Biochemistry 1993, 32, 13277-1 3287
13277
A 13C and 2H Nuclear Magnetic Resonance Study of Phosphatidylcholine/ Cholesterol Interactions: Characterization of Liquid-Gel Phases? T.-H. Huang,: C. W. B. Lee,s S. K. Das Gupta,* A. Blume,ll and R. G. Griffin’,# Francis Bitter National Magnet Laboratory and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Institute of Biomedical Sciences, Academia Sinica, Taipei 11 529, Taiwan, and Department of Chemistry, Universitiit Kaiserslautern, 0-6750 Kaiserslautern, Germany Received September 2, 1993” ABSTRACT: A detailed study on the structure, dynamics, and thermodynamic behavior of phosphatidylcholine/ cholesterol (PC/CHOL) mixtures was undertaken using differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance (NMR) spectroscopy. DSC thermograms of mixtures of cholesterol (CHOL) with 1,2-dipalmitoyl-sn-phosphatidylcholine(DPPC), 1,2-distearoyl-sn-phosphatidylcholine (DSPC), and 1,2-diarachidoyl-sn-phosphatidylcholine(DAPC) showed a broadening of the first-order liquid crystalline transition and a decrease in the transition enthalpy, indicating a gradual loss of gel cooperativity for high CHOL concentrations. DPPC and DSPC were labeled with 13Cat the carbonyl group of the sn-2 chain and 2Hwas introduced into the middle of the sn-2 chain at the 6- and 12-position for DPPC and DSPC, respectively. The 13C and 2H N M R spectra of each labeled lipid were studied as a function of temperature and CHOL concentration. The residual quadrupole splitting in the *HN M R spectra, A v Q ~ , was analyzed as a function of temperature and composition. For CHOL concentrations less than 30 mol %, a precipitous change in A V Qoccurs ~ near the chain melting temperature of the phospholipid. Further increases in CHOL concentration broaden the transition and shift the midpoint to higher temperature, indicating the presence of a new phase at higher CHOL contents. Moreover, at a given temperature, A V Q ~ increases with increasing cholesterol content, which indicates a more ordered structure. The 13C NMR spectra in the gel state consisted of a superposition of two components which can be attributed to both gel-like and fluid phospholipid domains in the bilayer. This two-component spectrum can be simulated quantitatively with a two-parameter chemical exchange model, which permits the fraction of each form and the exchange rate to be determined as a function of temperature and composition. At high CHOL contents the line width of the fluid component broadens, suggesting an increase in the exchange rate between the domains. These results were interpreted in terms of a temperature composition diagram with one region LBr,two regions LGI and LGII, and one liquid crystalline region L,, with LG denoting “liquid-gel” type phases. Liquid-gel phases correspond to phases with increased order in the hydrocarbon chains (in comparison to that of the pure PC bilayer in the L, phase) combined with fast limit axial diffusion that averages the I3CN M R spectrum to a “fluidlike” line. These phases are similar to those found in phosphatidylethanolamine/ cholesterol (PE/CHOL) mixtures [Blume, A., & Griffin, R. G. (1981) Biochemistry 24, 62301 and are in agreement with the results of Vist and Davis [(1990) Biochemistry 29, 4511.
-
Cholesterol (CHOL)’ can be found in many biological membranes at very high concentrations, up to 50 mol 96 in the case of the human erythrocyte. Although its function in the membrane is not well understood, it is postulated to maintain membrane integrity under a variety of physiological conditions by mediating membrane fluidity. Consequently, its interaction with other membrane components, such as phospholipids, is of interest. Since biological membranes are heterogeneous entities, consisting of different lipid species together with a variety of proteins, the use of a model membrane system ~
t This research was supported by the National Institutes of Health (GM-25505 and RR-00995). C.W.B.L. was supported in part by a fellowship from the National Sciences and Engineering Research Council of Canada. A.B. was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. Academia Sinica. 5 MIT. 11 Universitit Kaiserslautern. e Abstract published in Advance ACS Abstracts, November 15,1993. Abbreviations: CHOL, cholesterol; DAPC, 1,2-diarachidoyl-snphosphatidylcholine;DPPC, 1,2-dipalmitoyl-sn-phosphatidylcholine;DSPC, 1,2-distearoyl-sn-phosphatidylcholine;PC, phosphatidylcholine; PE, phosphatidylethanolamine;DSC, differential scanning calorimetry;EPR, electron paramagnetic resonance; NMR, nuclear magnetic resonance; Tm.lipid chain melting temperature.
