177
Biochimica et Biophysics @ ElsevierlNorth-Holland
Acta,
489
Biomedical
(1977)
177-190
Press
BBA 57078
HYDROCARBON
CHAIN DISORDER
IN LIPID BILAYERS
TEMPERATURE DEPENDENT RAMAN SPECTRA PHOSPHATIDYLCHOLINE-WATER GELS
NEHAMA
YELLIN
* and IRA W. LEVIN
OF 1,2-DIACYL
**
Laboratory of Chemical Physics, National Institute of Arthritis, Metabolism Diseases, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)
(Received
April 12th,
and Digestive
1977)
Summary Vibrational Raman spectra of multilayers of 1,2-dimyristoyl, 1,2-dipalmitoyl and 1,2-distearoyl phosphatidylcholine were recorded as a function of temperature for the gel phase from -180°C to values slightly below the pretransition temperature. Temperature profiles for band intensity ratios involving the acyl chain C-C stretching modes define a characteristic temperature T, which denotes the onset of hydrocarbon chain trans-gauche isomerization in the gel. T, for the dimyristoyl (Cl0 chain lengths), dipalmitoyl (Cl6 chain lengths) and distearoyl (Cl8 chain lengths) systems are approximately -40, -40 and 5”C, respectively. The higher T, value for distearoyl phosphatidylcholine is associated with increasing interactions between the terminal chain areas of the individual monolayers forming the bilayer unit. This increased interaction between bilayer halves is presumably a consequence of the larger angle through which the hydrocarbon chains are tilted in the Cl8 phospholipid gel. Temperature-dependent peak height intensities for the 3000-2800 C-H stretching region provide convenient probes for quantitatively describing the increase in lateral chain packing disorder as the gel assemblies are warmed. The temperature profiles for various methylene mode peak height ratios indicate that both the inter- (lateral packing) and intramolecular (tram-gauche) disordering processes are related and are initiated at nearly the same temperatures for each lipid system. Increases in the van ‘t Hoff enthalpies which result from the disordering of the lipid gel matrix by a lattice expansion were estimated for to be approximately 1.5, 1.2 and 3.2 kcal/ the Ci4, Ci, and Cl8 phospholipids mol, respectively.
* Present address: Sores Nuclear Research Center, ** To whom correspondence should be addressed.
Yavne,
Israel.
178
Introduction For a variety of biomembranes, the gel bilayer form is uniquely characterized by the introduction of lateral domains of aggregated particles. This phase separation process is presumably induced by a liquid crystalline - gel phase transition in which the lamellar phospholipids acquire a high degree of both inter- and intramolecular order as the lipid matrix solidifies from its fluid, high temperature state (see refs. 1-3 and additional references therein). In an effort to clarify the specific lipid conformational changes and packing rearrangements that are involved in gel formation, we recently applied vibrational Raman spectroscopic probes toward describing lipid assemblies in a variety of liposoma1 preparations [ 4,5]. Specifically, for a series of multilamellar phospholipid bilayers dispersions we emphasized the remaining conformational tram-gauche disorder, or relative flexibility, inherent in the lipid hydrocarbon chains at temperatures significantly below the bilayer phase transition [5]. In these earlier studies [ 51, t,he deviations from an ideal all-trans lipid chain matrix were estimated from variations in spectral intensity of the temperature dependent carbon-carbon (C-C) stretching modes for the lipid. In order to define more completely the lipid-lipid interactions relating both to the dynamics of gel formation and to the stability of the zones of phase segregated lipids, we examine in the present study the low temperature spectral behavior of several structurally sensitive vibrational Raman features, in addition to chain stretching displacements, of bilayer gels of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholine-water liposomes. The various spectral changes are interpreted in terms of concomitant intrachain and lateral intermolecular disordering effects within the hydrocarbon portion of the phospholipid matrix. Further, differences in the temperature dependence of specific spectral transitions for the various gel assemblies are discussed in terms of additional intermolecular interactions arising between the adjacent monolayers defining the discrete bilayer unit, Experimental High purity samples of 1,2-dimyristoyl DL-phosphatidylcholine, 1,2-dipalmitoy1 DL-phosphatidylcholine and 1,2-distearoyl DL-phosphatidylcholine were obtained from Sigma Chemical Company. Since no major spectral contaminants were observed, the samples were used without further purification. Multilamillar samples, containing 31- -36% water, were prepared by heating thoroughly mixed phospholipid-water dispersions to about 10” C above the primary phase transition temperature. Homogeneous gels were obtained by repeating the heating and agitation procedures several times. Raman spectra were recorded with a modified Cary Model 81 spectrophotometer equipped with a Coherent Radiation Model 52 argon ion laser operating at 400 mW at 514.5 nm. The spectral resolution was approximately 3.5 cm-‘. Absolute spectral frequencies, calibrated for individual traces with atomic argon lines, are determined to 22 cm-‘. Spectra of annealed samples were recorded using either a thermostated capillary or a variable temperature cryostat which has been described previously
179
[ 4,6]. Temperatures, estimated to ?3“C, were monitored by a copper-constantan thermocouple inserted within the gel as near as possible to the laser beam transit. Samples were suitably equilibrated after each temperature change prior to recording their Raman spectra. For assessing spectral differences in the 1150-1000 cm-’ CC stretching region as a function of temperature, band area ratios of the 1062, 1085-1090 and approx. 1130 cm-’ transitions were taken relative to the CH, twisting mode, which appears as an isolated spectral transition at 1295 cm-‘. Band area measurements, determined with a planimeter after correcting for overlapping contours, are estimated to be accurate to ?lO%. Curve resolution is extremely complicated for the vibrational modes in the 3000-2800 cm-’ region; consequently, only peak height intensity ratios were determined for these spectral transitions. Differential calorimetry was performed with a Perkin-Elmer DSC-2 on samples of l-2 mg total weight. Scan rates of 2.5 deg. C/min. were employed. Results Raman spectra of multilayers of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholine, with hydrocarbon chain lengths of CIA, C,, and C,s, respectively, were recorded as a function of temperature for the gel phase from -180°C to points just preceeding the lower transition, or pretransition, temperature. The various spectral regions examined primarily reflected acyl chain
TABLE
I
FREQUENCIES GEL
(cm-‘)
FORMS
WATER
OF
RAMAN
TRANSITIONS DIPALMITOYL
AS
A FUNCTION
AND
OF
DISTEAROYL
TEMPERATURE
FOR
THE
PHOSPHATIDYLCHOLINE-
DISPERSIONS
Dimyristoyl
-__ Dipalmitoyl
phospha-
phospha-
10°C
Distearoyl
Assignment
phospha-
tidylcholine ______
tidylcholine
tidylcholine -17ec
OF
DIMYRISTOYL.
-18O’C
33cc
-18O’C
45OC
2959
2959
2959
2960
__2959
2960
(CHJ)
C-H
choline
asym.
(CH3)
stretch
C-H
sym.
stretch 2934
2934
2934
2934
2934
2934
(CH3)
Cm-H
sym.
2879
2879
2879
2879
2879
2879
(CHz)
C-H
asym.
2844
2844
2844
2844
2844
2844
(CHz)
C-H
sym.
1466
1454
1470
1453
1468
1455
1440.5
1437
1441
1436
1441.5
1436
CHZ
deformation
1295
CH2
twist
CHZ
rock
**
1423
* *
1421
1295
1295
1295
1178 1127 _
1130.5
1122
1295
1295
1062
*
Overlapped
* * Shoulder
1127
_
** 1103
1090-1085 1062
1062
1062
‘bans
1127.5
1132 *
1100
109&1085 1062
**
1175
1123
** *
1092
stretch stretch
1420
1176
1129
stretch
1122
**
Gauche
1100
*
‘frans
1088
Gauche
1062
?‘runs
(+ CH3
rock)
C-C
stretch
form form form
C-C C-C
form form
C-C C-C
stretch stretch stretch stretch
+
180
vibrations; namely, the 3000-2800 cm-’ CH stretching region, the 1450 cm-’ CH, deformation region, the 1295 CH,, twisting region and the 1150-1000 cm-’ C-C skeletal stretching region. As an example of the general spectral characteristics, Fig. 1 displays survey Raman spectra of distearoyl phosphatidylcholine-water dispersions at -180 and +39”C. Although both temperatures lie within the gel phase for this multilamellar system, spectral characteristics differ significantly over this temperature span. Similar reversible temperaturedependent spectral behavior is noted for the dimyristoyl and dipalmitoyl phosphatidylcholine-water systems. Table I summarizes the observed Raman vibrational frequencies and corresponding vibrational assignments. A. 1350-l 000 cm-’ region Three acyl chain all-truns C-C stretching modes are assigned to the spectral features at approx. 1130, approx. 1100 and 1062 cm-‘. The intense 1062 cm-’ line is both chain length and temperature insensitive, whereas the intense 1130 cm-’ and weaker 1100 cm- ’ lines are functions of both temperature and chain length. Two C-C stretching modes which reflect gauche conformers formed at higher temperature are also recorded in this spectral region at 1090-1085 cm-’ and at 1122 cm-‘. Below the primary gel-liquid crystalline phase transition the 1090-1085 cm-’ feature is partially obscured by the 1100 cm-’ all-trans C-C stretching mode; while above the phase transition, the 1090-1085 cm-’ feature predominates. The 1122 cm-’ line appears as a shoulder on the 1130 cm-’ transition for the spectral resolution used in these experiments [ 51. The contributions to the 1090-1085 and 1062 cm-’ areas from temperature changes involving the weak, underlying PO:- and CO stretching modes were neglected in the contour decompositions for this region. At -18O”C, noguuche conformers are observed (see Fig. 1). As the temperature increases, the all-truns modes lose intensity while the features associated with gauche rotamers simultaneously gain intensity. In addition, frequency shifts accompany the intensity changes for these modes. The trends for these spectral changes are analogous to the spectral changes reflected by the systems as they pass through their respective gel-liquid crystalline phase transitions, except that the spectral changes through the gel temperature span are not as
DISTEAROYL
PHOSPHATIDYL
CHOLINE-WATER
MULTILAYERS
Fig. 1. Survey Raman spectra of disteamyl phosphatidylcholine-water gels in the hydrocarbon twisting and C-C skeletal stretching region (A), CH2 deformation region (B) and the methylene methyl C-H stretching region (C). Spectra were recorded at -180°C (a) and 39’C (b).
