J. Phys. Chem. 1993,97, 29462951
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31P-NMRand Calorimetric Studies of the Law-Temperature Behavior of Three 19F-Labeled Dimyristoy lphosphatidylcholines Susan R. Dowd,' J u l i ~M. Sturtevant,* Virgil Simplaceanu,? and Chien Ho'J Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 1521 3, and Department of Chemistry, Yale University, New Haven, Connecticut 06.51 1 Received: September 30, 1992; In Final Form: November 25, 1992
The thermotropic phase behavior of three difluorodimyristoylphosphatidylcholines,containing a difluoromethylene group in the 2-acyl chain a t either the 4-, 8-, or 12-position, has been studied by differential scanning calorimetry (DSC) and by 31Pnuclear magnetic resonance (NMR) spectroscopy at temperatures at and below their normal gel to liquid crystalline transition. The fluorinated lipids have been found to behave in a manner similar to their protonated counterpart with the exception of the 4,4-difluoro isomer. The 4,4-substituted isomer, 1-myristoyl-2-(4,4-difluoromyristoyl)-sn-glycero-3-phosphocholine (2-[4,4-F2]DMPC), appears by j*P N M R upon cooling to 10 deg below the main (Pf to La)phase transition to rapidly achieve a crystalline subgel phase. The same anomalous behavior of rapid conversion to the crystalline (L,) phase is seen by DSC upon cooling the 2-[4,4-F2]DMPC lipid to 5 OC. By both DSC and NMR, the few hours taken for conversion of the 2-[4,4-F2]DMPC to a solid phase is unusually short when compared either to the other fluorolipids or to unsubstituted saturated straight-chain diacylphosphatidylcholines,which generally take a day or more for conversion to their solid forms.
Introduction The use of selective I9F-labeling coupled with 19F-NMR spectroscopy offers a number of advantages in investigating the structural properties and dynamics of phospholipids dispersed as sonicated vesicles, multilayered liposomes, or oriented bilayers. By using a variety of solution and solid-state NMR techniques such as multipulse and spin-lattice relaxation in both the laboratory and rotating frame, we have gained information regarding the structure and dynamic properties of a variety of 19F-substituted DMPCs as well as their interactions with cholesterol and The thermotropic behavior of 1,2-bis(difluoromyristoyl)phosphatidylcholinescontaining two fluorine atoms in either the 4-, 8-, or 12-position of both acyl chains as observed by DSC has previously been reported by our laboratorie~.~.~ The isomer with fluorines in the 4-position of the acyl chains has a higher main transition temperature than dimyristoylphosphatidylcholine (DMPC) itself and the transition shows a pronounced hysteresis. The 1,2-di(8,8-Fz)-and 1,2-di(12,12-F2)DMPClipids have main transition temperatures below that of the parent compound and appear to have normal reversible thermotropic behavior. All these three fluorinated lipids have transition enthalpies approximately twice that of DMPC. In order to define further the thermotropic phase behavior of three additional fluorinated DMPC lipids, which contain a difluoromethylene group only in the 2-acyl chain at either the 4-, 8-, or 12-position, we have carried out studies by DSC and by 31P-NMR spectroscopy. Attempts to characterize the lowtemperature transitions of the fluorolipids by 19F-NMR spectroscopy were not informativefor reasons detailed in the Appendix.
Experimental Section Materials. The preparation of l-myristoyl-2-(4,4-difluoromyristoyl)-sn-glycerol-3-phosphocholine(2- [4,4-F2]DMPC), 1-myristoyl-2-(8,8-difluoromyristoyl)-sn-glycerol-3-phosphocholine (2-[8,8-F2]DMPC), and l-myristoyl-2-(12,lZdifluoromyristoyl)-sn-glycerol-3-phosphocholine (2- [ 12,12-F2]DMPC) has To whom correspondence should be addressed. Carnegie Mellon University. Yale University.
