J . Phys. Chem. 1987, 91, 5286-5291
5286
shielding of the carbon nuclei.44 Figure 12 shows that the resonances of the methylene carbons adjacent to the nitrogen atom are shifted downfield when octanol is added. If the observation is interpreted in terms of surfactant headgroup tilting, octanol decreases the angle of the headgroup with reference to the normal of the micelle. The O H group of octanol is probably located close to the positively charged nitrogen atoom on the micelle surface, which may affect the headgroup orientation. However, since the magnitude of the chemical shift change is rather small, other sources, such as contributions from changes in intermolecular interactions or solvent interactions, may be of importance. Figures 12 and 13 show that the effect on the chemical shifts of the surfactant headgroup is the same, independent of alcohol. Differences in magnitudes of the chemical shift changes on the headgroup can be related to the different alcohol molar ratios used. 5. Conclusions The interpretation of I3C relaxation and NOE data leads to (44) De Weerd, R.; De Haan, J.; Van den Ven, L.; Buck, H. J . Phys. Chem. 1982, 86, 2528.
the conclusion that the zwitterionic headgroup DAPS is more firmly anchored at the micellar surface as compared to headgroups of ionic surfactants, and that its local motions are also more restricted. Furthermore, the changes in I3C chemical shifts upon micellization indicate that the headgroup is tilted relative to the normal of the micellar surface. Addition of octanol has a large stabilizing effect of the micelle, decreasing the free surfactant concentration and increasing the micellar aggregation number. The influence of octanol on the order parameter and fast correlation time profiles is minor, demonstrating that the local molecular properties depend only weakly on the environment. However, I3C chemical shift measurements demonstrate that octanol does induce conformational changes of micellized DAPS.
Acknowledgment. We thank Professor M. Almgren and Dr. 0. Soderman for helpful discussions. Financial support from the Swedish Natural Sciences Research Council is gratefully acknowledged. The Knut and Alice Wallenberg foundation is thanked for a grant that financed the purchase of the XL-300 spectrometer. Registry No. DAPS, 15163-36-7; octanol, 11 1-87-5.
Photophysical Behavior in Spread Monolayers. Dansyl Fluorescence as a Probe for Polarity at the Air-Water Interface F. Grieser, P. Thistlethwaite,* R. Urquhart, Department of Physical Chemistry, University of Melbourne, Parkville, Victoria, 3052, Australia
and L. K. Patterson Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (Received: February 17, 1987)
The emission spectrum of N - [ 5-(dimethylamino)naphthalene-I-sulfonyl]dihexadecylamine(dansyldihexadecylamine) in monolayers at the air-water interface has been studied. In some cases sudden shifts in the dansyl emission can be correlated with particular features of the surface pressure-area isotherms. These spectral shifts can be explained in terms of a change in the conformation of the head group on the surface and with aggregation of the dansyldihexadecylamine. In other cases the dansyl emission shows a blue shift with increasing compression that can be associated with reduced head-group hydration.
Introduction Over the past 10 years there has been widespread use of fluorescent probes aimed at characterizing the interfaces of micelles, vesicles, and membranes, as well as active sites in biological macromolecules.’“ Shifts in the pK, of acid-base indicators away from the value in bulk solution have been used to determine the surface potentials of micellar ~ y s t e m s . ~In , ~such studies it has been customary to assign part of the pK, shift to “nonelectrostatic” factors. This nonelectrostatic effect has often been associated, in a somewhat vague way, with “the difference in dielectric properties” between water and the interfacial region. While there are many reasons to think that the dielectric constant itself is not a particularly appropriate parameter in this context, there has been general agreement in assigning to the interfacial region a (1) Law, K. Y . Phorochem. Photobiol. 1981, 33, 799. (2) Kalyanasundaram, K.; Thomas, J. K. J. Phys. Chem. 1977,81, 2176. (3) Azzi, A. Q.Rev. Biophys. 1975, 8, 237. (4) Loew, L. M.; Cohen, L. B.; Salzberg, B. M.; Obaid, A. L.; Bezanilla, F . Biophys. J . 1985, 47, 71. (5) Walsh Kinnally, K.; Tedeschi, H.; Maloff, B. L. Biochemistry 1978, 17, 3419. (6) Dederen, J. C.; Covsemans, L.; De Schryver, F. C.; Van Dormael, A. Photochem. Photobiol. 1979, 30, 443. (7) Drummond, C. J.; Warr, G. G.; Grieser, F.; Ninham, B. W.; Fennel1 Evans, D. J . Phys. Chem. 1985, 89, 2103. (8) Fernandez, M. S.; Fromherz, P. J . Phys. Chem. 1977, 81, 1755.
