Detergent solubilization of lipid bilayers: a balance of driving forces

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Detergent solubilization of lipidbilayers: a balance of driving forcesDov Lichtenberg1, Hasna Ahyayauch2, Alicia Alonso2, and Felix M. Goni2

1 Tel Aviv University, Sackler School of Medicine, Department of Physiology and Pharmacology, Ramat Aviv, 69978 Tel Aviv, Israel2 Unidad de Biofısica (CSIC, UPV/EHU) and Departamento de Bioquımica, Universidad del Paıs Vasco, P.O. Box 644, 48080 Bilbao,

Spain

Although detergents are routine tools in biomembraneresearch, their use remains empirical. We propose thatsolubilization is the result of a balance between twoparameters: (i) the energy associated with bending ofphospholipid monolayers into a curved micellar surface,and (ii) the energy associated with filling the void in thecenter of the resultant mixed micelle. In this review, weshow that reliable data on the phase boundaries, andtheir dependence on various conditions, are consistentwith this hypothesis, even if the data might have beeninterpreted differently. Although most of the experimen-tal data discussed here were obtained with the non-ionicdetergent Triton X-100, the conclusions should be appli-cable to a wide variety of detergents.

The role of detergents in membrane researchA biological membrane is a hydrophobic barrier betweentwo aqueous compartments. Although membrane compo-sition varies considerably, there are several common attri-butes to all biomembranes. An important common featureis that their structural matrix is made of water-insolubleamphiphiles (see Glossary), particularly phospholipids andamphiphilic proteins, which are very difficult to dissociate.

Much of the available data on membrane compositioncome from thermodynamically stable, isotropic aqueoussolutions, obtained on mixing of the membranes withwater-soluble amphiphiles called surfactants or detergents(Figure 1a) [1–3]. Above a specific concentration, called thecritical micellar concentration (cmc), these compounds self-assemble in the form of micelles. When mixed with lamel-lar structures (membranes) made of insoluble amphi-philes, mixed assemblies are formed whose structuredepends on the molar ratio of soluble-to-insoluble amphi-philes: below a critical value of this ratio (Re

sat) the mixedassemblies are lamellar, whereas above another criticalratio, Re

sol (both Re values are more precisely definedbelow), the membranes are transformed into detergent–lipid–protein mixed micelles (Figure 1b) [4–7].

This process, denoted solubilization, allows identificationand characterization of solubilized micellar proteins. Usingsufficient detergent and phospholipid, mixed micelles can bemade to either have one membrane protein molecule ornone. Under these conditions, standard methods can be

used to purify membrane proteins, to crystallize them, orto reconstitute them into proteoliposomes.

In spite of its importance, detergent solubilization ofbiomembranes has not been systematically studied be-cause of the complex composition of biomembranes. There-fore, much work has been devoted to solubilization of therelatively simple model membranes known as liposomes.Although these lipid bilayers are indeed simpler thanbiomembranes, their solubilization is not very simpleand many basic questions have not yet been answered.A better understanding of how detergents operate wouldlead to more efficient methods of membrane protein puri-fication and reconstitution [1,2,8–10].

The most widely used detergent in membrane studieshas been the non-ionic polyoxyethylene detergent Triton X-100 (denoted here as Triton), an inhomogeneous detergentcontaining polyoxyethylene moieties of several lengths. Inthe present review we focus on the solubilization of lipo-somes by Triton. Data regarding solubilization by otherdetergents are also discussed when relevant. In our inter-pretation, the available, and often disperse, data obtainedusing a variety of detergents are consistent with the gen-eral conclusions of the review.

Review

Glossary

Amphiphile: molecule that contains both hydrophylic and lipophylic moieties.

Detergent: water-soluble amphiphile with the capacity to solubilize lipids or

other hydrophobic molecules.

Gel phase: lamellar lipid phase. Lipids in the gel phase are characterized by

very low translational and rotational diffusion rates, and by a high degree of

hydrocarbon chain order.

Kinetic trap: relatively stable but not true equilibrium state giving the

misleading impression that the system has reached equilibrium.

Liquid crystalline or fluid phase: lamellar lipid phase in which lipids have full

freedom of diffusional, rotational, and conformational motions within the

bilayer plane.

Liquid-ordered phase: cholesterol-containing lamellar phase in which the lipids

are packed more tightly than in the liquid crystalline state but less tightly than

in the gel phase. The liquid-ordered phase is stabilized by intercalation of the

flat, rigid cholesterol molecule among the more flexible phospholipids.

Micelles: spherical or ellipsoidal aggregates of amphiphiles, dispersed in an

aqueous solvent. Pure (detergent only) and mixed (lipid and detergent)

micelles can occur.

Surfactants: molecules that, when added to an aqueous medium, decrease the

surface pressure at the air–water interface. Most biomolecules, and all

detergents, are surfactants. Surfactants and detergents are often, if not very

accurately, used as synonyms.

