Thermodynamic Characterization of Phospholipids Dispersed

in Ethylammonium Nitrate (a Fused Salt)

W. T A M U R A - L I S , *'~ L. J. L I S , t AND P. J. Q U I N N {
*Obstetrics/Gynecology Research, University of Nebraska Medical Center, Omaha, Nebraska 68105-1065;
t Division of Ontology~Hematology, Chicago Medical School, North Chicago, Illinois 60064; and {.Department of
Biochemistry, King's College, London, Kensington Campus, London W8 7AH, United Kingdom
Received March 25, 1991; accepted August 7, 1991

The phases and transition sequences and mechanisms for phospholipids dispersed in fused salt (ethyl-
ammonium nitrate, EAN) were examined via calorimetry, X-ray diffraction, and freeze-fractureelectron
microscopy. In most cases, complex and hysteretic phase behavior as a function of temperature was
observed or inferred. Dilauroyl-, dimyristoyl-, and dipalmitoylphophatidylcholine; dipalmitoylphospha-
tidyletbanolamine; and dipalmitoylphosphatadic acid were shown to produce nonlamellar phases above
Tm when dispersed in 80% (vol/wt) EAN. However, the lipids distearoylphosphatidylcholine and di-
palmitoylphosphatidylglycerolwere found to produce lamellar phases over the temperature range studied,
0-80°C. In general, phospholipids dispersed in EAN were shown to favor curved surfaces, including the
formation of the HH phase. There is some indication that the degree of curvature produced may be
headgroup or acyl chain dependent. The complete characterization of these systems must involve studies
of the influence of the complete thermal history (i.e., equilibration times and temperatures, rate of change
of temperature with time) of the sample as well as composition and sample temperature. © 1992Academic
Press, Inc.

INTRODUCTION wide variety o f organic solvents ( 3 - 5 ) and 1

The characterization o f lipids in water has
encompassed over 30 years o f continuous
study. N e w or i m p r o v e d techniques, and the
increase in our knowledge o f sample handling
and thermal history, have increased our un-
derstanding about metastability and hysteresis
in these systems. Unfortunately, the integral
role o f water in the determination o f the struc-
tures, transition mechanism, etc., in lipid
model m e m b r a n e systems c a n n o t be ade-
quately described unless comparisons are
m a d e with systems involving a wide variety o f
solvents. Recent efforts have clearly established
that certain salt solutions (including fused salt)
and organic solvents ( 1, 2) modified the pack-
ing within gel bilayer phases and influenced
the transitions between bilayer and nonbilayer
phases. Specifically, it has been shown that a
1To whom correspondence should be addressed at
16006 Arbor Street, Omaha, NE 68130.
M K S C N (6, 7) caused the interdigitation o f
chains in the gel bilayer phase, whereas ethyl-
a m m o n i u m nitrate ( E A N ) 2 induced the for-
m a t i o n o f nonbilayer phases in dipalmitoyl-
phosphatidylcholine ( D P P C ) (8, 9) and di-
palmitoylphosphatidylethanolamine ( D P P E )
(10) at temperatures that normally favor bi-
layer phases in water.
In addition, previous studies have examined
the effect o f substituting water with E A N as
the "solvent" for D P P C (8, 9) and distea-
roylphosphatidylcholine ( D S P C ) ( 1 1 ). Sam-
ples o f D P P C were shown to produce micellar
phases in low vol% E A N (8) and H . phases
2 Abbreviations used: DSC, differential scanning calo-
rimetry; EAN, Ethylammonium nitrate; DLPC, Dilau-
roylphosphatidylcholine; DMPC, Dimyristoylphospha-
tidylcholine; DPPC, Dipalmitoylphosphatidylcholine;
DSPC, Distearoylphosphatidylcholine; DPPE, Dipalmi-
toylphosphatidylethanolamine; DPPG, Dipalmitoylphos-
phatidylglycerol; DPPA, Dipalmitoylphosphatidic acid.