CHZ and
181
1130 _ 6
i 1
+-__.._
1100 L
L -200
m%c
~100
~50
_
II ~.~~
0
-
+50
T (OC)
Fig. 2. Temperature-dependence Plots of the 2 113 2 cm-l if I 2 9 5 cm-l, 1 I o 62 cm-1 1112 9 5 cm-’ 11295 cm-~ intensity ratios and the 1132 cm-1 and ap~rox. 1090 CIII-’ frequency shifts.
1Igauche/
marked. For the distearoyl phosphatidylcholine-water gel system, Fig. 2 displays the band area intensity ratios for the 1132 and 1062 cm-’ all-truns C-C stretching modes and for the 1088 cm-’ gauche rotamer C-C stretching mode relative to the 1295 cm-’ CH2 twisting vibration. (The 1295 cm-’ transition provides a suitable reference band area as its bandwidth remains invariant to gel temperature and to hydrocarbon chainlength.) The most marked change occurs at which the in the &auche/11295 cm- t ratio at 5°C; that is, the temperature stretching modes for the gauche rotamers at 1088 cm-’ first appear. The inflections, also at approximately 5”C, in plots for the 1132 and 1062 cm-’ intensity ratios (Fig. 2) reflect the simultaneous loss in intensity in the all-truns modes. Since the temperature corresponding to the points of inflection is associated with the formation of gauche conformers, we define this value as T,. In general, similar behavior is observed in the data for the dimyristoyl and dipalmitoyl phosphatidylcholine gels. The curves representing the temperature dependent behavior for IgaUche/11z95 cm- 1 for these systems indicate that the values for T, for the Cl4 and Cl6 chain systems are equivalent (--40”(Z), but are significantly lower than the 5°C value found for the distearoyl (Cl8 chain) system. The results from the various intensity ratio curves are summarized in Table II. With the exception of the dipalmitoyl Ilo62 cm- 1 ratio (and perhaps the dimyrisat which the all-trans markers toy1 11130 cm- I intensity ratio) the temperatures lose intensity closely correspond, as expected, to the temperatures for which the gauche markers first appear. Although the shifts in absolute frequency for the 1130 cm-’ all-trans C-C stretching mode are not as sensitive a conformational change parameter for the gel as the correspondmg 11130cm- 1/1,~~~o-1 intensity ratios, slight inflections
I1
18
Distearoyl phosphatidylcholine-Hz0 -._I --^
+ 15
I15
*
in frequency
1090-1085
+ 15
i 15
~.
+ 15
t 15
___
+5 !: 10
-5
-40
..-
_-..
-10 _.._~~
+ 10
--45 +_15
--45 I 15
?I2844 cm-’
h2879 cm-‘/
--
***
_lll_
+10 z 10
____-
C5?
15
--40 ?r 15
+ 15
-45
f 15
_.~_~
cm-’
-46
-.
&‘I440
..___I_-_
-.-_
****
OF SPEClFIC
--45 F I5
h2844 cm-’ ~_._____
_-.
BEHAVIOR
h2930 em-l /
DEPENDENT
INTEN-
m the CH-J deformation
region
I refers to a band arcil measuremrnt. component
transition. of the 1440 cm-’
C--C gauche stretchmg
of temperature
hydrocarbon
as a function
cm-’
dipalmitoyl and dlsttsarovl phosphatidylcholine-eater multilayers are 13.5. 34.0 and 49.1 C. Tm2 for dimyristoyl. dipalmltoyl and distcaroyl phosphatidvfrhollttr-eater multilayers aw 23.7.
-._-___
+5 + 10
-40
-20
cm-’
‘1295
11295 cm-’
1
THE TEMPERATURE
JlOix? cm-’
_--
7’g ( C) FROM
11130 cm-!/
dimyristoyl, temperatures
+5 ? 10
-40
-40
11295 cm-’ .--... .__--._
Lower phase transition temperatures ‘/‘ml for respectiveiy (ref. 14). Primary phase transition 41.8 and 58.2”C. respectively (ref. 14). ** lgaucbe represents plots for the intensity of the * * * h represents the peak height intensity. **** Au1440 cm-I represents the plot for the change
1G
-_~
Dipaimitoyl phosphatidylcholine-HZU
-___..
per lipid
~._
TEMPERATURES
‘eauche/ **
ISOMERlZATION SHIFTS
c atoms
14
*
Dimyristoyl phosphatidylcholine-H*O
Gel system
I_-______-
VALUES FOR ?'RANS.GAC:CIfE SITY RATIOS AND FREQUENCY
TABLE
183
r 1445 1
Cl4
Cl6 CHAIN
Distearovl Phowhatldylcholine
Cl8
LENGTH
Fig, 3. (A) Dependence of the phasr transition temperature T,z and trans-guuchc~ isomeriution ature Tg upon hydrocarbon chain length. (Data for 7’,2 are taken from ref. 14.) (B) Dependence tilt 0 upon hydrocarbon chain length for gels with 25”; water. (Data from ref. 15.) Fig. 4. Temperature-dependence phatidyle:~oline, dipalmitoyl range -18O’C
temperof chain
plots of the CH2 deformation mode at 1440 cm-l for distearoyl nhosphospahtidylcholinc and dimyristoyl phosphatidylcholine-water gels in the
< 7‘ < ‘I’ml.
are noted near T,. Fig. 2 displays the 1130 and 1103 cm-’ frequency shift plots for the distearoyl phosphatidylcholine gel assembiy. For the three phospholipid gels, Fig. 3A compares the chain length dependence of Tmz, the primary phase transition temperature, and T,, two processes which reflect, to quite different extents, intramolecular chain disordering. Although Tg follows a non linear pattern, 1p,2 exhibits a linear dependence upon hydrocarbon chain length. The intensity of the 1176 cm-’ transition, assigned to a CH2 rocking mode, is also temperature sensitive. At T > Tg the band intensity decreases rapidly and finally disappears as T approaches the lower transition temperature. B. 1450 cm-’ region For temperatures just below the phase transition, a strong Raman feature with a prominent shoulder is observed in the 1450 cm-’ region for the CH, methylene deformation modes. On cooling, the frequencies shift upward and change in relative intensity simultaneous with the appearance of a new feature at about 1422 cm-’ (see Fig. 1 and Table I). Severe overlapping of band contours precludes accurate intensity measurements. Frequency shift plots for the bilayer-water systems as a function of temperature, however, do suggest a change in behavior at T,, as shown in Fig. 4. C. 3000-2800 cm-’ region Since curve resolution in the methylene and methyl 3000-2800 cm-’ C-H stretching region is ex~aordin~ily complex, the various bilayer inter and intramolecular disordering processes were monitored through changes in the relative peak heights of the 2844, 2879 and 2934 cm -l transitions. These features are
184
180
90
-55
T (“Cl
0
50
Fig. 5. Temptrature-dependence plots for the Peak height ratios I12930 cm-l/h28q4 cm-l and hZ879 cm-l /h2~44 cm-l. Tg is estimated from the intrrsection of the two nearly linear sets of data Points. 0. data from the present work; , thr, Phase transition data from Yellin, N. and Bulkin, B.J.. unpublished.