Thermotropic phase transition: transition between two thermodynamic phases

driven by heat absorption or release. Examples are the transition between

liquid and vapor water and, in the context of this review, the transition between

the gel and liquid-crystalline phases of a phospholipid bilayer.

Corresponding author: Goni, F.M. ([email protected])Keywords: detergent(s); solubilization; liposomes; bilayers; mixed micelles;spontaneous curvature; phospholipids; cholesterol.

0968-0004/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2012.11.005 Trends in Biochemical Sciences, February 2013, Vol. 38, No. 2 85


Characterization of detergent solubilizationIn most studies, liposome dispersions were mixed withdetergent solutions and the resulting systems were ana-lyzed by spectrophotometric methods, most commonlylight scattering. The initial effect of detergent additionon light scattering by the lipid dispersion depends onthe state of aggregation of the lipids and the vesicle size.Specifically, addition of detergent to small unilamellarvesicles (SUVs) results in increased scattering due todetergent-induced vesicle size growth. However, for largeunilamellar vesicles (LUVs) the vesicle size can eitherremain unaltered or be slightly reduced prior to the onsetof solubilization because of transitional solubilization andsubsequent reconstitution. Furthermore, phospholipidsundergo a thermal transition from a gel phase, in whichthe phospholipids are tightly packed, to a less orderedliquid-crystalline state. This transition affects the parti-tioning of detergents between bilayers and water, and thepacking within the detergent-containing bilayers, as dis-cussed below.

Hence, as increasing amounts of detergent are added toa lipid dispersion, the light scattering either remainsconstant or even increases until, at a critical point, itbegins to decrease. This is the onset of solubilization(Figure 2a, Dt

sat). As the detergent concentrationincreases, another critical point is eventually reached atthe completion of solubilization (Dt

sol), beyond which lightscattering does not further decrease.

The (apparently linear) dependence of the lattertwo critical points on the phospholipid concentration

(Figure 2b) gives rise to a simple phase diagram. Theslope of each of these [detergent]/[lipid] lines is the ratiobetween the concentration of detergent in mixed lipid–detergent assemblies and the concentration of phospho-lipid at the points of onset and completion of solubiliza-tion. These two parameters are designated Re

sat andRe

sol, respectively. Re refers to the effective (as opposedto total) [detergent]/[lipid] molar ratio [11], which is thefraction of total (or added) detergent concentration thatresides in mixed assemblies under equilibrium condi-tions. The distinction is necessary because in a mem-brane–detergent–water system the detergent partitionsbetween the membrane and water [12]. In the rangebetween Re

sat and Resol, phospholipid bilayers saturated

with detergent coexist with detergent micelles saturatedwith lipid, implying that the energy of detergent-saturated phospholipid bilayers (of a composition givenby Re

sat) is of the same order of magnitude as the energyof phospholipid-containing mixed micelles [13,14] of acomposition given by Re

sol.

AssemblyMolecularstructure

(a)

(b)

Packing parameter

Curvature

≈ 1 ≈ 0

≈ 1/3 > 0

+Solubiliza�on

Recons�tu�on

(a li pid)

(a detergent)

( - detergent )

TiBS

Figure 1. Self-assemblies in mixtures of lipids and detergents. (a) Molecular

geometries and assemblies of pure lipids or pure detergents. The molecular

geometry of the lipid (or detergent) determines the mode of assembly:

approximately cylindrical lipid molecules give rise in water to flat bilayers,

whereas a conical detergent leads to highly curved micelles. In both cases

hydrocarbon–water contacts are minimized. (b) Membrane solubilization and

reconstitution by addition and removal of detergents. When flat bilayers (which

can include proteins) interact with curved detergent micelles, lipid–protein–

detergent mixed micelles are formed in a process called solubilization. The

phenomenon can be reversed by removing the detergent (reconstitution).

[Detergent]

Ligh

t sca

�er

ing

[Lipid]

[Det

erge

nt]

Bilayers

Micelles

Coexistence

(a)

(b)

cmc ReSat

D tSat

DtSol

DtSat Dt

Sol

ReSol

DwSat

DwSol

TiBS

Figure 2. Characterization of lipid–detergent mixtures. (a) Schematic description of

membrane solubilization as monitored by changes in light scattering of the

membrane suspension. Dtsat and Dt

sol correspond to the total (as opposed to

effective) detergent concentrations causing the onset and completion of

solubilization, respectively, at a given membrane concentration. (b) Dependence of

Dtsat and Dt

sol on the lipid concentration. The resulting lines are boundaries for the

coexistence of bilayers and mixed micelles in aqueous lipid–detergent mixtures. The

slopes of these lines are Resat and Re

sol, corresponding to the effective detergent–lipid

molar ratios causing the onset and completion of membrane solubilization. Dwsat and

Dwsol are the concentrations of monomeric detergent in water at the onset and

completion of membrane solubilization, respectively. They are both lower than the

detergent critical micellar concentration (cmc).

