Solubilization of phospholipids by detergents structural and kinetic aspects

Biochimica et Biophysica Acta, 737 (1983) 285-304

285

Elsevier Biomedical Press

BBA 85246

S O L U B I L I Z A T I O N OF P H O S P H O L I P I D S BY D E T E R G E N T S S T R U C T U R A L A N D KINETIC A S P E C T S DOV L I C H T E N B E R G a, R O B E R T J. R O B S O N b,. and E D W A R D A. D E N N I S b.**

a Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-A viv University, Ramat- A viv, Tel-A viv 69978 (Israel) and b Department of Chemistry, University of California at San Diego, La Jolla, CA 92093 (U.S.A.) (Received November 4th, 1982)

Contents I.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286

I1.

Scope of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286

III.

Basic definitions and considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Amphiphiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Micelles C. Solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Role of equilibration in solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286 286 287 288 288

IV.

Aggregation states of detergents in aqueous media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Detergents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Micelle structure

289 289 290

V.

Aggregation states of phospholipids in aqueous media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phospholipid vesicle structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

291 291 293

VI.

Aggregation states of phospholipid-detergent mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Formation of mixed micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mixed micelle structure C. Effective detergent concentration for mixed micelle formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

294 294 295 297

VII.

Approaches to the solubilization of phospholipids with detergents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Incorporation of detergent into phospholipid vesicles leading to solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Selective solubilization of lipids and proteins in biomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Choice of detergent for solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

298 298 300 301

* Present address: Chevron Research Company, Richmond, CA 94802, U.S.A. ** To whom correspondence should be addressed. Abbreviations: CMC, critical micellization concentration; MLV, multilamellar vesicles; SUV, small unilamellar vesicles; 0 3 0 4 - 4 1 5 7 / 8 3 / 0 0 0 0 - 0 0 0 0 / $ 0 3 . 0 0 © 1983 Elsevier Science Publishers

PC, phosphatidylcholine; Rt, ratio of total detergent to phospholipid; Re, effective molar ratio of detergent to phospholisol pid; R e , R e at complete solubilization; R TM, R e at the onset of the lamellar/micelle transition; R~, R e in micelles; and R~, R e in solubilizate aggregate structures.

286

VIII. Addendum

..............................................................................

Acknowledgements References

............................................................................

..................................................................................

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

II. Scope of the review The goal of this review is to summarize the data available on the solubilization of phospholipids by detergents with special emphasis on the solubilizing power of various detergents, the time it takes for equilibrium to be reached in the solubilization, and the structure of the detergent-phospholipid mixed micelles formed. This review is not intended to be comprehensive. Rather, it describes those observations which we feel contribute to an understanding of these issues. In order to accomplish this, we will first discuss and define amphiphiles, micelles, solubilization, and equilibrium which are basic to the subject. Then we will describe the normal aggregation states of pure detergents and

301 301 302

pure phospholipids. Finally, a description of the structure of phospholipid-detergent mixed micelles and some suggested approaches to the solubilization of phospholipids will be discussed. Detailed reviews are available which deal with the properties of detergents used for membrane solubilization [1], micelle formation in general [2-4], and the interaction of detergents with proteins and the use of detergents for the solubilization of membranes [5,6]. Furthermore, 'facts relevant to the choice of detergents for particular experiments' have been summarized and the general conclusion was reached that ' t h e optimal detergent for a particular membrane or membrane protein has to be found empirically' [1]. This certainly is the present state of the art in the field of membrane solubilization, in spite of the large body of data available on solubilization in many systems. Nonetheless, progress in understanding the process of solubilization of phospholipids and sphingolipids in many detergent systems has been made and we wish to summarize our current understanding of this subject. We hope that this information will be helpful in dealing most effectively with membrane solubilization problems and while we will refer to the effect of detergent solubilization on membrane proteins and approaches to membrane 'reconstitution', comprehensive coverage of these subjects is well beyond the scope intended for this review.

111. Basic definitions and considerations

IliA. Amphiphiles The term amphipathy (Greek: amphi = on both sides, of both kinds, dual; pathos = feeling) which describes 'the possession of both feelings' toward water, was first described in the now classical book on paraffin chain salts by Hartley [7]. For an interesting recent perspective, see Ref. 8. Amphipath has now generally been replaced by the word amphiphile (Greek: philos = strong affinity or at-

287 traction, liking, loving). This property describes the presence in the same molecule of both polar and non-polar moieties. In aqueous solution these moieties are manifested as hydrophilic or lipophobic portions, and hydrophobic or lipophilic portions. The hydrophilic groups can be charged (anionic, cationic, zwitterionic) or uncharged but polar (polyoxyethylene, polyhydroxy residues, etc.). The hydrophobic groups are usually hydrocarbon, and are aliphatic chains, polycyclic moieties or aromatic groups that are sparingly soluble in water. The hydrophobic portions have a low solubility in water because of the 'hydrophobic effect' [3]. Water-water hydrogen bonds would have to be broken by the insertion of a hydrocarbon into the medium. It should be stressed that the primary origin of the hydrophobic effect is not the attraction of the nonpolar groups, but the prevention of the disruption of the strong attractive forces of water molecules that would have to occur were the hydrophobi,~ chains to be dissolved in water. Amphiphiles have been classified by their behavior in aqueous solution [9]. One class is that of the insoluble amphiphiles. It can be divided into two sub-groups: that of the non-swelling amphiphiles, which include the triacylglycerols, diacylglycerols, long-chain fatty acids, and cholesterol and that of the swelling amphiphiles, which include most phospholipids, monoacylglycerols, and some glycolipids. Both of these sub-groups form stable monolayers at the air/water interface but they differ in that the non-swelling amphiphiles are not present at all in bulk aqueous solution, whereas the swelling amphiphiles form lamellar liquid crystals. A second class is that of the soluble amphiphiles. It can also be divided into two subgroups: soluble amphiphiles with lyotropic mesomorphism (the ability to form cubic, hexagonal, or lamellar liquid crystal structures at high concentrations) and soluble amphiphiles without lyotropic mesomorphism. The molecules in the first subgroup form an unstable monolayer (i.e., they are in equilibrium with bulk solution) and above a critical concentration (denoted as the critical micellization concentration, CMC) and temperature exist as micelles. Examples include salts of long chain fatty acids, lysolecithin, gangliosides, and most anionic, cationic, and nonionic detergents. The

second sub-group of soluble amphiphiles also form unstable monolayers but do not form liquid crystals at high concentrations (probably because of their bulky cyclic or aromatic moieties [6]); micelles, however, are formed above a critical concentration. Examples of this subclass include the bile salts, saponins, and various drugs such as chlorpromazine. Although the above considerations have been developed for lipids, membrane proteins can also be considered as insoluble amphiphiles since when lipid or detergent is removed from them in aqueous solution, they form insoluble precipitates. IIIB. Micelles

Many amphiphiles aggregate in aqueous media to form 'micelles'. The word micelle has several definitions, but in this review it refers to aggregates formed spontaneously in aqueous solutions of amphiphilic compounds and specifically excludes those aggregates which form an internal compartment of aqueous media; these will be referred to as lipid vesicles (sometimes as liposomes). The term micelle was first used by McBain [10] in describing aggregates of soaps and detergents in aqueous solution. The characteristic feature of micelle forming compounds is their 'amphipathic' or 'amphiphilic' nature, whereby the amphipaths are aggregated with the hydrophobic portions shielded from contact with water yet the hydrophilic portions remain wetted. Micelles can be thought of in a general sense as aggregates with a liquid-like core and with their ionic or polar nonionic moieties exposed to water [7]. Upon transfer of the monomer into the micelle, there is loss of hydrocarbon/water interfacial energy since the hydrophobic chain is in contact with other chains and mainly sequestered from water. The transfer also means that the local water structure, where the hydrocarbon moiety was originally located, is decreased, resulting in an increase in entropy (and thus a decrease in free energy). Factors opposing micelle formation include electrostatic repulsion of charged headgroups, repulsion or unfavorable solvation of the bulky polar groups in nonionic surfactants, and loss in translational degrees of freedom (giving a negative entropy change) for the monomer. For a more complete discussion, see Ref. 1 1.