*
consiting of a single phospholipid species can provide insight into the physical effects of CHOL. Numerous studies on the physical properties of CHOL/ lipid bilayers have been published [for reviews, see Demel and De Kruijff (1976), Huang (1977), Melchior (1982), Yeagle (1985), and Cullis et al. (1985)l. Early investigations on mixtures of PC and CHOL revealed that CHOL condenses fluid phospholipid monolayerswhile fluidizing solid ones (Shah & Shulman, 1967). Subsequent DSC studies have shown that CHOL broadens the gel-to-liquid crystalline phase transition, as well as decreasing the calorimetrically observed transition enthalpy (Ladbrooke et al., 1968; Mabrey et al., 1978; Estep et al., 1978). Thus, CHOL was postulated to order the liquid crystalline phase and disorder the gel phase. As discussed below, this view is only partially correct. On the molecular level, the details of the PC/CHOL interaction have been examined by a variety of techniques including EPR (Shimshick & McConnell, 1973a,b; Recktenwald & McConnell, 198 I), fluorescence polarization (Rubenstein et al., 1967; Calhoun & Shipley, 1979; Rand et al., 1980), electron diffraction (Hui & He, 1983), neutron scattering (Mortensen et al., 1988), freeze-fracture electron microscopy (Copeland & McConnell, 1980; Lentz et al., 1980), and NMR (Gally et al., 1976; Haberkorn et al., 1977; Brown
0006-2960/93/0432- 1 3277%04.00/0 0 1993 American Chemical Society
13278 Biochemistry, Vol. 32, No.48, 1993 & Seelig, 1978; Wittebort et al., 1982; Vist & Davis, 1990). These studies demonstrated the condensing effect of CHOL in the La phase. In particular, for T > Tmof the pure lipid, addition of CHOL leads to an incease in the orientational order of the phospholipid hydrocarbon chains. In addition, CHOL enhances rotational diffusion of the lipids in the bilayer. Several of these reports have suggested the formation of specific phospholipid-cholesterol complexes [for a summary, see Lentz et al. (1980)], but a number of stoichiometries for these complexes have been proposed, as diverse as 7.5, 20, 22, 25, 30,33,47, and 50 mol 5% CHOL/mol of lipid. Other studies revealed no complex formation (Stockton & Smith, 1976; Jacobs & Oldfield, 1979), while yet others proposed multiple complexes (Lentz et al., 1980). Partial PC/CHOL phase diagrams have been constructed (Shimshick & McConnell, 1973a,b; Gershfeld, 1978; Lentz et al., 1980; Rand et al., 1980; Recktenwald & McConnell, 1981; Blume & Griffin, 1982; Mortensen et al., 1988), and different experimental techniques have led to different proposed phases. However, recent investigations (Vist & Davis, 1990; Ipsen et al., 1988) have converged on a more consistent interpretation of the PC/CHOL system. In this study, we have employed a combination of calorimetry and 2Hand 13Csolid-stateNMR to examine the phase equilibria and dynamical behavior of the PC bilayer in the presence of varying concentrations of CHOL. The DSC experiments constitute a systematic study of phospholipids with differing hydrocarbon chain lengths and are in general agreementwith previouslypublished results. 2HNMRspectra were useful in characterizing the La phase but exhibited complex behavior in the gel phases. In contrast, the 13CNMR results are particularly informative of gel phase behavior. Previously published results (Wittebort et al., 1981, 1982; Blume et al., 1982; Blume & Griffin, 1982) have shown that, in going from the gel-to-liquid crystalline state, the 13C spectrum of the sn-2 carbonyl position in glycerophospholipids transforms from an axially symmetric powder spectrum to an isotropic line. This spectral change is due to rotational diffusion and a conformationalchange at the glycerol backbone region of the lipid. Furthermore, the