assigned to the methylene C-H symmetric stretching, methylene C-H asymmetric stretching and methyl C-H symmetric stretching modes [ 71, respectively. As the gel undergoes intramolecular chain disorder, the 2934 cm-’ transition appears on the edge of a broad 2925 cm-’ feature which arises from the infrared active methylene asymmetric stretching modes [23]. Fig, 5 presents the peak height ratios for h2879 cm-~ /h2844 cm-~ and h2934 cm-1 /h2844 cm-~ for dipalmitoyl phosphatidylcholine-water multilayers. The use of the 2844 cm-’ peak height as an internal reference is partially restricted as the intensity of this feature is temperature dependent. Since phase transition behavior can be determined from peak height plots analogous to those described above (Yellin and Bulkin, unpublished), our confidence in using these specific parameters, rather than band area measurements, to reflect bilayer dynamics is increased. Using the data recorded by Yellin and Bulkin (unpublished), we extend the temperature dependence plots in Fig. 5 through the phase transition region (35-60°C) for comparison purposes. An inflection below the phase transition temperatures is observed in both the h28,9 _-z and hZVX4cm-~ intensity plots. (The inflection point was estimated from extrapolations of the two linear regions characteristic of each intensity ratio (see Fig. 5).) Analogous behavior below the phase transition region also occurs in plots for the dimyristoyl and distearoyl phosphatidylcholine-water systems. Inflection points, obtained from the appropriate plots for the three gel assemblies and summarized in Table II, correspond within the range of error to T, values determined from the temperature profiles for the C-C stretching modes. Discussion The purpose of recording the temperature dependence of the vibrational Raman spectra of phospholipid gels is to identify more clearly the specific structural features involved in bilayer reorganization. In particul~, we are interested first in assessing the contributions to molecular disorder from interchain packing considerations and, separately, from intrachain conformational changes
185
and then in relating the various aspects of chain disorder to one another and to general bilayer gel properties. In recording the Raman spectra of the gels from liquid nitrogen temperatures (approx. -180°C) to slightly below the lower transition temperature T,~, no changes were observed in the spectral features assigned to the hydrocarbon portions of the dimyristoyl and dipalmitoyl bilayers around 0°C. Since this temperature region would correspond to the melting of the water phase, we infer that changes in the polar head group environment of the bilayer gel are not further reflected by either conformational changes within the acyl chains or further lateral disordering between chains. Although the distearoyl phospatidylcholine-water multilayers display inflections for various temperature profiles at approx. 5°C (Figs. 2, 4 and 5), we associate these changes for the Cls system with the onset of hydrocarbon chain gauche conformers (vide supra). At the lowest temperatures studied (-18O”C), the hydrocarbon chains of annealed samples reflect a highly ordered, all-trans packing configuration (see the general discussion regarding the all-trans chain conformation in gels in refs. 4, S-12). Since significant spectral intensity changes occur in the 1150-1000 cm-’ C-C stretching region for T < T,, (Figs. 1 and 2) which are similar, but occur to a lesser extent than the spectral changes during the upper phase transition Tm2 (Yellin and Bulkin, unpublished, and refs. 11-13) we associate the intensity and frequency changes, which begin at a characteristic T, (Table 11) for each lipid system, with the intramolecular disordering processes accompanying acyl chain tram-gauche isomerization. From studies of the C-C stretching modes and a determination of the van ‘t Hoff enthalpy differences M,n between rotational isomers [ 51, the formation in the gel of gauche conformers is assumed to originate toward the center of the bilayer about the locus of the acyl chain terminal methyl group. The gradual change in spectral intensities from T, to approx. Tml, which occurs over a 60, 75 and 45 deg. C temperature range for the C1+ Cl6 and Cl8 chain lipids, respectively (Table II and Figs. 2, 4 and 5), and the absence of an additional cooperative phase change from -180°C < T < Tml (f rom both the Raman and differential scanning calorimetric results) suggest that the rotational isomers are distributed throughout the bilayer planes as opposed to the formation of phase separated all-trans and liquid crystalline domains. (For separate hydrocarbon all-trans and liquid crystalline areas to occur within pure lipid bilayers, the rotationally disordered lipids would presumably be initially located about defects in the gel matrix.) The nonlinear dependence of values for T,, the temperature at which hydrocarbon chain gauche isomers first appear in the gel, with respect to acyl chain length is shown in Fig. 3. For comparison purposes, the phase transition values Tmz [ 141, also plotted in the figure as a function of chain length, display a linear behavior. (We note that the nonlinear behavior for T, is quite analogous to plots for the van ‘t Hoff tram-gauche isomerization enthalpies M,n for the three bilayer systems [5].) In their extensive X-ray diffraction studies on a the variety of phospholipid-water phases, Tardieu et al. [15] determined angle of tilt 8 of the lipid chains to the normal to the bilayer plane. A plot of 6, for dipalmitoyl, dimyristoyl and distearoyl phosphatidylcholine systems containing approx. 25% water, (slightly less water than the experimental systems used in the present study), yields the same general nonlinear behavior found for
186
Tg and AH,, (Fig. 3B). As a consequence of the lipid chain tilt, Tardieu et al. [ 151 proposed an “interlocking” mechanism primarily between the terminal methyl groups of the two respective monolayers forming the bilayer unit. Although the chains for a given lipid molecule may not overlap exactly, as in dilauroyl phosphatidylethanolamine [ 161, the larger angle of tilt for the distearoyl lipid chains, as compared to the dimyristoyl and dipalmitoyl systems [ 151, leads to a greater interaction between the terminal chain areas, toward the bilayer center, of the individual monolayers. This increased interaction between contiguous monolayers of the distearoyl system is then consistent with the higher temperature (5” C as compared to -40°C for the dipalmitoyl and dimyristoyl systems, respectively) required for the formation of acyl chain rotamers in Cl8 gels. The additional bilayer chain interactions, accompanying increases in both chain tilt and average head group area [ 151, may lead to a partially strained lipid matrix [ 151 which would allow for an increase in the number of gauche bonds per lipid chain to relieve the induced stresses upon the bilayer. The larger observed AH,, values for the distearoyl system in comparison to the dimyristoyl and dipalmitoyl phospholipids [ 51 reflects this predicted increase in number of rotational isomers per molecule. Having characterized the intramolecular trans-gauche isomerization process from the CX chain stretching region of the spectrum, we are now in a position to interpret additional changes in other spectral regions in terms of a superposition of both lateral chain disorder and deviations from an all-trans chain conformation. Spectral changes in the 1450 cm-’ region appear to be related to interchain interactions, as noted, for example, in anhydrous dilauroyl phosphatidylethanolamine [ 171 and polyethylene [ 181. Since no acyl chain transgauche isomerization occurs in the dilauroyl lipid crystal for T < T,, these intensity variations were attributed to an increase in both the thermal amplitudes of the hydrogen atoms and their positional disorder which accompany increasing temperatures in the solid [ 171. The doublet at 1421 and 1441 cm-’ in the dilauroyl system occurs in polyethylene at low temperatures at 1414 and 1441 cm-’ [ 181. In polyethylene, Boerio and Koenig assign the 1414 cm-’ feature to a crystal field component of the 1441 cm-’ A,, deformation mode [ 181. The phospholipid-water gels studied here exhibit the same general behavior in the 1450 cm-’ region as the dilauroyl solid system except that, in addition to an increase in the thermal motions of the CH, hydrogens, the gels also undergo chain rotational isomerization at these temperatures. Therefore, although the spectral changes in this region arise primarily from interchain interactions, slight inflections in temperature plots displaying the frequency shift for the intense 1440 cm- ’ feature (Fig. 4) for the three gel systems generally parallel the temperatures for the onset of chain trans-gauche isomerization. The severely overlapped contours, particularly at warmer gel temperatures, preclude reliable intensity measurements for individual spectral components in this region. However, a plot for the distearoyl phosphatidylcholine gel of the ratio of the total area of the 1450 cm-’ region to the area of the 1295 cm-’ CH, twisting transition, 11450 cm-1/I1295 cm -I, for temperatures in the range -180 to 50°C yields a broad maximum at approx. 15 + 15°C. Since the maximum occurs in the range of Tg, we are apparently observing the effects upon the vibrational spectrum from both the lateral disordering of the matrix and the
formation of chain rotational conformers. Analogous plots for the dimyristoyl and dipalmitoyl systems were not as clearly defined. For dipalmitoyl gels, for example, a broad maximum approx. -50 ? 25°C occurs in the ZldsO cn,-l/ Z129s cm-l plots, In the temperature interval from -25 to +35”C the intensity ratio decreases gradually to about 15% of the maximum value. The 3000-2800 cm-’ methyl and methylene C-H stretching region primarily reflects the packing arrangements of the lipid chains (Yellin and Bulkin, unpublished and refs. 4, 17, 19-22). The disorder created by acyl chain rotational isomers is, of course, also reflected by intensity changes in this spectral region. As shown in Fig. 5, values for T, determined from the temperature behavior of the 2879 and 2934 cm-’ features correspond well with those determined from the 1100 cm- ’ C-C stretching region. Since the plots in Fig. 5 from approx. -180 to -40°C are nearly linear, the lipid lattice apparently releases its lateral packing constraints simultaneously with the formation of gauche rotamers at the center of the bilayer. Fig. 5 also contains the values, determined in a previous study (Yellin and Bulkin, unpublished), for dipalmitoyl phosphatidylcholine multilayers from 27 to 60°C; that is, intensity ratios corresponding to temperatures which span the lower and primary phase transition points. The change of h,,,, c.-l/h2844 c,,,- I during the main phase transition m2 is about twice the change from T, < T < T,,. For the h,,,. cm-~/h2844 cm-~ ratio most of the change occurs for T, < T < T,,, while relatively small, but abrupt, changes arise during the phase transitions ml and m2. The dramatic increase in intensity in the 2934 cm-’ feature, assigned in part to a chain methyl symmetric C-H stretching motion [ 71, stems from the underlying, nominally Raman inactive, but infrared active, methylene asymmetric C-H stretching modes at approx. 2925 cm-‘. These modes acquire Raman activity as the inter and intramolecular disordering processes reduce the local all-trans C&, chain symmetry (for even numbers of carbon atoms) in the gel below T, [ 231. Intensity changes specific to the methyl C-H stretching feature at 2934 cm-‘, compared to the overall intensity changes throughout the 2925 cm-’ region, are difficult to distinguish. Since spectral evidence suggests, however, that the intensity of the isolated methyl vibrational transition is relatively insensitive to its environment in either the gel or liquid crystalline state [ 231, the use of the intensity change at 2934 cm-’ as an index to monitor chain disorder is a reasonable approximation. Since the C-H vibrational stretching region reflects primarily the lateral packing characteristics of the hydrocarbon chains, a value for the interaction energy between chains may be estimated from the temperature behavior of the spectral features. Specifically, we determine the increase in enthalpy AHinternal in the gel resulting from the disordering of the lipid matrix from the integrated form of the van ‘t Hoff equation expressed as ln(Zdisordered/Zordcred) = -AHi,_ ternal/RT + C. In this relation Iordered and Zdisordercd represent the Raman peak intensities for the appropriate spectral transitions which are proportional, respectively, to the concentration of all-trans chain forms (situated within an ordered lattice array) and to the concentration of chain forms whose packing arrangements have been disrupted by both rotational isomerization and relief of lateral crystal constraints. R represents the gas constant, while T represents the temperature range from about T, to Tml. AHinternal is the enthalpy increase
188
arising from changes in the intermolecular attractive and repulsive forces which stem from the lateral expansion of the lipid matrix. (The determination of van ‘t Hoff truns-gauche enthalpies from the temperature behavior of the hydrocarbon C-C stretching modes has been discussed in detail [5,24].) From the 3000-2800 cm-’ region we identify the CH* asymmetric stretching mode at 2879 cm-’ with the ordered domains of all-trans chain forms. The intensity of this feature decreases with increasing temperature. The 2934 cm-’ feature with its increase in apparent intensity, due to the underlying methylene asymmetric stretching modes discussed above, is associated with the loss of crystal symmetry and increase of lateral disorder arising from reduced chain interactions. The results of the temperature dependent van ‘t Hoff plots for the three phospholipid systems appear in Fig. 6. dipalmitoyl and distearoyl phosphoValues for AHinternal for the dimyristoyl, lipid gels are 1.5, 1.2 and 3.2 kcal/mol, respectively. Since peak heights, with no corrections for the overlapping contours, were used, it is difficult to estimate a range of error for these values. (Although we recognize the approximate nature of these values, we note, however, that the data are quite reproducible
69%
; x w q
-2.0
-
Olpalmltoyl
!