Review Trends in Biochemical Sciences February 2013, Vol. 38, No. 2

86


Notably, back-extrapolation of the phase boundaries toa phosphatidylcholine concentration of [PtdCho]=0 leadsto two different intercepts, Dw

sat and Dwsol (Figure 2b),

namely the concentrations of detergent monomers in wa-ter at the saturation and solubilization points, respective-ly. They are not equal, indicating that at very low lipidconcentrations the critical detergent concentrations arenot linear functions of [PtdCho] [3,15,16]. The concentra-tion of detergent monomers in water Dw increases withthe total detergent concentration Dt and remains almostconstant above Re

sat.In the vesicle range, an increase in Dt results in an

increase in both Re and Dw, as expected on the basis of thepartition coefficient K:

K ¼ ½Db=ðL þ DtÞ�=Dw; (1)

where L is the lipid concentration and Db is the concen-tration of membrane-bound detergent. An equation can bederived from the above that describes Re as a function of Kand Dw:

Re ¼ KDw=ð1 � KDwÞ: (2)

This implies that the value of K can be evaluated on thebasis of detergent partition data as a function of L and Dt.This has been experimentally demonstrated for the case ofoctyl glucoside partition into bilayers [14]. Furthermore,an equation can be derived that describes Re as a functionof the concentrations of lipid and detergent and the parti-tion coefficient K [16]. Above Re

sat, added detergent causesphase transformation, that is, solubilization. We attributethis change to saturation of the bilayer with detergent,although we cannot rule out the possibility that the aque-ous solution is saturated by detergent monomers.

Phase diagrams and the need for kinetic studiesPhase boundaries (Figure 2b) are meaningful only forsystems at equilibrium. It is important to keep in mindthat a steady-state structure might reflect kinetic traps;that is, relatively stable but not real equilibrium states.Therefore, a claim that solubilization requires more deter-gent because of difficulties in penetration is not strictlytrue because, given enough time, solubilization wouldoccur. The traditional way to ensure that a given systemis at equilibrium is to show that mixtures of equal compo-sition prepared differently are identical. For instance,mixtures of a given composition obtained by codispersalof lipid and detergent in aqueous solutions should behavethe same as mixtures of the same composition obtained bymixing lipid and detergent dispersions that have beenindividually prepared. The few published investigationsin which such experiments were conducted lend strongsupport to the conclusion that the systems studied were atequilibrium [17].

The least we should do to rule out the possibility that theapparent phase boundaries reflect kinetic traps ratherthan equilibrium states is to show that the mixturesstudied are measured under steady-state conditions, asdetected by monitoring the time dependence of a physico-chemical property. The important issues of the mechanismand kinetics of solubilization are beyond the scope of the

present review, yet kinetic studies are important to sup-port the validity of the thermodynamic aspects discussedhere.

Solubilization of biomembranes or unilamellar phos-pholipid liposomal bilayers by detergents occurs in threesteps [12]. First, at sub-solubilizing detergent concentra-tions, the detergent partitions between the liposomes andthe solution according to the partition coefficient K. Sec-ond, at higher detergent concentrations, micellizationoccurs, beginning at Re

sat and ending at Resol. Third,

following completion of solubilization, when the detergentconcentration is higher than that required for completesolubilization, addition of more detergent results in areduction in the size of mixed micelles. The term solubili-zation in fact relates to the second step, which is probablythe rate-limiting step of the overall process. This step cantake from seconds to many days, depending on the compo-sition of the system and the temperature [17–24].

Kinetic studies are particularly important when equili-bration is very slow, as in experiments conducted at lowtemperature with gel-phase lipids [17] or/and when theprotocol of the solubilization experiment involves titrationof multilamellar liposomes (MLVs) by a detergent solution,for example, deoxycholate [25] or Triton [26]. MLV titra-tion with a detergent solution initially results in partition-ing of the added detergent between water and theoutermost bilayer, so that the detergent:lipid ratio in thisbilayer is sufficient to solubilize it even when the deter-gent:lipid ratio in the whole system is much lower than theratio corresponding to the onset of solubilization of theentire lipid population. Equilibration of the system occursvia peeling (i.e., micellization) of the lipid bilayers, oneafter the other, followed by repartition of detergent mole-cules. When the detergent concentration is insufficient tosolubilize any more lipid, equilibration again involvesrepartition of detergent, eventually yielding detergent-containing bilayers. We propose that the most seriouscauses of irreproducible results are probably inappropriateexperimental protocols that use non-equilibrium condi-tions and/or insensitive methods. Hence, the next part ofthe review is a critical evaluation of the methods used toassess published data.

