J. Phys. Chem. 1992,96, 5093-5096 (4) Christenson, H. K. J. Chem. Phys. 1983, 78,6906. (5) Christenson, H. K. J. Chem. Soc., Faraday Trans. 1 1984,80, 1933. (6) Christenson, H. K.; Horn, R. G.Chem. Scr. 1984, 25, 37. (7) Pashley, R.M.; Israelachvili,J. N. J. Colloid Interface Sci. 1984,101, 511. (8) van Megan, W.; Snook, I. K. J. Chem. SOC.,Faraday Trans. 2 1979, 75, 1095. (9) Snook, I. K.; van Megan, W. J . Chem. Phys. 1980, 72, 2907. (10) Lane, J. E.; Spurling, T. H. Chem. Phys. Lett. 1979,67, 107; Ausr. J. Chem. 1980,33, 231. (1 1) Grimson, M. J.; G. Rickayzen, G.;Richmond, P. Mol. Phys. 1980, 39, 61. (12) Mitchell, D. J.; Ninham, B. W.; Pailthorpe, B. A. Chem. Phys. Lett. 1977.51, 257. Chan, D. Y. C.; Mitchell, D. J.; Ninham, B. W.; Pailthorpe, B. A. J. Chem. Soc., Faraday Trans. 2 1980, 76, 776. (13) Karlstram, G.Chem. Scr. 1985, 25, 89. (14) Magda, J. J.; Tirell, M.; Davies, H. T. J. Chem. Phys. 1985,83,1888. (15) Luzar, A.; Bratko, D.; Blum, L. J. Chem. Phys. 1987, 86, 2955. (16) Kjellander, R.; Sarman, S. Mol. Phys. 1991, 74, 665. (17) Plischke, M.; Henderson, D. Proc. R. Soc. London, A 1986,404,323. (18) Henderson, D.; Lozada-Cassou, M. J . Colloid Interface Sci. 1986, 114, 180. (19) Belloni, L. Chem. Phys. 1985, 99, 43. (20) Zhou, Y.;Stell, G.J.-Chem. Phys. 1990, 92, 5533. (21) Lozada-Cassou,M.;Diaz-Herrera, E. J. Chem. Phys. 1990,92, 1194.
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(22) Sarman, S . J. Chem. Phys. 1990, 92, 4447. (23) Attard. P.: Patev. G. N. J . Chem. Phvs. 1990. 92. 4970. (24) Attard; P.i Beraid, D. R.; Ursenbach, C. P.; Patey, G . N. Phys. Rev. A 1991, 44, 8224. (25) Attard, P. J. Chem. Phys. 1989, 91. 3083. (26) Attard, P.; Wei, D.; Patey, G. N.; Torrie, G. M. J. Chem. Phys. 1990, 93, 7360. (27) Vanderlick, T. K.; Scriven, L. E.; Davis, H. T. Colloids and Surfaces; Elsevier: Amsterdam, 1991; Vol. 52, p 9. (28) Israelachvili, J. N.; Adams, G.E. J. Chem. SOC.,Faraday Trans. 1 1978, 74, 975. (29) Parker, J. L.; Christenson, H. K.; Ninham, B. W. Reu. Sci. Instrum. 1989,60, 3135. (30) Parker, J. L. Lungmuir 1992,8, 551. (31) Parker, J. L.; Stewart, A. M. Prog. Colloid Polym. Sci., in press. (32) Derjaguin, B. V. Kolloid 2.1934, 69, 155. (33) White, L. R. J. Colloid Interface Sci. 1983, 95, 286. (34) Christenson, H. K.; Blom, C. E. J. Chem. Phys. 1987, 86, 419. (35) Stewart, A. M.; Christenson, H. K. Meas. Sci. Technol. 1990,1,1301. (36) Parker, J. L.; Stewart, A. M. Manuscript in preparation. (37) Israelachvili, J. N. J. Colloidal Interface Sci. 1973, 44, 259. (38) Attard, P.; Parker, J. L. Manuscript in preparation. (39) Handbook of Chemistry and Physics, 57th ed.;Weast, R. C., Ed.; CRC Press: Cleveland, 1976. (40) Christenson, H. K. PhD Thesis, Austalian National University, 1983.
