7726
J . Phys. Chem. 1990, 94, 1126-7130
2H NMR Spectroscopy of Oriented Phospholipid Bilayers in the Gel Phase Wigand Hubnert and Alfred BIume*** Institut f u r Physikalische Chemie, Uniuersitat Freiburg, Albertstrasse 23a, D- 7800 Freiburg, West Germany (Received: September 2, 1988; I n Final Form: March 14, 1990)
Macroscopically oriented bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero3-phosphoethanolamine (DMPE), specifically deuterated at the 4-position of the fatty acyl chains, have been prepared on microscope cover glasses. ZHNMR spectra were recorded as a function of the angle of orientation of the glass plates with respect to the magnetic field. Spectra of lipids in the liquid-crystalline phase display the characteristic dependence of the quadrupole splitting on the angle of orientation. Spectra of DPPC in the gel phase indicate a tilt of the molecular axis of 20° and a conical distribution for the director axis, as expected for a multidomain sample of DPPC in the Lgphase. Analysis of the spectra of oriented DPPC in the gel phase indicated that the correlation times for reorientational motions are in the intermediate exchange regime. This effect and the simulation problems arising from the complicated distribution function for the director axes precluded the successful simulation of the observed spectra. Spectra of DMPE in the gel phase indicate a parallel orientation of the director axis with respect to the bilayer normal as expected, as DMPE forms an L, gel phase. Spectra of DMPE taken at different orientations with respect to the magnetic field could be quantitatively simulated with a reorientational model allowing for 3-fold rotational jumps,trans-gauche isomerization,and a Gaussian distribution of director axes with a half width of 18'. This distribution is caused by surface undulations of the oriented bilayers.
Introduction
Lipid bilayer membrana are widely used as model systems for biological membranes, and numerous techniques have been applied to characterize their physicochemical behavior. One of the most powerful methods to study structure and dynamics of lipid bilayer systems is 2H N M R spectro~copy.~-~ With this method it is possible to study intramolecular dynamics of lipid molecules in great detail, as different parts of the molecules under study can be labeled specifically with 2H via chemical synthesis. Combination of different pulse techniques together with relaxation time measurements makes it possible to study lipid dynamics over a broad frequency range.s*6 For lipids in the gel state the fluctuations of the C-ZH bonds are in a time range comparable to the reciprocal of the quadrupole coupling. In this frequency range the 2HNMR line shapes are particularly sensitive to the rate and mechanism of the particular reorientational motion of the C-ZH bond. Line shape analysis using appropriate simulation programs can give a wealth of information on the motional behavior of lipids in the gel ~ t a t e . ~ . ~This - ' ~ has been applied to pure and mixed lipid systems and to study lipid-ion and lipid-protein interaction~.'-'~ For lipids in the liquid-crystalline state the reorientational frequencies are too high to affect the line shape as the correlation times of the various motional modes are shorter than lO-'s. In this case the quadrupole splittings give only information on the order parameters of the various segments of the acyl chains.I However, lipid dynamics can also be studied in this frequency range by spin-lattice relaxation measurements.6J8-zz Despite the success in simulating gel-state spectra of lipids, situations can arise where the observed line shapes cannot be unambiguously simulated by using just one particular motional model. Though in principle the recording of spectra at different pulse separations of the quadrupole echo sequence or the application of two-dimensional methods can give additional information to discriminate between different reorientational modes, these techniques are very time consuming and in the case of specifically labeled phospholipids very difficult to perform because of the low spin concentrations in the sample^.*^^^^ The use of macroscopically oriented lipid bilayers offers an alternative approach of obtaining additional information, because with oriented systems spectra can be recorded with different orientations of the lipid lamellae with respect to the external magnetic field.22~z5~26 This technique has been used quite ex-
'
Present address: National Research Council of Canada, Division of Chemistry, I 0 0 Sussex Drive, Ottawa KIA OR6, Canada. *New address: Fachbereich Chemie, Universitat Kaiserslautern, ErwinSchrMinger-Strasse, D-6750 Kaiserslautern, West Germany.
