J. Phys. Chem. 1992, 96,6849-6852
6849
Electron Spin Resonance Line Shape Analysis of x-Doxylstearlc Acid Spin Probes in Dioctadecyldimethyiammonlum Chloride Vesicles Peter J. Bratt and Larry Kevan+ Department of Chemistry, University of Houston, Houston, Texas 77204-5641 (Received: April 15, 1992)
Electron spin resonance spectra of a series of x-doxylstearic acids (x = 5, 7, 10, 12, and 16) solubilized in cationic dioctadecyldimethylammoniumchloride vesicles were recorded over 298-328 K and analyzed by employing spectral line simulation. The spectra from the vesicle phase are well reproduced by using the microscopic order-macroscopic disorder model of Freed and co-workers with Brownian rotational diffusion. The partial averaging of the magnetic interactions by local anisotropic motions in the vesicles is quantified mainly by an order parameter (S) and the rotational diffusion rate perpendicular to the alkyl chain (R,).The simulations show that the doxy1 position becomes progressively less ordered (S decreases) as x increases up to x = 12 but becomes more ordered at x = 16. This suggests a U-shaped bent conformation of the doxylstearic acid alkyl chain. R , shows a similar, but inverse, correlation; that is, R , increases, plateaus, and decreases with increasing x. The U-shaped conformation of the spin probe alkyl chain in liquid vesicle solutions is the same as deduced previously in frozen vesicle solutions measured by electron spin echo modulation spectroscopy.
Introduction Biological membranes are of fundamental importance in the biochemistry of living systems as they provide suitable microenvironments for the controlled transport of solutes. Biological membranes are a highly anisotropic medium composed of regularly packed amphiphilic molecules with a polar headgroup and a hydrophobic tail. In an aqueous environment these molecules form bilayers where the packing of the constituent molecules is determined by the lipid headgroupwater interactions, the intramolecular interactions of the alkyl chains, and the geometrical shape of the molecule. The complexities of natural membranes suggest the use of model systems, and synthetic surfactant vesicles are believed to be the simplest functional membrane model.’ Such model systems have been used to investigate the storage of light energy by photoinduced charge separation of solutes,14 where the photoefficiency observed is in part dependent upon the internal bilayer structure of the vesicle. One of the best characterized vesicle systems is that composed of dioctadecyldimethylammonium chloride (DODAC) molecules.’ C18H37\ C18H37’
+,CH3C1-
N
‘CH3
DODAC
Considerable insight into the behavior of membranes has been obtained from electron spin resonance (ESR) studies. Because the membrane is not itself paramagnetic, it is necessary to dope the membrane with a suitable paramagnetic probe molecule. Stable nitroxide radicals have been widely used as spin probes in studies of biological membranes and membrane mimetic syst e m ~ . ~Among -~ others, the x-doxylstearic acids of the general formula shown below have turned out to be particularly useful, for they are sparingly soluble in water but can be readily incorporated into heterogeneous aqueous systems using amphiphilic molecules.8 HOOC(CH~)~-Z--C-(CHZ),~-FH~ 0’ ‘NO
actions. More realistic correlation times are obtained by splitting the molecular motion into fast tumbling and slow tumbling components where the slow tumbling component is characterized by an order parameter which quantifies the local averaging of the anisotropic interactions.18-*’ However, a careful consideration of how the motional dynamics affects the ESR line shape suggests that a singltcomponent model can account for the ESR line shape in detai1.22-z6 ESR spectra obtained from amphiphilic spin probes in micelles and lipid bilayers are typically in the intermediate motional regime where the motions are on the time scale of the modulated interaction, with the spectral line shape arising from both static and dynamic effect^.^ In such cases the evaluation of the molecular correlation times by a full line shape analysis is necessary. Recently, a suite of programs written by Schneider and Freed has been made available for the personal computerz7 suitable for simulating ESR spectra under these conditions. In a previous study28electron spin echo modulation (ESEM) spectrcwcopy was used to observe the interaction of x-doxylstearic acids (where x = 5,7,10,12, and 16) with deuterium in deuterated water at the vesicle interface in frozen solutions of cationic dioctadecyldimethylammonium chloride (DODAC) vesicles. The deuterium modulation depth is inversely related to the average interaction distance between the unpaired electron spin and deuterium in D20. It was observed that the modulation depth versus x-doxy1 shows a decrease with increasing x, a minimum near x = 10-12, and then a substantial increase for x = 16.28This can be interpreted in terms of a U-shaped bending of the stearic acid alkyl chain. The observation of ESEM requires a frozen solution so that the anisotropic hyperfine interactions are not averaged to zero. In this work we have used ESR line shape analysis of x-doxylstearic acid spin probes in cationic DODAC vesicles in the liquid state to analyze the motional freedom of the x-doxy1 group versus x and have related the degree of motional freedom to relative locations of the x-doxy1 group from the interface. This allows a comparison of differential x-doxyl positions in frozen and liquid solutions. Interestingly, the trends in the differential positions are the same. ExperimentrrlSection
