reveal kff values that range from 3 to 15 s-1 for LUV and from 0.1 to 1.0 s-1 for GUV, depending upon temperature and FA type. For these same vesicles, dissociation rate constants range from 4 to 40 s-1 for LUV and from 0.3 to 2.5 s-1 for GUV. In all instances, the rate constant for flip-flop is smaller than koff, and because the rate of binding is greater than the rate of transport, we conclude that flip-flop is the rate-limiting step for transport. These results demonstrate that (1) kff and koff are smaller for GUV than for LUV, (2) the rate constants increase with FA type according to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Comparison of the flip-flop rates determined for GUV with values estimated from previously reported metabolic rates for cardiac myocytes, raises the possibility that flip-flop across the lipid phase alone may not be able to support metabolic requirements.     

Quantitative studies on the melittin-induced leakage mechanism of lipid vesicles

We have investigated, both experimentally and theoretically, the efflux of carboxyfluorescein (a self-quenching fluorescent dye) from vesicles of different sizes and lipid species (POPC, DOPC) after having added the bee venom peptide melittin. This comprises quantitative analyses regarding the extent of lipid-associated peptide, the mode as well as the temporal progress of dye release and the possible leakage mechanism. Our results indicate a graded efflux characterized by a single-pore retention factor reflecting the formation of pores whose lifetimes are rather small (millisecond range). The observed fluorescence signal arising from the dequenching of effluent dye has been converted to the number of pore openings over the course of time. All the resulting curves exhibit a pronounced slowing down of the pore formation rate revealing two distinct relaxation steps at about 20 and 200 s, respectively, being largely independent of vesicle type and peptide to lipid ratio. The pore formation rate itself increases in proportion to the amount of membrane bound peptide. We give a quantitative account of our experimental findings based on a novel reaction scheme applicable to any of our various liposome systems. It implies that the pore formation rate is controlled by a passage through two intermediate monomeric peptide states. These states are thought to become well populated in the initial stage of lipid bilayer perturbation, but would practically die out after some time owing to a restabilization of the membrane system.     

The formation and annealing of structural defects in lipid bilayer vesicles

It is shown that sonication of phospholipid-water dispersions below the crystalline leads to liquid crystalline phase transition temperature (Tc) produces bilayer vesicles with structural defects within the bilayer membrane, which permit rapid permeation of ions and catalyze vesicle-vesicle fusion. These structural defects are annihilated simply by annealing the vesicle suspension above Tc. The rate of annealing was found to be slow, of the order of an hour for T = 3 degrees C above Tc, but annealing is complete within 10 min for T = 10 degrees C above Tc. It is proposed that these structural defects are fault-dislocations in the bilayer structure, which arise from a population defect in the distribution of the lipid molecules between the outer and inner monolayers, when small bilayer fragments reassemble to form the small bilayer vesicles during the sonication procedure. Such a population defect can only be remedied by lipid transport via the inside in equilibrium outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 degrees C above the phase transition.     

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).     

Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP

The gene encoding the secondary multidrug transporter LmrP of Lactococcus lactis was heterologously expressed in Escherichia coli. The energetics and mechanism of drug extrusion mediated by LmrP were studied in membrane vesicles of E. coli. LmrP-mediated extrusion of tetraphenyl phosphonium (TPP+) from right-side-out membrane vesicles and uptake of the fluorescent membrane probe 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) into inside-out membrane vesicles are driven by the membrane potential (Deltapsi) and the transmembrane proton gradient (DeltapH), pointing to an electrogenic drug/proton antiport mechanism. Ethidium bromide, a substrate for LmrP, inhibited the LmrP-mediated TPP+ extrusion from right-side-out membrane vesicles, showing that LmrP is capable of transporting structurally unrelated drugs. Kinetic analysis of LmrP-mediated TMA-DPH transport revealed a direct relation between the transport rate and the amount of TMA-DPH associated with the cytoplasmic leaflet of the lipid bilayer. This observation indicates that drugs are extruded from the inner leaflet of the cytoplasmic membrane into the external medium. This is the first report that shows that drug extrusion by a secondary multidrug resistance (MDR) transporter occurs by a "hydrophobic vacuum cleaner" mechanism in a similar way as was proposed for the primary lactococcal MDR transporter, LmrA.     