200
0021-9797/92 $3.00
Copyright © I992 by Academic Press, Inc.
All rights of reproduction in any form reserved. Journal q/Cr~lloid and lntelface Science, Vol. 150, No. 1, April 1992
 PHOSPHOLIPIDS IN FUSED SALT
in higher vol% EAN (9) when above the gel-
to-liquid-crystal phase transition. The subgel
phase of DPPC in EAN was argued to have
an acyl chain packing (9) different than that
of DPPC in water (12). Our previous X-ray
diffraction study (9) also indicated that the
subgel phase in this system transformed di-
rectly to the Hn phase when heated at 10°C/
min. The thermal properties of this system
were also examined and showed unique meta-
stable/hysteretic phase behavior when the
sample was cooled and reheated. The single
transition (Lc --~ HI1) observed during the ini-
tial heating scan was not reversible on cooling
and a supercooled hysterefic transition was re-
corded. Further heating cycles produced two
endotherms and one exotherm that eventually
became two endothermic transitions as the
sample equilibrated for longer periods above
Tm. The presence of two endotherms separated
by an exotherm can only be rationalized by
proposing that a more ordered phase is pro-
duced in the exothermic process. One can hy-
pothesize that an L~ --~ L~ transition occurs
during the first endotherm, which is followed
by, or occurs simultaneously with, an L~ --~
Lc transition during the exothermic process.
The Lc phase can be proposed as the inter-
mediate formed by the exothermic process be-
cause it is a more ordered structure and it can
transform directly into the HII phase, which
would occur during the second endothermic
process. The change in the ordering of the acyl
chains of the L~ phase that would be required
to form the HII phase may not be thermody-
namically favored. Samples of DSPC, how-
ever, were less dramatically affected by the
presence of EAN. Both bilayer structure and
thermodynamic parameters appeared to differ
only slightly from that of DSPC in water ( 11 ).
We have focused our attention on a calo-
rimetric study of the effect of ethylammonium
nitrate on a variety of single- and double-
component phospholipid systems. Particular
attention has been paid to the elucidation of
the effect of headgroup, lipid acyl chains, sol-
vent concentration, calorimeter scan rate, and
equilibrium history. The nature of these tran-
201
sitions can be inferred by comparison with our
previous structural studies on DPPC (9) and
DPPE (10) in EAN and those of others on
DPPC (8) and DSPC ( 11 ) in EAN. However,
we have also determined some high temper-
ature phase structures using freeze-fracture
electron microscopy and examined the tran-
sition sequences and nonequilibrium phases
in some lipid systems using real-time X-ray
diffraction techniques. In addition, real-time
X-ray diffraction can provide information
about phase transitions that do not produce a
large enough enthalpy to register during a ca-
lorimetric scan (13). It can be shown that the
unique combination of solvent polarity and
hydrogen bonding establishes conditions that
do not favor the gel and liquid crystal bilayer
phases in these systems.
MATERIALS AND METHODS
All phospholipids were obtained from
Avanti Polar Lipids (Birmingham, AL) and
used without further purification. N-Ethylam-
monium nitrate (EAN) was a gift from Pro-
fessor Barry Ninham. The purification of EAN
has been described in Evans et al. ( 11 ). Lipid
dispersions were prepared by suspending the
lipid in distilled water or EAN (1:4 wt/vol
ratio) at 80°C until a uniform phase was ob-
served. All dispersions were then equilibrated
at ~ 0 ° C for over 1 week. Samples destined
for calorimetric measurements were hermet-
ically sealed in aluminum pans. From 2 to 10
samples were examined for each system.
Transition temperatures and ethalpies were
measured by a Perkin-Elmer DSC-2C. Anal-
yses of the measured thermograms were made
with a Perkin-Elmer thermal analysis data
station using the supplied standard program
to determine the peak enthalpy and onset
temperature of the transition. This system
provides a greater degree of precision than the
DSC-2C with a chart recorder. Enthalpy per
unit area was calibrated using indium
(99.999% pure). The heating and cooling rate
was 2.5°C/min, except where noted, for sam-
ples and calibration. Transition temperatures
Journal of Colloid and Interface Science, VoL 150, No. 1, April 1992