Methods used in solubilization studies: scope andlimitationsThe critical values Re

sat and Resol can be estimated using a

large number of experimental tools, including microscopic,spectrophotometric, and calorimetric approaches, asreviewed by Goni and Alonso [2]. The various methodshave different sensitivities for the two ratios. Of specialinterest are two complementary methods. Specifically, theonset of solubilization is best detected by NMR, becausehigh-resolution signals are only generated by micelles[25,27]; the NMR signal of bilayer nuclei is broadenedalmost beyond detection (Figure 3a). By contrast, lightscattering is a more reliable tool for evaluating the com-pletion of solubilization, because it is sensitive to largerparticles and therefore reflects the completion of micelliza-tion much better than the beginning [28]. Turbidity, mea-sured as the apparent absorbance at 400–500 nm on aspectrophotometer, is often used as a substitute for light


87


scattering measurements. However, if the mixture doesnot become transparent on addition of more Triton, theremight be a very small fraction of detergent-resistant mem-branes (DRMs) that contribute to light scattering morethan the micelles, even if most of the particulate material ismicellar. Under such conditions, it is essential to fraction-ate the dispersions to avoid errors in determining thephase boundaries, in spite of possible changes to thesystem by such fractionation.

Determining the value of Resat solely on the basis of

turbidity measurements has led to particularly question-able conclusions. For Triton and similar detergents, lightscattering is particularly unreliable because above a giventemperature the turbidity might increase because of inter-actions in phospholipid–Triton mixed micelles. The tem-perature at which a sudden increase in turbidity of thelipid-detergent suspension occurs is known as the cloudpoint. Specifically, the cloud point of pure Triton is >608C,but it is lower in phospholipid-containing micelles [29].Thus, higher turbidity does not necessarily mean that themixture contains less solubilized (micellar) phospholipids.

In this case, other methods should be employed, such asthose that use the absorbance of a dye or the fluorescenceintensity of a suitable probe [28].

Isothermal titration calorimetry is another valuabletool for detecting phase boundaries [23,30] and gainingknowledge on the heat associated with composition-in-duced phase transformations. Specific structural informa-tion can be derived from cryo-transmission electronmicroscopy [31,32]. Although this technique is limited interms of its capability to yield quantitative results, it is themost direct method for studying the structure of mixedaggregates. More recent methodologies used to study mem-brane solubilization by detergents include atomic forcemicroscopy [33,34], detergent-induced changes in mem-brane order, measured as diphenylhexatriene fluorescencepolarization [35], and optical microscopy of giant unilamel-lar vesicles [36].

In summary, great care is required in choosing theoptimal methods and procedures that best fit the processbeing studied. Notably, different methods are optimal fordetermination of the onset and completion of solubiliza-tion. An understanding of the scope and limitations ofmethods that yield apparent ambiguities is essential indeciding which experimental results should be used toascertain the attributes of solubilization.

Self-assembly in mixtures of amphiphilesSelf-assembly of amphiphiles occurs to prevent exposure oftheir hydrophobic moieties to water [37]. Intuitively, thenature of self-assembly is governed by the molecular struc-ture of the amphiphile: when the molecular structure iscylindrical (i.e., it can be idealized as a cylinder), as is thecase for the most-studied phospholipids, the molecularvolume (V) is approximately equal to the product of thepolar surface cross-area (S) and the length (l) of the hydro-phobic chain [38]. Hence, the packing parameter p [p=V/(S�l)] will be 1.0 [38,39]. Such amphiphiles are likely toform self-assemblies that are flat bilayers because suchpacking yields minimal exposure of the hydrophobic chainsto water (Figure 1).

The molecular structure of Triton, and of most otherdetergents, can be idealized as a cone, so that the volume ofthe molecule is smaller than the product of the polarsurface area and the length of the extended chain. Hence,the packing parameter for conical amphiphiles is less than1.0 and the exposure of the hydrophobic chains to water isminimal when curved micelles are formed instead of la-mellae [38,39].

Mixing phospholipids with a detergent will yield layersthat are not ideal for either of the two amphiphiles. In-stead, the two components are forced by entropy to residein mixed aggregates. Prior to being solubilized, the bilayersretain their lamellar structure, but as the detergent:lipidratio in the bilayers increases, holes of increasing diameterare formed [40,41]. The end product of micellization isalmost always thread-like long cylinders whose surfaceis positively curved. The alternative, less recent descrip-tion of mixed micelles in terms of a disc model could havebeen expected, particularly for bile salt–phospholipid mix-tures, because the discoidal self-assemblies can allow theformation of a phospholipid-rich almost flat bilayer covered

10 5 0 −5 −10 −15 −20 ppm

CerPCho PtdCho

(d)

(c)

(b)

(a)

TiBS

Figure 3. Membrane solubilization monitored by NMR. (a–c) 31P NMR spectra of

phosphatidylcholine (PtdCho)/sphingomyelin (CerPCho) mixtures (1:1 molar ratio)

in the presence of different concentrations of Triton. The effective detergent:lipid

molecular ratio (Re) is (a) 0, (b) 0.5, and (c) 1.3. Solubilization is indicated by the

presence of anisotropic (0 shift) narrow signals when detergent is added [41]. (d)31P NMR spectrum of a PtdCho/CerPCho/cholesterol (Chol) mixture (47.5:47.5:5.0

molar ratio) in the presence of Triton at Re 1.0 [41]. Note that partial solubilization

in the presence of Chol involves preferential solubilization of PtdCho over

CerPCho.


