2998
J. Phys. Chem. 1981, 85,2998-3003
Steady-State and Time-Resolved Fluorescence Studies on Energy Transfer in Anionic Surfactant Membranes Kojl Kano;
Hlrofuml Kawazuml, and Tellchlro Ogawa
Department of Molecular Science and Technology, Graduate School of Engineering Sciences, Kyushu lJnivers& Fukuoka 8 12, Japan (Received: May 27, 198 1)
Extremely efficient singlet-singletenergy transfer from pyrene to proflavin has been observed in dicetyl phosphate membrane suspensions. The acceptor-concentrationdependence on the energy-transfer efficiency and the nonexponential decay curves of pyrene fluorescence were interpreted in terms of the theory of two-dimensional energy transfer without effect of diffusion. The effects of the membrane structure on the energy-transfer probability have been discussed.
Introduction Recent studies have revealed that some amphiphiles with single and/or double alkyl chains form biomembrane-like aggregates.l Dialkyldimethylammonium halides are typical amphiphiles which form bilayer membranes in their dilute aqueous solutions. The characteri z a t i ~ n ~and - ~ the catalytic effectsb8 of these cationic surfactant membranes have been studied. Most of the sonic suspensions of the dialkyldimethylammoniumhalides provide multilamellar vesicles, and each of them has a characteristic gel-liquid crystalline phase transition temperature (TJ.The cationic surfactant membranes accelerate the base-catalyzed hydrolyses of the ester^,^?^ the decarboxylation of 6-nitrobenzisoxazole-3-carboxylate,’ and the energy transfer from lysopyrene to pyranine.s In contrast with the cationic surfactant membranes, little has been reported on the formation and the characterization of anionic surfactant membranes. Mortara et al. have found the formation of dicetyl phosphate (DCP) vesicles by electron microscopy and gel filtration when DCP is sonicated in water at 55 0C.9 Czarniecki and Breslow have characterized the didodecyl phosphate membrane by using a photochemical probe technique $nd concluded that the cationic surfactant molecules in their bilayer membrane vesicles are considerably disordered.1° Fendler and his co-workers reported the effects of the DCP aggregates on the photoinduced electron transferal1 The present study deals with the effects of the DCP membranes on the fluorescence energy transfer. Forster-type energy transfer12J3 as well as collisional (1)For review, see: Kunitake, T. J. Macromol. Sei., Chem. 1979,A13, 587402. Fendler, J. H.Acc. Chem. Res. 1980,13,7-13. (2)Kajiyama, T.; Kumano, A.; Takayanagi, M.; Okahata, Y.; Kunitake, T. Chem. Lett. 1979,645-8. (3)Nagamura, T.;Mihara, S.; Okahata, Y.; Kunitake, T.; Matsuo, T. Ber. Bunsenges. Phys. Chem. 1978,82,1093-8. (4)Kano, K.; Romero, A,; Djermouni, B.; Ache, H. J.; Fendler, J. H. J. Am. Chem. SOC. 1979,101,4030-7. (5)Kunitake, T.; Sakamoto, T. J. Am. Chem. SOC. 1978,100,4615-7. (6)Okahata, Y.;Ando, R.; Kunitake, T. Bull. Chem. SOC. J p n . 1979, 52,3647-53. (7)Kunitake, T.;Okahata, Y.; Ando, R.; Shinkai, S.; Hirakawa, S. J. Am. Chem. SOC. 1980,102,7877-81. (8)Nomura, T.;Escabi-Prez, J. R.; Sunamoto, J.; Fendler, J. H. J.Am. Chem. SOC. 1980,102,1484-8. (9) Mortara, R. A.; Quina, F. H.; Chaimovich, H. Biochem. Biophys. Res. Commun. 1978,81,1080-6. (10)Czarniecki, M. F.; Breslow, R. J. Am. Chem. SOC.1979, 101, 36754. (11)Escabi-Prez, J. R.;Romero, A.; Lukac, S.; Fendler, J. H. J . Am. Chem. SOC. 1979,101,2231-3. (12)Aso, Y.;Kano, K.; Matsuo, T. Biochim. Biophys. Acta 1980,599, 403-16.