288

II1C. Solubilization Most amphiphiles in biological membranes including phospholipids, cholesterol, and membrane proteins are insoluble amphiphiles. Much of the recent progress in the purification, characterization and reconstitution of these membrane components is due to the possibility of solubilizing them in the form of mixed micelles with soluble amphiphiles. This process can be defined [11] as ' t h e preparation of a thermodynamically stable isotropic solution of a substance normally insoluble or very slightly soluble in a given solvent by the introduction of an additional amphiphilic component or components.' In phospholipid-detergent mixtures, the state of aggregation, at equilibrium, of course depends on the components and composition of the mixture. Detailed phase diagrams have been documented for some phospholipid-detergent-water mixed systems [12] and the molecular details of various mixed micelles have Been described [6,13-18]. Most studies on solubilization in these systems have been devoted to structural aspects, namely to the structure of mixed micelles obtained when sufficiently high detergent concentrations are used to form mixed micelles. These studies were conducted on mixtures at equilibrium. Such a state is probably reached relatively fast if the solubilization is done by adding the detergent to unilamellar phospholipid vesicles or by co-dissolving a phospholipid-detergent mixture obtained by co-lyophilizing a mixed solution of the components in a common organic solvent [t7]. On the other hand, if the detergent solutions are added either to dried phospholipids or to phospholipid multilamellar vesicles, the rate of solubilization is not necessarily rapid. It certainly depends on detergent concentration [17] and temperature may also be an important variable especially at low detergent:lipid ratios. At equilibrium, the size and shape of detergentphospholipid mixed micelles depend on the ratio of detergent to lipid in the micelles. This ratio differs from the total bulk ratio of detergent to phospholipid concentration, defined as R,, because some of the detergent is present as monomers in solution and for detergents with high C M C values this can be important. (The monomer

concentration of phospholipids is so low [3] that it is neglected in this discussion). The concentration of detergent in detergent-lipid mixed micelles will be equal to the difference between the total detergent concentration and its monomer concentration in that mixture. The monomer concentration may be quite different from the C M C determined for the detergent alone. We can define R e as the 'effective' ratio of detergent to phospholipid in aggregates as shown in Eqn. 1. Re =

[detergent] - [detergent monomers] [phospholipid]

Thus, when all of the phospholipid is solubilized and at equilibrium, R e would correspond to the effective ratio of detergent to phospholipid in the mixed micelles. This of course is not the ratio of detergent to phospholipid in each and every micelle, since some distribution of ratios certainly exists [19]. However, the average ratio is equal to R e and normal (Poisson or Gaussian) distributions of solubilizate in the mixed micelles can generally be assumed. Of course, the size and shape of the resulting mixed micelles may also depend on R e.

IlID. Role of equilibration in solubilization When phospholipid multibilayers or smectic mesophases are solubilized by detergents, only the molecules of the outermost bilayers are initially available for interaction with added detergents. Consequently, when transformation of these bilayers into mixed micelles occurs, the composition of the micelles may be different from that of the whole dispersion and their structure may be different from that expected at equilibrium. The rate and final state of equilibration of such systems depend on the detergent concentration. For sodium deoxycholate and phosphatidylcholine (PC), it is accomplished in less than 10 min. at 37°C when the detergent to phospholipid R e is greater than 4 : 1 , but for lower ratios the equilibration takes hours [17]. This is important when phospholipids are solubilized for various studies in which the results may be affected by the structure of the mixed micelles. In membrane reconstitution experiments, the homogeneity of vesicles formed upon removal of the detergent depends on that of the mixed micel-

289 ratios, it is possible to just add an aqueous solution of the detergent to dry phospholipid employing agitation and heating to approach equilibrium quickly.

lar dispersion [20]. In turn, homogeneity of the mixed micelles can be expected to be greater at equilibrium than at any other state. When phospholipids are solubilized in order for them to be substrates of soluble enzymes (most studies have been done with phospholipases [21,22]), the rate of enzymatic reaction may depend on the structure of the micelles in which the substrate is contained and the reproducibility of such enzymatic studies will therefore depend upon whether or not the system is at equilibrium. In practical terms, this suggests that equilibrium states of mixed micelle preparations be used for all experiments. To be sure of obtaining such states, the components may be first co-lyophilized, or the final mixture may be co-sonicated. At high detergent to phospholipid

IV. Aggregation states of detergents in aqueous media

IVA. Detergents Detergents which have proven useful for the solubilization of phospholipids form micelles themselves in aqueous solution. There are numerous excellent discussions of micelles available [2-4,23-30]. The soluble amphiphiles which form

TABLE I COMMON SOLUBLEAMPHIPHILES Adapted from Refs. 5, 6, 11. Chemical name

Structural formula

Anionic

Sodium dodecylsulfate Potassium laurate Sodium cholate

CH3(CH2)I1OSO3 Na + CH3(CH2)toCOO- K + ~ HO'" v

Cationic

Cetyltrimethylammoniumbromide Dodecylammoniumchloride

.

COO- Na +

v I-.OH +

CH3(CH2)IsN(CH3)3BrCH3(CH2)IINH3CI

Zwinerionic

Dodecyl-N-betaine Lysophosphatidylcholine

CH3(CH2) ilfi.I(CH3)2(CH2)2COORCOOCH2 I HOCH O I II + C H 2° P O C H 2CH2 N ( C H 3 )3 I O

Nonionic p-t-Octylphenyl polyoxyethylene ether

(CH3)3CCH2C(CH3)2- ' ~

Dodecyl octaoxyethylene ether

CH3(CH2)II(OCH2CH2)sOH

O(CH2CH20)nH

290 micelles are also often referred to in the literature as surface-active agents, soaps, or surfactants. Amphipathic compounds include common anionic compounds (e.g., sodium alkyl sulfates, potassium alkanoates), cationic compounds (e.g., alkyl trimethylammonium bromides, alkylamine hydrochlorides), nonionic compounds (e.g., alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers, alkyl pyranosides), and zwitterionic compounds (e.g., lyso-phosphatidylcholine, sulfobetaines). Examples are given in Table I. More complete information can be found in Ref. 1. IVB. Micelle structure

