J. Phys. Chem. 1993,97, 1364-1310
7364
Behavior of a Pyrene-Labeled Phospholipid in Monolayers of Dimyristoyl-L-a-phosphatidylcholine at the Gas-Water Interface: A Fluorescence Quenching Study Frank Caruso, Franz Grieser,' and Peter J. Thistletbwaite School of Chemistry, University of Melbourne, Parkville 3052, Australia
Mats Almgren, Erik Wistus, and Emad Mukhtar Department of Physical Chemistry, University of Uppsala. S - 751 21 Uppsala. Sweden Received: March 16, 1993
The behavior of N-( 1-pyrenesulfonyl)dipalmitoyl-L-a-phosphatidylethanolamine (pyrene-DPPE) embedded in dimyristoyl-L-a-phosphatidylcholine(DMPC) monolayers at the gas-water interface has been examined by surface pressurearea isotherm measurements and steady-state and time-resolved fluorescence spectroscopy. The pyrene moiety of pyreneDPPE markedly alters the packing characteristics from those of the pure lipid DPPE, contributing significantly to the area per molecule. Steady-state fluorescence spectra showed monomer emission only, and a nonlinear increase in fluorescence intensity with concentration of pyreneDPPE was observed, which can be attributed to oxygen quenching. Time-resolved fluorescence measurements yielded single-exponential decays for the pyrene chromophore, providing evidence that pyrene-DPPE was not aggregated in the monolayer film. The lifetime of pyrene-DPPE was also found to increase with monolayer compression. The presence of iodide ions in the subphase efficiently quenched the fluorescence of pyreneDPPE in the monolayer. The degree of quenching was found to be independent of the molecular packing density, suggesting that the pyrene chromophore is located in the headgroup region of the monolayer. Measurements of the steady-state fluorescence intensity as a function of area per molecule showed pyrene-DPPE to be strongly susceptible to oxygen quenching. The degree of oxygen quenching was found to decrease with compression, accounting for the increase in the fluorescence lifetime of the excited pyrene with increasing surface pressure.
Introduction The main constituents of naturally occurring biological membranes are lipids, proteins, and carbohydrates, together with water. Membranes are highly selective permeable barriers, with the lipids providing the basic barrier and a fluid boundary to the cell.1-2 The structures of membranes are based on a lipid bilayer stabilized by a balance of hydrophilic and hydrophobic interactions. The molecular packing density and structure of these lipids influence the structure and function of the membrane they form, An understanding of how these lipids form membrane systems, along with the effects the molecular packing and configuration of the lipids have on the permeability of chemical species crossing the aqueous-lipid or gas-lipid boundaries, is of great importance in membrane and lipid research. The gas-water monolayer presents an attractive model system for biological lipid-water interfaces, and this has stimulated numerous studies on the monolayers of synthetic phospholipids at the gas-liquid interface.3-12 It facilitates thestudy of a number of parameters including the nature and packing characteristics of the lipid molecules, as well as the nature of the subphase. These parameters affect the barrier properties of the system. The permeability of membranes increases as the degree of unsaturation along the hydrocarbon chain increases,13 preventing tight molecular packing of the chains. This effect can be readily investigated in the gas-liquid monolayer. Pyrene and several of its derivatives have been widely used as fluorescent probes to study the microenvironments of organized media such as micelles,1&16 microem~lsions,1~J* bilayers,l9 and mon0layers.~9.~~ It is an attractive probe since it has a relatively long fluorescence lifetime and a high quantum yield of fluorescence, properties which makeit suitable for fluorescence quenching studies. In addition, its emission spectrum reflects the nature of its environment. The local environment of certain pyrene-
* To whom correspondence should be addressed. 0022-3654/93/2091-1364$04.00/0
containing molecules may be probedvia the ratio of the intensities of the third and first vibronic bands ( Z I ~ I / ~ I )of the monomer emission which is dependent upon polarity2'J2and the fluorescence lifetime which decreases the increasing polarity.23 Pyrene is also known to form excimers at a diffusion-controlled rate, and its excimer formation has often been used to investigate the fluidity of artificial and biological membranes.2k26 The location of the pyrene moiety within the membrane can be controlled by covalently linking the pyrene chromophore to a phospholipid. Various pyrene-labeled phospholipids, with the pyrene moiety in either the headgroup or the alkyl chain of the lipid, have been used to study lateral diffusion, phase transitions, and phase separation, as well as lipid organization in gas-water monolayers and membra ne^.^.^.*^-^^ The fluorescenceprobe used in this study is N-( 1-pyrenesulfonyl)dipalmitoyl-L-a-phosphatidylethanolamine (pyreneDPPE), which has the pyrene moiety covalently linked to the amino group of phosphatidylethanolamine. The pyrene moiety is thus expected to be located in the monolayer headgroupwater interface. In this paper we examine the interaction of pyreneDPPE embedded in dimyristoyl-L-a-phosphatidylcholine(DMPC) monolayers with the fluorescence quenchers iodide, located in the subphase, and oxygen, which is present in the subphase, the monolayer, and the gas phase above the monolayer. By manipulating lipid organization by compression of the monolayer, we have been able to investigate gas-monolayer and aqueousmonolayer quenching interactions at various levels of lipid organization.
