Review Spectroscopic techniques in the study of membrane solubilization … · 2017-01-03 · Review Spectroscopic techniques in the study of membrane solubilization, reconstitution

Review

Spectroscopic techniques in the study of membrane solubilization,reconstitution and permeabilization by detergents

Felix M. Gon¬i, Alicia Alonso *Unidad de Biof|sica (CSIC-UPV/EHU) and Departamento de Bioqu|mica, Universidad del Pa|s Vasco, Aptdo. 644, 48080 Bilbao, Spain

Abstract

This review focuses on the use of spectroscopic techniques for the study of membrane solubilization, reconstitution, andpermeabilization by detergents. Turbidity and light scattering,visible and infrared spectroscopic methods, fluorescence,nuclear magnetic resonance, electron spin resonance and X-ray diffraction are examined from the point of view of theirapplicability to the above detergent-mediated phenomena. A short introduction is provided about each of the techniques,and references are given for further study. ß 2000 Elsevier Science B.V. All rights reserved.

Keywords: Biomembrane; Detergent; Surfactant; Light scattering; Spectroscopy; Fluorescence; NMR; ESR

1. Introduction and scope

Detergents, like many other biochemical tools,have been extensively used on a semiempirical basis.Simultaneous, parallel rather than converging studieshave attempted to establish the foundations for arational use of surfactants, on the basis of biophys-ical studies of the molecular interactions of lipids anddetergents. Spectroscopic techniques provide a widevariety of enormously powerful methods, that can,

when used in wise combination with each other orwith non-spectroscopic techniques, supply a uniquelydetailed view of the process of detergent-mediatedbilayer solubilization and reconstitution.

This brief paper is intended to provide the readerwith an overview of methods and results. A detailedexplanation of the more technical aspects of the spec-troscopic procedures, let alone their physical founda-tions, would certainly exceed the limits and purposeof the article. Moreover, a comprehensive review ofthe relevant literature would be almost useless for thesheer volume and repetitiousness of data. Instead, anumber of spectroscopic techniques have been se-lected on the basis of their proven applicability inthe study of membrane^surfactant interactions.Each of them will be discussed brie£y, particularlywith respect to their advantages and disadvantages inthe study of membrane solubilization. A few repre-sentative references will be cited, some preferencebeing given to the more recent and easily accessibleones, even if this does not always make justice to thepioneers in some areas.

The review is focused on the process of membrane

0005-2736 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 4 1 5 7 ( 0 0 ) 0 0 0 1 1 - 3

Abbreviations: ANS, 8-anilino-1-naphthalenesulfonate;ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; C12E8, octylo-(ethylene oxide) dodecylether; DPH, diphenylhexatriene; DPX,p-xylenebis(pyridinium bromide); EPR, electron paramagneticresonance; IR, infrared; Laurdan, 6-dodecanoyl-2-dimethylami-nonaphthalene; NMR, nuclear magnetic resonance; NPN, N-phenylnaphthylamine; SDS, sodium dodecylsulfate; TEMPO,2,2,6,6-tetramethylpiperidine-N-oxyl ; TMA-DPH, 1-(4-trimethyl-aminophenyl)-6-phenyl-1,3,5-hexatriene; Triton, poly(ethyleneoxide)-t-octylphenyl ether

* Corresponding author. Fax: +34-94-464-8500;E-mail : [email protected]

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solubilization and reconstitution by detergents, in-cluding detergent binding to membranes, and subse-quent events leading to the complete transformationof the lipid bilayer into lipid^detergent mixed mi-celles, and, conversely, the removal of detergentfrom mixed micelles and eventual reformation of abilayer in reconstitution experiments. In addition,surfactant-induced permeabilization of membraneswill be discussed separately. Phenomena of micelliza-tion of pure surfactants will not be considered. Spec-troscopic studies on the structure of proteins in mi-celles or bicelles will not be reviewed either. Focusingon a relatively narrow series of events is expected toincrease the usefulness of the present overview. Thissubject has been (brie£y) dealt with previously byLasch [1], as part of his review on the interactionof detergents with lipid vesicles.

2. Turbidimetry, light scattering

Turbidimetry and light scattering are discussed to-gether because, in spite of the di¡erences in the re-quired instrumentation, they are both based on thesame phenomenon, i.e., the scattering of electromag-netic radiation by many particles, and are both usedfor similar purposes. A good elementary descriptionof the principles involved may be found in the text-book by Van Holde et al. [2]. In practice, an increasein turbidity (or light scattering) is interpreted as anincrease in size of the particles in suspension, and theopposite is assumed to occur when turbidity de-creases. This is true as far as the wavelength of theincident radiation is larger than the particle size, i.e.,when the so-called `Rayleigh condition' prevails. Thisis not always the case with certain systems, as will beshown below. However in most cases a decrease inturbidity/light scattering can be safely taken as anindication of membrane solubilization.

2.1. Turbidity measurements

Turbidity is commonly measured in spectropho-tometers, using a wavelength that will not be ab-sorbed by the particles (membranes, micelles) in sus-pension, so that absorbance does not compete withlight scattering. A lower wavelength will increase theintensity of the scattered light, but if it becomes

equal or smaller than the average particle size thenthe `Rayleigh condition' will not hold. A compromiseis usually reached with wavelengths in the 400^500nm range. The simplest instrument will be the bestfor this purpose, since more sophisticated spectro-photometers are usually designed to prevent scat-tered light from reaching the detector.

The assessment of membrane solubilization as adecrease in suspension turbidity is perhaps the bestknown example of a spectroscopic method as appliedto the study of membrane^surfactant interactions. Itwould be impossible to give here even the shortestselection of speci¢c applications, so large is theirnumber. It should be mentioned, however, that tur-bidimetric studies of membrane solubilization werethe basis of the ingenious method developed by Lich-tenberg [3] and Schurtenberger et al. [4] for the quan-titative evaluation of membrane-bound and free de-tergent, thus of è¡ective' detergent/lipid ratios inmembrane solubilization (see also the paper by Lich-tenberg et al. [5], in this issue). In those studies, total(i.e., free+membrane-bound) detergent concentra-tions inducing membrane disruption were measuredat varying membrane concentrations, and e¡ectivedetergent/lipid ratios deduced from the slope of `totaldetergent concentration producing solubilization vs.membrane concentration' plots.