 202
were reproducible between equivalent scans
on similar samples within +0.5 °C while tran-
sition enthalpies varied by less than +5%.
X-ray diffraction patterns were obtained
using the 0.150-nm X-radiation at station 7.2/
3 of the synchrotron radiation source at the
SERC Daresbury Laboratory (14). A cylin-
drically bent single crystal of Ge (15 ) and a
long float mirror were used for monochro-
matization and horizontal focusing, providing
about 2 × 10 9 photons s -~ clown a 0.2-mm
collimator at 2.0 GeV and 100 to 200 mA of
electron beam current. A Keele flat plate cam-
era was used with a sample path of 1 mm.
Scattered X-rays were recorded on a linear de-
tector constructed at the Daresbury Labora-
tory. The dead time between data acquisition
frames was 50 #s with a temporal resolution
of 2 s for each frame. X-ray scattering has been
plotted as a function of reciprocal space ( S =
1/d = 2 sin 0/~) using Teflon (d --- 0.488 mm)
as a calibration standard (16). All mesophase
and subcell spacings were calculated using
Bragg's Law (17). Lamellar phases are rec-
ognized by the characteristic 1:2:3... ratio for
the spacing of the X-ray scattering peaks while
hexagonal phases are recognized by the char-
acteristic 1:V3:2... ratio. X-ray patterns,
however, cannot be used to distinguish be-
tween hexagonal arrays of lipid cylinders (HI)
or water cylinders (HH). Phospholipids are
known to only form HII phases.
Temperature scans were produced by water
baths connected internally to the sample
mount of the X-ray camera. The temperature
of the sample was monitored internally using
a thermocouple placed adjacent to the sample
in the X-ray sample holder.
Before electron microscopic examination,
lipid dispersions were equilibrated at the re-
quired quench temperature for at least 15 min
prior to freezing in a slurry of nitrogen. T h e
frozen samples were fractured at - 1 1 5 ° C in
a Poleron freeze-fracture device and shadowed
with platinum and carbon immediately after
fracture. The replicas were washed with chlo-
roform/methanol (2:1 by vol) and examined
with a Philips EM 301 electron microscope.
Jmtrnal q/'Colloid and lnterj~u'e Science. Vol. 150, No. 1, April 1992
TAMURA-LIS, LIS, A N D Q U I N N
RESULTS A N D DISCUSSION
Calorimetric data were collected for DPPC
samples in various vol% EAN. In these and
all other experiments, the scan rate was 2.5°C/
min. We have recently collected X-ray dif-
fraction patterns of the initial phase which in-
dicate that it indeed has an Lc structure. Char-
acteristic acyl chain wide-angle scattering
peaks at 0.377 and 0.404 were obtained. These
data indicated a somewhat closer packing for
the DP15C in EAN phase than that previously
observed for DPPC in water (12). The first
important observation is that the reduction of
the scan rate from the previously described
10 oC / min (9) enables two endothermic tran-
sitions at ~ 5 4 and 59°C to occur on initial
heating from the subgel to liquid crystal phase
for DPPC in 80% (vol/wt) EAN (see Table
I). It was clear that slowing the transition rate
allowed for the appearance of a tentatively as-
signed L~ phase between the Lo and Hn phases
in this system. It can be inferred that more
time a n d / o r thermal energy than obtained at
10°C/min is required to allow the Lc ~ L ,
phase transition to proceed before the for-
mation of the HH phase when a scan rate of
2.5 ° C / m i n is used. The enthalpies for the two
transitions (Lc --~ L , and L~ --~ HII) observed
when the scan rate was 2 . 5 ° C / m i n were ap-
proximately the same as that obtained for the
one transition (Lc ~ Hn) when 10°C/min
was used. It can be concluded that the Lc --~
H~ transition is an equivalent but alternate
pathway to the usual two-stage L¢ ~ L , and
L~ --~ HH transition sequence. However, the
characteristic two-endotherm and one-exo-
therm transition were retained upon reheating,
even at this scan rate. It can be assumed that
the reheating thermogram was again charac-
teristic of an L~ ~ (L,) --~ Lc -~ Hn transition
sequence that could involve second-order
phase transitions.
The influence o f acyl chain length on the
phase transitions for PCs in 80 wt% EAN or
water is shown in Table I. In all cases, the
samples were equilibrated at 0°C before ex-
amination. Our calorimetric results indicated
 PHOSPHOLIPIDS IN FUSED SALT 203