x-doxylslearic acid
Considerable insight into the motional dynamics of amphiphilic systems can be obtained from several magnetic resonance techniques. One such technique is the use of amphiphilic ESR-active spin probesei6 which “mimic” the motional behavior of the surrounding environment. If the correlation time is calculated using a simple isotropic model,17 the value obtained is often unrealistically due to local averaging of the anisotropic interOQ22-3654/92/2096-6849%03.00/0
Dioctadecyldimethylammonium chloride was prepared from dioctadecyldmethylammonium bromide purchased from Aldrich Chemical Co. and ion-exchanged as described previo~sly.~~ The x-doxylstearic acids (x-DSA) were purchased from Sigma Chemical Co. and were used without further purification. They were stored as 0.4 mM stock solutions in chloroform at 10 OC. In order to form vesicle solutions, thin films were formed from 18 mM solutions of DODAC in chloroform. The films were then 0 1992 American Chemical Society
6850 The Journal of Physical Chemistry, Vol. 96, No. 16, 1992
sonicated in water using a Fisher Model 300 sonic dismembrator operated at 35% output power with a 4-mm microtip at 53 f 2 OC. The vesicle solutions were added to previously evaporated spin probe films and were allowed to stand for 24 h. This method solubilized the spin probe in the vesicle near the interface2’ The samples were purged with nitrogen and introduced into 1-mm4.d. by 2-mm4.d. Suprasil quartz tubes and flame-sealed. The ESR samples were equilibrated for 1 week at 10 OC before ESR spectroscopy. The label concentration was 18 p M so that the amphiphile-to-label ratio was 1000/ 1 in all samples. ESR spectra were recorded with a Bruker ESP 300 ESR spectrometer operating at X-band with 100-kHz magnetic field modulation at 77 K and 0.2-mW microwave power to avoid power saturation. The magnetic fields were measured with a Varian E400 nuclear magnetic resonance gaussmeter, and the microwave frequencies in the 9-GHz range were directly measured with a Hewlet-Packard 5350B microwave frequency counter. The resulting ESR spectra were transferred to an IBM compatible 33-MHz 486 personal computer for off-line analysis. Computer simulations for the ESR line shape analysis were performed on the same personal computer using the program from Schneider and Freed.27
Theory A collection of spin probes localized within a spherical vesicle may be described as a random sample, in which collections of bilayer fragments are dispersed chaotically in space?O However, within a given fragment, the liquid crystalline director is oriented uniformly in space, implying substantial ‘microscopic order” in a morphology which may be described as ‘macroscopically disordered”. Freed and co-workers denoted this model as the MOMD (micrampic ordet-macrampic disorder)22-26 model. In this model rotational diffusion rates parallel and perpendicular to the alkyl chain, an order parameter, and the relative orientation of the rotational diffusion and magnetic tensor axes are the independent variables employed in the ESR line shape analysis. Local ordering at the microscopic level means that the spin probe reorients in the presence of an orienting potential, A, imposed by the surrounding molecules in the vesicle bilayer which is characterized by a director d. Because there is no macroscopic order in the vesicle solution, d will be distributed at random relative to the external magnetic field B. In order to calculate the appearance of the ESR spectrum, it is necessary to sum spectra weighted by sin B for all angles B between B and d. These simulations involve four principal coordinate systems:26 (1) the laboratory axes determined by B, x,y,z; (2) the local environmental axes determined by d, x’’~”,z’’; (3) the molecular axes determined by the principal axes for molecular rotational diffusion, x’J’,~’;and (4) the molecular axes determined by the principal axes of the magnetic hyperfine A and g tensors, x”’,Y When the alkyl chains of the spin probe are fully aligned with the alkyl chains of surfactant molecules in the vesicle, the x‘,y’,z’ axes are coincident with the xff,yff,z’’axes; that is, the molecular rotational diffusion axes are coincident with the local environmental axes determined by d. Since the spin probe molecules are approximately cylindrical, the molecular rotational diffusion tensor is axial so the orientation of the z components of the diffusion axes relative to the magnetic tensor a x e (X’~~J’’~,Z”’)is specified by a polar angle q. In the simulation model \k is a parameter that gives the tilt angle of the rotational diffusion axes relative to the magnetic tensor axes. In order to simulate an ESR spectrum according to the MOMD model,%the following parameters are then employed: the principal values of the A and g tensors which are known,31the inhomogeneous line width (taken as 1.5 GZ6J1),the components of the rotational diffusion tensor R,, and R , which are parallel and perpendicular to z’(the axis of molecular rotational diffusion), the orienting potential X which determines the order parameter S, and the diffusion tilt angle e.The order parameter S is related to the orienting potential X by (1) and (2) where P ( Y ) is an orientational distribution function and 0‘ is the polar angle which r r : ~ f f f .
