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diacylglycerol lipid mixtures
Chemistry and Physics of Lipids, 56 (1990) 149--158 Elsevier Scientific Publishers Ireland Ltd.
149
Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures Sun-Yung Chen and Kwan Hon Cheng Physics Department, Texas Tech University, Lubbock, TX 79409 (U.S.A.) (Received March 30th, 1990; revision received May 25th, 1990; accepted August 27th, 1990)
Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfaciai region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylgiycerol (DG). Infrared vibrational frequencies of the CH 2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L0-L) and the L, to inverted hexagonal (L -Hu) phase transitions were observed at 'x,15° and 55°C, respectively. As the DG content increased gradually from 0% to 15%, the Lo-HI~ phase transition temperature decreased drasticaily while the LFL ° phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at ~ I 0 ° C . For the composition study at 23°C, the La-H u transition occurred at ~6----10% DG as indicated by abrupt increases in both the CH 2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, ~-paimit~y~-2-[[2-[4-(6-pheny~-trans-~'3~5-hexatrieny~)pheny~]ethy~]carb~ny~]~3-sn-ph~sphatidy~ch~ine~ in these PE/ DG mixtures were observed at the Lo-HH phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientationai packing of the lipids were altered at the composition-induced L-H~j transition, and agreed with a previous temperature-induced L - H u transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025--1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L,-H, phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the Lo-H~ transition.
Keywords: FTIR; fluorescence; phosphatidylethanolamine; diacylglycerol; phase transition; hydration.
Introduction The molecular structure and dynamics of lipids in various lamellar and non-lamellar phases have been studied extensively for many years. Structural techniques, such as X-ray diffraction [1] and electron microscopy [2] provide significant information relating to the geometrical symmetry and lattice dimensions of the ordered phases. On the other hand, spectroscopic techniques provide molecular information on the Correspondence to: Kwan Hon Cheng, Physics Department, Texas Tech University, Lubbock, TX 79409, U.S.A.
orientation and dynamics of the lipid and water molecules in those ordered phases. Generally speaking, spectroscopic techniques employed in lipid research can be classified into three major categories according to their respective time scales. They are (1) nuclear magnetic resonance (NMR) [3], (2) fluorescence or electron spin paramagnetic resonance (ESR) [4] and (3) vibrational spectroscopy (Infrared and Raman) [5]. NMR provides molecular information which represents averages of all possible motions that are faster than milli- or microsecond. The interpretation of NMR data, both steady state and relaxation measurements,
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
150 usually involves assumptions of dynamics models, such as dipolar relaxation theories and uncoupling of the slow motions from the fast motions. Fluorescence and ESR are techniques in the nanosecond scale and require the use of extrinsic probe molecules. Vibrational spectroscopy is sensitive to the nature of chemical bonding and the fast conformational changes of chemical groups such as the trans-gauche isomerization. This technique is therefore sensitive to the fast motion down to the 10-15 second regime. It is fair to say that each technique has its own advantages and possible drawbacks. Therefore, in order to get a comprehensive picture of the lipid dynamics, different techniques should be applied. In recent years, the application of more than one spectroscopic technique to tackle a single problem has been noticed. A combination of NMR and infrared spectroscopy to investigate the ion-lipid interaction of membranes [3] represents one of few known examples. This study involves a combination of two spectroscopy techniques, time-resolved fluorescence and infrared. Recently, we have applied time-resolved fluorescence technique to study the molecular dynamics of both pure unsaturated phosphatidylethanolamine (PE) and mixtures of PE with other lipids in various lamellar and non-lamellar phases. In those studies, the order parameter and rotational diffusion of the acyl chains of lipids, as well as the conformation and hydration of the lipid/water interface have been investigated by using lipophilic and water soluble fluorescent probes [6--8]. It is known that similar molecular information regarding the acyl chains and lipid/water interface may also be obtained by the use of infrared spectroscopy [9]. The vibration frequencies of the CH 2 symmetric stretching and the C = O stretching of the lipid chains represent two convenient parameters to examine the above lipid properties. The molecular mechanism governing the nonlamellar phase formation is a subject of research interest in our laboratory. A binary lipid mixture consisting of PE and diacylglycerol (DG) was examined in this study. The phase behavior of this binary mixture has been determined by dif-
ferential scanning calorimetry, 3~P-NMR and Xray diffraction [1,10,11]. DG is a major breakdown product of the cell membrane phosphatidyl inositol turnover during the signal transduction process of cells. Its role in perturbing the lamellar phases of both phospatidylcholine and PE has been well documented [1,2,10--12]. In addition, the correlation of its lamellar perturbation behavior and its role in the membrane-related functional activation has recently been investigated [2]. Yet the molecular mechanism of DG in perturbing the lipid polymorphism is still speculative. This study aims at understanding the molecular dynamics of the PE/DG lipid system in the 10-9- and 10-~5-s time scales. Both the lipid fatty acyl motional order as well as the lipid/water interfacial conformation and hydration of this lipid system in lamellar and non-lamellar phases were examined. Materials and Methods
Sample preparation PE transphosphatidylated from egg phosphatidylcholine (PC) (TPE) and 1,2-dioleoyl-sn-glycerol (diolein) were obtained from Avanti Polar Lipids (Birmingham, AL). 1-palmitoyl-2-[[2[4-(6phenyl-trans-l,3,5-hexatrienyl) phenyl] ethyl]carbonyl]-3-sn-PC (DPH-PC) and 8,l-anilinonaphthalenes sulfonate acid (ANS) were obtained from Molecular Probes, Inc. (Eugene, OR). All lipids were stored in chloroform at - 3 0 ° C and were used without further purification. For the ANS fluorescence measurements, the concentration of ANS was 1 × 10-7 M. For the DPH-PC fluorescence measurements, the fluorescent probe and lipid were first mixed in chloroform (spectroscopic grade). The molar ratio of DPH-PC to total lipid was 0.2%. The mixture was dried under a gentle nitrogen stream in a clean pyrex tube and further kept in vacuum for at least 4 h to ensure complete removal of the residual chloroform. The thin film formed on the tube was then hydrated with an aqueous buffer (100 mM NaCI, 10 mM TES and 2 mM EDTA pH = 7.4) at 4°C. The suspension thus formed was vortexed rigorously and
151 placed under mild sonication in a bath sonicator for a few seconds. The resulting suspension was then incubated overnight at 4°C in the dark to ensure proper hydration. For the fluorescence measurements, a further dilution of the lipid sample to ~0.15 pg//~l was performed. The sample temperature was controlled by an external water-jet circulator connected to a thermalstat cell.
Fluorescence spectroscopy
cence decay profile. However, the fluorescence intensity decay profiles of DPH-PC in PE/DG binary mixtures were fitted by a double-exponential function [6,7].