0022-3654/90/2094-1726$02.50/0
tensively in ESR and fluorescence spectroscopy. For powder samples the line shapes are a superposition of subspectra of all different director orientations. In oriented samples the director orientation can be selected by the macroscopic orientation of the sample with respect to the magnetic field. The model used for the simulation must then describe all subspectra equally well. In this way it is in principle possible to distinguish between different motional modes. The use of oriented systems is therefore of considerable advantage. However, in the case of 2H N M R spectroscopy there are some experimental limitations. These are due to low sigSeelig, J. Q. Reo. Biophys. 1977, IO, 353. Griffin, R. G. Methods Enzymol. 1981, 72, 108. Smith, I. C. P.; Jarrell, H. C. Acc. Chem. Res. 1983, 16, 266. Smith, R. L.; Oldfield, E. Science 1984, 225, 280. Spiess, H. W. Ado. Polym. Sci. 1985, 66, 23. Brown, M. F. J . Chem. Phys. 1982, 77, 1576. Huang, T. H.; Skarjune, R. P.; Wittebort, R. J.; Griffin, R. G.; Oldfield, E. J. Am. Chem. SOC.1980, 102, 7377. (8) Wittebort, R. J.; Olejniczak, E. T.; Griffin, R. G. J. Chem. Phys. 1987, 86, 541 1. (9) Meier, P.; Ohmes, E.; Kothe, G.; Blume, A,; Weidner, J.; Eibl, H. J . Phys. Chem. 1983,87,4904. (IO) Meier, P.; Ohmes, E.; Kothe, G. J. Chem. Phys. 1986, 85, 3592. (1 I ) Rice, D. M.; Blume, A.; Herzfeld, J.; Wittebort, R.J.; Huang, T. H.; Das Gupta, S. K.; Griffin, R. G. In Biomolecular Stereodynamics; Sarma, P. H., Ed.; Adenine Press: New York, 1981; Vol 11, p 255. (12) Blume, A.; Rice, D. M.; Wittebort, R. J.; Griffin, R. G. Biochemistry (I) (2) (3) (4) (5) (6) (7)
1982, 21, 6220. ( I 3) Blume, A.; Griffin, R. G. Biochemistry 1982, 21, 6230. (14) Blume, A.; Wittebort, R. J.; Das Gupta, S. K.;Griffin, R. G. Biochemistry 1982, 21, 6243. (15) Akutsu, H.; Seelig, J. Biochemistry 1981, 20, 7366. (16) Huang, T. H.; Blume, A.; Das Gupta, S. K.; Griffin, R. G. Biophys. J . 1988, 54, 173.
(17) Meier, P.; Sachse, J.-H.; Brophy, P. J.; Marsh, D.; Kothe, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 3704. (18) Kimmich, R.; Schnur, G.; Scheuermann, A. Chem. Phys. Lipids 1983, 32, 271. (19) Brown, M. F.; Ribeiro, A. A,; Williams, G. D. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4325. (20) Williams, G. D.; Beach, J. M.; Dodd, S. W.; Brown, M. F. J. Am. Chem. SOC.1985, 107. 6868. (21) Siminovitch, D. J.; Olejniczak, E. T.; Ruocco, M. J.; Das Gupta, S. K.; Griffin, R. G. Chem. Phys. Lett. 1985, 119, 251. (22) Jarrell, H. C.; Smith, I. C. P.; Jovall, P. A,; Mantsch, H. H.; Siminovitch, D. J. J . Chem. Phys. 1988, 88, 1260. (23) Schmidt, C.; Bliimich, B.; Wefing, S.; Kaufmann, S.; Spiess, H. W. Ber. Bunsen-Ges. Phys. Chem. 1987,91, 1141. (24) Muller, K.; Schleicher, A.; Kothe, G. Proceedings of the 9th European Experimental N M R Conference; Bad Aussee, Austria 1988, p 57 (25) Hiibner, W.; Blume, A. Proceedings of the 9th European Experimental NMR Conference; Bad Aussee, Austria 1988, p 91. (26) Blume, A.; Hlibner, W.; Miiller, M.; Biuerle, H. D. Ber. Bunsen-Ges. Phys. Chem. 1988, 92, 964.