Always on the basis of turbidimetric studies, de laMaza, Parra and co-workers have re¢ned the abovequantitative measurements to obtain reliable esti-mates of other parameters of bilayer solubilizationby detergents, particularly membrane/water partitioncoe¤cients (see, e.g., [6]). Following a similar meth-odology, the decrease in turbidity that accompaniesdetergent solubilization of algal (Phormidium) mem-branes was used by Ochoa de Alda et al. [7] to de-duce changes in free (non-membrane bound) deter-gent during solubilization.

Early studies on turbidity changes induced by Tri-ton X-100 in sonicated liposomes [8,9] showed that,at subsolubilizing concentrations, the detergent in-duced an increase in turbidity. The observation,that has been repeatedly con¢rmed a for a varietyof detergents and vesicular systems (see, e.g.,[10,11]) was interpreted, with the help of electronmicroscopy, in terms of vesicle lysis and reassemblyinto larger, multilamellar structures. The latter, uponincreasing detergent concentration, disintegrate into

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lipid^detergent mixed micelles, with the expectedconcomitant decrease in suspension turbidity.

Otherwise, with systems consisting initially of lip-id/detergent mixed micelles, surfactant removal leadsto an increase in suspension turbidity, due to thereconstitution of bilayered structures. A simple butinformative quantitative treatment of these data hasbeen put forward by Almog et al. [12].

2.2. Light scattering

When unquali¢ed, the expression `light scattering'refers usually to 90³-scattering under steady-stateconditions. (The so-called dynamic light scatteringis discussed in the next section.) Measurements arecarried out most often in conventional spectro£uo-rometers, with both excitation and emission mono-chromators set at the same wavelength (in the 400^600 nm range) and slits wide open. Light scattering isfar more sensitive than turbidity, thus it can be rec-ommended when the concentration of the sample issmall. Also its higher sensitivity allows the use ofhigher wavelengths, thus ensuring Rayleigh condi-tions for larger particle sizes. Examples of the appli-cation of static light scattering in the study of modeland biomembranes can be found in [13,14].

2.3. Dynamic light scattering

This technique is also known as quasielastic lightscattering and photon correlation spectroscopy,among other names. In dynamic light scattering,the intensity of scattered light is monitored in themicrosecond time range domain, versus the secondin static light scattering. The Brownian motion of theparticles induces broadening of the spectrum, in away that is related to their size and shape [15]. Com-mercially available dynamic light scattering instru-ments provide direct information on average particlesize, thus their applicability to solubilization and re-constitution studies. Speci¢c applications include thecharacterization of sphingomyelin^Triton X-305mixed micelles [16], phosphatidylcholine^Triton X-100 mixed micelles [17], myelin basic protein^dode-cylphosphocholine complexes [18], and the e¡ect oftemperature on the bilayer^micelle transition in thephosphatidylcholine^C12E8 system [19], to mentionbut a few examples.

2.4. Time-resolved studies

Kinetic studies of the detergent-induced changes inturbidity or light scattering can provide unique clueson the mechanism of detergent e¡ects. This sort ofwork has been carried out with spectrophotometersequipped with stopped-£ow sampling accessories.These devices allow fast mixing of detergent andmembrane dispersions, so that events occurring inthe millisecond time scale can be conveniently moni-tored. Elamrani and Blume [20] used this techniqueto describe the kinetics of lysophosphatidylcholineincorporation into phosphatidylcholine vesicles(half-times on the order of 50^500 ms), and the slow-er lysophosphatidylcholine-induced vesicle aggrega-tion and/or fusion. With a similar experimental ap-proach, Alonso et al. [21] showed that, upon mixingof liposomes with Triton X-100, a small increase inturbidity occurred in the ¢rst 100 ms, that was at-tributed to detergent insertion in the bilayer. Then,sonicated unilamellar vesicles underwent `fusion' (orrather lysis and reassembly) in the next 20^40 s, asshown by a large increase in turbidity. Multilamellarvesicles, however, after detergent uptake, gave rise tolipid/detergent mixed micelles in a slow process thatcould take hours to be completed. Similar resultswere obtained with C12E8 by Edwards and Almgren[11]. More recently, Saez-Cirion et al. [22] have usedthis technique to show the in£uence of the lipidphase structure on the solubilization abilities of asurfactant. In particular, a £uid-ordered phaseformed by dimyristoylphosphatidylcholine and cho-lesterol (65:35 mole ratio) at 50³C was found to bemuch more resistant to solubilization than a liquidcrystalline phase formed by pure phosphatidylcholineat the same temperatures (Fig. 1). An application ofstopped-£ow kinetics to the solubilization of a cellmembrane (Halobacterium purple membrane) can befound in Viguera et al. [23].

Although poly(ethyleneglycol) is not a detergent,some of its e¡ects on membranes are very similar tothose of surfactants. In particular, poly(ethylenegly-col) induces aggregation, lipid exchange and fusionof sonicated phospholipid vesicles [24,25]. The ki-netics of vesicle aggregation induced by this polymerhas been studied by the stopped-£ow technique byViguera et al. [26]. The results include a descriptionof anomalous light scattering due to non-Rayleigh

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conditions, when vesicle aggregation leads to par-ticles larger than the incident light wavelength.

3. Absorption spectroscopy: visible

No commonly used detergent absorbs electromag-netic radiation in the visible range, thus the applica-tion of absorption spectroscopy is limited to mem-branes containing either natural or externally addedchromophores. The former is the case of light-trans-ducing membranes. In particular, bacteriorhodopsin,the light-absorbing pigment of Halobacterium purple

membranes has been extensively used as a built-inprobe in the study of membrane^surfactant interac-tions [27^29]. Gonzalez-Man¬as and co-workers [30]established a relationship between binding of TritonX-100, spectral changes and purple membrane solu-bilization, showing that the detergent has a complexe¡ect on the retinal chromophore, modifying its mi-croenvironment (detected as a blue-shift in the ab-sorption maximum) and promoting hydrolysis of theretinal-bacteriorhodopsin Schi¡'s base (observed as adecrease in speci¢c absorbance or bleaching). Mem-brane solubilization is also accompanied by retinalisomerization, from all-trans to 13-cis.

This isomerization occurs as well in the absence ofa surfactant when bacteriorhodopsin is transferredfrom light to dark environments. Gonzalez-Man¬aset al. [31,32] showed that Triton X-100 in£uencesthe kinetics of purple membrane dark adaptation,and that bacteriorhodopsin is more readily solubi-lized from dark-adapted than from light-adaptedmembranes. Subsequently, Meyer et al. [33] madegood use of visible spectroscopy to describe the sol-ubilization steps of dark-adapted purple membraneby Triton X-100.