0022-3654/93/2097-2946$04.00/0
been described previously.* Thin-layer chromatographyindicated the purity of the lipids to be at least 98%. Preparation of Samples. Samples of lipid dispersed in excess H20 were prepared by vortexing lipid and water together vigorously, in appropriate ratios, at approximately 40-50 OC, until a stable milky white dispersion was achieved. Differential!hnnhg Calorimetry. All calorimetricscans were made on a Model DASM-1M instrument9 with a lipid concentration equal to 1 mg mL-' and a scan rate of 0.25 K min-I. In cases where the sample was cooled between scans, the cooling rate was the rate characteristic for the DASM-lM, roughly an hour being required to cool from 30 to 5 OC. NMR Measurements. The "P-NMR spectra were acquired on a Bruker AM-300 spectrometer operating at 121.5 MHz for 31P. The samples for the NMR were prepared from 50-80 mg of lipid dispersed in 0.4 mL of HzO and spectra were typically taken with a spectral width of 62.5 KHz and 3000 scans. A pulse width of 6 ~s (-4OO rotation angle), a relaxation delay of 1 s, and full-power proton decoupling (10-12 W) were employed. The temperature was controlled by using a Bruker variabletemperature unit.
Results and Discussion Phosphatidylcholine lipids are known to go through several phase transitions below their main transition temperature ( Tm). For example, dipalmitoylphosphatidylcholine (DPPC) shows several low-temperaturetransitions, which have been well-studied and characterized by a variety of means, including X-ray diffraction,lOJ1DSC,1z-15time-resolved X-ray d i f f r a ~ t i o n , ~ ~ , ' ~ Fourier-transform infrared (FT-IR) spectroscopy,l*J9and I3Cand 3IP-NMR spectroscopy.20-21 These studies have shown that DPPC has two transitions below its main transition temperature (T, = 41 "C), the first around 34 OC, the pretransition (Tprc), and the second around 18 OC, the subtransition (Tsub). For increasing temperature, the pretransition involves a transition from a lamellar gel to a gel state (Lp to Pp) characterized by an increase in the acyl chain and headgroup mobility. The subtransition first observed by Sturtevant and co-workersI2using DSC involves a transition from the lowest temperaturecrystalline state to the lamellar gel state (L, Lp). The L p to P.y transition does not appear to involve a loss or gain in the hydration of the
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0 1993 American Chemical Society
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 2941
Behavior of I9F-Labeled Dimyristoylphosphatidylcholines
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TemperaturePC Figure 1. Main phase transition of 2-[12,12-F2]DMPC(curve A) and of 2-[8,8-F2]DMPC(curve B) observed by DSC at a lipid concentration of 1 mg mL-l and a scan rate of 0.25 K m i d . The solid lipids were suspended in water by vortexing at 40-50 O C and were praumably present in the form of multilamellar vesicles. The dashed curves (- -) show the best fit to the observed data (solid curves, -) obtained on the basis of a first-orderphase transition" of a lipidcontaminatedwith a small amount of water-insoluble impurity forming ideal solutions in both lipid phases.2s
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lipid, while both the main transition and the subtransition do involve changes in the number of water molecules around the lipid headgroup.' I R u m and Shipleyll observed by X-ray diffraction that, in the initial 2-12-h portion of the cooling period needed to achieve the subgel phase, the bilayer periodicity slowly shrinksto a limiting value. At the limiting value, only the water molecules tightly bound to the polar lipid interface remain in the subgel phase (estimated at 11 water molecules per lipid). Each of the techniques used to study the subgel state and its formation on cooling from the Lg' phase has indicated that achieving the crystalline form is a slow process. Using DSC, reports by Wu et a1.,l3Silvius et aLZzand Tristram-Nagle et al.I4 all indicate that achieving the subgel state is incubation time dependent for DPPC and that the two kinetic forms present initially convert over long storage times to a single form. Lewis and McElhaneyI9 have observed even further changes by FT-IR when the lipid is held at low temperatures for extended periods. Earlier studies from one of our laboratories using 19F-NMR line-shapeanalysisaand rotating-frame relaxation measurementsz3 have shown 2-[ 12,12-Fz]DMPC, 2-[8,8-Fz]DMPC, and 2-[4,4FzlDMPC to be sensitive probes of model membrane bilayers without causing undue perturbations from the presence of the fluorine labels. In order to study further the physical properties of these lipids, DSC and 3'P-NMR spectroscopy have been used to examine the behavior of the phosphorus headgroup and the phase behavior of the flourinated lipids at temperatures around and below their main transitions. The 2-[4,4-F2]DMPC shows more complex thermotropic behavior than either the 8,8-F2 or 12,12-F2 isomer as observed by both techniques.
