Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.     

Solubilization of multilamellar liposomes of egg yolk lecithin by the bile salt sodiumtaurodeoxycholate and the effect of cholesterol--a rapid-ultrafiltration study

The solubilization of multilamellar egg yolk lecithin liposomes by sodiumtaurodeoxycholate in aqueous phase was studied by ultrafiltration as a function of time, bile salt and cholesterol concentration. The corresponding equilibrium states were analysed. Complete solubilization was achieved at total bile salt/lecithin molar mixing ratios of approximately 5. The minimum ratio to start solubilization was 0.1, corresponding to a free bile salt concentration of only 5% of the critical micelle concentration (CMC). Mean equilibrium constants for the partition of bile salts between non-filterable aggregates and filterable mixed micelles and also the free bile salt concentration were determined. Sodiumtaurodeoxycholate had a higher affinity for small mixed micelles than for lamellar mixed aggregates especially in the presence of cholesterol, which reduces the degree and rate of the solubilization process. A non-homogeneous distribution of bile salts in the lipid phase was detected at low bile salt concentrations.     

Liposomes, disks, and spherical micelles: aggregate structure in mixtures of gel phase phosphatidylcholines and poly(ethylene glycol)-phospholipids

Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the 'equilibrium partition model', the dependence of the 'solubilizing detergent concentration' on the lipid concentration intersects with the lipid axis at -1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical ('solubilizing') effective ratio depends upon the nature of detergent less than any of these two factors.     

Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles

Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.     

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.     

The Mechanism of Detergent Solubilization of Lipid Bilayers

Multiple data are available on the self-assembly of mixtures of bilayer-forming amphiphiles, particularly phospholipids and micelle-forming amphiphiles, commonly denoted detergents. The structure of such mixed assemblies has been thoroughly investigated, described in phase diagrams, and theoretically rationalized in terms of the balance between the large spontaneous curvature of the curvophilic detergent and the curvophobic phospholipids. In this critical review, we discuss the mechanism of this process and try to explain the actual mechanism involved in solubilization. Interestingly, membrane solubilization by some detergents is relatively slow and the common attribute of these detergents is that their trans-bilayer movement, commonly denoted flip-flop, is very slow. Only detergents that can flip into the inner monolayer cause relatively rapid solubilization of detergent-saturated bilayers. This occurs via the following sequence of events: 1), relatively rapid penetration of detergent monomers into the outer monolayer; 2), trans-membrane equilibration of detergent monomers between the two monolayers; 3), saturation of the bilayer by detergents and consequent permeabilization of the membrane; and 4), transition of the whole bilayer to thread-like mixed micelles. When the detergent cannot flip to the inner monolayer, the outer monolayer becomes unstable due to mass imbalance between the monolayers and inclusion of the curvophilic detergent molecules in a flat surface. Consequently, the outer monolayer forms mixed micellar structures within the outer monolayer. Shedding of these micelles into the aqueous solution results in partial solubilization. The consequent leakage of detergent into the liposome results in trans-membrane equilibration of detergent and subsequent micellization through the rapid bilayer-saturation mechanism.     

Intermediate structures in the cholate-phosphatidylcholine vesicle-micelle transition

The vesicle-micelle transition of egg phosphatidylcholine (PC) and sodium cholate was described by comparing cryo-transmission electron microscopic (cryo-TEM) images of the structures formed to the associated turbidity changes. These experiments were designed to identify the morphology of the intermediates between vesicles and small spheroidal mixed micelles. With increasing cholate concentration, the vesicular structures changed size and more multilamellar vesicles were seen. Between the apparent upper and lower phase boundaries, three structures were observed: open vesicles, large bilayer sheets (twenty to several hundred nanometers in diameter), and long (150-300 nm) flexible cylindrical micelles. The cylindrical micelles evolved from the edges of the bilayer sheets. At higher relative cholate concentration, the phase boundary was sharply defined by optical clarification of the egg PC-cholate mixtures. Cryo-TEM revealed only small spheroidal mixed micelles at this transition. These results provide the first direct evidence of the structural pathway or of molecular intermediates between a lamellar and a micellar state. Understanding these specific intermediates and the transitions between them is essential to developing reconstitution protocols and properly analyzing either activity or structural data obtained from cholate-dispersed membrane proteins.     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, R_e. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the ‘equilibrium partition model’, the dependence of the ‘solubilizing detergent concentration’ on the lipid concentration intersects with the lipid axis at −1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical (‘solubilizing’) effective ratio depends upon the nature of detergent less than any of these two factors.     

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilizing effects caused by the nonionic surfactant dodecylmaltoside in phosphatidylcholine liposomes.

