J . Phys. Chem. 1987, 91, 4625-4627 as functions of viscosity. The overall process was found to have little or no energy of activation around 300 K. The comparison of rotational relaxation and proton-transfer rates has revealed that they are strongly correlated, indicating that the proton transfer is mediated by solvent frictional forces. A reasonable fit of the data is obtained if it is assumed that the proton-transfer rate is the sum of the rotational relaxation rate for the solute and the Debye relaxation rate for the solvent at each viscosity. We believe
4625
these experiments constitute the first direct measurements of a reaction rate which implicates a sum of two rotational diffusion processes.
Acknowledgment. This research was supported by a grant from the Division of Materials Research of the National Science Foundation (DMR-85-07740). Registry No. HMPB,2440-22-4
Structures and Mechanisms of Llpid Phase Transitions in Nonaqueous Media: Dlpalmitoylphosphatidylcholine in Fused Salt W. Tamura-Lis, L. J. Lis,* Department of Physics and The Liquid Crystal Institute, Kent State University, Kent, Ohio 44242
and P. J. Quinn Department of Biochemistry, King’s College London, London W8 7AH, U.K. (Received: December 1, 1986; In Final Form: May 4, 1987)
The mechanisms of the single phase transition observed for L-dipalmitoylphosphatidylcholinedispersed in excess fused salt, ethylammonium nitrate (EAN), was examined with time-resolved X-ray diffraction. The samples were allowed to equilibrate at 0 OC for over 4 days to induce the subgel state. The subgel state characterized by an orthorhombic acyl chain subcell converts directly to the hexagonal subcell characteristic of the disordered acyl chain state. This transformation involves the continuous loosening of the subcell packing until the disordered state is induced. A concomitant change in the mesophase unit cell is observed which is characterized as a transition from a multilamellar array of bilayers to an hexagonal array of cylinders.
Introduction The interaction between phospholipids and water is currently receiving a great deal of attention as we strive to understand how lipid structures and biological membranes are stable in this solvent. This interest has also spawned a renewed effort in the study of phospholipid structures and phase transitions in nonaqueous solvents. In particular, the replacement of water by organic solvents such as glycerol’-’ and “dry” solvents such as trehaloseM has been shown to alter the bilayer structure, packing, and phase-transition parameters for systems containing phosphatidylcholines. A uniquely different type of solvent used in previous studies7v8was ethylammonium nitrate (EAN), a fused salt of low melting temperature, which provides a purely ionic (although highly polar) medium. Thus the role of ionic bonding between head groups can be studied and contrasted to that of hydrogen bonding in water or similar polar solvents. The effect of replacing water with EAN was first reported,’ using bilayers made from distearoylphosphatidylcholine. It was found that the presence of EAN had little effect on the main (gel to liquid crystal) phase transition in DSPC, although there was some evidence of an increase in the pretransition temperature. A further observation was that the d spacing for the L, phase in a 1:l (by weight) mixture of EAN and DSPC was drastically reduced when compared with a fully hydrated DSPC bilayer. The acyl chain packing was found to be hexagonal subcells with a sharp ~
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(1) McDaniel, R. V.; McIntosh, T. J.; Simon, S. A. Biochim. Biophys. Acta 1983, 731, 97. (2) Rowe, E. S. Biochemistry 1983, 22, 5299. (3) Simon, S. A.; McIntosh, T. J. Biochim. Biophys. Acta 1984, 773, 169. (4) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science (Washington, D.C.) 1984, 223, 701. (5) Lee, C. W. B.; Waugh, J. S.; Griffin, R. G. Biochemistry 1986, 25, 3731. (6) Finegold, L.; Singer, M. A. Biochim. Biophys. Acta 1986, 855, 417. (7) Evans, D. F.; Kaler, E. W.; Benton, W. J. J . Phys. Chem. 1983, 87, 533. (8) OLeary, T. J.; Levin, I. W. J . Phys. Chem. 1984, 88, 4074. ~~
0022-3654/87/2091-4625$01.50/0