88


by detergent-rich curved edges on the perimeter of the disc[25,42–44]. At least for Triton and structurally relateddetergents, we favor the description of solubilization interms of a hypothetical series of two steps (formation andsealing of hollow mixed micelles) because the product ofthis series is elongated rather than discoidal micelles.Furthermore, theoretical considerations demonstratedthat mixed discs can be expected to be more stable thancylinders only under non-relevant conditions [15,16].

Another more commonly used approach to the self-assembly of amphiphiles is based on the spontaneouscurvature, which is defined as the reciprocal of the radiusof the surface along which a given amphiphile assembles[13,14]. Unlike the packing parameter, the spontaneousradius of curvature can be experimentally determined [45].Cylindrical molecules are curvophobic, in that they tend toaggregate along flat surfaces whose spontaneous radius ofcurvature is infinite (spontaneous curvature is zero).Amphiphiles with a conical molecular structure are curvo-philic. Their packing parameter can be either less than orgreater than 1.0. Those with p<1 (relatively large headgroup) tend to aggregate in the form of micelles and theirspontaneous of curvature is positive and equal to theradius of their spherical micelles. A different type of am-phiphile with a conical molecular geometry but a relativelysmall head group ( p>1) tends to form hexagonal (tubular)structures of negative spontaneous curvature (not shown).

On the basis of these considerations, we can view thestate of aggregation in lipid–detergent mixtures as a bal-ance between the favored geometries of the components.When an amphiphile is forced to reside along a surface ofcurvature different from its spontaneous curvature, localinstability occurs. Sometimes the amphiphile is describedas being frustrated. In these terms, a mixture of a curvo-phobic phospholipid and a curvophilic detergent formsmixed aggregates in which the instabilities caused by bothamphiphiles are canceled, as depicted schematically inFigure 1b. The average curvature of a mixed aggregatecan be expected to be a weighted average of the spontane-ous curvature of the detergent and lipid, as discussedbelow.

Temperature dependence of the solubilization of liquid-crystalline bilayersBoth Re

sat and Resol depend on the length and unsatura-

tion of the hydrophobic chains, as well as the phasetransition and temperature (Table 1). Abundant dataare available on the effect of temperature on the solubili-zation of fluid, or liquid-crystalline, bilayers. The results ofthese studies [17,46,47] are key to understanding theeffects of other factors. For instance, the detergent con-centration required to solubilize liquid-crystallinebilayers increases on heating of the system. In comparisonto the differences between gel and liquid-crystallinebilayers, the change due to heating of liquid-crystallineliposomes is small but reproducible.

Our interpretation of these results is based on theassumption that the concentration of detergent requiredfor solubilization increases with the difference in curvaturebetween the two amphiphiles. Hence, on heating-inducedexpansion of the phospholipid hydrocarbon moiety relative

to the polar head group, the spontaneous curvature ofthe phospholipid becomes more negative, so that moredetergent is needed to solubilize the bilayers at highertemperatures. Furthermore, the cross-sectional area of thedetergent hydrophobic moiety increases and thus the de-tergent becomes less conical, which means that more of it isrequired for solubilization [17,47,48]. Many experimentsconfirm this approach, showing that the major contributionto the change in both Re

sat and Resol of liquid-crystalline

bilayers is the change in spontaneous curvature. In otherwords, bending of a nearly flat monolayer made of phos-pholipid becomes more difficult (and hence requires moreenergy) with increasing temperature. Other forces appar-ently play only minor roles.

From the available data, many of them summarized inTable 1, we can generalize that the detergent concentra-tion required for solubilization of liquid-crystallinebilayers is: (i) an increasing function of lipid disorder;(ii) an increasing function of unsaturation; (iii) a slightlydecreasing function of chain length; and (iv) lower forsphingomyelin (CerPCho) (0.5 Triton:CerPCho) than forPtdCho (2 Triton:PtdCho) [29]. In addition, at a detergentconcentration sufficient for partial solubilization ofPtdCho–CerPCho mixed bilayers, CerPCho-rich mixedmicelles appear before PtdCho-rich mixed micelles(Figure 3a–c). Interestingly, the concentration requiredfor solubilization of cholesterol (Chol)-containing bilayersis an increasing function of Chol [49] and much higherdetergent concentrations are required for CerPCho thanfor PtdCho in the solubilization of CerPCho–PtdCho–Cholbilayers [48–50]. This means that Chol stabilizes CerPChoagainst solubilization, probably because of the formation ofa CerPCho–Chol complex of large negative curvature. Thiscould be the basis of the DRM effect.