DSC Measurements Figure 1 shows the main phase transition for 2-[8,8-F2]DMPC (13.5 "C, curve 9 ) and 2-[12,12-Fz]DMPC (12.9 "C, curve A). In each case, the transition occurs well below the temperature at which the unsubstituted lipid melts (23.9 "C). In the figure, the solid curves show the observed data and the dashed CUNW the data calculated on the basis of a first-order phase transitionz4of a lipid contaminated with a small amount of water-insoluble forming ideal solutions in both of the lipid phases. Table I lists the parameters that give the best fits and for comparison the parameters obtained with a suspension of DMPC. Each lipid showsdeviationsof the best-fit curve from the observed curve in the regions of the tails of the transitions. This type of
deviation is always observed with lipids, due presumably to a distribution of cooperative unit sizes, including some material having small cooperative units with correspondingly broadened transitions. In addition, curve B for the 2-[8,8-F2]DMPC shows an extremely broad high-temperature tail of unkown origin, which was observed with two independent preparations of the lipid. The 2- [4,4-Fz]DMPC behaves in a totally unexpected manner, It appears in our DSC studies to have two mutually exclusive transitions, both of which yield on heating the liquid crystalline form of the lipid. One of those transitions (curve A in Figure 2) is very sharp and is centered at 18.4 "C. It is exhibited by lipid which has been cooled from temperatures above 30 OC to 12-16 "C. The other peak (curve B in Figure 2) is much broader, asymmetric and centered at about 24.6 "C. It is shown by lipid that has been cooled from above 30 "C to 5 "C or lower. Cooling to temperatures between 5 and 12 "C leads to both transitions with diminished enthalpies, as shown in Figure 3. The lower transition is reversible in the sense that it is again seen with lipid cooled to 12-16 "C, whereas the upper transition can be reversed only by cooling to 5 "C or lower. We have seen no significant effects due to the length of time that the lipid was held in either cooling range. The dashed curves in Figure 2 are the results of fitting the experimental data to theoretical curves. The model used for curve A is the same as that used for the 8,8- and 12,124somers and leads to the parameters given in Table I. The pronounced asymmetry of curve B would require the assumption of a totally unreasonable level of impurity in this model. The dashed curves in B are obtained by assuming a process involving independent two-state transitions for two different solid phases, with the parameters listed in Table I. It should be noted that this fit to the observed data is consistent with, but does not prove, the existenceof two solid phases. For example, a similar result could be obtained if a single phase undergoes a transition consisting of two independent or sequential two-state steps. The transition at 18.4 "C (curve A in Figure 2) is presumably the Pg' to L,, or main, phase transition, with the small shoulder at 18 "C, the Lg' to Pg' transition, or pretransition. Since the transition at 24.6 "C (curve B in Figure 2) involves a phase that is formed on cooling to a relatively low temperature, it is assumed that this is an L, to L, transition, or subtransition, although it occurs at a higher temperature than the main transition. A similar occurrence of the subtransition at a temperature above that of the main transition has been reported for dilauroylphosphatidylethanolamine.2"z8 A most unusual feature of the phase giving the 24.6 "C transition, if it is indeed the subgel phase, is that it is completely formed within the time required to cool the suspension in the calorimeter, namely, 30-60 min, whereas with other lipids for which the subgel phase has been observed, a lowtemperature period of hours or days is required.
