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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

A. Amphiphiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
B. Micelles 287
C. Solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
D. Role of equilibration in solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

IV. Aggregation states of detergents in aqueous media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

A. Detergents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
B. Micelle structure 290

V. Aggregation states of phospholipids in aqueous media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

A. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
B. Phospholipid vesicle structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

VI. Aggregation states of phospholipid-detergent mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294

A. Formation of mixed micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
B. Mixed micelle structure 295
C. Effective detergent concentration for mixed micelle formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

VII. Approaches to the solubilization of phospholipids with detergents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

A. Incorporation of detergent into phospholipid vesicles leading to solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
B. Selective solubilization of lipids and proteins in biomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
C. Choice of detergent for solubilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

* Present address: Chevron Research Company, Richmond, PC, phosphatidylcholine; Rt, ratio of total detergent to phos-
CA 94802, U.S.A. pholipid; Re, effective molar ratio of detergent to phospholi-
** To whom correspondence should be addressed. sol
pid; R e , R e at complete solubilization; R TM, R e at the onset of
Abbreviations: CMC, critical micellization concentration; the lamellar/micelle transition; R~, R e in micelles; and R~, R e
MLV, multilamellar vesicles; SUV, small unilamellar vesicles; in solubilizate aggregate structures.

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

286

VIII. Addendum .............................................................................. 301

Acknowledgements ............................................................................ 301

References .................................................................................. 302

1. Summary pure phospholipids. Finally, a description of the

structure of phospholipid-detergent mixed micelles
Most amphiphiles in biological membranes in- and some suggested approaches to the solubiliza-
cluding phospholipids, steroids, and membrane tion of phospholipids will be discussed.
proteins are insoluble amphiphiles and would form Detailed reviews are available which deal with
liquid crystals or insoluble precipitates alone in the properties of detergents used for membrane
aqueous media. Detergents are soluble amphiphiles solubilization [1], micelle formation in general
and above a critical concentration and tempera- [2-4], and the interaction of detergents with pro-
ture form micelles of various sizes and shapes. teins and the use of detergents for the solubiliza-
Much of the recent progress in studying the in- tion of membranes [5,6]. Furthermore, 'facts rele-
soluble amphiphiles is due to the formation of vant to the choice of detergents for particular
thermodynamically stable isotropic solutions of experiments' have been summarized and the gen-
these compounds in the presence of detergents. eral conclusion was reached that ' t h e optimal de-
This process, which is commonly denoted as tergent for a particular membrane or membrane
'solubilization,' involves transformation of lamel- protein has to be found empirically' [1]. This cer-
lar structures into mixed micelles. The information tainly is the present state of the art in the field of
available to date on the solubilization of phos- membrane solubilization, in spite of the large body
pholipids, which constitute the lipid skeleton of of data available on solubilization in many sys-
biomembranes, by the common detergents is dis- tems. Nonetheless, progress in understanding the
cussed in this review, both with respect to the process of solubilization of phospholipids and
kinetics of this process and the structure of the sphingolipids in many detergent systems has been
various phospholipid-detergent mixed micelles made and we wish to summarize our current un-
formed. It is hoped that this discussion will lead to derstanding of this subject. We hope that this
somewhat more useful, although still necessarily information will be helpful in dealing most effec-
fairly empirical, approaches to the solubilization tively with membrane solubilization problems and
of phospholipids by detergents. while we will refer to the effect of detergent solu-
bilization on membrane proteins and approaches
II. Scope of the review to membrane 'reconstitution', comprehensive cov-
erage of these subjects is well beyond the scope
The goal of this review is to summarize the data intended for this review.
available on the solubilization of phospholipids by
detergents with special emphasis on the solubiliz- 111. Basic definitions and considerations
ing power of various detergents, the time it takes
for equilibrium to be reached in the solubilization, IliA. Amphiphiles
and the structure of the detergent-phospholipid
mixed micelles formed. This review is not intended The term amphipathy (Greek: amphi = on both
to be comprehensive. Rather, it describes those sides, of both kinds, dual; pathos = feeling) which
observations which we feel contribute to an under- describes 'the possession of both feelings' toward
standing of these issues. In order to accomplish water, was first described in the now classical book
this, we will first discuss and define amphiphiles, on paraffin chain salts by Hartley [7]. For an
micelles, solubilization, and equilibrium which are interesting recent perspective, see Ref. 8. Amphi-
basic to the subject. Then we will describe the path has now generally been replaced by the word
normal aggregation states of pure detergents and amphiphile (Greek: philos = strong affinity or at-