D 1990 American Chemical Societv
Oriented Phospholipid Bilayers
The Journal of Physical Chemistry, Vol. 94, No. 19, 1990 7727
nal-to-noise ratio when specifically labeled lipids are used, as it is difficult to get enough oriented material, which is deposited on glass slides, into the limited volume of an N M R sample tube. For recording spectra of lipids in the liquid-crystalline phase these problems are less severe, as the lines are sharp due to fast motional averaging. However, experiments with lipids in the gel phase are much more difficult, as the spectra are very wide. When the reorientational motions approach the intermediate exchange regime, the quadrupole echo spectra show characteristic losses in intensity, which lead to further decrease of the signal-tenoise ratio. Despite these problems we succeeded in obtaining 2H N M R spectra of oriented 1,2(4,4-2H2)DPPC and 1,2(4,4-2H2)DMPE in the gel ~ t a t e . In ~ ~this . ~paper ~ we will describe an analysis of the gel-state spectra of oriented DPPC bilayers and the results of line-shape simulations for gel-state spectra of DMPE.
Materials and Methods Materials. 1,2(4,4-2H2)DPPC was prepared from glycerophosphorylcholine CdC12 complex by reacylation with the fatty acid anhydride of [4,4-2H2]palmitic acid using 4-(dimethylamino)pyridine as a ~ a t a l y s t . ~ 'Similarly 1,2(4,4-2H2)DMPC was prepared from [4,4-2H2]myristicacid anhydride. The specifically labeled fatty acids were synthesized as described previously.'2*28 1,2(4,4-2H2)DMPEwas prepared from the corresponding DMPC by transphosphatidylizationwith phospholipase D in a buffer containing ethanolamine hydro~hloride.~~ All phospholipids were purified by column chromatography on silica gel (Kieselgel60, reinst, Merck, Darmstadt, West Germany) using a CHC13/MeOH gradient. Purity was tested by TLC and differential scanning calorimetry using a DASM- 1 DSC calorimeter.30-3' Methods. Oriented bilayers were prepared on microscope cover slides from a lipid solution in organic solvent. The lipids were hydrated over the vapor phase of deuterium depleted water. Approximately 20-25 glass slides were stacked together and sealed in a IO-" NMR tube. Samples were annealed at temperatures above the gel to liquid-crystalline phase transition for several hours. The degree of hydration could be roughly controlled by the length of time of hydration over the water vapor. In the case of DPPC excess hydration resulted in poorer orientation. A sample hydrated to only ca. lO-l5% (w/w) gave much better oriented bilayers. With the DMPE sample sufficient hydration was easily obtained, though the amount of oriented material was somewhat less than for DPPC. The multilamellar powder sample of DMPE was prepared by adding the same amount of deuterium-depleted water to ca. 80 mg of dry 1,2(4,4-2H2)DMPEin a 5-mm sample tube and equilibrating the sample at 70 OC for 1 h. 2H NMR spectra were recorded with a Bruker CXP 300 NMR spectrometer using the quadrupole echo sequence with a pulse separation of 40 ps. Echo decays were accumulated with a dwell time of 0.5 ps. Spectra of the powder samples were obtained by using a probe with a 5-mm solenoid. For this probe the 90° pulse length was ca. 2.5 ps. In the case of the oriented sample a IO-" solenoid had to be used. For this coil the 90° pulse length was 5.5 1 s . For the oriented sample 20000-180000 echo decays had to be accumulated to get reasonable signal-to-noise ratios, particularly for lipids in the gel phase. The 1 0 " sample tube could be adjusted to different angles of orientation with respect to the external field. Spectra of oriented samples were recorded in the following manner: for each of the four different orientations the sample was first heated to 70 OC and the spectrum for the liquid-crystalline phase was recorded after temperature equilibration. Then the sample was cooled to 30 O C , and the spectrum for the (27) Gupta, C. M.; Radakrishnan, R.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 43 15. (28) Das Gupta, S.K.;Rice, D. M.; Griffin, R. G. J . Lipid Res. 1982, 23, 197.
