Langmuir 1994,10, 2613-2618
2613
Photoreduction of Alkylmethylviologens in Dipalmitoylphosphatidylcholine Vesicles: Effect of the Pendent Alkyl Chain Length and the Addition of Cholesterol on the Net Photoyield Hugh J. D. McManus, Young So0 Kang, Masato Sakaguchi, and Larry Kevan* Department of Chemistry, University of Houston, Houston, Texas 77204-5641 Received March 17,1994. I n Final Form: May 6,1994@ A comprehensive electron magnetic resonance investigation was undertaken on a series of alkylmethylviologens which were solubilized into a DzO suspension of dipalmitoylphosphatidylcholinevesicles with and without added cholesterol. These samples were photoirradiated at 77 K with ultraviolet light, and the radical yield was determined through electron spin resonance spectroscopy. The location of the photoreduced specieswas determined by means of both deuterium electron spin-echomodulation (ESEM) spectroscopy at 4 K and proton electron nuclear double resonance (ENDOR)spectroscopy at 140 K. The effect of added cholesterol on the photoyield was correlated with the location of the photoreduced radical within the lipid bilayer. Addition of cholesterol disrupts the interfacial region of the vesicles, allowing increased water penetration which serves to enhance the radical yield. As the pendent alkyl chain length is increased, the photoreduction yield increases and deuterium ESEM and proton ENDOR show that the viologen moiety is "pulled" deeper into the bilayer. This is consistent with the interpretation that deeper solubilization decreases the rate of electron back-transfer which effects an increased photoreduction yield.
Introduction Increasing attention has turned to the initial, crucial event in photosynthesis-sustainable photoinduced charge separation. This event occurs in a membrane-bound proteidchlorophyll complex-the photosynthetic reaction center. 1-8 By segregating the charge transfer species across a n electrostatic barrier, redox biochemistry proceeds almost free of any interference from electron backtransfer. Work in this laboratory has focused on the unique structural aspects of this biological membrane which serve to enhance the p h ~ t o y i e l d . ~ -Using '~ simple mimetic systems such as dihexadecyl phosphate vesicles coupled with techniques such as electron nuclear double resonance (ENDOR)or electron spin resonance modulation (ESEM)," the optimal environment for microheterogenous photochemistry can be investigated.ls Since micelles and
* Abstract published in Advance A C S Abstracts, June 15, 1994. (1)Fox, M. S. Photochem. Photobiol. 1990, 52, 617. (2) Kevan, L. Int. Rev. Phys. Chem. 1990, 9, 307. (3) Griitzel, M. Heterogeneous Photochemical Electron Transfer; CRC: Boca Raton, FL, 1988. (4) Katz, J. J.; Hindman, J. C. In Photoinduced Conversion and Storage of Solar Energy; Connolly, J. S., Ed.; Academic: New York, 1981; p 27. (5) Fendler, J. H. ACC.Chem. Res. 1980, 13, 7. (6) Calvin, M. Photochem. Photobiol. 1983, 37, 349. (7) Hiff. T.: Kevan. L. Colloids Surf. 1980. 45. 185. (8) Hiromitsu. I.: Kevan. L. J . Phvk. Chem. 1989. 93. 3218. (9) Kang, Y. 6.;Baglioni, P.; McManus, H. J.; Kevan, L. J . Phys. Chem. 1991,95, 7944. (10)Bratt, P.; Kang, Y. S.; Kevan, L. J . Phys. Chem. 1992,96,5629. (11) Baglioni, P.;Rivera-Minten,E.;Kevan, L. J . Phys. Chem. 1988, 92.4726. (12) Baglioni, P.;Kevan, L. Prog. Colloid.Polym. Sci. 1988, 76,183. (13) Rivara-Minten, E.; Baglioni, P.; Kevan, L. J . Phys. Chem. 1988, 92, 2613. (14) Baglioni, P.; Kevan, L. J . Chem. SOC., Faraday Trans. 1 1988, 84, 467. (15) Li, A. S. W.; Kevan, L. Arab. J . Sci. Eng. 1988,13, 147. (16) Plonka, A.; Kevan, L. J . Phys. Chem. 1985, 89, 2087. (17) Fendler, J. H. Membrane Mimetic Chemistry;Wiley: New York, 1982; Chapter 2. (18) McManus, H. J. D.; Kevan, L. J . Phys. Chem. 1991,95,10172. 1981, (19)Narayana, P. A,;Li, A. S. W.;Kevan, L. J . Am. Chem. SOC. 103,3603. (20) Li, A. S. W.; Kevan, L. J . Am. Chem. SOC.1983, 105, 5752. (21) Bachman,L.; Dasch, W.;Kotter, P. Ber. Bunsen-Ges. Phys. Chem. 19981,85, 883.