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 lipo-
phobic 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 hydro-
carbon, and are aliphatic chains, polycyclic moie-
ties 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 preven-
tion 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 be-
havior 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 amphi-
philes, which include the triacylglycerols, di-
acylglycerols, long-chain fatty acids, and choles-
terol 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 sub-
groups: soluble amphiphiles with lyotropic meso-
morphism (the ability to form cubic, hexagonal, or
lamellar liquid crystal structures at high concentra-
tions) and soluble amphiphiles without lyotropic
mesomorphism. The molecules in the first sub-
group form an unstable monolayer (i.e., they are in
equilibrium with bulk solution) and above a criti-
cal concentration (denoted as the critical micelliza-
tion 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
287
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 concentra-
tion. Examples of this subclass include the bile
salts, saponins, and various drugs such as chlor-
promazine. 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 aggre-
gates formed spontaneously in aqueous solutions
of amphiphilic compounds and specifically ex-
cludes those aggregates which form an internal
compartment of aqueous media; these will be re-
ferred 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 hydro-
philic 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 non-
ionic 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 in-
cluding phospholipids, cholesterol, and membrane
proteins are insoluble amphiphiles. Much of the
recent progress in the purification, characteriza-
tion and reconstitution of these membrane compo-
nents 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 insolu-
ble or very slightly soluble in a given solvent by
the introduction of an additional amphiphilic
component or components.' In phospholipid-de-
tergent mixtures, the state of aggregation, at equi-
librium, of course depends on the components and
composition of the mixture. Detailed phase dia-
grams have been documented for some phos-
pholipid-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 con-
ducted on mixtures at equilibrium. Such a state is
probably reached relatively fast if the solubiliza-
tion is done by adding the detergent to unilamellar
phospholipid vesicles or by co-dissolving a phos-
pholipid-detergent mixture obtained by co-lyophi-
lizing 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 concentra-
tion [17] and temperature may also be an im-
portant variable especially at low detergent:lipid
ratios.
At equilibrium, the size and shape of detergent-
phospholipid 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,, be-
cause some of the detergent is present as mono-
mers 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 deter-
gent 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.
[detergent] - [detergent monomers]
Re = [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 bi-
layers into mixed micelles occurs, the composition
of the micelles may be different from that of the
whole dispersion and their structure may be differ-
ent 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

lar dispersion [20]. In turn, homogeneity of the
mixed micelles can be expected to be greater at
equilibrium than at any other state. When phos-
pholipids 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
ratios, it is possible to just add an aqueous solu-
tion of the detergent to dry phospholipid employ-
ing agitation and heating to approach equilibrium
quickly.
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 numer-
ous 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 CH3(CH2)I1OSO3 Na +
Potassium laurate CH3(CH2)toCOO- K +
Sodium cholate

~ . COO- Na +

HO'" v v I-.OH
Cationic +
Cetyltrimethylammoniumbromide CH3(CH2)IsN(CH3)3Br-
Dodecylammoniumchloride CH3(CH2)IINH3CI
Zwinerionic
Dodecyl-N-betaine CH3(CH2) ilfi.I(CH3)2(CH2)2COO-
Lysophosphatidylcholine RCOOCH2
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- ' ~ O(CH2CH20)nH