The interaction of the nonionic surfactant dodecylmaltoside (DM) with phosphatidylcholine liposomes was investigated. Permeability alterations were detected as a change in 5(6)-carboxyfluorescein released from the interior of vesicles and bilayer solubilization as a decrease in the static light scattered by liposome suspensions. This surfactant showed higher capacity to saturate and solubilize PC liposomes and greater affinity with these structures than those reported for the octyl glucoside. At subsolubilizing level an initial maximum in the bilayer/water partitioning (K) followed by an abrupt decrease of this parameter occurred as the effective molar ratio of surfactant to phospholipid in bilayers (Re) rose. However, at solubilizing level a direct dependence was established between both parameters. A direct correlation took place in the initial interaction steps (Re up to 0.28) between the growth of vesicles, their fluidity, and Re. A similar direct dependence was established during solubilization (Re range from 0.9 to 1.7) between the decrease in both the surfactant-PC aggregate size, the light scattering of the system, and Re (composition of aggregates). The fact that the free DM concentration at subsolubilizing and solubilizing levels showed values lower than and similar to its critical micelle concentration indicates that permeability alterations and solubilization were determined, respectively, by the action of surfactant monomer and by the formation of mixed micelles.     

Phospholipid vesicle solubilization and reconstitution by detergents. Symmetrical analysis of the two processes using octaethylene glycol mono-n-dodecyl ether

The processes of liposome solubilization and reconstitution were studied by using n-dodecyl octaethylene glycol monoether (C12E8). The solubilization of large unilamellar liposomes prepared by reverse-phase evaporation was systematically investigated by turbidity, 31P nuclear magnetic resonance, and centrifugation experiments. The solubilization process is well described by the three-stage model previously proposed for other detergents, and our results further demonstrate the validity of some of the postulates related to this model. In stage I, the detergent distributes between the bilayers and the aqueous solution with a partition coefficient of 1.6 mM-1. In stage II, the detergent-saturated liposomes convert into mixed micelles, the conversion being complete by stage III where all the phospholipids are present as mixed micelles. The agreement between the three methods was excellent, and the results allowed quantitative determination of the effective detergent to phospholipid ratios at which the lamellar to micellar transformation begins and is complete, which amounted to 0.66 and 2.2 (mol/mol), respectively. Furthermore, compositional analysis determined from centrifugation experiments directly demonstrate that the properties of detergent-saturated liposomes and mixed micelles remain constant throughout most of stage II: the C12E8 to phospholipid ratios in the pelleted vesicles and in micelles are constant during stage II and similar to the ratios at which stage II was initiated and complete, respectively. On the other hand, bilayer formation upon detergent removal from mixed C12E8-phospholipid micelles by SM2 Bio-Beads is demonstrated to be the symmetrical opposite of bilayer solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of amphiphilic surfactant LDAO on the solubilization of DOPC vesicles and on the activity of Ca(2+)-ATPase reconstituted in DOPC vesicles

Solubilization of large unilamellar 1,2-dioleoylphosphatidylcholine (DOPC) vesicles by N-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was studied using turbidimetry. From turbidity data, the LDAO partition coefficient between the aqueous phase and DOPC bilayers was obtained. Using this partition coefficient, the LDAO:DOPC molar ratio in the bilayer was calculated and effects of LDAO on the bilayer stability, bilayer thickness and on the phosphohydrolase activity of sarcoplasmic reticulum Ca(2+) transporting ATPase (SERCA) reconstituted into DOPC were compared at the same LDAO:DOPC molar ratios in the bilayer. The sequence "bilayers in vesicles - bilayer fragments (flat mixed micelles) - tubular mixed micelles - globular mixed micelles" was suggested for the solubilization mechanism of DOPC vesicles from the combined turbidimetric and small-angle neutron scattering (SANS) results. The effective molecular packing parameter delta = 0.5, corresponding to the mixed bilayer - mixed tubular micelle transition, was calculated from fragmental DOPC and LDAO volumes at the molar ratio LDAO:DOPC = 2.00 in bilayers, in the middle of transition region observed earlier experimentally by small-angle neutron scattering (SANS). The bilayer thickness decrease induced by LDAO in DOPC observed by SANS did not result in the SERCA phosphohydrolase activity decrease and this indicates that some other factors compensated this bilayer effect of LDAO. The ATPase activity decrease at higher LDAO concentrations was caused by the bilayer deformation. This deformation resulted in the formation of non-bilayer aggregates in LDAO+DOPC system.     

Formation of supported phospholipid bilayers via co-adsorption with beta-D-dodecyl maltoside

We have investigated the formation of supported model membranes via the adsorption of phospholipid-surfactant mixtures at the Si-water interface by specular neutron reflection. The adsorption of mixed micelles of the nonionic surfactant beta-D-dodecyl maltoside and DOPC or POPC was determined as a function of bulk concentration, and using d25-beta-D-dodecyl maltoside, the composition of DOPC and POPC bilayers was determined. Bilayer thicknesses of 39+/-3 A for DOPC and 41+/-3 A for POPC agree well with data from bulk lamellar phases for both lipids, and the average area per lipid molecule can be varied from 62 to 115 A2 by varying the bulk concentrations used. The amount of surfactant in the bilayer is very sensitive to the bulk volume-to-surface area ratio, but it can be fully eliminated by ensuring a sufficiently large dilution/rinsing volume of the solution.     