TABLE I

Thermodynamic Properties of Phospholipids Dispersed in Water or EAN (80% vol/wt)

Tt H, T2 H2 T~ //3
Lipid Solvent (°C) (kJ/mol) (°C) (kJ/tool) (°C) (kJ/mol)

DLPC H20 -3.2 0.37 0.6 0.17

-5.1 0.49 4.8 0.24
-3.1 0.39 2.1 0.16
EAN 27.2 2.70
6.5 2.17
26.3 2.63
DMPC H20 15.6 0.12 24.0 7.30
EAN 42.5 4.80
20.8 2.09
22.3 1.71 24.0 -2.93 45.6 3.56
DPPC H20 20.7 0.60 35.0 0.23 41.0 1.73
29.8 0.12 40.3 1.07
35.3 0.16 40.9 1.02
EAN 53.8 1.80 58.6 3.43
38.1 2.66
43.0 2.30 45.0 -3.91 58.6 4.52
DSPC H20 26.7 0.36 50.2 0.22 53.9 1.93
53.2 1.84
49.4 0.17 53.9 1.81
EAN 68.9 5.21
52.9 1.40 57.2 1.26
57.7 3.00
DPPE H20 25.9 a 38.2 a 63.8 a
63.5
62.7
EAN 38.9 a 56.7
53.7
56.2
DPPG H20 23.2 1.64 38.5 0.19 41.2 1.89
35.3 0.08 40.7 1.99
38.5 0.17 41.1 1.87
EAN 46.9 2.82
46.4 2.31 44.2 0.06
47.1 2.43
DPPA H20 63.4 1.90
63.7 1.89
63.4 1.90
EAN 91.7 --

a Weight of sample not known.

Note.The data are generally presented for the initial heating, cooling, and reheating scans. The transition temperatures
and enthalpies are subscripted 1, 2, or 3 to indicate the first, second, or third calorimetric peak observed during the
heating or cooling scan. All scan rates were 2.5 °C/min.

t h e p r e s e n c e o f a single e n d o t h e r m b e l o w 0 ° C
and a broad endotherm above 0°C. These
t r a n s i t i o n s c a n b e i n t e r p r e t e d as solely i n v o l v -
ing bilayer phases (18). In the presence of
E A N , a single reversible t r a n s i t i o n was again
o b s e r v e d b u t at a t e m p e r a t u r e ~ 2 0 ° C higher
t h a n t h a t for d i l a u r o y l p h o s p h a t i d y l c h o l i n e
(DLPC) in H20. Freeze-fracture electron mi-
croscopy conclusively showed that the higher
temperature phase structures for DLPC in