Bratt and Kevan 5-DSA in DODAC 328K
I+
Figure 1. ESR spectra of 5-doxylstearic acid in DODAC vesicles as a function of temperature.
n
7-DSA in DODAC
Figure 2. ESR spectra of 7-doxylstearic acid in DODAC vesicles as a function of temperature. 10-DSA in DODAC
Figwe 3. ESR spectra of 10-doxylstearic acid in DODAC vesicles as a function of temperature.
defines the orientation of z‘in the xf”y”’zff’ frame. The calculational details have been g i ~ e n . ~ ~ ~ ~ S = ((3 cos2 e’
- 1)) = 1P(ef)[!12(3 cos2 8‘ - l)] sin Bf de’ (1)
P(8’) = exp[f/,X(3cos2 e’-’ - l)] Sexp[J/J(3 cos2 8l-l - l)] sin 0‘ de’ (2)
Results Experimental ESR spectra as a function of temperature at 298, 308,318, and 328 K of the various x-DSA/DODAC sytems are shown in Figures 1-5. Line shape simulations have been done as described above. The static g and A tensors for a doxy1 group are coincident, and are taken from the literature as g, = 2.0088, gvv = 2.0061, g,, = 2.0027, A, = 6.26 G, Ayy = 5.85 G, and A,
The Journal of Physical Chemistry, Vol. 96, No. 16, 1992 6851
x-Doxylstearic Acid Spin Probes in DODAC
I
I
12-DSA in DODAC
v 3269
Figure 4. ESR spectra of 12-doxylstearicacid in DODAC vesicles as a function of temperature.
n n
3289
3309
Gauss Figure 6. Experimental (solid lines) and calculated (dotted lines) ESR spectra of 5-, 7-, lo-, 12-, and 16-doxylstearic acids solubilized in DODAC vesicles.
16-DSA in DODAC
1&DSA/DODAC
:,
Fast Tumbling Component
Figwe 5. ESR spectra of 16-doxylstearicacid in DODAC vesicles as a function of temperature.
TABLE I: Dynmical Parametem Obtained for Variolro x-Doxylsterric Acids in DODAC Vesicles at 298 K X
5
7 io 12 16
R,, s-I 0.22 x io8 0.27 X 10 0.29 x io8 0.29 X lo8 0.22 X lo8
R,s-' 0.19 x 109 0.21 X lo9 0.21 x 109 0.21 X IO9 0.21 X lo9
3269
3289
3309
S
q, deg
Gauss
0.60 0.33 0.22 0.06 0.44
39 39 39 39 39
Figure 7. Experimental (solid line) and calculated (dotted line) spectra of 16-doxylstearic acid in DODAC vesicles at 308 K. The calculation is an average of 54% of a slow tumbling spectrum (MOMD model) at 298 K and 46% of a fast tumbling spectrum at 328 K. The pure slow and fast tumbling spectra are also shown.