-1.5
, -0.5 1:
65%
Phosphabdylchollne
Dlmyrlstoyl Phosphabdylcholine
3.5
Hz0
4.0
order-disorder
processes
4.5
(OKI
6. Temperature-dependence
dimyristoyl
+ 35%
\
IITXlO’ Fig.
H20
.
3.0 ular
+ 31%
.
plots for
phospahtidylcholine-water
of ln(h2
distearoyl gels.
9 34
cm-
I //12 8 79
phosphatidylcholine.
cm-
1) for
hydrocarbon
dipalmitoyl
chain
intermolec-
phosphatidylcholine
and
189
and that the precision within the van ‘t Hoff plots (Fig. 6), in a least-squares sense, is quite good,) We now compare the experimental van ‘t Hoff enthalpies to relatively crude calculated estimates for the increase in internal energy required to expand to bilayer gel below T, , . In this calculation we assume that the all-truns chains are initially 4.8 K apart, while the terminal portions of the chain which form gauche configurations at higher gel temperatures are 5.3 n apart 1251. In addition, for the segments of the chains remaining in the all-trans form, we assume that a lateral expansion of the lipid lattice increases the interchain distances to 5.0 A [ 261. Thus, the model is that of a laterally expanded lattice bilayer with areas of occasional intramolecular disorder stemming from the formation of gauche rotamers near the bilayer center. We ignore in the calculation contributions to the disorder of the system from simple rotation of the lipid chains about their long axes. The expression used for the attractive potential, derived by Salem [ 271, is that for two infinitely long all-trans chains; namely, Uatt = -1.240 . lo3 (N/r”) kcal/mol, where N is the number of carbon atoms in the portion of the chain under consideration and r is the appropriate interchain distance. For the repulsive potential U,,, in these calculations we simply take u rep = 0.25 Uatt. This is approximately the relationship determined by McClure is his treatment for CzI paraffin chains in crystals below the rotational phase transition temperature [ 281. Again, for simplicity, we assume the same analytical form for the interaction energy between chain segments containing the gauche bonds as that for the parallel all-trans segments. (McCammon and Deutch [ 291 elected, however, to reduce the interaction energy between slightly disordered chains by about 6% from the all-trans values.) Following McCammon and Deutch [ 291, we take the effective chain length equal to N-2, as a consequence of chain overlap [ 161, and treat the methyl groups as methylene units. The number of gauche isomers per chain for each lipid system in the gel state was taken from ref. 5. With these approximations, we obtain for the dimyristoyl, dipalmitoyl and distearoyl systems iw values of 1.6, 1.9 and 2.5 kcal/mol, respectively, values in good agreement with the enthalpies determined from the experimental van ‘t Hoff expressions. The experimental values suggest that the Cl4 and Clb systems are somewhat analogous Mintrmal in behavior to one another, but differ from the Cl8 system. In contrast to the experimental enthalpies, the calculated values suggest a more gradual change in AHinternal between all three chain types. Since the calculation omits an explicit interaction term coupling the two monolayers, the enthalpy changes involved in the disordering effects toward the bilayer center may be significantly underestimated, particularly, for the distearoyl matrix. In summary, the temperature-dependent Raman spectra for particularly the C-H stretching region illustrates the inseparability between inter and intrachain contributions to hydrocarbon disorder in 1,2-diacyl phosphatidylcholinewater gels. Since changes in the various intensity ratios for C-H stretching transitions primarily reflect an expansion of the lipid matrix, Fig. 5 demonstrates for the Cl6 system the decrease in lateral chain packing order as vibrational modes between adjacent acyl chains uncouple. Thus, for dipalmitoyl phosphatidylcholine-water dispersions (Fig. 5), for example, the decrease in hzsT9 2.07 to 1.33 represents the disordering of the crystalline cm-llhm4 cm- I from
190
lattice beginning at approximately -45” C. Since trans-gauche isomerization also begins about -40°C in the Cl6 system, we conclude that the separate matrix events involving lattice expansion and chain rotational isomerization are closely related and occur simultaneously perhaps to relieve the constraints dictated by the interlocking of the methyl groups along the bilayer center. Coincident with the temperature for the onset of trans-gauche isomerization, the increase for the C,, system to approx. -10 to 10°C (Table II) for the inflection of the C-H stretching mode intensity ratios suggests that the angles of chain tilt markedly affect the temperature for which both the lateral packing and intrachain conformational characteristics are altered. Acknowledgment One of us (N.Y.) acknowledges the support of Health Visiting Fellow appointment.
provided
by a National
Institutes
References 1
Ifackenbrock.
2
Verkleij,
A.J.
Verkleij,
A.J.,
3
Acta
C.R.. and
M. and
Chau,
P.H.J.Th.
Ververgaert.
R.M.
(1976)
(1975)
Ann.
Van
P.H.J..
Deenen,
4
Spiker,
Jr.,
YeIIin,
N. and
6
Levin.
7
Spiker,
8
Rothman,
9
Levine,
R.C.
I.W.
lishing
and
Levin,
Levin,
(1969)
Jr.,
I.W.
I.W.
(1976)
(1977)
Biophys.
Physical
L.L.M.
and
Levin,
(1973)
Acta
I.W.
and
Acta
Chem.
26.
Elbers,
P.F.
Acta
455.
560-575
Acta
388.
361-373
455,
466484
101-122
(1972)
Biochim.
Biophys.
J. Theor.
(1972)
Prog.
(1972)
Biomembranes
Biophys.
New
1157-1160
Biochim.
Biol.
H.
Biophys.
16.642447
25A.
(1975)
Y.K.
Corporation,
Biochim.
Biochem.
Spectrochim.
R.C. J.E.
Traiible,
38, Mol. 3.
Biophys.
l-16 Biol.
24,
(F.
Lippert,
J.L.
and
Peticolas,
W.L.
(1972)
Biochim.
Lippert,
J.L.
and
Peticolas,
W.L.
(1971)
Proc.
13
Spiker,
I.W.
(1976)
Biochim.
14
Hinz,
15
Tardieu,
H.J.
16
Hitchcock,
R.C. and
A.,
and
Levin.
Sturtevant,
Luzzati, P.B.,
Kreuzer
and
J.F.G.
Slegers,
Eds.),
Plenum
Pub-
York
11
Jr.,
l-74
197-227
12
71.
Biochim. Rev.
288.326-332
5
10
HBchli, Ververgaert.
J.M.
V. and
Mason,
(1972)
Reman,
R.,
J. Biol. F.C.
Thomas,
Biophys.
Nat.
Sci..
Biophys. Chem.
(1973)
K.M.
Acta.
Acad.
Acta
247,
J. Mol.
and
282.
8-17
U.S.A.
68,
433,
1572-1576
457-468
60714075 Biol.
Shipley,
75.
711-733
G.G.
(1974)
Proc.
Nat.
Acad.
Sci..
U.S.A.
3036-3040
17
Mendelsohn,
18
Boerio.
F.J.
19
Larsson,
20
Mendelsohn,
K.
21
Brown,
22
Lamson.
R.,
Sunder,
S. and
and
Koenig,
J.L.
(1973) R.,
K.G.,
Rand,
M. and
Lcvin,
23
Bunow, Yellin,
N. and J.F.
25
Nagle, Shapiro,
27
Salem,
Levin,
(1973)
E. and
28
McClure,
29
McCammon,
D.W.
W.L. R.P. I.W.
I.W.
(1968) J.A.
and
Brown,
(1977) Phys.
J. Chem. Deutch,
58,
(1975)
Biochim.
Phys.
52,
Lipids
10,
H.J. E.
(1976) (1973)
Acta Acta Acta
Biophys. Biophys.
326, 487.
245-255 388-394
468,
490-494
Science
47,
252-264
J. CoIloid
and
Interface
38-49
37,2100-2113 Phys.
J.M.
49.1830-1839
(1975)
J. Am.
Acta
413,
329-340
419.
563-569
165-176
Biochem.
Biophys. Biophys.
Biophys.
3425-3431 Biochim.
Biophys.
Biochim. Biochim.
Phys.
of
Biochim.
(1977)
S. (1974)
J. Chem.
Physics
(1973)
H.J.
J. Chem.
Bernstein,
and
J. Chem. Ohki,
L. (1962)
and
S. and
Peticolas,
K. and
24 26
Chemistry Sunder,
Bernstein,
(1970)
Chem.
Sot.
97,
66754681
Acta Res.
Commun.