0022-36S4/87/2091-S286.%01 . S O / O
“polarity” lower than that of bulk Studies with polarity-sensitive probes have tended to support the conclusions of the pK, studies.’s2 It is not clear whether this reduced polarity is entirely explicable on the basis of the reduced concentration of water in an interface consisting of water and head group^,^ or whether there is an additional contribution associated with water structuring, due to the proximity of the hydrocarbon tail group^.^.'^ Alteration to the H-bond structure of the interfacial water by H-bonding head groups might also be significant. The polarity probe approach can in principle be applied to air-water monolayer films. The use of spread monolayers is of particular interest due to the extra degree of freedom provided by the ability to continually compress the monolayer and alter in a controlled way the interactions among components of the layer. Furthermore, this model system may, by means of force-area isotherms, be monitored for changes in molecular organization and even phase transitions. Recent work has demonstrated the feasibility of making steady-state fluorescence spectral measurements as well as time-resolved fluorescence measurements on air-water mono(9) Mukerjee, P.; Cardinal, J. R.; Desai, N. R. In Micellization, Solubilization and Microemulsions; Mittal, K. L., Ed.; Plenum: New York, 1977; Vol. 1 , p 241. (10) Ramachandran, C.; Pyter, R. A,; Mukerjee, P. J . Phys. Chem. 1982, 86, 3198.
0 1987 American Chemical Society
The Journal of Physical Chemistry, Vol. 91, No. 20, 1987 5287
Polarity at the Air-Water Interface
3
so2 I
-N H R--I-0-CH
I H ?C-
0
I1
7
0 - -0-
C HZ-CH~-;CH~)~ 40
bDOL R = DPL R =
* PJVVQ~A
Figure 1. Structures of dansyldihexadecylamineand various lipids.
Accordingly, we have been interested in studying the response of a widely used fluorescent polarity probe to incorporation in various monolayers and to variation in surface pressure. The essential question we wish to address is whether a polaritysensitive fluorescent probe located at the air-water interface ‘‘sees’’ a polarity lower than that of bulk water and, furthermore, how this polarity varies with the nature of the monolayer and the degree of compression. It might be anticipated that the solvation of head groups would be altered as the monolayer is compressed. Polarity probes of the aminonaphthalenesulfonate type which have been shown to be sensitive to H-bonding as well as p ~ l a r i t y ’might ~,~~ thus sense such changes. Changes in the orientation or position of the probe molecule in the monolayer with compression might also be detected. The molecule l-(dimethylamino)-5-naphthalenesulfonate (dansyl) has been widely used in polarity studies in biological systems.1619 The emission maximum shifts from ca. 440 nm in chloroform to ca. 510 nm in water, corresponding to a Stokes shift variation from 8200 to 11 400 cm-I,I9 This large polarity sensitivity, together with the compactness of the molecule, suggests dansyl would be a suitable probe for monolayer polarity studies. Dansyl has the additional advantages, for this sort of study, of high fluorescence quantum yield in all solvents and moderately high molar absorpti~ity.]~ These latter two factors are important considerations in monolayer studies where the fluorescence levels are very low. It has already been demonstrated that the emission maximum of N-dansylhexadecylamine attached to dipalmitoylphosphatidylcholine vesicles is sensitive to the gel/liquid-crystal phase transition.*O In the present work the long-chain derivative, N- [ 5-(dimethy1amino)naphthalene- 1-sulfonyl]dihexadecylamine (dansyldihexadecylamine)was studied alone and diluted in various other monolayers.
Experimental Section Dansyldihexadecylamine (DDHA) and N-[5-(dimethylamin0)naphthalene-1-sulfonyl]dipalmitoyl-L-a-phosphatidylethanolamine triethylammonium salt (dansyl-DPPE) were obtained from Molecular Probes. Dioleoyllecithin (DOL), dipalmitoyllecithin (DPL), and distearoylphosphatidic acid were (11) Subramanian, R.; Patterson, L. K. J . Phys. Chem. 1985, 89, 1202. (12) Agrawal, M. L.; Chauvet, J.-P.;Patterson, L. K. J. Phys. Chem. 1985, 89, 2979.