(29) Eibl, H.; Kovatchev, S.Methods Enzymol. 1981, 72, 632. (30) Privalov, P.; Plotnikov, V. V.;Filimonov, V. V. J . Chem. Thermodynam. 1975, 7,41. (31) Blume, A. Biochemistry 1983, 22, 5436.
1.2 lk4-02) DPPC
c ~
75
1,214.h-0,I DMPE
1
0 [kHz1
75
I
L
-15
0 lkHz1
75
Figure 1. 2H NMR spectra of oriented bilayers of 1,2(4,4-2Hz)DPPC and 1,2(4,4-2Hz)DMPEat a temperature of 70 O C . @ = angle between glass plate normal and magnetic field. Note the powder pattern of residual unoriented material particularly visible for @ = 0'. gel phase was recorded at the same angle of orientation. Changes in the quality of orientation were checked by repeatedly recording spectra of the liquid-crystalline phase. Orientation quality did not change during the course of the experiment. Spectral simulations were performed using the six-site jump model described before.'**J2 All calculations were run on a Sperry Univac 1100/80 of the Rechenzentrum of the University of Frei burg.
Results and Discussion Liquid-Crystalline Bilayers. For controlling the quality of the macroscopic orientation of the bilayers, we recorded spectra of lipids in the liquid-crystalline phase. Figure 1 shows spectra of oriented bilayers of 1,2(4,4-2H2)DPPCand 1,2(4,4-2H2)DMPE at a temperature of 70 OC where both systems are in their liquid-crystalline state. Spectra were recorded a t different angles fl of the bilayer normal with respect to the magnetic field. As expected from theory, the spacing between the two lines varies with the angle of orientation. For bilayers in the liquid-crystalline phase all motions with the exceptions of slow collective order fluctuation^^^ are in the fast limit on the deuterium time scale, and the director axis and bilayer normal are parallel to each other. The line separation AvQ(p) can then be accurately described by the equation AvQ(P) = ?"(e2qQ/h).'/2(3 cos2 P
- l)*SmoI
with /3 being the angle of orientation of the director axis or bilayer normal with respect to the exterhal field and (e2qQ/h) the quadrupole coupling constant (168 kHz for an aliphatic C-2H bond). For 1,2(4,4-2H2)DMPEthe order parameter S,, was determined from a powder sample to be 0.51 (not shown). This agrees with the value of S,,, calculated from AvQ as a function of p. For 1,2(4,4-ZH2)DPPCthe order parameter SmOI for hydrated unoriented bilayers at T = 70 OC is 0.34 (not shown). For the oriented sample, however, a much higher value of 0.54 was found. Measurements of the transition temperature of the oriented bilayers gave a value of 60-62 "C for T,, ca. 20 OC higher than T , of DPPC in excess water (41.5 "C). From the phase diagram (32) Power, L.; Pershan, P. S.Eiophys. J . 1977, 20, 137.