vesicles retain their shape upon rapid f r e e ~ i n g , ' ~ -the ~l information gained using ESEM provides insight into the structural features that promote the initial charge separation. Recent studies in organic aggregates have examined the photochemical properties of chlorophyll a when it functions as the electron Changing the vesicle surface charge, the surfactant chain length, or the addition of cosurfactants such as alcohols or cholesterol can affect the photoionization yield. Addition of cholesterol to phospholipid vesicle systems has been shown to influence the kinetics of electron transfer reactions across the bilayedwater interface,26which in turn affects the radical yield. Dimethylviologen is a n important electron acceptor which has been the focus of much recent a t t e n t i ~ n . ~ ' - ~ l Much interest has focused on the ability of methylviologen to act as a n electron relay in solar energy conversion systems32and to produce hydrogen from water when in the presence of catalysts, such as colloidal platinum or h y d r ~ g e n a s eand , ~ ~on its activity as a herbicide.34 Much work has also been carried out on the mechanism for photoreduction of d i m e t h y l v i ~ l o g e n , ~ with ~ - ~ a~ recent (22) Hiromitsu, I.; Kevan, L. J. Am. Chem. SOC.1987, 109, 4501. (23) Hiff, T.; Kevan L. J. Phys. Chem. 1988,92, 3982. (24) Hiff, T.; Kevan, L. J . Phys. Chem. 1989, 93, 2069. (251 Hiff, T.; Kevan, L. J . Phys. Chem. 1989, 93, 3227. (26) Ford, W. E.; Tollin, T. Photochem. Photobiol. 1984, 40, 249. (27)Yoon, K. B.; Kochi, J. K. J . Am. Chem. SOC.1988, 110, 6568. (28)Yoon, K.; Kochi, J. K. J . Am. Chem. SOC. 1989, 111, 1128. (29) Bockman, T. M.; Kochi, J. K. J. Org. Chem. 1990, 55, 4127. (30) Turbeville, W.;Robins, D. S.; Dutta, P. J . Phys. Chem. 1992,96, 5024. (31) Dutta. P. K.: Turbeville. W. J.Phvs. Chem. 1992. 96. 9410. (32) Borghello, E.;Kiwi, J.;Pelizetti, E.yVisia,M.;Gratzel,M.Nature fLondon) 1981,289, 158. (33) Kalyanasundaram, K.; Porter, G. Proc. R.SOC.London,A 1978, 364, 29. (34) Dodge, A. D. Endeavour 1971,30, 130. (35) McKellar, J. F.; Turner, P. H. Photochem. Photobiol. 1971,13, 437. (36) Ebbesen, T. W.; Levey, G.; Patterson, L. K. Nature (London) 1982,298,545. (37) Levey, G.; Ebbesen, T. W. J . Phys. Chem. 1983,87,829. (38) Ebbesen, T. W.; Ferraudi, G. J. Phys. Chem. 1983, 87, 3717. (39) Ebbesen, T. W.; Manring, L. E.;Peters, K. S. J. Am. Chem. SOC. 1984,106, 7400.