(ii) Fluorescence anisotropy decay measurements For time-resolved fluorescence anisotropy decay measurements, both excitation and emission polarizers were used. The same emission filter as in the lifetime measurement was used in here. The differential polarized phase angle (6± till) and the ratio of polarized modulation amplitudes (M±/Mu) were measured at different modulation frequencies (1 to 230 MHz). The subscripts H and Z refer to the directions of the polarization axis of the emission polarizer that are parallel and perpendicular to the vertical axis, respectively. The polarization axis of the excitation polarizer in our system was always set in the vertical direction. A first-order approximation model [16] was used to fit the frequency domain data. The anisotropy decay function has the following form: -
Fluorescence measurements were performed on an ISS frequency domain cross-correlation fluorometer (Champaign, IL). The excitation source was a cw He-Cd laser (Liconix 4240NB) with an output of 17 mW at 325 nm. The operational principle of this continuously variable frequency phase-modulation fluorometer has been described elsewhere [13,14].
(i) Intensity decay measurements Because the light exiting from the pockel cell (electro-optical device) is vertically polarized, a UV polarizer with the polarization axis set at 35°C with respect to the vertical was placed in the excitation beam to eliminate the rotational diffusion effect [15]. The fluorescence signals of either DPH-PC or ANS were measured and compared with a non-fluorescent glycogen solution (reference). A lower wavelength cutoff filter (model 3-72, Corning Glass-Works, Corning, NY) was used to remove the excitation light from the fluorescent signal and no filter was used for the reference sample. The phase delay (dF - 6s) and modulation ratio (MF/Ms) were measured at different modulation frequencies ranging from 1 to 230 MHz. Here dv and tis represent the phase delay of the signal by the fluorescent sample and that by the reference sample, respectively, and M F and M s represent the intensity modulation values of the fluorescent sample and that of the reference. The fitting of the frequency domain data was based on the minimization of the reduced chisquares according to the Marquardt algorithm [13]. Both single- and multi-exponential decay functions were employed to fit the ANS fluores-
-
r(t) = (r0 - r®)exp ( - t / t )
+ r
(1)
where r 0 and r® are the initial and residual anisotropy values and t r is the rotational correlation time. From the fitted parameters (r0, r® and tr), the normalized residual anisotropy (r®/ro) and initial slope (b(O)/ro) of the anisotropy decay function were then calculated.
FTIR spectroscopy For the FTIR measurements, the lipid samples were prepared under an identical sample preparation procedure as the fluorescence experiments but in the absence of fluorophores. The lipid was concentrated by centrifugation at 15,000 g for 20 min at 4°C. The lipid pellet was then applied to two CaF2 windows separated by a 25/am teflon spacer. The sandwich CaF 2 which contained the lipid film with uniform thickness was then mounted to a thermostated cell (Wilmad H200). The temperature of the cell was regulated by an external circulating bath (Haake F3C) and was recorded by a thermistor (YSI 200) which
152
was glued directly to the window mount. Spectra were recorded with an Analect RFX-40 Fourier Transform Infrared spectrometer equipped with a liquid nitrogen cooled mercury-cadmium-telluride detector. Usually 500 interferograms were collected for each temperature. The interferograms were apodized with a Norton-Beer medium function and fourier transformed with three levels of zero filling. The background spectra, which were obtained when the sample cell was present but in the absence of lipid film, were subtracted from the sample spectra. The spectral resolution was better than 2 cm -~. Results
FTIR spectra of PE/DG lipid mixtures were measured at different temperatures and for different lipid compositions. A typical FTIR lipids spectrum (407o DG at 23°C) is shown in Fig. 1.
The CH 2 stretching (2800---3000 cm-~), C = O stretching (1700m1800 cm-~), O--H bending (1600--1700 cm-~), CH 2 bending (1400--1500 cm-t) and P = O stretching (1200--1250 cm -l) regions were clearly observed. The vibrational bands below 1200 cm-~ were attributed to lipid headgroup vibrations and other collective vibrations of the lipid acyl chains. The frequency of the C H 2 symmetric stretching band (2850--2854 cm -t) was determined as a function of temperature (0--70°C) for lipid mixtures with different DG contents (0--16070 DG). Figure 2A shows the spectra of three representative DG concentrations (0, 4 and 15070 DG). For 007o DG, the C H 2 symmetric stretching frequency exhibited two distinct transitions at 12--20°C and 52--58°C. At those transitions, abrupt increases in the frequencies were found. For 4°70 DG, two abrupt transitions were also observed. However, the temperature ranges at which those abrupt changes occurred were downshifted to 10
~BSORBANCE 3.000
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WAVENUMBER(cm-1) Fig. 1. T y p i c a l F T I R s p e c t r u m o f a fully h y d r a t e d P E / D G m i x t u r e (DG = 4 % ) at 23 °C.
I 1400
,
I 1200
I I 000
153
2868
C-H atretching(cm-1)
C-O stretching(cm-1)
174,<
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A 2855 1742
2854 1740 2852
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2848 0
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t 40
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1738 70
TEMPERATURE(C)
0
] 10
I 20
t 30
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TEMPERATURE(C)
Fig. 2. Temperature dependence of the CH~ symmetric stretching frequency (panel A) and the C = O stretching (panel B) of PE/ DG mixtures with 0% (@), 4°10 (v1) and 15% (O) DG.
- - 1 8 ° C and 2 8 - - 3 2 ° C . At 15% DG, only one transition at 8 - - 1 5 ° C was observed. The frequency of the C = O stretching band (1736--1744 cm -~) was also determined as a function of temperature for different D G contents. Again Fig. 2B showed the spectra for three representative D G concentrations. For 0070 DG, the C = O vibrational frequency exhibited two transitions at 10--20°C and 50--58°C. It was f o u n d that the C = O frequency decreased abruptly at the low temperature transition while increased abruptly at the higher temperature transition. Note that the C = O frequencies at high temperatures ( > 5 8 ° C ) were almost the same as those at low temperatures (0--10°C). Similar observations of two distinct transitions were found for 4070 DG. However the high temperature transition was downshifted to 2 5 - 38°C. No shift in the low temperature transition was observed. Interestingly, the C = O stretching frequencies were constant at all temperatures for 15070 DG. Besides the temperature-induced transition, the composition-induced transition of P E / D G was also investigated. The variations of the fie-
quencies o f the C H 2 symmetric (Fig. 3A), C = O (Fig. 3B) and O = P = O antisymmetric (Fig. 3B) stretching bands with D G composition at 23°C were illustrated. The frequencies o f the C H 2 and C = O vibrational bands exhibited abrupt increases at 'o6--10°70 DG. On the contrary, the O = P = O antisymmetric stretching frequencies were constant for all D G contents. For both the C H 2 symmetric stretching and C = O stretching frequencies, the higher temperature transition was found to decrease more prominently than the lower temperature transition as the D G content increased gradually from 007o to 1607o. This was illustrated in a phase diagram as shown in Fig. 4. The transition temperature ranges in the phase diagram were determined from the C H 2 symmetric stretching frequency versus temperature measurements (see Fig. 2). The phase boundaries corresponding to the Lp-Lo and L : H . transitions are shown. At "o 1007o DG, a merge o f these two phase boundaries occurred at "ol0°C. A similar phase diagram was also obtained by using the C = O stretching frequencies (results not shown). From time-resolved fluorescence anisotropy
154
C=O stretching (cm-1)
C-H stretching (cm-1) 2858
P-O stretching (cm-1)
1744
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i
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Fig. 3. Lipid composition dependence of the CH 2 symmetric stretching ([3) (panel A); C = O stretching (O) and O = P = O anti-
symmetric stretching (D) (panel B) of PE/DG mixtures at 23 °C.