7728 The Journal of Physical Chemistry, Vol. 94, No. 19, I990 of DPPC3354we estimated the water content of our oriented sample to be between 10 and 12% (w/w), as T , increases, when the water content drops below 25%.34 The higher order parameter of the oriented DPPC sample can be explained by the fact that at 70 'C the oriented sample is only 10 OC above T,, whereas for the powder sample the difference is 30 O C . The known temperature dependence of Sd and the somewhat tighter packing due to lower hydration both contribute to the higher Sd value for the oriented sample. As mentioned in the Material and Methods section excess water gave poorer orientation of the bilayers, so we had to compromise and decided to use the sample with lower hydration but better orientation. The DMPE sample seemed to be fully hydrated as it showed the correct transition temperature of 49 'C also found for multilamellar dispersions. It is known that phosphatidylethanolamines need less water for full hydration and that the transition temperature becomes independent at a water content of ca. 7 molecules of water/lipid molecule (ca. 20%).35,36 In all spectra residual powder patterns of unoriented material are visible, m a t clearly in the spectra taken at an orientation with j3 = 0'. For DMPE the amount of unoriented material is slightly larger than for DPPC. For spectra of lipids in the liquid-crystalline phase these residual powder patterns did not disturb the evaluation of the line splittings. For spectra of lipids in the gel phase, however, they had to be removed by spectral subtraction (see below). With the same sample of oriented 1,2(4,4-2H2)DPPCwe also determined the relaxation time T , Zas a function of j3 using the inversion recovery sequence. In accordance with the findings of others22,37v38 almost no angular dependence of T I Zcould be detected. At a frequency of 46.1 MHz we found the following values: 28.5 f 1 ms for j3 = Oo, 29 f 1 ms for j3 = 57', and 27.5 f 1 ms for j3 = 90'. This missing TI, anisotropy seems to be fortuitious. Speyer et aL3* could show that in cerebrosides and probably also in phosphatidylcholines the T I , anisotropy depends on the label position and can be positive or negative depending on whether the label is in the upper or lower part of the hydrocarbon chain. In PCs the orientation dependence of TIZseems to be almost zero at position 4. Gel-Phase Bilayers. Whereas good quality spectra of oriented lipids in the liquid-crystalline state can be fairly easy obtained, this is much more difficult for bilayers in the gel state. The gel-state spectra are much wider to begin with, so that problems of power roll-off due to the relatively long 90' pulse necessary for the IO-" coil are more s e v e ~ e . ~In, ~addition ~ the signalto-noise ratio becomes very low when the molecular motions are in the intermediate exchange regime as the echo amplitudes are drastically reduced due to the short T2e.5*8940 Finally, unoriented material giving rise to an overlapping powder pattern complicates the quantitative analysis of the spectra. Nevertheless we succeeded in obtaining spectra of sufficient quality for a line-shape analysis. DPPC. Spectra of oriented bilayers of 1,2(4,4- H2)DPPC in the gel phase ( T = 30 "C) as a function of the angle of orientation j3 are shown in Figure 2. At this temperature DPPC adopts an L, phase with slightly tilted chains.41 Spectra recorded at j3 = 0' show two broad lines with a spacing of 96 kHz for DPPC. The residual powder pattern of unoriented material is clearly visible but relatively small in the DPPC sample, in agreement with the spectra of DPPC in the liqyid-crystalline phase. For a DPPC powder sample the separation AvQ, is ca. 112 kHz at 30 'C (not shown), whereas in the oriented sample the splitting at j3 = 0' is only 96 kHz. There are two possible explanations (33) Albon, N. J . Chem. Phys. 1983, 78, 4676. (34) Seddon, J. M.; Cevc, G.;Marsh, D. Biochemistry 1983, 22, 1280. (35) Cevc, G.;Marsh, D. Biophys. J . 1985, 47, 27. (36) Rommel, E.; Noack, F.; Meier, P.; Kothe, G.J . Phys. Chem. 1988, 92, 298 1. (37) Mayer, C.; GrBbner, G.;Miiller, K.; Weisz, K.; Kothe, G . Chem. Phys. Lett. 1990, 165, 155. (38) Speyer, J. B.; Weber, R. T.;Das Gupta, S.K.; Griffin, R. G.Biochemistry 1989, 28, 9569. (39) Bloom,M.; Davis, J. H.; Valic, M. I. Can. J . Phys. 1980, 58, 1510. (40) Spiess, H. W.; Sillescu, H. J . Magn. Reson. 1981, 42, 381. (41) Tardieu, A.; Luzzati, V.;Reman, F. C. J . Mol. Bioi. 1973, 75, 71 I .
Hiibner and Blume 1,214.L-021 DMPE
h \
p.56'
L
-l
,
l
,
l
,
1
.
I
.