fluorescence quenching1s15 may provide useful information not only on the binding sites of chromophores in membranes but also on the dynamic behavior of membranes. In this study, pyrene, which is expected to be located at the hydrophobic alkyl chain region of the DCP membrane, was used as an energy donor, and proflavin, a water-soluble cationic dye, was used as an energy acceptor. Proflavin should be bound electrostatically to the anionic head group of the DCP membrane. Steady-state and nanosecond time-resolved fluorometry was used to analyze the energy transfer. Since the fluorescence lifetime of pyrene in the absence of an energy acceptor (quencher) is quite long, the fluorescence decay curves of an energy donor can be measured easily by using a pulsed nitrogen laser as an exciting light source. Experimental Section Materials. DCP and synthetic dipalmitoyl-D,L-aphosphatidylcholine (DPPC) were used as received from Sigma Chemical Co. The purity of DCP was determined by elemental analysis to be satisfactory. Pyrene (Nakarai) was carefully purified by means of silica gel column chromatography with cyclohexane as an eluent. Proflavin (3,6-diaminoacridinehemisulfate, Wako) was recrystallized from aqueous methanol. 1,3-Di(l-pyrenyl)propane(P3) was prepared and purified by H. Goto in this laboratory according to the procedures described in the literature.18 SpectroscopicMeasurements. Uncorrected fluorescence emission and excitation spectra were measured on a Hitachi 650-10s spectrofluorometer whose cell compartment was thermostated. Corrected emission spectra for determining the fluorescence quantum yield (af)of pyrene were measured on a Hitachi 650-409 spectrofluorometer with a microcomputer. The fluorescence decay curves were obtained by using a pulsed nitrogen laser (10-ns pulse width) as described in the previous paper.16 Absorption spectra were taken on a Jasco UVIDEC 505 spectrophotometer. The negative-stained electron micrographs of the DCP aggregates were taken on a Hitachi H-500 electron microscope. Preparation of DCP and DPPC Membranes. In a Pyrex test tube, 0.4 mL of 1 X M pyrene in chloroform was (13)Haigh, E. A.; Thulborn, K. R.; Sawyer, W. H. Biochemistry 1979, 18,3525-32. (14)Kano, K.;Kawazumi, H.; Ogawa, T.; Sunamoto, J. Chem. Phys. Lett. 1980,74, 511-4. (15)Kano, K.; Kawazumi, H.; Ogawa, T.; Sunamoto, J. J. Phys. Chem., in press. (16)Zachariasse, K. A.;Kuhnle, W. 2.Phys. Chern. (Frankfurt am Main) 1976,101,267-76.
0022-365418112085-2998$01.2510 0 1981 American Chemical Societv
Energy Transfer in Anlonic Surfactant Membranes
The Journal of Physical Cfmmlstw,Vol. 85, No. 20, 1981 2999
A,""'
Figure 2. Absorptkm (-)
-
and uncorrected emission spectra (- -)
of pyrene and proflavin in sodium dodecyl sulfate (0.2 M) micellar sdutbns. The emspectrum of pyrene (5 X 10.' M) and pdlavk, (2 X lod M) were measured upon excitation at 338 and 456 nm. respectively. -7.
N@aibsiaii-
of dicetyl phosphate membrane.
'
ac'ij)electron miaographo
placed and chloroform was removed by a stream of nitrogen gas. To the residue in the tube were added 21.9 mg (4 X lo6 mol) of DCP and 5 mL of pH 8.0 Tris buffer (0.05 MI, and the mixture was sonicated with a bath-type sonicator (Bransonic 12,50 w) at 55-60 "C untiJ the solution became almost clear. During sonication, the solution was bubbled with nitrogen gas. The resulting DCP solution was diluted to 20 mL with the same buffer. The final concentrations of pyrene and DCP were 2 X 104 and 2 X M, respectively. The method of preparation of pyrene-embedding DPPC liposomes was the same as that described in the previous In this case, pH 7.0 phosphate buffer (0.01 M) containing 0.1 M NaCl was used in place of the Tris buffer. The final concentrations of pyrene and DPPC were 5 X 10" and 1 X M, respectively. In these two systems, pyrene shows only monomer emission upon excitation, no excimer emission being observed. Energy Transfer. After addition of a n appropriate volume of aqueous proflavin stuck solution (55 X ltT3mL), the membrane suspension (2 mL) was resonicated at 55-60 OC for several minutes. The sample placed in a side arm of a quartz cell (1-cm optical path) was cooled on an ice bath, and the system was evacuated to remove air before admitting the nitrogen gas prior to the fluorescence measurements. The emission spectra were measured upon excitation of pyrene molecules (energy donor) at 338 nm. The excitation spectra were monitored at 510 nm, which is the emission maximum of proflavin (energy acceptor). The energy. transfer efficiency (E, 7'0)was determined from the ratio of the fluorescence intensity of pyrene in the presence of proflavin to that in its absence: $7 E = lOO(1 - l / l o ) (1) The fluorescence intensity in the presence of proflavin, I, was corrected for the reabsorption of pyrene fluorescence due to proflavin by the method described in the literature.l* Since the optical densities of the samples for energy-transfer experiments in the DCP membrane sus(17) Thomaa, D.D.;Carlson, W. F.;Stryer, L. Roc. Natl. Acad. Sci. U.S.A. 1978, 75, 5746-50. (18)E~tep,T. N.; Thompaon, T. E. Biophys. J. 1979, 26, 195-208.
pensions were less that 0.1, an inner-filter effect could he negligible. All of the present experiments were undertaken at 25 "C unless otherwise noted.