31P-NMRStudie~ The 3'P-NMR spectra obtained at 121.5 MHz for fully hydrated samples of DMPC, 2-[ 12,12-F2]DMPC, 2-[8,8-F& DMPC, and 2-[4,4-Fz]DMPC dispersed as multilamellar liposomes as a function of temperature are illustrated in Figure 4. Samples were cooled from above their highest transition temperatures to around 10 deg below that temperature. The manner in which the resonance lifie shapes for three of the four lipids broaden as the temperatures decreases is very similar. Presumably the broadening is due to a decrease in motion without large changes in the conformation of the phosphorus-containingheadgroup. As discussed in the Appendix, I9F-NMR spectra of the three fluorolipidsobtained at temperatures below their phase transitions show only broadened humps, which are uninformative. By IlPNMR spectroscopy, which is sensitive to the environment of the phosphorus-containing headgroup, the 2-[4,4-F~] DMPC is seen to have undergone definite changes. The striking difference
Dowd et al.
The Journal of Physical Chemistry, Vol. 97, No. 12, 1993
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TABLE I: Panmeters Obtained in Fitting Phase Transition Data for DMPC and F-Labeled DMPCs Ah-!, cal mol-'
T m , "C 23.90 13.47 12.94 18.40 24.20, 24.78
lipid DMPC 2- [8,8-F2]DMPC 2-[ 12,12-Fz]DMPC 2-[4,4-F2]DMPC (curve A) 2-[4,4-F2]DMPC (curve B, components 1,2)
curve fitting
planimeter
A H V ~kcal , mol-'
5.64 5.27 5.41 6.13 9.18, 12.68
7.18 7.24 8.45 7.57 22.13e
2730 1020 2200 2000 530,850
Kb
X2: mol fractn 0.008 0.010 0.0018 0.005
0.885 0.956 0.957 1.057
*Total mole fraction of impurity in the lipid. Ratio of mole fractions of impurity in gel and liquid crystal phases. Total enthalpy. 20
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18
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20 22 Temperature ('C)
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24
26
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Figure 4. Temperature dependence of 121.4-MHz "P-NMR spectra of phospholipidsdispened in H20 (3070 w/w): (A) DMPC; (B) 2412.12FzIDMPC; (C) 2-[8,8-F2]DMPC;(D) 2-[4,4-F2]DMPC. Samplcs were placed in the magnet at a temperature above the liquid crystalline phasetransition temperature and slowly cooled successively to the indicated temperatures (in "C). Spectra were acquired with full-power proton decoupling and are scaled to the same height for purposes of illustration.
.......... I
2.5
5.0
7.5 10.0 Temperature ('C)
-tm
0
PPY
Figure 2. DSC traces of the two mutually exclusive transitions shown by 2-[4,4-Fz]DMPC. The sharp transition (curve A) was observed with lipid cooled from above 30 "C to 12-16 "C and is presumably the main phase transition with the low-temperature shoulder being thepretransition. The broad transition (curve B) is observed with lipid cooled from above 30 OC to below 5 "C and appears to be the subtransition normally observed at temperatures below the main transition. The dashed curve (- -) is a fit to the observed data by using the same model as used for curve fitting in Figure 1. Curve B is too broad to be tit to such a model; in this case a fit to two independent two-state transitions was obtained. 25
-
Im
12.5
15.0
~
7 -
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I 0
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-100
Figure 3. Enthalpies, determined by planimeter integration, of the two transitions shown by DSC for 2-[4,4-FzlDMPC as a function of the temperature to which the lipid is cooled after being heated to 30 "C. The enthalpy of the sharp, low-temperature transition has been multiplied by four. As indicated, if the lipid is cooled to temperatures within the range 5-12 "C, both transitions are seen with diminished enthalpies.
