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A 31P- and 2H-NMR study on lecithins in liquid crystalline polyoxyethylene detergents
Chemistry and Physics of Lipids, 34 (1983) 65-80 Elsevier Scientific Publishers Ireland Ltd.
65
A 3tp. A N D 2 H - N M R S T U D Y O N L E C I T H I N S I N L I Q U I D CRYSTALLINE POLYOXYETHYLENE DETERGENTS
KLAUSBEYER lnslitut flir Physikalische Biochemie tier Universitiit Miinchen, Goethestrasse 33, 8000 Miinchen 2 (Federal Republic of Germany) Received January 7th, 1983 accepted May 20th, 1983
revision received May 19th, 1983
Phosphatidylcholines were incorporated into hexagonal liquid crystalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecyletherwith D20. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the heptagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyi chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the 31P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from 3tP-NMR. At the same time 2H-NMR exhibits a new signal from I~O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems. Keywords: NMR; lecithin; hexagonal; liquid crystalline; detergents.
Introduction T h e d i s a s s e m b l y of b i o l o g i c a l m e m b r a n e s a i m i n g at t h e i s o l a t i o n of p a r t i c u l a r m e m b r a n e p r o t e i n s a n d r e c o n s t i t u t i o n of t h e i r f u n c t i o n r e q u i r e s t h e f o r m a t i o n of m i x e d p h o s p h o l i p i d - d e t e r g e n t micelles. A l t h o u g h t h e a p p l i c a t i o n of d e t e r g e n t s is a s t a n d a r d t e c h n i q u e in m e m b r a n e b i o c h e m i s t r y , t h e i n t e r n a l s t r u c t u r e of m i x e d m i c e l l e s r e m a i n s a m a t t e r of c o n t r o v e r s y [ 1 - 3 ] . T h i s is m a i n l y d u e to t h e r a p i d i s o t r o p i c t u m b l i n g of m i c e l l e s in a q u e o u s s o l u t i o n r e n d e r i n g s t r u c t u r e sensitive m e t h o d s such as X - r a y diffraction o r p h o s p h o r u s a n d d e u t e r i u m N M R i n a p p l i c a b l e . Thus, w h e r e a s t h e t i m e a v e r a g e d m i c e l l e size a n d s h a p e a r e easily d e t e r m i n e d [4], t h e m o d e of i n t e r n a l m o l e c u l a r p a c k i n g r e m a i n s u n c l e a r . R e s t r i c t e d a n i s o t r o p i c m o l e c u l a r m o t i o n p a r t i a l l y p r e v e n t s this a v e r a g i n g in l i q u i d c r y s t a l l i n e p h a s e s f o r m e d b y a l m o s t all a m p h i p h i l e s at low w a t e r c o n t e n t , in c o n t r a s t t o m i c e l l a r a g g r e g a t e s . H e x a g o n a l s t r u c t u r e s , f r e q u e n t l y 0009-3084/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
66 found at detergent concentrations above 40%, by wt, may be considered as consisting of densely packed micelles of rather elongated shape. Hence it seems to be justified to incorporate small amounts of phospholipid into a hexagonal liquid crystalline detergent phase in order to gain insight into the internal structure of mixed phospholipid-detergent micelles. In the present contribution, phase structures and phase transitions of choline phospholipids incorporated into hexagonal liquid crystalline lattices of oxyethylene detergents have been studied by 31p_ and 2H-NMR. Both nuclei can give structural information due to the anisotropy of phosphorus chemical shifts and deuterium quadrupole couplings, respectively [5,6]. The host phases for the incorporation of the phospholipids were made up of Triton X-100 and octaethyleneglycoldodecylether (C12E8), both detergents being of widespread use in membrane biochemistry. In a previous paper [7] the phase structures of the system Triton X-100-D20 have been studied in detail by 2H-NMR. Similar to Triton, C12E8 forms a stable hexagonal liquid crystalline phase as shown here in the same way as for Triton X-100. In mixed phospholipid membranes the thermotropic liquid crystal to gel phase transition of one of the components eventually leads to lateral phase separation [8]. A similar behavior was observed in the present study when phospholipids with saturated acyl moieties were incorporated into the detergent host phases. Unexpectedly, after long time incubation below the transition temperature of the phospholipids, the 31p-NMR signal disappeared almost completely, whereas in the 2H-NMR spectrum from bound D20 a new signal appeared. This behavior, never observed before in mixed lecithin-detergent systems, will be explained tentatively in terms of aggregation and dehydration of the phospholipid headgroups, in analogy to the formation of a coagel in fatty acid-water systems. Materials and methods
Chemicals L-a-l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), L-a-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and L-a-distearoyl-sn-glycero-3phosphocholine (DSPC) were obtained from Sigma (D-8000 M/inchen), ~o,to'-d6-DPPC was from Serdary Research Lab., Inc. (London, Ontario, N6G 2R7, Canada). Egg lecithin was prepared as previously described [9]. All phospholipids were checked for purity by thin-layer chromatography. Triton X-100 was obtained from Sigma and C12E8from The Kouyoh Trading Co., Ltd. (Kyodo Bldg., 4-1,2-Chome, Iwamoto-Cho, Chiyoda-Ku, Tokyo, Japan).
Methods The components of the liquid crystalline phospholipid-detergent-D20 samples were weighed directly into NMR tubes of 10mm o.d. and
67 thoroughly mixed by shaking above the respective phase transitions of the detergents and phospholipids. The total weight of the samples was always 600-700 mg. Sealing by parafilm was sufficient to avoid appreciable water evaporation during the mixing procedure. The samples were stored for at least 1 week prior to the NMR experiments. The partial phase diagram of the system C~2Es-D20 was constructed by 2H-NMR as described previously [7]. In the present case only the boundary between the hexagonal phase and the surrounding two phase region was determined. The hexagonal nature of the liquid crystalline structure was confirmed by polarizing microscopy. NMR-measurements were performed with a Bruker SXP 4-100 spectrometer. All 3~p-NMR spectra (36.4 MHz) were broadband decoupled at a constant decoupling field strength of about 0.15 mT as determined by the method of Ernst [10]. The pulse repetition time was 1 s-~ and the pulse angle =70°C. The 2H-NMR spectra (13.8 MHz) were obtained by single 90° pulses or, in the case of deuterium-labeled lipids, by the quadrupole echo method [11]. The temperature was controlled with a digital thermometer [7]. Results
Incorporation of a small amount of lecithin into the hexagonal phase of Triton X-100 [7] leads to cylindrical motion of the phospholipid molecules (see Fig. 1, upper left spectrum). This can be concluded from the line shape of the 31p-NMR signal (see Ref. 5 for a detailed review). The effective chemical shift difference of 23 ppm is almost exactly half the value found in a multilamellar egg lecithin dispersion in excess water at the same temperature. Obviously the lecithin molecules adopt the cylindrical structure of the detergent host phase, whereas the conformation and mobility of the headgroup seem to be hardly affected by the surrounding detergent molecules. In the same sample the 2H-NMR spectrum of the bound deuterium oxide was obtained. The powder pattern (Fig. 1, upper right spectrum) reflects the anisotropic diffusion of the water molecules along the cylinder geometry. An almost identical quadrupole splitting as in the mixed system has been found earlier in the pure detergent phase at the same temperature and water content [7]. At increased molar ratios of lecithin/Triton, the 31p spectra reveal the appearance of a separate lamellar phase (Fig. 1). Phase separation is also manifest in the corresponding 2H-NMR spectra. At a lecithin/detergent molar ratio of 2, clearly two powder patterns are superimposed, whereas at lower ratios the two components are less distinct. In principle it should be possible by computer simulation of the 31p_ and 2H-spectra to evaluate the composition of the co-existing phases. However,
68 Tnfon/Egg Lecilhln mo[/mot
3,g5
I
50 ppm
500Hz
Fig. 1. 36.4 MHz-3LP- (left column) and 13.8MI-Iz-2H-NMR spectra (right column) of the system Triton X-100-egg lecithin-D20. Temperature ll°C. The mole fraction of D20 was held constant in all samples at XD:O= 0.967. superposition of simulated 3~P- signals showed that the experimental alp_ spectra deviated slightly from the ideal line shape in the magic angle region leading to uncertainties in the integral ratio of the two components. This deviation may be due to structural distortions in the boundary zones between the phase domains. Nevertheless, it could be estimated that the lamellar phase is always enriched in phospholipid. It could be argued that the incorporation of lecithin into the hexagonal Triton structure is due to the bulky hydrophilic isooctyl moiety of this detergent. Further, Triton is rather inhomogeneous with respect to its oxyethylene chain. In order to see whether the structure of the Triton phospholipid mixtures and the observed phase separations are due to these features of Triton, the interaction of lecithins with the commercially available h o m o g e n e o u s detergent C~2E8 has been studied; C12E8, like Triton and several other oxyethylene detergents [12], forms an extended hexagonal phase, as shown in Fig. 2. A similar but not identical phase boundary has