appearance of the isotropicline, which is diagnostic for fluid lipids, can be induced by the addition of CHOL. The model system used in this study consist of binary mixtures of DPPC/CHOL and DSPC/CHOL, two phospholipids with hydrocarbon chains of differing lengths. It is necessary to study both species because one of the primary physical effects used to detect phase changes is thesn-2 13C=0 spectrum, which transforms to a narrow line in the La phase. However, in the PPIphase, the line shape consists of a sharp line superimposed upon a powder spectrum similar to that induced by CHOL in the gel phase, an effect which could complicate the interpretation of the spectra. In contrast to DPPC, DSPC has a narrow P Bregion ~ and the sharp line is due almost exclusively to the addition of CHOL. The amount of CHOL was varied from 0 to 50 mol 5% and a wide temperature range was sampled. 2HNMR experiments were performed with specificallydeuterated PC, either 2-[6,6-2H2]DPPC or 2-[12,12-2H2]DSPC. 13CNMR experiments utilized sn-2 carbonyl-labeled PC, 2-[ 1-13C]DPPC or 2-[ 113C]DSPC. The experimentally derived temperature/ composition diagram is compared with results from earlier studies as well as with theoretical descriptions of the PC/ CHOL bilayer.
EXPERIMENTAL PROCEDURES Materials. Cholesterol, DPPC, DSPC, and DAPC were obtained from Sigma Chemical Co. (St. Louis, MO, or
Huang et al. Deisenhofen, Germany). [ 1-13C]Palmiticand stearic acids were purchased from CambridgeIsotope Labs (Wobum, MA). 2H-Labeled fatty acids were synthesized according to previouslypublishedmethods (Das Gupta et al., 1982). 13C-and 2H-labeledphosphatidylcholineswere synthesized by acylation of the corresponding lysophosphatidylcholine with the fatty acid anhydride using N,N-dimethyl-4-aminopyridineas a catalyst (Gupta et al., 1977). Purity was routinely checked with TLC on silica gel plates both before and after NMR measurements. DSC Sample Preparation. Phospholipid suspensionswith and without CHOL were prepared by mixing appropriate amounts of chloroform stock solution of CHOL with the respective phospholipid. Chloroform was removed rapidly under a stream of N2 gas at 50 OC. Residual chloroform was removed under high vacuum for at least 12 h. After addition of doubly glass-distilledwater, the sampleswere first vortexed at temperatures above the phase transition of the lipid and then sonicated for 2-5 min using an MSE lOOW ultrasonic distintegrator. After further equilibration at temperatures above 2" for 1 h, the suspensions were placed in the calorimetric cell. NMR Sample Preparation. Samples for the NMR experiments consisted of approximately 50 mg of PC together with the appropriateamount of CHOL, which were codissolved in chloroform and evaporated under a stream of dry N2 gas. Residual solvent traces were removed under a vacuum of 1 p 2 Torr. The samples were dispersed in an equal weight of H20 (2H-depletedH2O for 2H-labeledsamples) and sealed in 7-mm glass tubes. Calorimetry. DSC measurements were performed as previously described (Blume, 1980, 1983) using a Privalov DASM-1 M DSC calorimeter. For the detection of the broad transitions in lipid/cholesterol mixtures the establishment of a highly reproducible base line is important. The base line obtained with pure buffer or water was subtracted from the curves obtained with the lipid dispersions prior to data evaluation. Lipid concentrations varied between 1 and 8 mg/ mL, depending on cholesterol content. The lipid concentrations were determined by phosphorus analysis using a modified procedure after Hague and Bright (1941). A scan rate of 1 OC/min was used for all experiments. Data evaluation and simulation were performed using the ORIGIN DSC software supplied by MicroCal, Inc. (Amherst, MA). For the simulation of overlapping DSC peaks a two-state model is used, with thecalorimetric AH,thevan't Hoff AH,and the transition temperature as variable parameters for each peak. Nonlinear least-squares fitting is accomplished using the LevenbergMarquardt algorithm. NMR Spectroscopy. 