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Langmuir 1993,9, 3142-3148
Lateral Diffusion of Amphiphiles in Fatty Acid Monolayers at the Air-Water Interface: A Steady-State and Time-Resolved Fluorescence Quenching Study Frank Caruso, Franz Grieser, and Peter J. Thistlethwaite' School of Chemistry, University of Melbourne, Parkville 3052, Australia
Mats Almgren Department of Physical Chemistry, University of Uppsala, S-751 21 Uppsala, Sweden Received March 26,1993. I n Final Form: June 17,1999 Lateral diffusion of amphiphilic molecules has been investigated in oleic acid (OA) monolayers at the ail-water interface by studying the fluorescence quenching of N-(l-pyrenylsulfonyl)dipalmitoyl-L-aphosphatidylethanolamine (pyrenyl-DPPE) by 4-(N~-dimethyl-N-hexadecylammonium)-2,2,6,~te~~ethylpiperidine-1-oxy1iodide (CAT-16). Both steady-state and time-resolved techniques were employed to yield data which show clear evidence for the features predicted for diffusion-controlled reactions in two dimensions. Analysis of the steady-state quenching behavior as a function of quencher concentration produced nonlinear Stem-Volmer plots. The time-resolved measurements showed single-exponential behavior for the pyrene chromophore in the absence of quencher, providing evidence that it is not aggregated in the monolayer film. In the presence of quencher the decays were nonexponential. The lateral diffusion coefficients were found to decrease with increasing surface pressure. This result reflects an expeded decrease of monolayer fluidity upon compression.
Introduction The phenomenon of lipid diffusion in biological membranes is of fundamental importance in biochemistry since it reflects the degree of membrane fluidity and is related to processes such as electron transfer,' receptor mediation: membranes have and photoreceptor f u n ~ t i o n . Model ~ often proven to be very useful for obtaining detailed information on many physical and functional properties of membranes. There have been a number of experimental studies of lateral diffusion in natural membranes,lp4p5and related model systems such as phospholipid bilayers614 and spread mon01ayers.l"~~ The diffusion of lipid molecules in membranes is visualized as a two-dimensional
* To whom correspondence should be addressed.
* Abstract published in Advance ACS Abstracts, August 15,1993. (1) Hackenbrock, C. R. Trends Biochem. Sei. 1981,6,151.
(2) Paatan, I. H.; Willingham, M. C. Science 1981,214,504.
(3) Liebman, P. A.; Pugh, E. N., Jr. Vision Res. 1979,19, 375. (4) Cherry, R. J. Biochim. Biophys. Acta 1979,559, 289. (5) Peters, R. Cell Biol. Znt. Rep. 1981,5, 733. (6)Trauble, H.; Sackmann, E. J.Am. Chem. SOC.1972, 94,4499. (7) Devaux, P.; McConnell, H. M. J. Am. Chem. SOC.1972,94,4475. (8) Wu, E.-S.; Jacobson, K.; Papahajopoulos, D. Biochemistry 1977, 16, 3936. (9) Galla, H.-J.; Sackmann, E. Ber. Bunsen-Ges. Phys. Chem. 1974, 78, 949. (10) Galla,H.-J.; Sackmann, E. Biochim. Biophys. Acta 1974, 339, 103. (11)Scandella, C. J.; Devaux, P.; McConnell, H. M. h o c . Natl. Acad. Sci. U.S.A. 1972, 69, 2056. (12) Kano, K.; Kawazumi,H.; Ogawa, T.; Sunamoto, J. J.Phys. Chem. 1981,85, 2204. (13) Vanderkooi, J. M.; Fischkoff, S.;Andrich, M.; Podo, F.; Owen, C. S. J. Chem. Phys. 1975,63, 3661. (14) Miller, D. D.; Evans, D. F. J. Phys. Chem. 1989,93, 323. (15) Stroeve, P.; Miller, I. Biochim. Biophys. Acta 1975, 401, 157. (16) Teissie, J.;Tocanne, J.-F.;Baudras, A. Eur. J. Biochem. 1978,83, 77. (17) Loughran, T.; Hatlee, M. D.; Patterson, L. K.; Kozak, J. J.Chem. Phys. 1980, 72, 5791. (18)Subramanian, R.; Patterson, L. K. J. Am. Chem. SOC.1985,107, 5820. (19) Peters, R.; Beck, K. h o c . Nutl. Acad. Sci. U.S.A. 1983,80,7183. (20) Kim, S.; Yu, Hyuk. J. Phys. Chem. 1992, 96, 4034. (21) Meller, P.;Peters, R.; Ringadorf, H. Colloid Polym. Sci. 1989,267, 97. (22) Bohorquez, M.; Patterson, L. K. J. Phys. Chem. 1988,92, 1835.