reflection at 4.1 and a broad reflection at 4.0 A for the L, and La phases, respectively. The surface areas for the DSPC head groups in EAN of both the L, and L, phases were similar (ca. 80 A*) when calculated with the L, and L, d spacings. These results contrast with previous structural information determined for DSPC bilayers in water, in which, for example, a surface area of 52 A2 is reported for L,. However, it could not be determined whether these observations were influenced solely by the solvent character or whether the amount of solvent present also had some influence. A later study using Raman spectroscopy examined the dispersion of dipalmitoylphosphatidylcholine(DPPC) in EAN.* Only a single phase transition was observed at 59.5 “C, which was characterized as an acyl chain transition from an ordered orthorhombic subcell to a disordered hexagonal subcell, rather than the usual multiple phase transitions observed for DPPC dispersed in water. Another difference between DPPC dispersed in EAN vs. DPPC in water was the inferred presence of a micellar (in EAN) rather than a lamellar (in water) phase above the liquid crystal phase transition temperature. The basis for the assignment of the high-temperature micellar phase for DPPC in EAN was the degree of disruption of the Fermi resonance of the 1440-cm-’ Raman band as it influenced the relative intensities of symmetric and asymmetric C-H stretch modes. Recently, high-intensity X-ray beams at synchrotron sources have been used to determine lipid mesophase and acyl chain structures at high r e s o l u t i ~ n ,as~ ~well ~ ~ as the kinetics and mechanisms of a variety of phase transitions.”-I6 In this report, DPPC bilayers dispersed in EAN (20% w/v DPPC/EAN) were (9) Caffrey, M. J.; Feigenson, G. W. Biochemistry 1984, 23, 323. (10) Tenchov, B.; Lis, L. J.; Quinn, P.J. Biochim. Biophys. Acta 1987, 897, 143. (1 1) Caffrey, M.; Bilderback, D. H. Biophys. J . 1984, 45, 627. (12) Caffrey, M. Biochemistry 1985, 24, 4826. (13) Ranck, J. L. Chem. Phys. Lipids 1983, 32, 251. (14) Laggner, P. Top. Curr. Chem., in press. (15) Lis, L. J.; Quinn, P. J. Biochim. Biophys. Acta 1986, 862, 81. (16) Quinn, P. J.; Lis, L. J. J . Colloid Interface Sci. 1987, 115, 220.
0 1987 American Chemical Society
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The Journal of Physical Chemistry, Vol. 91, No. 17, 1987
Tamura-Lis et al.
examined by using X-ray diffraction techniques at the Daresbury (U. K.) Synchrotron Laboratory. The lipid-EAN mixtures were prepared by methods similar to those previously described'O which cause the formation of the subgel phase in L-DPPC bilayers in excess water. Our results for DPPC bilayers dispersed in EAN are compared with those of DPPC bilayers dispersed in water over the temperature range in which the subgel, pretransition, and main phase transitions have been observed for DPPC in water. Differential scanning calorimetry was used to examine possible hysteresis in the various transitions studied.
Materials and Methods 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine was purchased from Fluka, AG. The lipid was checked by thin-layer and gas chromatographies and found to be at least 99% pure. Ethylammonium nitrate (EAN) was prepared as previously described7 and was provided by Prof. B. Ninham. Specifically, EAN was prepared by adding 3 M nitric acid to a cooled 25% aqueous solution of ethylamine such that a slight excess of amine remained. Most of the water was removed with a rotary evaporator; final drying was achieved with a lyophilizer. The anhydrous fused salt was then dissolved in acetonitrile, mixed with activated charcoal, filtered, recrystallized, and redried in the lyophilizer. Weighed portions of DPPC and EAN were combined in glass vials and heated to -80 "C for 30 min. The samples were then stored in a sealed container at 0 "C for 4 days before X-ray examination. Similar samples for calorimetric analysis were prepared, and equilibration was achieved in sealed aluminum sample pans. In all cases, mixtures were made at 80% v/w EAN / DPPC. Thermograms were obtained by using a Perkin-Elmer DSC-2C with data collection and analysis done via a Perkin-Elmer data station. Temperature and enthalpy were calibrated by using indium. Temperature scans were run at 10 OC-min-l between the temperature limits of 275 and 350 K. X-ray experiments were carried out by using a monochromatic (0.150 nm) focussed X-ray beam at station 7.25 and the Daresbury Synchrotron Laboratory as previously de~cribed.'