The critical micellization concentration (CMC) [31] is a very important factor in detergent studies. It has been defined as the narrow concentration range of surfactants below which no micelles are detected and above which virtually all additional surfactant forms micelles. The CMC determination is usually based on a property whose rate of change with concentration is different above and below this concentration range such as surface tension, conductivity, dye solubilization, ultraviolet absorption, optical rotation, etc. The value of the CMC might therefore depend on the physical property chosen for measurement, and a unique CMC may not be obtained. Nevertheless, the highly co-operative nature of self-association makes the CMC very narrow in most cases. The free amphiphile concentration in equilibrium with the micelles changes slowly with the concentration of micelles [3]. At the same time, the number of surfactant molecules comprising a micelle (aggregation number) generally increases. The CMC is usually sharp enough that the phase-separation model for micelle formation [32] can be used. Mittal and Mukerjee [33] provide a critical discussion of CMCs and a compilation of CMCs for hundreds of compounds in aqueous solutions has been prepared by Mukerjee and Mysels [34]. The molecular organization of amphiphiles within micelles has been under study for some time. McBain and Hoffman [35] considered a lamellar micelle structure [36]. The basic features for the more widely accepted spherical (or globular) ionic micelle in aqueous solvent systems were first discussed by Hartley [7]. The micellar core is

a liquid-like hydrocarbon [37,38], and the surface consists of the polar headgroups. Some percentage of the counterions of the salt are dynamically bound to the micelle. The headgroup and counterions in the immediate vicinity comprise the compact Stern layer (a few angstroms thick). Beyond this is the diffuse Gouy-Chapman double layer, containing the bulk of the counterions and extending up to several hundred angstroms. The ionic micelle should not be looked upon as a perfectly spherical hydrocarbon droplet with a charged surface. The interface is probably rough [39,40], the methylene groups may be partially wet by water [41,42], and geometrical constraints are not always satisfied by purely spherical models. The aliphatic chains contain both all-trans and partially gauche conformations [43,44]. There is Brownian translation as well as Brownian motions of rotation of the entire micelle [8]. The molecular weights of micelles have been determined by many methods. One of the most commonly used is light scattering. Measurement of turbidity as a function of concentration gives the molecular weight [45]. Other methods include ultracentrifugation, viscometry, membrane osmometry [46], and gel filtration [47-49]. The magnitude of the headgroup repulsion is one determinant of micelle size. For example, an increase in ionic strength results in a dramatic increase in aggregation number probably due to a decrease in the repulsion between ionic headgroups. An increase in size of the nonpolar group, as in an elongation of the alkyl chain, also influences micelle size since the hydrophobic nature of the surfactant has been increased. An increase in concentration favors larger micelle sizes with ionic surfactants. Micelles are often considered homogeneous in size, but in fact the aggregation number is centered around a mean and the size distribution can be rather large. Micellar molecular weights, aggregation numbers, and C M C s for some common surfactants are listed in Table II. It should be noted that these properties depend greatly on experimental conditions such as temperature, pH, ionic strength, concentration and the presence of various additives. For a more extensive compilation of each detergent class, the reader is referred to the review by Helenius et al. [1]. Nonionic detergents which are particularly use-

291 TABLE II MICELLAR WEIGHTS, A G G R E G A T I O N NUMBERS A N D CMC FOR SELECTED SURFACTANTS

and provide many advantages for membrane solubilization [ 1,6,16].

From Ref. 2, 5, 6, 34, 50. PEG, poly(oxyethylene glycol) (polydisperse preparation).

V. Aggregation states of phospholipids in aqueous media

Surfactant

VA. Phospholipids

Anionic Sodium Sodium Sodium Sodium Sodium Sodium

decyl sulfate dodecyl sulfate tetradecyl sulfate dodecanoate cholate deoxycholate

Cationic Decyltrimethylammonium bromide Cetyltrimethylammonium bromide Dodecylammonium chloride

Agg No.

Micellar weight

CMC (mM)

50 62 138 56 3 7

14000 18000 44000 12 000 1400 3 000

33 8.2 2.1 24 14 5

48

13 000

65

169

62 000

0.92

55

12000

Zwitterionic: Lyso phosphatidylcholine N-Dodecyl betaine N-Tetradecyl betaine

15

181 87 130

92 000 26000 43 000

0.80 0.06

Nonionic Triton X-100 PEG( 10)nonyl phenol PEG(15)nonyl phenol PEG(20)nonyl phenol PEG(30)nonyl phenol PEG(50)nonyl phenol PEG(6)dodecanol PEG(8)dodecanol PEG( 12)dodecanol PEG(18)dodecanol PEG(30)dodecanol PEG(39)dodecanol PEG(67)dodecanol

140 100 80 62 55 20 400 123 81 51 55 19 7

90000 66 000 70000 68 000 82000 48 000 180000 68 000 59 000 51000 82000 35 000 21000

0.24 0.08 0.11 0.15 0.2 0.28 0.09 0.11 0.09 0.08 0.08 -

ful for membrane solubilization differ from ionic detergents due to the polar headgroups. The polar portion of a nonionic surfactant consists of an uncharged polar group such as polyoxyethylene chain [50] and for this class of surfactants, the polar group is usually much larger than the hydrophobic tail. Because of this, the solution properties [51] of these surfactants are quite different from those of the ionic surfactants for which classical micellar behavior and structure are derived [3,40]

Biological lipids are a heterogeneous collection of amphiphiles that have in common their low solubility in water. The structures of some representative lipids are given in Table III. In this review, the term phospholipid will be used to encompass both glycerophospholipids and sphingolipids. For phospholipids, particular molecular species within classes are grouped together regardless of the fatty acid composition. For a particular phospholipid with a given headgroup, the variation in physical, chemical, and biological properties is the result of the wide variation of fatty acyl chain compositions. Typically, naturally occurring phospholipids are composed of about half unsaturated fatty acyl chains mostly on the 2-position of glycerol and half saturated chains mostly on the 1-position of the glycerol, although there are exceptions (the membranes of lung aveoli contain a large percentage of dipalmitoyl phosphatidylcholine). Because most solubilization studies have been done with phosphatidylcholine (PC), Table IV lists some commercially available synthetic and naturally occurring PCs along with their usual physical state in water and their thermotropic phase transition temperatures (Tm). In aqueous dispersions, phospholipids assume an aggregation state which primarily depends on the phospholipid fatty acid chain length and concentration. Thus, synthetic PC with two identical fatty acid residues exists in an aqueous medium as monomer if the acyl chain contains 4 carbon atoms or less [52], whereas at concentrations above the CMC, the predominant form of PC with chains of 6 - 8 carbon atoms is micellar [53,55], and that of PC with longer paraffinic chains is lamellar [54]. The latter phospholipids are no doubt the most interesting from the point of view of their biological relevance. These long chain phospholipids exhibit several different hydrated phases, a property called lyotropic mesomorphism. When a limiting concentration of water is reached, further addition

292 T A B L E III COMMON

BIOLOGICAL

LIPIDS

For CnH ~, when n = 14 a n d x = 28, the fatty acid is palmitic acid and when n = 16 a n d x = 30, the fatty acid is oleic acid. Class/example