Dodecyl octaoxyethylene ether CH3(CH2)II(OCH2CH2)sOH

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 tri-
methylammonium bromides, alkylamine hydro-
chlorides), nonionic compounds (e.g., alkylphenyl
polyoxyethylene ethers, alkyl polyoxyethylene
ethers, alkyl pyranosides), and zwitterionic com-
pounds (e.g., lyso-phosphatidylcholine, sulfobe-
taines). Examples are given in Table I. More com-
plete 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 determina-
tion 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, ultra-
violet absorption, optical rotation, etc. The value
of the CMC might therefore depend on the physi-
cal 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 (aggre-
gation 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 discus-
sion 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 globu-
lar) 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 counter-
ions in the immediate vicinity comprise the com-
pact Stern layer (a few angstroms thick). Beyond
this is the diffuse Gouy-Chapman double layer,
containing the bulk of the counterions and extend-
ing 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 par-
tially 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 ul-
tracentrifugation, viscometry, membrane osmome-
try [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 aggre-
gation 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, aggre-
gation 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 ex-
perimental conditions such as temperature, pH,
ionic strength, concentration and the presence of
various additives. For a more extensive compi-
lation 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 and provide many advantages for membrane

MICELLAR WEIGHTS, A G G R E G A T I O N NUMBERS solubilization [ 1,6,16].
A N D CMC FOR SELECTED SURFACTANTS
From Ref. 2, 5, 6, 34, 50. PEG, poly(oxyethylene glycol) V. Aggregation states of phospholipids in aqueous
(polydisperse preparation). media
Surfactant Agg Micellar CMC
VA. Phospholipids
No. weight (mM)

Anionic Biological lipids are a heterogeneous collection

Sodium decyl sulfate 50 14000 33 of amphiphiles that have in common their low
Sodium dodecyl sulfate 62 18000 8.2 solubility in water. The structures of some repre-
Sodium tetradecyl sulfate 138 44000 2.1
sentative lipids are given in Table III. In this
Sodium dodecanoate 56 12 000 24
Sodium cholate 3 1400 14 review, the term phospholipid will be used to
Sodium deoxycholate 7 3 000 5 encompass both glycerophospholipids and sphin-
Cationic golipids. For phospholipids, particular molecular
Decyltrimethylammonium species within classes are grouped together regard-
bromide 48 13 000 65 less of the fatty acid composition. For a particular
Cetyltrimethylammonium phospholipid with a given headgroup, the varia-
bromide 169 62 000 0.92
tion in physical, chemical, and biological proper-
Dodecylammonium
chloride 55 12000 15 ties is the result of the wide variation of fatty acyl
chain compositions. Typically, naturally occurring
Zwitterionic:
Lyso phosphatidylcholine 181 92 000 phospholipids are composed of about half un-
N-Dodecyl betaine 87 26000 0.80 saturated fatty acyl chains mostly on the 2-posi-
N-Tetradecyl betaine 130 43 000 0.06 tion of glycerol and half saturated chains mostly
Nonionic on the 1-position of the glycerol, although there
Triton X-100 140 90000 0.24 are exceptions (the membranes of lung aveoli con-
PEG( 10)nonyl phenol 100 66 000 0.08 tain a large percentage of dipalmitoyl phosphati-
PEG(15)nonyl phenol 80 70000 0.11
dylcholine). Because most solubilization studies
PEG(20)nonyl phenol 62 68 000 0.15
PEG(30)nonyl phenol 55 82000 0.2 have been done with phosphatidylcholine (PC),
PEG(50)nonyl phenol 20 48 000 0.28 Table IV lists some commercially available syn-
PEG(6)dodecanol 400 180000 0.09 thetic and naturally occurring PCs along with their
PEG(8)dodecanol 123 68 000 0.11 usual physical state in water and their thermo-
PEG( 12)dodecanol 81 59 000 0.09
tropic phase transition temperatures (Tm).
PEG(18)dodecanol 51 51000 0.08
PEG(30)dodecanol 55 82000 0.08 In aqueous dispersions, phospholipids assume
PEG(39)dodecanol 19 35 000 - an aggregation state which primarily depends on
PEG(67)dodecanol 7 21000 - the phospholipid fatty acid chain length and con-
centration. Thus, synthetic PC with two identical
fatty acid residues exists in an aqueous medium as
monomer if the acyl chain contains 4 carbon atoms
ful for membrane solubilization differ from ionic or less [52], whereas at concentrations above the
detergents due to the polar headgroups. The polar CMC, the predominant form of PC with chains of
portion of a nonionic surfactant consists of an 6 - 8 carbon atoms is micellar [53,55], and that of
uncharged polar group such as polyoxyethylene PC with longer paraffinic chains is lamellar [54].
chain [50] and for this class of surfactants, the The latter phospholipids are no doubt the most
polar group is usually much larger than the hydro- interesting from the point of view of their biologi-
phobic tail. Because of this, the solution properties cal relevance. These long chain phospholipids ex-
[51] of these surfactants are quite different from hibit several different hydrated phases, a property
those of the ionic surfactants for which classical called lyotropic mesomorphism. When a limiting
micellar behavior and structure are derived [3,40] 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 CH3(CH 2)I4COOH