69
,
i
i
40
50
w
60
70
W% C12E 8
Fig. 2. Hexagonal phase of the system C12Es-D20 as determined by 2H*NMR [7]. Only the boundary of the one phase region was determined. The temperature was varied in steps of 1°C and the phase boundary was defined by the appearance of an isotropic 2H-NMR signal superimposed on the powder pattern. been found in the system C12Es-H20 by Shinoda [13]. The difference may be due to the heterogeneity in the oxyethylene chain of the detergent used in the earlier study. Egg lecithin fits into this phase as into the corresponding Triton structure. This demonstrates that the accommodation of the phospholipid into the hexagonal detergent phase is due to the general capability of these detergents to form mixed micelles rather than being a special property of Triton. The superposition of a hexagonal and a lamellar 31P-signal was also demonstrated by a saturation transfer experiment [14,15]. Application of a 40 ms D A N T E pulse train sufficient for partial saturation of the lamellar signal as indicated in Fig. 3 revealed the high field shoulder of the hexagonal signal and had no influence on the intensity of the hexagonal peak. Assuming a lateral diffusion constant of 2 × 10-Scm2s -1 for the phospholipid molecules in the liquid crystalline state [16], this means that the lamellar and hexagonal phase regions must be at least about 4000 ,~ in diameter. Also the superimposed 2H-NMR spectra of bound O20 reflect this spatial separation of the two co-existing phase structures. Increasing temperature in both ternary mixtures leads to preferred formation of the lamellar structure, as shown for the system T r i t o n - e g g lecithin-D20 at a Triton/phospholipid molar ratio of 2 (Fig. 4). Complete transition of the phospholipid molecules to the lamellar state at 26°C as evidenced in the 31P-spectrum is accompanied by the disappearance of the 2H-NMR powder pattern exhibiting the smaller splitting pertinent to the hexagonal phase. Thus at this temperature the entire sample is homo-
70
without
with
decoupting
r
d
I 50 ppm
Fig. 3. Left spectrum : 31p-NMR of a mixture of 200 mg C12Es and 139.4 mg egg lecithin, molar ratio Cs2Ea/lecithin = 2. D20 content 43.5%, by wt, corresponding to Xo~o = 0.959. Temperature 25°C. Right spectrum: Saturation transfer 31p-NMR spectrum ( D A N T E ) of the same sample. Saturation at the spectral position indicated by the arrow. The pulse sequence was [(P1 - D1)N1- D2 - P2 - acquisition]N~ with PI = 0.5 Ixs; D1 = 200 ~ts; NI = 200; /92 = 10 ~xs; />2 = 10 O-s; N2 = 500. P denotes pulse width; D, delay time and N, no. of repetitions. Trlton/Egg Lecithn : 2 mot/mot
20% I
/
\
,i
-I°[
/
/f ./'
J /
~ -~-.-. _if ~'~ F
50 ppm
~,~ 4
,
(
500 Hz
Fig. 4. Temperature dependence of 3tp. and corresponding 2H-NMR spectra in a liquid crystalline mixture of Triton and egg lecithin (for sample composition cf. bottom spectra in Fig. 1).
71 geneously lamellar. At the same detergent/lecithin ratio this transformation temperature is about 35°C in the C12Es-egg lecithin-D20 system. The small cross-sectional diameter and the high curvature of the rod-like structure in the hexagonal detergent phase suggests that the average conformation of the acyl chains of the incorporated phosphatidylcholine is rather different from what is known about phospholipid bilayers in the liquid crystalline state. In order to examine the effect of geometrical constraints on the fatty acid order dipalmitoyllecithin selectively deuterated in the terminal methyl groups (to, to'-d6-DPPC) was incorporated into a hexagonal C12EsH 2 0 sample. The quadrupole splitting of the methyl deuterons of the acyl moieties in multilamellar to,to'-d6-DPPC is 2.67 kHz at 45°C (see Fig. 5). At the same temperature, to,to'-d6-DPPC in the hexagonal phase of C12E8 exhibits a splitting of 0.84kHz (Fig. 5). This is significantly less than the value of 1.35 kHz to be expected on the basis of cylindrical phospholipid motion [6] indicating that the acyl moieties are disordered at least at the end of the chains. Macroscopically homogeneous and translucent samples were obtained when the synthetic lecithins DMPC, D P P C or DSPC above their respective thermotropic phase transitions were mixed with Triton or Ca2E8 and the appropriate amounts of D20 were added to yield the hexagonal phases. Upon cooling, these samples became turbid. At a molar ratio of detergent/phospholipid of 6 the onset of this turbidity was found about 8-12°C
I
I
2 kHz
Fig. 5. 13.8MHz-2H-NMR spectra of t0,t0'-d6-DPPC at 45°C. (A) 50% in H20. (]3) In the hexagonal liquid crystalline phase of C12Es. Molar ratio detergent/phospholipid= 6/1. The central signal originates most likely from natural abundance deuterium in I-t20 in the outer coaxial NMR tube.
72
below the thermotropic phase transition of the pure lipids, irrespective of the host detergent (Fig. 6). 31p-NMR spectra in this temperature region revealed the co-existence of phospholipid molecules in the hexagonal liquid crystalline and in a lamellar state characterized by restricted headgroup mobility (Fig. 7). The line shapes of proton decoupled gel phase 31p spectra of
O • •
•
•
•
.
Q
° .
°
DMPC
"
E
DPPC
" DSPC
0 I
0
o ul
S
I
1
0
Q
0 0 0 0
#
<:{
2
DMPC
I
•
.
•
DPPC
°°°°o
°.
;
1~
1~
1~
2~
2~
3;
' 3'4 ' 3~
~2
4~ "c
Fig. 6. Temperature dependence of turbidity in liquid crystalline mixtures of synthetic phospholipids with C12Es (A) and Triton X-100 (B). Molar ratio detergent/phospholipid = 6/1. Water mol fraction was 0.959 in (A) and 0.967 in (B). Absorbance was measured at 366 nm in flame sealed ampoules after equilibration for 20 min at the temperatures indicated.
E12E8 / DPP[ 6 mol./mo[
26 o[
~ - ~ /
10%
4V~¢~;/~°y
'~::~
50ppm
Fig. 7. Phase transition of D P P C in C12Es as indicated by 31P-NMR.
73 the lecithins in excess water were virtually identical to those found in the detergent mixtures. This suggests that the lipid molecules in these mixtures segregate by lateral diffusion to form ordered gel phase domains. Furthermore, the t e m p e r a t u r e d e p e n d e n c e of 31p signal widths at half height of D P P C and D S P C in aqueous dispersion and in the Triton and C12E8 mixtures showed a rather good correlation (see Fig. 8). Also in the 13C-NMR spectra of d e t e r g e n t - p h o s p h o l i p i d mixtures below the t e m p e r a t u r e of the phase separation, the resonances from the acyl chains were b r o a d e n e d beyond detectability (data not shown). This confirms the idea that the segregated phospholipid is self-associated in almost pure form and is endowed with the same residual motional f r e e d o m as ordinary gel phase lecithins in excess water. In contrast to the egg lecithin-detergent mixtures at high egg lecithin content (cf. Fig. 1), 2H-NMR did not show separate powder patterns from bound D 2 0 even after completion of the phospholipid crystallization as indicated by 31p-NMR. T h e quadrupole splittings in mixtures of the lecithins with C12E8 were nearly identical to those found in the pure hexagonal detergent mesophase. It must be noted, however, that the phospholipidb o u n d water c o m p o n e n t may remain undetectable due to the low water binding capacity of the lecithin headgroup. On the other hand, averaging of the 2H spectrum due to rapid water exchange between the gel phase and the hexagonal liquid crystalline regions would suggest that the phospholipid clusters are small c o m p a r e d to the diffusion length per unit time of the water molecules. Surprisingly, after storage of mixtures of C12E8 with D S P C or D P P C for several weeks at 4°C, the intensity of the 3~p spectra recorded at the storage
A
o•
~=26-
o
2~,~ 22"6 2.o1.8-
16-
in • DPPC 1 oDSPCJ C12E8
a
• DPPC] T X o OSPCJ • OPPC'[ OSPCJ Suspension
o'ss
o
d9
0'gs
TIT c (°K/OK)
Fig. 8. Full 31p line width at half height of the gel phase signal vs. reduced temperature for various synthetic phospholipids in detergent liquid crystalline host phases or in aqueous suspension. Superimposed 'hexagonal' 3tp signals <30% of the total signal intensity had no effect on the line width. Tc = temperature of the thermotropic phase transition of the respective pure phospholipid. The decoupling field strength was held constant at 150 gT.