Experimental Details Materials. N-( 1 -Pyrenesulfonyl)dipalmitoyl-~-a-phosphatidylethanolamine (pyrene-DPPE) was purchased from Molecular Probes Inc. Dipalmitoyl-L-a-phosphatidylethanolamine (DPPE) and dimyristoyl-L-a-phosphatidylcholine (DMPC) were obtained from Sigma Chemical Co. Sodium perchlorate (AR grade) was 0 1993 American Chemical Society
Behavior of Pyrene-DPPE in Monolayers of DMPC purchased from Merck and potassium iodide (AR grade) from Aldrich. All chemicals were used without further purification. All nonaqueous solvents were spectroscopic grade and were obtained from Ajax chemicals or Merck. "Milli-Q" water was used to prepare the subphase (conductivity ( K ) < 1 p S cm-l, surface tension (yo) = 72 mN m-' at 25 "C). Chloroform was used as the spreading solution for all monolayer experiments. Solution Spectroscopic Measurements. Solution fluorescence experiments were performed on a Perkin-Elmer LS-5B spectrofluorimeter and solution absorption experiments on a data processor controlled Hitachi 150-20 spectrophotometer. Surface Pressure-Area Measurements. Surface pressure-area ( P A ) measurements of the pure amphiphile monolayers were conductedona 57.9 X 13.5cm2poly(tetrafluoroethylene)(PTFE) Langmuir trough, with a PTFE barrier, driven at a compression rate of 0.03 nm2 molecule-' min-1. A 59.7 cm X 16.5 cm2 PTFE Langmuir trough with a compression rate of 0.05 nm2molecule-' min-l and a 47.0 X 15.0 cm2 PTFE Langmuir trough (KSV2200) (compression rate 0.05 nm2 molecule-1 min-1) equipped with a quartz window in the bottom were used for steady-state monolayer fluorescence experiments. The KSV-2200 trough was also used for time-resolved monolayer fluorescenceexperiments. Experiments which involved maintaining a nitrogen atmosphere over the monolayer were performed on a Fromhen-type round trough enclosed in a chamber which could be flushed with watersaturated nitrogen. The pressure in the chamber was kept slightly above the atmospheric pressure. The isotherms were measured discontinuously with the barrier stoppingwhen either the pressure increased by 2 mN m-l or the mean area per molecule changed by 0.02 nm2. The surface pressure was recorded after a relaxation time of 10 s. The u-A isotherms measured in this way were identical to those measured with continuous compression. All surface pressure-area measurements were made by the Wilhelmy hanging plate method." For a-A measurements of the pure amphiphiles and steady-state monolayer fluorescence measurements, a 4.3-cm mica plate suspended from a Shinkoh 2-g capacity strain gauge was used. The apparent changes in weight with monolayer compression were converted to voltages by the strain gauge and recorded on an Apple Macintosh PC. A 2.0-cm-wide strip of filter paper was used as the plate for the N2 experiments. Time-resolved monolayer fluorescence measurements were made using a 3.0-cm roughened platinum plate suspended from a Cahn microbalance. The change in voltage from the microbalance was monitored by a KSV-2200 trough controller and recorded on an IBM PC with software from KSV, Helsinki. Experiments were initiated by filling the trough with the appropriatesubphase. Approximately 101' molecules from 1 mM chloroform solutions mixed to the desired ratio were spread dropwise onto the subphase, using a 100-pL SGE syringe. The solvent was then allowed to evaporate for 10 min, after which the monolayer was compressed as desired. Steady-State Monolayer Fluorescence Measurements. A number of experimental setups were used to perform steady-state fluorescencemeasurements. The first employed a Perkin-Elmer LS-5 luminescence spectrophotometer,and details of the complete system have been given previously.3 Briefly, two silica fiber optic bundles were used to transfer the exciting light and the fluorescenceto and from the monolayer. Since the fluorescence signal from the monolayer was small, it was necessary to subtract the background signal due to scatter from the subphase of the exciting light and/or fluorescence from the PTFE. The background signal, monitored at the same emission wavelength as that of pyrene-DPPE, was recorded for 10 min, averaged, and then subtracted from the fluorescence signal when the monolayer was present. In the second setup, a silica lens and mirrors were used to focus the excitation light from a pulsed (20 Hz) nitrogen laser (L =