Q743-7463/94I241Q-2613$04.5QlQ 0 1994 American Chemical Society
2614 Langmuir, Vol. 10, No. 8, 1994
McManus et al.
study of micellar and vesicular systems highlighting the involvement of t h e charged headgroup as the electron source in micelles and vesicles.40 In this work, the photoreduction of a series of alkylmethylviologen molecules, A P , in frozen dipalmitoylphosphatidylcholine (DPPC) vesicle suspensions is investigated as a function of the alkyl chain length of the electron acceptor. This is correlated with information on the location of the photoproduced radical within the vesicle structure. The relative location of the photoproduced radical with respect to the bilayedwater interface is determined with ESEM,and the relative extent to which the viologen cation is embedded into the hydrocarbon surface of the vesicle is investigated through ENDOR spectroscopy. Finally, t h e effect of adding varying concentrations of cholesterol to DPPC on both the location and photoreduction yield of alkylviologen is shown.
Sample Preparation. Stock 3 mM aqueous (DzO)solutions of the alkylviologens (AV) were prepared under nitrogen and stored in the dark at 3 "C. The concentration of the stock solutions was checked periodically using optical absorption spectroscopy. Vesicle suspensions were prepared by a method reported else~here.'8.41-~~ A chloroform solution of the DPPC surfactant and cholesterol (if added) was placed in a centrifuge tube evaporated under a stream of argon gas to form a thin film. The thin film was then thermostated in a rotary evaporator at 57 3 "C and flushed with argon for 30 min, followed by evacuation 3 "C ifor 1h. A 20 mM solution of Tris(Cl)/DzOat pH 7.8 at 57 = was added to the centrifuge tube. The suspension was thermostated under nitrogen at 57 f 3 "C for 10 min and allowed to "swell". Sonication at 57 f 3 "C under a nitrogen atmosphere was carried out using a Fisher Model 300 sonic dismembrator operated at 35% relative output power through a 4-mm-0.d. tungsten microtip. Sonications were performed in three 12-min periods, separated by 10-min rest periods. After sonication, the 0, 9, and 23 mol% cholesterol suspensions were clear; however, the 33 mol % cholesterol solutions were very slightly turbid. Increasing the sonication time or power for the 33 mol % solutions did not cause the solution to turn clear. Following sonication, 385-pL aliquots of the surfactant suspension were introduced into each of five, clean l/2-dram vials. Then, 15pLof the required 3 mMstock aqueous (D20)AVsolution was introduced onto the inner wall of the sample vial. The vials were agitated for 1min using a Fisher Model K-550-G VortexGenie. The mixtures were allowed t o stand for 2 h to equilibrate, and then three 100-pLportions of each suspension were placed
into separate 3-mm-0.d.by 2-mm-i.d.Suprasil quartz tubes which were sealed at one end. Once filled, the suspensions were rapidly frozen by plunging the sample cell into liquid nitrogen and were stored at 77 K until use. Experiments were carried out on the samples within 12h of their preparation. Precautions were taken at all times to ensure that the samples were never exposed to atmospheric oxygen. When possible, sample preparations were carried out under red light. The effective concentrations of surfactant and AV+ were 12 and 0.122 mM, respectively. These concentrations amount to a ratio of 1AV+ molecule to 107 surfactant molecules. Sample Photolysis. Photoirradiation of the frozen samples was carried out for 10 min at 77 K with a 300-W Cermax xenon lamp (LX 300 U V ) ; the power supply used was from ILC Technology. The irradiation light passed through a 10-cmwater filter and a Corning glass filter, no. 7-54 (70%transmittance at 310 nm). The relative transmission of the filter combination at the absorbance band maximum of the viologens was ap,1 = 257 nm;44CSV+,CsV+,C I O V , proximately 20% (W+, C12V+, C16V+,,A = 257-262 nm).46 During photoirradiation the dewar was mechanically rotated at 4 rpm to ensure complete illumination of the sample. The average light power at the sample position, measured with a YSI Kettering Model 65 radiometer, was (1.1f 0.1) x lo3 W m-2. Magnetic Resonance Experiments. Electron spin resonance (ESR) spectra were recorded at X-band using a Bruker ESP 300 spectrometer with 100-kHz field modulation. The irradiated sample cell was placed in a quartz ESR dewar (Wilmad Glass