~A cylindrically bent single crystal of Gel8 and a long float mirror were used for monochromatization and horizontal focusing, providing 2 X lo9 photons/s down a 0.2-mm collimator at 2.0 GeV and 200-300 d of electron beam current. A Keele flat-plate camera was used with an areal detector modified to act as a linear detector, constructed at Daresbury. A dead time between data-acquisition frames was 50 ps. Patterns were calibrated by using Teflon (0.48 nm) as a ~ t a n d a r d . ' ~All mesophase and subcell spacings were calculated according to Bragg's Law.*O All analysis was done with the OTOKO program available at the Daresbury Laboratory. Temperature jumps and scans were produced by water baths connected internally to the sample mount of the X-ray camera. The temperature of the sample was monitored internally by a thermocouple placed adjacent to the sample in the X-ray sample holder. Results and Discussion The thermal characteristics of L-dipalmitoylphosphatidylcholine dispersed in 80% (v/w) ethylammonium nitrate and equilibrated at 0 O C for over 4 days are shown in Figure 1. This figure represents consecutive differential scanning calorimetric thermograms recorded at a scan rate of 10 "C-min-'. The initial heating of the sample produced a single thermogram with an onset temperature of -58 "C. The subsequent cooling also indicated only a single thermogram with an onset temperature, however, of -47 OC. It can be inferred from comparison with our previous (17) Nave, C.; Helliwell, J. R.; Moore, P. R.; Thompson, A. W.; Worgan, J. S.; Greenall, R. J.; Miller, A,; Burley, S. K.; Bradshaw, J.; Pigram, W. J.; Fuller, W.; Siddons, D. P.; Deutsch, M.; Tregear, R. T. J . Appl. Crystallogr. 1985, 18, 396. (18) Helliwell, J. R.; Greenough, T. J.; Carr, P. D.; Rule, S. A.; Moore, P. A.; Thompson, A. W.; Worgan, J. S. J . Phys. E 1982, 15, 1363. (19) Burns, C. W.; Howell, E. R. Nature (London) 1954, 174, 549. (20) Levine, Y . K. Prog. Surf.Sci. 1972, 3, 279.
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TEMPERATUREVK ) Figure 1. Differential scanning calorimetric thermogram of a multilamellar dispersion of L-dipalmitoylphosphatidylcholinein ethylammonium nitrate stored for 4 days at 0 OC: (a) first heating at a scan rate of 10 OC/min; (b) cooling at a scan rate of 10 OC/min; and ic) reheating at a scan rate of 10 OC/min.
S (r") Figure 2. Three-dimensionalplot of X-ray scattering intensity vs. reciprocal spacing as a function of temperature for a sample of L-dipalmitoylphosphatidylcholine dispersed in ethylammonium nitrate equilibrated for 4 days at 0 OC. The sample was heated from 15 OC at a rate of 10 OCmin-'. Diffraction patterns were accumulated over 1.7 s. Every tenth recorded diffraction pattern is shown.
time-resolved X-ray diffraction examination of phase transitions involving DPPC in waterlo that the supercooling observed during this initial cooling cycle is due to the nucleation of a gel phase in lieu of the initial subgel phase. In fact, subsequent reheating scans show two endothermic transitions with onset temperatures of -45 and -55 OC separated by an exothermic transition at -50 "C. Only when the sample is allowed to equilibrate above the transition temperature for approximately 1 h does one observe a reduction of the -55 "C endotherm, an elimination of the exothermic transition, and an increase in the -45 OC endotherm. Equilibration of the sample at room temperature or below for an extended period (days) allows the subgel phase to reform. A complete calorimetric study of the effect of varying amounts of EAN on phosphatidylcholines and phosphatidylethanolamines is currently under way.
The Journal of Physical Chemistry, Vol. 91, No. 17, 1987 4627
Lipid Phase Transition of DPPC in Fused Salt
An examination of the individual frames within our data set indicates that a single phase transition is observed at -57 OC. During this transformation, the mesophase acyl chain subcell changes continuously with the 0.487-nm diffraction peak shifting to 0.494 nm and the 0.754-nm peak broadening unit it completely disappears. The disordering of the subcell axis represented by the 0.754-nm peak and the change in the a, axis defined in part by the 0.494-nm peak is characteristic of a weakly first-order or second-order transition.22
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Figure 3. Plot of representative X-ray scattering profiles from L-dipalmitoylphosphatidylcholine dispersed in excess ethylammonium nitrate during first heating through the main phase transition at 10 OC-min-l.