Structure

Fatty acids Palmitic acid Glycerides

CH3(CH 2)I4COOH

Diglyceride

CH3(C nH¢)COOCH

CH3(C nH¢)COOCH 2

I

Glycerophospholipids

CH2OH C H 3( C . H x ) C O O C H 2

I CH3(C nH ~)COOCH

f CH 20 POX

I

O

Phosphatidic acid Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidylglycerol Sphingolipids

X=H X = CH2CH2N(CH3) 3 X = CH2CH2NH 2 X = CH2CH(NH2)(COOH ) X = CH2CHOHCH2OH CH3(CH2)I2CH = CHCHOH

I Sphingomyelin

C H 3 ( C " H ~') C O N H C H

I

O

II

C H 2 0 P O C H 2 C H 2 N ( C H 3 )3

I O

Steroids Cholesterol

results in a two-phase system of water and the maximally hydrated lamellar phase. The lyotropic phases also show thermotropic mesomorphism at certain temperatures. The main transition temperature (Tm) is called the thermotropic phase transition and is associated with a transformation from a liquid crystalline phase to a gel phase. Although pretransition temperatures have been associated with certain changes, in general, a disordering of the hydrocarbon chains in the interior of the bilayer (and concomitant fluidity or microviscosity changes) results when the temperature of an aque-

ous dispersion of phospholipid exceeds the phase transition temperature [56-59]. The Tm depends on the phospholipid headgroup, chain length, and degree of unsaturation. The transition temperatures for some selected PCs are included in Table IV. While phase transition temperatures are available for other phospholipid head group classes, they are not included here as these phospholipids have regretably not yet been carefully studied with regard to detergent solubilization. The Tm must be considered when solubilizing saturated phospholipids.

293 TABLE IV

PHOSPHOUPID •

SELECTED PHYSICAL PROPERTIES OF DIACYL PHOSPHATIDYLCHOLINES State in water actually depends on concentration. Acyl chain length

PC name

State in water

2 3 4 6 7 8 12 14 16 18 22 18 : 1 mixture

diacetyl PC dipropionyl PC dibutyroyl PC dihexanoyl PC diheptanoyl PC dioctanoyl PC dilauryl PC dimyristoyl PC dipalmitoyl PC distearoyl PC dibehenoyl PC dioleoyl PC egg PC

monomer monomer monomer micelle micelle micelle bilayer bilayer bilayer bilayer bilayer bilayer bilayer

Tma (°C)

0 23 41 58 75 - 22 - 11

Ref.

52 52 52 53 53 53 54 54 54 54 54 54 54

a Thermotropic phase transition.

VB. Phospholipid vesicle structure W h e n PC is p l a c e d in excess water at a t e m p e r a t u r e a b o v e its t h e r m o t r o p i c phase transition, myelin tubes (macroscopic, coaxial, cylindrical sheets) are formed. G e n t l e d i s r u p t i o n of these ' t u b e s ' lead to the f o r m a t i o n of m u l t i l a m e l l a r vesicles ( M L V ) s o m e t i m e s called l i p o s o m e s or ' B a n g h a m s o m e s ' [60] c o m p o s e d of spherical concentric lamellae. T h e l i p o s o m e s have m o l e c u l a r weights in the billions. T h e y are illustrated schem a t i c a l l y in Fig. 1 along with the o t h e r structures f o r m e d by p h o s p h a t i d y l c h o l i n e . The size of the multibilayers, the n u m b e r of lamellae a n d the a q u e o u s v o l u m e e n t r a p p e d within t h e m d e p e n d on e x p e r i m e n t a l c o n d i t i o n s of p r e p a r a t i o n . Typically, the average r a d i u s of the l i p o s o m e s is a b o u t 0.7 /~M [61] a n d the average n u m b e r of lamellae is a b o u t 7 - 1 0 [62]. O n l y a b o u t 10-15% of the phosp h o l i p i d c o n t a i n e d in these ' o n i o n skin' aggregates is c o n t a i n e d in the o u t e r m o s t bilayer a n d is exp o s e d to externally a d d e d solubilizing agents. However, larger l i p o s o m e s ( 0 . 5 - 1 0 jam in d i a m e ter) with fewer lamellae can be o b t a i n e d b y perm i t t i n g a thinly s p r e a d layer of h y d r a t e d phosp h o l i p i d s to swell slowly in distilled water [63].

MLV >to,o00 ~

AIR

LUV 500-2000 J,

SUV 250

H20

"20

MONOLAYER

MI CELLE

INVERSE MPCELLE

Fig. I. Aggregated structures formed by phosphatidylcholine are illustrated schematically. For vesicles, their diameters are also indicated. Multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) have such large diameters that the outer surface, compared to the cross sectional area of a phospholipid molecule, is relatively flat. For small unilamellar vesicles (SUV), however, the outer surface is highly curved as illustrated. Phospholipids form monolayers at the air/water interface. Synthetic phospholipids with short fatty acid chains can form micelles in aqueous solution without the addition of detergents. In certain solvent systems, natural phospholipids form reverse micelles or oil/water microemulsions with a small amount of water in the central core. Adapted with permission from Ref. 16.

Moreover, several ' m e c h a n i c a l techniques' have b e e n d e v e l o p e d to reduce the n u m b e r of lamellae in liposomes. Thus, extrusion of liquid crystalline M L V ' s t h r o u g h p o l y c a r b o n a t e filters results in the f o r m a t i o n of smaller vesicles, with fewer lamellae ( a b o u t 1-4). T h e size of the vesicles a p p r o a c h e s the p o r e d i a m e t e r of the filters [61,64]. Extrusion o f M L V s t h r o u g h a F r e n c h pressure cell is even m o r e effective in reducing the size a n d f o r m e d vesicles are essentially u n i l a m e l l a r [65-67]. Small (220 ,~ d i a m e t e r ) u n i l a m e l l a r vesicles (SUV), obt a i n e d by ultrasonic i r r a d i a t i o n of m u l t i l a m e l l a r d i s p e r s i o n s [68,69] have been used extensively for m o d e l - m e m b r a n e studies. A l t e r n a t i v e m e t h o d s of vesicle p r e p a r a t i o n have also been d e v e l o p e d a n d vesicles of a whole variety of c o m p o s i t i o n s a n d sizes can be p r e p a r e d b y

294

these methods. Several methods are based on altering the size of vesicles prepared by sonication, which occurs either spontaneously in the gel phase [70-72] or upon the addition of Ca 2+ to negatively charged vesicles [73]. Other methods are based on the injection of phospholipid solutions in organic solvents (ethanol, ethyl ether or petroleum ether) into aqueous media [74], followed by removal of the organic solvent. The size distribution of the resultant unilamellar vesicles depends on the organic solvent used, the lipid composition and concentration and other experimental details, such as the temperature and the ionic strength. Several other methods are based on the solubilization of the phospholipids by detergents followed by removal of the detergent, which also results in the spontaneous formation of unilamellar vesicles. The size distribution of these vesicles depends on the detergent and lipid used as well as on the concentration of the two components and the rate of detergent removal. Although the details of these dependencies are not known, the procedures based on removal of detergents from dispersions of solubilized lipids can be used empirically to produce vesicle dispersions of various compositions and sizes. For a recent review of this subject, see Szoka and Papahadjopoulos [75]. In summary, solubilization studies generally start with one of the vesicle structures illustrated in Fig. 1 and since most solubilization studies to date have utilized PC as the phospholipid, we have not discussed other types of liquid crystalline aggregates formed by phosphatidylethanolamines and the anionic phospholipids.