TEMPERATURE(C) 70
60
60
H:, 40
30
20
Z..x 10
Lo I
I
I
I
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DG tool% Fig. 4. The phase diagram of PE/DG lipid mixtures as con-
structed from the CH 2 symmetric stretching frequency versus temperature measurements (see Fig. 2). The starting and completion temperatures of the Lp-Lo and Lo-H. phase transitions were represented by (D, O) and (<>, A), respectively.
16
decay measurements, the normalized residual anisotropy (~/ro) and initial slope (b(O)/r o) of the fluorescence anisotropy decay function of DPH-PC were determined as a function of temperature from 20 to 65 °C and shown in Figs. 5A and 5B, respectively. The fluorescence lifetime decay of DPH-PC was required to calculate the anisotropy decay parameters from the frequency domain data. Using a double-exponentiai decay function, two distinct lifetimes of DPH-PC were obtained for all temperatures. The long lifetime component (,~ 5.5 ns) represented a major intensity fraction (>0.95) of the decay profile of DPH-PC. The presence of a minor short lifetime component (~2.0 ns) indicated the existence of some unavoidable photodecay products of the probes during sample preparation. For 0070 DG, an abrupt decrease in r®/r o was observed at ,~55°C. For 4070 DG, the transition was downshifted to ,~30°C. Using arrhenius plots, abrupt declines in the values of ;,(O)/r o were observed at similar temperatures for 0070 and 4070 DG. From time-resolved fluorescence intensity decay measurements, the lifetimes of ANS in PE/DG mixtures were obtained for different DG
155
L O G ~'(O)/ro
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1 / T × 1 0 0 0 (K-')
Fig. 5. Plots of fluorescence anisotropy decay parameters r l r o (panel A) and log~0/(0)/r0 (panel B) as a function of temperature for DPH-PC in PE/DG mixtures with 0% ( 1 ) and 4e/0 (O) DG. The uncertainty of each measurement is indicated by the error bar.
compositions at 23 °C. Various multi-exponential decay functions were used to fit the frequency domain data. As judged by the chi-square values of the fits (Table I), the two- and three-exponential functions were found to provide better fits to the data than the one-exponential fit. For the three-exponential fit, the intensity fraction of the third component was very small (~,0.01) and the lifetime uncertainty of that component was very large (-130 ns). Interestingly, both twoand three-exponential fits gave short lifetime components in the range of 100--200 ps. The long lifetime component of the two-exponential fit and the middle lifetime component of the three-exponential fit for 0% DG were always
higher than that for 16% DG. Figure 6 shows the changes of the two lifetime components obtained from the two-exponential fit for different DG compositions. At low DG contents (0-6%), the long lifetime component was around 6 ns. A slight increase in the lifetime component was observed at around 8--10% DG. Thereafter the lifetime remained at ~ 7 ns for DG contents from 11% to 16%. The short components werf found to remain essentially constant and independent of DG content. Discussion
TPE in aqueous dispersions exhibits two ther-
TABLE I Lifetime heterogeneity of ANS in a PE/DG mixture (DG = 2%) at 23°C. The frequency domain data were fitted by one-, twoand three-exponential decay functions. The lifetime components were represented by Ts and their intensity fractions by fs. The reduced chisquares (X2) of all the fits were also shown.
T,
f,
~
f~
5.225 ± 0.136 0.218 ± 0.031 0.213 ± 0.040
1.0 0.097 ± 0.003 0.097 ± 0.004
5.629 ± 0.034 5.582 ± 1.315
0.903 0.889 ± 0.959
~
8.571 ± 132.22
f,
x2
0.014
321.10 8.07 8.64
156
lO
Tl(ne)
T2(n$)
8
4
3
7
6
1
/ 0
69
n,
0
2
q 4
q 6
[] , O,
8
DG
i [] L 10
12
mole
%
0
Fig. 6. F l u o r e s c e n c e l i f e t i m e h e t e r o g e n e i t y o f A N S in P E / D G m i x t u r e s as a f u n c t i o n o f D G c o n t e n t a t 2 3 ° C . The resolved lifetime c o m p o n e n t s T, ( n ) a n d T2 (O) were s h o w n .
motropic phase transitions, lamellar gel to lamellar liquid crystalline ( L : L ) and Lo to inverted hexagonal (L:Hl[), at ~15 and 55°C, respectively. The temperatures of these phase transitions have been determined by using differential scanning calorimetry [17], 3tP-NMR [18], infrared [9] and X-ray diffraction [1]. The frequencies of the CH 2 symmetric and C - O stretching bands exhibit abrupt changes at both the L#-L and L : H , transitions. The increase in the frequency of CH~ symmetric stretching band at both phase transitions suggests that the amount of gauche rotamers in the lipid acyl chains increases as the lipids undergo thermotropic phase transitions. On the other hand, the C = O stretching frequency decreases at the L:L+ transition but increases at the Lo-H. transition. These differential changes of C = O stretching frequency have been attributed to the alterations of the local conformation of the lipids at the ester carbonyl regions [18] and of the accessibility of water to the lipid/water interface [19]. These observed changes in both the CH 2 and C = O vibrational modes with temperature for pure TPE agree with the earlier results obtained by Casal and Mantsch [9].