I
0 100 i kHzl Figure 2. 2H N M R spectra of oriented bilayers of 1,2(4,4-2H2)DPPC and 1,2(4,4- H2)DMPE at a temperature of 30 O C . j3 = angle between glass plate normal and magnetic field. -100
0
100
-100
IkHzl
for this discrepancy. If the reorientational motion around the molecular axis is in the fast limit, then the director axis must be tilted with respect to the bilayer normal by an angle a of ca. 18-20' calculated from the (3 cos2 a - 1) dependence of the line splittings. This would be in agreement with previous X-ray results on the L, phase that show a variable tilt angle between 10 and 30%water content.33*41v42Our value of 18-20' for DPPC with 10-12% water would be inside the range of values reported by other^.^^,^^ The other extreme is the rigid-limit case. With the assumption of an isotropic distribution of the C 2 H vectors around the molecular axis, a tilt angle of 20' would result in a distribution of the orientations of the C-2H vectors in an angular range between 70 and l 10'. The spectrum would then consist of a "powder pattern" of lines with separations ranging between 115 and 92 kHz. This shows that motional averaging around the long axis ' 0 does not lead to a drastic change in the line shape when j3 = . As the line shapes of unoriented DPPC bilayers indicate motions with correlation times between lo4 and lo-' s,9,10*14 which are close to the fast limit regime, the assumption of the rigid limit case seems not to be justified. All DPPC molecules in the sample seem to adopt the same tilt angle. As we do not have a monodomain sample, a conical distribution of the molecular axis around the bilayer normal can be deduced. For j3 = 90' and fast limit axial diffusion this conical distribution would lead to a distribution of director axes over an angular range of 90 f 20'. The maximal width of the powder pattern for the orientation with j3 = 90' would then be 56 kHz, half the value of the separation of the outer edges of the DPPC powder pattern. In the rigid limit case we would get a distribution for the C-2H vectors between 20 and 160' and thus a maximal width of the powder pattern of ca. 190 kHz. We actually observe a value of 112 kHz for j3 = 90°, which is between the two extremes. For the other orientations the expected widths of the 'powder patterns" in the fast limit case can be calculated to 48 kHz for B = 58' and 102 kHz for j3 = 34'. Inspection of Figure 2 shows that the measured values are again much larger, namely 104 kHz for j3 = 58' and 108 kHz for j3 = 34'. There are several possibilities for the discrepancies between the estimated widths of the powder patterns for the two extreme cases, the fast limit and the rigid limit case, and the actually measured values. The reorientational motions are in the intermediate exchange regime, or the assumed distribution of director axes is not correct, or more complicated reorientational motions than simple ~~
(42) Stamatoff, J . B.; Graddick, W. F.; Powers, L.; Moncton, 0. E. Biophys. J . 1979, 25, 253.
The Journal of Physical Chemistry, Vol. 94, No. 19, 1990 7729
Oriented Phospholipid Bilayers
I
I
I
-in
Dol
IN
[ kHz 1 Figure 3. Bottom: *H NMR spectrum of multibilayers of 1,2(4,42H2)DMPEat T = 30 "C.Top: simulated spectrum; see text for sim-
ulation parameters. rotational jumps about the molecular axis plus additional transgauche isomerization have to be assumed. The first explanation seems likely, as we observe low signal-to-noise ratios, indicating intensity losses due to motional correlation times in the intermediate exchange regime.s*8*40 However, the other effect may also play a role. Because of these multiple effects the simulations become very complicated and we did not succeed up to now to get good fits between calculated and experimental spectra. So in the case of DPPC in the gel phase the analysis of the line shapes of the oriented samples had to be postponed. DMPE. For 1,2(4,4-2H2)DMPEthe separation of the two lines at j3 = 0' (1 10 kHz) agrees with the separation of the outer edges AuQIof the powder sample (see Figures 2 and 3) indicating that for this lipid the molecular axis is indeed parallel to the bilayer normal as expected for a phospholipid forming an L, phase.43 As the DMPE powder pattern and the spectra of the oriented sample have relatively high signal-to-noise ratios, motional averaging around the molecular axis must be fast. In this case line-shape analysis is much easier than for DPPC. As can be seen from Figure 3 the separation of the two lines varies as expected for a director orientation parallel to the bilayer normal. At j3= 90' the line separation is 55 kHz, half the value of the separation determined