VI. Aggregation states of phospholipid-detergent mixtures VIA. Formation of mixed micelles If complete phase diagrams for all of the commonly employed detergent/phospholipid/water systems as a function of temperature were available, one could approach a complete description of the aggregation states which occur in the mixtures. Unfortunately, they are not generally available. However, if we restrict ourselves to systems containing a large excess of water and ambient to physiological temperatures, some information is

available on several of the more commonly employed detergents. Almost always, studies have been carried out on phosphatidylcholine as the lipid and there is need for detailed studies on other phospholipids. However, the interaction of soluble amphiphiles (such as SDS or Triton X-100) with insoluble amphiphiles (such as phospholipids) even in excess water, is complex. When the soluble amphiphile concentration is low, the detergent is found associated with the phospholipid bilayers without much loss in general structure of the bilayer [14,76,77]. Above a certain concentration, some detergents may lead to increased permeability of the membranes [17] prior to having any effect on their general structure. As the concentration of detergent in the bilayer is increased beyond a critical lamellar/micellar transition concentration, mixed micelles are formed. One goal of this review is to summarize studies on the formation and structure of these mixed micelles, as they have a direct application to the solubilization of biological membranes by detergents and to the study of lipolytic enzymes where detergent/phospholipid mixed micelles are used as the substrate matrix. When a detergent is added to phospholipid bilayers in an aqueous milieu, the detergent distributes between the bilayers and the solution. Studies on the interaction of sodium taurocholate, SDS and octyl glucoside with PC bilayers suggest that the concentration of the free detergent remains below the CMC of the pure detergent [6,78,79]. Thermodynamic arguments [3] also suggest that the concentration of monomeric detergent in equilibrium with p h o s p h o l i p i d / d e t e r g e n t mixtures remains below the CMC of the pure detergent. The thermodynamic treatment is similar to that of liquid-vapor equilibria of liquid solutions. It also implies that the monomer concentration of the phospholipid is lowered. However, for long-chain diacyl phospholipids the monomer concentration is so low [3,80] that it can be ignored for most purposes. Evidence from permeability studies on black lipid films in the presence of detergents [81,82], and from fluorescent probe binding [83] and anesthetic binding [84] to liposomes indicates that the bilayers can accommodate some detergent without being disrupted, although there may be other changes. P C / l y s o P C / w a t e r ternary phase diagrams [85] and P C / s o d i u m cholate/water

295

ternary phase diagrams [86] indicate that phospholipid bilayers can incorporate surfactants to some degree and still retain the overall lamellar structure. Recently, N M R techniques have been employed to study the effect of small amounts of detergent on the liquid crystalline phases including bilayer and hexagonal arrangements [87-90]. Mixed micelles begin to form only after the phospholipid bilayers are saturated with detergent. A mixture of detergent-saturated bilayers and phospholipid-saturated mixed micelles should exist until enough detergent is added to convert all the bilayers into mixed micelles [6,14[. Then with further addition of detergent, the micelles become smaller and more dilute in phospholipid as illustrated schematically in Fig. 2. Thus, the state of aggregation in a mixture of phospholipid and detergent depends on the ratio of the components as well as on the temperature, ionic strength, and many other factors.

BILAYERS

+

H20

0

MICELLES

BILAYERS + MICELLES

2

MOLARRATIO (TRITON X-IOO/PHOSPHATIDYLCHOLINE)-X Fig. 2. Schematic diagram of the average composition of the phases formed by Triton X-100 (T) and egg phosphatidylcholine (P) in the presence of an excess of water. This is shown as a function of the molar ratio ( X ) of Triton/phospholipid. For simplicity, the stoichiometry of the phospholipid bilayers (B) in the presence of an excess of Triton is assumed to be l : 1 and the stoichiometry of Triton micelles (M) in the presence of an excess of phospholipid is assumed to be 2:1. The monomer concentration of phospholipid and Triton is negligible and is not indicated. Thus, R t = R e defined herein. Reproduced with permission from Ref. 14.

VIB. M i x e d micelle structure

Information is available on the structure, shape and size of some detergent-phospholipid mixed systems. Among the detergents most widely used in membrane solubilization and reconstitution experiments are the bile salts, the most commonly employed being sodium cholate and sodium deoxycholate. These naturally occurring amphiphiles form small micelles in aqueous media and are capable of solubilizing large quantities of phospholipids (up to about 2 mole phospholipid per mole bile salt) by forming mixed micelles with them. It has been suggested that the basic structure of the mixed micelles is a disk or a phospholipid bilayer, sealed on its hydrophobic edges by bile salt molecules [12,79]. The bilayer part is not necessarily flat so that the micelles may deviate from a disk shape [17]. However, for a disk structure the size and the packing of phospholipid molecules within it would be a function of the molar ratio of bile salt to phospholipid. Decreasing this ratio results in the formation of larger mixed micelles with a hydrodynamic radius of up

to 100 A [20]. Recent studies by Mazer et al. [91] of mixed bile salt-phosphatidylcholine micelles under a variety of experimental conditions using quasielastic light-scattering has led to a modification of the original model of Small [12]. They suggest that bile salts of a fixed stoichiometry are also included within the interior of the bilayer to form a 'mixed bilayer disk' in addition to serving on the perimeter of the disk as originally proposed by Small and co-workers [12]. Both views of these mixed micelles are illustrated schematically in Fig. 3. For mixed micelles with other bile salts, see reference 92. Recent X-ray studies by Mtiller [93] and ultraviolet spectral studies by Claffey and Holzbach [94] present evidence to suggest that while a mixed disk model may be correct at low bile salt to PC ratios, structural dimorphism actually occurs and at higher molar ratios (e.g., bile salt to phospholipid ratios of 3 : 1) the micelles have a centrosymmetric arrangement and a nearly spherical shape [93]. Mazer et al. [91] had suggested that at high bile salt concentrations two kinds of micelles coexist, mixed disk plus pure bile salt micelles. (see Addendum.)

296 A.

SMALL MOOEL

: ~ 1 1 ~ :~ L ' ~ ! ! ~ ECI

"MIXED DISC" MODEL

B.

~ 1 ~ i 1 ~

.... ~ / ~ l ~

LONGITUDINAL

CROSS S[CTIONAt

.......

Lt¢lral~

phospholipid, if the general micelle structure is preserved, then the resulting mixed micelles would be either classical oblate ellipsoids or nonclassical spherical micelles as illustrated in Fig. 4. Light-

~

Fig. 3. Schematic models for the structure of the bile salt-phosphatidylcholine mixed micelle, shown in longitudinal (cut through the disk diameter) and cross section (cut through middle of the hydrocarbon steroid parts and fatty acid chains of bile salts and phosphatidylcholine, respectively). The closed circles and ovals represent the nonionic polar groups of the molecules, and the open circles with negative and positive signs represent the ionic polar parts of the molecules. Both Small's mixed micellar model (A) and Mazer, Benedek and Carey's mixed disk (spelled disc in the figure) model (B) are shown. Note that hydrogen-bonded bile salts are incorporated within the interior of the bilayer in the mixed disk model. Reproduced with permission from Ref. 91.