The Journal of Physical Chemistry, Vol. 97, NO. 28, 1993 7365 337 nm) (Laser Science, Inc., WSL-337ND) onto the monolayer. The exciting light then passed through a quartz window in the bottom of the trough and into a black box, which acted as a light sink, reducing the intensity of the scattered light. The emission was collected and transmitted to an optical multichannel analyzer (OMA, EE&G Model 1460) by means of a silica optical fiber, positioned normal to the interface. Typical exposure times were 20 s. Details are described elsewhere.20 Spectra obtained using this experimental setup are shown in Figure 2. The experimental apparatus for measuring the fluorescenceof monolayer films under a Nz-saturated atmosphere has been described previously.32 Briefly, two optical fiber bundles (excitation fiber positioned at 30' to the air-water interface and emission fiber positioned normal to the interface) were used to convey the exciting light and fluorescence to and from the monolayer. The fluorescencesignal was collected over 10 s using photon-counting techniques. All excitation and fluorescence spectra were obtained by subtracting the background signal from the subphase from the monolayer fluorescence signal. The normalized intensity values were calculated by multiplying the absolute intensity values by the corresponding area per molecule. Fluorescence intensity curves as a function of monolayer compression were obtained using this experimental setup (Figure 8). Time-ResolvedMonolayer Fluorescence Measurements. Fluorescence decay curves were measured by the time-correlated single-photon-countingmethod.33 The excitation light (320 nm) was produced by a frequency-doubled mode-locked DCM dye laser (Spectra Physics Model SP 375 and 3443) synchronously pumped by a modelocked Nd:YAG laser (Spectra Physics Model S P 3800). The exciting light was focused onto the monolayer by means of a lens and mirrors and passed through a quartz window in the bottom of the trough. The emission from the monolayer was focused by a fused silica lens onto a Hamamatsu (Model R15640) microchannel plate photomultiplier tube, having passed through a polarizer set so as to remove distortion of decay curves by rotational relaxation. The wavelength of observation (400 nm) was determined by an Oriel narrow-band-pass filter and an Oriel low-fluorescence cutoff filter. The electronics and analysis software have been described previously.20 The time-resolved fluorescence measurements showed a weak fast decay component, attributable to fluorescence from the PTFE trough. This background decay was subtracted from the measured decays to yield the true decay curves. A photodiode (Elfa Ltd., Model BPW 34) connected to a voltage/frequency converter and counter monitored the exciting light and gated the detection system to ensure the background and monolayer were recorded with the same overall excitation intensity. Typical exposure times were 20 min.
Results Surface Pressure-Area Isotherms. The surface pressurearea ( P A ) isotherms of the pure amphiphiles DPPE, DMPC, and pyreneDPPE obtained by continuous compression on a 0.1 M NaC104 subphase are shown in Figure 1. They are identical to those measured on a pure water subphase. Both DMPC and pyrene-DPPE exhibit isotherms indicative of a single liquidexpanded (LE) phase throughout the whole range of pressure, with no obvious kinks or plateaus suggestive of phase transition^.^^ DPPE, however, exhibits an isotherm indicative of a condensed phase, and this behavior can be attributed to ethanolamine lipids forming intermolecular hydrogen bonds as well as to the absence of a repulsive net charge.29.35 This behavior has also been observed for various other alkyl chain pho~phatidylethanolamines.2~-3~ The DPPE isotherm yields a limiting area (the extrapolated area at zero pressure for the condensed region) of 0.45 nm2 molecule-', which is in close agreement with values reported in the literature.lOJ1 Figure 1 shows that the covalent linking of the pyrene moiety of pyreneDPPE to the amino group of phosphatidylethanolamine
Caruso et al.