An interesting correlation between bacteriorho-dopsin solubilization and spectral e¡ects in the visi-ble range was observed by del R|o et al. [34]. Ob-serving the e¡ects of a wide variety of detergents,these authors conclude that surfactants containingthe cholane ring (cholate, taurocholate, CHAPS)are virtually unable to solubilize native bacteriorho-dopsin, and they modify but slightly its absorptionspectrum. Conversely, `linear' detergents (Triton X-

6

Fig. 1. Solubilization of lipid bilayers by the non-ionic deter-gent C12E8, as seen by (A) steady-state and (B,C) time-resolvedchanges in suspension turbidity. Two lipid systems are com-pared, one in the LK liquid crystalline phase (pure DMPC at62³C, panel A dotted line, and panel B), and the other one inthe LoK liquid-ordered phase (DMPC/cholesterol, 70:30 moleratio, at 65³C, panel A continuous line, and panel C). Total lip-id concentration is 1 mM. In panel A, lipid vesicles and deter-gent are left to equilibrate for 2 h before measuring turbidity asabsorbance at 500 nm. In panels B,C, equal volumes of lipidvesicles and detergent are mixed in a stopped-£ow apparatus togive a detergent/lipid molar ratio of 5.15:1. Note that the liq-uid-ordered phase appears to be more resistant to solubilizationunder these conditions. From [21].

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100, sodium dodecylsulfate, octylglucoside) that solu-bilize bacteriorhodopsin also produce bleaching, par-tial or total, of the protein chromophore. These dataare probably indicating that a certain £exibility, thatcholane-derived amphiphiles do not posses, is re-quired in the surfactant molecule, in order to elicitthe double process of protein solubilization andbleaching. Structure^function correlations of thistype are not often found in the detergent literature.

The purple membrane of Halobacterium is quiteunique in that its solubilization by detergents is farfrom being a rapid phenomenon. It may take hoursor even days to be completed, when the correspond-ing process takes place within seconds in most othermembranes. The slow kinetics was noted by Dencherand Heyn [35] and by Casadio et al. [36], and studiedin more detail by Viguera et al. [23]. It should benoted that the `purple membrane' consists actuallyof patches of a two-dimensional crystalline array ofbacteriorhodopsin and lipids interspersed in the bac-terium plasma membrane. When puri¢ed, the purplemembrane exists in aqueous suspension in the formof £at sheets, their rigidity preventing them to formvesicles, so that in their perimeter hydrocarbon^water contacts occur. Viguera et al. [23] suggested,from a combination of equilibrium and stopped-£owmeasurements, that the tight crystalline organizationof the purple membrane prevented the insertion ofdetergent monomers in the bulk of the lipid bilayer;instead, the surfactant would bind the periphery ofthe membrane `patches', i.e., the hydrocarbon^watercontact region, and solubilization would take placegradually, from the periphery towards the core of themembrane fragments, at a progressively lower rate asthe amounts of free detergent and detergent-bindingsites are decreased by the previous solubilizationsteps. This complex nature of purple membrane in-teraction with surfactants was also noted by Tan andBirge [37] who studied the solubilization and bleach-ing of bacteriorhodopsin by a series of alkyl-ammo-nium surfactants using time-resolved absorptionspectroscopy in the time scale of minutes.

Rhodopsin is an integral membrane protein foundin the cells of the eye retina. Like bacteriorhodopsin,it contains retinal as a chromophore. Several studiesof rod membrane solubilization and reconstitutionhave made use of its property of absorbing visiblelight [38,39].

3.1. Dyes as spectroscopic probes

Dyes have not been commonly used in the study ofdetergent-membrane interactions, in contrast withthe extensive use of £uorescent or spin probes (seebelow). However, Kaschny and Gon¬i [40] studiedthree dyes, namely merocyanine-540, pinacyanolchloride and Nile red, that could be easily incorpo-rated into membranes. Upon addition of surfactants,these optical probes showed characteristic spectralchanges, each at speci¢c lipid/detergent ratios, sug-gesting that they were reporting on di¡erent aspectsof membrane^surfactant interaction (Fig. 2). The ab-sorption spectrum of merocyanine-540 has a complexshape, and it can be decomposed into bands assignedrespectively to dye monomers, dimers, or large ag-gregates [41]. The spectral parameters (Vmax and in-tegrated intensity) of each component can be sepa-rately analyzed and their variation studied as afunction of detergent/membrane ratios. Gonzalez-Man¬as et al. [42] applied merocyanine-540 to thestudy of the solubilization of phosphatidylcholine bi-layers by Triton X-100, and were able to detect spec-tral changes signaling a variety of events along abroad range of detergent/lipid ratios, from TritonX-100 incorporation into the bilayer to the forma-tion of mixed micelles. The data in Fig. 2 demon-strate the applicability of dyes as spectroscopicprobes in detergent studies.

4. Absorption spectroscopy: infrared

Biological infrared spectroscopy was renovated inthe early eighties, when the new generation of Four-ier-transform infrared (FT-IR) spectrometers becamewidely available. Today all commercially availableinstruments are of the FT-type, thus the techniqueshould better be called again simply infrared (IR), inspite of the charm that the FT letters appear to havefor some users. The great advantage of the `new' IRspectrometers is that spectra can be taken underhighly repetitive conditions, have them digitalizedand operated with. In turn, this allows the subtrac-tion of the water component from spectra of biolog-ical material in aqueous media, thus removing themain di¤culty in the application of IR to biologicalsamples. Basic information on the uses of IR in the

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context of lipids and membranes can be found inArrondo and Gon¬i [43].