(13) Grieser, F.; Thistlethwaite, P. J.; Triandos, P. J . Am. Chem. Soc. 1986, 108, 3844. (14) Drew, J.; Thistlethwaite, P. J.; Woolfe, G. Chem. Phys. Lett. 1983, 96, 296. (15) Sadkowski, P. J.; Fleming, G. R. Chem. Phys. 1980, 54, 79. (16) Brand, L.; Gohlke, J. R. Annu. Reu. Eiochem. 1972, 41, 843. (17) Turner, D. C.; Brand, L. Biochemistry 1968, 7, 3381. (18) D d y , M. C.; Gotto, A. M.; Smith, L. C. Biochemistry 1982, 21, 28. (19) Li, Y.-H.; Chan, L.-M.; Tyer, L.; Moody, R. T.; Himel, C. M.; Hercules, D. M. J . Am. Chem. SOC.1975, 97, 3 1 1 8. (20) Iwamoto, K.; Sunamoto, J., Bull. Chem. SOC.Jpn. 1981, 54, 399.
60
80
A(hnolec)
Figure 2. Surface pressure-area isotherm ( T = 295 K) for dansyldihexadecylamine. (Inset) DDHA emission spectra at areas per molecule of (1) 66.1 A2, (2) 42.1 AZ,and (3) 31.8 A2. In this and the following figures the points at which spectra were recorded are indicated on the
isotherms. obtained from PL Biochemicals. All compounds were used as received. The structures of the probe and the various lipids are given in Figure 1. Measurements of pressure-area isotherms and fluorescence spectra were made on two different systems, one located at the Radiation Laboratory, University of Notre Dame, and the other at Melbourne University. Both systems involved a rectangular Teflon Langmuir trough with a motor-driven Teflon barrier. In both cases exciting light and fluorescence were conveyed between fluorometer and monolayer by fiber optic bundles. All spectra were corrected for scattering from the trough and aqueous subphase. All emission spectra were excited at 340 nm. In the Notre Dame system surface pressure measurements were made by the Wilhelmy plate method (using a 1-cm2piece of filter paper) with a Cahn 2000 electrobalance. The flu rometer was a Spex Fluorolog, photon counting model, giving spectra free from distortion by wavelength dependence of photomultiplier sensitivity. The Melbourne system again used the Wilhelmy plate method but with a mica plate and a Shinkoh 2 g capacity strain gauge as surface pressure transducer. The fluorometer was a PerkinElmer LS-5, microprocessor-controlled model, capable of multiple scanning and signal averaging. The usual procedure was to record 30 scans of both background and fluorescence in order to achieve a satisfactory signal-to-noise ratio. In a typical isotherm determination, on the order of lo1’ molecules were spread on the trough surface and the monolayer was compressed at ca. 3 A2 molecule-] m i d . All water was Milli-Q filtered. The efficacy of the cleaning procedures adopted was checked by recording the isotherm for stearic acid. All of the isotherms with the exception of that for the DDHA/stearic acid system were recorded in a thermally insulated cabinet flushed with water-saturated nitrogen. The temperature was accurately monitored by means of a thermocouple. The spreading solvent for DDHA and dansyl-DPPE was hexane. DPL and DOL solution were made up in chloroform and distearoylphosphatidic acid in hexane acidified with a trace of glacial acetic acid. For the mixed monolayers the two stock solutions were mixed so that the spreading solution was 20 vol % hexane in chloroform. The spreading solvents were allowed to evaporate prior to recording of the isotherms.
Results Dansyldihexadecylamine (DDHA) alone exhibits an unusual surface pressure-area isotherm (Figure 2 ) characterized by two surface pressure plateaux, of which the first is the more pronounced. Other evidence to be presented later suggests that this behavior can be associated with a change in the configuration of DDHA on the surface which is accompanied by a tendency toward aggregation. The first slowing in the rate of surface pressure rise occurred at ca. 9 mN m-l and an average area per molecule of 68 A2. The emission spectrum at various surface pressure values is shown in
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The Journal of Physical Chemistry, Vol. 91, No. 20, 1987
Grieser et al.