54,
358-368
Biochimica et Biophysics @ ElsevierlNorth-Holland
Acta,
489
Biomedical
(1977)
177-190
Press
BBA 57078
HYDROCARBON
CHAIN DISORDER
IN LIPID BILAYERS
TEMPERATURE DEPENDENT RAMAN SPECTRA PHOSPHATIDYLCHOLINE-WATER GELS
NEHAMA
YELLIN
* and IRA W. LEVIN
OF 1,2-DIACYL
**
Laboratory of Chemical Physics, National Institute of Arthritis, Metabolism Diseases, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)
(Received
April 12th,
and Digestive
1977)
Summary Vibrational Raman spectra of multilayers of 1,2-dimyristoyl, 1,2-dipalmitoyl and 1,2-distearoyl phosphatidylcholine were recorded as a function of temperature for the gel phase from -180°C to values slightly below the pretransition temperature. Temperature profiles for band intensity ratios involving the acyl chain C-C stretching modes define a characteristic temperature T, which denotes the onset of hydrocarbon chain trans-gauche isomerization in the gel. T, for the dimyristoyl (Cl0 chain lengths), dipalmitoyl (Cl6 chain lengths) and distearoyl (Cl8 chain lengths) systems are approximately -40, -40 and 5”C, respectively. The higher T, value for distearoyl phosphatidylcholine is associated with increasing interactions between the terminal chain areas of the individual monolayers forming the bilayer unit. This increased interaction between bilayer halves is presumably a consequence of the larger angle through which the hydrocarbon chains are tilted in the Cl8 phospholipid gel. Temperature-dependent peak height intensities for the 3000-2800 C-H stretching region provide convenient probes for quantitatively describing the increase in lateral chain packing disorder as the gel assemblies are warmed. The temperature profiles for various methylene mode peak height ratios indicate that both the inter- (lateral packing) and intramolecular (tram-gauche) disordering processes are related and are initiated at nearly the same temperatures for each lipid system. Increases in the van ‘t Hoff enthalpies which result from the disordering of the lipid gel matrix by a lattice expansion were estimated for to be approximately 1.5, 1.2 and 3.2 kcal/ the Ci4, Ci, and Cl8 phospholipids mol, respectively.
* Present address: Sores Nuclear Research Center, ** To whom correspondence should be addressed.
Yavne,
Israel.
178
Introduction For a variety of biomembranes, the gel bilayer form is uniquely characterized by the introduction of lateral domains of aggregated particles. This phase separation process is presumably induced by a liquid crystalline - gel phase transition in which the lamellar phospholipids acquire a high degree of both inter- and intramolecular order as the lipid matrix solidifies from its fluid, high temperature state (see refs. 1-3 and additional references therein). In an effort to clarify the specific lipid conformational changes and packing rearrangements that are involved in gel formation, we recently applied vibrational Raman spectroscopic probes toward describing lipid assemblies in a variety of liposoma1 preparations [ 4,5]. Specifically, for a series of multilamellar phospholipid bilayers dispersions we emphasized the remaining conformational tram-gauche disorder, or relative flexibility, inherent in the lipid hydrocarbon chains at temperatures significantly below the bilayer phase transition [5]. In these earlier studies [ 51, t,he deviations from an ideal all-trans lipid chain matrix were estimated from variations in spectral intensity of the temperature dependent carbon-carbon (C-C) stretching modes for the lipid. In order to define more completely the lipid-lipid interactions relating both to the dynamics of gel formation and to the stability of the zones of phase segregated lipids, we examine in the present study the low temperature spectral behavior of several structurally sensitive vibrational Raman features, in addition to chain stretching displacements, of bilayer gels of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholine-water liposomes. The various spectral changes are interpreted in terms of concomitant intrachain and lateral intermolecular disordering effects within the hydrocarbon portion of the phospholipid matrix. Further, differences in the temperature dependence of specific spectral transitions for the various gel assemblies are discussed in terms of additional intermolecular interactions arising between the adjacent monolayers defining the discrete bilayer unit, Experimental High purity samples of 1,2-dimyristoyl DL-phosphatidylcholine, 1,2-dipalmitoy1 DL-phosphatidylcholine and 1,2-distearoyl DL-phosphatidylcholine were obtained from Sigma Chemical Company. Since no major spectral contaminants were observed, the samples were used without further purification. Multilamillar samples, containing 31- -36% water, were prepared by heating thoroughly mixed phospholipid-water dispersions to about 10” C above the primary phase transition temperature. Homogeneous gels were obtained by repeating the heating and agitation procedures several times. Raman spectra were recorded with a modified Cary Model 81 spectrophotometer equipped with a Coherent Radiation Model 52 argon ion laser operating at 400 mW at 514.5 nm. The spectral resolution was approximately 3.5 cm-‘. Absolute spectral frequencies, calibrated for individual traces with atomic argon lines, are determined to 22 cm-‘. Spectra of annealed samples were recorded using either a thermostated capillary or a variable temperature cryostat which has been described previously
179
[ 4,6]. Temperatures, estimated to ?3“C, were monitored by a copper-constantan thermocouple inserted within the gel as near as possible to the laser beam transit. Samples were suitably equilibrated after each temperature change prior to recording their Raman spectra. For assessing spectral differences in the 1150-1000 cm-’ CC stretching region as a function of temperature, band area ratios of the 1062, 1085-1090 and approx. 1130 cm-’ transitions were taken relative to the CH, twisting mode, which appears as an isolated spectral transition at 1295 cm-‘. Band area measurements, determined with a planimeter after correcting for overlapping contours, are estimated to be accurate to ?lO%. Curve resolution is extremely complicated for the vibrational modes in the 3000-2800 cm-’ region; consequently, only peak height intensity ratios were determined for these spectral transitions. Differential calorimetry was performed with a Perkin-Elmer DSC-2 on samples of l-2 mg total weight. Scan rates of 2.5 deg. C/min. were employed. Results Raman spectra of multilayers of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholine, with hydrocarbon chain lengths of CIA, C,, and C,s, respectively, were recorded as a function of temperature for the gel phase from -180°C to points just preceeding the lower transition, or pretransition, temperature. The various spectral regions examined primarily reflected acyl chain
TABLE
I
FREQUENCIES GEL
(cm-‘)
FORMS
WATER
OF
RAMAN
TRANSITIONS DIPALMITOYL
AS
A FUNCTION
AND
OF
DISTEAROYL
TEMPERATURE
FOR
THE
PHOSPHATIDYLCHOLINE-
DISPERSIONS
Dimyristoyl
-__ Dipalmitoyl
phospha-
phospha-
10°C
Distearoyl
Assignment
phospha-
tidylcholine ______
tidylcholine
tidylcholine -17ec
OF
DIMYRISTOYL.
-18O’C
33cc
-18O’C
45OC
2959
2959
2959
2960
__2959
2960
(CHJ)
C-H
choline
asym.
(CH3)
stretch
C-H
sym.
stretch 2934
2934
2934
2934
2934
2934
(CH3)
Cm-H
sym.
2879
2879
2879
2879
2879
2879
(CHz)
C-H
asym.
2844
2844
2844
2844
2844
2844
(CHz)
C-H
sym.
1466
1454
1470
1453
1468
1455
1440.5
1437
1441
1436
1441.5
1436
CHZ
deformation
1295
CH2
twist
CHZ
rock
**
1423
* *
1421
1295
1295
1295
1178 1127 _
1130.5
1122
1295
1295
1062
*
Overlapped
* * Shoulder
1127
_
** 1103
1090-1085 1062
1062
1062
‘bans
1127.5
1132 *
1100
109&1085 1062
**
1175
1123
** *
1092
stretch stretch
1420
1176
1129
stretch
1122
**
Gauche
1100
*
‘frans
1088
Gauche
1062
?‘runs
(+ CH3
rock)
C-C
stretch
form form form
C-C C-C
form form
C-C C-C
stretch stretch stretch stretch
+
180
vibrations; namely, the 3000-2800 cm-’ CH stretching region, the 1450 cm-’ CH, deformation region, the 1295 CH,, twisting region and the 1150-1000 cm-’ C-C skeletal stretching region. As an example of the general spectral characteristics, Fig. 1 displays survey Raman spectra of distearoyl phosphatidylcholine-water dispersions at -180 and +39”C. Although both temperatures lie within the gel phase for this multilamellar system, spectral characteristics differ significantly over this temperature span. Similar reversible temperaturedependent spectral behavior is noted for the dimyristoyl and dipalmitoyl phosphatidylcholine-water systems. Table I summarizes the observed Raman vibrational frequencies and corresponding vibrational assignments. A. 1350-l 000 cm-’ region Three acyl chain all-truns C-C stretching modes are assigned to the spectral features at approx. 1130, approx. 1100 and 1062 cm-‘. The intense 1062 cm-’ line is both chain length and temperature insensitive, whereas the intense 1130 cm-’ and weaker 1100 cm- ’ lines are functions of both temperature and chain length. Two C-C stretching modes which reflect gauche conformers formed at higher temperature are also recorded in this spectral region at 1090-1085 cm-’ and at 1122 cm-‘. Below the primary gel-liquid crystalline phase transition the 1090-1085 cm-’ feature is partially obscured by the 1100 cm-’ all-trans C-C stretching mode; while above the phase transition, the 1090-1085 cm-’ feature predominates. The 1122 cm-’ line appears as a shoulder on the 1130 cm-’ transition for the spectral resolution used in these experiments [ 51. The contributions to the 1090-1085 and 1062 cm-’ areas from temperature changes involving the weak, underlying PO:- and CO stretching modes were neglected in the contour decompositions for this region. At -18O”C, noguuche conformers are observed (see Fig. 1). As the temperature increases, the all-truns modes lose intensity while the features associated with gauche rotamers simultaneously gain intensity. In addition, frequency shifts accompany the intensity changes for these modes. The trends for these spectral changes are analogous to the spectral changes reflected by the systems as they pass through their respective gel-liquid crystalline phase transitions, except that the spectral changes through the gel temperature span are not as
DISTEAROYL
PHOSPHATIDYL
CHOLINE-WATER
MULTILAYERS
Fig. 1. Survey Raman spectra of disteamyl phosphatidylcholine-water gels in the hydrocarbon twisting and C-C skeletal stretching region (A), CH2 deformation region (B) and the methylene methyl C-H stretching region (C). Spectra were recorded at -180°C (a) and 39’C (b).