Electron Spin Echo Modulation Studies of Doxylstearic Acid Spin Probes in Frozen Dlhexadecyl Phosphate and Dioctadecyldlmethylammonium Chloride Vesicles: Interaction of the Spin Probe with Deuterated Water and Effects of Cholesterol Addition Peter Bratt, Hugh J. D. McManus, and Larry Kevan* Department of Chemistry, University of Houston, Houston, Texas 77204-5641 (Received: January 21, 1992)
Electron spin echo studies have been carried out for a series of x-doxylstearic acid (x = 5, 7, 10, 12, and 16) and 3doxyl-5a-cholestane spin probes in frozen deuterated aqueous solutions of anionic dihexadecyl phosphate (DHP) vesicles and cationic dioctadecyldimethylammoniumchloride (DODAC) vesicles. Modulation effects due to interactions of the spin probe nitroxide group with D 2 0at the vesicle interface give distancebased information which can be used to draw conclusions about the ordering and chain packing of the vesicles. It is found that the normalized modulation depths are greater for DHP than DODAC vesicles, indicating that the DHP vesicle structure is less tightly packed with less chain bending for the doxy1 moiety at x = 16. Upon addition of cholesterol to DHP vesicles, the degree of hydration of the spin probes near the interface is increased while the degree of hydration of the spin probes located deeper inside the bilayer is decreased. Upon addition of cholesterol to DODAC vesicles, the degree of hydration is demeased for all spin probes up to 50 mol % cholesterol concentration above which the vesicle begins to lose its structure and D2O can penetrate within the bilayer.
Introduction The photoinduced charge separations of photoionizable solutes in organized molecular assemblies such as micelles, reversed micelles, vesicles, and microemulsions are model systems for the storage of light energy.I4 In many ways these model systems parallel natural photosynthesis, in which a chlorophyll solute in a lipid bilayer is photoionized, resulting in electron transfer. In particular, vesicles have been widely used to mimic natural membra ne^.^^^ Photoionization and net charge separation can be significantly influenced by the surfactant head group,'^^ counterion?3l0 alkyl chain length,''-'3 and addition of s a l t ~ . ' ~ The J~ addition of slightly water-soluble compounds such as alcohols and cholesterol also modifies the assembly interface and partially controls the charge separation e f f i ~ i e n c y . ' ~ ' ~ Stable nitroxide radicals have found wide use as spin probes in studies of biological membranes and membrane mimetic systems.2w22 The x-doxylstearic acids of the general formula shown in Figure 1 are especially useful, due to their low solubility in water and tendency to be incorporated into heterogeneous aqueous systems containing amphiphilic molecules.23 Electron spin echo modulation (ESEM) spectroscopy has found considerable use as means of measuring the interaction of a radical
located within a frozen organized molecular assembly with the surrounding deuterium in D 2 0 of the bulk water phase at the assembly i n t e r f a ~ e . ' ~ +In* ~a previous study using electron spin resonance (ESR)and ESEM, the alkyl chain length effect on the photoionization yields of a series of alkylphenothiazine derivatives in three vesicle systems was dwribed.I2 In the present work, the effect of cholesterol addition upon photoinduced charge separation within vesicles is studied systematically with ESEM, and the relative locations of doxylstearic acid and cholestane spin probes in positively charged DODAC vesicles and negatively charged DHP vesicles are determined. The results are discussed in terms of the hydration of the vesicle interface and the ordering of the surfactant aggregates. Experimental Section Dihexadecyl phosphate (DHP) was purchased from Sigma Chemical Co. and was used without further purification. Dioctadecyldimethylammonium chloride (DODAC) was prepared as described previously.I2 The spin-labels 5-, 7-, lo-, 12-, and 16doxylstearic acid and 3-doxyl-5a-cholestane were obtained from Sigma and used as received. They were stored as stock solutions in chloroform a t -10 "C.
0022-3654/92/2096-5093$03.00/00 1992 American Chemical Society
Bratt et al.