13C and 2H NMR spectra were obtained on a home-built spectrometer operating at 73.9 MHz for 13C,45.1 MHz for 2H, and 296 MHz for 'H. 2H NMR spectra were obtained with a quadrupole echo sequence (Davis et al., 1976) with a 7r/2 pulse of less than 2.0 ps. 13CNMR spectra were obtained with either a Hahn echo or crosspolarization, with a 180° refocusing pulse to minimize base line distortion (Pines et al., 1973; Griffin, 1981). The 13C ?r/2 pulse was 3 ps. Temperature control to 0.5 OC was achieved with a gas flow system described elsewhere (Wittebort et al., 1981). 13C spectra are referenced to external tetramethylsilane (TMS). RESULTS
Differential Scanning Calorimetry. The DSC scans displayed in Figure 1 show the well-known effects of CHOL on the phase transition of phospholipid bilayers. With increasing
Biochemistry, Vol. 32, No. 48, 1993
PC/Cholesterol Interactions
I 1 i
l ' l ' l ' l ' l ' l ' l ' l ' l
'
l
~
l
'
l
~
l
~
l
~
l
13279 ~
l
~
l
DAPCKHOL
-
0% 5% 10 % 15 % 20% 25% 30 % 40 %
2 A
1
1
1
I
1
1
1
1
1
l
I
I
I
I
I
I
10%
0% 5%
-
- 15% 20 z
-
5 2 0-
%
% %
40% 50 %
50 % 1
0% 5%
-
1
50 96 1 , 1 1 1 1 1 , 1 , 1 1 1 1 1 1 1
-
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90
0 10 20 30 40 50 60 70 80 90
TemperaturePC
TemperaturePC
TemperaturePC
FIGURE1: DSC thermograms of DPPC/CHOL, DSPC and DAPC/CHOL mixtures as a function of CHOL content.
4 m
-
3000
-
-0
DAPC 20% CHOL
2000
a
DSPC . 20% CHOL
0-
1000
-
,
' DI 20% CHOL
1
oD l P+:,
,
,
30% CHOL
1
0 0
20
40
60
TemperaturePC
80
0
20
40
60
80
TemperaturePC
FIGURE 2: DSC thermograms of DPPC, DSPC, and DAPC with 20 and 30 mol % CHOL. The experimental peaks were simulated employing the procedure described in the text using a narrow and a broad-component (see Discussion).
CHOL content, the phase transition is broadened, and the transition enthalpy is descreased and is finally totally abolished. Due to the scan rate of 1 OC/min, the sharp peaks for dispersions with low CHOL content are slightly broadened due to the instrumental dead time. At higher CHOL content this effect is negligible and does not affect the shape of the DSC curves. While the general qualitative behavior for all three phospholipids is the same, closer inspection of the DSC scans of DPPC, DSPC, and DAPC/CHOL reveals distinct differences in transition behavior, which are obviously related to the chain length of the phospholipid. Figure 2 shows the DSC peaks of DPPC, DSPC, and DAPC at 20 and 30 mol % CHOL content. Significant differences in the shift of the temperatures for the onset and completion of melting induced by CHOL are evident. For all three lipids the peaks can be decomposed into a sharper and a very broad component. For DPPC/CHOL, the temperature at which melting is complete is shifted to much higher temperatures with increasing CHOL content, in agreement with all previous studies (Ladbrooke et al., 1968; Estep et al., 1978; Mabrey et al., 1978; Vist & Davis, 1990). This effect is not as pronounced in DSPC/ CHOL and is almost completelyeliminated in DAPC/CHOL. The temperature at the end of the broad peaks is ca. 65-70 OC in all three cases. Only the temperatures at the beginning of the peaks are somewhat different. The area and halfwidth of the sharp component is clearly dependent on the
2
i
00
10
20
30
40
50
Mole% Cholesterol
FIGURE 3: Variation of the total transition enthalpy, including the pretransition and the broad component, with CHOL concentration for different PC/CHOL mixtures.
