Langmuir 1989,5, 883-885 3u
2u
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Time under water (minutes)
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Figure 2. Transfer ratio of lead stearate as a function of the time the film has been under water. This time is equal to 2ND/v,where N is the cycle number, u the dipping rate, and D the distance between the position under consideration and the top edge of the deposited film. Data from Figure 2 of Peng et ala? 0 , N = 5; 0 , N = 6;X, N = 7 ; m, N = 8; 0,N = 9. Figure 1. Schematic presentation of X-type film formation. Id shows the situation after the first downward trip; 2u shows the situation after the second upward trip, etc. The picture after the first upward trip (lu) is not shown. It is to be noted that on the upward trip deposition occurs only on molecules with their head toward the water. This way half of “the disorder” disappears during the upward trip.
Furthermore, it should be noted that when esters are deposited pure X-type films are formed.BJ X-ray analysis showed that in these cases the structure is head tail head tail, etc. The explanation could be that there is good adherence between the long chain and the small head (6) Stenhagen, E. Trans. Faraday SOC.1938,34, 1328. (7) Enkelmann, V.;Lando, J. B. J. Polym. Sei. 1977,15, 1843.
group and not so much between head groups.2 Hence, in a qualitative sense we do understand the experimental results obtained by Peng et al. about the Xtype character of their LB films. These results also support Honig’s theory on the mechanism of X-type film formation. In ref 2 a theory is presented on how the transfer ratio depends on the various interactions of the deposited molecules with each other and with water. This, however, is an equilibrium theory that tells only what should happen if one waits long enough to establish equilibrium. In order to produce a quantitative explanation of the data presented in ref 1, a kinetic theory is needed.
Ordering of Azobenzene-Containing Amphiphile on Two-Dimensional Lattice of Bacteriorhodopsin Tatsuo Katsura Research Institute for Polymers & Textiles, 1 - 1 - 4 Higashi, Tsukuba, Ibaraki 305, Japan Received March 1, 1989 A bilayer-forming, non optically active cationic dye, 1, mixed with reconstituted bacteriorhodopsin (BR)-lipid vesicle systems showed induced positive and negative CD bands in its 357-nm absorption band, only when BR aggregated into the same hexagonal lattice as in the purple membrane of Halobacterium halobium. This lattice-dependent induced CD suggests ordering of dye molecules on the two-dimensional array of BR.
Introduction Efforts have been made to understand the mechanism of adsorption and spontaneous organization of amphiphilic molecules on solid surfaces, in order to develop new methods of preparing two-dimensional organic assemblies.’ Metals and metal oxides are usually used as solid surfaces, on which interaction sites for chemisorption of amphiphilic molecules are fixed. However, in the study of spontaneous organization of amphiphilic molecules on a surface, it will be interesting to introduce a surface where charged interaction sites form a two-dimensional array in a controllable way. (1) Allara, D.; Nuzzo, R. G . Langmuir 1988,1,45-52.
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Bacteriorhodopsin (BR), of which function is known as a light-driven proton pump,2 forms almost perfect twodimensional crystals in the purple membrane (PM) of Halobacterium h a l ~ b i u m . ~Furthermore, it has been shown that BR, when incorporated into lipid vesicles, changes its aggregational state if the lipid undergoes a phase t r a n ~ i t i o n . ~ Above the transition temperature T,, BR is in the monomeric state, and below T,, BR aggregates (2)Stoeckenius, W.; Bogomolni, R. A. Annu. Rev. Biochem. 1982,52, 587-616. (3)(a) Henderson, R. J . Mol. Biol. 1975,93,123-138. (b) Blaurock, A. E. J. Mol. B i d . 1975,93,139-158. (4) (a) Heyn, M. P.; Cherry, R. J.; Dencher, N. A. Biochemistry 1981, 20,840-849. (b) Cherry, R. J.; Muller, U.; Henderson, R.; Heyn, M. P. J. Mol. Biol. 1978,121,283-298.
0 1989 American Chemical Society
884 Langmuir, Vol. 5 , No. 3, 1989
Letters
-4 I
-5
400 500 Wavelength (nm)
300
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Figure 1. Effect of temperature on the CD spectrum of 1 in the
BR-DMPC-DHPC vesicle systems. The molar ratio of 1 to BR was 14 and that of lipid to BR was 54. The spectra were recorded with a JASCO 5-600 spectropolarimeter using a water-jacketed 10-mm cell: -, 25 "C; -, 20 "C; ---,15 "C; ---, 10 "C; --, 5 "C. into the same hexagonal lattice (P3symmetry) as in the PM; this change can be easily monitored by observing the CD spectrum of the retinylidene chromophore of BR.4 Thus, BR-lipid vesicles provide a well-characterized system in which charged sites of BR form a regular surface lattice in a controllable fashion. This letter reports lattice-dependent ordering of amphiphilic molecules at the surface of BR-lipid vesicles. Such ordering was manifested by induced circular dichroism (CD) in the absorption band of a non optically active dye. Bilayer-forming cationic a m ~ h i p h i l ep, ~- [( W (trimethylammonio)pentyl)oxy]-p'-(dodecy1oxy)azobenzene bromide (1)) an azobenzene-containing dye, was used.
c H,(C
H
o
9 N
7 H3
=
N 0-(C ~ HJ~-
v-CH,B!