74 t e m p e r a t u r e without reheating was drastically reduced. U p o n equilibration of the sample at higher temperatures but well below the t e m p e r a t u r e where the onset in turbidity was observed (cf. Fig. 6) a 'hexagonal' 31p signal a p p e a r e d (Fig. 9, left column). Cooling from 19.5°C led to transformation of most of this 'hexagonal' into a 'gel phase' signal. Incubation of the sample for 1 min at 45°C to restore the h o m o g e n e o u s hexagonal phase and subsequent cooling resulted in the same sip-spectrum as obtained immediately after sample preparation (Fig. 9, right column, see also Fig. 7). The reduction of the observable 3~p signal may be tentatively ascribed to strong immobilization of the lecithin headgroups leading to a rigid limit powder pattern and to enhanced dipolar broadening. Unfortunately the 31p line shape could not be evaluated under the experimental conditions employed. Sufficient signal to noise and the correct 3~p line shape would only be available in a cross polarization experiment at very high proton decoupling power [17]. In the D20-2H spectrum of the same sample as in Fig. 9, after 2 - 3 weeks at 4°C, a broad singlet was superimposed on the quadrupole powder pattern. OPPC in C12E8
after' storage at 4"E 3.5 "[
I/
after' Dncubahon at 19,5 "C
,'~
115
after Lncubahon at ~00 "C
11,5"[ 0,5
19,5"C
~
~
Fig. 9. Effect of long-time incubation (2 weeks at 4°C) on the 3~p line shape in the system C12Es-DPPC-D20. Molar ratio C12Ea/DPPC= 6/1. The mole fraction of D20 was Xtho = 0.959. Prior to the NMR experiments the sample was equilibrated for 30 min at the temperatures indicated. Heating to 40°C (lower right spectrum) was for 1 min only.
75 Again, after heating to 40°C the original powder pattern was restored (Fig. 10). No loss in total signal intensity could be detected after the storage period in contrast to the phosphorus spectra. The 2H spin lattice relaxation time of both D20 components has been measured. The signal intensity of the components could be determined separately after the 180-r-90 sequence by subtraction of an appropriate computer simulated Lorentzian line. The isotropic water signal was found to relax more rapidly than the splitted signal. At 4.4°C relaxation times of 0.026 s and 0.042 s were found. After rehomogenization of the sample by heating to 45°C as described above, T1 was 0.040 s. The anisotropic motion of D20 molecules in a liquid crystalline phase can be described in terms of a two correlation times model. Due to this model, spin lattice relaxation is determined by rapid flipping about the bisector of the water molecule, whereas the slow tumbling of this axis does not contribute to T1 [18]. Thus the decrease in Tt and, at the same time, the disappearance of the quadrupole splitting signify that in the new environment the water molecules undergo slow isotropic instead of rapid unisotropic motion. The amount of water corresponding to the singlet D20 signal in Fig. 10 was evaluated also by subtraction of a Lorentzian line of appropriate intensity and width from the compound experimental 2H spectrum so as to obtain the pure powder pattern (Table I). The isotropic signal corresponds to 30-40 molecules of D20 per phospholipid headgroup. This may be after storage at 4%
/~
~
/~
0%
40%
_/ after incubct,on
500 Hz Fig. ]0. Storage effect on the EH-NMR spectra o[ bound D20. Sample and storage conditions as in Fig. 9. Incubation at 40°C for I rain.
76 TABLE I R E L A T I V E A M O U N T S O F D 2 0 IN D I F F E R E N T M O T I O N A L S T A T E S A F T E R L O N G T I M E I N C U B A T I O N O F DSPC/CI2Es/D20 S A M P L E S CI2Es/DSPC (mol/mol) ~
Ao/A Ib
D20(I)/DSPC (mol/mol) ~
D~O(Q)/C12E8 (tool/tool) c
6.0 8.1 9.7 11.9 15.1 19.8
4.0 5.6 6.8 7.5 9.7 10.6
33.0 33.6 31.7 35.6 35.4 41.0
21.8 22.3 22.5 22.4 22.6 22.0
aThe mole fraction of D 2 0 was held constant in all samples at Xa-zo = bRatio of the integrated intensities of the pure powder pattern and pure powder pattern was obtained by c o m p u t e r subtraction of a appropriate line width and intensity. CD20(l); D 2 0 ( Q ) , a m o u n t of D 2 0 corresponding to the isotropic pattern, respectively.
0.959. the isotropic signal. T h e simulated Lorentzian of line and to the powder
compared to the number of about 9 water molecules tightly bound to gel phase phospholipid as reported earlier [19]. Thus more than the amount of water hydrating the phospholipid headgroups has been liberated upon phospholipid aggregation. At the same time the hydration of the detergent remains nearly constant at about 22 tool D20 per mol of C12E8, corresponding to a detergent concentration of about 55% by wt (cf. Fig. 2).