In the presence of DG, the temperatures of both L#-L+ and Lo-H. transitions are downshifted. Since the frequency shift of C = O stretching mode is both conformation and hydration related, the assignments of the onset and completion of the two thermotropic phase transitions rely on the CH 2 stretching frequency. The phase diagram (Fig. 3) thus generated shows two interesting features. First, the Lo-H. phase boundary declines drastically with increasing DG content. Second, the L:Lo boundary decreases only slightly with DG. Owing to the relative difference in the slopes of the two phase boundaries, a point in the phase diagram is reached at which a merge of the above two phase boundaries occurs. This observation suggests that a direct transition from the L# to H . phase may occur at •10°70 DG. This suggestion is supported by the fact that only one transition in the CH~ stretching frequency was found for DG > 10%. In addition, no abrupt changes in the C = O stretching frequency with temperature are evident at those high DG contents. Of course, one still cannot exclude the possibility that the intermediate region (i.e., 8--20 °C and DG > 10%) in the phase diagram represents coexisting L J L J H , phases. It is interesting to mention that a direct L : H , transition behavior has also been observed in the salt-induced phase transitions of some saturated PE lipids [20]. Recently, we have been interested in applying time-resolved fluorescence spectroscopy to investigate the molecular dynamics of lipids and water in non-bilayer phases [6--8,211. Note that fluorescence spectroscopy provides information in the nanosecond time regime. Hence within the fluorescence time scale of the probes used in here ( * 6 ns), the rotational motions (wobbling) of the probe with respect to the normal axis of the lipids as well as to the long symmetry axis of lipid cylindrical tubes (for the case of HII phase) give rise to the anisotropic decay of the probe [6,7,21], while the orientational distribution of the probe governs the level of the residual anisotropy [16]. Using a first order approximation, the normalized initial slope of the decay function (~(0)/r0) and the residual anisotropy (r®/r 0)
157
reflect the averaged rotational rate of the probe and the global orientational distribution of the probe. The latter further contains the information of both the local order packing and packing symmetry of the lipid mixture and therefore is very sensitive to the L-Hxl transition [6]. Upon comparing the results from fluorescence with that from infrared, it is clear that the temperatures corresponding to abrupt changes in the ~(O)/r o and r®/r o agree well with that in the CH 2 and C = O stretching frequencies for both 0O7o and 4o'/0 DG. In the composition study, the frequencies of CH z and C = O stretching modes increase abruptly at '~6---10°10 DG, signifying the L-HI~ transition. A time-resolved fluorescence study on this composition-induced transition has also been performed (manuscript in preparation). From that study, abrupt changes in the fluorescence parameters, i.e., ~(O)/r o and r J r o, are evident at 6--10~0 DG and therefore agree with the infrared results. The frequency increase of C = O stretching mode could be due to hydration and conformational changes at the lipid/water interfacial region. The frequency of the O = P = O antisymmetric stretching mode is known to be sensitive to the hydration of the phosphate group [22,23]. The lack of any change in the O = P = O antisymmetric stretching frequency as a function of DG content indicates that the hydration of the lipid phosphate group remains unaltered at the L - H ~ transition. Similar conclusion was drawn by Casal and Mantsch 0984). However, one still can not exclude the possibility of hydration changes at the carbonyl region of the lipid/water interface, because of the different locations of the phosphate and carbonyl groups. Time-resolved fluorescence spectroscopy was also employed to examine further the conformation and hydration properties of the composition-induced Lo-H, phase transition of P E / D G system. In the ANS lifetime vs. DG010 plot, an abrupt increase in the long lifetime component (T ~ 5 - - 7 ns) of ANS is clearly evident. According to our previous interpretation, this increase in the fluorescent lifetime may be attributed to
an increase in the surface hydration of the lipid/ water interface [8]. Using a three-exponential fit, a longer lifetime component (T > 8 ns) was obtained. However, the fraction of this component is negligibly small (see Table I). This suggests that the majority of ANS probes are located in the bulk water phase and the hydrated layer of the lipid/water interface. Similar observations were reported for the temperatureinduced L-Hi[ phase transition studies of dioleoyl PE and TPE [8]. The increase in the surface hydration of lipids in the H . phase may be explained by an increase in the spacing between PE molecules clue to the presence of DG. This larger PE headgroup spacing may disrupt the intermolecular hydrogen bonding among PE headgroups and thereby increase the binding affinity and/or number of water to the interfaciai region. Using the water adsorption isotherm method, a similar conclusion of the hydration behavior of PE mixtures containing PC or cholesterol was drawn [24]. An alternative explanation for the ANS fluorescence lifetime result is the possible conformationai change at the lipid/water interface induced by DG. The interfaciai region of P E / DG may allow the ANS molecules to partition deeper into the glycerol backbone region as the DG content increases. This more hydrophobic environment of the glycerol backbone region causes a longer fluorescence lifetime of ANS. Our FTIR and fluorescence results, therefore, suggest that significant perturbations of the hydration and conformation of the lipid/water interfacial region occur at the Lo-H, transition of the PE/DG system. In conclusion, this study has demonstrated that significant changes in the lipid acyl chains and lipid/water interfacial regions occur at either the temperature-induced or compositioninduced L - H . transition of the P E / D G mixtures. Both fluorescence and infrared results appear to complement one another and provide molecular information in two different time scales, i.e., 10-9 and 10-15, respectively. A direct transition from the L~ to H . phase is predicted for DG > 100k from the phase diagram.
158
Acknowledgements This work was supported by grants from the National Cancer Institute (CA 47610) and the Robert A. Welch Research Foundation (D-1158) to K.H.C.S.Y.C. is an awardee of the Robert A. Welch scholarship. References 1 2 3 4 5 6 7 8 9 10
S. Das and R.P. Rand (1986) Biochemistry 25, 2882-2889. K.H. Cheng and S.W. Hui (1986) Arch. Biochim. Biophys. 244, 382--386. H.L. Casal, H.H. Mantsch and H. Hauser (1989) Biochim. Biophys. Acta 982, 228--236. K.H. Cheng, J.R. Lepock, S.W. Hui and P.L. Yeagle (1986) J. Biol. Chem. 261, 5081--5087. K.E. Reily, A.J. Mautone and R. Mendelsohn (1989) Biochemistry 28, 7368--7373. K.H. Cheng (1989) Biophys. J. 55, 1025--1031. K.H. Cheng (1989) Chem. Phys. Lipids 51, 137--145. K.H. Cheng (1990) Chem. Phys. Lipids 53, 191--202. H.L. Casal and H.H. Mantsch (1984) Biochim. Biophys. Acta 779, 381---401. R.M. Epand (1985) Biochemistry 24, 7092--7095.
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R.M. Epand, R.F. Epand and C.R.D. Lancaster (1988) Biochim. Biophys. Acta 945, 161--166. 12 H. Ellens, D.P. Siegel, D. Alford, P. Yeagle, L. Boni, L.J. Lis, P.J. Quinn and J. Bentz (1989) Biochemistry 28, 3692--3703. 13 E. Gratton, D.M. Jameson and R.D. Hall (1984) Annu. Rev. Biophys. Bioen8. 13, 105--124. 14 J.R. Lakowicz and B.P. Maliwal (1985) Biophys. Chem. 21, 61--78. 15 R.D. Spencer and G. Weber (1970) J. Chem. Phys. 52, 1654--1663. 16 W.B. Van Der Meer, H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx and H. Schr6der (1984) Biophys. J. 46, 515--523. 17 K.H. Cheng, P.L. Yeagle, J.R. Lepock and S.W. Hui (1986) Biophys. J. 49, 324a. 18 P.M. Green, J.T. Mason, T.J. O'Leary and I.W. Levin (1987) J. Phys. Chem. 91, 5099--5103. 19 A. Blume, O. Hubner and G. Messner (1988) Biochemistry 27, 8239--8249. 20 J.M. Seddon, G. Cevc and D. Marsh (1983) Biochemistry 22, 1280---1289. 21 K.H. Cheng (1989) in: E. Roland Menzel (Ed.), Fluorescence Detection III, Proc. SPIE 1054, 160--167. 22 H.L. Casal and H.H. Mantsch (1987) Biochemistry 26, 4408--4416. 23 H.L. Casal, H.H. Mantsch, F. Paltauf and H. Hauser (1987) Biochim. Biophys. Acta 919, 275--286. 24 D. Marsh (1989) Biophys. J. 55, 1093--1100.