Cryo-transmission electron microscopy of a superstructure of fluid dioleoylphosphatidylcholine (DOPC) membranes

Using cryo-transmission electron microscopy, we have obtained abundant and reproducible evidence for a superstructure of dioleoylphosphatidylcholine (DOPC) bilayers. Dispersions of vesicles were prepared by gentle shaking of a 2% suspension of DOPC in water followed in part by extrusion through a porous technical membrane. Sampling and cryofixation took place at various times within 3 weeks after the preparation. From the micrographs we infer that the small fraction of vesicles enclosing one another develop passages (connections) between the bilayers. In contrast, the superstructure is basically a feature of disconnected membranes. Among its modifications are isolated membrane bends or folds and a grainy membrane texture with a minimal grain spacing of 4-6 nm. In the extruded dispersions the passages and the superstructure seem to be formed mostly within the first day. The fraction of smooth and unilamellar vesicles is large at all times and in all dispersions.     

A conformational model for the action of general anesthetics at the membrane level. II. Experimental observations on the effects of anesthetics on lipid fluidity and lipid protein interactions

We have investigated the effect of general anesthetics (the normal alcohol series up to pentanol, halothane, pentrane, ether, chloroform, and ketamine) on lipid fluidity of phospholipid vesicles and mitochondrial and erythrocyte membranes by using spin labels and fluorescent probes. The spin labels used (5- and 16-doxyl stearic acids) show that all anesthetics tested have a slight fluidizing effect on lipid vesicles but induce a very strong increase in mobility of spin labels in mitochondria and lower in erythrocyte ghosts. These results are interpreted as a labilization of lipid protein interactions at all depths in the bilayer. The fluorescent molecules ANS and NPN, which probe the glycerol region and the core of the bilayer respectively, show a decrease of fluorescence induced by alcohols, halothane, ether, chloroform in both lipid vesicles and membranes. The decrease of fluorescence is due to decreased quantum yield as shown by double reciprocal plots of probe fluorescence against membrane concentration. The fluorescence decrease is interpreted mainly as an increase in fluidity of the lipid bilayer and not as an increase of polarity of the probe environment. The effect of ketamine is that of fluidization in the bilayer core (NPN) but of increased rigidity in the glycerol region (ANS) perhaps due to the amphipathic character of this anesthetic, that is supposed to bind in the polar region of the bilayer. Pentrane also induces fluidization in the bilayer core (NPN) but has a peculiar effect near the surface (ANS): in lipid vesicles it induces a fluorescence decrease, whereas an increase is seen in mitochondrial membranes. These complex effects are considered as the result of some specific change in the lipid protein interactions in the region probed by ANS. The effects of anesthetics on maximal NPN fluorescence (Fo) have been usually found to be stronger in mitochondrial membranes than in lipid vesicles, thus confirming the results of the spin label studies, showing a labilization of lipid protein interactions induced by anesthetics. The effects on Fo of ANS, however, appear to be stronger in lipid vesicles than in membranes. These findings indicate that the presence of the proteins counteracts the perturbation induced by anesthetics at the level of the membrane surface, in contrast with the disruption of lipid protein interactions observed in the membrane hydrophobic areas.     

Structural studies of polymer-cushioned lipid bilayers

The structure of softly supported polymer-cushioned lipid bilayers, prepared in two different ways at the quartz-solution interface, were determined using neutron reflectometry. The polymer cushion consisted of a thin layer of branched, cationic polyethyleneimine (PEI), and the bilayers were formed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. When vesicles were first allowed to adsorb to a bare quartz substrate, an almost perfect bilayer formed. When the polymer was then added to the aqueous solution, it appeared to diffuse beneath this bilayer, effectively lifting it from the substrate. In contrast, if the polymer layer is adsorbed first to the bare quartz substrate followed by addition of vesicles to the solution, there is very little interaction of the vesicles with the polymer layer, and the result is a complex structure most likely consisting of patchy multilayers or adsorbed vesicles.     

Interaction of lipoprotein lipase with homogeneous lipid emulsions

The central function of lipoprotein lipase (LpL) is to hydrolyze triacylglycerols in chylomicrons and very low density lipoproteins. We have examined the binding of purified milk lipoprotein lipase to homogeneous synthetic lipid emulsions. Emulsions composed of either naturally occurring ester-linked lipids or the non-hydrolyzable ether analogues were prepared by sonication and pressure extrusion, and fractionated by sucrose density gradient centrifugation. Flotation analysis using the analytical ultracentrifuge indicated that the individual fractions were relatively homogeneous with respect to size with flotation coefficients and molecular weights for the separated fractions ranging from 100 to 1100 S and 5.2 x 10(7) to 6.0 x 10(8), respectively. Purified milk lipoprotein lipase bound with high affinity and in a saturable manner to emulsions prepared from the non-hydrolyzable ether-linked lipid analogues of 1-oleoyl, 2-palmitoyl phosphatidylcholine and triolein. At low concentrations of LpL, the enzyme caused aggregation of the emulsion particles by interparticle cross-linking. At higher LpL concentrations, the flotation coefficient of the emulsions decreased significantly with a concomitant increase in particle density. At saturation, the number of LpL monomers bound to lipid particles of radii 67, 75, and 79 nm was 1315, 1449, and 1466, respectively. The results demonstrate close packing of LpL on the lipid surface and are consistent with there being little disruption to the overall structure of the emulsion particle.     