Palmitic acid
Glycerides CH3(C nH¢)COOCH 2
I
Diglyceride CH3(C nH¢)COOCH

CH2OH
Glycerophospholipids 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 X=H
Phosphatidylcholine X = CH2CH2N(CH3) 3
Phosphatidylethanolamine X = CH2CH2NH 2
Phosphatidylserine X = CH2CH(NH2)(COOH )
Phosphatidylglycerol X = CH2CHOHCH2OH
Sphingolipids CH3(CH2)I2CH = CHCHOH
I
Sphingomyelin C H 3 ( C " H ~') C O N H C H O
I 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 temper-
ature (Tm) is called the thermotropic phase transi-
tion 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 bi-
layer (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 tempera-
tures for some selected PCs are included in Table
IV. While phase transition temperatures are avail-
able 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 phos-
pholipids.
 293

TABLE IV PHOSPHOUPID •

SELECTED PHYSICAL PROPERTIES OF DIACYL PHOS-

PHATIDYLCHOLINES
State in water actually depends on concentration.

Acyl PC name State in Tma Ref.

chain water (°C)
length

2 diacetyl PC monomer 52
3 dipropionyl PC monomer 52
4 dibutyroyl PC monomer 52 MLV LUV SUV
>to,o00 ~ 500-2000 J, 250
6 dihexanoyl PC micelle 53
7 diheptanoyl PC micelle 53
8 dioctanoyl PC micelle 53
AIR H20
12 dilauryl PC bilayer 0 54
14 dimyristoyl PC bilayer 23 54 "20
16 dipalmitoyl PC bilayer 41 54
18 distearoyl PC bilayer 58 54
22 dibehenoyl PC bilayer 75 54 MONOLAYER MI CELLE INVERSE
MPCELLE
18 : 1 dioleoyl PC bilayer - 22 54
mixture egg PC bilayer - 11 54 Fig. I. Aggregated structures formed by phosphatidylcholine
are illustrated schematically. For vesicles, their diameters are
a Thermotropic phase transition. also indicated. Multilamellar vesicles (MLV) and large un-
ilamellar vesicles (LUV) have such large diameters that the
outer surface, compared to the cross sectional area of a phos-
pholipid molecule, is relatively flat. For small unilamellar
VB. Phospholipid vesicle structure vesicles (SUV), however, the outer surface is highly curved as
illustrated. Phospholipids form monolayers at the air/water
W h e n PC is p l a c e d in excess water at a t e m p e r - interface. Synthetic phospholipids with short fatty acid chains
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, can form micelles in aqueous solution without the addition of
detergents. In certain solvent systems, natural phospholipids
myelin tubes (macroscopic, coaxial, cylindrical
form reverse micelles or oil/water microemulsions with a small
sheets) are formed. G e n t l e d i s r u p t i o n of these amount of water in the central core. Adapted with permission
' 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 from Ref. 16.
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 con-
centric lamellae. T h e l i p o s o m e s have m o l e c u l a r Moreover, several ' m e c h a n i c a l techniques' have
weights in the billions. T h e y are illustrated sche- b e e n d e v e l o p e d to reduce the n u m b e r of lamellae
m a t i c a l l y in Fig. 1 along with the o t h e r structures in liposomes. Thus, extrusion of liquid crystalline
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 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
multibilayers, the n u m b e r of lamellae a n d the f o r m a t i o n of smaller vesicles, with fewer lamellae
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 ( a b o u t 1-4). T h e size of the vesicles a p p r o a c h e s
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 p o r e d i a m e t e r of the filters [61,64]. Extrusion
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 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 [61] a n d the average n u m b e r of lamellae is m o r e effective in reducing the size a n d f o r m e d
a b o u t 7 - 1 0 [62]. O n l y a b o u t 10-15% of the phos- vesicles are essentially u n i l a m e l l a r [65-67]. Small
p 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 (220 ,~ d i a m e t e r ) u n i l a m e l l a r vesicles (SUV), ob-
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 ex- t 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
p o s e d to externally a d d e d solubilizing agents. d i s p e r s i o n s [68,69] have been used extensively for
However, larger l i p o s o m e s ( 0 . 5 - 1 0 jam in d i a m e - m o d e l - m e m b r a n e studies.
ter) with fewer lamellae can be o b t a i n e d b y per- 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
m i t t i n g a thinly s p r e a d layer of h y d r a t e d phos- also been d e v e l o p e d a n d vesicles of a whole variety
p h o l i p i d s to swell slowly in distilled water [63]. 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 alter-
ing 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 re-
moval 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 con-
centration 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 pro-
duce 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 com-
monly employed detergent/phospholipid/water
systems as a function of temperature were availa-
ble, 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 con-
taining a large excess of water and ambient to
physiological temperatures, some information is
available on several of the more commonly em-
ployed 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 bi-
layer [14,76,77]. Above a certain concentration,
some detergents may lead to increased permeabil-
ity of the membranes [17] prior to having any
effect on their general structure. As the concentra-
tion of detergent in the bilayer is increased beyond
a critical lamellar/micellar transition concentra-
tion, 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 biologi-
cal 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 dis-
tributes 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 re-
mains below the CMC of the pure detergent
[6,78,79]. Thermodynamic arguments [3] also sug-
gest that the concentration of monomeric deter-
gent 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 solu-
tions. It also implies that the monomer concentra-
tion of the phospholipid is lowered. However, for
long-chain diacyl phospholipids the monomer con-
centration 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 bin-
ding [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