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Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures Sun-Yung Chen and Kwan Hon Cheng Physics Department, Texas Tech University, Lubbock, TX 79409 (U.S.A.) (Received March 30th, 1990; revision received May 25th, 1990; accepted August 27th, 1990)
Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfaciai region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylgiycerol (DG). Infrared vibrational frequencies of the CH 2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L0-L) and the L, to inverted hexagonal (L -Hu) phase transitions were observed at 'x,15° and 55°C, respectively. As the DG content increased gradually from 0% to 15%, the Lo-HI~ phase transition temperature decreased drasticaily while the LFL ° phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at ~ I 0 ° C . For the composition study at 23°C, the La-H u transition occurred at ~6----10% DG as indicated by abrupt increases in both the CH 2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, ~-paimit~y~-2-[[2-[4-(6-pheny~-trans-~'3~5-hexatrieny~)pheny~]ethy~]carb~ny~]~3-sn-ph~sphatidy~ch~ine~ in these PE/ DG mixtures were observed at the Lo-HH phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientationai packing of the lipids were altered at the composition-induced L-H~j transition, and agreed with a previous temperature-induced L - H u transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025--1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L,-H, phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the Lo-H~ transition.
Keywords: FTIR; fluorescence; phosphatidylethanolamine; diacylglycerol; phase transition; hydration.
Introduction The molecular structure and dynamics of lipids in various lamellar and non-lamellar phases have been studied extensively for many years. Structural techniques, such as X-ray diffraction [1] and electron microscopy [2] provide significant information relating to the geometrical symmetry and lattice dimensions of the ordered phases. On the other hand, spectroscopic techniques provide molecular information on the Correspondence to: Kwan Hon Cheng, Physics Department, Texas Tech University, Lubbock, TX 79409, U.S.A.
orientation and dynamics of the lipid and water molecules in those ordered phases. Generally speaking, spectroscopic techniques employed in lipid research can be classified into three major categories according to their respective time scales. They are (1) nuclear magnetic resonance (NMR) [3], (2) fluorescence or electron spin paramagnetic resonance (ESR) [4] and (3) vibrational spectroscopy (Infrared and Raman) [5]. NMR provides molecular information which represents averages of all possible motions that are faster than milli- or microsecond. The interpretation of NMR data, both steady state and relaxation measurements,
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150 usually involves assumptions of dynamics models, such as dipolar relaxation theories and uncoupling of the slow motions from the fast motions. Fluorescence and ESR are techniques in the nanosecond scale and require the use of extrinsic probe molecules. Vibrational spectroscopy is sensitive to the nature of chemical bonding and the fast conformational changes of chemical groups such as the trans-gauche isomerization. This technique is therefore sensitive to the fast motion down to the 10-15 second regime. It is fair to say that each technique has its own advantages and possible drawbacks. Therefore, in order to get a comprehensive picture of the lipid dynamics, different techniques should be applied. In recent years, the application of more than one spectroscopic technique to tackle a single problem has been noticed. A combination of NMR and infrared spectroscopy to investigate the ion-lipid interaction of membranes [3] represents one of few known examples. This study involves a combination of two spectroscopy techniques, time-resolved fluorescence and infrared. Recently, we have applied time-resolved fluorescence technique to study the molecular dynamics of both pure unsaturated phosphatidylethanolamine (PE) and mixtures of PE with other lipids in various lamellar and non-lamellar phases. In those studies, the order parameter and rotational diffusion of the acyl chains of lipids, as well as the conformation and hydration of the lipid/water interface have been investigated by using lipophilic and water soluble fluorescent probes [6--8]. It is known that similar molecular information regarding the acyl chains and lipid/water interface may also be obtained by the use of infrared spectroscopy [9]. The vibration frequencies of the CH 2 symmetric stretching and the C = O stretching of the lipid chains represent two convenient parameters to examine the above lipid properties. The molecular mechanism governing the nonlamellar phase formation is a subject of research interest in our laboratory. A binary lipid mixture consisting of PE and diacylglycerol (DG) was examined in this study. The phase behavior of this binary mixture has been determined by dif-
ferential scanning calorimetry, 3~P-NMR and Xray diffraction [1,10,11]. DG is a major breakdown product of the cell membrane phosphatidyl inositol turnover during the signal transduction process of cells. Its role in perturbing the lamellar phases of both phospatidylcholine and PE has been well documented [1,2,10--12]. In addition, the correlation of its lamellar perturbation behavior and its role in the membrane-related functional activation has recently been investigated [2]. Yet the molecular mechanism of DG in perturbing the lipid polymorphism is still speculative. This study aims at understanding the molecular dynamics of the PE/DG lipid system in the 10-9- and 10-~5-s time scales. Both the lipid fatty acyl motional order as well as the lipid/water interfacial conformation and hydration of this lipid system in lamellar and non-lamellar phases were examined. Materials and Methods
Sample preparation PE transphosphatidylated from egg phosphatidylcholine (PC) (TPE) and 1,2-dioleoyl-sn-glycerol (diolein) were obtained from Avanti Polar Lipids (Birmingham, AL). 1-palmitoyl-2-[[2[4-(6phenyl-trans-l,3,5-hexatrienyl) phenyl] ethyl]carbonyl]-3-sn-PC (DPH-PC) and 8,l-anilinonaphthalenes sulfonate acid (ANS) were obtained from Molecular Probes, Inc. (Eugene, OR). All lipids were stored in chloroform at - 3 0 ° C and were used without further purification. For the ANS fluorescence measurements, the concentration of ANS was 1 × 10-7 M. For the DPH-PC fluorescence measurements, the fluorescent probe and lipid were first mixed in chloroform (spectroscopic grade). The molar ratio of DPH-PC to total lipid was 0.2%. The mixture was dried under a gentle nitrogen stream in a clean pyrex tube and further kept in vacuum for at least 4 h to ensure complete removal of the residual chloroform. The thin film formed on the tube was then hydrated with an aqueous buffer (100 mM NaCI, 10 mM TES and 2 mM EDTA pH = 7.4) at 4°C. The suspension thus formed was vortexed rigorously and
151 placed under mild sonication in a bath sonicator for a few seconds. The resulting suspension was then incubated overnight at 4°C in the dark to ensure proper hydration. For the fluorescence measurements, a further dilution of the lipid sample to ~0.15 pg//~l was performed. The sample temperature was controlled by an external water-jet circulator connected to a thermalstat cell.
Fluorescence spectroscopy
cence decay profile. However, the fluorescence intensity decay profiles of DPH-PC in PE/DG binary mixtures were fitted by a double-exponential function [6,7].
(ii) Fluorescence anisotropy decay measurements For time-resolved fluorescence anisotropy decay measurements, both excitation and emission polarizers were used. The same emission filter as in the lifetime measurement was used in here. The differential polarized phase angle (6± till) and the ratio of polarized modulation amplitudes (M±/Mu) were measured at different modulation frequencies (1 to 230 MHz). The subscripts H and Z refer to the directions of the polarization axis of the emission polarizer that are parallel and perpendicular to the vertical axis, respectively. The polarization axis of the excitation polarizer in our system was always set in the vertical direction. A first-order approximation model [16] was used to fit the frequency domain data. The anisotropy decay function has the following form: -
Fluorescence measurements were performed on an ISS frequency domain cross-correlation fluorometer (Champaign, IL). The excitation source was a cw He-Cd laser (Liconix 4240NB) with an output of 17 mW at 325 nm. The operational principle of this continuously variable frequency phase-modulation fluorometer has been described elsewhere [13,14].