problem with the lipids diffusing within the plane of the membrane. Studies of fluorescence quenching in twodimensionalmodel systems are of considerable interest as they can provide valuable information on the diffusional process in more complex structures, such as membranes, bilayers, and vesicles. The air-water monolayer is a particularly attractive model system for studying dynamic processes occurring in restricted molecular geometries. Various parameters such as molecular packing density, molecular composition, and molecular shape, and the nature of the subphase can be controlled, and the effect of these parameters on lateral diffusion can be readily examined. Early studies of lateral diffusion in monolayers spread at the air-water interface have yielded lateral diffusion coefficients a t least an order of magnitude larger than those reported for lipid bilayers and membranes,lS1' whereas more recent studies indicate that there is little difference between lateral diffusion in spread monolayers and b i l a y e r ~ . l g ~ ~ In recent years, a number of different approaches have been used to study lateral diffusion in spread monolayers. The most often used technique is that of fluorescence recovery after photobleaching (FRAP),495J6J921which involves monitoring the recovery of fluorescence due to fluorophores diffusing into a photobleached region. This technique, however, is complicated by minute temperature gradients which cause surface flow at the air-water interface which can perturb the radius of the photobleached region. An accurate value of this radius is required for calculation of a reliable value of the diffusion coefficient. In measurements of lipid probe diffusion, where the bleaching pulse must be applied quickly, it is also necessary to limit the fluorophore concentration carefully to minimize local heating which may perturb the results.l9." An alternative approach has been to derive the mutual diffusion coefficient from bimolecular reaction rate data ~~~~
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(23) Carum,F.;Grieaer, F.; Murphy, A.;Thistlethwaite,P. J.; Urquhart, R.; Almgren, M.; W i s h , E. J. Am. Chem. SOC.1991,113,4838. (24) Axelrod, D. Biophys. J. 1977, 18, 129.
0~43-~463/93/2~09-3~~2~~4.o0/0 0 1993 American Chemical Society
Diffusion of Amphiphiles in Fatty Acid Monolayers
Langmuir, Vol. 9, No. 11, 1993 3143
2200 trough was also used for time-resolved monolayer fluoresfor pyrene excimer formation. This technique has been cence experiments. used in a number of surfactant systems such as memAll surface pressure-area measurements were made by the branes,1° bilayers: and m ~ n o l a y e r s . ' ~ JThe ~ ~ ~pyrene ~ Wilhelmy hanging plate method.n For r-A measurements of excimer method is complicated by the possibility of dissociation of the e ~ c i m e r . This ~ ~ p o s ~ i b i l i t y ~is J ~ * ~ ~the pure amphiphiles and steady-state monolayer fluoreacence measurements, a 4.3-cm mica plate suspended from a Shinkoh commonly neglected, even though to positively exclude it 2-g-capacity strain gauge was used. The apparent changes in is difficult. Moreover, previous work using the pyrene weight with monolayer compreasion were converted to voltages excimer system has often been interpreted in terms of a by the strain gauge and recorded on an Apple Macintosh PC. A simple bimolecularrate constant. This, as has been shown 3.0-cm roughened platinum plate suspended from a Cahn by Owen,26is an oversimplification. microbalance was used in the time-resolved monolayer fluorescence measurements. The change in voltage from the microbalThe fluorescence quenching approach employed in this ance was monitored by a KSV-2200trough controllerand recorded study is free from the experimental difficulties present in on an Il3M PC with software from KSV, Helsinki. the FRAP technique, and unlike the pyrene excimer Experiments were initiated by f i i i g the trough with the formation method, it involves an irreversiblefluorescence appropriate subphase. Approximately 10'' moleculesfrom 1mM quenching reaction. It therefore provides an attractive chloroform solutions mixed to the desired ratio were spread technique for the determination of lateral diffusion dropwise onto the subphase, using a 100-pL SGE syringe. The coefficients in air-water monolayers. The possibility that solvent was then allowed to evaporate for 10 min, after which the the presence of probe