PPM Figures. 121.4-MHz 3'P-NMRspectrumat 12 "Cof2-[4,4-F2]DMPC dispersed in H20 (38:62 w/w) after incubation at 12 OC for 2 weeks. The relatively sharp peak centered at 0 ppm is assumed to arise from small micelles present in the sample. Spectra were acquired with full-power proton decoupling: experimental (solid line) and theoretical asymmetric powder pattern (dotted line).
observed for spectra taken between 19 and 14 OC reflects a change from an axially symmetric powder pattern to one approaching an axially asymmetric pattern. The fluorolipid seems to have achieved the line shape characteristic of an anhydrous solid at a temperature rather close to its T,. In an effort to investigate more closely the behavior of the 2-[4,4-Fz]DMPC around its lowest transition, a hydrated multilayer sample was stored at 12 OC for 2 weeks and then placed in the magnet at 12 OC. The resulting spectrum is shown in Figure 5 . Except for the sharp peak centered at 0 ppm, which probably arises from micelles trapped in the sample, the observed spectrum coincides well with the theoretical asymmetric powder
pattern (dotted line) spectrum of an anhydrous solid, consistent with the L, phase, and is identical with that seen at 14 OC in Figure 4D. The 2-[4,4-F2]DMPC sample was warmed to 15 OC and, after 30 min, a second spectrum was taken over a 50-min period. The spectrum at 15 OC (results not shown) is identical with the 12 OC spectrum. Further warming to 18 (results not shown) and 21 OC gives rise to spectra that appear to show a mixture of a solid and a gel or liquid-crystalline phase (Figure 6). The portion characteristic of the L, phase slowly increases with increasing temperature until 24 O C , where only it remains (Figure 7). The DSC measurements also show that a phase transition occurs at 24.6 OC.
Behavior of I9F-Labeled Dimyristoylphosphatidylcholines
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The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 2949
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F i w e 6 . 121.4-MHz3'P-NMRspectrumof2-[4,4-F2]DMPCdispersed in H20 (38:62 w/w) after warming to 21 OC from 12 ' C . The spectrum was acquired with full-power proton decoupling.
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F i p e 7 . 121.4-MHz31P-NMRspectrumof 2-[4,4-Fz]DMPCdispcrsed in H20 (38:62 w/w) after warming to 24 O C from 21 'C. The spectrum was acquired with full-power proton decoupling.
As previously mentioned, in the subgel or crystalline solid state, the phosphate headgroup of DPPC appears to have no more than 11 water molecules bound.I0J1 In the La phase, the headgroup has been determined to have at least 25 water molecules bound." The results for the 2- [4,4-F2]DMPC imply that, over a very short temperature range (from 15 to 24 "C), the phosphate headgroup becomes completely mobile and fully hydrated. That the transition to the Laphase occurs during the time period (30 min) for temperature equilibration is not unexpected in view of the finding by Tenchov et and discussed by Caffrey'O that as observed by synchrotron X-ray diffraction less than 10 s is needed for complete hydrationa2*The 2-[4,4-F2]DMPC, however, is the only one of the straight-chain fluorinated isomers studied whose subgel phase appears to occur so close to its main transition. Figure 7 shows the spectrum of 2-[4,4-F2]DMPC dispersed in H 2 0 at 24 "C, which illustrates the axially symmetric powder pattern line shape typically found by 3IP N M R for a phospholipid dispersed in H20 as multilayer liposomes in the liquid crystalline state. In order to study the time course of the conversion of 2-[4,4-F2]DMPC in H20 from the Lato the subgel state and to duplicate the conditions used in the DSC experiments, a sample of lipid in H20 at 27 "C was placed in the NMR probe precooled to 12 "C. After an initial spectrum, four spectra were acquired with a 30-min waiting period between sets and an accumulation time of 17 min. As seen in Figure 8A, the beginnings of the conversion to a broader line component can be discerned in the
1
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-1 00
Figure 8. Time course of the 121.4-MHz "P-NMR spectra of 2-[4,4F21DMPC dispersed in H20 (38:62 w/w) acquired while cooling from 27 O C to 21 O C . The sample at 27 OC as placed in the magnet precooled to 12 O C . Spectra were accumulated with full-power proton decoupling over a 17-min period with a 30-min waiting period between subsequent acquisitions. Total time elapsed: (A) 20 min; (e) 47 min; (C) 74 min; (D) 101 min; (E) 118 min. Spectra are scaled to the same height for purposcs of illustration.