Journal of Colloid and Interface Scit,nce, Vol. 150,No. I, April 1992

204
EAN involved nonlamellar phases, i.e., Hn and
micellar (Fig. 1 ). DMPC in water has been
shown to typically produce a pre- and main
transition solely involving bilayers (19-22).
The substitution of EAN for H20 allowed
dimyristoylphosphatidylcholine ( D M P C ) to
produce thermograms similar to DPPC in
EAN, even on reheating. The high tempera-
ture phase for DMPC in EAN was again shown
by freeze-fracture electron microscopy to in-
volve nonlamellar, i.e., amorphous structures
(Fig. 1 ). Finally, DSPC in H20 typically pro-
duced sub-, pre-, and main transitions that
have been shown to involve bilayer phases (21,
22). The substitution of EAN for H20 resulted
in the elimination of the sub- and pretransition
and the increase in the main transition tem-
perature (Tin). Although the Tm changed on
reheating, there was no evidence of the char-
acteristic two endotherms separated by an ex-
otherm produced on reheating by DMPC and
DPPC in EAN. Previous X-ray diffraction (13)
showed conclusively that the high temperature
DSPC in EAN phase is a bilayer. In all cases
the transitions observed in EAN had higher
enthalpies than those observed in H20. It can
be concluded that the length of the acyl chain
is a factor in the EAN-induced formation of
nonlamellar phases in phosphatidylcholines.
Phosphatidylcholines with 12, 14, or 16 carbon
linkages in their acyl chain can be induced to
form nonlamellar phases while PCs with 18
carbon linkages form only lamellar phases. It
could be expected that acyl chain length will
also affect the ability of EAN to induce non-
lamellar phases in other lipid systems.
The effect of varying headgroups on the ca-
lorimetric features for dipalmitoylphospholi-
pid derivatives in 80% (vol/wt) EAN is shown
in Table I. The thermograms and phases for
DPPC in water and EAN were discussed
above. Previously (10) we have shown that
the presence of EAN reduces the DPPE Le --~
Hn phase transition temperature by over 40°C
FIG. 1. Electronmicrographsof(a) DLPC in EAN quenched from 54°C, (b) DMPC in EAN quenched
from 74°C, and (c) DPPA in EAN quenched from 104°C. Bars represent 10 t~m.
Journal of Colloid and Interface Science, Vol. 150, No. 1, April 1992
TAMURA-LIS, LIS, AND QUINN
from that observed in water. The heating ther-
mogram for the main transition (L~ --~ Hn)
was reversible in this system. The initial heat-
ing calorimetry scan for dipalmitoylphospha-
tidylglycerol ( D P P G ) in water showns the
presence of three endotherms that can be cor-
related with sub-, pre-, and main phase tran-
sitions, as deduced from the similar tracings
of DPPC in water. Upon subsequent cooling
and reheating, only the pre- and main phase
transitions were reversible. The substitution
of EAN for water causes D P P G to undergo a
single reversible phase transition in the tem-
perature range studied. The phase relation-
ships as deduced by calorimetry for dipalmi-
toylphosphatidic acid (DPPA) in water and
EAN are less complex. Only a single reversible
phase transition was observed in either system.
The transition temperature for the single
transformation in D P P A - E A N dispersions
was ~ 3 0 ° C higher than that observed for
DPPA in water. When charged lipids were ex-
amined in EAN the high temperature phase
was not consistently nonlamellar. Freeze-
fracture electron microscopy showed the pres-
ence of an Hn phase for DPPA (Fig. 1 ) while
a bilayer phase existed for D P P G (data not
shown). In both cases, the lipid does not form
nonbilayer phases in H20 over the tempera-
ture range studied (23, 24). We have exam-
ined, in detail, the phase transitions undergone
by D P P G in H20 and EAN using real-time
X-ray diffraction (Fig. 2) to clarify the above
calorimetric results. The data for D P P G in
water has been previously reported (27). The
subgel phase (25, 26) was induced as the start-
ing phase in this system with a mesophase (d-
spacing) repeat of 5.61 nm and acyl chain dif-
fraction peaks at 0.400 and 0.422 nm. Real-
time X-ray diffraction clearly showed the acyl
chain transitions involved in the sub- and
main phase transitions for D P P G in the pres-
ence of water. The subgel-to-gel phase tran-
sition involved a continuous or second-order
PHOSPHOLIPIDS IN FUSED SALT 205

Journal (~fCotloid and lnterjoce Sctence, Vol. 150, No. 1, April 1992
 206 TAMURA-LIS, LIS, AND QUINN

7
a b

z
z w
w I--
I- z
z

O~lllll )

FIG. 2. X-ray diffraction patterns collected for DPPG in EAN undergoing (a) an initial linear heating
scan of 8.1 ° C / m i n and (b) a linear reheating scan of 7.1 °C/min. Every tenth frame of 2 s duration of a
total data set of 255 frames is shown. (Insets) Every twenty-fifth frame of the data Set is plotted as a function
of inverse repeat spacing.