The structural changes that occur upon the initial heating at 10 OGmin-l of a dispersion of L-dipalmitoylphosphatidylcholine in 80% (v/w) EAN after equilibration at 0 O C for over 4 days are shown in Figure 2. Diffraction patterns representing every tenth frame of a total of 255 frames recorded at 1.7 s/frame is shown as calibrated in reciprocal units to a Teflon standard. The initial bilayer structure is determined to have a mesophase d spacing of 5.8 nm, which transforms during the single observed phase change to an hexagonal array of cylinders (H1J with a d spacing of 5.08 nm. It can be hypothesized that the dramatic difference in the gel to liquid crystal phases in the DPPC/EAN system relative to the fully hydrated DPPC systemlo is due to a change in the interactions between adjacent lipid molecules in the presence of an ionic (although polar) rather than a strictly polar solvent. The previously inferred high-temperature micellar phase for DPPC in EAN (1:l by weight) was not observed. In our sample, which contained a greater amount of EAN, a cylindrical phase was determined to be present. These observations are consistent with results from phospholipid/water systems2I which indicate that a reduction in the amount of solvent results in the preferential formation of micellar rather than cylindrical mesophases. The wide-angle diffraction peak characteristic of the acyl chain subcell was also analyzed for this scan (Figure 3). Two major peaks at 0.754 and 0.487 nm and two shoulder peaks at 0.628 and 0.542 nm were observed in the subgel state. An orthorhombic subcell for the subgel-state acyl chains can be inferred if it is assumed that the shoulder peaks are artifacts of the splining routine used to reduce the noise in our patterns. This assumption is consistent with the previous Raman studySs The diffraction peaks at 0.754 and 0.487 nm can then be assigned to the (020) and (210) planes within the subcell.1° The equivalent unit cell dimensions, a, and b,, for this subcell would be 2.38 nm and 0.97 nm, respectively. It has been previously shownI0 that the subgel phase for fully hydrated DPPC can be indexed to an orthorhombic unit cell with dimensions of a, = 1.050 nm and 6, = 0.750 nm. The acyl chain subcell structure for DPPC in EAN is thus larger than that for DPPC in water and may be due to a greater disorder in the acyl chains and/or a greater repulsion between DPPC head groups immersed in EAN. The acyl chain subcell for the DPPC liquid crystal bilayer phase in EAN is an hexagonal lattice of dimension 0.494 nm, which is again larger than that observed for DPPC bilayers in water.1° (21) Eriksson, P.-0.; Rilfors, L.; Lindblom, G.; Arvidson, G. Chem. Phys. Lipids 1985, 37, 351.
Conclusions In summary, a transition involving a subgel to liquid crystalline transition was observed for DPPC bilayers in EAN. This type of transition has not been previously observed for DPPC multilamellar arrays in water lo or even “dispersed” by a trehalose c ~ m p l e x a t i o n . ~The ~ disordering of the acyl chains forces this system to transform from the subgel bilayer structure to an HII structure without an intermediate L,, L,, or L, form. The failure of these gel-state phases to appear may be an indication that the gel-state bilayer is indeed a metastable phase occurring between the thermodynamically stable L, and L, phases only when water or perhaps polar solvents are present. The acyl chain subcells were found to be larger for DPPC in EAN when compared with DPPC in water above and below the transition temperature. This spreading of the phosphatidylcholine head groups may allow the HIIphase to be the thermodynamically stable high-temperature liquid crystal phase. The thermodynamic implications of the reduction in the lateral spreading pressure between lipid head groups has been discussed by O’Leary and L e v h 8 The calorimetric results for DPPC dispersed in EAN can now be discussed in terms of the above-mentioned structures and previous observations for DSPC’ and DPPC’ in EAN and DPPC in water.I0 On initial heating, a sample of DPPC in EAN which has been equilibrated below the main phase transition temperature undergoes a transition from the L, to.H, phase (or some other nonlamellar phase, depending on the amount of solvent in the system). The initial cooling can be interpreted to be an H, to gel-state lamellar (L,, L,, or P,) phase transition, since it is highly unlikely that the L, phase would form under these conditions; this phase usually requires a number of days at low temperature to induce its formation. It is interesting to speculate on possible structures involved in subsequent heating and cooling cycles. The two endotherms observed on reheating could be representative of the gel bilayer to L, and L, to HII phase transitions. The appearance of an exotherm after the first endotherm indicates a reordering within the acyl chain packing that would be inconsistent with this scheme. The qualitative observation that incubation of the sample above 60 OC for extended periods of time results in an increase in the first endotherm and a decrease in the second endotherm upon subsequent reheating could be interpreted to indicate an HI1to L, transition if the phase sequence evolves from gel bilayer to L, to HI,. However, these speculations can only be resolved by further time-resolved X-ray diffraction experiments. It is apparent that any intermediate phase would be the consquence of a transition into or between metastable states. Further experiments examining the influence of varying amounts of EAN or of other polar solvents of DPPC structures and transitions are necessary to determine the role of water in the stability of the L,, P,,, and La phases. Acknowledgment. This research was partially supported by a grant from the Science and Engineering Reseach Council (U.K.). (22) Landau, L. S.; Lifschitz, E. M. Sfafistical Physics; Peraamon: London, 1959. (23) Tenchov, B.; Lis, L. J.; Quinn, P. J. Biochemistry, to be submitted.