lower portion of the spectrum. Within a period of 2.5 h, the conversion to a spectrum (Figure 8E)identical with that shown in Figure 5 seems to be complete. Again, a sharp component centered at 0 ppm, which probably arises from micelles trapped in the sample, is apparent. The cause of the rapid reorganization and dehydration of the lipid headgroup and, by extension, packing of the acyl chains cannot be readily determined. There are at least two possible explanations for the behavior observed. The
2950 The Journal of Physical Chemistry, Vol. 97, No. 12, 1993
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Figure 9. Temperature dependence of 282.4-MHz I9F-NMR spectra of 2-[4,4-F2]DMPC dispersed in H20 (31:69 w/w), stored at 14 'C and warmed successively to (B) 19 'C and (A) 27 'C.
two fluorine atoms in 2-[4,4-F2]DMPC may perturb the acyl chains in such a way as to lead more readily to the all-trans acyl chain configuration, which may be necessary for the closest packing of the chains in the crystalline state, than is possible for the other fluorolipids or for DMPC itself. Alternatively, the presence of two fluorine atoms with van der Waals radii that are 12.5%larger than that of a hydrogen atom may perturb the acyl chain packing in such a manner that the lipid headgroups more readily align to achieve the crystalline state. The present results do not allow us to discriminate between the various possibilities. Previous work using I9F-NMR spectroscopy at temperatures at and above Tmfailed to reveal any anomalous behavior for the 2-[4,4-F2]DMPC lipid while in the La phase.31 A number of unsuccessful attempts have been made to detect a difference between the "P-NMR spectra taken of a sample stored at 12 "C and one that had been stored for months at -10 OCover thecourseofwarming toabove27 OC (resultsnot shown). No detectable difference could be observed. Unlike the results obtained by DSC, which are sensitive to overall structural changes in the whole lipid, no suggestion of two different forms for the crystalline state is detected by 31PNMR, which reports only on the environment of the headgroupphosphorus. Though additional changes may be taking place upon long-term storage at low temperature, either 31P-NMRspectroscopyis not sensitiveenough to observe them or the changes occur in the packing of the acyl chains without affecting the motions of the headgroup. As noted earlier, the formation of the subgel or crystalline state has been shown to be a complex, slow kinetic process for saturated, straight-chain phosphatidyl~holines.~~J~ DSC and 31P NMR studies that include results for methyl-branched anteisoacylphosphatidylcholines containing odd-numbered carbon chains have been reported by Lewis et al.32 Some of the anteisoacylphosphatidylcholine lipids were found to form a 'condensed" phase similar to the subgel phase of saturated linearchain phosphatidylcholines, but achieved far more rapidly. Specifically, holding 1,2-bis( 14-methylpalmitoy1)phosphatidylcholine at 0 OC for 30 min gave rise to a 3lP-NMR resonance line shape typical of slow axially asymmetric motion, indicative of the formation of a solid. The width of the axially asymmetric spectral line was 150 ppm, compared to the 200 ppm width normally seen for the phosphate group of DPPC at very low t e m p e r a t ~ r e s . ~Lewis ~ et al.ls have suggested that, in the gel phase ofthe chain lipids, greater mobility in the phosphate containing headgroups and looser packing for the acyl chains occurs than for the straight-chain