Mixed micelles of Triton X-100 and phosphatidylcholine have been extensively investigated. At high Triton/phospholipid ratios, the size of the mixed micelles as determined by gel chromatography appears to be increased over that of pure Triton micelles proportionally to the amount of the phospholipid present [49]. Geometrical constraints of the Triton molecule suggest that an oblate ellipsoid is preferred over a prolate ellipsoid for the structure of the Triton micelle [51]. These calculations assume that the structure of the polyoxyethylene-containing detergent micelles follows classical divisions of a hydrophobic core surrounded by a polar region; however, it is possible for portions of the oxyethylene chain which comprise a large portion of the volume of the micelle to actually be embedded in the' hydrophobic core.' If this occurs then the hydrophobic core region can in fact take on a spherical shape and the non-classical micelle may be more spherical than ellipsoidal. With the addition of a small amount of

b( Fig. 4. Schematic view of the oblate ellipsoid model (a) and spherical model (b) for a mixed micelle containing the nonionic surfactant Triton X-100 and a low molecular fraction of phospholipid ( < 0.1). The micelle model shapes were calculated [51] based on a Stokes' radius of 44 A at 40°C and a hydration (taken from the value for the Triton X-100 micelle at 25°C) of 1.3 g w a t e r / g Triton. Using volume/density calculations for the hydrophobic core, (a) is a classical micelle with the shape of an oblate ellipsoid with an approximately 2: l axial ratio. For the spherical micelle model (b), the octylphenyl groups cannot pack in a spherical core to form a classical micelle. Therefore, in this model some oxyethylene units must be included in the hydrophobic core. It is not possible to precisely calculate the arrangement of groups in this non-classical model because one does not have the limits imposed by a distinct hydrophobic/ hydrophilic boundary, but all of the octylphenyl groups are shown in the core plus the relevant portion of the oxyethylene chains that are attached to the octylphenyl groups. It is assumed that the hydrophilic region extends one oxyethylene chain length (16 ~,) beyond the hydrophobic core making the radius of the whole micelle about 44 /~. Reproduced with permission from Ref. 49.

297 scattering studies [95] on gangliosides, which form large micelles themselves in the absence of detergent and on mixed micelles with nonionic detergents [96] indicate that the molecular weight of the mixed micelles decreases smoothly toward that of pure detergent micelles at high detergent to ganglioside ratios. Another system which has been investigated in a rather detailed fashion [15,18,97,98] is that of sphingomyelin and Triton X-100. In this system, Yedgar et al. [15] suggested that sphingomyelin bilayers exist when the Triton to sphingomyelin ratio is lower than 0.3 whereas at higher ratios the system is essentially micellar. For molar ratios between 0.5 and 4, they propose that only mixed micelles are present in the dispersion [15]. Each of these mixed micelles would contain about 200 Triton molecules with 50-400 sphingomyelin molecules. The shape of these micelles has been suggested to be oblate ellipsoids, the long axes of which decreases with increasing Triton to sphingomyelin molar ratio. For mixtures at molar ratios greater than 4, ultracentrifugation data was interpreted by these authors to suggest that mixed micelles of Triton to sphingomyelin at a ratio of 4 : 1 coexist with smaller pure Triton micelles [15]. However, the composition of the smaller Triton micelles was not determined in the ultracentrifugation studies. Other data by Robson and Dennis [98] suggest that these mixed micelles do contain some sphingomyelin and that the distributions may depend on fractionation of the polydisperse Triton and on the Tm of the sphingomyelin used as is the case with dipalmitoyl PC. Robson and Dennis [98] conclude that pure Triton micelles and mixed micelles do not coexist. While complete studies are not available on the effect of Tm on solubilization, the temperature at which phospholipid is solubilized may be critical with saturated phospholipids such as dipalmitoyl PC [77,98]. (see Addendum).

date allows us to generalize that above some effective detergent to phospholipid molar ratio, all of the phospholipid present as multibilayers, vesicles, etc. is converted to mixed micelles; this minimal R e for complete solubilization in which all of the lipid is in mixed micelles, will be referred to as RSOl This would normally correspond to the amount of detergent necessary to form an optically clear solution consisting of stable isotropic mixed micelles and has been measured by a variety of techniques such as light-scattering or NMR-integrated intensities. Thus R s°~ should correspond to the empirically observed R e for a particular system. Another important quantity would be R T M which would correspond to the R e at the beginning of the critical solubilization process when the bilayers are saturated with detergent and the onset of the lamellar to micelle transition occurs to convert the phospholipid into mixed micelles. In Fig. 2, X = R t = R e. R T M would correspond to X = 1 and R S°L would correspond to X = 2. In between g T M and R~°l, both bilayers saturated with detergent and mixed micelles saturated with phospholipid coexist. In this region, the effective detergent to phospholipid ratio in the aggregated structures formed by the solubilizate (bilayers for PC), R~, is defined by Eqn. 2.

VIC. Effective detergent concentration for mixed micelle formation

R m_

In subsection IIIC, we discussed how for structural purposes the ratio of detergent to phospholipid, Re, in the solubilized mixed micelles should take into account the monomer concentration of detergent. (See Eqn. 1) Information on the formation of lipid detergent mixed micelles available to

e

•

A~

e

,

[ detergent] _ [ detergent ] _ [ detergentin ] J t monomersj / micelles R~ [ phospholipid]_ [ phospholipidin ] [ micelles J

(2)

Note that we used the symbol 's' for 'solubilizate aggregates' so that the nomenclature could also be used for phospholipids that may form structures other than bilayers. The detergent to phospholipid ratio in the mixed micelle, Rem, is defined by Eqn. 3. [ detergent] - [ detergent ] _ [ detergentin ] I.monomersj solubilizateaggregatesJ solubilizateaggregates (3) R~e and Rem should each be fairly constant in the region between RSeat and RSe°1. At detergent ratios above -'eRS°l, the R e in the micelles, R~, generally increases with increasing total detergent to phos-

298

pholipid, R t- For a homogeneous surfactant which forms monodisperse micelles solubilizing monodisperse particles of a single phospholipid, the R T M and RSe°j should be sharp. However, for the usual situation of polydisperse micelles (even for a homogeneous single species detergent) with a distribution of micelle sizes, R T M and R s°l may be less sharply defined. Thus, the range of R e values in which both phospholipid particles (multibilayers, vesicles, etc.) and mixed micelles coexist would center around a mean with the upper limit corresponding to the R~°l observed experimentally. From a practical standpoint, R~°l is the point at which one could be assured of complete solubilization. Of course, if solubilization efficiency depends on micelle size, the larger the distribution of sizes, the greater the R~°l that would be observed. The R T M may be much more difficult to detect experimentally, but it may be estimated by some physical methods. Ideally, RSeat and R s°l would be determined by examining a series of detergent/phospholipid mixtures at varying total mole ratios, R t, with a suitable physical method that could detect changes in the aggregation state. For each mixture, the monomer concentration of detergent would be determined and this would be used to calculate R e according to Eqn. 1 and the R e corresponding to R T M and R~°l would be found. In practice, it is difficult to determine the monomer concentration of detergent in phospholipid/detergent mixtures. In some cases, it may be possible to assume that the CMC of the pure detergent approximates the monomer concentration in order to calculate R e for the mixtures. In subsection VIIA, examples are given where this approximation was used as well as when the monomer concentration was experimentally determined. However, a complete analysis of the validity of this approximation is not available. One advantage of the nonionic detergents with very low CMCs is that at high detergent concentrations, the contribution of monomers to R e would be negligible and R e and R t can be assumed equivalent.