Solubilization of bilayers in the gel stateMuch fewer data are available on the solubilization of gel-phase bilayers. Furthermore, several published investiga-tions contain questionable results because some experi-ments used mixtures that were not at equilibrium.Moreover, the methods used were sometimes inappropri-ate for the questions asked. The main reason for our doubtsis that solubilization of gel-phase bilayers is very slow(much slower than the solubilization of liquid-crystallinebilayers), which implies that the mixtures studied were notat equilibrium and thus might have given rise to artifacts.

In spite of the limitations, two generalizations can bemade [17,34,47]: (i) compared to liquid-crystalline bilayers,less detergent is required for both the onset and completionof solubilization of gel-phase bilayers; and (ii) within therange of gel-phase temperatures, the detergent concentra-tion required for solubilization decreases with increasingtemperature.

We suggest that the concentration of detergent neededto solubilize gel-phase bilayers is lower than that needed tosolubilize liquid-crystalline bilayers because the spontane-ous curvature of the phospholipid is less negative in themore tightly packed gel-phase bilayers. Apart from thebending of phospholipid monolayers, the other contributorto the energy of solubilization is the energy associated withfilling the void in the center of the newly formed mixed


89


Table 1. Factors that affect lipid solubilization by Triton X-100a

Factor Lipid Conditions Onset Solubilization Effectb Remarks Refs

Phase Temp. (-C) Dtsat Re

sat D50 Dtsol Re

sol

Chain length DiLauPtdCho (C12:0) Fluid (LC) 45 2.6 4.8

#

[D] required to

solubilize LC liposomes

[24]

DiMyrPtdCho (C14:0) 45 2.3 3.5 decreases with chain length

DiPamPtdCho (C16:0) 45 2.3

DiMyrPtdCho (C14:0) Gel Tm–20c 0.32

"

[D] required to solubilize

liposomes in the gel phase

increases with chain length

[47]

DiPamPtdCho (C16:0) Tm–20c 1.7

DiStePtdCho (C18:0) Tm–20c >5

Chain

unsaturation

DiMyrPtdCho (C14:0) Fluid 45 3.0 3.8

"


LC liposomes

[24]

DiDMyrPtdCho (C14:1) 45 2.3 4.8 increases with chain

unsaturation

DiOlePtdCho (C18:1) Fluid 20 1.25 3.9

DiLnPtdCho (C18:2) 20 1.5 4.0 "DiaLnnPtdCho (C18:3) 20 1.5 4.8

DiPamPtdCho (C16:0) Fluid 45 2.3 3.6 "DiDPamPtdCho (C16:1) 45 3.0 4.8

Cholesterol

addition

PamOlePtdCho Fluid 4 1.75

"

[D] required to

solubilize liposomes

[48]

PamOlePtdCho/Chol (65/35) Lo 4 6 increases with the

cholesterol

DiPamPtdCho Gel 37 0.5 concentration in the bilayer

DiPamPtdCho/Chol (65/35) Lo 37 9

PtdCho/CerPCho (50/50) Domains 25 0.368 1.36

"

[D] required to


[49]

PtdCho/CerPCho/Chol (40/40/20) Lo 25 0.477 3.87 increases with

cholesterol addition

PtdCho/CerPCho (60/40) Domains 4 0.5

"

[D] required to


[19]

PtdCho/CerPCho/Chol (60/20/20) Lo 4 2.8 increases with liposome

cholesterol content

Temperature DiPamPtdCho Gel 37 <0.2 0.28

"[D] required to solubilize

LC liposomes

[17]

Fluid 47 0.31 1.81 increases with temperature

DiMyrPtdCho Fluid 30 0.88

"[47]

37 1.55

47 2.28

DiPendecPtdCho Fluid 30 0.37

"[47]

37 0.72

47 1.54

DiStePtdCho Fluid 51 0.28 " [47]

59 0.35

DiPamPtdCho Gel 23 1.73 9.73

#


gel-phase

[17]

26 0.34 1.74 liposomes decreases

as a function of T

DiMyrPtdCho Gel 4 0.32 # [47]

20 0.28

DiPendecPtdCho Gel 4 0.65

#20 0.52

30 0.37

DiStePtdCho Gel 37 >5

#47 0.68

51 0.28

Cis–trans

isomerism

DiOlePtdCho (cis) Fluid 45 3.6 4.5 The cis isomer requires

higher [D]

[24]

DiElaPtdCho (trans) 1.5 2.5 than the trans isomer

aAbbreviations: CerPCho, sphyngomyelin; Chol, cholesterol; DiElaPtdCho, dielaidoyl phosphatidylcholine; DiLauPtdCho, dilauroyl phosphatidylcholine; DiLnPtdCho,

dilinoleoyl phosphatidylcholine; DiaLnnPtdCho, dilinolenoyl phosphatidylcholine; DiMyrPtdCho, dimyristoyl phosphatidylcholine; DiDMyrPtdCho, dimyristoleoyl phos-

phatidylcholine; DiOlePtdCho, dioleoyl phosphatidylcholine; DiPamPtdCho, dipalmitoyl phosphatidylcholine; DiDPamPtdCho, dipalmitoleoyl phosphatidylcholine; DiPen-

decPtdCho, dipentadecanoyl phosphatidylcholine; DiStePtdCho, distearoyl phosphatidylcholine; PamOlePtdCho, palmitoyl oleoyl phosphatidylcholine; LC, liquid

crystalline, or fluid; Lo, liquid ordered; [D], detergent concentration.

bEffect of factor on solubilization.

c208C below Tm.