Discussion
From extensive X-ray studies [20,21] it is known that hexagonal structures in pure lecithins form only at rather high temperatures (>100°C) and very low hydration (<4% by wt of water). In excess water these phospholipids exhibit a variety of lamellar structures (for a review see Ref. 8). When mixed with liquid crystalline non-ionic detergents, the phosphatidylcholines obviously fit easily into the two-dimensional hexagonal lattice. In contrast, phosphatidylethanolamines and certain phospholipid mixtures undergo lamellar-hexagonal phase transitions also in excess water. The hexagonal phase formed by these lipids are generally of the H,-type where the headgroups are directed towards a water channel in the center of the cylindrical~structure with the acyl chains facing outwards. On the other hand it it known from earlier work that in the hexagonal ('middle') phase of oxyethylene detergents the hydrophilic tails extend in the opposite direction even in the presence of small amounts of foreign molecules [22,23]. The headgroups of the phosphatidylcholine molecules incorporated into the
77 hexagonal detergent host phase are most likely located in the oxyethylene layer of the detergent cylinders with the water of hydration exchanging rapidly between oxyethylene and choline sites. This means that the phospholipid, when solubilized in the hexagonal detergent clusters, is in the Hi-state. Thus, at low concentration the phospholipid molecules are forced to assume the structure of the detergent host phase, in agreement with the traditional picture of mixed phospholipid-detergent micelles [24]. At higher lecithin concentration the situation is reversed in as much as now the phospholipid determines the phase structure, resulting in lamellar aggregates. This behavior may be tentatively explained in terms of geometry. It has been suggested [25,26] that a dimensionless packing parameter v/ao'lc determines the structure of the aggregates, where v denotes the volume and Ic the maximum length of the hydrophobic chains and a0 the cross-sectional area of the hydrophilic headgroup of the amphiphilic molecules at minimum surface free energy. Rod-like structures are favored if ~< v/ao" lc <½, whereas at ratios >~, lamellar structures will form. Thus addition of increasing amounts of phospholipids with rather voluminous fatty acid chains to a hexagonal detergent phase can be expected to result in a hexagonal-lamellar phase transition. Weakening of water binding to the hydrophilic headgroup and thereby decreasing surface area a0 could explain the preference of the lamellar structure at elevated temperatures. Although the behavior of the system egg lecithin-Triton X-100 or egg lecithin-C12E8 is in qualitative agreement with an interpretation along these lines, the question remains how the rather long phospholipid acyl chains fit into the hydrophobic core of the rod-like structure. On the basis of the 3~P chemical shift anisotropy observed it can be concluded that the motional behavior of the lecithin headgroups in the hexagonal phase is not very different from that in the ordinary liquid crystalline Lo~ phase. It has been shown recently that the headgroup structure of lecithins is identical in micelles and bilayers [27,28]. It appears reasonable to assume that this is true also in the hexagonal liquid crystalline mixture, although this is not borne out by the results of 3~p-NMR, due to the ambiguity inherent in the non-axial chemical shift tensor of the 31p nucleus [5]. The diameter of the hydrophobic core of the rod-like structures in the Triton X-100 host phase can be calculated to be about 20/~ assuming a circular cross-section [29]. The thickness of the egg lecithin bilayer in the liquid crystalline state is about 38/~ at a water content of 40%, by wt, and 25°C [20]. Thus radial intercalation of lecithin molecules in the hexagonal structure seems to be unlikely. In C~2E8 the diameter of the hydrophobic core is larger than in Triton (about 24-28 ~ , assuming a circular cross-section) but again too small to allow radial arrangement of the phospholipid molecules in the detergent matrix [30]. Alternatively, an elliptical cross-section of
78 the rods may be assumed where the chain lengths of the acyl moieties in the phospholipid determines the long axis. Flipping of the phospholipids about 180/~ at a rate ~>103s would lead to the observed 'hexagonal' alp. NMR pattern. However, this is in contrast to phospholipid flip-flop rates in bilayer membranes which are about 8 orders of magnitude slower [31]. A non-classical micelle model has been recently proposed by Fromherz [3]. In this model parallel correlated bilayer blocks are arranged so as to form a cubic structure with a similar perimeter as the classical micelle. Extending this idea to the hexagonal liquid crystalline phase it is conceivable that phospholipids replace some of the detergent molecules without any extreme bending of the acyl chains. The observation of reduced quadrupole splitting in DPPC deuterated in the terminal methyl groups of the long chains does not contradict this model since the chain ends may have more conformational freedom in the block arrangement than in the extended bilayers. Additional experiments, in particular T~ measurements, are needed to examine whether the decrease in 'fatty acid order' is accompanied by an increase in chain mobility. The occurrence of lateral phase separation in mixed lipid bilayers upon crystallization of one membrane component has been established by a number of physical techniques. When the chain length of the lipids differs by more than 4 C-atoms or when lipids with saturated and unsaturated acyl chains are mixed, immiscibility in the gel phase and monotectic behavior are commonly observed [32]. It is conceivable that the systems under investigation here behave analogously. It has been noted, however, by Lee [33] that complete immiscibility in the gel phase must be considered as a limiting case and the appearance of a eutectic composition very close to one of the pure components is more probable. Thus, although segregation of pure gel phase lecithin seems plausible, an admixture of a trace of the detergent to the gel phase patches cannot be excluded. The direct transition from the hexagonal liquid crystalline solution to the lamellar gel state requires lateral diffusion of the phospholipid molecules along the axis of the rod-like clusters and parallel alignment of the acyl chains in the primary crystal nuclei. Whereas unhindered lateral diffusion can be assumed, the formation of small gel phase patches seems to be unfavorable due to the large free energy contribution of the domain boundaries. Thus it is not surprising that the presumably rather small primary gel phase patches are thermodynamically metastable and undergo a slow secondary association. This process proceeds within a few days whereas the formation~of the primary gel phase patches is complete within a few minutes. The different rates may be due to the very different diffusion rates of single lecithin molecules and gel phase clusters in the viscous liquid crystalline environment. A large decrease in entropy is to be expected on gel phase formation if the phospholipid molecules are constrained in a crowded
79 conformation by radial intercalation into the hexagonal detergent structure. In bilayers, on the contrary, the corresponding entropy change per methylene group is only about one half that found in bulk hydrocarbons [34]. Again, the above mentioned block model [3], where parallel alignment of small units is supposed, could explain the ease of the transition. Yet no experimental evidence for this model is available to date. The conspicuous disappearance of the 31p-signal after long time incubation below the transition temperature can be explained tentatively by strong immobilization of the headgroups. In fully hydrated samples of DPPC headgroup immobilization has been observed only at about -10°C, i.e. 50°C below the phase transition [35]. Thus the loss of headgroup motion must have to do with the low water content of the mixed liquid crystalline system. 31p-NMR of anhydrous DPPC at 15°C shows the characteristic rigid lattice line shape to be expected for a chemical shift tensor with three different principal components. 'Upon addition of more than 3 water molecules per headgroup this spectrum transforms to the anisotropic signal typical for rapid axial motion [36]. This signal is insensitive to further water addition and changes only slightly with temperature. Thus the 'bound water' liquifies the lecithin headgroup dipoles. Likewise, the lack of 31p intensity in the lecithin-detergent systems under low power decoupling conditions and, at the same time, the appearance of an isotropic D20 line suggests that upon secondary aggregation of the gel phase lipid the headgroups become dehydrated and thereupon locked by strong dipole-dipole interaction. The observation of headgroup immobilization and isotropic water motion may be ascribed to the formation of a coagel in analogy to the behavior of hydrated long chain fatty acids [20]. In some cases the hydrated gel phase of these compounds is metastable and water will be squeezed,oiat of the lipid lamellae. The released water merges into small pockets where it undergoes isotropic tumbling [37]. After long-time incubation the amount of water in these pockets obviously exceeds the water quantity released from the phospholipid headgroups, whereas the detergent hydration reaches a constant value of about 2.8 molecules of D20 per oxyethylene group (cf. Table I). Thus the final state of the system may be determined by the free energy minimum ensuing from the optimal hydration of the hexagonal liquid crystalline detergent phase.
Acknowledgment The author is grateful to Prof. M. Klingenberg for valuable discussions and constant support. Also the excellent technical assistance of Renate Lafuntal and Liselotte Schmidt-Erhard is gratefully acknowledged. The work was supported by the Deutsche Forschungsgemeinschaft.
8O
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
H. Wennerstr6m and B. Lindman, Phys. Rep., 52 (1979) 1. R.L. Fisher and D.G. Oakenfull, Chem. Soc. Rev., 6 (1977) 25. P. Fromherz, Ber. Bunsenges. Phys. Chem., 85 (1981) 891. C. Tanford, The Hydrophobic Effect, John Wiley, New York, 2nd edn., 1980, p. 79. J. Seelig, Biochim. Biophys. Acta, 505 (1978) 105. J. Seelig, Q. Rev. Biophys., 10 (1977) 353. K. Beyer, J. Colloid Interface SCi., 86 (1982) 73. H. Hauser and M.C. Phillips, in: D.A. Cadenhead and J.F. Danielli (Eds.), Surface and Membrane Science, Vol. 13, Academic Press, New York, 1979, p. 297. K. Beyer and M. Klingenberg, Biochemistry, 17 (1978) 1424. R.R. Ernst, J. Chem. Phys., 45 (1966) 3845. J.H. Davis, K.R. Jeffrey, M. Bloom, M.I. Valic and T.P. Higgs, Chem. Phys. Lett., 42 (1976) 390. H. Kelker and R. Hatz, Handbook of Liquid Crystals, Verlag Chemic, Weinheim, 1980, Chap. 10 and 11. K. Shinoda, J. Colloid Interface Sci., 34 (1970) 278. G. Morris and R. Freeman, J. Magn. Reson., 29 (1978) 433. B. DeKruijff, G.A. Morris and P.R. Cullis, Biochim. Biophys. Acta, 598 (1980) 206. P. Devaux and H.M. McConnell, J. Am. Chem. Soc., 94 (1972) 4475. R.G. Griffin, J. Am. Chem. Soc., 98 (1976) 851. B.A. Cornell, J.M. Pope and G.J. Troup, Chem. Phys. Lipids, 13 (1974) 183. N.J. Salsbury, A. Darke and D. Chapman, Chem. Phys. Lipids, 8 (1972) 142. V. Luzatti, in: D. Chapman (Ed.), Biological Membranes, Physical Fact and Function, Academic Press, London, 1968, p. 71. A. Tardieu and V. Luzatti, J. Mol. Biol., 75 (1973) 711. A. Skoulios, Adv. Colloid. Interface Sci., 1 (1967) 79. P. Ekwall, L. Mandell and K. Fontell, Acta Chem: Scand., 22 (1968) 373. A.A. Ribeiro and E.A. Dennis, Biochemistry, 14 (1975) 3746. J.N. Israelachvili, D.J. Mitchell and B.W. Ninham, J. Chem. Soc. Faraday Trans. II, 72 (1976) 1525. J.N. Israelachvili, S. Mar~elja and R.G. Horn, Q. Rev. Biophys., 13 (1980) 121. H. Hauser, W. Guyer, I. Pascher, P. Skrabal and S. Sundell, Biochemistry, 19 (1980) 366. N. Gains and H. Hauser, Biochim. Biophys. Acta, 646 (1981) 211. R.J. Robson and E.A. Dennis, J. Phys. Chem., 81 (1977) 1075. C. Tanford, J. Nozaki and M.F. Rohde, J. Phys. Chem., 81 (1977) 1555. J.E. Rothman and J. Lenard, Science, 195 (1977) 743. M.C. Phillips, B.D. Ladbrooke and D. Chapman, Biochim. Biophys. Acta, 196 (1970) 35. A.G. Lee, Biochim. Biophys. Acta, 472 (1977) 285. M.C. Phillips, R.M. Williams and D. Chapman, Chem. Phys. Lipids, 3 (1969) 234. R.G. Griffin, L. Powers and P.S. Pershan, Biochemistry, 17 (1978) 2718. R.G. Griffin, J. Am. Chem. Soc., 98 (1976) 851. K. Abdollal, E.E. Burnell and M.I. Valic, Chem. Phys. Lipids, 20 (1977) 115.