The IR spectrum provides information on vibra-tional energy states from all kinds of chemical group-ings belonging to lipids, proteins, surfactants, etc.This is why IR is one of the few techniques thatcan provide comprehensive information on all thecomponents of a membranous system. When appliedto membrane solubilization and reconstitution, thevarious authors have indeed taken advantage of

this fact, focusing on one or the other componentas required. Gon¬i et al. [44] provided some of theearly data in this ¢eld, analyzing the interaction ofsaturated phosphatidylcholines with Triton X-100:The IR spectral position of the CH2 stretching bandsis a good indicator of hydrocarbon chain order, theband being shifted to higher wavenumbers when `dis-order' (i.e., proportion of gauche conformers) in-creases. Typically, a large increase is observed whensaturated phospholipid bilayers undergo the gel-to-

Fig. 2. E¡ects of Triton X-100 on various spectral parameters of phosphatidylcholine bilayers (or of spectroscopic probes therein),with an emphasis on the e¡ects on optical probes. The horizontal lines show the range of lipid/detergent mole ratios at which a par-ticular e¡ect is detected, the sign U indicates the approximate lipid/detergent ratio at which a half-maximal e¡ect is observed. MLV,multilamellar vesicles; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSC, di¡erential scanning cal-orimetry; MER, merocyanine 540; PIN, pinacyanol chloride; NIL, Nile red. From [39].

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£uid transition. Gon¬i et al. [44] showed that TritonX-100 increased the disorder of the acyl chains thusfacilitating the gel-£uid transitions. A similar e¡ectwas observed by Bayerl et al. [45] with sodium deox-ycholate as surfactant. Moreover, at high surfactant/lipid ratios (S:L = 2) deoxycholate was found tobroaden the transition temperature range, and, inter-estingly, increase the proportion of gauche conform-ers in the gel state, while decreasing them in the £uidstate. The latter phenomenon, not seen with TritonX-100, is reminiscent of the e¡ect of cholesterol [46]and must be due to the rigid cholane ring of deoxy-cholate.

Using a more complex system, namely mitochon-drial complex III^phospholipid^Triton X-100 mixedmicelles, and the reconstituted membranes obtainedafter detergent elimination from the mixed micelles,Valpuesta et al. [47] were able to show that surfac-tant removal led to an important rearrangement inthe methylene stretching vibrations of protein andphospholipids. In the same system, those authors de-tected as well an in£uence of detergent on the polarregions of membrane phospholipids, in the form ofchanges in the 1000^1300 cm31 spectral region, cor-responding to phosphate stretching vibration.

In the studies mentioned above, CH2 vibrationsignals arising from lipid, detergent, and eventuallyprotein could not be distinguished from each other.This problem can be circumvented by using isotopicderivatives of some of the components in the mix-ture. In particular, the use of deuterated lipids hasbeen helpful in this context, because the C^D stretch-ing vibrations appear in a spectral region (2050^2250cm31) that is both separate from the one correspond-ing to C^H vibrations (2800^3000 cm31) and unen-cumbered by other IR signals. Echabe et al. [48]studied mixtures of perdeuterated-chain saturatedphosphatidylcholine with natural palmitoylcarnitineor palmitoyl-coenzyme A, two well known metabolicintermediates, the former possessing surfactant prop-erties. The IR data show that palmitoylcarnitinemixes well with the bilayer, and undergoes an or-der-disorder transition together with the main tran-sition of the pure phospholipid, an e¡ect that re-minds of surfactants of the lysophospholipid type[49]. In contrast, under the same conditions palmito-yl-coenzyme A smears out the phospholipid transi-tion, a phenomenon attributed by Echabe et al. [48]

to bilayer interactions with the bulky coenzyme Apolar moiety. More recently, Ma«dler et al. [50]have made use of perdeuterated-chain dimyristoyl-phosphatidylcholine in mixtures with C12E4 to con-struct, with the combined use of IR and di¡erentialscanning calorimetry, a detailed phase diagram of thelipid^detergent system in excess water.

Detergent and membrane components can also beseparately studied by IR picking up vibrationalmodes that correspond only to one class of mole-cules. This was the approach of Pistorius et al. [51]who detected detergent-speci¢c bands in cholate anddodecylmaltoside and used them to quantify the re-sidual amount of detergent left in reconstituted pro-teoliposomes by ratioing the detergent-speci¢c band(carboxylate band of cholate at 1397 cm31, carbohy-drate CÔ stretching band of dodecylmaltoside at1150 cm31) to the lipid-speci¢c phosphate (asymmet-ric PÔ) vibration band at 1235 cm31. Detergent/phospholipid ratios as low as 1:10 could be esti-mated by this method.

The perturbation of proteins, the other main mem-brane component, by detergents has also beenstudied by infrared spectroscopy. For the applicationof IR to membrane proteins see Arrondo and Gon¬i[52]. Arrondo and co-workers, in two di¡erent mem-braneous systems, namely natural sarcoplasmic retic-ulum membranes [53] and reconstituted mitochon-drial complex III [47], have observed that additionof detergent (SDS or Triton X-100) is accompaniedby a decrease in the proportion of protein L-sheetstructure, and the opposite happens when the deter-gent is removed.

5. Fluorescence spectroscopy

The term £uorescence spectroscopy encompasses awide variety of techniques having in common theirbeing based on the phenomenon of £uorescence.Fluorescence techniques are extremely versatile and,like most luminescence techniques, highly sensitive.There is hardly an area of molecular biology thathas not bene¢ted from the use of £uorescence inone or another form, and membrane-detergent inter-actions are not an exception. The book by Lakowicz[54] is an excellent source of information on £uores-cence spectroscopy at an introductory/intermediate

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level. From a more practical point of view, the Mo-lecular Probes web site (www.probes.com) o¡ers awealth of useful information.

Usually each £uorescence probe, or family ofthem, provides a particular kind of information.Therefore, it is not inadequate to discuss the variousforms and applications of £uorescence by looking atthe main probes used in the ¢eld of membranes.ANS (8-anilino-1-naphthalenesulfonate) is com-monly used as a £uorescent probe in membrane stud-ies because, when transferred from a polar (e.g.,aqueous) to non-polar (e.g., membrane or micellar)environment, its £uorescence emission increases no-toriously in intensity, while being shifted to lowerwavelengths. This makes ANS suitable, for instance,in the determination of critical micellar concentra-tions of detergents. DeGrip et al. [39] have usedANS to measure the removal of surfactant from de-tergent/lipid/protein mixed micelles using cyclodex-trins, in a novel procedure for the preparation ofreconstituted proteoliposomes. Alonso et al. [55] per-formed a parallel study of membrane^detergent in-teractions using ANS, believed to be located near thelipid^water or detergent^water interface, and N-phen-yl-1-naphthylamine (NPN), that partitions deeplyinto the hydrophobic matrix of bilayers and micelles.ANS, but not NPN, reported on a release of environ-

mental constraints, i.e., a £uidization, upon additionof surfactant to preformed bilayers.