molecules alters the host monolayer monolayer was compressed as desired. properties cannot be totally excluded; however, the Steady-State Monolayer Fluorescence Measurements. experiments undertaken show no macroscopic evidence Steady-state fluorescencemeasurements were performed on two for this in the monolayer systems investigated. different systems. The fiist, located at The university of using the fluorescence quenching Melbourne, employed a Perkin-Elmer LS-6 luminescence specIn a previous trophotometer, and details of the complete system have been approach, we observed lateral diffusion in dioleoyl-L-agiven previously.B Briefly, two silica fiber optic bundles were phosphatidylcholine (DOPC) monolayers by monitoring used to transfer the exciting light and the fluorescence to and the fluorescence quenching of a pyrene-bearing lipid by from the monolayer. Since the fluorescence signal from the an amphiphilicquencher. The lateral diffusion coefficient monolayer was small, it was necessaryto subtract the background was found to decrease with increasing surface pressure signal due to scatter from the subphase of the exciting light and/ and to approach in magnitude those observed in lipid or fluorescence from the PTFE. The background signal, monbilayers and membranes. itored at the same emission wavelengthas that of pyrenyl-DPPE, In order to gain a better kderstanding of lateral was recorded for 10 min, averaged, and then subtracted from the fluorescence signal when the monolayer was present. Fluoresdiffusion in monolayers,we have examined the same probe/ cence intensity curves as a function of monolayer compression quencher couple in fatty acid monolayers over an extended were obtained using this experimental setup (Figures 2 and 6). surface pressure range. The saturated (stearic) and In the second setup (University of Uppsala), a silica lens and unsaturated (oleic) fatty acids were chosen in order to mirrors were used to focus the excitation light from a pulsed (20 study the factors which govern the miscibility of N41pyrenylsulfony1)dipalmitoyl-L-cu-phosphatidylethanola- Hz) nitrogen laser (hm= 337 nm) (Laser Science, Inc., WSL337ND) onto the monolayer. The exciting light then passed mine (pyrenyl-DPPE) and 4-(N,N-dimethyl-N-hexade- through a quartz window in the bottom of the trough and into cyla"onium)-2,2,6,6-tetramethylpiperidine-l-oxyliodide a black box, which acted as a light sink, reducing the intensity (CAT-16) in the monolayer. We provide further evidence of the scattered light. The emissionwas collectedand transmittad that the results obtained from the fluorescencequenching to an optical multichannel analyzer (OMA,EE & G Model 1460) technique employed can be adequately interpreted within by means of a silica optical fiber, poeitioned at a right angle to the surface. Typical exposure times were 20 8. Details are the theoretical framework of diffusion-controlledquenchdescribed elsewhere.% Spectra obtained using this experimental ing in a two-dimensional environment.
setup are shown in Figures 3 and 4. T h 0 - % 6 O l V e d Monolayer Fluorescence Measurement 6. Fluorescencedecay curves were measured by the time-correlated Materials. N-(l-Pyrenylsulfonyl)dipalmitoyl-L-a-phosphati- single-photon-countingmethod.% The decay measurements were dylethanolamine (pyrenyl-DPPE) and 4(N,N-dimethyl-N-hexamade at the University of Uppsala using frequency-doubled (320 decylammonium)-2,2,6,6-tetramethylpiperidine-l-oxyliodide nm) radiation from a DCM dye laser (Spectra Physics Models (CAT-16) were purchased from Molecular Probes Inc. Stearic SP 375 and 3445) synchronously pumped by a mode-locked, N d acid (SA), obtained from Sigma Chemical Co., was purified by YAG laser (Spectra Physics Model SP 3800). The exciting light recrystallization from ethanol three times. Oleic acid (OA) was was focused onto the monolayer by means of a lens and mirrors purchased from Aldrichand sodium perchlorate (AR grade) from and passed through a quartz window in the bottom of the trough. Merck. Unless otherwise stated, all chemicals were used as The emission from the monolayer was focused by a fused silica received. All nonaqueous solvents were spectroscopic grade and lens onto a Hamamatsu (Model R15640) microchannel-plate were obtaind from Ajax chemicals or Merck. "Milli-B" water photomultiplier tube, having passed through a polarizer set so was used to prepare the subphase (conductivity