CHZ and
181
1130 _ 6
i 1
+-__.._
1100 L
L -200
m%c
~100
~50
_
II ~.~~
0
-
+50
T (OC)
Fig. 2. Temperature-dependence Plots of the 2 113 2 cm-l if I 2 9 5 cm-l, 1 I o 62 cm-1 1112 9 5 cm-’ 11295 cm-~ intensity ratios and the 1132 cm-1 and ap~rox. 1090 CIII-’ frequency shifts.
1Igauche/
marked. For the distearoyl phosphatidylcholine-water gel system, Fig. 2 displays the band area intensity ratios for the 1132 and 1062 cm-’ all-truns C-C stretching modes and for the 1088 cm-’ gauche rotamer C-C stretching mode relative to the 1295 cm-’ CH2 twisting vibration. (The 1295 cm-’ transition provides a suitable reference band area as its bandwidth remains invariant to gel temperature and to hydrocarbon chainlength.) The most marked change occurs at which the in the &auche/11295 cm- t ratio at 5°C; that is, the temperature stretching modes for the gauche rotamers at 1088 cm-’ first appear. The inflections, also at approximately 5”C, in plots for the 1132 and 1062 cm-’ intensity ratios (Fig. 2) reflect the simultaneous loss in intensity in the all-truns modes. Since the temperature corresponding to the points of inflection is associated with the formation of gauche conformers, we define this value as T,. In general, similar behavior is observed in the data for the dimyristoyl and dipalmitoyl phosphatidylcholine gels. The curves representing the temperature dependent behavior for IgaUche/11z95 cm- 1 for these systems indicate that the values for T, for the Cl4 and Cl6 chain systems are equivalent (--40”(Z), but are significantly lower than the 5°C value found for the distearoyl (Cl8 chain) system. The results from the various intensity ratio curves are summarized in Table II. With the exception of the dipalmitoyl Ilo62 cm- 1 ratio (and perhaps the dimyrisat which the all-trans markers toy1 11130 cm- I intensity ratio) the temperatures lose intensity closely correspond, as expected, to the temperatures for which the gauche markers first appear. Although the shifts in absolute frequency for the 1130 cm-’ all-trans C-C stretching mode are not as sensitive a conformational change parameter for the gel as the correspondmg 11130cm- 1/1,~~~o-1 intensity ratios, slight inflections
I1
18
Distearoyl phosphatidylcholine-Hz0 -._I --^
+ 15
I15
*
in frequency
1090-1085
+ 15
i 15
~.
+ 15
t 15
___
+5 !: 10
-5
-40
..-
_-..
-10 _.._~~
+ 10
--45 +_15
--45 I 15
?I2844 cm-’
h2879 cm-‘/
--
***
_lll_
+10 z 10
____-
C5?
15
--40 ?r 15
+ 15
-45
f 15
_.~_~
cm-’
-46
-.
&‘I440
..___I_-_
-.-_
****
OF SPEClFIC
--45 F I5
h2844 cm-’ ~_._____
_-.
BEHAVIOR
h2930 em-l /
DEPENDENT
INTEN-
m the CH-J deformation
region
I refers to a band arcil measuremrnt. component
transition. of the 1440 cm-’
C--C gauche stretchmg
of temperature
hydrocarbon
as a function
cm-’
dipalmitoyl and dlsttsarovl phosphatidylcholine-eater multilayers are 13.5. 34.0 and 49.1 C. Tm2 for dimyristoyl. dipalmltoyl and distcaroyl phosphatidvfrhollttr-eater multilayers aw 23.7.
-._-___
+5 + 10
-40
-20
cm-’
‘1295
11295 cm-’
1
THE TEMPERATURE
JlOix? cm-’
_--
7’g ( C) FROM
11130 cm-!/
dimyristoyl, temperatures
+5 ? 10
-40
-40
11295 cm-’ .--... .__--._
Lower phase transition temperatures ‘/‘ml for respectiveiy (ref. 14). Primary phase transition 41.8 and 58.2”C. respectively (ref. 14). ** lgaucbe represents plots for the intensity of the * * * h represents the peak height intensity. **** Au1440 cm-I represents the plot for the change
1G
-_~
Dipaimitoyl phosphatidylcholine-HZU
-___..
per lipid
~._
TEMPERATURES
‘eauche/ **
ISOMERlZATION SHIFTS
c atoms
14
*
Dimyristoyl phosphatidylcholine-H*O
Gel system
I_-______-
VALUES FOR ?'RANS.GAC:CIfE SITY RATIOS AND FREQUENCY
TABLE
183
r 1445 1
Cl4
Cl6 CHAIN
Distearovl Phowhatldylcholine
Cl8
LENGTH
Fig, 3. (A) Dependence of the phasr transition temperature T,z and trans-guuchc~ isomeriution ature Tg upon hydrocarbon chain length. (Data for 7’,2 are taken from ref. 14.) (B) Dependence tilt 0 upon hydrocarbon chain length for gels with 25”; water. (Data from ref. 15.) Fig. 4. Temperature-dependence phatidyle:~oline, dipalmitoyl range -18O’C
temperof chain
plots of the CH2 deformation mode at 1440 cm-l for distearoyl nhosphospahtidylcholinc and dimyristoyl phosphatidylcholine-water gels in the
< 7‘ < ‘I’ml.
are noted near T,. Fig. 2 displays the 1130 and 1103 cm-’ frequency shift plots for the distearoyl phosphatidylcholine gel assembiy. For the three phospholipid gels, Fig. 3A compares the chain length dependence of Tmz, the primary phase transition temperature, and T,, two processes which reflect, to quite different extents, intramolecular chain disordering. Although Tg follows a non linear pattern, 1p,2 exhibits a linear dependence upon hydrocarbon chain length. The intensity of the 1176 cm-’ transition, assigned to a CH2 rocking mode, is also temperature sensitive. At T > Tg the band intensity decreases rapidly and finally disappears as T approaches the lower transition temperature. B. 1450 cm-’ region For temperatures just below the phase transition, a strong Raman feature with a prominent shoulder is observed in the 1450 cm-’ region for the CH, methylene deformation modes. On cooling, the frequencies shift upward and change in relative intensity simultaneous with the appearance of a new feature at about 1422 cm-’ (see Fig. 1 and Table I). Severe overlapping of band contours precludes accurate intensity measurements. Frequency shift plots for the bilayer-water systems as a function of temperature, however, do suggest a change in behavior at T,, as shown in Fig. 4. C. 3000-2800 cm-’ region Since curve resolution in the methylene and methyl 3000-2800 cm-’ C-H stretching region is ex~aordin~ily complex, the various bilayer inter and intramolecular disordering processes were monitored through changes in the relative peak heights of the 2844, 2879 and 2934 cm -l transitions. These features are
184
180
90
-55
T (“Cl
0
50
Fig. 5. Temptrature-dependence plots for the Peak height ratios I12930 cm-l/h28q4 cm-l and hZ879 cm-l /h2~44 cm-l. Tg is estimated from the intrrsection of the two nearly linear sets of data Points. 0. data from the present work; , thr, Phase transition data from Yellin, N. and Bulkin, B.J.. unpublished.