11
‘f Stokes shift
io
( ’ 0 0 0cm-’1
9
ii
loi
(mN m‘)
6
50
Figure 3. Variation of Stokes shift for DDHA with solvent ET(30) parameter. 0 , dioxane/water mixtures. A, other solvents: 1, hexane; 2, chloroform; 3, dimethylformamide;4, acetonitrile; 5, butanol; 6, eth-
anol: 7 methanol. Figure 2 (inset). The emission maximum is at ca. 553 nm at a surface pressure of 9.4 m N m-I and shifts to 527 nm at a surface pressure of 17.1 mN m-’ (area per molecule 42.1 A2). At the same time, the intensity of the emission rises, the rise being approximately that expected on the basis of the number of molecules per unit area, which of course changes with compression. In an attempt to relate the observed emission maximum to polarity, the absorption and emission spectra of very dilute solutions of DDHA were recorded for a range of solvents and dioxane/water mixtures. The results are shown in Figure 3, where the Stokes shift (taken as the difference between the frequencies of the absorption and emission maxima) is plotted against solvent ET(30) parameter.21 As the solvent changes from hexane to ethanol, the Stokes shift rises approximately linearly. Acetonitrile and dimethylformamide give somewhat larger Stokes shifts than would be expected from their ET(30) values. The behavior in dioxane/water mixtures is similar at low ET(30) values. However, at an ET(30) value of ca. 50 kcal mol-] in the mixed solvent, the Stokes shift stops rising and further ET(30) increase leads to a sharp fall. At the same point the absorption spectrum changes. The absorption maximum shifts to ca. 329 nm and now shows a slower tailing off to longer wavelengths. In the pure solvents, and the dioxane/water mixtures below an ET(30) of 50 kcal mol-], the absorption maximum stays approximately constant at ca. 340 nm. The choice of the ET(30) parameter as the abscissa in Figure 3 is based on the previous observation that the Stokes shift of the dansyl chromophore correlates well with this parameter.I9 The correlation with the dielectric constant is less good, although the behavior is qualitatively similar in that the same initial rise followed by a sudden drop is seen. We attribute this sudden drop in Stokes shift to the formation of aggregates. The sudden change in the absorption spectrum is also compatible with this idea. Moreover, aggregate formation in H20-MeOH solutions of N-dansylhexadecylamine has already been reported above a water volume fraction of 0.4.20 The isotherm obtained for 22.7 mol % DDHA in dioleoyllecithin (DOL) is shown in Figure 4. A concentration of DDHA of 20 mol % is necessary to achieve an adequate fluorescence intensity. In this case the isotherm is similar to that obtained for DOL alone but shows an inflection at a surface pressure of 52 m N m-l, corresponding to an area per molecule of 74 A2. The highest surface pressure recorded was 68 m N m-l, at which the area per molecule was 63 A2. This is only slightly less than the area found for pure DOL.22 In this case, as Figure 4(inset) shows, the dansyl emission maximum remains constant at 550 nm until quite high surface pressures and then shifts to 520 nm after the inflection. (21) Reichardt, C. Angew. Chem., Int. Ed. Engl. 1979,18, 98. (22) Patterson, L K., unpublished data
100
150
A (@molec) Figure 4. Surface pressure-area isotherm ( T = 295 K) for 22.7 mol 94 DDHA in DOL. (Inset) DDHA emission spectra at average areas per molecule of (1) 97 A2,(2) 79 AZ,and (3) 63.4 A2.
I
I 40
60
60
A (ff/molec) Figure 5. Surface pressure-area isotherm ( T = 296 K) for 25 mol 9 i DDHA in DPL. (Inset) DDHA emission spectra at average areas per molecule of (1) 66.3 A2, (2) 50.8 A2, and ( 3 ) 40.2 A2. Figure 5 shows the isotherm obtained for a monolayer consisting of 25 mol % DDHA in dipalmitoyllecithin (DPL). The isotherm exhibits inflection points at 25 mN m-l (area 62.8 A2), 35.6 mN m-I (area 54.6 A2), 53 mN m-* (area 45.1 A2), and 57.6 mN m-l (area 44.0 A*). The first slowing in the rate of surface pressure rise is similar to, but less pronounced than, one that occurs in pure DPL and which is associated with a phase t r a n s i t i ~ n . * ~It