transition in the acyl chain packing from a
rectangular subcell into a hexagonal subcell
with a repeat of 0.406 nm. The gel-to-liquid-
crystalline phase transition proceeded by a
first-order or two-state transition mechanism
between the L~ phase (d-spacing of 5.61 nm
and acyl chain scattering at 0.415 nm) and the
Lo phase (d-spacing of 6.44 n m and acyl chain
scattering at 0.434 nm). The pretransition was
observed to involve a continuous expansion
of the L~ acyl chain packing from 0.406 to
0.415 nm. Similar acyl chain transitions were
observed for all three phase transitions on re-
heating as observed in the initial heating.
These observations indicated the presence of
a subtransition of low enthalpy on reheating.
The presence of EAN in this system resulted
in the appearance of a single continuous sec-
ond-order D P P G transition on both heating
and reheating involving the Lc (d-spacing of
7.05 n m and acyl chain scattering at 0.376
and 0.402 nm) to Le (d-spacing of 6.72 n m
and acyl chain scattering at 0.407 nm) phase
transition. There was no X-ray diffraction ev-
idence for the formation of any disordered
chain phase for D P P G in EAN over the tem-
perature range studied during either the heat-
ing or the reheating scans. The observed
Journal of Colloid and Interface Science, Vol. 150,No. I, April 1992
D P P G in EAN Lc --~ L~ phase transition was
25°C higher than that observed for the same
phase transition when D P P G was hydrated by
water. It cannot be ruled out that the pre- and
main phase transitions for D P P G in EAN oc-
cur at temperatures greater than the upper
temperature limit of these calorimetric and X-
ray diffraction experiments, i.e., 90°C. These
results are consistent with the previous calo-
rimetry and indicate that the D P P G bilayer
phase is greatly stabilized in the presence of
EAN.
CONCLUSIONS
A number of general conclusions can be
made about the effect o f substitution of EAN
for water in single lipid systems. In all cases,
the enthalpies for the observed transitions are
greater in EAN than in water. This observation
would usually indicate a greater packing den-
sity for the lipid than was observed in appro-
priate X-ray diffraction patterns for DPPC.
The presence of EAN may modify the repul-
sive forces between headgroups causing a
closer molecular packing within the lipid
phase. In most cases the structural rearrange-
ment of the low temperature bilayer phase
caused by EAN results in a nonlamellar phase
 PHOSPHOLIPIDS IN FUSED SALT
forming at higher temperatures. The possible
cause of this phenomenon has been discussed
elsewhere (5, 9, 10). It can be inferred that a
change in the spontaneous radius of curvature
due to a preference for closer lipid packing
may be the driving force favoring the forma-
tion of nonlamellar phases. In most cases, the
phases and transitions observed for lipids in
EAN are dependent on sample history and
equilibration. In certain systems, the presence
of two endotherms separated by an exotherm
during a heating calorimetry scan may be an
indicator of the formation of high temperature
nonlamellar phases. It will be difficult then to
produce equilibrium phase diagrams for these
systems. Therefore, diagrams relating com-
position, temperature, equilibration time, and
scan rate are essential.
Specific cases in which the above observa-
tions were not valid include DSPC and DPPG
in EAN. Since both lipids are known to pack
in relatively dense acyl chain subcells in water,
the presence of EAN may have a minimal ef-
fect on the low temperature phase structure.
Thus these lipids will follow bilayer phase se-
quences. Some influence on the packing
within these phases is indicated by the obser-
vation of greater transition enthalpies when
EAN is present.
ACKNOWLEDGMENTS
We thank Dr. A. P. R. Brain for obtaining electron
micrographs of freeze-fractured samples for various lipids
in EAN, the stafl'ofthe Daresbury Synchrotron Laboratory
for help in obtaining the X-ray diffraction data, and the
Glen H. Brown Liquid Crystal Institute at Kent State
University for use of the calorimeter.
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