assigned to the methylene C-H symmetric stretching, methylene C-H asymmetric stretching and methyl C-H symmetric stretching modes [ 71, respectively. As the gel undergoes intramolecular chain disorder, the 2934 cm-’ transition appears on the edge of a broad 2925 cm-’ feature which arises from the infrared active methylene asymmetric stretching modes [23]. Fig, 5 presents the peak height ratios for h2879 cm-~ /h2844 cm-~ and h2934 cm-1 /h2844 cm-~ for dipalmitoyl phosphatidylcholine-water multilayers. The use of the 2844 cm-’ peak height as an internal reference is partially restricted as the intensity of this feature is temperature dependent. Since phase transition behavior can be determined from peak height plots analogous to those described above (Yellin and Bulkin, unpublished), our confidence in using these specific parameters, rather than band area measurements, to reflect bilayer dynamics is increased. Using the data recorded by Yellin and Bulkin (unpublished), we extend the temperature dependence plots in Fig. 5 through the phase transition region (35-60°C) for comparison purposes. An inflection below the phase transition temperatures is observed in both the h28,9 _-z and hZVX4cm-~ intensity plots. (The inflection point was estimated from extrapolations of the two linear regions characteristic of each intensity ratio (see Fig. 5).) Analogous behavior below the phase transition region also occurs in plots for the dimyristoyl and distearoyl phosphatidylcholine-water systems. Inflection points, obtained from the appropriate plots for the three gel assemblies and summarized in Table II, correspond within the range of error to T, values determined from the temperature profiles for the C-C stretching modes. Discussion The purpose of recording the temperature dependence of the vibrational Raman spectra of phospholipid gels is to identify more clearly the specific structural features involved in bilayer reorganization. In particul~, we are interested first in assessing the contributions to molecular disorder from interchain packing considerations and, separately, from intrachain conformational changes
185
and then in relating the various aspects of chain disorder to one another and to general bilayer gel properties. In recording the Raman spectra of the gels from liquid nitrogen temperatures (approx. -180°C) to slightly below the lower transition temperature T,~, no changes were observed in the spectral features assigned to the hydrocarbon portions of the dimyristoyl and dipalmitoyl bilayers around 0°C. Since this temperature region would correspond to the melting of the water phase, we infer that changes in the polar head group environment of the bilayer gel are not further reflected by either conformational changes within the acyl chains or further lateral disordering between chains. Although the distearoyl phospatidylcholine-water multilayers display inflections for various temperature profiles at approx. 5°C (Figs. 2, 4 and 5), we associate these changes for the Cls system with the onset of hydrocarbon chain gauche conformers (vide supra). At the lowest temperatures studied (-18O”C), the hydrocarbon chains of annealed samples reflect a highly ordered, all-trans packing configuration (see the general discussion regarding the all-trans chain conformation in gels in refs. 4, S-12). Since significant spectral intensity changes occur in the 1150-1000 cm-’ C-C stretching region for T < T,, (Figs. 1 and 2) which are similar, but occur to a lesser extent than the spectral changes during the upper phase transition Tm2 (Yellin and Bulkin, unpublished, and refs. 11-13) we associate the intensity and frequency changes, which begin at a characteristic T, (Table 11) for each lipid system, with the intramolecular disordering processes accompanying acyl chain tram-gauche isomerization. From studies of the C-C stretching modes and a determination of the van ‘t Hoff enthalpy differences M,n between rotational isomers [ 51, the formation in the gel of gauche conformers is assumed to originate toward the center of the bilayer about the locus of the acyl chain terminal methyl group. The gradual change in spectral intensities from T, to approx. Tml, which occurs over a 60, 75 and 45 deg. C temperature range for the C1+ Cl6 and Cl8 chain lipids, respectively (Table II and Figs. 2, 4 and 5), and the absence of an additional cooperative phase change from -180°C < T < Tml (f rom both the Raman and differential scanning calorimetric results) suggest that the rotational isomers are distributed throughout the bilayer planes as opposed to the formation of phase separated all-trans and liquid crystalline domains. (For separate hydrocarbon all-trans and liquid crystalline areas to occur within pure lipid bilayers, the rotationally disordered lipids would presumably be initially located about defects in the gel matrix.) The nonlinear dependence of values for T,, the temperature at which hydrocarbon chain gauche isomers first appear in the gel, with respect to acyl chain length is shown in Fig. 3. For comparison purposes, the phase transition values Tmz [ 141, also plotted in the figure as a function of chain length, display a linear behavior. (We note that the nonlinear behavior for T, is quite analogous to plots for the van ‘t Hoff tram-gauche isomerization enthalpies M,n for the three bilayer systems [5].) In their extensive X-ray diffraction studies on a the variety of phospholipid-water phases, Tardieu et al. [15] determined angle of tilt 8 of the lipid chains to the normal to the bilayer plane. A plot of 6, for dipalmitoyl, dimyristoyl and distearoyl phosphatidylcholine systems containing approx. 25% water, (slightly less water than the experimental systems used in the present study), yields the same general nonlinear behavior found for
186
Tg and AH,, (Fig. 3B). As a consequence of the lipid chain tilt, Tardieu et al. [ 151 proposed an “interlocking” mechanism primarily between the terminal methyl groups of the two respective monolayers forming the bilayer unit. Although the chains for a given lipid molecule may not overlap exactly, as in dilauroyl phosphatidylethanolamine [ 161, the larger angle of tilt for the distearoyl lipid chains, as compared to the dimyristoyl and dipalmitoyl systems [ 151, leads to a greater interaction between the terminal chain areas, toward the bilayer center, of the individual monolayers. This increased interaction between contiguous monolayers of the distearoyl system is then consistent with the higher temperature (5” C as compared to -40°C for the dipalmitoyl and dimyristoyl systems, respectively) required for the formation of acyl chain rotamers in Cl8 gels. The additional bilayer chain interactions, accompanying increases in both chain tilt and average head group area [ 151, may lead to a partially strained lipid matrix [ 151 which would allow for an increase in the number of gauche bonds per lipid chain to relieve the induced stresses upon the bilayer. The larger observed AH,, values for the distearoyl system in comparison to the dimyristoyl and dipalmitoyl phospholipids [ 51 reflects this predicted increase in number of rotational isomers per molecule. Having characterized the intramolecular trans-gauche isomerization process from the CX chain stretching region of the spectrum, we are now in a position to interpret additional changes in other spectral regions in terms of a superposition of both lateral chain disorder and deviations from an all-trans chain conformation. Spectral changes in the 1450 cm-’ region appear to be related to interchain interactions, as noted, for example, in anhydrous dilauroyl phosphatidylethanolamine [ 171 and polyethylene [ 181. Since no acyl chain transgauche isomerization occurs in the dilauroyl lipid crystal for T < T,, these intensity variations were attributed to an increase in both the thermal amplitudes of the hydrogen atoms and their positional disorder which accompany increasing temperatures in the solid [ 171. The doublet at 1421 and 1441 cm-’ in the dilauroyl system occurs in polyethylene at low temperatures at 1414 and 1441 cm-’ [ 181. In polyethylene, Boerio and Koenig assign the 1414 cm-’ feature to a crystal field component of the 1441 cm-’ A,, deformation mode [ 181. The phospholipid-water gels studied here exhibit the same general behavior in the 1450 cm-’ region as the dilauroyl solid system except that, in addition to an increase in the thermal motions of the CH, hydrogens, the gels also undergo chain rotational isomerization at these temperatures. Therefore, although the spectral changes in this region arise primarily from interchain interactions, slight inflections in temperature plots displaying the frequency shift for the intense 1440 cm- ’ feature (Fig. 4) for the three gel systems generally parallel the temperatures for the onset of chain trans-gauche isomerization. The severely overlapped contours, particularly at warmer gel temperatures, preclude reliable intensity measurements for individual spectral components in this region. However, a plot for the distearoyl phosphatidylcholine gel of the ratio of the total area of the 1450 cm-’ region to the area of the 1295 cm-’ CH, twisting transition, 11450 cm-1/I1295 cm -I, for temperatures in the range -180 to 50°C yields a broad maximum at approx. 15 + 15°C. Since the maximum occurs in the range of Tg, we are apparently observing the effects upon the vibrational spectrum from both the lateral disordering of the matrix and the
formation of chain rotational conformers. Analogous plots for the dimyristoyl and dipalmitoyl systems were not as clearly defined. For dipalmitoyl gels, for example, a broad maximum approx. -50 ? 25°C occurs in the ZldsO cn,-l/ Z129s cm-l plots, In the temperature interval from -25 to +35”C the intensity ratio decreases gradually to about 15% of the maximum value. The 3000-2800 cm-’ methyl and methylene C-H stretching region primarily reflects the packing arrangements of the lipid chains (Yellin and Bulkin, unpublished and refs. 4, 17, 19-22). The disorder created by acyl chain rotational isomers is, of course, also reflected by intensity changes in this spectral region. As shown in Fig. 5, values for T, determined from the temperature behavior of the 2879 and 2934 cm-’ features correspond well with those determined from the 1100 cm- ’ C-C stretching region. Since the plots in Fig. 5 from approx. -180 to -40°C are nearly linear, the lipid lattice apparently releases its lateral packing constraints simultaneously with the formation of gauche rotamers at the center of the bilayer. Fig. 5 also contains the values, determined in a previous study (Yellin and Bulkin, unpublished), for dipalmitoyl phosphatidylcholine multilayers from 27 to 60°C; that is, intensity ratios corresponding to temperatures which span the lower and primary phase transition points. The change of h,,,, c.-l/h2844 c,,,- I during the main phase transition m2 is about twice the change from T, < T < T,,. For the h,,,. cm-~/h2844 cm-~ ratio most of the change occurs for T, < T < T,,, while relatively small, but abrupt, changes arise during the phase transitions ml and m2. The dramatic increase in intensity in the 2934 cm-’ feature, assigned in part to a chain methyl symmetric C-H stretching motion [ 71, stems from the underlying, nominally Raman inactive, but infrared active, methylene asymmetric C-H stretching modes at approx. 2925 cm-‘. These modes acquire Raman activity as the inter and intramolecular disordering processes reduce the local all-trans C&, chain symmetry (for even numbers of carbon atoms) in the gel below T, [ 231. Intensity changes specific to the methyl C-H stretching feature at 2934 cm-‘, compared to the overall intensity changes throughout the 2925 cm-’ region, are difficult to distinguish. Since spectral evidence suggests, however, that the intensity of the isolated methyl vibrational transition is relatively insensitive to its environment in either the gel or liquid crystalline state [ 231, the use of the intensity change at 2934 cm-’ as an index to monitor chain disorder is a reasonable approximation. Since the C-H vibrational stretching region reflects primarily the lateral packing characteristics of the hydrocarbon chains, a value for the interaction energy between chains may be estimated from the temperature behavior of the spectral features. Specifically, we determine the increase in enthalpy AHinternal in the gel resulting from the disordering of the lipid matrix from the integrated form of the van ‘t Hoff equation expressed as ln(Zdisordered/Zordcred) = -AHi,_ ternal/RT + C. In this relation Iordered and Zdisordercd represent the Raman peak intensities for the appropriate spectral transitions which are proportional, respectively, to the concentration of all-trans chain forms (situated within an ordered lattice array) and to the concentration of chain forms whose packing arrangements have been disrupted by both rotational isomerization and relief of lateral crystal constraints. R represents the gas constant, while T represents the temperature range from about T, to Tml. AHinternal is the enthalpy increase
188
arising from changes in the intermolecular attractive and repulsive forces which stem from the lateral expansion of the lipid matrix. (The determination of van ‘t Hoff truns-gauche enthalpies from the temperature behavior of the hydrocarbon C-C stretching modes has been discussed in detail [5,24].) From the 3000-2800 cm-’ region we identify the CH* asymmetric stretching mode at 2879 cm-’ with the ordered domains of all-trans chain forms. The intensity of this feature decreases with increasing temperature. The 2934 cm-’ feature with its increase in apparent intensity, due to the underlying methylene asymmetric stretching modes discussed above, is associated with the loss of crystal symmetry and increase of lateral disorder arising from reduced chain interactions. The results of the temperature dependent van ‘t Hoff plots for the three phospholipid systems appear in Fig. 6. dipalmitoyl and distearoyl phosphoValues for AHinternal for the dimyristoyl, lipid gels are 1.5, 1.2 and 3.2 kcal/mol, respectively. Since peak heights, with no corrections for the overlapping contours, were used, it is difficult to estimate a range of error for these values. (Although we recognize the approximate nature of these values, we note, however, that the data are quite reproducible
69%
; x w q
-2.0
-
Olpalmltoyl
!