7366 The Journal of Physical Chemistry, Vol. 97, No. 28, 1993
I
1 h
r
E
J
50
-
I
-a l l \ 40
30
m
70
5
60
Y
\
-
I-
v)
50 c
.-C
40
Q
20
-
10
-
0
Area per molecule (nm') Figure 1. Surface pressurearea isotherms of the pure amphiphile monolayers on a 0.1 M NaC104 subphase at 20 k 1 OC: (a) DPPE, (b)
DMPC, and (c) pyrene-DPPE.
markedly alters the surface pressure-area characteristics from those of dipalmitoyl-L-a-phosphatidylethanolamine (DPPE). The T-A isotherm for pyrene-DPPE shows an onset of positive surface pressure at a significantly larger area per molecule (ca. 1.4 nm2) than DPPE (ca. 0.5 nm2). At a surface pressure of 30 m N m-l, pyreneDPPE occupies an area per molecule of 0.90 nm2 compared to 0.4 nm2 for DPPE. In addition, the collapse pressure of ca. 50 mN m-1 for DPPE is noticeably more than that for pyreneDPPE (ca. 40 mN m-I), indicating tighter packing of the molecules. Substitution of pyrene is known to lower the phase transition temperature of the derivatives as compared to the pure pho~pholipids,2~.29 and this may explain the absence of the solidliquid transition in the pyrene-DPPE isotherm. The substitution of pyrene moieties into other phospholipids has also been observed to affect the packing characteri~tics.~.30In summary, the substitution of pyrene is the dominant influence on the packing, resulting in a significant increase in the effective surface area over the entire pressure range. The mixing behavior of various binary phospholipid monolayers has been extensively detailed by Dorfler.36 It was found that if two phospholipids individually display liquid-expanded T-A behavior, mixtures of these phospholipids in any proportion will produce miscibility in the monolayer. This has also been observed more recently in a study of Forster energy transfer in phospholipid air-water m0nolayers.3~The T-A isotherm of 2 mol % pyreneDPPE in DMPC is virtually unchanged from that of pure DMPC (Figure 8), suggesting homogeneous mixing of the two monolayer components. This observation is confirmed by only monomer emission being seen in the fluorescence emission spectrum (Figure 2) and the single-exponential behavior of pyrene-DPPE in the monolayer film (Figure 4). It is important tonote that all experiments have been performed below the chain-melting phase transition temperature for DMPC (24 OC).34 Steady-State Fluorescence. The excitation (emission 400 nm) and fluorescence (excitation 337 nm) spectra for a 2 mol % pyrene DPPE/DMPC monolayer, recorded at different surface pressures, are shown in Figure 2. The excitation spectrum of pyrene-DPPE displays a major peak around 350 nm which is identical, within experimental error, to that observed in methanol solution (data not shown), DOPC monolayer^,^ and DMPC vesicles.38 The fluorescence spectrum of pyrene-DPPE in DMPC monolayers exhibitspeaksat 378,397,and417nm (shoulder). Onlymonomer emission is seen, and there is no change in the structure of the spectrum upon compression. The peak positions of the emission spectrum are in agreement with values reported in the literature.9839 The spectrum is also similar to that observed in DMPC vesicles with the exception of the broad excimer band around 550 nm seen in the latter case. The absence of excimer emission indicates
30 Q 0 v)
20
2
10
E
300
325
350
375
400
425
450
475
500
Wavelength (nm) Figure 2. Steady-stateexcitation and fluorescence spectra of a 2 mol 96
pyrene-DPPE/DMPC monolayer: (a) Excitation spectrum collected at an average area per molecule of 0.60 nm2, ,&,= 400 nm. Fluorescence spectra were collected at different surface pressures, bx= 337 nm; (b) a = 5 mN m-l, (c) T = 15 mN m-I, (d) a = 25 mN m-1. (e) a = 35 mN m-l. Each emission spectrum gives a point of fluorescence intensity as shown in Figure 3. All spectra were recorded at 20 1 OC. Subphase: 0.1 M NaCIO4.