Results Electron Microscopy of DCP Membrane. Figure 1 shows the negative-stained (phosphotungsticacid) electron micrographs of the DCP membrane which was prepared by sonicating DCP in pH 8.0 Tris buffer. A small multilamellar structure with a length of 200-700 A and a ~ which is different from thickness of 50-200 A w a observed, the result reported by Mortara et a1.9 Mortara et al. ohserved the formation of unilamellar vesicles with an average diameter of 500 A when DCP in water is sonicated (an immersion-type sonicator, 80-90 W) and centrifuged (1h, 8000g).9 Although we tried to reproduce the result of Mortara et al. by using a bath-type Sonicator (50 W), no optically clear suspension was obtained. The diffrence in intensity of sonicators and in medium (water and Tris buffer) may cause the difference in structure of the DCP membranes. Determinution of Critical Transfer Distance. The absorption and emission spectra of pyrene and proflavin in sodium dodecyl sulfate (SDS) micellar solutions are shown in Figure 2. The overlap of the absorption spectrum of proflavin with the emission spectrum of pyrene predicts that the singlethinglet energy transfer occurs from pyrene in the excited state to proflavin in the ground state. The critical transfer distance (Ro) is given by the following Forster equation: 14-21
where P is an orientation factor, usually taken as 2 / 3 for a random distribution, +f is the absolute fluorescence quantum yield of the donor, n is the refractive index of the medium, N is Avogadro's number, and fD(u) and cA(u) are the spectral distributions of emission of the donor and of absorption of the acceptor, respectively, on a wavenumber scale. For calculating Ro, we used Kz = 2/3, n = 1.34, and = 0.53. Since the absorption spectrum of proflavin in the DCP membrane suspension was somewhat incorrect because of weak light scattering, we determined (19) Fbrster, T.2.Naturforsck. A 1949, 4, 321-7. (20) Fbrster, T. Discuss. Faraday Soe. 1959.27, 7-17. (211 Fbmter, T. In "ModernQuantum Chemistry";Sinanoglu,O., Ed.: Academic Press: New Yoik, 1965, p 93.
3000
The Journal of Physlcal Chemistry, Vol. 85, No. 20, 1981
Kano et ai. a
0,b
20
30
40
Temperature , 'c
50
60
Figure 3. Fluorescence intensity ratios of excimer to monomer for 1,3dl(l-pyrenyl)propane (5 X lo-' M) in (0) dicetyi phosphate (2 X lo4 M) and (A) dipalmitoyiphosphatidyichoiine(1 X lo3 M) membrane suspensions as a function of temperature. The probe was excited at 332 nm, and the excimer and monomer emission Intensities were measured at 480 and 378 nm, respectively,under anaerobic conditions.
the Ro value for the pyrene-proflavin system in SDS micellar solution. Both emission and absorption spectra in the SDS micellar solutions were virtually identical with those in the DCP membrane suspensions. The fluorescence quantum yield of pyrene in the SDS micellar solution (af= 0.53) was determined by using quinine sulfate (1X lo4 M in 1.0 M H2S04)as a standard. The calculated Ro value was 38 A. Determination of Gel-Liquid Crystalline Phase Transition Temperature of DCP Membrane. A gel-liquid crystalline phase transition temperature, T,, is one of the fundamental physical parameters for studying biological and artificial membranes. It has been demonstrated that intramolecular excimer and exciplex systems are sensitive Then probes for determining the T, of a we determined the TC)sof the DCP and DPPC membranes by using 1,3-di(l-pyrenyl)propane(PJ as a probe. Figure
p3 3 shows the ratios of the fluorescence intensities of the intramolecular excimer to those of the monomer of P3 ( I E / I M ) as a function of temperature. The I E / I M values in the case of the DPPC sonicated liposomes abruptly changed at 41 "C, which corresponds to the T, of the DPPC liposome. On the other hand, a gradual increase in the I E / I M values was observed above 35 "C in the case of the DCP membrane, indicating that the DCP membrane has a wide temperature range where the gel and liquid crystalline phases are separated from each other. Since all experiments of energy transfer were carried out at 25 "C, the DCP membranes should be in the gel phase under the experimental conditions. (22) Georgescauld, D.; DesmasBz, J. P.; Lapouyade, R.; Babeau, A.; Richard, H.; Winnik, M. Photochern. Photobiol. 1980, 31, 539-45. (23) Zachariasse, K. A.; Kuhnle, W.; Weller, A. Chern.Phys. Lett. 1980, 73,6-11. (24) Kano, K.; Goto, H.; Ogawa, T. Chern. Lett. 1981, 653-6.