ternary phase diagrams [86] indicate that phos-
pholipid 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 fur-
ther addition of detergent, the micelles become
smaller and more dilute in phospholipid as il-
lustrated schematically in Fig. 2. Thus, the state of
aggregation in a mixture of phospholipid and de-
tergent depends on the ratio of the components as
well as on the temperature, ionic strength, and
many other factors.
BILAYERS BILAYERS MICELLES
+ +
MICELLES
H20
0 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 phosphatidylcho-
line (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.
295
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 ex-
periments are the bile salts, the most commonly
employed being sodium cholate and sodium de-
oxycholate. These naturally occurring amphiphiles
form small micelles in aqueous media and are
capable of solubilizing large quantities of phos-
pholipids (up to about 2 mole phospholipid per
mole bile salt) by forming mixed micelles with
them. It has been suggested that the basic struc-
ture of the mixed micelles is a disk or a phos-
pholipid bilayer, sealed on its hydrophobic edges
by bile salt molecules [12,79]. The bilayer part is
not necessarily flat so that the micelles may de-
viate 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. Decreas-
ing 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 quasielas-
tic 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 perime-
ter 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 ultra-
violet 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 phospholi-
pid ratios of 3 : 1) the micelles have a centrosym-
metric 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 B. "MIXED DISC" MODEL phospholipid, if the general micelle structure is
preserved, then the resulting mixed micelles would
: ~ 1 1 ~ ~ 1 ~ i 1 ~ be either classical oblate ellipsoids or nonclassical
:~ L ' ~ ! ! ~
ECI
.... ~ / ~ l ~
LONGITUDINAL .......
Lt¢lral~ spherical micelles as illustrated in Fig. 4. Light-