chain length of the phosphatidylcholine, the DAPC/CHOL mixture displaying the largest area for the sharp component with the smallest half-width. Similar trends are observed at higher CHOL content. Interestingly, the width of the transition at 40 mol 5% CHOL, being mainly determined by the broad component, is the same for all three systems, extending roughly from below 10 to approximately 90 OC (not shown). Only the heat capacity maxima are at different temperatures, 42 OC for DPPC, 50 OC for DPPC, and 60 OC for DAPC with 40% CHOL, respectively. The plots of the transition enthalpy AH versus CHOL content in Figure 3 show that the transition of DPPC is completely eliminated at 50 mol %, whereas for 1:l DAPC/ CHOL, a broad transition with an enthalpy of ca. 4 kcal/mol is still visible. This is similar for 1:1 DSPC/CHOL, although the transition is here only half as large. Deuterium N M R Spectra of PCICHOL Mixtures. The temperature dependenceof 2- [ 12,l2-2H2]DSPC/5O wt % H2O dispersions in the presence of various CHOL concentrations is shown in Figure 4. Spectra for 2-[6,6-2Hz]DPPC/50 wt
Huang et al.
13280 Biochemistry, Vol. 32. No. 48, 1993 (c) 30
( 8 ) 20 Mole%
(A) 0 Mple%
M MM
-
Mole%
( 0 ) 50 Mole%
M
h
A
M J-L A
h
A
M
A
M
h
-
0
-200 200
45
40
h
35
H
30 25
N
-2d0 200
0
Frequency
0
-200 200
0
50
i 40
0
b
--
I
200
T ('C)
30 204
0
200
--
DPPC
30 40 Mole % Cholesterol
10
20
1 I
50
(kHz)
FIGURE 4: Experimental 45.1-MHz zH NMR spectra of 2-[12,122Hz]DSPC/CHOLmixtures in 50 wt %, zH-depletedHzO at various temperatures and CHOL compositions. Spectra were obtained with
a quadrupole echo sequence and plotted on an absolute intensity
scale.
I 50
5
40
i
0 30
(A)
60 i;
B -1
104 0
50
I
50
40
FIGURE6: Residual quadrupole splitting, A v Q ~as , a function of CHOL concentration. (A) 2-[6,6-2H2]DPPC/CHOL (0)30 OC, (A) 40 OC, (0) 41 OC, (0) 43 OC, (A) 50 OC, (m) 60 OC. (B) 2-[12,12-2H2]DSPC/CHOL: (0)50 OC, (A) 54 OC,( 0 )55 OC, (0) 60 OC, (A) 75 OC.
U
b'
20 30 40 Mole % Cholesterol
10
301 CV'
0
10 20
30 40 50 60
Temperature (' C)
50
3
40
-1
0
30
-01
50
40
60
70
Temperature ( C)
FIGURE 5: Residual quadrupole splitting, A v Q ~as, LI function of temperature. (A) 2-[6,6-2H2]DPPC/CHOL: (0)0 mol % CHOL, (A) 10 mol % CHOL, ( 0 ) 15 mol % CHOL, (0)20 mol % CHOL, ( A ) 3 0 m o l 9% C H O L , (B) 5 0 mol % C H O L . ( B ) 2-[12,12-*Hz]DSPC/CHOL: (0)0 mol % CHOL, (A) 10 mol % CHOL, (0) 18 mol % CHOL, (0)24 mol 8 CHOL, (A) 30 mol %I CHOL, (B) 40 mol % CHOL, (V) 50 mol % CHOL.