Co.) which was filled with liquid nitrogen and secured in a TEloz cavity. The finger of the dewar was placed at the microwave magnetic field maximum. The microwave power was maintained at 1.97 mW, and the microwave frequency was measured with a Hewlett-Packard 5350Bfrequency counter. The magnetic field was monitored with a Bruker ER 032M Hall effect field controller. The standard spectrometer settings used to determined the yield from ESR experiments were 0.272-mT modulation field amplitude, 20-mT sweep width, 9 scan accumulations, 42-s scan time, 140-ms time constant, microwave frequency 9.52 GHz, and 5 x lo4receiver gain. DPPH was used periodically as a g-factor reference before and after all experiments. Two-pulse electron spin-echo (ESE) signals were recorded a t 4.2 K with a home-built spin-echo spectrometer which was operated at X-band.46s47The microwave frequencywas measured with a Hewlett-Packard 5342A microwave frequency counter, and the magnetic field was monitored with a Varian F501 gaussmeter. A Nicolet 12/80 minicomputer interfaced t o the ESE spectrometer was used both to control the microwave pulse sequences and in the acquisition of the ESEM decay signals. Microwave pulse widths of 40 and 80 ns were used during the experiment. Once obtained, the ESE data were transferred to an IBMcompatible 486 based microcomputer for later, off-line analysis. The normalized two-pulse deuteron modulation depth was measured by a method previously described.18 Proton matrix ENDOR spectra were obtained at 140 K using an ER 300 ESR spectrometer interfaced with a Bruker ENDOR unit. A Bruker ER 4111 VT nitrogen flow variable-temperature unit was used to control and monitor the temperature in the ENDOR cavity. The temperature remainedwithin a 0.5 "C range for all experiments. The microwave power level used to saturate the ESR transition was 2 mW. The radiofrequency ($ I power was held at 100 Wand was frequency modulated at a rate of 12.5 kHz. The resulting microwave response at the center of the desaturated ESR line was synchronously detected at the rf modulation frequency,resulting in a first-derivative presentation of the ENDOR spectrum. The line width was determined from the peak-to-peak separation of the first-derivative spectrum. Typical instrument settings for ENDOR experiments were 158kHz modulation depth, 6-MHz sweep width, 10.5-s sweep time, 41-ms time constant, 1 x lo4receiver gain, and 120-W rfpower.
(40)McManus, H. J. D.; Kang, Y. S.;Kevan, L. J.Phys.Chem. 1992, 96,2274. (41)Huang, C.Biochemistry 1969,8,344. (42)Oettmeirer,W.; Norris, J. R.; Katz, J. J. 2.Naturforsch. 1976, 31c, 163. (43)Lim, Y.Y.;Fendler, J. H. J . Am. Chem. SOC.1979,101,4023.
(44)Watanabe, T.;Honda, K J.Phys. Chem. 1982,86,2617. (45)Thompson, D. H. P.; Barrette, W. C., Jr.; Hurst, J. K. J . Am. Chem. SOC. 1987,109,2003. (46)Ichikawa, T.;Kevan, L.; Narayana, P. A. J.Phys.Chem. 1979, 83,3378. (47)Narayana, P.A.;Kevan, L. Magn. Reson. Reu. 1983,7,239.
Experimental Section Reagents. Dipalmitoylphosphatidylcholine(DPPC)was purchased from Sigma Chemical Co. and was used without further and trispurification. Methylviologendichloride hydrate (W+) [(hydroxymethyl)aminolmethane(Tris),Gold label, 99.9+%, were obtained from Aldrich Chemical Co. Cholesterol, 2.0 N hydrochloric acid, and l,l-diphenyl-2-picrylhydrazyl(DPPH) were supplied by Sigma Chemical. The cholesterol was recrystallized from absolute ethanol and from ether. Deuterium oxide (DzO) was obtained from Aldrich (99.9 atom % D). The viologens N-hexyl-N'-methyl-4,4'-bipyridinium dichloride(Cap'), N-octylN-methyl-4,4'-bipyridinium dichloride (CaV+), N-decyl-Nmethyl-4,4'-bipyridinium dichloride (CloV+), N-dodecyl-N'methyL4,4'-bipyridinium dichloride (C12Vf), and N-hexadecylN'-methyl-4,4'-bipyridiniumdichloride (C16V+)were furnished by D. H. P.Thompson and J. K. Hurst or the Oregon Graduate Research Center. The structure of the alkylviologensis as follows:
*
~~
~
Photoreduction of Alkylmethylviologens
Langmuir, Vol. 10, No. 8, 1994 2615 0.58
1.00
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Alkyl Chain Length Figure 1. Normalized photoreduction yield from the ESR intensity at 77 K plotted as a function of the pendent alkyl chain length for a series ofN-alkylviologens solubilized in DPPC 0 mol % cholesterol; (0) 9 vesicles with added cholesterol: (0) mol % cholesterol; (W) 23 mol % cholesterol; (0)33 mol % cholesterol.