Laurdan (6-dodecanoyl-2-dimethylaminonaphtha-lene) is a relatively new addition to the collectionof £uorescence probes [56]. This molecule is partic-ularly sensitive to environmental polarity, with a verylarge red shift in the emission maximum when trans-ferred to polar solvents, and virtually no £uorescencein water. In membranes, Laurdan is only found inthe bilayer interior. These properties make it appro-priate for detecting phase changes, as well as forma-tion of microdomains in membranes [56]. Heerklotzet al. [57] have used Laurdan to measure the parti-tion of oligo(ethylene oxide) dodecylethers (C12E2ÿ8)between phospholipid membranes and the aqueousphase (Fig. 3). The experimental data so obtainedallowed these authors to analyze solubilization interms of several useful solubilization parameters,namely the limiting detergent fraction in the mem-branes, the minimal detergent fraction in mixed mi-celles, and the critical detergent concentration inwater.

Pyrene derivatives are used in membrane studiesbecause, under favorable conditions, they can giverise to non-covalent dimers (èxcimers') within thelipid matrix. Excimers have di¡erent £uorescentproperties than monomers (their emission spectra isred-shifted), so that they can be easily detected. Ex-cimer formation is highly dependent on membrane£uidity. Yegutkin [58], using a pyrene probe, ob-served an increased £uidity in rat liver and adiposeplasma membrane upon addition of Triton X-100 atconcentrations above the `critical micellar concentra-tion'. In a di¡erent context, Zhou and Roberts [59]used pyrene-labeled diacylglycerol molecules to studytheir solubilization by a variety of detergents, withthe aim of understanding certain kinetic aspects ofenzymes that utilize or produce diacylglycerols.

With some probes, the anisotropy of £uorescenceemission is used to monitor membrane £uidity. Forthis purpose, polarized excitation light is used, andemission is measured at both parallel and perpendic-ular planes with respect to excitation. The `depolari-zation' of £uorescence emission can be related to themotion of the probe in its speci¢c environment. Di-phenylhexatriene (DPH) is frequently used as a hy-drophobic environment probe in polarization studies.The trimethylammonium derivative (TMA-DPH)

Fig. 3. An example of the use of the £uorescence probe Laur-dan in the study of lipid^detergent interactions. The £uores-cence of Laurdan is usually expressed in terms of a `generalizedpolarization' (GP) parameter, that is computed on the basis ofthe £uorescence intensities at two predetermined wavelengths[55]. The ¢gure shows the Laurdan generalized polarization in1-palmitoyl-2-oleoylphosphatidylcholine/C12E4 mixtures versusthe total mole fraction (xt) of the detergent. Lipid concentra-tions are 5 WM (F), 10 WM (b), 15 WM (R), 25 WM (S),45 WM (E), 100 WM (+), 200 WM (U). From [56].

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provides information on the lipid^water interfaces,where it resides. Koga et al. [60] provided an inter-esting example of the use of DPH and TMA-DPH inmembrane^detergent studies. With a variety of sur-factants of the Span and Tween families applied atsubsolubilizing concentrations on rat intestinalbrush-border membrane vesicles, DPH reported adecreased anisotropy (increased £uidity) for all deter-gents used. In contrast, the anisotropy of the inter-facial probe TMA-DPH decreased with those deter-gents with a lower hydrophile/lipophile balance (e.g.,Tween-81), but increased with the more hydrophilicones (e.g., Tween-20), probably re£ecting the loca-tion of these surfactants within the bilayer.

Advanced £uorescence instrumentation allows themeasurement of parameters such as time-resolved£uorescence anisotropy, and lifetimes of £uorescenceemission. Das [61] applied these powerful methods tothe study of cholate/phospholipid mixtures in thepresence of the probe 3,3P-diethyloxadicarbocyanineiodide, to observe that, in lipid^detergent mixed mi-celles, gradual removal of cholate from the mediumleads to a large increase in rotational correlationtime, corresponding to the formation of vesicles.

Fluorescence by resonance energy transfer is verysensitive to changes in relative orientation or distancebetween two molecules. In this relatively simple tech-nique, two separate £uorescence probes are used, adonor and an acceptor. The system is excited at afrequency that will be absorbed by the donor, butnot by the acceptor. Then, under certain circumstan-ces, requiring short distance and adequate relativeorientation, energy transfer occurs between donorand acceptor, and, when the former is excited, thelatter emits £uorescence at its own characteristicwavelength. Naturally many surfactant-dependentevents in membranes are re£ected by the e¤ciencyof energy transfer between the appropriate mole-cules. Ollivon et al. [62] made good use of this phe-nomenon to describe in detail both the solubilizationand reconstitution of phosphatidylcholine vesicles inthe presence of octylglucoside. Two £uorescent de-rivatives, respectively N-(7-nitro-1,2,3,-benzoxadia-zol-4-yl) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine were the acceptor anddonor molecules.

In the example above, the £uorophore was alwaysan externally added molecule, or probe. This is not

always required since some biological molecules ex-hibit £uorescence by themselves. Tryptophanyl resi-dues in proteins are particularly useful in this respect,their £uorescence being referred to as ìntrinsic' pro-tein £uorescence. The following studies are based onTrp £uorescence and its quenching, or attenuation,by speci¢c reagents. In the above-mentioned investi-gation on mitochondrial complex III^Triton X-100^phospholipid mixed micelles [47] the authors detectedchanges in Trp £uorescence emission as the surfac-tant was added or removed, suggestive of changes inthe protein environment. Moreover, the £uorescentcenters accessible to the water-soluble quencher io-dide, probably located in the outermost protein re-gion, did not experience, on the average, an impor-tant change in microenvironment upon removal ofdetergent. However a signi¢cant proportion of theTrp residues `sensing' the less polar quencher acryl-amide became less readily available to this reagent inthe absence of Triton X-100.