5094 The Journal of Physical Chemistry, Voi. 96, No. 12, 1992
n H
O
Cholestane/DHP
W
Cholesterol
3-Doxy l-Sa-Cholestane 50 mole % Choiesteroi
10 mole % Cholesterol
0 mole % Cholesleroi
DODAC x-Doxylstearic Acid Figure 1. Structures of compounds used.
A
I
DHP
0.3
x-DSA/DHP
1.3
2.3
Figure 3. Two-pulse electron spin echo decay envelopes recorded at 4.2 K for the 3-doxyl-5a-cholestanespin probe in DHP vesicle solutions as a function of cholesterol concentration. The base lines have been offset vertically to avoid overlap.
: O0.
0.8
1.8
1.3
7,PS
x = 5
0.3
0.8
1.8
-34
2.3
7,PS
F g u e 2. Two-pulse electron spin echo decay envelopes recorded at 4.2 K for x-doxylstearic acid spin probes in DHP vesicle solutions. The base lines have been offset vertically to avoid overlap.
In order to form vesicle solutions, thin films were formed from solutions of the surfactant and cholesterol in chloroform. The films were then sonicated in D20using a Fisher Model 300 sonic dismembrator operated a t 35% relative output power with a 4mm-0.d. microtip.. DODAC vesicles in D 2 0 were formed by sonication for 15 min at 53 f 2 0C.6 DHP vesicles were formed in 20 mM tris(hydroxymethy1)aminomethane (TRIS) buffer solution adjusted to pH = 7.8 with hydrochloric acid after sonication for 20 min a t 71 f 2 0C.25,26 The vesicle solutions were added to previously evaporated spin probe films and were allowed to stand for 24 h. This method solubilized the spin probes near the interface of the vesicle.I8 The respective concentrations of surfactants and spin probes were 18 and 0.18 mM, which corresponds to a 1OO:l surfactant-to-probe ratio. ESR spectra were recorded at 77 K with a Bruker ESP 300 spectrometer operating a t X-band with 100-kHz magnetic field modulation and 0.2-mW microwave power to avoid power saturation. The magnetic fields were measured with a Varian E-500 nuclear magnetic resonance gaussmeter, and the microwave frequencies in the 9-GHz range were directly measured with a Hewlett-Packard 5350B microwave frequency counter. Two-pulse electron spin echo decay curves were observed at 4.2 K at 9 GHz and 330 mT with a home-built spectrometer*’ for the M I= 0 14Nhyperfine line of the powder spectrum of the samples. Three ESEM decay curves were measured and the results averaged to obtain error statistics. The ESEM data was transferred to an IBM compatible 386-SX PC for off-line data analysis. Results Two-pulse electron spin echo decay envelopes were recorded from the M,= 0 I4N hyperfine transition of the nitroxide electron spin resonance spectrum. The decay envelopes as a function of
Figure 4. Dependence of the normalized deuterium modulation depth on the position of the doxy1 group in x-doxylstearic acid spin probes and on the mole percent cholesterol in DHP vesicle solutions.
I
.-0
6 0.35
cholestane/DHP
I
1
I
I
1 10 20 30 40 50 $0 Cholesterol, Mole %
Figure 5. Dependence of the normalized deuterium modulation depth on the mole percent of cholesterol incorporated into DHP vesicles for the
3-doxyl-Sa-cholestanespin probe.
the x-doxy1 probe in DHP vesicles are shown in Figure 2. To illustrate the effect of cholesterol upon the ESE decay envelopes, the ESE decay envelopes for cholestane as a function of added cholesterol are shown in Figure 3. All of the echo d a y envelopes display modulation with a period of 0.08 ps which is due to weak hyperfine interactions of the nitroxide groups with protons from the surfactant alkyl chains and a second, deeper modulation with a period of 0.55 ccs which is due to interactions of the radical center with deuterium from D 2 0which is located at the vesicle interface. The normalized deuterium modulation depth is defined as the vertical distance from the extrapolated unmodulated decay curve
Doxylstearic Acid Spin Probes in DHP and DODAC
- 0.5 1
I
Doxy1 Position X
Figure 6. Dependence of the normalized deuterium modulation depth on the position of the doxyl group in x-doxylstearic acid spin probes in DODAC vesicle solutions. x-DSAIDODAC 16-DSA
\
c
O"'
0
1
I
10 20 30 40 50 $0 Cholesterol, Mol %
Figwe 7. Dependence of the normalized deuterium modulation depth on the mole percent of cholcatcrol incorporated into DODAC vesicles for the 5- and 16doxylstearic acid spin probes.