% HzO dispersions and various CHOL concentrations exhibit trends very similar to those of DSPC (not shown). In the absence of CHOL and at low temperatures, a round-top spectrum of 120-kHz breadth was observed, which transforms to a cone at room temperature. In the Pat phase, the spectrum becomes axially symmetric with a parallel splitting of 1 16kHz,and the P,q La phase transition, the spectrum collapses to a Pake doublet with a splitting A V Qof~ 30 kHz. Addition of CHOL alters the spectra in several respects. First, as the CHOL concentration is increased, the narrow Pake doublet spectrum is observed at progressively lower temperatures. However, the size of the splitting A V Q is~ different from that of pure DPPC and increaseswith increasing [CHOL]. The appearance of the Pake doublet line shape is a clear indication that the lipids are performing fast rotations around the molecular axis. So, with respect to rotational diffusion they are "fluid". The temperature dependence of A V Qfor ~ various CHOL concentrationsis shown in Figure 5 , panels A and B for DPPC and DSPC, respectively. For CHOL concentrations of C20 mol 96,thepakedoublet componentisobserved at temperatures
-
-
very close to the T m of the pure lipid and appears as a superposition on top of the gel-phase powder pattern. Also, ~ fairly constant but decrease the values for A V Q remain liquid crystal phase transition. precipitously at the gel However, for high [CHOL] the decrease is linear with no sharp changes. The concentration dependence of A V Q at ~ constant temperature is shown in Figure 6. There is an observable increase ~ as the CHOL concentration is increased. in the A V Qvalues However, this effect is highly dependent on the temperature. In the La phase at high temperatures (50-60 OC for DPPC and 60-75 OC for DSPC), the splitting increases almost linearly with [CHOL]. At lower temperatures (but still in the La phase) the increase resembles a sigmoidal curve, with the most rapid increases occurring at 20 mol % CHOL. At temperatures below T m of pure DPPC, yet another behavior is observed. For DPPC at 30 OC, A V Qincreases ~ up to 30 mol % CHOL and then decreases slightly for higher CHOL ~ fairly concentrations. For DSPC at 50 OC, A V Q stays constant for [CHOL] up to 20 mol % and then drops off for higher [CHOL]. I3C N M R Spectra of PCICHOL Mixtures. The temperaturedependenceof 2-[ 1-13C]DSPC/50wt % H2Odispenions in the presence of various CHOL concentrations is shown in Figure 7. Spectra of 2-[ 1-13C]DPPC/CHOL dispersions are qualitatively similar in appearance (not shown), The spectral region of interest occurs downfield where the powder pattern from the I3C=0 group is located. The I3C=0 spectra of the pure PC shown in Figure 7A were previously discussed in detail (Wittebort et al., 1981, 1982). Briefly, the axially asymmetric powder spectrum of the dry powder sample (ull = 260.5 ppm, u22 = 140.5 ppm, 433 = 117.5 ppm relative to TMS) is characteristic of immobilized phospholipid molecules. Upon dispersion in excess H20, an axially symmetric spectrum (Au 1! 110 ppm) is observed, indicating the onset of rotational diffusion of the whole phospholipid molecule. The reduced breadth is the result of motional averaging according to the approximation
-
60
-
-
Biochemistry, Vol. 32, No. 48, 1993 13281
PC/Cholesterol Interactions
(A) 0 Mole o/o Cholesterol
T ("C)
A L 54
A A A A nn A nn
50
47 35
135
0
25
20
m
-135 7270
10 1
0
Chem. Shift (ppm)
Chem. Shift (ppm)
L
b
3
5- %
0
270
Chem. Shift (ppm)