The phenomenon of £uorescence quenching wasgiven a novel use in the ¢eld of membrane^surfactantinteractions by de Foresta et al. [63,64] who usedbrominated lipids and detergents with that purpose.Bromine, either pure or in combination, is a power-ful £uorescence quencher. Brominated lipids can beeasily prepared by the addition of Br2 to unsaturatedfatty acyl chains. Starting with lipids containing dou-ble bonds at various positions along the chain, acollection of molecules may be obtained that, whenincorporated into a bilayer, will contain a quenchingagent located at speci¢c depths. De Foresta et al. [63]applied this procedure to study the delipidationcaused by C12E8 or dodecylmaltoside on sarcoplas-mic reticulum Ca2�-ATPase. In the absence of sur-factant, the intrinsic £uorescence of the ATPase islow, because it is quenched by the bromine atomsin the lipids. Delipidation is accompanied by an in-crease of Trp £uorescence. Parallel studies of enzymefunction showed that full activity required that theATPase hydrophobic surface be occupied by phos-pholipids; binding of only a few detergent moleculeson that hydrophobic surface appeared to be su¤cientto inhibit the enzyme. In a more recent study, deForesta et al. [64] used brominated analogs of dode-cylmaltoside and of 2-O-laurylsucrose. With thissystem detergents caused, even at subsolubilizingconcentrations, quenching of ATPase intrinsic £uo-

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rescence, a phenomenon that could be reverted byaddition of excess non-brominated surfactant. Itwill be interesting to see further developments usingthis experimental approach.

One important point, before leaving this section on£uorescence techniques, is the problem presented inthis kind of studies by contaminant £uorescence aris-ing from reagents or labware. The sensitivity of thetechnique is such that otherwise `pure' reagents maybe inadequate for this purpose. Ironically, one of themain sources of £uorescence contaminations are thedetergents themselves. The aromatic ring of TritonX-100 may give rise to some £uorescence, and a hy-drogenated version of the detergent, otherwise verysimilar in its solubilizing properties, is commerciallyavailable (see [23] for an application). In other occa-sions, £uorescent impurities accompany the surfac-tant preparations. Dijkstra et al. [65] have publisheda method for the removal of £uorescent impuritiesfrom surfactants, in order to use these molecules inconjuction with studies of protein intrinsic £uores-cence.

6. Nuclear magnetic resonance (NMR)

Like £uorescence, nuclear magnetic resonance isextremely versatile, and has found applications inalmost every ¢eld of biology, chemistry, and evenmedicine. The technique reports essentially on themobility of certain nuclei (1H, 2H, 13C, 31P, etc.)that have the property of becoming oriented withrespect to an externally applied magnetic ¢eld. Interand intramolecular interactions modify the local ef-fects of the magnetic ¢eld on a given nucleus, thuscontributing to the richness and complexity of thespectra. NMR can provide very detailed structuraland kinetic information, its main drawback beingprobably its inherent low sensitivity, that makesmandatory the use of large (or very large) amountsof sample for compensation. For an introduction tothe principles and applications of NMR the readermay refer to, e.g., Campbell and Dwek [66], or Sand-ers and Hunter [67].

After the pioneering work by Ribeiro and Dennis[68,69] describing the structure and dynamics of Tri-ton X-100^phospholipid mixed micelles by 1H-NMRand 13C-NMR, two nuclei have been most often used

in the study of membrane^surfactant interactions,namely 31P and 2H. The former occurs naturally inlipids, while deuterium has to be introduced by or-ganic or biological synthesis in the molecules to bestudied, its natural abundance and NMR sensitivitybeing quite low. 31P is particularly useful because it isnaturally present in phospholipids and membranes.The correlation time of 31P nuclei is such that in cellmembranes or large vesicles, the nuclei appear immo-bile, and a broad powder spectrum is produced. Con-versely, in micelles, or in sonicated vesicles, the rapidtumbling of the particles averages out the spectralanisotropy, and 31P nuclei give o¡ a narrow, sym-metric, isotropic signal. Thus vesicle^micelle transi-tions are very easily detected by 31P-NMR. In addi-tion, the technique allows quantitation of theisotropic and anisotropic components, so that solu-bilization and reconstitution can be accurately moni-tored in this way.

31P-NMR was used by Jackson et al. [70] to de-scribe the solubilization of large unilamellar phos-pholipid vesicles by octylglucoside, and later in sim-ilar studies with other surfactants by Gon¬i et al. [44],Paternostre et al. [71], and Otten et al. [19]. Thesestudies show, in addition, a very good correlationbetween 31P-NMR and other methods for detectingvesicle^micelle transitions. 31P-NMR and turbiditywere used in conjunction by King and Marsh [72]to describe the polymorphic behavior of lysophos-phatidylcholine in poly(ethylene glycol)^water mix-tures. Valpuesta et al. [47] took advantage of thistechnique to observe the conversion of mitochondrialcomplex III^phospholipid^Triton X-100 mixed mi-celles into membranous structures upon removal ofdetergent.

2H-NMR was probably introduced in the ¢eld ofmembrane solubilization by detergents in a study byGon¬i et al. [44], using saturated phosphatidylcholinescontaining perdeuterated acyl chains in the form ofmultilamellar vesicles, that were solubilized by TritonX-100. 2H-NMR provides a direct and convenientmeasure of static order in the alkyl chains, sincethe order parameter is related to the quadrupolarsplitting in the spectrum. Subsolubilizing concentra-tions of Triton X-100 (detergent/lipid ratios below1:3) produced a decrease in quadrupolar splitting,thus in acyl chain order. This parameter did notchange at detergent/lipid ratios between 1:3 and

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1:1, while, at higher detergent concentrations, thequadrupolar splitting collapsed, and an isotropic sig-nal appeared instead, marking the vesicle-to-micelletransition. A good agreement was found between 2H-NMR, 31P-NMR, turbidimetric and calorimetric ob-servations of this phenomenon.

A similar combination of techniques was appliedby Otten et al. [19] to study the phosphatidylcholine^C12E8^water system (Fig. 4). Interestingly, these au-thors incorporated temperature changes in their sol-ubilization studies, including conditions above andbelow the gel^£uid transition temperatures of thepure phospholipids. At a phospholipid/detergent mo-lar ratio of 2:1, membranes oriented in the magnetic¢eld could be observed above the transition temper-ature, while cooling the mixture below Tc led to mi-

celle formation. The temperature dependence of 2H-NMR segmental order parameters of the acyl chainsallowed these authors to conclude that the bilayer^micelle transition is the result of an imbalance be-tween the chain and head group repulsion forces.