*
0
0.01
0.02
0.03
0.04
[pyrene-DPPE] (molecules nm' ') Figure 3. Steady-state fluorescence intensity as a function of surface concentration of 2 mol % pyrene-DPPE in a DMPC monolayer. The X, fluorescence intensity was measured at T = 5 , 15,25, and 35 "-1. = 337 nm and he,, = 397 nm. Temperature = 20 1 OC. Subphase: 0.1 M NaC104.
that pyrene-DPPE is homogeneously distributed within the monolayer film. This is in agreement with the observations made from T-A isotherms and time-resolved fluorescence measurements. The fluorescence spectra show an increase in intensity as the monolayer is compressed. By taking the fluorescence intensity values from Figure 2 at 397 nm and plotting them against the surface concentration of pyrene-DPPE molecules, a nonlinear plot is obtained, as shown in Figure 3. This plot clearly shows that the fluorescence intensity of the pyrene-DPPE molecules does not increase linearly with surface concentration. This is consistent with the steady-state fluorescence measured with continuous compression (Figure 8). A linear plot of fluorescence intensity versus surface concentration usually indicates that the fluorescing molecules do not interact with one another and suggests that concentration quenching is not occ~rring.3~ The upward curvature from linearity shown in Figure 3, which is associated with an increase in lifetime upon compression (see later), was earlier observed in DOPC monolayers and was attributed to the decline in the effective polarity of the environment as the monolayer was c~mpressed.~ As will be shown later, this is not the real cause for the lifetime variation. The nonlinear increase of fluorescence intensity with surface concentration of pyrene-
The Journal of Physical Chemistry, Vol. 97, No. 28, I993 7367
Behavior of Pyrene-DPPE in Monolayers of DMPC
1 0'
In c
c
3
0
u
1 o3
1 01
t
1 0'
'1 L 0
5
10
15
20
25
35
30
Time (ns) Figure 4. Fluortscence decays of a 2 mol 5% pyrenc-DPPE/DMPC monolayer at various surface pressures: (a) r = 5 mN m-', (b) r = 15 mN m-l, (c) r = 25 mN m-l. Subphase: 0.1 M NaC104. X, = 320 nm and &, 400 nm. Temperature = 20 1 OC. All fluorescence decays have been normalized to the same maximum intensity.
-
*
15 I
cE
-
1 0'
0
40
30
20
40
50
Time (ns) Figure 6. Fluorescence decays of a 2 mol % pyreneDPPE/DMPC monolayer with various concentrationsof iodide ions at r = 25 mN m-I: (a) [I-] =OmM,(b) [I-] = 4mM,(c) [I-] = 15 mM,(d) [I-] = 50mM, (e) [I-] = 100 mM. Subphase: 0.1 M NaC104. X, = 320 nm and #&, = 400 nm. Temperature = 20 1 OC. All fluorescence decays have been normalized to the same maximum intensity.
*
1
A
141 13
TABLE I: Decay Parameters of a 2 mol % Pyrene-DPPE/ DMPC Monolayer Measured at Different Surface Pressures and Various Concentrations of Iodide Ions (X, = 320 nm, X, = 400 nm, Temperature = 20 f 1 "C) r/mNm-l [I-l/mM Ala q/ns A2' Tl/ns x2 5
15 I
0
10
5
10
15
20
25
30
35
40
Surface wessure (mN m")
25
Figure 5. Plot of the fluorescence decay lifetimesas a function of surface
pressure of a 2 mol % pyrene-DPPE/DMPC monolayer measured at 400 nm (X, = 320 nm) on 0.1 M NaClO4. Temperature = 20 f 1 'C.