CROSS S[CTIONAt ~

Fig. 3. Schematic models for the structure of the bile salt-phos-

phatidylcholine 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

b(
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 phos-

phatidylcholine have been extensively investigated.
At high Triton/phospholipid ratios, the size of the
mixed micelles as determined by gel chromatogra-
phy appears to be increased over that of pure
Triton micelles proportionally to the amount of
the phospholipid present [49]. Geometrical con-
straints 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 poly-
oxyethylene-containing detergent micelles follows
classical divisions of a hydrophobic core sur-
rounded by a polar region; however, it is possible
for portions of the oxyethylene chain which com-
prise 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
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 phos-
pholipid ( < 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 as-
sumed 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.

scattering studies [95] on gangliosides, which form
large micelles themselves in the absence of deter-
gent and on mixed micelles with nonionic deter-
gents [96] indicate that the molecular weight of the
mixed micelles decreases smoothly toward that of
pure detergent micelles at high detergent to gang-
lioside 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 ultracentrifuga-
tion 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 solubi-
lized may be critical with saturated phospholipids
such as dipalmitoyl PC [77,98]. (see Addendum).
VIC. Effective detergent concentration for mixed
micelle formation
In subsection IIIC, we discussed how for struct-
ural purposes the ratio of detergent to phospholi-
pid, Re, in the solubilized mixed micelles should
take into account the monomer concentration of
detergent. (See Eqn. 1) Information on the forma-
tion of lipid detergent mixed micelles available to
297
date allows us to generalize that above some effec-
tive 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
e •
amount of detergent necessary to form an opti-
cally clear solution consisting of stable isotropic
mixed micelles and has been measured by a variety
of techniques such as light-scattering or NMR-in-
tegrated 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 begin-
ning 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.
A~ e ,
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.
[ detergent] _ [ detergent ] _ [ detergentin ]
J t monomersj / micelles
R~ - (2)
[ phospholipid]_ [ phospholipidin ]
[ micelles J
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
R m_
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 mono-
disperse 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 ho-
mogeneous single species detergent) with a distri-
bution 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 corre-
sponding to the R~°l observed experimentally.
From a practical standpoint, R~°l is the point at
which one could be assured of complete solubiliza-
tion. 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 experi-
mentally, but it may be estimated by some physi-
cal methods.
Ideally, RSeat and R s°l would be determined by
examining a series of detergent/phospholipid mix-
tures 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 de-
termined 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 experi-
mentally determined. However, a complete analy-
sis of the validity of this approximation is not
available. One advantage of the nonionic deter-
gents 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 phospholi-
pids 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 effec-
tive ratio of detergent to lipid in the bilayer ex-
ceeds 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 subsec-
tions IIIC and VIC, the ratio might differ consid-
erably from the total ratio of the components in
the dispersion since some detergent would be solu-
ble 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 intermicel-
lar 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 in-
crease in size when PC is solubilized. The mono-
mer concentration of cholate increases signifi-
cantly 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 al-
lowing 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 relation-
ship between the mole ratio of bile salt to phos-