A related study was performed by Wenk et al. [73],combining 2H-NMR and calorimetry in exploringthe phosphatidylcholine-octylglucoside system. 1-Pal-mitoyl-2-oleoyl-phosphatidylcholine was selectivelydeuterated at the headgroup segments and at di¡er-ent positions of the fatty acyl chains. Measurementsof the quadrupolar splittings indicated that octylglu-coside had almost no in£uence on the lipid head-group region, even at near solubilizing concentra-tions. In contrast, the £uctuations of fatty acylchain segments located in the inner part of the bi-layer, increased strongly with increasing surfactantconcentrations. It is noteworthy that, while Ottenet al. [19] found that C12E8 signi¢cantly disorderedthe headgroup region of the phospholipid at a 2:1lipid/detergent ratio, an equivalent proportion of oc-tylglucoside leaves the headgroup unchanged [73],which could suggest a di¡erent orientation of thesetwo surfactants relative to the phospholipid bilayer,or perhaps be due to the di¡erent size of their polarhead groups.

7. Electron spin resonance (ESR)

This resonance technique, also known as electronparamagnetic resonance (EPR), is based on the ori-entation of unpaired electrons, rather than nuclei, ina magnetic ¢eld. Since the electron magnetic momentis much higher than that of any nuclei, the inherentsensitivity of ESR is correspondingly higher thanthat of NMR. The di¤culty here is that neither bio-molecules (apart from some metalloproteins), nor de-tergents contain unpaired electrons, thus the use ofspin label probes is necessary. 2,2,6,6-tetramethylpi-peridine-N-oxyl (TEMPO), N-oxyl-4P,4P-dimethylox-azolidine (Doxyl) an their derivatives are most oftenused in membrane studies. Both TEMPO and Doxylprobes can be chemically linked to lipid or detergentmolecules at a variety of positions, so that the mem-brane bilayer may be explored at various depths. Theapplications of ESR to biomembranes have been re-viewed by Marsh [74].

Fig. 4. 2H-NMR spectra (left column) and 31P-NMR spectra(right column) of pure alkyl-chain perdeuterated DMPC(DMPC-d54) (a), and of DMPC-d54/C12E8 mixtures at molar ra-tios 3:1 (b), 2:1 (c), 1.5:1 (d), 1:1 (e). Note that the solubiliza-tion is marked by the abolition of spectral anisotropy and theappearance of narrow symmetric signals in both NMR tech-niques. From [18].

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Spin labels are highly sensitive to molecular mo-tion, including the changes in molecular order thataccompany phase transitions, hence their applicabil-ity to studies on membrane^detergent interactions. Infact, a large number of ESR studies have been car-ried out using a variety of membranes and surfac-tants. The invariable result is that detergent incorpo-ration into the bilayer increases the moleculardisorder and mobility of the hydrocarbon chains.To mention but a few speci¢c examples, Sersen etal. [75] analyzed the interactions of bactericidal sur-factants with liposomes prepared from Escherichiacoli isolated lipids. A correlation was observed be-tween the extent of membrane perturbation and thebactericidal potency of N-(1-methyldodecyl)-N,N,N-trimethylammonium bromide and N-(1-methyldodec-yl)-N,N-dimethylamine oxide. The authors proposeda model on the location of surfactant molecules inthe bilayer, according to which at low concentrationsdetergent molecules occupy structural defects in thebilayer, and only when these are ¢lled up do thesurfactants penetrate the bilayer core, expand it lat-erally, and increase the proportion of gauche confor-mations in the hydrocarbon chains.

Glover et al. [76] have also explored by ESR thesurfactant action on bacterial membranes, in this caseusing whole bacterial cells, and a variety of deter-gents. While observing in all cases the usual deter-gent-induced increase in £uidity, no correlation wasseen in this case between enhanced membrane £uidityand biocidal activity. It should be noted, however,that Glover et al. studied surfactants belonging todi¡erent chemical families (quaternary ammoniumcompounds, alkylsulfates, etc.) so that a strict phys-ico-biological correlation is perhaps not to be ex-pected. It is interesting in this respect that Galembecket al. [77], studying the e¡ects of a series of poly-(oxyethylene)n nonylphenols, with n = 9.5^100, onerythrocyte membranes, observed in all cases an in-creased mobility of spin-labeled stearic acid upon ad-dition of the amphiphiles, although some of thesemolecules were hemolytic, while others actually pro-tected erythrocytes against hypotonic hemolysis. Itappears that, while detergents increase membrane £u-idity, this is not enough by itself to cause membranedisruption, instead an imbalance between interactionsat the hydrocarbon chain and polar headgroup levelsis required for bilayer disruption to occur.

The importance of the stratum corneum in skincare and dermatology treatments has been put for-ward repeatedly in the recent years (see paper byLopez et al. in this issue). Kawasaki et al. [78] exam-ined the response of the stratum corneum lipid bi-layer to anionic surfactants (sodium dodecylsulfateand sodium dodecylglutamate) by ESR. The ex-pected increase in mobility was observed, and, inthis case, it appears to be correlated to a decreasein the skin barrier function.

A particularly detailed study of membrane^surfac-tant interactions by ESR was carried out by Gallovaet al. [79]. These authors prepared oriented phospha-tidylcholine bilayers and could calculate various or-der parameters from the angular dependence of thespectral parameters. Hexyltrimethylammonium wasestimated to increase the probability of gauche con-formations of the lipid chains by about 13^14%, anddecrease the e¡ective energy di¡erence between thetrans and gauche conformations by about 420^480J/mol, at a lipid/detergent 2:1 molar ratio. The re-sults are in special agreement with the `free volumemodel', according to which the surfactant orients it-self in the membrane, parallel to the phospholipids.Insertion of the detergent increases the packing den-sity of lipids at the headgroup level, while a freevolume is formed at the alkyl chain level. The freevolume is ¢lled in, due to increased trans^gaucheisomerization (see also [75]).

The sensitivity of spin label probes to molecularmotion has also been applied to the study of protein^surfactant interaction, and particularly to the incor-poration of membrane or amphipathic proteins todetergent micelles. This is the case of studies of bind-ing of myelin basic protein to dodecylphosphocho-line micelles [18], binding of human growth hormoneor human interferon-Q to Brij or Tween [80], or bind-ing of human tissue factor (rhTF) to C12E8 micelles[81]. The early study by Holladay and Wilder [82] onsomatostatin-detergent interactions is singular in thatthe protein itself was spin-labeled. In all of thesecases, ESR was shown to be an excellent methodfor measuring detergent^protein stoichiometriesand, in some occasions, also for exploring the topol-ogy of the resulting complexes.