0.90
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.-* 0 0
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mol % Cholesterol Figure 2. Normalized photoreduction yield from the ESR intensity at 77 K plotted as a function of added cholesterol for a series of N-alkylviologenssolubilized in DPPC vesicles: (0) c1x (0) c6- (A) C S y (W) cloy (0)c12x (A)ClSV. Each ENDOR spectrum was accumulated up to 256 times to improve the signal-to-noiseratio.
Results The normalized doubly integrated ESR signal intensities for all experiments are the average offour experiments with a standard deviation of less than 5%. These results are plotted in Figures 1 and 2. Figure 1 shows the photoyield a s a function of pendent alkyl chain length, while Figure 2 illustrates these data as a function of added cholesterol. The photoyield data were normalized to the
10
15
Alkyl Chain Length Figure 3. Normalized D-modulation depth at 4 K plotted as a function of the pendent alkyl chain length for a series of N-alkylviologens solubilized in DPPC vesicles with added 9 mol % cholesterol at 4.2 K (0) 0 mol % cholesterol; (0) cholesterol: (W) 23 mol % cholesterol;(0)33 mol % cholesterol.
largest value obtained for C16Vwith 23 mol % cholesterol; the photoyields are stable a t 77 K. Theg-factor (g = 2.0033), line width (AHpp= 1.7 mT), and pale blue color of each of the irradiated viologen samples correspond favorably with previous reports on this s y ~ t e m . ' ~No , ~ESR ~ signal was observed from any sample before irradiation, or from photoirradiated samples containing only DPPC and/or cholesterol. As was previously reported, no appreciable surfactant radical ESR signal was observed for alkylviologens in DPPC. The normalized deuterium modulation depths for the sample series are also the results of four different experiments and have a standard deviation of less than 5%. Figures 3 and 4 show the normalized modulation depth results plotted as a function ofthe alkyl chain length and the mole percent of added cholesterol, respectively. ENDOR experiments at 140 K produced a spectrum with a single line centered a t 14.38 MHz, which corresponds to the Larmor precession frequency for a proton in a 0.3377-T magnetic field. The ENDOR line widths have standard deviations of less than 3%from averaging the results of up to nine experiments. Figures 5 and 6 show plots of the ENDOR line width as a function of the alkyl chain length of the substituted viologen and mole percent of cholesterol, respectively. Figure 7 shows the ENDOR spectra for a series of alkylviologensin DPPC vesicles with 33 mol % cholesterol. The upper spectrum is for CleV, while the bottom spectrum is for C1V. The scan width for these experiments was reduced to 4 MHz. The ESR, ESE, and ENDOR data show a number of trends. When the photoyield is examined as a function of the alkyl chain length, a monotonic increase is observed. When these data are plotted as a function of the mole percent of added cholesterol, the photoyield shows a steady but slight increase as a function of added cholesterol up to, but not including, the highest concentration used in these studies. The ESEM results show analogous trends. The modulation depth decreases as the pendent alkyl chain (48)Sakaguchi, M.; Kevan L. J.Phys. Chem. 1989,93,6039.
McManus et al.
2616 Langmuir, Vol. 10, No. 8, 1994 0.70 1
0.42
0.50 4 I 0 10 20 0
10
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40
mol % Cholesterol Figure 4. Normalized D-modulation depth at 4 K plotted as a function of added cholesterol for a series of N-alkylviologens CSv, (A) c ~ v(W) , solubilized in DPPC vesicles: (0)c9, (0) CIOV(0)CIZV(A)c16v.