A .nm
h .nm
Figure 4. Changes in the fluorescence emission (a) and excitation spectra (b) upon addition of proflavin into the pyrene-embeddlng dicetyl phosphate (2 X M) membrane in pH 8.0 Tris buffer (0.05 M) at 25 OC. Pyrene (2 X lo-' M) was excited at 338 nm under anaerobic conditions. The concentrations (XIOd M) of profhvin were as follows: 0, 1.0, 2.4, 4.0, 6.0, 8.4, and 11.6. The excitation spectra were monitored at 510 nm. The intensities of the excitation peaks corresponding to the absorption maxima of pyrene increased with increasing profiavin concentrations.
TABLE I: Degrees of Fluorescence Polarization of Roflavin at 510 nm under Various Conditions at 25 "Ca system 106[proflavin], M Pb
H*O
DCP without pyrene DCP with pyrene DPPC without pyrene DPPC with pyrene
8 8 8 40 40
0.007 0.24 0.022 0.039 0.034
a The concentrations of pyrene in the DCP ( 2 X lo-' M ) and DPPC (1 X lo-' M ) membrane systems were 2 X lo-' and 5 x 10.' M, respectively. Degrees of fluorescence polarization were determined according to the following equation: P = [Iii II - ( I i i i . ~ i i i ) I i ~ ) l /iI.~ Uiii.Iiii)IiiL ~!~~ In all cases, the fluorophores were excited at 338 nm.
Energy-Transfer-Steady-State Fluorescence Measurements. The effective fluorescence quenching of pyrene accompanying an appearance of the proflavin fluorescence was observed upon addition of proflavin into the pyreneembedding DCP membrane suspensions (Figure 4a). In the excitation spectra monitored at 510 nm (Amm of the proflavin emission), the peaks corresponding to the pyrene absorption maxima were obviously observed at 322 and 338 nm (Figure 4b), suggesting that the energy transfer from excited pyrene to proflavin occurs. The well-depolarized fluorescence from sensitized proflavin also suggests that the singlet energy of pyrene transfers to randomly distributed proflavin. The degree of the fluorescence polarization (P)was determined by the procedures described in the The results are shown in Table I. The P value in the DCP membrane suspension in the absence of pyrene (0.24)is significantly larger than that in water (0.007). The electrostatic binding of cationic proflavin to the negatively charged head group of the DCP membrane may restrict the motion of proflavin. On the other hand, the fluorescence of proflavin (25) Price, J. M.; Howerton, H. K. AppE. Opt. 1962, 1, 521-33. (26) Shinitzky, M.; Dianoux, A X . ; Weber, G. Biochemistry 1971,10, 2106-13.
The Journal of Physical Chemistry, Vol. 85, No. 20, 1981 3001
Energy Transfer in Anionic Surfactant Membranes
TABLE 11: Fluorescence Quenching Parameters for Various Systems at 25 "C fluorophore
quencher
medium
pyrene pyrene pyrene pyrene pyrene
proflavin Ni2tC proflavin proflavin DMAS~
MeOH DCP DCP DPPC DPPC
Ksv," M-' 3200 2870 233000 34000 170
kp,a M-' s-' 1.2 x 1O'O 9.6 x 109 6.0 X 10" 8.7 X 10" 4.6 X 10'
a Ksv and k , represent the Stern-Volmer and fluorescence quenching rate constants, respectively. quenching; ET = energy transfer. NiSO, was used. p-N,N-Dimethylanilinesulfonate.
type of quenchingb
CQ CQ ET ET CQ CQ = collisional
2
0 loo
C (Accepters per R. ) 0.2 0.4 0.6 0.8
7
v-
0
0
0
1
2
4 6 8 [~roftavine~l~', M
10
2
[~rof1avine1105,~
Flgure 5. Energy-transfer efficiencies ( E ) for the pyrene-proflavin system in the dicetyi phosphate membrane suspensions at 25 O C . The solM line is the energy-transfer efficiency calculated by using eq 7 and 1 (see text).