Dowd et al. lipids. By analogy, the fact that 2-[4,4-Fz]DMPC has a baseline 3'P-NMR linewidth of 180 ppm at 14 OC suggests that the full rigid-lattice limit has almost been reached for the fluorinated lipid within 10 deg of its main transition temperature. The indication of two phases coexisting in the subgel lipid, as seen by DSC (Figure 3), is also consistent with the findings for the methylbranched chain lipids. In summary, by DSC and 31P-NMRspectroscopy two of the fluorinated lipids, 2-[8,8-Fz]DMPC and 2-[ 12,12-F2]DMPC, exhibit phase behavior that is analogous to that shown by DMPC itself. The substitution of a difluoromethylene group for the methylene group at the 4-position in the 2-acyl chain of DMPC leads to anomalous behavior on cooling. The DSC results indicate that the calorimetric transitions of the lipid are dependent on its cooling history. Calorimetry measures the system as a whole, while 31P-NMRspectroscopy can only observe the interactions of the 31P-containingheadgroup with its surroundings. As noted before, at least two possibilities exist to explain the results found. The first is that the CF2 group may facilitate a rapid conversion to the all-trans configuration that could be necessary to achieve the best packing of the acyl chains and thesubsequent dehydration of the headgroup for the transition to the L, phase. The second is that the bulkier CF2 group perturbs the packing of the acyl chain causing the headgroups to be constrained to a conformation that is closer to that of the crystalline state than for other fluoro or straight-chain lipids. The DSC measurements may be indicating that additionalrearrangementsin the acyl chain packing have to occur before a "true" L, phase is achieved.
Acknowledgment. We express our admiration to Dr. Harden M. McConnell for his outstanding contributions to science. In particular, his pioneering research in using spectroscopicprobes to investigate structural and dynamic properties of lipids and proteins has had a major impact on this paper. We thank Dr. S. Tristram-Nagle of Carnegie Mellon University for her suggestion that the observed transition at 24.6 OC could be the subtransition. This research was supported in part by research grants from the National Institutes of Health (GM-04725 to J.M.S. and GM-26874 to C.H.) and the National Science Foundation (DMS 8810329 to J.M.S.). A P w x The results obtained for the fluorolipids when 19F-NMR spectroscopy was used to examine their behavior at temperatures below their main transition temperatures are illustrated by Figure 9. This figure shows the 19F-NMR spectra taken of a fully hydrated sample of 2-[4,4-Fz]DMPC warmed slowly in the magnet from 14 to 27 OC, where (B) is obtained at 19 OC and (A) at 27 OC. Since all three fluorolipids exhibited similar line shapes (broad humps), which are not conducive to useful interpretation, examination of the behavior of the 2-acyl chain by I9F NMR was not pursued. The 19F-NMR spectra were acquired as described previou~ly.~~
Notes
References
Bio,.(1) Chem. Oldfield, 1980,E.; 255, Lee,1652-1 R. W. 1655. K.; Meadows, M.;Dowd, S.R.; Ho. C. J. (2) Ho, C.; Dowd, S.R.; Poet, J. F. M. Curr. Top. Bianerg. 1985,14, 53-95. (3) Wu. W.-G.; Dowd. S. R.; Simplawnu, V.; Peng, Z.-y.; Ho, C. Bioe*emisrry 1985* 2g' 7153-7161. (4) Peng, Z.-y.;Simplaceanu, V.; Dowd, S.R.; Ho, C. Proc. Natl. Acad. sei. U.S,A 1989.86.8758-8762. (5) Peng, Z:-y.;'Tjandra, N.; Simplaceanu, V.; Ho. C. Blophys. J. 1989,
569877-885. ( 6 ) Sturtevant, J. M.; Ho, C.; Reiman, A. h o c . Natl. Acad. Sci. U.S.A. 1979. 76., ~~2239-2243. (7) A reviewer of this paper made the interesting suggcation that the ~~
~
1,2-bis(difluoromyristoyl)phosphatidylcholinesstudied in an earlier pap+ may have k e n forming the LS phase instead of the Lr phase assumed at that time. It may be noted that our 1979 paper6preceded by 1 year the discovery12 of the subtransition. We hope to explore this matter further at a latter date.