65
A 3tp. A N D 2 H - N M R S T U D Y O N L E C I T H I N S I N L I Q U I D CRYSTALLINE POLYOXYETHYLENE DETERGENTS
KLAUSBEYER lnslitut flir Physikalische Biochemie tier Universitiit Miinchen, Goethestrasse 33, 8000 Miinchen 2 (Federal Republic of Germany) Received January 7th, 1983 accepted May 20th, 1983
revision received May 19th, 1983
Phosphatidylcholines were incorporated into hexagonal liquid crystalline mixtures of the non-ionic detergents Triton X-100 and octaethyleneglycoldodecyletherwith D20. It is shown by nuclear magnetic resonance (NMR) that the phospholipids adopt the heptagonal liquid crystalline structure of the detergent host lattice. The anisotropic motion of the phospholipid headgroups seems to be unaffected, whereas the acyl chains are disordered. Increasing phospholipid concentration leads to separation of a lamellar phase. The lamellar structure is also preferred at elevated temperatures. Phosphatidylcholines with saturated acyi chains undergo a transition from the hexagonal liquid crystalline to an ordered lamellar state. The shape of the 31P-NMR signals suggests that pure gel phase phospholipid separates out. The headgroup region of this gel phase phospholipid becomes immobilized after a few weeks of storage below the transition temperature as judged from 3tP-NMR. At the same time 2H-NMR exhibits a new signal from I~O undergoing slow isotropic motion. This behavior bears resemblance to the formation of a coagel in fatty acid-water systems. Keywords: NMR; lecithin; hexagonal; liquid crystalline; detergents.
Introduction T h e d i s a s s e m b l y of b i o l o g i c a l m e m b r a n e s a i m i n g at t h e i s o l a t i o n of p a r t i c u l a r m e m b r a n e p r o t e i n s a n d r e c o n s t i t u t i o n of t h e i r f u n c t i o n r e q u i r e s t h e f o r m a t i o n of m i x e d p h o s p h o l i p i d - d e t e r g e n t micelles. A l t h o u g h t h e a p p l i c a t i o n of d e t e r g e n t s is a s t a n d a r d t e c h n i q u e in m e m b r a n e b i o c h e m i s t r y , t h e i n t e r n a l s t r u c t u r e of m i x e d m i c e l l e s r e m a i n s a m a t t e r of c o n t r o v e r s y [ 1 - 3 ] . T h i s is m a i n l y d u e to t h e r a p i d i s o t r o p i c t u m b l i n g of m i c e l l e s in a q u e o u s s o l u t i o n r e n d e r i n g s t r u c t u r e sensitive m e t h o d s such as X - r a y diffraction o r p h o s p h o r u s a n d d e u t e r i u m N M R i n a p p l i c a b l e . Thus, w h e r e a s t h e t i m e a v e r a g e d m i c e l l e size a n d s h a p e a r e easily d e t e r m i n e d [4], t h e m o d e of i n t e r n a l m o l e c u l a r p a c k i n g r e m a i n s u n c l e a r . R e s t r i c t e d a n i s o t r o p i c m o l e c u l a r m o t i o n p a r t i a l l y p r e v e n t s this a v e r a g i n g in l i q u i d c r y s t a l l i n e p h a s e s f o r m e d b y a l m o s t all a m p h i p h i l e s at low w a t e r c o n t e n t , in c o n t r a s t t o m i c e l l a r a g g r e g a t e s . H e x a g o n a l s t r u c t u r e s , f r e q u e n t l y 0009-3084/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
66 found at detergent concentrations above 40%, by wt, may be considered as consisting of densely packed micelles of rather elongated shape. Hence it seems to be justified to incorporate small amounts of phospholipid into a hexagonal liquid crystalline detergent phase in order to gain insight into the internal structure of mixed phospholipid-detergent micelles. In the present contribution, phase structures and phase transitions of choline phospholipids incorporated into hexagonal liquid crystalline lattices of oxyethylene detergents have been studied by 31p_ and 2H-NMR. Both nuclei can give structural information due to the anisotropy of phosphorus chemical shifts and deuterium quadrupole couplings, respectively [5,6]. The host phases for the incorporation of the phospholipids were made up of Triton X-100 and octaethyleneglycoldodecylether (C12E8), both detergents being of widespread use in membrane biochemistry. In a previous paper [7] the phase structures of the system Triton X-100-D20 have been studied in detail by 2H-NMR. Similar to Triton, C12E8 forms a stable hexagonal liquid crystalline phase as shown here in the same way as for Triton X-100. In mixed phospholipid membranes the thermotropic liquid crystal to gel phase transition of one of the components eventually leads to lateral phase separation [8]. A similar behavior was observed in the present study when phospholipids with saturated acyl moieties were incorporated into the detergent host phases. Unexpectedly, after long time incubation below the transition temperature of the phospholipids, the 31p-NMR signal disappeared almost completely, whereas in the 2H-NMR spectrum from bound D20 a new signal appeared. This behavior, never observed before in mixed lecithin-detergent systems, will be explained tentatively in terms of aggregation and dehydration of the phospholipid headgroups, in analogy to the formation of a coagel in fatty acid-water systems. Materials and methods
Chemicals L-a-l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), L-a-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and L-a-distearoyl-sn-glycero-3phosphocholine (DSPC) were obtained from Sigma (D-8000 M/inchen), ~o,to'-d6-DPPC was from Serdary Research Lab., Inc. (London, Ontario, N6G 2R7, Canada). Egg lecithin was prepared as previously described [9]. All phospholipids were checked for purity by thin-layer chromatography. Triton X-100 was obtained from Sigma and C12E8from The Kouyoh Trading Co., Ltd. (Kyodo Bldg., 4-1,2-Chome, Iwamoto-Cho, Chiyoda-Ku, Tokyo, Japan).