35
DPPE seen in Figure 3 is due to an increase in the lifetime of pyreneDPPE with compression, which, in turn, is due to a decrease in 0 2 quenching in the more compressed monolayer. Time-Resolved Fluorescence. Fluorescence decay data were fitted to either a single or a double exponential of the form
I(t) = A, exp(-t/.,)
+ A, exp(-t/TJ
where Z ( t ) denotes the fluorescence intensity at time t , Al and A2 are the preexponential factors, and T I and T Z are the fluorescence lifetimes. Figure 4 shows the fluorescence decay behavior of a 2 mol % pyreneDPPE/DMPC monolayer at surface pressures of 5 , 15, and 25 mN m-1. The fluorescence follows single-exppnential decay kinetics, which confirms that pyrene-DPPE is not aggregated in the monolayer film. The single-exponential behavior is characteristic of a single class of fluorophore in a homogeneous environment. Figure 5 shows there is an approximately linear relationship between the lifetime of pyrene-DPPE and the surface pressure. The lifetime increases from a value of 6.4ns at 5 m N m-1 to 13.2 ns at 35 mN m-1, and the values for different surface pressures are given in Table I. In order to establish whether the position of the pyrene moiety in the film changed with compression of the monolayer, fluorescence quenching measurements of aqueous iodide with the excited pyrene-DPPE were performed. The addition of sodium iodide to the subphase did not significantly affect the packing of the molecules, and in all cases
0 4 15 50 100 0 4 15 50 100 0 4 15 50 100 0 4 15 50 100
1.00 0.16 0.24 0.37 0.40 1.00 0.14 0.32 0.39 0.53 1.00 0.22 0.35 0.40 0.59 1.00 0.19 0.31 0.46 0.63
6.4 2.3 1.7 1.7 1.5 8.8 2.3 2.1 1.5 1.3 11.2 2.9 2.1 1.8 1.2 13.2 3.5 2.5 1.6 1.2
0.84 0.76 0.63 0.60
5.7 4.9 4.4 4.0
0.86 0.68 0.61 0.47
7.9 6.7 5.3 4.5
0.78 0.65 0.60 0.41
10.7 8.2 6.5 4.9
0.81 0.69 0.54 0.37
12.6 10.7 6.7 5.4
1.2 1.1 1.1 1.1 1.2 1.2 1.1 1.1 1.3 1.2 1.2 1.1 1.2 1.4 1.4 1.1 1.1 1.2 1.1 1.4
a The sum of the fraction of the components has been normalized to unity.
the surface pressurearea isotherms were within f0.02 nm2 molecule-' of each other in the absence or presence of iodide quencher. The fluorescence decay curves for the quenching of p y r e n e DPPE, diluted into a DMPC monolayer, by various concentrations of iodide, at a surface pressure of 15 mN m-l are shown in Figure 6. These measurements show, as expected, that pyrene-DPPE is strongly quenched by the iodide ions in the subphase. The decays as a result of iodide quenching are nonexponential and can be described by a double exponential of the form given by eq 1. The resultsof the fitting are shown in Table I. Examination of Table I shows a short lifetime of about 2 k 1 ns and a longer lifetime between 4 and 12.6 ns, which, for a given iodide concentration, increases as the surface pressure is increased. Table I also shows that increasing the concentration of iodide quencher results in increased quenching. Several groups have investigated the subphase iodide quenching of fluorescence of pyrene,20 pyrene-bearing lipids,w and porphyrin41 probe molecules embedded in various matrix monolayers at the air-water and N2-water interface. In each of the above studies, nonexponential fluorescence decay curves were obtained
7368 The Journal of Physical Chemistry, Vol. 97, No. 28, 199'3
7
TABLE II: Pyrene-DPPE Fluorescence Lifetimes, Air-Saturated Oxygen Concentrations, and Quenching Rate Constants in Various Solvents at 20 f 1 O C [021 x 1031 k, x i o l y rf/nsb MC M-I s-I solvent rf/nso 2.17 1.9 & 0.2 methanol 13.8 f 0.5 33.3 f 0.5 33.6 f 0.5 2.11 1.6 & 0.2 ethanol 15.5 f 0.6 1.84 1.3 f 0.2 1-butanol 18.6 & 0.7 33.7 & 0.5 0 Air-saturatedsolutions. b Nitrogen-bubbled solutions. Reference 45. d Calculated using eq 2 (see text).
i
+I I
o 1 0
5
Caruso et al.