to the first minimum in the modulated curve divided by the distance from the unmodulated curve to the base line.28 Figure 4 shows the dependence of the normalized modulation depth2" as a function of the position of the doxyl moiety and the concentration of added cholesterol for the DHP/x-DSAlcholesterol system. In Figure 5 the effect of cholesterol addition upon the normalized modulation depths of 3-doxyl-Sa-cholestane is seen. Figure 6 shows the dependence of the normalized deuterium modulation depths for the x-doxylstearic acid spin probes in DODAC/D20. The effect of cholesterol addition to this system is shown in Figure 7 for x = 5 and x = 16 x-doxylstearic acids.
Discussion It has been shown that upon rapid freezing the micelle or vesicle structure is r e t a i ~ ~ e dand ~ ~that, " ~ at 4.2 K, the ESE sensitivity is enhanced and the magnetic relaxation times are longer than in the liquid state.31 The normalized deuterium modulation depth is dependent upon the number and distance of deuterium nuclei in the vicinity of the paramagnetic center. Typically, modulation from the deuterium nuclei can only be detected for a physically reasonable number of nuclei within a distance of 0.6 nm from the unpaired electron?' Therefore, the normalized modulation depth gives semiquantitative data relating to the distance of the paramagnetic center from bulk water at the interface. DHP Vesicles. From Figure 4 it can be seen that the normalized modulation depth decreases quite dramatically as the doxyl group is incorporated further from the carboxylic acid group moiety in the stearic acid alkyl chain. Unlike the results observed for the dipalmitoylphosphatidylcholine/DzOsystem,l" it can be seen that for 16-doxylstearic acid there is no interaction with the surrounding D20. In order to explain these results, we firstly consider the likely solubilization sites and the conformations of the spin probes. It has been shown that the carboxylic acid group tends to be solubilized near the vesicular interface with the hydrocarbon tail extending into the lipophilic core. It can be seen from Figure 4 that the probability of interaction between the probe and DzO
The Journal of Physical Chemistry, Vol. 96, No. 12, I992 5095 decreases with increasing x. This suggests that the probe is in an extended conformation with little chain bending as was observed by Hiff and Kcvanla for the dipalmitoylphosphatidylcholine/DzO system. However, the value of the normalized modulation depth for 5doxylstearic acid in the dipalmitoylphosphatidylcholine/D20 system (0.17) is much lower than that obtained for the DHP/D20 system (0.29). This suggests that DHP vesicles are more hydrated at their interface and hence less ordered than dipalmitoylphogphatidylcholine vesicles. General Effect of Cholesterol Addition. Upon the addition of cholesterol, two differing trends may be discerned from Figure 4. For the spin probes where the doxyl moiety is localized close to the carboxylic acid group (x = 5 and 7), the addition of cholesterol has the effect of increasing the normalized modulation depths from 0.29 to 0.40. Conversely, the effect of cholesterol addition for the spin probes where the doxyl moiety is localized further away from the carboxylic group (x = 10, 12, 16) is to reduce the normalized modulation depths. A major constituent of biological membranes is cholesterol. Therefore, lipid-cholesterol interactions are of considerable interest. However, the role of sterols in membrane structure and function as yet remains somewhat unclear. In general, the addition of cholesterol to a lipid bilayer has the effect of increasing the order p a ~ m e t e P + ~and ' decreases the probability of trans-gauche isomerism. Experiments with headgroup spin-label~'~have indicated decmsed motional freedom at low concentrations of added cholesterol to the Laphase of dimyristoylphosphatidylcholine and increased motional freedom at higher concentrations as the physical spaces between the headgroups increase. At concentrations between 25 and 40 mol %, a two-phase region seems to exist in dimyristoylphosphatidylcholinebilayers!'