Anomalies of nanosecond ultrasonic relaxation in the lipid bilayer transition

The frequency dependence of ultrasonic velocity as well as absorption in a suspension of sonicated dipalmitoylphosphatidylcholine vesicles was measured by a differential ultrasonic resonator. The frequency was scanned between 1.3 and 13 MHz and the temperature was varied from 25 to 47 degrees C. A pronounced relaxation was observed in the time range of 10 ns. The data were analyzed assuming a single relaxation which appeared to be a good approximation. The relaxation time as well as relaxation strength increased anomalously in the vicinity of the gel-to-liquid crystal transition of 41.5 degrees C. This result represents the first definite evidence of the critical slowing down in the lipid bilayer and is discussed in terms of the Landau theory of phase transition. The possible biological significance of the mechanical relaxation is also presented.     

Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein

The gene for the microsomal triglyceride transfer protein (MTP) is defective in subjects with the genetic disease abetalipoproteinemia, indicating that MTP is essential for the assembly of apolipoprotein B containing lipoproteins. In vitro, MTP is a lipid molecule binding protein that catalyzes lipid transport between membranes by a shuttle mechanism. In this study, the lipid binding properties of MTP were examined. MTP was incubated with donor phosphatidylcholine vesicles of varying neutral lipid composition. MTP was subsequently reisolated by ultracentrifugation, and MTP-bound lipid was quantitated. When the triolein content of the vesicles was increased up to 4 mol %, neutral lipid binding to MTP increased proportionately, while phosphatidylcholine binding appeared to remain constant around two molecules per MTP. Using phosphatidylcholine emulsions containing 60 mol % triolein as the donor particles resulted in only a slight increase in triolein binding to MTP. The highest triolein:MTP ratio observed was (0.20-0.25):1. Differences in the neutral and phospholipid binding properties of MTP were observed by measuring the transport of lipid from MTP to acceptor vesicles. Transport of triolein was rapid and complete, while phosphatidylcholine transport was biphasic, containing rapid and slow phases. These results indicated that MTP contains more than one class of lipid molecule binding site. Measurements of fluorescent lipid transport from donor vesicles to MTP supported this hypothesis. The transport of pyrene-labeled triglyceride from donor particles to MTP was rapid, while phosphatidylcholine transfer had fast and slow phases. From these data, we propose that MTP contains at least two distinct classes of lipid molecule binding sites that differ in function. The fast site or sites are responsible for lipid transport.     

Binding of pertussis toxin to lipid vesicles containing glycolipids

The binding of pertussis toxin and its B oligomer to lipid vesicles containing glycosphingolipids was studied. Both pertussis toxin and the B oligomer bound to lipid vesicles containing ganglioside GD1a. Binding of pertussis toxin to these vesicles decreased upon treatment of the vesicles with neuraminidase, suggesting that sialic acid residues are important for efficient binding of the toxin to GD1a.     

Inhibitory effect of ethanolamine plasmalogen on iron- and copper-dependent lipid peroxidation

The effect of ethanolamine plasmalogen (EtnPm) on lipid peroxidation was investigated in liposomal suspension of egg yolk phosphatidylcholine. EtnPm inhibited iron- and copper-dependent peroxidation in the presence of preformed hydroperoxides, although it was not effective for radical initiator mediated lipid peroxidation. EtnPm resulted in complete binding of iron to liposomal lipids, suggesting that EtnPm exerts its inhibitory effect on lipid peroxidation through inhibiting preformed peroxide decomposition by trapping transition metal ions.     

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.     

Interaction of wheat alpha-thionin with large unilamellar vesicles

The interaction of the wheat antibacterial peptide alpha-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat alpha-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethylene glycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-aminonaphthalene-1,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.     

Erythrocyte hemolysis by detergents

The numbers of Triton X-100 and sodium dodecyl sulfate molecules required to form respective pores were estimated from the relationship between the detergent concentrations and the rates of fast and slow hemolysis components. It has been found that the slow hemolysis component evoked by Triton X-100 is related to the existence of two different pores. It is shown that the fast hemolysis component induced by sodium dodecyl sulfate is associated with the modification of phosphatidylcholine which determines the break in the Arrhenius plots of the hemolysis rate within the range of 20 degrees C. The shape of hemolysis kinetic curves and the dependence of hemolytic parameters on the detergent concentration and temperature are discussed based on the concept of hemolysis caused by the formation of pores in various membrane lipid regions and by releasing vesicles from erythrocytes.     