(i) Intensity decay measurements Because the light exiting from the pockel cell (electro-optical device) is vertically polarized, a UV polarizer with the polarization axis set at 35°C with respect to the vertical was placed in the excitation beam to eliminate the rotational diffusion effect [15]. The fluorescence signals of either DPH-PC or ANS were measured and compared with a non-fluorescent glycogen solution (reference). A lower wavelength cutoff filter (model 3-72, Corning Glass-Works, Corning, NY) was used to remove the excitation light from the fluorescent signal and no filter was used for the reference sample. The phase delay (dF - 6s) and modulation ratio (MF/Ms) were measured at different modulation frequencies ranging from 1 to 230 MHz. Here dv and tis represent the phase delay of the signal by the fluorescent sample and that by the reference sample, respectively, and M F and M s represent the intensity modulation values of the fluorescent sample and that of the reference. The fitting of the frequency domain data was based on the minimization of the reduced chisquares according to the Marquardt algorithm [13]. Both single- and multi-exponential decay functions were employed to fit the ANS fluores-
-
r(t) = (r0 - r®)exp ( - t / t )
+ r
(1)
where r 0 and r® are the initial and residual anisotropy values and t r is the rotational correlation time. From the fitted parameters (r0, r® and tr), the normalized residual anisotropy (r®/ro) and initial slope (b(O)/ro) of the anisotropy decay function were then calculated.
FTIR spectroscopy For the FTIR measurements, the lipid samples were prepared under an identical sample preparation procedure as the fluorescence experiments but in the absence of fluorophores. The lipid was concentrated by centrifugation at 15,000 g for 20 min at 4°C. The lipid pellet was then applied to two CaF2 windows separated by a 25/am teflon spacer. The sandwich CaF 2 which contained the lipid film with uniform thickness was then mounted to a thermostated cell (Wilmad H200). The temperature of the cell was regulated by an external circulating bath (Haake F3C) and was recorded by a thermistor (YSI 200) which
152
was glued directly to the window mount. Spectra were recorded with an Analect RFX-40 Fourier Transform Infrared spectrometer equipped with a liquid nitrogen cooled mercury-cadmium-telluride detector. Usually 500 interferograms were collected for each temperature. The interferograms were apodized with a Norton-Beer medium function and fourier transformed with three levels of zero filling. The background spectra, which were obtained when the sample cell was present but in the absence of lipid film, were subtracted from the sample spectra. The spectral resolution was better than 2 cm -~. Results
FTIR spectra of PE/DG lipid mixtures were measured at different temperatures and for different lipid compositions. A typical FTIR lipids spectrum (407o DG at 23°C) is shown in Fig. 1.
The CH 2 stretching (2800---3000 cm-~), C = O stretching (1700m1800 cm-~), O--H bending (1600--1700 cm-~), CH 2 bending (1400--1500 cm-t) and P = O stretching (1200--1250 cm -l) regions were clearly observed. The vibrational bands below 1200 cm-~ were attributed to lipid headgroup vibrations and other collective vibrations of the lipid acyl chains. The frequency of the C H 2 symmetric stretching band (2850--2854 cm -t) was determined as a function of temperature (0--70°C) for lipid mixtures with different DG contents (0--16070 DG). Figure 2A shows the spectra of three representative DG concentrations (0, 4 and 15070 DG). For 007o DG, the C H 2 symmetric stretching frequency exhibited two distinct transitions at 12--20°C and 52--58°C. At those transitions, abrupt increases in the frequencies were found. For 4°70 DG, two abrupt transitions were also observed. However, the temperature ranges at which those abrupt changes occurred were downshifted to 10
~BSORBANCE 3.000
--
2.500 2.000
1.500
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--
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, °'°°~IoI."
esoo
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WAVENUMBER(cm-1) Fig. 1. T y p i c a l F T I R s p e c t r u m o f a fully h y d r a t e d P E / D G m i x t u r e (DG = 4 % ) at 23 °C.
I 1400
,
I 1200
I I 000
153
2868
C-H atretching(cm-1)
C-O stretching(cm-1)
174,<
B
A 2855 1742
2854 1740 2852
1738 2850 I
2848 0
I 10
I 20
I 30
t 40
I 50
I 60
1738 70
TEMPERATURE(C)
0
] 10
I 20
t 30
I 40
I 50
I 60
70
TEMPERATURE(C)
Fig. 2. Temperature dependence of the CH~ symmetric stretching frequency (panel A) and the C = O stretching (panel B) of PE/ DG mixtures with 0% (@), 4°10 (v1) and 15% (O) DG.
- - 1 8 ° C and 2 8 - - 3 2 ° C . At 15% DG, only one transition at 8 - - 1 5 ° C was observed. The frequency of the C = O stretching band (1736--1744 cm -~) was also determined as a function of temperature for different D G contents. Again Fig. 2B showed the spectra for three representative D G concentrations. For 0070 DG, the C = O vibrational frequency exhibited two transitions at 10--20°C and 50--58°C. It was f o u n d that the C = O frequency decreased abruptly at the low temperature transition while increased abruptly at the higher temperature transition. Note that the C = O frequencies at high temperatures ( > 5 8 ° C ) were almost the same as those at low temperatures (0--10°C). Similar observations of two distinct transitions were found for 4070 DG. However the high temperature transition was downshifted to 2 5 - 38°C. No shift in the low temperature transition was observed. Interestingly, the C = O stretching frequencies were constant at all temperatures for 15070 DG. Besides the temperature-induced transition, the composition-induced transition of P E / D G was also investigated. The variations of the fie-
quencies o f the C H 2 symmetric (Fig. 3A), C = O (Fig. 3B) and O = P = O antisymmetric (Fig. 3B) stretching bands with D G composition at 23°C were illustrated. The frequencies o f the C H 2 and C = O vibrational bands exhibited abrupt increases at 'o6--10°70 DG. On the contrary, the O = P = O antisymmetric stretching frequencies were constant for all D G contents. For both the C H 2 symmetric stretching and C = O stretching frequencies, the higher temperature transition was found to decrease more prominently than the lower temperature transition as the D G content increased gradually from 007o to 1607o. This was illustrated in a phase diagram as shown in Fig. 4. The transition temperature ranges in the phase diagram were determined from the C H 2 symmetric stretching frequency versus temperature measurements (see Fig. 2). The phase boundaries corresponding to the Lp-Lo and L : H . transitions are shown. At "o 1007o DG, a merge o f these two phase boundaries occurred at "ol0°C. A similar phase diagram was also obtained by using the C = O stretching frequencies (results not shown). From time-resolved fluorescence anisotropy
154
C=O stretching (cm-1)
C-H stretching (cm-1) 2858
P-O stretching (cm-1)
1744
A
1228
B
2856
1742
1220
1740
1224
0 2854
2883
f
1222
1738 0
0
2882
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o
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i
,
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i
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i
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8
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DG tool%
~
~
~
i
i
i
~
~
i
0
2
4
6
8
10
12
14
18
1218
DG mol%
Fig. 3. Lipid composition dependence of the CH 2 symmetric stretching ([3) (panel A); C = O stretching (O) and O = P = O anti-
symmetric stretching (D) (panel B) of PE/DG mixtures at 23 °C.