VII. Approaches to the solubilization of phospholipids with detergents VIIA. Incorporation of detergent into phospholipid vesicles leading to solubtlization When unilamellar vesicles or membranes are exposed to a detergent, an equilibrium state, which depends on the lipid and detergent as well as on their relative concentrations, can be approached relatively fast [17]. Transformation of all of the vesicles into mixed micelles occurs when the effective ratio of detergent to lipid in the bilayer exceeds a critical level R sol e . This ratio of course depends on the detergent and the lipid but in most cases studied has been found to be in the range of a molar ratio of 0.5 to 3.0. As indicated in subsections IIIC and VIC, the ratio might differ considerably from the total ratio of the components in the dispersion since some detergent would be soluble in the dispersion as monomers. Shankland [99] recognized this in his light scattering studies on PC-cholate mixed micelles, where he was able to determine the free cholate concentration in the presence of mixed micelles. He found that the monomer concentration (referred to as intermicellar concentration) increases significantly with total cholate concentration so the R e would be as much as a factor of 2 to 3 different from the ratio of total PC to cholate in the solution. Sodium cholate forms relatively small charged micelles, which increase in size when PC is solubilized. The monomer concentration of cholate increases significantly as the total cholate concentration is raised above the CMC. The CMC is quite salt dependent and Shankland [99] found that high concentrations of NaC1 lowered the monomer concentration allowing more cholate to be incorporated into the mixed micelles which decreases their average size. This would correspond to an increase in R e. More recently, Duane [100,101] extended these studies to human bile and used equilibrium dialysis to assess the intermicellar concentration of bile salt in equilibrium with mixed micelles of PC and bile salts. This method would not differentiate bile salt monomers from small bile salt micelles and this may also be the case for light scattering, but Duane [100] was able to quantitate the relationship between the mole ratio of bile salt to phos-

299 pholipid and the intermicellar concentration of bile salt for both human bile and cholate; the results with cholate agreed with those of Shankland [99] obtained by light scattering. Recently, it has been shown [78] that upon the addition of the nonionic detergent octylglucoside to large unilamellar vesicles of egg PC, the following sequence of events occurs: low (sub-solubilizing) levels of the detergent partition between the lipid and aqueous phases, with a partition coefficient of about 60 favoring the lipid phase. Introduction of subsolubilizing levels of octylglucoside into the PC vesicles results in increased turbidity of the vesicle dispersion, probably due to expansion of the PC bilayers a n d / o r aggregation and fusion of the vesicles. At the same time, the detergent also causes fluidization of the bilayer, as indicated by the reduction of anisotropy of the emission fluorescence of 6-carboxyfluoresceine contained in the membrane. At higher octylglucoside concentrations, the turbidity curves peak and then rapidly decrease, as the vesicles are solubilized. The octylglucoside to PC ratio at which conversion to mixed micelles begins (turbidity starts de° creasing) depends on the lipid concentration. When it was assumed that the C M C of octylglucoside approximates the monomer concentration of octylglucoside for the purposes of calculating Re, and the C M C of octylglucoside is subtracted from the total octylglucoside concentration and the turbidity is plotted as a function of Re, the turbidity peaks at about R e = 1.5 before decreasing rapidly, independent of the total phospholipid concentration. Complete solubilization is only obtained at about R e = 3.0. Over the range of 1.5 < R e > 3.0, a mixture of mixed micelles and vesicles co-exists giving rise to 31P-NMR signals [78] which are composed of a sharp signal due to micellar PC [102,103] superimposed on a broader signal from vesicular PC [104]. In order to interpret these findings, Jackson et al. [78] assumed that on the average any given vesicle will be ruptured when the octylglucoside to PC ratio in it approaches a value of about 2.0. As implied from the discussion of Mimms et al. [19], when octylglucoside is added to preformed vesicles it can be distributed between the vesicles so that the critical ratio will be reached in some vesicles at an average R e = 1.5, whereas

for other vesicles this critical ratio will only be obtained at an average R e as high as 3.0, which corresponds to R s°l Of course, if the octylglucoside monomer concentration does actually increase some with increased total octylglucoside, as occurs with the charged cholate [99], then the true RSe°l values would be slightly lower. For any mixture of a detergent and a phospholipid, the R s°l appears to depend on both the phospholipid and the detergent. As an example, solubilization of sphingomyelin by Triton X-100 is reported to occur at a total molar ratio of detergent to sphingomyelin, Rt, of about 0.3 [15] whereas egg PC is solubilized by the same detergent only at a total molar ratio of about 1.5 [13]. Solubilization of egg PC by the anionic detergent SDS occurs at a very similar ratio [105] in spite of the very different (much higher) C M C of this detergent in comparison to Triton X-100. Of course for SDS, the R t may depend on concentration due to its higher CMC than Triton, and the use of R e rather than R t was not considered at the time of those experiments. The relevance of the detergent's C M C to the critical solubilizing ratio is not clear at this time. There is the observation that the bile salts cholate and sodium deoxycholate rupture egg PC bilayers at total bile salt to PC ratios of 0.5 and 0.7, respectively [12]. This is in spite of the large difference in CMCs between the two bile salts. In other words, the CMC of a detergent does not necessarily correlate with its solubilizing power of R s°~ but sufficient data on R~°1 for various detergents must be obtained before these questions can be resolved. Clearly, the amount of detergent that a phospholipid bilayer can accommodate depends on the packing of the phospholipid molecules within the bilayer, the nature of detergent, and the detergentphospholipid interactions within the bilayer. Thus, sphingomyelin molecules, being less soluble than PC, are probably packed in their bilayers tighter than PC and, as a consequence, sphingomyelin bilayers can accommodate fewer molecules of Triton than PC bilayers before saturation is reached and solubilization starts [18]. Interaction of bile salt molecules with bilayers may involve introduction of polar hydroxyl groups into the bilayers' hydrophobic core, which will have a destabilizing effect on bile salt-phospholipid mixed vesicles. This - ' e

"

A~

e

,

300 may be the reason for the relatively low R~°~ of bile salt for which mixtures with phospholipids are transformed into micellar structures. Moreover, it can explain the lower molar ratio (0.5) obtained for cholate, as compared to sodium deoxycholate (0.7) as a result of the additional destabilizing effect of the third hydroxyl group present in cholate but not in sodium deoxycholate.