Short-chain phospholipids as detergents

The physico-chemical properties of short-chain phosphatidylcholine are reviewed to the extent that its biological activity as a mild detergent can be rationalized. Long-chain diacylphosphatidylcholines are typical membrane phospholipids that form preferentially smectic lamellar phases (bilayers) when dispersed in water. In contrast, the preferred phase of the short-chain analogues dispersed in excess water is the micellar phase. The preferred conformation and the dynamics of short-chain phosphatidylcholines in the monomeric and micellar state present in H(2)O are discussed. The motionally averaged conformation of short-chain phosphatidylcholines is then compared to the single-crystal structures of membrane lipids. The main conclusion emerging is that in terms of preferred conformation and motional averaging short-chain phosphatidylcholines closely resemble their long-chain analogues. The dispersing power of short-chain phospholipids is emphasized in the second part of the review. Evidence is presented to show that this class of compounds is superior to most other detergents used in the solubilization of membrane proteins and the reconstitution of the solubilized proteins to artificial membrane systems (proteoliposomes). The prominent feature of the solubilization/reconstitution of integral membrane proteins by short-chain PC is the retention of the native protein structure and hence the protein function. Due to their special detergent-like properties, short-chain PC lend themselves very well not only to membrane solubilization but also to the purification of integral membrane proteins. The retention of the native protein structure in the solubilized state, i.e. in mixed micelles consisting of the integral membrane protein, intrinsic membrane lipids and short-chain PC, is rationalized. It is hypothesized that short-chain PC interacts primarily with the lipid bilayer of a membrane and very little if at all with the membrane proteins. In this way, the membrane protein remains associated with its preferred intrinsic membrane lipids and retains its native structure and its function.     

Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate

The mechanisms governing the solubilization by Triton X-100, octyl glucoside, and sodium cholate of large unilamellar liposomes prepared by reverse-phase evaporation were investigated. The solubilization process is described by the three-stage model previously proposed for these detergents [Lichtenberg, D., Robson, R.J., & Dennis, E.A.(1983) Biochim. Biophys. Acta 737, 285-304]. In stage I, detergent monomers are incorporated into the phospholipid bilayers until they saturate the liposomes. At that point, i.e., stage II, mixed phospholipid-detergent micelles begin to form. By stage III, the lamellar to micellar transition is complete and all the phospholipids are present as mixed micelles. The turbidity of liposome preparations was systematically measured as a function of the amount of detergent added for a wide range of phospholipid concentrations (from 0.25 to 20 mM phospholipid). The results allowed a quantitative determination of RSat, the effective detergent to lipid molar ratios in the saturated liposomes, which were 0.64, 1.3, and 0.30 for Triton X-100, octyl glucoside, and sodium cholate, respectively. The corresponding ratios in the mixed micelles, RSol, were 2.5, 3.8, and 0.9 mol of detergent/mol of phospholipid. The monomer concentrations of the three detergents in the aqueous phase were also determined at the lamellar to micellar transitions (0.18, 17, and 2.8 mM, respectively). These transitions were also investigated by 31P NMR spectroscopy, and complete agreement was found with turbidity measurements. Freeze-fracture electron microscopy and permeability studies in the sublytic range of detergent concentrations indicated that during stage I of solubilization detergent partitioning between the aqueous phase and the lipid bilayer greatly affects the basic permeability of the liposomes without significantly changing the morphology of the preparations. A rough approximation of the partition coefficients was derived from the turbidity and permeability data (K = 3.5, 0.09, and 0.11 mM-1 for Triton X-100, octyl glucoside, and sodium cholate, respectively). It is concluded that when performed systematically, turbidity measurements constitute a very convenient and powerful technique for the quantitative study of the liposome solubilization process by detergents.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

Interaction of nonionic PEO-PPO diblock copolymers with lipid bilayers

The relationship between the molecular architecture of a series of poly(ethylene oxide)-b-poly(propylene oxide) (PEO-PPO) diblock copolymers and the nature of their interactions with lipid bilayers has been studied using small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC). The number of molecular repeat units in the hydrophobic PPO block has been found to be a critical determinant of the nature of diblock copolymer-lipid bilayer association. For dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-based biomembrane structures, polymers whose PPO chain length approximates that of the acyl chains of the lipid bilayer yield highly ordered, expanded lamellar structures consistent with well-integrated (into the lipid bilayer) PPO blocks. Shorter diblock copolymers produce mixed lamellar and nonlamellar mesophases. The thermotropic phase behavior of the polymer-doped membrane systems is highly influenced by the presence and molecular architecture of the diblock copolymer, as evidenced by shifting of the main phase transition to higher temperatures, broadening of the main transition, and the appearance of other features. Studies of temperature-induced changes in the mesophase structure for compositions prepared with well-integrated PEO-PPO polymers indicate that they undergo reversible changes to a nonlamellar structure as the temperature is lowered. Increasing either the number of repeat units in the PEO block or the polymer concentration promotes a greater degree of structural ordering.