TEMPERATURE(C) 70
60
60
H:, 40
30
20
Z..x 10
Lo I
I
I
I
0
4
8
12
DG tool% Fig. 4. The phase diagram of PE/DG lipid mixtures as con-
structed from the CH 2 symmetric stretching frequency versus temperature measurements (see Fig. 2). The starting and completion temperatures of the Lp-Lo and Lo-H. phase transitions were represented by (D, O) and (<>, A), respectively.
16
decay measurements, the normalized residual anisotropy (~/ro) and initial slope (b(O)/r o) of the fluorescence anisotropy decay function of DPH-PC were determined as a function of temperature from 20 to 65 °C and shown in Figs. 5A and 5B, respectively. The fluorescence lifetime decay of DPH-PC was required to calculate the anisotropy decay parameters from the frequency domain data. Using a double-exponentiai decay function, two distinct lifetimes of DPH-PC were obtained for all temperatures. The long lifetime component (,~ 5.5 ns) represented a major intensity fraction (>0.95) of the decay profile of DPH-PC. The presence of a minor short lifetime component (~2.0 ns) indicated the existence of some unavoidable photodecay products of the probes during sample preparation. For 0070 DG, an abrupt decrease in r®/r o was observed at ,~55°C. For 4070 DG, the transition was downshifted to ,~30°C. Using arrhenius plots, abrupt declines in the values of ;,(O)/r o were observed at similar temperatures for 0070 and 4070 DG. From time-resolved fluorescence intensity decay measurements, the lifetimes of ANS in PE/DG mixtures were obtained for different DG
155
L O G ~'(O)/ro
r®/ro 0.8 [
A
B 8.8
0.6
8.6 0.4 8.4
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0
20
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I
I
I
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30
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60
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66
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I
I
I
I
3
3.1
3.2
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TEMPERATURE(C)
3.4
1 / T × 1 0 0 0 (K-')
Fig. 5. Plots of fluorescence anisotropy decay parameters r l r o (panel A) and log~0/(0)/r0 (panel B) as a function of temperature for DPH-PC in PE/DG mixtures with 0% ( 1 ) and 4e/0 (O) DG. The uncertainty of each measurement is indicated by the error bar.
compositions at 23 °C. Various multi-exponential decay functions were used to fit the frequency domain data. As judged by the chi-square values of the fits (Table I), the two- and three-exponential functions were found to provide better fits to the data than the one-exponential fit. For the three-exponential fit, the intensity fraction of the third component was very small (~,0.01) and the lifetime uncertainty of that component was very large (-130 ns). Interestingly, both twoand three-exponential fits gave short lifetime components in the range of 100--200 ps. The long lifetime component of the two-exponential fit and the middle lifetime component of the three-exponential fit for 0% DG were always
higher than that for 16% DG. Figure 6 shows the changes of the two lifetime components obtained from the two-exponential fit for different DG compositions. At low DG contents (0-6%), the long lifetime component was around 6 ns. A slight increase in the lifetime component was observed at around 8--10% DG. Thereafter the lifetime remained at ~ 7 ns for DG contents from 11% to 16%. The short components werf found to remain essentially constant and independent of DG content. Discussion
TPE in aqueous dispersions exhibits two ther-
TABLE I Lifetime heterogeneity of ANS in a PE/DG mixture (DG = 2%) at 23°C. The frequency domain data were fitted by one-, twoand three-exponential decay functions. The lifetime components were represented by Ts and their intensity fractions by fs. The reduced chisquares (X2) of all the fits were also shown.
T,
f,
~
f~
5.225 ± 0.136 0.218 ± 0.031 0.213 ± 0.040
1.0 0.097 ± 0.003 0.097 ± 0.004
5.629 ± 0.034 5.582 ± 1.315
0.903 0.889 ± 0.959
~
8.571 ± 132.22
f,
x2
0.014
321.10 8.07 8.64
156
lO
Tl(ne)
T2(n$)
8
4
3
7
6
1
/ 0
69
n,
0
2
q 4
q 6
[] , O,
8
DG
i [] L 10
12
mole
%
0
Fig. 6. F l u o r e s c e n c e l i f e t i m e h e t e r o g e n e i t y o f A N S in P E / D G m i x t u r e s as a f u n c t i o n o f D G c o n t e n t a t 2 3 ° C . The resolved lifetime c o m p o n e n t s T, ( n ) a n d T2 (O) were s h o w n .
motropic phase transitions, lamellar gel to lamellar liquid crystalline ( L : L ) and Lo to inverted hexagonal (L:Hl[), at ~15 and 55°C, respectively. The temperatures of these phase transitions have been determined by using differential scanning calorimetry [17], 3tP-NMR [18], infrared [9] and X-ray diffraction [1]. The frequencies of the CH 2 symmetric and C - O stretching bands exhibit abrupt changes at both the L#-L and L : H , transitions. The increase in the frequency of CH~ symmetric stretching band at both phase transitions suggests that the amount of gauche rotamers in the lipid acyl chains increases as the lipids undergo thermotropic phase transitions. On the other hand, the C = O stretching frequency decreases at the L:L+ transition but increases at the Lo-H. transition. These differential changes of C = O stretching frequency have been attributed to the alterations of the local conformation of the lipids at the ester carbonyl regions [18] and of the accessibility of water to the lipid/water interface [19]. These observed changes in both the CH 2 and C = O vibrational modes with temperature for pure TPE agree with the earlier results obtained by Casal and Mantsch [9].