As mentioned above, some metalloproteins con-tain unpaired electrons that may give rise to ESRe¡ects. One representative example of how this prop-

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erty may be used to explore membrane^detergentinteractions is given in the work by Montoya et al.[83], showing that the photosystem II reaction centerundergoes a conformational change in the presenceof Triton X-100, so that the light-induced spin-po-larized triplet ESR signal is reduced. Interestingly,substitution of dodecylmaltoside for Triton X-100reverses the situation to the native state includingrecovery of the triplet ESR signal.

8. X-ray di¡raction

There is a distinguished tradition of surfactantstudies using X-ray di¡raction. In fact, the classicalstudies by Luzzati on soaps (reviewed in [84]) werethe basis for establishing fundamental concepts inmembrane structure, now widely accepted, the veryconcept of lipid bilayer among them. X-ray scatter-ing and di¡raction techniques have the notorious ad-vantage of being able to provide information on mo-lecular structure, sometimes at the atomic level.However they are highly specialized techniques, re-quiring lengthy and complex procedures for dataanalysis, that preclude their use in most routinework.

A recent study by Angelov et al. [85] uses X-raydi¡raction for examining the e¡ects of octylglucosideon three di¡erent lipids in excess water, namely di-oleoylphosphatidylethanolamine, monoolein and di-palmitoylphosphatidylcholine. When dispersed inwater, these lipids give rise respectively to invertedhexagonal (HII), bicontinuous cubic (Q224), and la-mellar gel (LL0) phases. At lipid/detergent ratios ap-proaching 1:1, octylglucoside destabilizes the HII

phase of the phosphatidylethanolamine, and theQ224 phase of monoolein, converting them into alamellar liquid crystalline (LK) phase. The gel phaseof dipalmitoylphosphatidylcholine is transformedinto a mixture of LK and micellar phases, both con-taining phospholipid and detergent (Fig. 5). The re-sults with dioleoylphosphatidylethanolamine andmonoolein are particularly interesting, because octyl-glucoside appears to form lamellar structures out ofthese essentially non-lamellar lipids, and this may berelevant in relation to membrane protein reconstitu-tion protocols using both non-lamellar lipids and oc-tylglucoside.

9. Assessing membrane permeabilization by detergents

In the previous sections, spectroscopic techniqueshave been described that allow the study of mem-brane solubilization and reconstitution. Howeversurfactants exert an additional e¡ect on bilayers,often at subsolubilizing concentrations, namely theycause the breakdown of the hydrophobic barrier andits permeabilization to solutes. This is a process that

Fig. 5. Scheme of the structural phase changes of lyotropicphases of dioleoylphosphatidylethanolamine (DOPE), mono-olein (MO), and DPPC upon mixing of these lipids with the de-tergent octylglucoside (OG) at a temperature of 30³C. The po-lar groups of OG are drawn in black. HII, inverted hexagonalphase; Q224, inverted bicontinuous cubic phase; LL0 , gel lamel-lar phase; LK, liquid crystalline lamellar phase; M, micellarphase. From [84].

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has not been studied in great detail, and several im-portant questions, e.g., the relationship between de-tergent/lipid ratios and size of the di¡using solutes,have not been addressed to date. We have publishedaccounts of the main procedures used to assess mem-brane leakage induced by soluble amphiphiles[86,87]. Three main spectroscopic methods havebeen applied in these kinds of studies, namely visible,£uorescence and ESR spectroscopy.

9.1. Visible spectroscopy

In a representative study [88], dry phospholipidsare swollen in bu¡er containing FeSCN2�. The re-sulting vesicles are made to react with a 20-fold ex-cess F3 in a stopped-£ow apparatus. External ironcomplexes react with £uoride during the mixing anddead time of the stopped-£ow experiment, thus theobserved absorption changes (in the visible range)re£ect ion di¡usion through the membranes. Addi-tion of the poly(ethylene oxide) detergent OP-10 in-creases membrane permeability to ions, as seen by adramatic decrease of the relaxation times of the re-action between FeSCN2� and F3 [88].

Similarly, glucose may be entrapped in liposomesand its release followed by a colorimetric method,e.g., glucose oxidase/peroxidase [89,90]. When deter-gents are tested on erythrocytes, release of hemoglo-bin (hemolysis) has been assayed by visible spectros-copy, after sedimentation of the detergent-treated redblood cells, in order to test the surfactant e¡ects onmembrane permeability [77].

9.2. Fluorescence techniques

Once again the high sensitivity of £uorescence de-tection, together with relatively accessible instrumen-tation, make this the technique of choice for detec-tion of vesicle leakage in most cases. Water-soluble£uorescence probes are entrapped in liposomes andthe non-entrapped fraction is removed by gel ¢ltra-tion. Probes used for this purpose include 6-carboxy-£uorescein [6,91], terbium/dipicolinic acid [25], andANTS/DPX [92]. It should be noted that theseprobes, and others that could be used, are notequally applicable to any problem, rather a carefulchoice has to be done in each case [87]. De la Maza,Parra and coworkers (see, e.g., [6,91]) have developed

a procedure, following the ideas of Lichtenberg [3]for solubilization, for the quantitative study of deter-gent-induced permeability. As a result, by measuringpermeabilization at varying membrane concentra-tions they were able to compute è¡ective' deter-gent/lipid ratios producing permeability alterationsat detergent concentrations well below those causingsolubilization. Fluorescence probe methods are com-monly used in connection with liposomes, but cellmembrane-derived vesicles have also been loadedwith water-soluble probes [93].

9.3. ESR spectroscopy

The high sensitivity of ESR also ¢nds a naturalapplication in this kind of study. In a typical appli-cation, Chan et al. [94] entrapped a water-solubleTEMPO derivative in liposomes, and reduced thenon-entrapped spin label with the membrane imper-meable reagent ascorbate. Reduced TEMPO doesnot give o¡ an ESR signal. Then these liposomeswere used to detect leakage caused by surface-activecomponents of the blood. In an earlier interestingwork, Miller and Barran [95] loaded macroconidiaof the fungus Fusarium sulphureum with Tempone,then observed the e¡ects of a cationic and an anionicsurfactant (respectively dodecylguanidinium acetateand sodium dodecylsulfate) on membrane permeabil-ity. These authors were able to distinguishbetween detergent-induced water uptake by thecells, membrane permeabilization to divalent cations,and inhibition of active transport function, depend-ing on the species of detergent, and on its concen-tration.

Acknowledgements

Experimental work in the authors' laboratory waspossible through the continuing help from the Span-ish Ministry of Education, the Basque Government,and the University of the Basque Country.

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