1
Experimental Section The purple membrane (PM) fragments were prepared from H. halobium strain MIRl cells as described by Oesterhelt and Stoeckenius.6 BR-lipid vesicles were prepared by a technique' in which dimyristoylphosphatidylcholine(DMPC, T,= 23 "C) was used as a long-chain lecithin and diheptanoylphosphatidylcholine (DHPC) as a short-chain lecithin. A small amount of ethanol solution of 1 (ca. 2 mM) was added into preformed BR-DMPC-DHPC vesicles suspended in 150 mM KCl. CD and UV-vis spectra were measured with a JASCO 5-600 Spectropolarimeterand a Shimazu MPS-2OOO spectrophotometer, respectively. Before spectroscopic measurement, 1was completely converted into the trans isomer by irradiating the sample with visible light through a Y-51 color filter (Toshiba). All amphiphiles used were obtained from Sogo Pharmaceutical Co. (Tokyo,Japan) and used without further purification. Results and Discussion Figure 1shows the temperature dependence of the CD spectrum of 1 and BR in the BR-DMPC-DHPC vesicle system. The CD band(s) of retinylidene chromophore in BR between 450 and 650 nm clearly demonstrates that BR molecules exist in the monomeric state at 25 "C and that they gradually aggregate into the hexagonal surface lattice (5) (a) Kunitake, T.; Okahata, Y. J. Am. Chem. SOC. 1977, 99, 3860-3861. (b) Kunitake, T.; Nakashima, M.; Shimomura, M.; Okahata, Y.; Kano, K.; Ogawa, T. J. Am. Chem. SOC.1980,102,6644-6646. (6)Oesterhelt, D.;Stoeckenius, W. Methods Enzymol. 1974, 31, 667-671. (7)Dencher, N.A. Biochemistry 1986,25,1195-1200.
400
500
6 00
700
Wavelength(nrn)
Figure 2. Effect of temperature on the UV-vis absorption spectrum of 1 in the BR-DMPC-DHPC vesicle systems. The same preparation as the CD measurements was used: -., 25 "C; --, 20 "C; -*-, 15 "C; ---,10 "C; -, 5 "C.
when the temperature is lowered from 25 to 5 0C.4 In parallel with the formation of BR aggregates, paired positive and negative CD bands having peaks a t 380 and 330 nm, respectively, appeared and increased their intensity; the negative 330-nm band developed and was superimposed on the existing band of BR. It should be noted that complete reversibility of this phenomenon was confirmed by raising the temperature from 5 to 30 "C. Since the UV absorption maximum of 1 exists at 357 nm, and appearance and disappearance of the CD bands were in parallel with the formation and disassembling of the hexagonal lattice of BR, it is natural to consider that the origin of the induced CD bands is related to the formation of some ordered structure of 1 on the surface lattice. Furthermore, the synchronous appearance of positive and negative CD bands strongly suggests that these bands are induced by the exciton coupling interaction8 between 1. The induction of CD bands through an exciton coupling mechanism requires some characteristic mode of interaction between dye molecules; random orientation of dye cannot induce CD bands. Furthermore, no induced CD will be expected if chromophores of 1bound to BR surface are coplanar or if they are oriented parallel or perpendicular to each other. A model for interpretation of the induced CD is as follows: (i) At higher temperature, a certain number of 1 are bound to the surface of monomeric BR molecules through electrostatic interaction and take an aggregated structure due to hydrophobic interaction between 1. In this state, there is no CD induced in the absorption band of dye molecules, but the absorption spectrum of 1 may be different from that of 1 merely dispersed in aqueous solution. (ii) As the temperature is lowered, clusters of 1 bound to different BR molecules are brought into members of regular array in consequence of the formation of a surface lattice of BR. This ordering of dye molecules generates an effective interaction between them and results in the induction of CD bands in the absorption band of 1, but it does not necessarily cause the substantial change in its absorption spectrum. It is worthwhile to mention that this model is an extension to two dimensions of the observation of Blout and Stryer? who first demonstrated that the helix formation of a single polypeptide molecule induces optical activity to a symmetrical dye bound to the (8) Tinoco, I., Jr. Radiat. Res. 1963,20,133-139. (9)(a) Blout, E.R.; Stryer, L. R o c . Natl. Acad. Sci. U.S.A. 1959,45, 1591-1593. (b) Stryer, L.;Blout, E. R. J. Am. Chen. SOC.1961,83, 1411-1418.