P
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mol % Cholesterol Figure 6. H-ENDOR line width at 140K plotted as a function of added cholesterolfor a series ofN-alkylviologenssolubilized c6y (A) CsV (W) CloV ( 0 )C12V in DPPC vesicles: (0)C 9 ; (0) (A)Cl6V.
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Alkyl Chain Length Figure 5. H-ENDOR line width at 140K plotted as a function of the pendent alkyl chain length for a series ofN-alkylviologens solubilizedin DPPC vesicles with added cholesterol: ( 0 )0 mol % cholesterol;(0) 9 mol %cholesterol;(W) 23 mol % cholesterol; ( 0 )33 mol % cholesterol.
is extended, reaching a maximum a t 12 carbons after which there is a slight increase. Further, the normalized modulation depth increases with added cholesterol. The ENDOR data complement the ESEM results. The HENDOR line width decreases with added cholesterol up to 33 mol %. When plotted as a function of the pendent alkyl chain length, the H-ENDOR line width increases by about 15%.
Discussion ESEM is a powerful technique for elucidation of the microenvironment of a n entrapped radical. The modula-
Figure 7. Proton matrix ENDOR spectra at 140 K for a series of N-alkylviologenssolubilized in DPPC vesicles with 33 mol % added cholesterol: (A) c16y (B) CIZV;(C) Cloy (D) CSV;(E) c6v; (F)C1V.
tion pattern has a period characteristic of the Larmor precession frequency of the surrounding paramagnetic nucleus or nuclei. Further, when coupled to a suitable model, ESEM data provide information about the relative location of these nuclei. ESEM spectroscopy gives a measure of the dipolar interaction between the paramagnetic alkylviologen and the water nearest molecules a t the vesicle interface. The normalized modulation depth of the ESE signal is directly related to both the mean viologedwater distance and the number of nearby magnetic nuclei.4g The trends in the ESEM data are most significant in this study. (49) Kevan, L.InModern Pulsed and Continuous-Wave Electron Spin Resonance; Kevan, L.,Bowman, M., Eds.; Wiley: New Pork, 1990; Chapter 5 .
Photoreduction of Alkylmethylviologens
Langmuir, Vol. 10, No. 8, 1994 2617
ENDOR can also be used to determine the relative cationic and anionic vesicular system^.'^,^^ The observalocations of the alkylviologen molecules within the DPPC tions can be explained by the effect that the extended vesicle. In solid disordered systems, such as the polychain has on the lipophilicity of the viologen cation. As the alkyl chain length becomes longer, the viologen cation crystalline samples examined in this study, a single becomes more lipophilic and is solubilized deeper into the ENDOR transition occurring at the free proton frequency DPPC vesicle interface. The chloride counterion of the is observed.50 This line is called the matrix ENDOR line viologen presumably remains near the vesicle interface. and results from purely dipolar interactions involving the This claim is supported by the ESEM and ENDOR data unpaired electron ofthe radical and those magnetic nuclei in Figures 3 and 5. This increased lipophilicity with alkyl located within a sphere of about 1-nm radius.51 The chain length is supported by the tendency of alkylviologens resonance signals observed in this study occurred near to form aggregates spontaneously in water.66 Deeper 14.38 MHz, which is the precession frequency for a proton solubilization serves to separate the nascent radical from in the 0.3377-T magnetic field. The matrix ENDOR line the electron donor (polar h e a d g r ~ u p )which , ~ ~ decreases width is inversely related to the average distance between the radical and the surrounding magnetic n u ~ l e i ; ~ ~ -the ~ ~rate of electron back-transfer, thus increasing the photoyield. The ESR results show an increase in photherefore, this first-derivative peak-to-peak separation toyield as the pendent chain is extended. The ESEM data gives a n indication as to the proton density in the vicinity indicate that the viologen moiety is located further from of the viologen moiety. the interfacial region as the pendant alkyl chain is The ESE modulation obtained from protons occurs at extended. The ENDOR results exhibit a n increase in line a comparatively high frequency and is shallow; no width as the chain length is extended which also indicate resonance due to D nuclei is observed in the proton matrix that the viologen moiety is pulled deeper into the interface ENDOR spectrum of the viologen radical. Therefore, in with longer pendant alkyl chains. this study, proton matrix ENDOR and deuterium ESEM Upon addition of cholesterol, however, the situation is are complementary techniques, since ESEM gives inforchanged. Cholesterol addition to DPPC causes a number mation about the location of the viologen with respect to of structural changes a t the i n t e r f a ~ e . Cholesterol ~~ the water (DzO)phase, whereas ENDOR provides insight disrupts the headgroup order, increasing the space into the proximity of the radical to the surfactant chains. between neighboring surfactant molecule^.