Methods The components of the liquid crystalline phospholipid-detergent-D20 samples were weighed directly into NMR tubes of 10mm o.d. and
67 thoroughly mixed by shaking above the respective phase transitions of the detergents and phospholipids. The total weight of the samples was always 600-700 mg. Sealing by parafilm was sufficient to avoid appreciable water evaporation during the mixing procedure. The samples were stored for at least 1 week prior to the NMR experiments. The partial phase diagram of the system C~2Es-D20 was constructed by 2H-NMR as described previously [7]. In the present case only the boundary between the hexagonal phase and the surrounding two phase region was determined. The hexagonal nature of the liquid crystalline structure was confirmed by polarizing microscopy. NMR-measurements were performed with a Bruker SXP 4-100 spectrometer. All 3~p-NMR spectra (36.4 MHz) were broadband decoupled at a constant decoupling field strength of about 0.15 mT as determined by the method of Ernst [10]. The pulse repetition time was 1 s-~ and the pulse angle =70°C. The 2H-NMR spectra (13.8 MHz) were obtained by single 90° pulses or, in the case of deuterium-labeled lipids, by the quadrupole echo method [11]. The temperature was controlled with a digital thermometer [7]. Results
Incorporation of a small amount of lecithin into the hexagonal phase of Triton X-100 [7] leads to cylindrical motion of the phospholipid molecules (see Fig. 1, upper left spectrum). This can be concluded from the line shape of the 31p-NMR signal (see Ref. 5 for a detailed review). The effective chemical shift difference of 23 ppm is almost exactly half the value found in a multilamellar egg lecithin dispersion in excess water at the same temperature. Obviously the lecithin molecules adopt the cylindrical structure of the detergent host phase, whereas the conformation and mobility of the headgroup seem to be hardly affected by the surrounding detergent molecules. In the same sample the 2H-NMR spectrum of the bound deuterium oxide was obtained. The powder pattern (Fig. 1, upper right spectrum) reflects the anisotropic diffusion of the water molecules along the cylinder geometry. An almost identical quadrupole splitting as in the mixed system has been found earlier in the pure detergent phase at the same temperature and water content [7]. At increased molar ratios of lecithin/Triton, the 31p spectra reveal the appearance of a separate lamellar phase (Fig. 1). Phase separation is also manifest in the corresponding 2H-NMR spectra. At a lecithin/detergent molar ratio of 2, clearly two powder patterns are superimposed, whereas at lower ratios the two components are less distinct. In principle it should be possible by computer simulation of the 31p_ and 2H-spectra to evaluate the composition of the co-existing phases. However,
68 Tnfon/Egg Lecilhln mo[/mot
3,g5
I
50 ppm
500Hz
Fig. 1. 36.4 MHz-3LP- (left column) and 13.8MI-Iz-2H-NMR spectra (right column) of the system Triton X-100-egg lecithin-D20. Temperature ll°C. The mole fraction of D20 was held constant in all samples at XD:O= 0.967. superposition of simulated 3~P- signals showed that the experimental alp_ spectra deviated slightly from the ideal line shape in the magic angle region leading to uncertainties in the integral ratio of the two components. This deviation may be due to structural distortions in the boundary zones between the phase domains. Nevertheless, it could be estimated that the lamellar phase is always enriched in phospholipid. It could be argued that the incorporation of lecithin into the hexagonal Triton structure is due to the bulky hydrophilic isooctyl moiety of this detergent. Further, Triton is rather inhomogeneous with respect to its oxyethylene chain. In order to see whether the structure of the Triton phospholipid mixtures and the observed phase separations are due to these features of Triton, the interaction of lecithins with the commercially available h o m o g e n e o u s detergent C~2E8 has been studied; C12E8, like Triton and several other oxyethylene detergents [12], forms an extended hexagonal phase, as shown in Fig. 2. A similar but not identical phase boundary has
69
,
i
i
40
50
w
60
70
W% C12E 8
Fig. 2. Hexagonal phase of the system C12Es-D20 as determined by 2H*NMR [7]. Only the boundary of the one phase region was determined. The temperature was varied in steps of 1°C and the phase boundary was defined by the appearance of an isotropic 2H-NMR signal superimposed on the powder pattern. been found in the system C12Es-H20 by Shinoda [13]. The difference may be due to the heterogeneity in the oxyethylene chain of the detergent used in the earlier study. Egg lecithin fits into this phase as into the corresponding Triton structure. This demonstrates that the accommodation of the phospholipid into the hexagonal detergent phase is due to the general capability of these detergents to form mixed micelles rather than being a special property of Triton. The superposition of a hexagonal and a lamellar 31P-signal was also demonstrated by a saturation transfer experiment [14,15]. Application of a 40 ms D A N T E pulse train sufficient for partial saturation of the lamellar signal as indicated in Fig. 3 revealed the high field shoulder of the hexagonal signal and had no influence on the intensity of the hexagonal peak. Assuming a lateral diffusion constant of 2 × 10-Scm2s -1 for the phospholipid molecules in the liquid crystalline state [16], this means that the lamellar and hexagonal phase regions must be at least about 4000 ,~ in diameter. Also the superimposed 2H-NMR spectra of bound O20 reflect this spatial separation of the two co-existing phase structures. Increasing temperature in both ternary mixtures leads to preferred formation of the lamellar structure, as shown for the system T r i t o n - e g g lecithin-D20 at a Triton/phospholipid molar ratio of 2 (Fig. 4). Complete transition of the phospholipid molecules to the lamellar state at 26°C as evidenced in the 31P-spectrum is accompanied by the disappearance of the 2H-NMR powder pattern exhibiting the smaller splitting pertinent to the hexagonal phase. Thus at this temperature the entire sample is homo-
70
without
with
decoupting
r
d
I 50 ppm
Fig. 3. Left spectrum : 31p-NMR of a mixture of 200 mg C12Es and 139.4 mg egg lecithin, molar ratio Cs2Ea/lecithin = 2. D20 content 43.5%, by wt, corresponding to Xo~o = 0.959. Temperature 25°C. Right spectrum: Saturation transfer 31p-NMR spectrum ( D A N T E ) of the same sample. Saturation at the spectral position indicated by the arrow. The pulse sequence was [(P1 - D1)N1- D2 - P2 - acquisition]N~ with PI = 0.5 Ixs; D1 = 200 ~ts; NI = 200; /92 = 10 ~xs; />2 = 10 O-s; N2 = 500. P denotes pulse width; D, delay time and N, no. of repetitions. Trlton/Egg Lecithn : 2 mot/mot
20% I
/
\
,i
-I°[
/
/f ./'
J /
~ -~-.-. _if ~'~ F
50 ppm
~,~ 4
,
(
500 Hz
Fig. 4. Temperature dependence of 3tp. and corresponding 2H-NMR spectra in a liquid crystalline mixture of Triton and egg lecithin (for sample composition cf. bottom spectra in Fig. 1).
71 geneously lamellar. At the same detergent/lecithin ratio this transformation temperature is about 35°C in the C12Es-egg lecithin-D20 system. The small cross-sectional diameter and the high curvature of the rod-like structure in the hexagonal detergent phase suggests that the average conformation of the acyl chains of the incorporated phosphatidylcholine is rather different from what is known about phospholipid bilayers in the liquid crystalline state. In order to examine the effect of geometrical constraints on the fatty acid order dipalmitoyllecithin selectively deuterated in the terminal methyl groups (to, to'-d6-DPPC) was incorporated into a hexagonal C12EsH 2 0 sample. The quadrupole splitting of the methyl deuterons of the acyl moieties in multilamellar to,to'-d6-DPPC is 2.67 kHz at 45°C (see Fig. 5). At the same temperature, to,to'-d6-DPPC in the hexagonal phase of C12E8 exhibits a splitting of 0.84kHz (Fig. 5). This is significantly less than the value of 1.35 kHz to be expected on the basis of cylindrical phospholipid motion [6] indicating that the acyl moieties are disordered at least at the end of the chains. Macroscopically homogeneous and translucent samples were obtained when the synthetic lecithins DMPC, D P P C or DSPC above their respective thermotropic phase transitions were mixed with Triton or Ca2E8 and the appropriate amounts of D20 were added to yield the hexagonal phases. Upon cooling, these samples became turbid. At a molar ratio of detergent/phospholipid of 6 the onset of this turbidity was found about 8-12°C
I
I
2 kHz
Fig. 5. 13.8MHz-2H-NMR spectra of t0,t0'-d6-DPPC at 45°C. (A) 50% in H20. (]3) In the hexagonal liquid crystalline phase of C12Es. Molar ratio detergent/phospholipid= 6/1. The central signal originates most likely from natural abundance deuterium in I-t20 in the outer coaxial NMR tube.
72
below the thermotropic phase transition of the pure lipids, irrespective of the host detergent (Fig. 6). 31p-NMR spectra in this temperature region revealed the co-existence of phospholipid molecules in the hexagonal liquid crystalline and in a lamellar state characterized by restricted headgroup mobility (Fig. 7). The line shapes of proton decoupled gel phase 31p spectra of
O • •
•
•
•
.
Q
° .