10
15
20
25
30
35
40
Surface pressure (mN m") Figure 7. Plot of kp, the rate constant for the quenching of 2 mol % pyrene-DPPE in a DMPC monolayer by various concentrationsof iodide ions, as a function of surface pressure: (0) 11-1 = 4 mM, {o)[I-] = 15 mM; ( 0 )[I-] = 50 mM; (A) [I-] = 100 mM. Excitation wavelength = 320 nm and emission wavelength = 400 nm. Temperature = 20 f 1
OC. for the iodide quenched cases. In energetically inhomogeneous, geometrically fractal, or low-dimensional (12)systems, nonexponential decays are observed rather than single-exponential behavior.42 For a dynamic quenching reaction in a homogeneous system where a diffusional steady state has been established, the relationship between the concentration of quencher, [Q], and the fluorescence lifetime (7)is given by the Stern-Volmer equation43 where 70 is the unquenched fluorescence lifetime and k, is the bimolecular quenching rate constant. The fast component observed in the decay curves for the quenching of pyrene-DPPE by iodide is similar to the fast background decay and is therefore difficult to determine accurately. Wistus et a1.,20 who studied the fluorescence quenching of pyrene in eicosanoic acid monolayers by subphase iodide ions, also observed a fast component of about 1.5 ns and attributed it to "static" quenching by iodide ions close to the pyrene molecules. Accordingly, the present results will be analyzed on the basis of the second (slower) decay component and on the further assumption that this component is the result of homogeneous bimolecular quenching. In reality, the geometry of the situation with the pyrene chromophore confined in the interfacial plane raises doubts as to whether the diffusional situation can be equated to that in homogeneous quenching in solution. The straightforward approach we chose to adopt is reasonable given that our purpose is to gain semiquantitative data on the quenching process, to enable comparison with other work, and to enable conclusions as to the location of the pyrene moiety. Values for the bimolecular quenching rate constants (k,), for various concentrations of iodide ions, were calculated using the Stern-Volmer equation. The k, values are in the range (1-5) X 109 M-1 s-l. These values are plotted as a function of surface pressure and are shown in Figure 7. The difference (nonsystematic) between the k,values, for different iodide concentrations, is largest at low surface pressures, and this may be attributed to the lifetime values being shorter at lower surface pressures, resulting in a greater error in these values. The values of k, for a concentration of 4 mM of iodide quencher show a larger spread, and this is most likely due to the small degree of quenching observed. As the monolayer is compressed, the k, values, for different concentrations of iodide ions, agree within experimental error. Essentially, Figure 6 shows that pyreneDPPE is efficiently quenched by iodide ions in the subphase with k, values of the
order of 109 M-1 s-1. Also, the data appear to show no general trend with increasing surface pressure, indicating that the pyrene moiety is equally accessible to the iodide ions at the different surface pressures studied. The kq values obtained in this study are in close agreement with those reported for the study of iodide subphase quenching of pyrene in eicosanoic acid monolayers (ca. 1.2 X lo9 M-I s-l), which showed pyrene to be located between the polar group of the surfactant molecules and the water subphase.20 They are about a factor of 3 larger than those observed by Bohorquez and Patterson40 for the subphase iodide quenching of pyrene-bearing lipids in uncompressed monolayers (T < 0 m N m-1) of DOPC at the N2-water interface ((3-4) X 108 M-1 s-1) and about an order of magnitude greater at surface pressures of 30 mN m-1 (1.5 X 108 M-1 s-I). In that case, the decreased quenching was ascribed to the diminished probe-iodide contact as the monolayer was compressed. The pyrene was in that case removed from the headgroup region of the molecules; Le., it was located in the alkyl part of the lipid and situated in the alkyl portion of the monolayer. If the pyrene chromophore was located in the alkyl part of the monolayer, a surface pressure (molecular packing density) dependence on the value of k, would be expected, as the increased molecular packing would reduce the possibility of iodide penetrating the matrix. The fact that such a dependence was not observed, and that pyrene-DPPE was efficiently quenched by iodide, suggests that the pyrene moiety of pyreneDPPE is located in the headgroup region of the monolayer at all levels of monolayer compression. Oxygen-Induced Fluorescence Quenching. Measurements of the degree of oxygen-induced fluorescence quenching of pyreneDPPE were performed at the air-water interface to confirm that the increase in lifetime of pyreneDPPE with compression is related to a variation in the interaction of the excited probe with oxygen. Oxygen is well known to be an efficient fluorescence quencher of ~ y r e n e .For ~ ~ example, the lifetime of pyrene in degassed ethanol is 475 ns,j9 with this value decreasing to about 135 ns in oxygen-saturated aqueous solutions.20 Measurements of fluorescence decay lifetimes in solvents of different polarity have been performed to determine whether pyreneDPPE is a polarity sensitive probe and to establish the efficiency of 02 quenching. The solvents used were methanol (MeOH), ethanol (EtOH), and butanol (BuOH). The fluorescence decays of pyreneDPPE in air-saturated solutions were all found to follow single-exponential kinetics. The decay times for pyrene-DPPE in the solvents are given in Table 11. The value of 15.5 ns in EtOH agrees well with the 14.9 ns (EtOH) reported by Kido and colleagues." Deoxygenated solutions of MeOH, EtOH, and BuOH yielded the same single-exponential lifetime, within experimental error, of 33(f 1) ns as shown in Table 11. These results clearly show that the differences observed in the pyreneDPPE lifetime in the airsaturated solvents are due to the different solubility of 0 2 in these solvents (see Table 11) and not due to any differences in solvent polarity. Figure 8 shows the surface pressure-area isotherm of a 1.5 mol % pyreneDPPE/DMPC monolayer and the normalized steady-state monomer fluorescence intensity as a function of monolayer compression for different N2 purging times. The
The Journal of Physical Chemistry, Vol. 97, No. 28, I993 7369
Behavior of Pyrene-DPPE in Monolayers of DMPC h
2
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gaseous O2
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30 20 aqueous O2
10 L
0 -
Figure 9. Stylized representation of
monolayer-aqueous system.