^^-^^ This Since both D-modulation and a n H-ENDOR line are increased spacing results in enhanced water penetration observed from each sample, the viologen radical is located into the hydrocarbon region of the bilayer.71 Cholesterol within about 0.6 nm from the bulk water phase and about addition can also lead to a n increase in size ofthe bilayer,72 1nm from the hydrocarbon surface of the vesicle. These and to an enhancement of the fluidity of the vesicle bilayer are the limiting distances over which such responses can which can lead to deeper penetration of solubilized be observed. Because the vesicle bilayer is essentially molecule^.^^^^^ impervious to ~ a t e r , ~the ~ - viologen ~l cation is located The ESEM and ENDOR results show a clear change as near the vesicle interface. cholesterol is introduced into these systems. The ESEM Studies show that a hydrocarbodwater interface exists data show that addition of cholesterol effects increased in surfactant aggregate@ with a more "ordered" arrangewater penetration into the bilayer interface (Figure 4). ment of alkyl tails nearer the interface compared to the The modulation depth in Figures 3 and 4 shows a near vesicle i n t e r i ~ r . The ~ ~ ,average ~~ proton density within a monotonic increase with the addition of cholesterol. sphere of about 1-nm radius centered a t the viologen Complementary changes occur in the ENDOR data. The moiety should increase on moving from the vesicle addition of cholesterol causes a slight decrease in the interface with deuterated water more into the hydrocarbon matrix ENDOR line width. The ENDOR results show phase ofthe vesicle interior. A n increase in the H-ENDOR that addition of cholesterol results in a decrease in the line width indicates a n increase in the proton density in proton density in the microenvironment of the viologen the vicinity of the viologen radical. moiety. This decrease is most likely due to increased deuterated water penetration into the bilayer. The trends In the absence of cholesterol, the ESR signal intensity in Figures 3 and 5 are clear. As the alkyl chain is extended, and ENDOR line width show a steady increase as the the viologen moiety is pulled deeper into the hydrocarbon pendent alkyl chain is extended, while the ESE modulation interior, further from the deuteron-rich aqueous phase. depth decreases. These results are consistent with The ESE modulation depth shows a steady decrease for previous reports of alkylviologen photoreduction in both all experiments while the ENDOR line width displays a concomitant increase. The decreasing polar nature ofthis (50) Hyde, J. S.; Rist, G. H.; Erikson, L. E. G. J . Phys. Chem. 1968, environment is reflected in the trend observed in the 72, 4269. ESEM data; likewise, the ENDOR data show that the (51)Kevan, L.; Kispert, L. D. Electron Spin Double Resonance Spectroscopy; Wiley: New York, 1976; p 239. microenvironment has an enhanced lipophilic character. (52) Helbert, J. N.; Wagner, B. E.; Poindexter, E. H.; Kevan, L. J . As cholesterol is added to each of the AV2+/DPPCvesicles, Polym. Sci., Polym. Phys. Ed. 1976, 13, 825. (53) Helbert, J.;Kevan, L.; Bales, B. L. J.Chem. Phys. 1972,57,723. (54) Helbert, J.; Kevan, L. J. Chem. Phys. 1973, 58, 1205. (55) Bales, B. L.; Schwartz, R. N.; Kevan, L. Chem. Phys. Lett. 1973, 22, 13. (56) Bales, B. L.; Schwartz, R. N.; Kevan, L. Ber. Bunsen-Ges. Phys. Chem. 1974, 78, 194. (57) Hase, H.; Ngo, F. Q.H.; Kevan, L. J.Chem. Phys. 1975,62,985. (58) Jain, M. K. In Introduction to Biological Membranes; Jain, M. J., Wagner, R. C., Eds.; Wiley: New York, 1980; Chapter 4. (59) Casal, H. J. J . Phys. Chem. 1989, 93, 4328. (60) Tanford, C. The Hydrophobic Effect: Formation ofMicelles and Biological Membranes, 2nd ed.; Wiley: New York, 1980; Chapter 11. (61) Buldt, G.; Gally, H.; Seelig, A.; Seelig, J.; Zaccai, G. Nature (London) 1978,271, 182. (62) Bendendouch, D.; Chen, S.-H.; Koehler, W. C. J . Phys. Chem. 1983,87, 153. (63) Gruen, D. W. R. J.Phys. Chem. 1985, 89, 146. (64) Gruen, D. W. R. J . Phys. Chem. 1986, 89, 153.