Behavior of 19F-Labeled Dimyristoylphosphatidylcholines (8) Engelsberg, M.; Dowd, S.R.;Simplaceanu, V.; Ho, C. Biochemistry 1982,21,69854989. (9) Privalov, P. L.; Plotniknov, V. V.; Filimonov, V. V. J. Chem. Thermodyn. 1975. 7, 4147. (IO) Ruocco, M. J.; Shipley, G. G. Biochim. Biophys. Acto 1982, 684, 59-66. (11) Ruocco, M. J.; Shipley, G. G. Biochim. Biophys. Acto 1982, 691, 309-320. ... ._.
(12) Chen, S.C.; Sturtevant, J. M.;Gaffney, B. J. Proc. Nod. Acod.Sci. U.S.A. 1980, 77, 5060-5063. (13) Wu, W.; Chong, P. L.-G.; Huang, C. Biophys. J . 1985,47,237-242. (14) Tristram-Nagle, S.;Wiener, M. C.; Yang, - C. P.; Nagle, - J. F. Bi&hemistry 1987, 23, 4288-4294. (1 5 ) Lewis, R.N. A. H.; Mak, N.; McElhaney, R. N. Biochemistry 1987, 26,6118-6126. (16) Tenchov, B. G.; Yao. H.; Hatta, I. Biophys. J. 1989.56,757-768. (17) Caffrey, M.; Fanger, G.; Magin, R.L.; Zhang, J. Biophys. J . 1990, 58, 677486. (18) Cameron, D. B.; Mantsch, H. H. Biophys. J . 1982, 38, 175-184. (19) Lewis, R.N. A. H.; McElhaney, R.N. Biochemistry 1990,29,79467953. (20) Fiildner, H. H. Biochemistry 1981, 20, 5707-5710. (21) Lewis, B. A.; Das Gupta, S.K.;Griffin, R.G. Biochemstry 1984,23, 1988-1993.
The Journal of Physical Chemistry, Vo1. 97, No. 12, 1993 2951 (22) Silvius. J. R.;Lyons, M.; Yeagle, P. L.; OLeary, T. J. Biochemistry 1985.24, 5388-5395. (23) Peng. Z.-y.; Simplaceanu, V.; Lowe, I. J.; Ho, C. Biophys. J . 1988, 54.81-95. (24) Albon, N.; Sturtevant, J. M. Proc. Nod. Acod. Sci. U.S.A.1978.75, 2258-2260. (25) Sturtevant. J. M. Proc. Nod. Acod. Sei. U.S.A. 1982, 79, 39633967. (26) Willtinson, D. A.; Nagle, J. F. Biochemistry 1981, 20, 187-192. (27) Chang, H.; Epand, R. M. Biochim. Biophys. Acto 1983, 728,319324. (28) Mantsch, H. H.; Hsi, S.C.; Butler, K. W.; Cameron, D. G. Biochim. Biophys. Acto 1983, 728, 325-330. (29) Tenchov, B. G.; Lis, L. J.; Quinn, P. J. Biochim. Biophys. Acto 1987, 897, 143-151. (30) Caffrey, M.Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 159186. (31) Dowd. S.R.;Simplaceanu, V.;Ho, C. Biochemistry 1984,23,61426146. (32) Lewis, R.N. A. H.; Sykes, B. D.; McElhaney, R.N. Biochemistry 1987.26.4036-4044. (33) Campbell, R. F.; Meirovitch, E.; Freed, J. H.J . Phys. Chem. 1979, 83, 525-533.
