Solubilization of phospholipids by detergents. Structural and kinetic aspects

Most amphiphiles in biological membranes including phospholipids, steroids, and membrane proteins are insoluble amphiphiles and would form liquid crystals or insoluble precipitates alone in aqueous media. Detergents are soluble amphiphiles and above a critical concentration and temperature form micelles of various sizes and shapes. Much of the recent progress in studying the insoluble amphiphiles is due to the formation of thermodynamically stable isotropic solutions of these compounds in the presence of detergents. This process, which is commonly denoted as "solubilization,' involves transformation of lamellar structures into mixed micelles. The information available to date on the solubilization of phospholipids, which constitute the lipid skeleton of biomembranes, by the common detergents is discussed in this review, both with respect to the kinetics of this process and the structure of the various phospholipid-detergent mixed micelles formed. It is hoped that this discussion will lead to somewhat more useful, although still necessarily fairly empirical, approaches to the solubilization of phospholipids by detergents.     

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

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)     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.     

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.     

Detergent organisation in solutions and in crystals of membrane proteins

The use of neutron scattering in studying the organisation of detergents in pure micelles, in protein/detergent mixed micelles and in crystals of membrane proteins, is reviewed. Small angle scattering has been used to study the size, shape and composition of pure and mixed protein/detergent micelles as well as the effects of adding small amphiphiles. The technique of contrast variation applied to single crystals is described and its application to the determination of the organization of detergent in single crystals of membrane proteins is discussed. A better understanding of protein/detergent interactions should help in producing crystals of membrane proteins more easily as well as clues to the nature of protein/lipid interactions in vivo.     

Membrane proteins, lipids and detergents: not just a soap opera

Studying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problems encountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successful solubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixed lipid/detergent systems. This review will concentrate on the methods currently available for efficient reconstitution and solubilization of membrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevant molecular properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein research is retaining the stability and function of the protein during solubilization, reconstitution and crystallization. We highlight some of the lessons learnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.     

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.     

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.     

Interaction of non-classical detergents with the mitochondrial porin. A new purification procedure and characterization of the pore-forming unit

The effect of different families of detergents on the solubilization and purification of the pore-forming protein (porin) of the mitochondrial outer membrane of bovine heart was investigated in detail. With Tritons, dimethylamine oxides and zwittergents, porin solubilization with respect to total mitochondrial membrane protein was more efficient with the more hydrophobic members of each series. With most detergents the protein eluted as protein-detergent micelles in the void volume of hydroxyapatite/celite columns. In contrast, the protein was bound to the column material and was eluted after the addition of salt to the elution buffer when the detergents octylglucoside, zwittergent Z-314 and lauryl(dimethyl)-amine oxide were used. The protein purified in the presence of the latter detergent had a higher pore-forming activity in lipid bilayer membranes compared to porin isolated in the presence of Triton X-100. The binding of porin to the hydroxyapatite/celite column was used to study the lipid content of the active pore-forming complex. The analysis revealed that the complex contained no phospholipid but rather five molecules of cholesterol/polypeptide chain.     

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.     

Detergent solubilization of phosphatidylcholine bilayers in the fluid state: Influence of the acyl chain structure

Seventeen different, chemically defined phosphatidylcholines, dispersed in aqueous medium in the form of large unilamellar vesicles, have been tested for solubilization by the non-ionic detergent Triton X-100. The temperatures (either 20 °C or 45 °C) were such that the bilayers were always in the liquid-disordered state. For each case, the solubilization parameters, D_on (total detergent: lipid mole ratio producing the onset of solubilization) and D₅₀ (total detergent: lipid mole ratio producing 50% solubilization), were determined under equilibrium conditions. Both parameters varied generally in parallel. When double bonds were introduced to the acyl chains, other factors remaining constant, solubilization became more difficult, i.e., more detergent was required. Cis-unsaturated phospholipids required more detergent than the corresponding trans-isomers. Increasing chain length in saturated phospholipids between C12 and C16 decreased moderately the detergent/lipid ratios causing solubilization. Acyl and alkyl phospholipids were equally susceptible to Triton X-100 solubilization. Lipid chain order, as measured by DPH fluorescence polarization, seemed to facilitate solubilization, perhaps because more ordered bilayers have a smaller capacity to accommodate detergent monomers without breaking down into lipid-detergent mixed micelles.     

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.     

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

Secondary structure of a truncated form of lecithin retinol acyltransferase in solution and evidence for its binding and hydrolytic action in monolayers

Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. The enzymatic activity of a truncated form of LRAT (tLRAT) which contains the residues required for catalysis but which is lacking N- and C-terminal hydrophobic segments has been shown to depend on the detergent used for its solubilization. Moreover, it is unknown whether tLRAT can bind membranes in the absence of these hydrophobic segments. The present study has allowed to measure the membrane binding and hydrolytic action of tLRAT in lipid monolayers by use of polarization modulation infrared reflection absorption spectroscopy and Brewster angle microscopy. Moreover, the proportion of the secondary structure components of tLRAT was determined in three different detergents by infrared absorption spectroscopy, vibrational circular dichroism and electronic circular dichroism which allowed to explain its detergent dependent activity. In addition, the secondary structure of tLRAT in the absence of detergent was very similar to that in Triton X-100 thus suggesting that, compared to the other detergents assayed, the secondary structure of this protein is very little perturbed by this detergent.     

Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X-100.

Effective detergent:lipid ratios (i.e. molar ratios in the mixed aggregates, vesicles or micelles) have been estimated for the solubilization of phosphatidylcholine vesicles by Triton X-100. Effective molar ratios are given for both the onset and the completion of bilayer solubilization; small unilamellar, large unilamellar and multilamellar vesicles have been used. Effective detergent:lipid ratios are independent of phospholipid concentration, and their use allows a deeper understanding of membrane-surfactant interactions.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Interaction of an amine oxide detergent with lecithin vesicles as studied by nuclear magnetic resonance

The interaction of an amine oxide detergent with single bilayer lecithin vesicles was investigated with proton and phosphorus magnetic resonance. The addition of the detergent micelles to vesicles suspensions leads to rapid detergent incorporation into the vesicle bilayer, resulting in a heterogenous vesicle population. Initially, some vesicles take up the equivalent of one detergent micelle, whereas others contain no detergent. Subsequently, the detergent is distributed between the vesicles by vesicle-vesicle collisions. This can be followed by the change in the Pr3+-shifted spectral positions of the detergent and lecithin head groups with time. From the intensity of the head-group signals, it can be concluded that after about 20 h the detergent is almost equally distributed between the outer and inner vesicle membrane monolayers. Vesicles obtained by cosonication of the detergent and lecithin take up metal ions. This ion permeability depends on the vesicle concentration and can be attributed to vesicle-vesicle or vesicle-mixed micelle collisions. Egg lecithin vesicles are stable against the detergent up to molar ratios of detergent to lecithin of 0.2--0.3. At larger ratios mixed micells and multibilayers are formed. Measurements of proton spin-lattice relaxation times confirmed that the internal architecture of the vesicle bilayer is almost unaffected by the incorporated detergent.