°
DMPC
"
E
DPPC
" DSPC
0 I
0
o ul
S
I
1
0
Q
0 0 0 0
#
<:{
2
DMPC
I
•
.
•
DPPC
°°°°o
°.
;
1~
1~
1~
2~
2~
3;
' 3'4 ' 3~
~2
4~ "c
Fig. 6. Temperature dependence of turbidity in liquid crystalline mixtures of synthetic phospholipids with C12Es (A) and Triton X-100 (B). Molar ratio detergent/phospholipid = 6/1. Water mol fraction was 0.959 in (A) and 0.967 in (B). Absorbance was measured at 366 nm in flame sealed ampoules after equilibration for 20 min at the temperatures indicated.
E12E8 / DPP[ 6 mol./mo[
26 o[
~ - ~ /
10%
4V~¢~;/~°y
'~::~
50ppm
Fig. 7. Phase transition of D P P C in C12Es as indicated by 31P-NMR.
73 the lecithins in excess water were virtually identical to those found in the detergent mixtures. This suggests that the lipid molecules in these mixtures segregate by lateral diffusion to form ordered gel phase domains. Furthermore, the t e m p e r a t u r e d e p e n d e n c e of 31p signal widths at half height of D P P C and D S P C in aqueous dispersion and in the Triton and C12E8 mixtures showed a rather good correlation (see Fig. 8). Also in the 13C-NMR spectra of d e t e r g e n t - p h o s p h o l i p i d mixtures below the t e m p e r a t u r e of the phase separation, the resonances from the acyl chains were b r o a d e n e d beyond detectability (data not shown). This confirms the idea that the segregated phospholipid is self-associated in almost pure form and is endowed with the same residual motional f r e e d o m as ordinary gel phase lecithins in excess water. In contrast to the egg lecithin-detergent mixtures at high egg lecithin content (cf. Fig. 1), 2H-NMR did not show separate powder patterns from bound D 2 0 even after completion of the phospholipid crystallization as indicated by 31p-NMR. T h e quadrupole splittings in mixtures of the lecithins with C12E8 were nearly identical to those found in the pure hexagonal detergent mesophase. It must be noted, however, that the phospholipidb o u n d water c o m p o n e n t may remain undetectable due to the low water binding capacity of the lecithin headgroup. On the other hand, averaging of the 2H spectrum due to rapid water exchange between the gel phase and the hexagonal liquid crystalline regions would suggest that the phospholipid clusters are small c o m p a r e d to the diffusion length per unit time of the water molecules. Surprisingly, after storage of mixtures of C12E8 with D S P C or D P P C for several weeks at 4°C, the intensity of the 3~p spectra recorded at the storage
A
o•
~=26-
o
2~,~ 22"6 2.o1.8-
16-
in • DPPC 1 oDSPCJ C12E8
a
• DPPC] T X o OSPCJ • OPPC'[ OSPCJ Suspension
o'ss
o
d9
0'gs
TIT c (°K/OK)
Fig. 8. Full 31p line width at half height of the gel phase signal vs. reduced temperature for various synthetic phospholipids in detergent liquid crystalline host phases or in aqueous suspension. Superimposed 'hexagonal' 3tp signals <30% of the total signal intensity had no effect on the line width. Tc = temperature of the thermotropic phase transition of the respective pure phospholipid. The decoupling field strength was held constant at 150 gT.
74 t e m p e r a t u r e without reheating was drastically reduced. U p o n equilibration of the sample at higher temperatures but well below the t e m p e r a t u r e where the onset in turbidity was observed (cf. Fig. 6) a 'hexagonal' 31p signal a p p e a r e d (Fig. 9, left column). Cooling from 19.5°C led to transformation of most of this 'hexagonal' into a 'gel phase' signal. Incubation of the sample for 1 min at 45°C to restore the h o m o g e n e o u s hexagonal phase and subsequent cooling resulted in the same sip-spectrum as obtained immediately after sample preparation (Fig. 9, right column, see also Fig. 7). The reduction of the observable 3~p signal may be tentatively ascribed to strong immobilization of the lecithin headgroups leading to a rigid limit powder pattern and to enhanced dipolar broadening. Unfortunately the 31p line shape could not be evaluated under the experimental conditions employed. Sufficient signal to noise and the correct 3~p line shape would only be available in a cross polarization experiment at very high proton decoupling power [17]. In the D20-2H spectrum of the same sample as in Fig. 9, after 2 - 3 weeks at 4°C, a broad singlet was superimposed on the quadrupole powder pattern. OPPC in C12E8
after' storage at 4"E 3.5 "[
I/
after' Dncubahon at 19,5 "C
,'~
115
after Lncubahon at ~00 "C
11,5"[ 0,5
19,5"C
~
~
Fig. 9. Effect of long-time incubation (2 weeks at 4°C) on the 3~p line shape in the system C12Es-DPPC-D20. Molar ratio C12Ea/DPPC= 6/1. The mole fraction of D20 was Xtho = 0.959. Prior to the NMR experiments the sample was equilibrated for 30 min at the temperatures indicated. Heating to 40°C (lower right spectrum) was for 1 min only.
75 Again, after heating to 40°C the original powder pattern was restored (Fig. 10). No loss in total signal intensity could be detected after the storage period in contrast to the phosphorus spectra. The 2H spin lattice relaxation time of both D20 components has been measured. The signal intensity of the components could be determined separately after the 180-r-90 sequence by subtraction of an appropriate computer simulated Lorentzian line. The isotropic water signal was found to relax more rapidly than the splitted signal. At 4.4°C relaxation times of 0.026 s and 0.042 s were found. After rehomogenization of the sample by heating to 45°C as described above, T1 was 0.040 s. The anisotropic motion of D20 molecules in a liquid crystalline phase can be described in terms of a two correlation times model. Due to this model, spin lattice relaxation is determined by rapid flipping about the bisector of the water molecule, whereas the slow tumbling of this axis does not contribute to T1 [18]. Thus the decrease in Tt and, at the same time, the disappearance of the quadrupole splitting signify that in the new environment the water molecules undergo slow isotropic instead of rapid unisotropic motion. The amount of water corresponding to the singlet D20 signal in Fig. 10 was evaluated also by subtraction of a Lorentzian line of appropriate intensity and width from the compound experimental 2H spectrum so as to obtain the pure powder pattern (Table I). The isotropic signal corresponds to 30-40 molecules of D20 per phospholipid headgroup. This may be after storage at 4%
/~
~
/~
0%
40%
_/ after incubct,on
500 Hz Fig. ]0. Storage effect on the EH-NMR spectra o[ bound D20. Sample and storage conditions as in Fig. 9. Incubation at 40°C for I rain.
76 TABLE I R E L A T I V E A M O U N T S O F D 2 0 IN D I F F E R E N T M O T I O N A L S T A T E S A F T E R L O N G T I M E I N C U B A T I O N O F DSPC/CI2Es/D20 S A M P L E S CI2Es/DSPC (mol/mol) ~
Ao/A Ib
D20(I)/DSPC (mol/mol) ~
D~O(Q)/C12E8 (tool/tool) c
6.0 8.1 9.7 11.9 15.1 19.8
4.0 5.6 6.8 7.5 9.7 10.6
33.0 33.6 31.7 35.6 35.4 41.0
21.8 22.3 22.5 22.4 22.6 22.0
aThe mole fraction of D 2 0 was held constant in all samples at Xa-zo = bRatio of the integrated intensities of the pure powder pattern and pure powder pattern was obtained by c o m p u t e r subtraction of a appropriate line width and intensity. CD20(l); D 2 0 ( Q ) , a m o u n t of D 2 0 corresponding to the isotropic pattern, respectively.
0.959. the isotropic signal. T h e simulated Lorentzian of line and to the powder
compared to the number of about 9 water molecules tightly bound to gel phase phospholipid as reported earlier [19]. Thus more than the amount of water hydrating the phospholipid headgroups has been liberated upon phospholipid aggregation. At the same time the hydration of the detergent remains nearly constant at about 22 tool D20 per mol of C12E8, corresponding to a detergent concentration of about 55% by wt (cf. Fig. 2).