VIIB. Selective solubilization of lipids and proteins in biomembranes Several aspects of the solubilization of phospholipid bilayers (and natural membranes) have not been systematically studied to date. A great need exists to develop a more detailed understanding of the principles governing membrane solubilization by detergents. In this review, we have emphasized the disruption of lipid bilayers by detergents to form detergent-phospholipid mixed micelles. However, it should be noted that in the process-of biological membrane solubilization, there is evidence, in some cases, that the initial step in the detergent attack on the membrane does not involve rupture of the membranes. Instead, some proteins (probably peripheral, membrane-associated, extrinsic proteins), are first solubilized in the form of detergent-protein aggregates. This 'extraction of membrane proteins' is followed, at higher detergent concentrations, by complete solubilization of the whole membrane, which results in a drastic decrease in particle size, with the protein and lipid sedimenting together on a sucrose gradient [ 106,107]. Furthermore, even membrane proteins may be extracted prior to 'complete solubilization' of the lipid. Thus, in the solubilization of retinal rod outer segment disks by octylglucoside, it has been shown that, at intermediate concentrations of the detergent, solubilization of the membrane-bound rhodopsin occurs prior to that of the phospholipids [108]. In addition, the use of low concentrations of several lysophosphatidylcholine analogues enabled Weltzien et al. [109] to selectively solubilize the acyl-CoA: lysolecithin acyltransferase from thymocyte plasma membranes. Such preferential extraction of proteins, which may be due to high affinity of the specific detergent for the extracted proteins, may be extremely valuable in the purifi-

cation of membrane proteins. Selective extraction of proteins, obtained by changing a variety of solubilization parameters [110-113] has previously provided a very helpful tool in extracting various active membrane proteins or removing inactive proteins prior to the solubilization of the remaining membrane fractions [114-117]. It is worth noting that a novel separation of integral membrane proteins from other hydrophilic proteins has been achieved by phase separation of Triton X-114 solubilized membranes above the cloud point of the detergent and this approach may also offer some advantages [ 118.]. Membrane lipids can also be selectively extracted by detergents, the selectivity being dependent on the detergent used as well as on the solubilized membranes [117,119-123]. As an example, the nonionic detergents Triton X-100 and Lubrol PX, at similar concentrations, extracted more phospholipids than proteins from rat liver and rabbit heart membranes, with some specificity for phosphatidylcholine compared to sphingomyelin [123]. The mechanism of this 'extraction procedure', as well as that of other selective solubilization processes is far from being understood and basic knowledge about the various stages of solubilization is still needed for a systematic use of differential solubilization in membrane protein purification. Another important aspect of membrane solubilization, as a tool in the purification of membrane proteins, is delipidation of the proteins. It is only at high detergent concentrations that fully solubilized membranes, which exist in the dispersion as lipid-protein-detergent complexes, gradually transform into fully delipidated protein-detergent mixed micelles [6]. This results in the reduction of the sedimentation coefficient and the size of the globular particles observed in electron microscopy. Purification of solubilized membrane proteins first requires this delipidation and then must be followed by separation of the protein-detergent and lipid-detergent mixed micelles by methods based on their size, density, charge, binding affinity and solvent partitioning [ 106,108,124]. At this stage, cooperative binding of the excess detergent to the protein might occur and this may be accompanied by a conformational change of the protein [3,125]. Such association is very com-

301 mon for ionic detergents (e.g., SDS) [ 126-128] but does not usually occur with nonionic detergents and bile salts [129-132]. Delipidation by the latter mild detergents results in the formation of mixed rnicelles in which the detergent molecules are bound to the hydrophobic domains of the amphiphilic protein in a micelle-like interaction. The membrane-orientation of the protein is very likely preserved in these aggregates, resulting in preservation of the protein conformation and activity [133]. In any event, delipidation of lipid-protein-detergent mixed aggregates can be based on selective solubilization of the lipid from these mixed aggregates. Basic understanding of this type of phospholipid solubilization requires a better understanding of the interaction of various lipids and proteins. Such an approach could lead to a more rational use of detergents to solubilize, dissect and recombine membrane components.

VIIC. Choice of detergent for solubilization The choice of a detergent for the solubilization of phospholipids (and membranes) is presently rather empirical. In most cases, solubilization studies have been carried out for the purpose of studying a specific property of the protein such as the enzymatic activity of a membranous protein or that of a lipolytic enzyme acting on the solubilized phospholipid substrate. Accordingly, the choice of a suitable detergent is usually based on its ability to preserve enzymatic activity. However, other factors have to be considered such as the capability of the detergent to disaggregate proteins [5], the possible alteration of the ionic character of native proteins, the possible interference of the detergent with physical and chemical assays of the solubilized compound, and the ease of detergent removal. At the present time, it is difficult to make any generalizations on the choice of detergents which would be equally applicable to all proteins or all phospholipids. It should however be noted that the nonionic detergents are usually much less harmful than the ionic ones in terms of denaturing solubilized proteins. Among the nonionic detergents, the group of alkyl saccharides appear to be especially non-destructive. Moreover, some members of the latter group of detergents have very high CMCs, so that

their removal is relatively easy. However, their sugar moieties might limit their usefulness to those cases in which sugars do not interfere with planned assays. Other nonionic detergents, especially those of the polyethoxy-type (e.g., Triton X-100) do not significantly affect most physical or chemical properties, but these detergents are less efficient at breaking protein-protein interactions and are not as easily removed, although this can be partially overcome [134]. Several ionic detergents are capable of breaking the latter interactions and the same is true for the zwitterionic N-alkyl sulfobetaines [ 135]. However, both of these groups of detergents are strongly denaturating. The recently developed zwitterionic derivative of cholic acid, 3-[(3cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS), [136,137] is very promising as it possesses 'the useful properties of both the sulfobetaine-type detergents and the bile salts anions,' although its utility has only been demonstrated in a limited number of systems. Hopefully, the development of new detergents as well as new understandings of the mechanisms of effective solubilization will aid further developments in this field.

Addendum Since preparation of this review, results of precision scanning calorimetry studies on phosphatidylcholine-bile salt mixed micelles have been reported [138]. These authors support the structural dimorphism conclusions of MOiler [93] that spherical mixed micelles occur at a n R t greater than about 2 which corresponds to an R e of about 1. Attention is also called to differences in the interaction above and below the Tm of saturated PC in large unilamellar vesicles with zwitterionic alk y l d i m e t h y l a m m o n i o h e x a n o a t e s which also solubilize PC to form mixed micelles at high concentrations [139].

Acknowledgements We wish to thank the National Science Foundation for support of this work under grant PCM 82-16963. Critical suggestions and stimulating discussions on solubilization with Dr. Karol J. Mysels and Professor Alan F. Hofmann at the University

302

of California at San Diego are gratefully acknowledged by E.A.D. References 1 Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. (1979) Methods Enzymol. 56, 734-749 2 Fendler, J.H., Fendler, E.J. (1975) Catalysis in Micellar and Macromolecular Systems, Academic Press, New York 3 Tanford, C. (1980) The Hydrophobic Effect: Formation of Micelles and Biological Membranes (2nd Edn.), Wiley-lnterscience, New York 4 WennerstrOm, H. and Lindman, B. (1979) Physics Rep. 52, 1-86

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