In the presence of DG, the temperatures of both L#-L+ and Lo-H. transitions are downshifted. Since the frequency shift of C = O stretching mode is both conformation and hydration related, the assignments of the onset and completion of the two thermotropic phase transitions rely on the CH 2 stretching frequency. The phase diagram (Fig. 3) thus generated shows two interesting features. First, the Lo-H. phase boundary declines drastically with increasing DG content. Second, the L:Lo boundary decreases only slightly with DG. Owing to the relative difference in the slopes of the two phase boundaries, a point in the phase diagram is reached at which a merge of the above two phase boundaries occurs. This observation suggests that a direct transition from the L# to H . phase may occur at •10°70 DG. This suggestion is supported by the fact that only one transition in the CH~ stretching frequency was found for DG > 10%. In addition, no abrupt changes in the C = O stretching frequency with temperature are evident at those high DG contents. Of course, one still cannot exclude the possibility that the intermediate region (i.e., 8--20 °C and DG > 10%) in the phase diagram represents coexisting L J L J H , phases. It is interesting to mention that a direct L : H , transition behavior has also been observed in the salt-induced phase transitions of some saturated PE lipids [20]. Recently, we have been interested in applying time-resolved fluorescence spectroscopy to investigate the molecular dynamics of lipids and water in non-bilayer phases [6--8,211. Note that fluorescence spectroscopy provides information in the nanosecond time regime. Hence within the fluorescence time scale of the probes used in here ( * 6 ns), the rotational motions (wobbling) of the probe with respect to the normal axis of the lipids as well as to the long symmetry axis of lipid cylindrical tubes (for the case of HII phase) give rise to the anisotropic decay of the probe [6,7,21], while the orientational distribution of the probe governs the level of the residual anisotropy [16]. Using a first order approximation, the normalized initial slope of the decay function (~(0)/r0) and the residual anisotropy (r®/r 0)
157
reflect the averaged rotational rate of the probe and the global orientational distribution of the probe. The latter further contains the information of both the local order packing and packing symmetry of the lipid mixture and therefore is very sensitive to the L-Hxl transition [6]. Upon comparing the results from fluorescence with that from infrared, it is clear that the temperatures corresponding to abrupt changes in the ~(O)/r o and r®/r o agree well with that in the CH 2 and C = O stretching frequencies for both 0O7o and 4o'/0 DG. In the composition study, the frequencies of CH z and C = O stretching modes increase abruptly at '~6---10°10 DG, signifying the L-HI~ transition. A time-resolved fluorescence study on this composition-induced transition has also been performed (manuscript in preparation). From that study, abrupt changes in the fluorescence parameters, i.e., ~(O)/r o and r J r o, are evident at 6--10~0 DG and therefore agree with the infrared results. The frequency increase of C = O stretching mode could be due to hydration and conformational changes at the lipid/water interfacial region. The frequency of the O = P = O antisymmetric stretching mode is known to be sensitive to the hydration of the phosphate group [22,23]. The lack of any change in the O = P = O antisymmetric stretching frequency as a function of DG content indicates that the hydration of the lipid phosphate group remains unaltered at the L - H ~ transition. Similar conclusion was drawn by Casal and Mantsch 0984). However, one still can not exclude the possibility of hydration changes at the carbonyl region of the lipid/water interface, because of the different locations of the phosphate and carbonyl groups. Time-resolved fluorescence spectroscopy was also employed to examine further the conformation and hydration properties of the composition-induced Lo-H, phase transition of P E / D G system. In the ANS lifetime vs. DG010 plot, an abrupt increase in the long lifetime component (T ~ 5 - - 7 ns) of ANS is clearly evident. According to our previous interpretation, this increase in the fluorescent lifetime may be attributed to
an increase in the surface hydration of the lipid/ water interface [8]. Using a three-exponential fit, a longer lifetime component (T > 8 ns) was obtained. However, the fraction of this component is negligibly small (see Table I). This suggests that the majority of ANS probes are located in the bulk water phase and the hydrated layer of the lipid/water interface. Similar observations were reported for the temperatureinduced L-Hi[ phase transition studies of dioleoyl PE and TPE [8]. The increase in the surface hydration of lipids in the H . phase may be explained by an increase in the spacing between PE molecules clue to the presence of DG. This larger PE headgroup spacing may disrupt the intermolecular hydrogen bonding among PE headgroups and thereby increase the binding affinity and/or number of water to the interfaciai region. Using the water adsorption isotherm method, a similar conclusion of the hydration behavior of PE mixtures containing PC or cholesterol was drawn [24]. An alternative explanation for the ANS fluorescence lifetime result is the possible conformationai change at the lipid/water interface induced by DG. The interfaciai region of P E / DG may allow the ANS molecules to partition deeper into the glycerol backbone region as the DG content increases. This more hydrophobic environment of the glycerol backbone region causes a longer fluorescence lifetime of ANS. Our FTIR and fluorescence results, therefore, suggest that significant perturbations of the hydration and conformation of the lipid/water interfacial region occur at the Lo-H, transition of the PE/DG system. In conclusion, this study has demonstrated that significant changes in the lipid acyl chains and lipid/water interfacial regions occur at either the temperature-induced or compositioninduced L - H . transition of the P E / D G mixtures. Both fluorescence and infrared results appear to complement one another and provide molecular information in two different time scales, i.e., 10-9 and 10-15, respectively. A direct transition from the L~ to H . phase is predicted for DG > 100k from the phase diagram.
158
Acknowledgements This work was supported by grants from the National Cancer Institute (CA 47610) and the Robert A. Welch Research Foundation (D-1158) to K.H.C.S.Y.C. is an awardee of the Robert A. Welch scholarship. References 1 2 3 4 5 6 7 8 9 10
S. Das and R.P. Rand (1986) Biochemistry 25, 2882-2889. K.H. Cheng and S.W. Hui (1986) Arch. Biochim. Biophys. 244, 382--386. H.L. Casal, H.H. Mantsch and H. Hauser (1989) Biochim. Biophys. Acta 982, 228--236. K.H. Cheng, J.R. Lepock, S.W. Hui and P.L. Yeagle (1986) J. Biol. Chem. 261, 5081--5087. K.E. Reily, A.J. Mautone and R. Mendelsohn (1989) Biochemistry 28, 7368--7373. K.H. Cheng (1989) Biophys. J. 55, 1025--1031. K.H. Cheng (1989) Chem. Phys. Lipids 51, 137--145. K.H. Cheng (1990) Chem. Phys. Lipids 53, 191--202. H.L. Casal and H.H. Mantsch (1984) Biochim. Biophys. Acta 779, 381---401. R.M. Epand (1985) Biochemistry 24, 7092--7095.
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R.M. Epand, R.F. Epand and C.R.D. Lancaster (1988) Biochim. Biophys. Acta 945, 161--166. 12 H. Ellens, D.P. Siegel, D. Alford, P. Yeagle, L. Boni, L.J. Lis, P.J. Quinn and J. Bentz (1989) Biochemistry 28, 3692--3703. 13 E. Gratton, D.M. Jameson and R.D. Hall (1984) Annu. Rev. Biophys. Bioen8. 13, 105--124. 14 J.R. Lakowicz and B.P. Maliwal (1985) Biophys. Chem. 21, 61--78. 15 R.D. Spencer and G. Weber (1970) J. Chem. Phys. 52, 1654--1663. 16 W.B. Van Der Meer, H. Pottel, W. Herreman, M. Ameloot, H. Hendrickx and H. Schr6der (1984) Biophys. J. 46, 515--523. 17 K.H. Cheng, P.L. Yeagle, J.R. Lepock and S.W. Hui (1986) Biophys. J. 49, 324a. 18 P.M. Green, J.T. Mason, T.J. O'Leary and I.W. Levin (1987) J. Phys. Chem. 91, 5099--5103. 19 A. Blume, O. Hubner and G. Messner (1988) Biochemistry 27, 8239--8249. 20 J.M. Seddon, G. Cevc and D. Marsh (1983) Biochemistry 22, 1280---1289. 21 K.H. Cheng (1989) in: E. Roland Menzel (Ed.), Fluorescence Detection III, Proc. SPIE 1054, 160--167. 22 H.L. Casal and H.H. Mantsch (1987) Biochemistry 26, 4408--4416. 23 H.L. Casal, H.H. Mantsch, F. Paltauf and H. Hauser (1987) Biochim. Biophys. Acta 919, 275--286. 24 D. Marsh (1989) Biophys. J. 55, 1093--1100.