90


micelles (Figure 4a) [17]. The process that leads to micelleswith no void (Figure 4c) is called interstitial energy or voidenergy. This description does not mean that a micelle witha void in the center ever exists at equilibrium; rather, thereis energy associated with the necessary filling of a theoret-ical, extremely unstable hollow micelle that leads to astructure that is not hollow.

We propose that (i) the effect of the interstitial energy onthe temperature dependence of solubilization opposes thatof the bending energy, (ii) the effects of temperature onboth Re

sat and Resol reflect a balance between the bending

energy and the interstitial energy, and (iii) the contribu-tion of the interstitial energy is more important in the gelstate than in the more flexible mixed micelles presentabove the thermal transition temperature.

The lower value of Resat for the solubilization of gel-phase

bilayers means either that the partition coefficient of deter-gent between gel-phase bilayers and the aqueous medium ishigher than that observed for liquid-crystalline bilayers, orthat the gel-phase bilayers can accommodate less detergentbefore the energy associated with lipid-containing mixed

micelles equals that of detergent-containing bilayers. Thefirst possibility can be ruled out on the basis that thepartition coefficient for Triton between gel-phase lipidsand water is equal to that obtained for liquid-crystallinebilayers [26]. Furthermore, a recent study lends strongsupport to the alternative possibility, showing that gel-phase lipids become saturated by detergent at lower deter-gent concentrations than those in the fluid phase [51]. Thereason why gel and fluid phases accommodate differentmaximal amounts of detergent is unclear at present. Onepossibility is that in the fluid state, the packing of lipidmolecules is sufficiently flexible to compensate for the dif-ferent spontaneous curvature of the detergent; therefore,the bilayer accommodates more detergent before breakingdown into mixed micelles.

Differential solubilizationLittle is known about the solubilization of bilayers made ofmore than one phospholipid. Of great interest is the solu-bilization of PtdCho and CerPCho in PtdCho–CerPCho–Chol bilayers, because liquid-ordered bilayers containingCerPCho and Chol are detergent-resistant. Some DRMsare believed to exist as membrane rafts [8,52]. The interestin Chol in the context of this discussion is that it rigidifiesthe packing of phospholipids in liquid-crystalline bilayersbut fluidizes the packing of gel-phase bilayers. When addedto gel-phase lipid, Chol transforms the gel phase intoliquid-ordered bilayers. The change in lipid packingchanges the spontaneous curvature of the lipid assemblyto a less negative value, thus reducing the concentration ofdetergent required for solubilization.

A 31P-NMR investigation revealed that in the absence ofChol, when Triton was added to PtdCho–CerPCho bilayersat a concentration sufficient for partial (but not complete)solubilization, the detergent-solubilized fraction was richin CerPCho [49], as expected from the smaller negativecurvature of CerPCho relative to PtdCho (Figure 4b). Cholreverses this relative susceptibility. Notably, Chol-contain-ing bilayers are more ordered than liquid-crystallinebilayers but less ordered than gel-phase bilayers.

In the presence of Chol in PtdCho–-CerPCho bilayers,the solubilized fraction of mixtures of PtdCho, CerPCho,and Chol is CerPCho-poor (compare Figure 3b to 3d). Theseobservations suggest that CerPCho–Chol mixtures have avery large negative spontaneous curvature, which makesthem resistant to detergent solubilization, as depictedschematically in Figure 4b. It has also been proposed thatthe reason why certain membrane fractions resist deter-gents is the preference of Chol for CerPCho over PtdCho[53,54].

How much detergent is needed to solubilizephospholipid bilayers?Assuming that the spontaneous curvature of mixedmicelles (Sm) is a weighted average of the spontaneouscurvature of the two amphiphiles (lipid and detergent), itcan be calculated as:

Sm ¼ Nl � Sl þ Nd � Sd; (3)

where Nl and Nd are the molar fractions of the lipid anddetergent, respectively (Nl+Nd=1.0), Sl is the spontaneous

(a)

(b)

(c)

Re

(i) (ii) (iii)

(iii)

(i) (ii)

sat

T

PtdCho TritonCerPCho

CerPCho-PtdCho

Spontaneous curvature0–1 +1

TiBS

Figure 4. The two forces in bilayer solubilization by detergents. (a) Temperature

(T) dependence of the two contributions to the critical detergent:lipid ratio (Resat):

(i) bending contribution; (ii) interstitial contribution; and (iii) sum of the two

contributions [40]. (b) phosphatidylcholine (PtdCho), sphingomyelin (CerPCho),

and Triton are shown along an axis of spontaneous curvature. The more negative

the lipid spontaneous curvature, the more detergent is required for its

solubilization (curvature values as shown are only approximations). (c)

Hypothetical monolayer deformations accompanying the bilayer-to-micelle

transformation: (i) initial flat state, (ii) intermediate state resulting from bending

of the monolayer and involving formation of a void, and (iii) final state resulting

from collapse of the void and formation of a hydrophobic core.