(65) Sakaguchi, M.; Kevan, L. J . Phys. Chem. 1991,95, 5996. (66) Wolszczal, M.; Stradowski, Cz. Radiat. Phys. Chem. 1989,33, 355. (67) Bratt, P. J.;McManus, H. J. D.; Kevan, L. J.Phys. Chem. 1992, 96, 5093. (68) Rubenstein, J. L. R.; Owicki,J . C.; McConnell,H. M. Biochemistry 1980,190,569. (69) Liposomes: From Physical Structure to TherapeuticApplicatwm;
Knight, C. G., Ed.; ElsevierNorth-Holland Biomedical Press: Amsterdam, 1981; Chapter 6. (70)Yeagle, P. L. Acc. Chem. Res. 1978, 11, 321. (71) Taylor, R. P.; Huang, C.-H.; Broccoli, A. Y.; Leak, L. Arch. Biochem. Biophys. 1977, 183, 83. (72) deKruijff, B.; Cullis, P. R.; Radda, G. K. Biochim. Biophys.Acta 1976,436, 729. (73) Shreier-Muccillo, S.;Marsh, D.; Dugas, H.; Schneider, H.; Smith, I. C. P. Chem. Phys. Lipids 1973, 10, 11.
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the ESE modulation depth increases. This growth in modulation depth implies steadily increasing water penetration. The ENDOR data show a decrease in the proton density in the microenvironment of the AV radical. As deuteron-rich water penetrates into the lipid bilayer, the proton density decreases which results in a decreased H-ENDOR line width. At the highest concentration of cholesterol used in this work, 33 mol %, the photoreduction yield drops slightly while the ESE modulation depth increases. In most experiments the H-ENDOR line width reaches a plateau. These results may be interpreted as a breakdown in the integrity of the vesicular interface. At these high cholesterol concentrations, the integrity of the interfacial region begins to be compromised and electron backtransfer increases. Attempts were made to load the vesicles with 50 mol % cholesterol; however, even with constant heating, the mixture remained very cloudy. Hence, the onset of vesicular breakdown appears to occur a t or above cholesterol concentrations of 33 mol % in DPPC vesicles.
Conclusions The effect of the pendent alkyl chain length and the addition of cholesterol on the photoreduction of a series of alkylviologens solubilized in DPPC vesicles was ex-
McManus et al. amined through ESR, ESEM, and ENDOR. The ESE and ENDOR data show that alkylviologens with the longest hydrocarbon tail are located deepest into the vesicle bilayer. The integrated ESR intensity shows a n increase in the photoreduction yield with increasing alkyl chain length of the alkylviologen. This increase seems interpretable as a decrease in the rate of electron back-transfer to the nascent radical. ESEM shows that adding cholesterol to these system effects greater water penetration into the bilayer region of the vesicle. A n analysis of the H-ENDOR data shows that this increased water (DzO) penetration effects a decrease in the proton density. The ENDOR and ESEM results are complementary and also show that a t the highest levels of cholesterol used (33 mol %)theintegrity ofthe vesicle structure is affected. Hence, a n enhancement of the photoyield is possible up to cholesterol concentrations of 23 mol % and is greatest for the alkylviologen with the longest pendent hydrocarbon tail.
Acknowledgment. This research was funded by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, U.S.Department of Energy. We thank D. H. P. Thompson and J. K. Hurst for the alkylviologen compounds.