= 33.46 G.31Spectra were calculated for 0 = lo, 3O, 6O, ..., 90° (31 spectra) and summed and weighted by sin 0 to obtain the final spectra for comparison with experiment. The calculation time for each final spectrum was about 25 min. Satisfactory fits could not be obtained with a diffusion tilt angle 0 = Oo; the best value of this parameter was 39O for 5-DSA/DODAC, and then this angle was found to be satisfactory for the other spectra. A diffusion tilt angle near 40' has been found previously for DSA spin-labels in polymers32and bilayers.33 It corresponds approximately to a gauche link in the DSA alkyl chain. The correlations between the simulated and experimental spectra for 5-, 7-,lo-, 12-, and 16-doxylstearic acid at 298 K solubilized in DODAC vesicles are shown in Figure 6. The model parameters for these fits at 298 K are summarized in Table I. It was found that the appearance of the simulated spectra was most sensitive to changes in S and moderately sensitive to changes in R,. With increasing temperature above 298 K it was possible to obtain acceptable fits between simulated and experimental data by varying RIionly, with the exception of 12-DSA and 16-DSA. For 16-DSA/DODAC, increasing temperature averages the anisotropy relatively faster than for the other x-DSAIDODAC systems. Simulations of the spectra above 298 K cannot be fit by the single-site MOMD model. However, they can be fit with a two-site model involving a summation of fast tumbling and slow tumbling spectra. The 328 K spectrum can be fit by an isotropic
I
I
10.4
I
P
I 1 -
0.35 r
v)
-0.3$
&-I I
tJ
O.' i 01 4
/
\
6
I
,
1
8
10
10.25
\+I
/
10.2 12
14
16
Doxy1 Position, x
Figure 8. S and RL parameters as determined by the ESR line shape simulations versus the x-doxy1 position for x-DSA in DODAC vesicles
compared with the normalized deuterium modulation depths from eltron spin echo data.28
tumbling model with R , and R,, = 0.46 X lo9 s-I, while the 298 K spectrum can be fit by a slow tumbling model (Table I). The
J. Phys. Chem. 1992,96,6852-6853
6852
intermediate spectra at 308 and 318 K can be fit with a sum of both slow and fast tumbling components as shown in Figure 7 for 308 K. Discussion The major results of these ESR spectral simulations are shown in Figure 8, which is a plot of the parameters S and R, versus the x-doxy1 position in the x-DSA/DODAC vesicle system. As x increasa,the order parameter Sdccreases in the range x = 5-12. This is consistent with more flexibility of the alkyl chain carbon to which the doxyl group is attached as the distance from the vesicle interface increases. However, at x = 16, S increases consistent with a U-shaped bending of the alkyl chain of x-DSA. The bending is likely a consequence of the doxyl group polarity and its position near the end of the stearic acid alkyl chain. This trend for liquid vesicle solutions is remarkably consistent with the trend of the xdoxyl position deduced infrozen DODAC vesicle systems from an analysis of deuterium modulation depths determined by electron spin echo spectroscopy.28 The inset to Figure 8 shows these data. These data were also interpreted as indicating a U-shaped bending of the 16-DSA alkyl chain. The trend of the R, parameter is also consistent with the S parameter trend. RL is slower in a more ordered environment and faster in a less ordered one. The decrease in R , for x = 16 is indicative of a U-shaped bend in the 16-DSA alkyl chain to put the doxyl group into a more ordered environment. The temperature dependence of the 16-DSA probe versus that of the 5-DSA and 7-DSA probes shows that the environment of the 16-doxy1group is not quite the same as that of the 5-doxy1 and 7-doxy1 groups. This difference is not shown by the 298 K spectra alone or by the S and R , parameters at 298 K. Since the 16-doxy1group is near the end of the stearic acid alkyl chain, one expects it to have greater motional fluctuation than a 5-doxy1 or 7-doxy1 group even if they are both near the same average distance from the vesicle interface. This difference becomes more detectable in the ESR spectra as the temperature increases above 298 K. Conclusions This study demonstrates that the ESR line shape of an xdoxylstearic acid spin probe located within DODAC vesicles between 298 and 328 K can be satisfactorily simulated with the microscopic order-macroscopic disorder model of Freed and coworkers. The simulated spectra were most sensitive to changes in the order parameter and the perpendicular component of the rotational diffusion rate R,. The trend in the order parameter with the x-doxy1 position approximately correlates with the deuterium electron spin echo modulation depths previously observed for frozen solutions of the x-DSAIDODAC system. It is concluded that the alkyl chain of 16-DSA is U-shaped in DODAC vesicles in both liquid and frozen solutions.
Acknowledgment. This research was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, US.Department of Energy. Regbtry No. 5-Doxylstearic acid, 29545-48-0; 7-doxylstearic acid, 40951-82-4; 10-dtoxylstearic acid, 50613-98-4; 12-doxylstearic acid, 29545-47-9; 16-doxylstearic acid, 53034-38-1; dioctadecyldimethylammonium chloride, 107-64-2.