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curvature of the lipid, and Sd is the spontaneous curvatureof the detergent.

The spontaneous curvature of a micelle-forming amphi-phile is positive and equal to the reciprocal of the size of themicelles it forms in aqueous solutions [55,56], whereas thespontaneous curvature of PtdCho is slightly negative [45].Hence, the phospholipid self-assembles in bilayers and aslong as the molar fraction of the detergent is much lowerthan that of the phospholipid, the effective spontaneouscurvature remains close to that of the lipid and the mixedassembly remains lamellar. Solubilization begins at acritical molar fraction at which the bilayer cannot accom-modate more detergent. At this point, the bilayer accumu-lates enough detergent so that the spontaneous curvatureof the mixed assemblies reaches a sufficiently positivevalue to adopt a micellar structure [15,17].

Previous studies have demonstrated that the saturatingdetergent concentration, given in terms of the molar frac-tion of detergent required to overcome the bending energy(Nd

sat), is approximately equal to the ratio between thespontaneous curvature of the lipid and that of the deter-gent. In terms of Re

sat,

Rsate =ð1 � Rsat

e Þ ¼ Sl=Sd (4)

and hence

Rsate ¼ Sl=ðSd þ SlÞ: (5)

If the radii of curvature of a given lipid–detergent pairare known, the latter approximation can be used to predicthow much detergent is needed to solubilize the given lipid.In the absence of reasonable estimates of the spontaneouscurvature, we can still use the rationale discussed above toexplain experimental observations such as the highersusceptibility of liposomes made of CerPCho in comparisonto PtdCho liposomes. Specifically, solubilization of CerP-Cho requires merely a quarter of the concentration ofTriton because its spontaneous curvature is less negativethan that of PtdCho.

Concluding remarksIn our search for a unifying hypothesis, we tried to explainthe available data (summarized in Table 1) on the basis ofthermodynamic considerations, without referring to eitherkinetic or mechanistic aspects. The factor whose effectshave been tested in the most detail is temperature (andthermal phase transitions). We think that our rationale ininterpreting these studies can be valid in assessing theeffect of other factors on the detergent concentrationsrequired for solubilization of lipid bilayers. Increasing inany way the difference between the spontaneous curvatureof the lipid and that of the detergent (i.e. the bendingenergy) results in an increase in the amount of detergentrequired for solubilization. This consideration explainsmost results obtained for liquid crystalline bilayers. Bycontrast, cooling of bilayers in the gel phase increases theconcentration of Triton necessary for solubilization, prob-ably because the void energy is greater than the bendingenergy (Figure 4).

Although the detailed mechanism of micellization ofmembranes and the micelle–bilayer phase boundariesand their dependence on composition and temperature

are not fully understood, some important points arehighlighted. (i) The phase boundaries in detergent solubi-lization studies should be based on equilibrium states andkinetic traps should be avoided. It is advisable to testwhether the state of aggregation of mixtures of identicalcomposition, prepared via different routes, is identical. (ii)The most sensitive methods should be used to determinethe onset and completion of solubilization (NMR for onset;light scattering for completion). (iii) Self-assembly in mix-tures of amphiphiles, and the resulting composition-in-duced lamellar–micellar transition, is a result of twoopposing factors: one is compensation between the proper-ties of the curvophilic detergent and that of the curvopho-bic phospholipid (described as a canceling of instabilities),and the other is the energy associated with filling the coreof curved mixed micelles. The latter energy is most impor-tant for gel phase lipids, whereas the energy of curvaturegoverns the solubilization of liquid-crystalline lipids. (iv)The detergent concentration needed for solubilizationdecreases with temperature for gel-phase lipids andincreases for liquid-crystalline lipids. (v) Partial solubili-zation of bilayers made of more than one component startswith the component whose spontaneous radius of curva-ture is highest. (vi) CerPCho–Chol complexes are respon-sible for the very low Triton solubilization susceptibility ofCerPCho in cell membranes.

AcknowledgmentsThis work was supported in part by grants BFU 2007-62062 (F.M.G.) andBFU 2011-28566 (A.A.) from the Spanish Ministry of Economy. We alsowish to thank the Lady Davis Fund and Bizkaia-Xede for financialsupport and Prof. Misha Kozlov for helpful discussions.

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Detergent solubilization of lipid bilayers: a balance of driving forces

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