(D) 24 Mole Yo Cholesterol T ("C)
0 7 135 "
0
'
.135 270
-
135
0
-135 270 7-0
1 135 0
.135 270
0
-270
Chem. Shift (ppm) Chem. Shift (ppm) Chem. Shift (ppm) FIGURE7: Experimental 73.9-MHz I3CNMR spectra of 2-[ 1-*3C]DSPC/CHOL mixtures in 50 wt % HzO at various temperatures and CHOL compositions. Spectra in the gel phase were obtained with cross-polarization, followed by a refocusing echo, and are plotted on an absolute intensity scale. 3 cos' e- 1 2 where AuRListhe breadth of the rigid lattice powder spectrum. Equation 1 is valid if (i) rotational diffusion is faster than yHoAu N lo4 s-l and (ii) AuRLis axially symmetric. Since the rigid lattice spectrum shows only a slight asymmetry, it can be reasonably represented by an axially symmetric spectrum of breath N 140 ppm. Above the main phase transition (Tm= 41.5 "C for DPPC and 55 "C for DSPC), the spectrum collapses to an isotropiclikeline of -5-1 0-ppm width, whereas in the Pr phase, a temperature-dependent superposition of the spectra was observed. The collapse of the powder pattern to a single line in the La phase can be explained by assuming that the sn-2 carbonyl of the phospholipid molecule undergoes a conformational change, so that the unique axis of the tensor is tilted to approximately the magic angle (e N 54.74O). Before proceeding to the PC/CHOL results, a clarification of the term "La-like component" is in order. In pure PC and PE bilayers, the sharp component in the l 3 C 4 spectrum is associated with the appearance of the La phase. The appearance of this sharp component is associated with a conformational change of the glycerol backbone so that it resembles that of the La phase lipid molecule. As previously noted, the collapse of the broad Lb component to a sharp line can be induced by the addition of CHOL at temperatures well below the main phase transition temperature (Blume & Griffin, 1982). RL
Au = Au
Addition of cholesterol (Figure 7B-F) enhances the population of the La-likecomponent in thegel phase. For example, for DSPC at 45 "C and 10 mol % CHOL a large fraction of the La-like component is evident, whereas it is a very minor component in the pure PC spectrum at 47 "C. Another feature of these spectra is the increased line width of the La-like component as the CHOL content is increased. This is due to increases in the rate of exchange between the two populations (Lr and La-like components) and will be discussed in further detail in the next section. These spectra have been successfully simulated by a model which assumes exchange between two components correspondingto the Lbfand La populations. The broad component was simulated by assuming a certain angle 0 between the unique axis of the 13Ctensor and the molecular axis, coupled with fast axial diffusion of the lipid molecule. The narrow component was simulated by assuming fast diffusion and an angle of 54.44" between the molecular axis and the unique axis of the tensor. Thus, the two main variable parameters in the simulations were the relative population of the La component,fLa, and the exchange rate, k,,, between the two components. Although the angle 0 was also varied to better fit the Lbt component, changes in 0 result in cosmetic changes in the line shape and do not significantly affectfLa or keX.The computer program used for these simulations has been previously described (Wittebort et al., 1987), and the parameters employed to fit the experimental spectra are listed in Tables I and 11.
13282 Biochemistry, Vol. 32, No. 48, 1993
Huang et al.