Discussion
From extensive X-ray studies [20,21] it is known that hexagonal structures in pure lecithins form only at rather high temperatures (>100°C) and very low hydration (<4% by wt of water). In excess water these phospholipids exhibit a variety of lamellar structures (for a review see Ref. 8). When mixed with liquid crystalline non-ionic detergents, the phosphatidylcholines obviously fit easily into the two-dimensional hexagonal lattice. In contrast, phosphatidylethanolamines and certain phospholipid mixtures undergo lamellar-hexagonal phase transitions also in excess water. The hexagonal phase formed by these lipids are generally of the H,-type where the headgroups are directed towards a water channel in the center of the cylindrical~structure with the acyl chains facing outwards. On the other hand it it known from earlier work that in the hexagonal ('middle') phase of oxyethylene detergents the hydrophilic tails extend in the opposite direction even in the presence of small amounts of foreign molecules [22,23]. The headgroups of the phosphatidylcholine molecules incorporated into the
77 hexagonal detergent host phase are most likely located in the oxyethylene layer of the detergent cylinders with the water of hydration exchanging rapidly between oxyethylene and choline sites. This means that the phospholipid, when solubilized in the hexagonal detergent clusters, is in the Hi-state. Thus, at low concentration the phospholipid molecules are forced to assume the structure of the detergent host phase, in agreement with the traditional picture of mixed phospholipid-detergent micelles [24]. At higher lecithin concentration the situation is reversed in as much as now the phospholipid determines the phase structure, resulting in lamellar aggregates. This behavior may be tentatively explained in terms of geometry. It has been suggested [25,26] that a dimensionless packing parameter v/ao'lc determines the structure of the aggregates, where v denotes the volume and Ic the maximum length of the hydrophobic chains and a0 the cross-sectional area of the hydrophilic headgroup of the amphiphilic molecules at minimum surface free energy. Rod-like structures are favored if ~< v/ao" lc <½, whereas at ratios >~, lamellar structures will form. Thus addition of increasing amounts of phospholipids with rather voluminous fatty acid chains to a hexagonal detergent phase can be expected to result in a hexagonal-lamellar phase transition. Weakening of water binding to the hydrophilic headgroup and thereby decreasing surface area a0 could explain the preference of the lamellar structure at elevated temperatures. Although the behavior of the system egg lecithin-Triton X-100 or egg lecithin-C12E8 is in qualitative agreement with an interpretation along these lines, the question remains how the rather long phospholipid acyl chains fit into the hydrophobic core of the rod-like structure. On the basis of the 3~P chemical shift anisotropy observed it can be concluded that the motional behavior of the lecithin headgroups in the hexagonal phase is not very different from that in the ordinary liquid crystalline Lo~ phase. It has been shown recently that the headgroup structure of lecithins is identical in micelles and bilayers [27,28]. It appears reasonable to assume that this is true also in the hexagonal liquid crystalline mixture, although this is not borne out by the results of 3~p-NMR, due to the ambiguity inherent in the non-axial chemical shift tensor of the 31p nucleus [5]. The diameter of the hydrophobic core of the rod-like structures in the Triton X-100 host phase can be calculated to be about 20/~ assuming a circular cross-section [29]. The thickness of the egg lecithin bilayer in the liquid crystalline state is about 38/~ at a water content of 40%, by wt, and 25°C [20]. Thus radial intercalation of lecithin molecules in the hexagonal structure seems to be unlikely. In C~2E8 the diameter of the hydrophobic core is larger than in Triton (about 24-28 ~ , assuming a circular cross-section) but again too small to allow radial arrangement of the phospholipid molecules in the detergent matrix [30]. Alternatively, an elliptical cross-section of
78 the rods may be assumed where the chain lengths of the acyl moieties in the phospholipid determines the long axis. Flipping of the phospholipids about 180/~ at a rate ~>103s would lead to the observed 'hexagonal' alp. NMR pattern. However, this is in contrast to phospholipid flip-flop rates in bilayer membranes which are about 8 orders of magnitude slower [31]. A non-classical micelle model has been recently proposed by Fromherz [3]. In this model parallel correlated bilayer blocks are arranged so as to form a cubic structure with a similar perimeter as the classical micelle. Extending this idea to the hexagonal liquid crystalline phase it is conceivable that phospholipids replace some of the detergent molecules without any extreme bending of the acyl chains. The observation of reduced quadrupole splitting in DPPC deuterated in the terminal methyl groups of the long chains does not contradict this model since the chain ends may have more conformational freedom in the block arrangement than in the extended bilayers. Additional experiments, in particular T~ measurements, are needed to examine whether the decrease in 'fatty acid order' is accompanied by an increase in chain mobility. The occurrence of lateral phase separation in mixed lipid bilayers upon crystallization of one membrane component has been established by a number of physical techniques. When the chain length of the lipids differs by more than 4 C-atoms or when lipids with saturated and unsaturated acyl chains are mixed, immiscibility in the gel phase and monotectic behavior are commonly observed [32]. It is conceivable that the systems under investigation here behave analogously. It has been noted, however, by Lee [33] that complete immiscibility in the gel phase must be considered as a limiting case and the appearance of a eutectic composition very close to one of the pure components is more probable. Thus, although segregation of pure gel phase lecithin seems plausible, an admixture of a trace of the detergent to the gel phase patches cannot be excluded. The direct transition from the hexagonal liquid crystalline solution to the lamellar gel state requires lateral diffusion of the phospholipid molecules along the axis of the rod-like clusters and parallel alignment of the acyl chains in the primary crystal nuclei. Whereas unhindered lateral diffusion can be assumed, the formation of small gel phase patches seems to be unfavorable due to the large free energy contribution of the domain boundaries. Thus it is not surprising that the presumably rather small primary gel phase patches are thermodynamically metastable and undergo a slow secondary association. This process proceeds within a few days whereas the formation~of the primary gel phase patches is complete within a few minutes. The different rates may be due to the very different diffusion rates of single lecithin molecules and gel phase clusters in the viscous liquid crystalline environment. A large decrease in entropy is to be expected on gel phase formation if the phospholipid molecules are constrained in a crowded
79 conformation by radial intercalation into the hexagonal detergent structure. In bilayers, on the contrary, the corresponding entropy change per methylene group is only about one half that found in bulk hydrocarbons [34]. Again, the above mentioned block model [3], where parallel alignment of small units is supposed, could explain the ease of the transition. Yet no experimental evidence for this model is available to date. The conspicuous disappearance of the 31p-signal after long time incubation below the transition temperature can be explained tentatively by strong immobilization of the headgroups. In fully hydrated samples of DPPC headgroup immobilization has been observed only at about -10°C, i.e. 50°C below the phase transition [35]. Thus the loss of headgroup motion must have to do with the low water content of the mixed liquid crystalline system. 31p-NMR of anhydrous DPPC at 15°C shows the characteristic rigid lattice line shape to be expected for a chemical shift tensor with three different principal components. 'Upon addition of more than 3 water molecules per headgroup this spectrum transforms to the anisotropic signal typical for rapid axial motion [36]. This signal is insensitive to further water addition and changes only slightly with temperature. Thus the 'bound water' liquifies the lecithin headgroup dipoles. Likewise, the lack of 31p intensity in the lecithin-detergent systems under low power decoupling conditions and, at the same time, the appearance of an isotropic D20 line suggests that upon secondary aggregation of the gel phase lipid the headgroups become dehydrated and thereupon locked by strong dipole-dipole interaction. The observation of headgroup immobilization and isotropic water motion may be ascribed to the formation of a coagel in analogy to the behavior of hydrated long chain fatty acids [20]. In some cases the hydrated gel phase of these compounds is metastable and water will be squeezed,oiat of the lipid lamellae. The released water merges into small pockets where it undergoes isotropic tumbling [37]. After long-time incubation the amount of water in these pockets obviously exceeds the water quantity released from the phospholipid headgroups, whereas the detergent hydration reaches a constant value of about 2.8 molecules of D20 per oxyethylene group (cf. Table I). Thus the final state of the system may be determined by the free energy minimum ensuing from the optimal hydration of the hexagonal liquid crystalline detergent phase.
Acknowledgment The author is grateful to Prof. M. Klingenberg for valuable discussions and constant support. Also the excellent technical assistance of Renate Lafuntal and Liselotte Schmidt-Erhard is gratefully acknowledged. The work was supported by the Deutsche Forschungsgemeinschaft.
8O
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