*42 These two phases consist of a cholesterol-rich phase characterized by lower mobility and more order and a cholesterol-poor phase which shows greater mobility. Further evidence to support this physical picture is obtained from the polarity profiles obtained for the L, phase of dipalmitoylphosphatidylcholine from the isotropic splitting factor.39 These results suggest that cholesterol is located at the interface and that it opens up the interface to allow more water penetration, while the interior of the vesicle becomes less polar as cholesterol is added. In short, previous work" has shown that addition of cholesterol has the effect of increasing the order parameter to produce a "chain stiffening" effect, thus allowing embedded molecules to diffuse more easily within the lipid bilayer. Therefore, for the spin probes where the doxyl moiety samples the interface ( x = 5 and 7) the increased normalized modulation depths suggest that the interface is more hydrated. It is known that addition of cholesterol can increase the size of vesicles by 40% due to the cholesterol molecule solubilizing in the interfacial region of the vesicle and increasing the mean distance between h e a d g r o ~ p s . ~ However, l~~~ for the spin probes where the doxyl moiety is located further from the carboxylic acid group (x = 10, 12,16), the "chain stiffening" effect prevents these paramagnetic centers from sampling the interface. Additional evidence for this physical picture may be gleaned from the behavior of the 3-doxyl-Sa-cholestane spin probe in DHP/DzO vesicles as a function of added cholesterol. The 3doxyl-5a-cholestane molecule is a spin probe resembling cholesterol and is thought to "mimic" the behavior of cholesterol. From Figure 5 it can be seen that the effect of cholestane parallels the behavior of the 5-doxylstearic acid spin probe which in turn suggests that cholesterol is located in the interfacial region of the vesicle. Effect of Cbolesterol Addition to M D A C Vesicles. DODAC vesicles are cationic vesicles. It can be seen from Figure 6 that the normalized modulation depths for the x-doxylstearic acids obtained in this study behave in a manner similar to that obtained by Hiff and Kevan'" which is very different to the behavior observed in DHP and dipalmitoylphosphatidylcholinevesicles. The curve shows a minimum at x = 7 and 10, indicating a degree of chain bending. It has been suggested by Hiff and KevanIa that this is due to enhanced packing of DODAC surfactant molecules preventing the weakly polar and bulky heterocyclic probe from penetrating deeply into the bilayer.
5096 The Journal of Physical Chemistry, Vol. 96, No. 12, I992
Upon addition of cholesterol the normalized modulation depths are reduced for 10-30 mol % cholesterol but are increased at 50 mol % cholesterol. This behavior is illustrated in Figure 7 for the 5- and 16-doxylstearic acids. These results may be explained in the following way. Initially, cholesterol has the effect of rigidifying the surfactant alkyl chains so that the spin probe can d i f f w deeper into the bilayer. This process occurs up to 30 mol % cholesterol. However, at a cholesterol concentration of 50 mol % the cholesterol has swollen the DODAC vesicle so much that D 2 0 can penetrate somewhat inside the bilayer structure and enhance the degree of hydration.
Conclusions This ESE study indicates that anionic DHP vesicles are more hydrated at their interface than are dipalmitoylphosphatidylcholine vesicles. Upon addition of cholesterol to DHP vesicles the interfacial region becomes more hydrated while the lipophilic core becomes less polar. Cationic DODAC vesicles are more hydrated than dipalmitoylphosphatidylcholine vesicles but less hydrated than DHP vesicles. Addition of cholesterol to DODAC vesicles locates the x-doxylstearic acids deeper within the vesicle to about 50 mol % cholesterol above which the vesicle begins to lose its structure and D20can penetrate within the vesicle interface. Acknowledgment. This research was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, U.S.Department of Energy. Re&@ NO.DHP,2197-63-9; DODAC,107-64-2; H20,7732-18-5; 5-doxylstearic acid, 29545-48-0; 7-doxylstearic acid, 40951-82-4; 10doxylstearic acid, 5061 3-98-4; 12-doxylstearic acid, 29545-47-9; 16doxylstearic acid, 53034-38-1; 3-doxyl-5a-cholestane, 18353-76-9; cholesterol, 57-88-5.