-1.5
, -0.5 1:
65%
Phosphabdylchollne
Dlmyrlstoyl Phosphabdylcholine
3.5
Hz0
4.0
order-disorder
processes
4.5
(OKI
6. Temperature-dependence
dimyristoyl
+ 35%
\
IITXlO’ Fig.
H20
.
3.0 ular
+ 31%
.
plots for
phospahtidylcholine-water
of ln(h2
distearoyl gels.
9 34
cm-
I //12 8 79
phosphatidylcholine.
cm-
1) for
hydrocarbon
dipalmitoyl
chain
intermolec-
phosphatidylcholine
and
189
and that the precision within the van ‘t Hoff plots (Fig. 6), in a least-squares sense, is quite good,) We now compare the experimental van ‘t Hoff enthalpies to relatively crude calculated estimates for the increase in internal energy required to expand to bilayer gel below T, , . In this calculation we assume that the all-truns chains are initially 4.8 K apart, while the terminal portions of the chain which form gauche configurations at higher gel temperatures are 5.3 n apart 1251. In addition, for the segments of the chains remaining in the all-trans form, we assume that a lateral expansion of the lipid lattice increases the interchain distances to 5.0 A [ 261. Thus, the model is that of a laterally expanded lattice bilayer with areas of occasional intramolecular disorder stemming from the formation of gauche rotamers near the bilayer center. We ignore in the calculation contributions to the disorder of the system from simple rotation of the lipid chains about their long axes. The expression used for the attractive potential, derived by Salem [ 271, is that for two infinitely long all-trans chains; namely, Uatt = -1.240 . lo3 (N/r”) kcal/mol, where N is the number of carbon atoms in the portion of the chain under consideration and r is the appropriate interchain distance. For the repulsive potential U,,, in these calculations we simply take u rep = 0.25 Uatt. This is approximately the relationship determined by McClure is his treatment for CzI paraffin chains in crystals below the rotational phase transition temperature [ 281. Again, for simplicity, we assume the same analytical form for the interaction energy between chain segments containing the gauche bonds as that for the parallel all-trans segments. (McCammon and Deutch [ 291 elected, however, to reduce the interaction energy between slightly disordered chains by about 6% from the all-trans values.) Following McCammon and Deutch [ 291, we take the effective chain length equal to N-2, as a consequence of chain overlap [ 161, and treat the methyl groups as methylene units. The number of gauche isomers per chain for each lipid system in the gel state was taken from ref. 5. With these approximations, we obtain for the dimyristoyl, dipalmitoyl and distearoyl systems iw values of 1.6, 1.9 and 2.5 kcal/mol, respectively, values in good agreement with the enthalpies determined from the experimental van ‘t Hoff expressions. The experimental values suggest that the Cl4 and Clb systems are somewhat analogous Mintrmal in behavior to one another, but differ from the Cl8 system. In contrast to the experimental enthalpies, the calculated values suggest a more gradual change in AHinternal between all three chain types. Since the calculation omits an explicit interaction term coupling the two monolayers, the enthalpy changes involved in the disordering effects toward the bilayer center may be significantly underestimated, particularly, for the distearoyl matrix. In summary, the temperature-dependent Raman spectra for particularly the C-H stretching region illustrates the inseparability between inter and intrachain contributions to hydrocarbon disorder in 1,2-diacyl phosphatidylcholinewater gels. Since changes in the various intensity ratios for C-H stretching transitions primarily reflect an expansion of the lipid matrix, Fig. 5 demonstrates for the Cl6 system the decrease in lateral chain packing order as vibrational modes between adjacent acyl chains uncouple. Thus, for dipalmitoyl phosphatidylcholine-water dispersions (Fig. 5), for example, the decrease in hzsT9 2.07 to 1.33 represents the disordering of the crystalline cm-llhm4 cm- I from
190
lattice beginning at approximately -45” C. Since trans-gauche isomerization also begins about -40°C in the Cl6 system, we conclude that the separate matrix events involving lattice expansion and chain rotational isomerization are closely related and occur simultaneously perhaps to relieve the constraints dictated by the interlocking of the methyl groups along the bilayer center. Coincident with the temperature for the onset of trans-gauche isomerization, the increase for the C,, system to approx. -10 to 10°C (Table II) for the inflection of the C-H stretching mode intensity ratios suggests that the angles of chain tilt markedly affect the temperature for which both the lateral packing and intrachain conformational characteristics are altered. Acknowledgment One of us (N.Y.) acknowledges the support of Health Visiting Fellow appointment.
provided
by a National
Institutes
References 1
Ifackenbrock.
2
Verkleij,
A.J.
Verkleij,
A.J.,
3
Acta
C.R.. and
M. and
Chau,
P.H.J.Th.
Ververgaert.
R.M.
(1976)
(1975)
Ann.
Van
P.H.J..
Deenen,
4
Spiker,
Jr.,
YeIIin,
N. and
6
Levin.
7
Spiker,
8
Rothman,
9
Levine,
R.C.
I.W.
lishing
and
Levin,
Levin,
(1969)
Jr.,
I.W.
I.W.
(1976)
(1977)
Biophys.
Physical
L.L.M.
and
Levin,
(1973)
Acta
I.W.
and
Acta
Chem.
26.
Elbers,
P.F.
Acta
455.
560-575
Acta
388.
361-373
455,
466484
101-122
(1972)
Biochim.
Biophys.
J. Theor.
(1972)
Prog.
(1972)
Biomembranes
Biophys.
New
1157-1160
Biochim.
Biol.
H.
Biophys.
16.642447
25A.
(1975)
Y.K.
Corporation,
Biochim.
Biochem.
Spectrochim.
R.C. J.E.
Traiible,
38, Mol. 3.
Biophys.
l-16 Biol.
24,
(F.
Lippert,
J.L.
and
Peticolas,
W.L.
(1972)
Biochim.
Lippert,
J.L.
and
Peticolas,
W.L.
(1971)
Proc.
13
Spiker,
I.W.
(1976)
Biochim.
14
Hinz,
15
Tardieu,
H.J.
16
Hitchcock,
R.C. and
A.,
and
Levin.
Sturtevant,
Luzzati, P.B.,
Kreuzer
and
J.F.G.
Slegers,
Eds.),
Plenum
Pub-
York
11
Jr.,
l-74
197-227
12
71.
Biochim. Rev.
288.326-332
5
10
HBchli, Ververgaert.
J.M.
V. and
Mason,
(1972)
Reman,
R.,
J. Biol. F.C.
Thomas,
Biophys.
Nat.
Sci..
Biophys. Chem.
(1973)
K.M.
Acta.
Acad.
Acta
247,
J. Mol.
and
282.
8-17
U.S.A.
68,
433,
1572-1576
457-468
60714075 Biol.
Shipley,
75.
711-733
G.G.
(1974)
Proc.
Nat.
Acad.
Sci..
U.S.A.
3036-3040
17
Mendelsohn,
18
Boerio.
F.J.
19
Larsson,
20
Mendelsohn,
K.
21
Brown,
22
Lamson.
R.,
Sunder,
S. and
and
Koenig,
J.L.
(1973) R.,
K.G.,
Rand,
M. and
Lcvin,
23
Bunow, Yellin,
N. and J.F.
25
Nagle, Shapiro,
27
Salem,
Levin,
(1973)
E. and
28
McClure,
29
McCammon,
D.W.
W.L. R.P. I.W.
I.W.
(1968) J.A.
and
Brown,
(1977) Phys.
J. Chem. Deutch,
58,
(1975)
Biochim.
Phys.
52,
Lipids
10,
H.J. E.
(1976) (1973)
Acta Acta Acta
Biophys. Biophys.
326, 487.
245-255 388-394
468,
490-494
Science
47,
252-264
J. CoIloid
and
Interface
38-49
37,2100-2113 Phys.
J.M.
49.1830-1839
(1975)
J. Am.
Acta
413,
329-340
419.
563-569
165-176
Biochem.
Biophys. Biophys.
Biophys.
3425-3431 Biochim.
Biophys.
Biochim. Biochim.
Phys.
of
Biochim.
(1977)
S. (1974)
J. Chem.
Physics
(1973)
H.J.
J. Chem.
Bernstein,
and
J. Chem. Ohki,
L. (1962)
and
S. and
Peticolas,
K. and
24 26
Chemistry Sunder,
Bernstein,
(1970)
Chem.
Sot.
97,
66754681
Acta Res.
Commun.
54,
358-368