References a d Notes (1) Fendler, J. H. Acc. Chem. Res. 1980, 13, 7. (2) Kalyanasudaram, K. Photochemistry in Microheterogeneous Systems; Academic: New York, 1987. (3) Hurley, J. K.; Tollin, G. Sol. Energy 1982, 28, 187. (4) Kevan, L. In Photoinduced Electron Transfer, Part B Fox, M., Chanon, M.. Eds.;Elsevier: Amsterdam, 1988; pp 329-384. (5) Freed, J. H. In Spin Labeling, Berliner, L. J., Ed.; Academic: New York, 1976; Chapter 3. (6) Fendler, J. H.; Fendler, E.J. Catalysis in Micelles and Macromolecular Systems; Academic: New York, 1975. (7) Marsh, D. In Membrane Spectroscopy; Grell, E., Ed.; Springer-Verlag: Berlin, 1981; Chapter 2. (8) Seelig, J.; Limacher, H.; Bader, P. J . Am. Chem. Soc. 1972,94,6364. (9) Waggoner, A. S.; Keith, A. D.; Griffith, 0. H. J . Phys. Chem. 1968, 72, 4129. (10) Povich, M. J.; Mann, J. A.; Kawamoto, A. J. J . Colloid Interface Sci. 1972, 41, 145. (1 1) Esposito, G.; Giglio, E.; Pavel, N. V.; Zanobi, A. J. Phys. Chem. 1987, 91, 356. (12) Hearing, G.; Luisi, P. L.; Hauser, H. J. Phys. Chem. 1988,92, 3574. (13) Emandes, J. R.; Schrier, S.; Chaimovich, H. Chem. Phys. Lipids 1976, 16, 19. (14) Yoshioka, H. Chem. Lorr. (Jpn.) 1977, 1477. (15) Baglioni, P.; Ottaviani, M. F.; Martini, G. J. Phys. Chem. 1986, 90, 5878. (16) Lasic, D. D.; Hauser, D. J. Phys. Chem. 1985, 89, 2648. (17) Schrier, S.; Polnaszek, C. F.; Smith, I. C. P. Biochim. Biophys. Acta 1978,515, 395. (18) Gaffney, B. J.; McConnell, H. M. J . Magn. Reson. 1974, 16. 1. (19) Berliner, L. J., Ed. Spin Labeling, Academic: New York, 1976. (20) Seelig, J. J . Am. Chem. SOC.1970, 92, 3881. (21) Hubbel, W. L.; McConnell, H. M.J . Am. Chem. Soc. 1971,93,314. (22) Moro, G.; Freed, J. H. J . Phys. Chem. 1980,84, 2837. (23) Moro, G.; Freed, J. H. J . Chem. Phys. 1981, 74, 3757. (24) Meirovitch, E.; Igner, D.; Igner, E.; Moro, G.; Freed, J. H. J. Chem. Phys. 1980, 84, 2459. (25) Meirovitch, E.; Freed, J. H. J . Phys. Chem. 1982, 77, 3915. (26) Meirovitch, E.; Nayeem, A.; Freed, J. H. J . Phys. Chem. 1984,88, 3454. (27) Schneider, D. J.; Freed, J. H. In Biological Magnetic Resonance; Berliner, L. J., Reuben, J., as. Plenum: ; New York, 1989; Vol. 8, Chapter 1. (28) Hiff. T.: Kevan. L.J . Phvs. Chem. 1989. 93. 1572. (29) Bratt, P. J.; Kang, Y. S.(Kevan, L.; Nakamura, H.; Matsuo, T. J . Phys. Chem. 1991,95,6399. (30) Seelig, J. In Spin Labeling, Berliner, L. J., Ed.; Academic: New York, 1976; Chapter 10. (31) Wikander, G.; Eriksson, P. 0.;Burnell, E. E.; Lindblom, G. J . Phys. Chem. 1990,94, 5964. (32) Mason, R. P.; Polnaszek, C. F.; Freed, J. H. J . Phys. Chem. 1974, 78, 1324. (33) Meirovitch, E.; Freed, J. H. J . Phys. Chem. 1980, 84, 3281.
COMMENTS
The Zeno Llne and the Radlal Dktrlbutlon Functlon at Contact Sir: Xu and Herschbach' have pointed out that the Zeno line, which is the locus of points in the T-p plane for which 2 = p / p k T = 1, is nearly linear for most normal fluids and that this linearity furnishes a constraint on equations of state for supercritical fluids and compressed liquids. They point out that the Song-Mason2 0022-3654/92/2096-6852$03.00/0
equation of state (EOS)for molecular fluids does not give a straight &no line, using C02as an example. In fact, neither does the simpler Song-Mason EOS for simple fluids, such as the noble gases.3 The Song-Mason equation contains a new strong principle of corresponding states in which an entire p v - T surface can be collapsed to a single curve: and the resulting EOS is appreciably more accurate than the original Song-Mason EOS. 0 1992 American Chemical Society