Nitric oxide and lipid peroxidation

Nitric oxide (NO) is a free radical produced enzymatically in biological systems from the guanidino group of L-arginine. Its large spectrum of biological effects is achieved through chemical interactions with different targets including oxygen (O2), superoxide (O2o-) and other oxygen reactive species (ROS), transition metals and thiols. Superoxide anions and other ROS have been reported to react with NO to produce peroxynitrite anions that can decompose to form nitrogen dioxide (NO2) and hydroxyl radial (OHo). Thus, NO has been reported to have a dual effect on lipid peroxidation (prooxidant via the peroxynitrite or antioxydant via the chelation of ROS). In the present study we have investigated in different models the in vitro and in vivo action of NO on lipid peroxidation. Copper-induced LDL oxidation were used as an in vitro model. Human LDL (100 micrograms ApoB/ml) were incubated in oxygene-saturated PBS buffer in presence or absence of Cu2+ (2.5 microM) with increasing concentrations of NO donnors (sodium nitroprussiate or nitroso-glutathione). LDL oxidation was monitored continuously for conjugated diene formation (234 nm) and 4-hydroxynonenal (HNE) accumulation. Exogenous NO prevents in a dose dependent manner the progress of copper-induced oxidation. Ischaemia-reperfusion injury (I/R), characterized by an overproduction of ROS, is used as an in vivo model. Anaesthetized rats were submitted to 1 hour renal ischaemia following by 2 hours of reperfusion. Sham-operated rats (SOP) were used as control. Lipid peroxidation was evaluated by measuring the HNE accumulated in rats kidneys in presence or absence of L-arginine or D-arginine infusion. L-arginine, but not D-arginine, enhances HNE accumulation in I/R but not in SOP (< 0.050 pmol/g tissue in SOP versus 0.6 nmol/g tissue in I/R), showing that, in this experimental conditions, NO produced from L-arginine, enhances the toxicity of ROS. This study shows that the pro- or antioxydant effects of NO are different in vivo and in vitro and could be driven by environmental conditions such as pH, relative concentrations of NO and ROS, ferryl species.     

Entropy-driven binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles

This study employed large unilamillar vesicles composed of purchased stratum corneum lipids to investigate the binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles. The partition coefficients of amino acids/dipeptides between the stratum corneum lipid vesicles and the acetate buffer were determined by HPLC. In addition, the binding/partition enthalpy of amino acids/dipeptides with the stratum corneum lipid vesicles was derived by directly measuring the binding/partition heat with isothermal titration calorimetry. According to the binding/petition Gibbs free energy and the binding/partition enthalpy, all the binding/partition of amino acids/dipeptides with the stratum corneum lipid vesicles is endothermic, implying an entropy-driven binding/partition. Also, the equilibrium binding/partition results demonstrate that the partition coefficients of amino acids/dipeptides do not correlate with the transdermal permeability. This finding suggests that either the interaction between the penetrants and the lipid bilayer between corneocytes may not be a determining step or that the paracellular path is not a dominant route of transdermal penetration.     

External surface and lamellarity of lipid vesicles: a practice-oriented set of assay methods

Three methods are presented for the determination of external surface of large lipid vesicles of different lamellarity with 2% absolute accuracy. These methods (referred to as EPR, NBD and TNBS assays) use different marker lipids which provide signals (electron paramagnetic resonance, fluorescence of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) residues, and UV absorption increase of 2,4,6-trinitrobenzenesulfonic acid after reaction with aminolipids, respectively). The signals change upon addition of different membrane-impermeant reagents due to reaction with marker lipids at the external vesicle surface. They were applied to the same vesicle samples, including unilamellar and multilamellar vesicles, both at two different lipid compositions. External surface data matched for the three assays within 2%, but only after appropriate redesign or adaptation of so far published procedures. Main improvements related to slow influx of reagents (TNBS and NBD assays) or to redistribution of marker lipids (EPR assay), obscuring determination of outer vesicle surface from fast reaction between reagent and readily accessible marker lipids. Furthermore, suitable strategies were found to obtain accurate 100% values (reaction of all marker lipids present), required to relate external vesicle surface to total surface. This included corrections for light scattering (NBD assay) and for turbidity (TNBS assay). These three methods appear to close a gap in the methodology to determine external surface of vesicles for typical practical needs. In particular, the reliability range of the NBD assay could be extended to marker lipid densities as low as 1 marker lipid per 3000 lipids.