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Bratt et al. (8) Jones, R. R. M.; Maldonado, R.; Szaajdzinska-Pietek, E.; Kevan, L. J . Phys. Chem. 1986, 90, 1126. (9) Plonka, A.; Kevan, L. J . Phys. Chem. 1985, 89, 2087. (10) Fang, Y.; Tollin, G. Phorochem. Phorobiol. 1984, 39, 685. (11) Narayana, P. A.; Li, A. S.W.; Kevan, L. J . Am. Chem. SOC.1982, 104,6502. (12) Bratt, P.; J. Kang, Y. S.;Kevan, L.; Nakamura, H.; Matsuo, T. J . Phys. Chem. 1991, 95,6399. (13) Kevan, L. Inr. Reo. Phys. Chem. 1990, 9, 307-328. (14) Plonka, A.; Kevan, L. J . Chem. Phys. 1985, 82, 4322. (15) Hiromitsu, I.; Kevan, L. J . Phys. Chem. 1986, 90, 3088. (16) Baglioni, P.; Kevan, L. J . Phys. Chem. 1987, 91, 2016. (17) Hiromitsu, I.; Kevan, L. J . Am. Chem. Soc. 1987, 109, 4501. (18) Hiff, T.; Kevan, L. J. Phys. Chem. 1989, 93, 1572. (19) Lanot, M. P.; Kevan, L. J . Phys. Chem. 1989, 93, 5280. (20) Berliner, L. J. Spin Lrrbelling Theory and Application; Academic Press: New York, 1976. (21) Fendler, J. H.; Fendler, E. J. Catalysis in Micelles and Macromolecular Systems; Academic Press: New York, 1975. (22) Marsh, D . In Membrane Spectroscopy; Grell, E., Ed.; SpringerVerlag: Berlin, 1981; p 51. (23) seclig, J.; Limacher, H.; Bader, P. J. Am. Chem. Soc. 1972,94,6364. (24) Kevan, L. Radiar. Phys. Chem., in press. (25) Colaneri, M.; Kevan, L.; Schmehl, R. J . Phys. Chem. 1989,93,397. (26) Sakaguchi, M.;Kevan, L. J . Phys. Chem. 1989, 93, 6039. (27) Ichikawa, T.; Kevan, L.; Narayana, P. A. J . Phys. Chem. 1979,83, 3378. (28) Szajdzinska-Retek, E.; Maldonado, R.; Kevan, L.; Berr, S.S.; Jones, R. R. M. J. Phys. Chem. 1985, 89, 154. (29) Narayana, P. A.; Li, A. S. W.; Kevan, L. J. Am. Chem. SOC.1981, 103, 3603. (30) Li, A. S. W.; Kevan, L. J. Am. Chem. SOC.1983, 105, 5752. (31) Kevan, L. In Time Domain Electron Spin Resonance; Kevan, L., Schwartz, R. N., Eds.; Wiley-Interscience: New York, 1979; Chapter 8. (32) Baglioni, P.; Ferroni, E.; Martini, G.; Ottaviani, M. F. J. Phys. Chem. 1982, 86, 3198. (33) Ramachandran, C.; Pyter, R. A.; Mukerjee, P. J. Phys. Chem. 1982, 86, 3198. (34) Butler, K. W.; Schrier-Muccillo, S.; Smith, I. C. P. Chem. Phys. Lipids 1973, 10, 11. (35) Ehrenburg, A.; Ericksson, L. E. G.; Shimoyama, Y. Biochim. Biophys. Acta 1978, 508, 213. (36) Hemminga, M. A. Chem. Phys. Lipids 1975, 14, 141. (37) Hemminga, M. A. Chem. Phys. Lipids 1975, 14, 151. (38) McConnell, H. M.; Owicki, J. C.; Rubenstein, J. L. R. Biochemistry 1980, 190, 569. (39) Marsh, D.; Watts, A. In Liposomes: From Physical Structure io Therapeutic Applicutions; Knight, C. G., Ed.; Elsevier: Amsterdam, 1981; Chapter 6. (40) Yeagle, P. L. Biochim. Biophys. Acta 1985,822, 267. (41) de Kruijff, B.; Cullis, P. R.; Redda, G. K. Biochim. Biophys. Acta 1976,436, 729. (42) Jonson, S. M. Biochim. Biophys. Acta 1973, 307, 27.