from the spectrum taken at j3 = Oo. At an angle close to the magic angle only a broad line is observed. The residual powder pattern of unoriented material can be easily removed by spectral subtraction. To this end we recorded the spectrum of a powder sample as shown in Figure 3. This powder pattern, in each case scaled to the same absolute intensity, was then subtracted from the experimental spectra of oriented 1,2(4,42H2)DMPEin Figure 2. The resulting spectra are shown in Figure 4. The powder pattern of Figure 3 can be simulated with a program applying the six-site jump model we have used and described previously for the simulation of powder spectra of 2(4,4-2H2)DPPE.8J2 It models long-axis rotation as a 3-fold rotational jump with sites 120' apart and trans-gauche isomerization as jumps from positions with an angle of 90' to the molecular long axis to an orientation with an angle of 35.3'. All three trans sites were equally populated, as were the three gauche sites. The total probability for the occupation of the gauche sites Pg is a variable parameter as are the rate constants R,, for the rotational jumps and Rtg for the jumps from a trans to a gauche site. For the simulated spectrum in Figure 3, top, the following parameters were used: Pg = 0.05, R,,, = 3.1 X IO6 s-l, RIg= 3 X IO5 s-I. With this parameter set the experimental spectrum can be simulated reasonably well as Figure 3 shows. With the same parameter set we also tried to simulate the spectra of oriented bilayers of 1,2(4,4-2H,)DMPE after subtraction of residual powder patterns. Before subtraction the powder pattern of Figure 3, recorded in a 5-mm tube with a 2.5-ps 90' pulse, was (43) McIntosh, T. J. Biophys. J . 1980, 29, 237.
I
-I*
[kHz1
I
I
8.1
IN
[kHz 1
Figure 4. Bottom: 2H NMR spectra of oriented bilayers of 1,2(4,42H2)DMPEat T = 30 "C after spectral subtraction of powder pattern
originating from unoriented material. Top: simulated spectra as a function of @; see text for simulation parameters. 8 = angle between glass plate normal and magnetic field. corrected with a frequency-dependent factor accounting for the larger power roll-off encountered with the 5.5-ps 90' pulse used with the 10-mm Despite some baseline distortions the quality of the spectra after subtraction is good enough to justify a line-shape analysis. In the first simulations we used a single orientation of the director axis with respect to the magnetic field. These simulations gave pairs of lines, which were too narrow. To simulate the relatively broad lines observed in the oriented sample, we then included a Gaussian distribution of director axes with a half-width of 18'. This was accomplished by calculating subspectra a t various angles ,8 with A@ = 2' and adding these subspectra with the appropriate scaling factors calculated for a Gaussian distribution of required half-width. With this distribution we got much better simulations as shown in Figure 4. We conclude that the surface of the bilayer, even in oriented samples, is not completely planar. These surface undulations are probably also present when the bilayers are in the liquid-crystalline state, but in this case rapid lateral diffusion over the characteristic distances of these cormgations lead to an additional averaging process, which is normally included in line-shape simulations in the form of a wobbling motion of the molecular a x i ~ . ~For J ~bilayers in the gel phase, lateral diffusion is not fast enough to lead to motional averaging. The different orientations have therefore to be included explicitly in the form of a distribution function. With this Gaussian distribution the spectra of oriented DMPE as a function of orientation can be simulated fairly accurately as Figure 4 shows. So for phosphatidylethanolamines in the LBphase the relatively simple six-site jump model allowing for rotational motion and trans-gauche isomerization is sufficient to quantitatively describe the 2H N M R spectra observed for oriented as well as for unoriented DMPE bilayers. As recent 2D 2H N M R spectra of the gel state of a glycolipid have shown, large angle jumps around the molecular axis are more likely in ordered systems than are diffusive axial motions.44 Thus it is not surprising that jump models can also describe the motions in the gel phase of other lipids. Summary and Conclusions 2H NMR studies on oriented bilayers of specifically deuterated phospholipids can in principle provide additional information on the motional dynamics of these molecules in their different phases. The recording of spectra of lipids in the liquid-crystalline phase (44) Auger, M.; Jarell, H.C. Chem. Phys. Leu. 1990, 165, 162.