in the DCP membrane containing pyrene molecules was markedly depolarized when the pyrene molecules were preferentially excited at 338 nm. These results are clearly interpreted in terms of the energy transfer from pyrene to proflavin which is randomly bound to the surfaces of the DCP membrane. The efficiencies of the energy transfer from excited pyrene to proflavin ( E )in the DCP and DPPC membrane systems are shown in Figures 5 and 6 as functions of the acceptor concentration. It was found that the very efficient energy transfer took place in the case of the DCP membrane while the E values were saturated at ca. 60% in the case of the DPPC liposomes. This difference in the energy-transfer efficiency can be interpreted in terms of the difference in the microscopic acceptor concentration between these two systems. As is suggested by a large P value of proflavin in the DCP membrane suspension, most proflavin molecules seem to be bound to the head group of the DCP membrane. By the way, the hydrophilic head group of DPPC is zwitterionic so that the binding of proflavin to the head group of DPPC liposomes is expected to be weaker. Indeed, the P value of proflavin in the presence of the DPPC liposomes (0.039) is considerably smaller than that in the presence of the DCP membrane (0.24, see Table I). The next problem is whether the collision of excited pyrene with proflavin occurs in the present system. To solve this problem, the fluorescence quenching of pyrene was carried out under various conditions. In homogeneous fluid solution, relative fluorescence intensity ( l o / I ) is correlated with quencher concentration ([Q]) by the following Stern-Volmer equation: (3) Io/I = 1 + KsdQl = 1 + kq~oEQ1
Figure 8. Energy-transfer efficiencies ( E ) for the pyrene-proflavln system in the dipalmitoyiphosphatidyichoiine liposome suspensions at 25 OC.
where Ksv and k , are the Stern-Volmer and quenching rate constants, respectively, and 7o is the fluorescence lifetime of fluorophore in the absence of quencher. In both vs. DCP and DPPC membrane systems, the plots of lo/l [Q] were deviated upward from the straight lines at higher proflavin concentrations. Then the apparent Ksv and k , values for these systems were determined from the linear relationship between I o / l vs. [Q] at lower proflavin concentrations. The results are summarized in Table 11. In the homogeneous methanolic solution, only fluorescence quenching of pyrene by proflavin was observed; no sensitized emission from proflavin was detected. The KsVand k , values were 3200 M-' and 1.2 X 1O'O M-', respectively, suggesting that diffusion-controlledquenching takes place. The fluorescent state of pyrene in the DCP membrane was efficiently deactivated by Ni2+,which should be concentrated at the surfaces of the DCP membrane by the Coulombic interaction. Meanwhile, the quenching rate constant (5 X 10" M-' s-') of the energy-transfer system (pyrene-proflavin-DCP) was 50-fold larger than those of the collisional quenching of the pyrene fluorescence by proflavin in methanol and by Ni2+in the DCP membrane suspension. Judging from these results, the collisional quenching of pyrene fluorescence by proflavin seems to be negligible in the DCP membrane system. In the lipid membrane system, the pyrene fluorescence was quenched by a water-soluble anionic quencher, p-N,N-dimethylanilinesulfonate (DMAS), in low efficiency (It, = 4.6 X los M-' s-l), while it was quenched by proflavin with the rate of 8.7 X 1O1O M-l s-l, which is much larger than the diffusion-controlled rate constant in water. The collisional quenching is, therefore, also negligible for the energy transfer in the DPPC liposomes. Energy-Transfer-Fluorescence Decay Curves. Figure 7 shows the fluorescence decay curves of pyrene in the DCP membrane suspensions in the presence and the abN
3002
The Journal of Physical Chemistry, Vol. 85, No. 20, 1981
Kano et al.
fluorescence decay curves, which are well fitted with the experimental ones.
L 0
100
200 300 400 Time, nsec.
500
'
Flgure 7. Fluorescence decay curves of pyrene in dicetyl phosphate membrane suspensions with and without profiavin at 25 O C . The concentrations (XlO-' M) of proflavin are 0,2.4, 4.0, 6.0, 11.6, and 16.0 from the top. The solid lines are theoretical curves calculated by using eq 6 (see text). The Cvalues used for fitting are 0, 0.106, 0.177, 0.266, 0.372, 0.513, and 0.708 from the top.
sence of proflavin. The decay curves were not single exponential; the decay rates change as a function of time. For three-dimensional singlet-singlet energy transfer in a rigid matrix, the decay of an excited donor molecule ( P ( t ) )can be represented by the following equation:19
p ( t ) = eXp[-(t/TD)
-
~ / ; ; N A R o ~ R(t/7D)1/2]