pholipid and the intermicellar concentration of
bile salt for both human bile and cholate; the
results with cholate agreed with those of Shank-
land [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 follow-
ing sequence of events occurs: low (sub-solubiliz-
ing) levels of the detergent partition between the
lipid and aqueous phases, with a partition coeffi-
cient of about 60 favoring the lipid phase. Intro-
duction of subsolubilizing levels of octylglucoside
into the PC vesicles results in increased turbidity
of the vesicle dispersion, probably due to expan-
sion of the PC bilayers a n d / o r aggregation and
fusion of the vesicles. At the same time, the deter-
gent 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 octylgluco-
side concentrations, the turbidity curves peak and
then rapidly decrease, as the vesicles are solubi-
lized.
The octylglucoside to PC ratio at which conver-
sion 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 oc-
tylglucoside 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 turbid-
ity peaks at about R e = 1.5 before decreasing
rapidly, independent of the total phospholipid
concentration. Complete solubilization is only ob-
tained 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
299
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 octylgluco-
- ' e "
side 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 phos-
pholipid, 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 de-
tergent to sphingomyelin, Rt, of about 0.3 [15]
whereas egg PC is solubilized by the same de-
tergent only at a total molar ratio of about 1.5
[13]. Solubilization of egg PC by the anionic de-
tergent 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 concentra-
tion 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
A~ e ,
R~°1 for various detergents must be obtained be-
fore these questions can be resolved.
Clearly, the amount of detergent that a phos-
pholipid bilayer can accommodate depends on the
packing of the phospholipid molecules within the
bilayer, the nature of detergent, and the detergent-
phospholipid 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 Tri-
ton than PC bilayers before saturation is reached
and solubilization starts [18]. Interaction of bile
salt molecules with bilayers may involve introduc-
tion of polar hydroxyl groups into the bilayers'
hydrophobic core, which will have a destabilizing
effect on bile salt-phospholipid mixed vesicles. This
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 phos-
pholipid bilayers (and natural membranes) have
not been systematically studied to date. A great
need exists to develop a more detailed understand-
ing of the principles governing membrane solubili-
zation by detergents. In this review, we have em-
phasized the disruption of lipid bilayers by de-
tergents 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-as-
sociated, extrinsic proteins), are first solubilized in
the form of detergent-protein aggregates. This 'ex-
traction of membrane proteins' is followed, at
higher detergent concentrations, by complete
solubilization of the whole membrane, which re-
sults 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 phospholi-
pids [108]. In addition, the use of low concentra-
tions of several lysophosphatidylcholine analogues
enabled Weltzien et al. [109] to selectively solubi-
lize 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 remain-
ing membrane fractions [114-117]. It is worth
noting that a novel separation of integral mem-
brane 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 ex-
tracted by detergents, the selectivity being depen-
dent on the detergent used as well as on the
solubilized membranes [117,119-123]. As an ex-
ample, 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 sphingomye-
lin [123]. The mechanism of this 'extraction proce-
dure', as well as that of other selective solubiliza-
tion processes is far from being understood and
basic knowledge about the various stages of solu-
bilization is still needed for a systematic use of
differential solubilization in membrane protein
purification.
Another important aspect of membrane solu-
bilization, as a tool in the purification of mem-
brane proteins, is delipidation of the proteins. It is
only at high detergent concentrations that fully
solubilized membranes, which exist in the disper-
sion as lipid-protein-detergent complexes, gradu-
ally transform into fully delipidated protein-deter-
gent mixed micelles [6]. This results in the reduc-
tion of the sedimentation coefficient and the size
of the globular particles observed in electron mi-
croscopy. Purification of solubilized membrane
proteins first requires this delipidation and then
must be followed by separation of the protein-de-
tergent and lipid-detergent mixed micelles by
methods based on their size, density, charge, bind-
ing 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-

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 ac-
tivity [133]. In any event, delipidation of lipid-pro-
tein-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, dis-
sect 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 stud-
ies have been carried out for the purpose of study-
ing 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 fac-
tors have to be considered such as the capability of
the detergent to disaggregate proteins [5], the pos-
sible alteration of the ionic character of native
proteins, the possible interference of the detergent
with physical and chemical assays of the solubi-
lized compound, and the ease of detergent re-
moval. 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-de-
structive. Moreover, some members of the latter
group of detergents have very high CMCs, so that
301
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 capa-
ble 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-[(3-
cholamidopropyl)dimethylammonio]- 1-propane-
sulfonate (CHAPS), [136,137] is very promising as
it possesses 'the useful properties of both the
sulfobetaine-type detergents and the bile salts an-
ions,' although its utility has only been dem-
onstrated in a limited number of systems. Hope-
fully, 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 pre-
cision scanning calorimetry studies on phosphati-
dylcholine-bile salt mixed micelles have been re-
ported [138]. These authors support the structural
dimorphism conclusions of MOiler [93] that spheri-
cal 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 inter-
action above and below the Tm of saturated PC in
large unilamellar vesicles with zwitterionic al-
k 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 con-
centrations [139].
Acknowledgements
We wish to thank the National Science Founda-
tion for support of this work under grant PCM
82-16963. Critical suggestions and stimulating dis-
cussions on solubilization with Dr. Karol J. Mysels
and Professor Alan F. Hofmann at the University
302
of California at San Diego are gratefully acknowl-
edged by E.A.D.
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