J . Phys. Chem. 1990, 94, 1130-7134
7730
is relatively unproblematic. As all motions are in the fast limit on the deuterium time scale, the two observed lines are very narrow, so that obtaining spectra with good signal-to-noise ratio presents no problems. For lipids in the liquid-crystalline phase it is thus even possible to perform more time-consuming pulse experiments, which are for instance necessary to determine the relaxation times T l z and Tlq.22*27*38 More difficult is the recording of spectra of lipids in the gel phase due to the great width of the spectra and the low signalto-noise ratios encountered. However, we succeeded in recording gel-phase spectra of oriented DPPC and DMPE. In the case of oriented DPPC we could not simulate the experimental spectra, due to the complexity of the system. Tilting of chains, reorientational correlation times close to the intermediate exchange regime, and undulations of the bilayer surface leading to very complex distribution functions for the director precluded successful line-shape simulations. Additional 2D 2H NMR spectra of powder samples in combination with T I Zand T2cexperiments on oriented systems could probably resolve the question, whether the previously used models for describing reorientational motions in gel-phase PC bilayers are correct.
For DMPE angular-dependent spectra of the gel phase could be recorded with relatively good quality. After spectral subtraction
of powder patterns originating from unoriented material, we succeeded in simulating the spectra with a simple six-site jump model used previously for the simulations of powder patterns of lipids in the gel phase. Agreement between simulated and experimental spectra could be improved when a Gaussian distribution of director axes was included. This indicates that the bilayers of oriented lipids in the gel state are distorted by surface undulations. With this paper we could show that despite experimental problems it is possible to obtain spectra of oriented lipids in the gel phase. This method can thus offer an alternative to other difficult and time-consuming methods, such as relaxation time measurements or 2D experiments, though a successful interpretation of the observed spectra is possible only when the reorientational motions are not of too great complexity. Acknowledgment. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 60, D-4) and the Fonds der Chemischen Ind.
Ion Chemistry at High Temperatures. 1. Thermochemistry of the Ammonium Ion from VariableTemperature EqWbrium Measurements. Proton Transfer, Association, and Decomposition Reactions in Ammonia, Isobutene, and tert-Butylamine Michael Meot-Ner (Mautner) and L. Wayne Sieck* Chemical Kinetics Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 (Received: September 14. 1989; I n Final Form: April 17, 1990)
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At 580-680 K, the proton-transfer reaction t-C4H9++ NH3 NH4+ + i-C4H8is in equilibrium in mixtures containing ammonia and isobutene. In the same mixtures, t-C4H9NH3+is also formed reversibly, and kinetic experiments identify the addition/thermal decomposition equilibrium NH4++ i-C4Hs + M t-C4H9NH3++ M. The decomposition of t-C4H9NH3+ is at the low-pressure limit, with rate constants of (1-14) X cm3/s, and E, = 29.1 kcal/mol. The thermally activated cm6/s and a negative temperature coefficient addition of NH4+ to i-C4H8shows third-order kinetics with k = (2-6) X of k = A T 1 0 . 8 .Equilibrium studies of the proton-transfer reaction yield AHo = -12.5 kcal/mol and ASo = -6.1 cal/(mol K). Referred to PA(i-C4H8)= 195.9 kcal/mol, these results yield PA(NH3) = 208.4 kcal/mol, at the high end of published values ranging from 202 to 210 kcal/mol (PA = proton affinity). For the addition reaction, the equilibrium studies yield AHo = -34.9 kcal/mol and ASo= -39.2 cal/(mol K). These results lead to PA(t-C4H9NH2)= 228.7 kcal/mol, compared with the recommended value of 221 kcal/mol.
Introduction The relative proton affinities (PA's) of hundreds of molecules have been measured and compiled.' Most measurements reported to date were obtained from equilibrium (1) measured at a single temperature (