Table I: Parameters Employed in the Computer Simulations of the sn-2 ‘ 3 C 4 Spectra for 2 4 1-I3C]DPPC/CHOL Mixtures (4
23 24 27 30 33 37 39 42
0
0
10 15 20 25 30 35 40
(4
(4
15
0
20
5 10 13 16 19 22 24 26 28 30 32.5 35.5 40 -10 0
(e)
5 10 15 20 30 40 -10 -5
30
0
5 10 20 30 -18 -10 -5 0 5 10 20
50
(0
~~~~
0.0 0.0 0.03 0.05 0.13 0.40 0.50 1.oo 0.0 0.04 0.10 0.25 0.28 0.40 0.55 1.oo 0.02 0.05 0.10 0.18 0.22 0.35 0.37 0.44 0.45 0.53 0.57 0.64 0.74 1.oo 0.02 0.04 0.10 0.22 0.33 0.55 1.oo 1.oo 0.0 0.09 0.28 0.40 0.50 0.80 1.oo 0.0 0.20 0.25 0.45 0.57 0.75 1.00
26 26 27 28 28 32 32 54.74 29 31 31 31 33 35 36 54.74 29 30 31 32 32 33 33 35 36 40 40 40 40 54.74 29 33 33 33 34 40 54.74 54.74 33 33 33 35 40 40 54.74 33 34 35 35 40 40 54.74
Table 11: Parameters Employed in the Computer Simulations of the sn-2 1 3 C 4Spectra for 2-[1-I3C]DSPC/CHOL Mixtures mol%CHOL
__ __ 9 16 45 200 150
_-
__
33 89 267 233 400 367
__
20 53 111 176 226 323 352 314 327 226 265 267 285
10
(4
24
_-
20 20 111 282 400
__
__ __
_98 389 533 500 2000
__
250 267 573 663 1500
_-
--
~
Figure 8 shows the change in the fraction of the La-like component,fLa, with temperature at various constant [CHOL] values. The temperature at which the spectrum showed only an isotropic 13CNMR spectrum CfLa = l), was obtained by extrapolation of the curves. Extrapolation to the other extreme where fLa = 0 provides the temperture at which a singlecomponent Lb or Lg’-like spectrum is observed. These temperatures are important in delineating the phase diagram for PC/CHOL mixtures. Figure 9 shows the variation of fLa with CHOL concentration at various constant temperatures for DPPC and DSPC. Again, these curves can be extrapolated to those CHOL concentrations where fLa = 0 and fLa = 1. The variation of the exchange rate, k,, with temperature and CHOL concentration is shown in Figures 10 and 11. As noted above, the 13C spectra exhibit exchange broadening between the Lb and La-like components at high [CHOL] and temperatures. This is reflected by an increase in the k,, values used in the simulations. PCICHOL Temperature Composition Diagrams. Temperature/composition diagrams were constructed from the
T(T) 42 48 50 54 57 0 20 25 30 35 40 45 50 55 0
(f)
50
15 20 30 35 40 45 50 55 60 0 10 14 20 25 30 35 40 50 60 0 10 15 20 25 30 35 40 50 60 0 5 10 15 20 30
fh
0
0.0 0.03 0.05 0.50 1.oo 0.0 0.04 0.04 0.10 0.18 0.33 0.43 0.60 1.00 0.0 0.0 0.04 0.06 0.15 0.25 0.35 0.47 0.55 0.60 1.oo 0.0 0.0 0.04 0.15 0.25 0.30 0.45 0.55 0.80 1.oo 0.04 0.08 0.19 0.25 0.35 0.45 0.55 0.65 1.oo 1.oo 0.0 0.05 0.25 0.40 0.75 1.oo
27 27 35 35 54.74 29 31 31 31 32 32 35 40 75.74 29 31 33 33 33 33 35 36 37 37 54.74 31 33 33 34 34 35 35 40 40 54.74 31 33 33 33 35 36 40 40 54.74 54.74 32 33 33 35 40 54.74
k,
(~-1)
__
16 16 28
__
33 33 78 132 200 226 375
_- -_ __ __
38 58 159 267 404 355 367 300
_-._ __
38 159 267 343 49 1 489 1200
.-
33 70 188 267 377 49 1 489 557
--
__ __
47 300 467 1200
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2Hand 13CNMRspectra for DPPC and DSPC in the presence of cholesterol. These results, which are qualitatively very similar, are shown in Figures 12 and 13. At low temperatures, the phospholipid exists in the Lg’ phase. The boundary for this phase was obtained from the 13C NMR results by extrapolation of the temperature and composition curves from Figures 8 and 9 to the fLa = 0 points. The boundary between the gel and liquid crystalline phases was obtained from examination of the 2H NMR results. For CHOL concentrations of