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polypeptide; the helix formation corresponds to the formation of the surface lattice in our model. The following results seem to validate this model. The temperature dependence of UV-vis absorption spectra of the BR-lipid vesicles is shown in Figure 2. As reported earlierpbthe absorption maximum of retinylidene chromophore a t around 550 nm shifted to longer wavelength with the monomer to trimer transition of BR. On the other hand, the UV absorption maximum of 1 in the BR-lipid vesicle systems persisted a t 357 nm in the whole temperature range studied; furthermore, the shape of the band, which is different from that of 1 dispersed either in ethanol or in 150 mM KC1, changed little. When 1 was added to the PM suspension, induced CD bands similar to the present observation appeared a t around 360 nm in addition to those of BR in the CD spectra.l0 In contrast with BR-lipid vesicle systems, the
induced CD bands were always observed between 5 and 30 O C ; this demonstrates again that the existence of the BR lattice is a prerequisite for the ordering of 1 and hence the observation of the induced CD bands.“ In conclusion, we have demonstrated that BR-lipid vesicle systems provide another aspect in the study of spontaneous organization of amphiphilic molecules, by showing a lattice-dependent ordering of amphiphilic dye molecules a t the surface of BR-lipid vesicles. Such ordering was manifested by the induced CD in the absorption band of non optically active dye. This phenomenon may be utilized to monitor an ordering state of proteins in biological membranes.
(10) Katsura, T., presented in part at the Sixth International Conference on Surface and Colloid Science, Hakone, Japan, June 1988, and will be reported separately.
(11)Mukohata and Ihara observed induced CD of diaminoacridine bound to the PM. Retinal proteins; VNU Science Press: 1987; pp
Acknowledgment. I am grateful to Drs. M. Ataka, H. Maeda, M. Ami, and T. Sakai for their helpful discussions.
195-204.
Comments Comments on “Pyrene Fluorescence as a Probe of Fluorocarbon Micelles and Their Mixed Micelles with Hydrocarbon Surfactants” The work of Kalyanasundaraml discusses the fluorescence of pyrene in micelles of fluorocarbon surfactants and in mixed micelles of fluorocarbon + hydrocarbon surfactants. In essence, the conclusions reached in this work are in agreement with those reached in the paper “Aggregation Behavior of Mixed Fluorocarbon and Hydrocarbon Surfactants in Aqueous Solution”, which we recently published.2 However, noting the limiting solubility of pyrene in fluorocarbon surfactant micelles, Kalyanasundaram rightly concludes that the pyrene excimer method cannot and should not be used for the determination of the micelle aggregation number, N,of fluorocarbon surfactants. Unfortunately, hia paper contains two statements which imply that we did use this method for measuring aggregation numbers of lithium perfluorooctanesulfonate (LiFOS) micelles. These statements are the following: (i) “During the preparation of this manuscript, Zana et a1.2 have reported on the use of pyrene fluorescence in perfluorooctanesulfonate micelles, and, except for the
(1)Kalyanasundaram, K. Langrnuir 1988,4,942. (2) Muto, Y.;Eaumi, K.; Meguro, K.; Zana, R. J. Colloid InterfaceSci. 1987,120, 162.
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formation and use of pyrene excimers, their results are in good agreement with ours” (page 942). (ii) “Very recently Zana et aL2 have used the pyrene excimer method to determine the aggregation number of perfluorooctanesulfonatemicelles. No information however has been provided on the concentration range of pyrene used or on the details of the excimer fluorescence” (page 944). These statements, together with the author’s description of his unsuccessful attempts to enforce excimer emission and his conclusion that the pyrene excimer method cannot be used with fluorocarbon surfactants, cast doubts on the values of the aggregation numbers of LiFOS micelles which we reported.2 We therefore feel compelled to correct the above two misquotations of our work by the following comments. (i) We had, of course, noticed the restricted solubility of pyrene in LiFOS micellar solutions. However, we state that “LiFOS solutions could solubilize a much larger amount of pyrene (than water), for instance more than 2 X M at [LiFOS] = 0.1 M”. Such a concentration gave rise to a negligible excimer emission but was sufficient for “performing time-resolved fluorescence studies of quenching of pyrene fluorescence by dodecylpyridinium ion in order to measure the aggregation number of LiFOS micelles” (quotations from page 164 of ref 2). (ii) As was just quoted, the pyrene excimer method was not used to measure LiFOS aggregation number. This is clearly stated in page 163 of our paper (ref 2) as follows: “The excimer studies were performed in the absence of 0 1989 American Chemical Society