0
0.4
0.6
0.8
1
1.2
0 2
equilibria in the gaseous-
1.4
Average area per molecule (nm2) Figure 8. Normalized steady-state fluorescence behavior of 1.5 mol 9% pyrene-DPPE in a DMPC monolayer during monolayer compression for different times of N2 purging: (a) surface pressure-area isotherm of 1.5 mol % pyrene-DPPE diluted into DMPC; (b), (c), and (d) are the fluorescence intensity versus area per molecule curves for different N2 purging times: (b) 0 h, Le., air atmosphere; (c) 1 h; (d) 3 h. Excitation wavelength = 320 nm and emission wavelength 400 nm. Temperature = 20 1 OC. Subphase: 0.1 M NaC104.
*
surface pressure-area isotherm is almost identical to that of pure DMPC (Figure 1) and shows liquid-expanded (LE) behavior. In all cases there is an abrupt rise in fluorescence intensity around 1.20 nm2 molecule-' and at a surface pressure below 0.1 m N m-1. The sharp rise in fluorescence intensity of pyrene-DPPE indicates that the LE phase of DMPC begins at larger average areas per molecule than indicated by the surface pressurearea isotherm. This has also been observed by others in the measurements of fluorescence intensity, surface potential, and surface reflection, as a function of the average area per molecule, for various monolayer systems.9 It is observed that, for pyreneDPPE diluted into DMPC at the air-water interface (curve b), the normalized fluorescence intensity increases as a function of monolayer compression. The fluorescence intensity maximum is at about 0.65 nm2molecule-l, and this value is 1.6 times larger than that measured at 1.10 nm2 molecule-'. A slight decrease in the fluorescence intensity is seen a t surface pressures above 30 m N m-l. This decrease is not due to excimer formation as measurements of the emission at the excimer maximum (500 nm) for pyrene-DPPE showed no evidenceof excimers. The time-resolved fluorescence experiments, as indicated by single-exponential lifetimes, and the steady-state fluorescence spectra (only monomer observed) provide further evidence for the absence of excimers. We are unable at present to explain the slight decrease in emission at high molecular packing densities. Curves c and d in Figure 8 show the fluorescence intensity of the mixed monolayer as a function of area per molecule, recorded after purging with N2 for 1 and 3 h, respectively. Figure 8 clearly shows that purging of the monolayer environment with N2 for 1 h significantly increases the fluorescence signal but that 3-h purging is required to saturate the gas phase, subphase, and monolayer with N2. (Increased purging times yielded the same curve as d.) At the N l w a t e r interface, the fluorescence intensity remains essentially constant below 30 mN m-l. The data presented in Figure 8 can be used to calculate Z N * / Z ~ ~which ~, can be used to examine the relative changes in the degree of oxygen quenching as a function of surface pressure. It is observed that, is about at surface pressures below 1 mN m-l, thevalue of 4, with this value decreasing to ca. 2.5 at a surface pressure of 25 mN m-1. This result shows that the oxygen-induced quenching of pyreneDPPE in the monolayer is surface pressure dependent. These results are in qualitative agreement with those reported by Bohorquez and Patterson,* who found the oxygen quenching rate constant to decrease as a function of molecular packing density
for pyrene-bearing probes in various phospholipid monolayers. Ahuja and MBbiusg have also reported a dependence of O2 quenching on the surface pressure and the acyl chain length of the lipid matrix molecules for a pyrene-labeled phospholipid in matrix monolayers. To discuss the role played by lipid organization in reducing the quenching, it is necessary to consider possible pathways by which the excited probe and 0 2 interact. The monolayer will be treated by the separate phase approach as adopted by Bohorquez and Patterson.* Since the iodide quenching results show the pyrene moiety to be located in the headgroup region of the monolayer a t all levels of monolayer compression, dynamic interaction of 0 2 with the pyrene chromophore is essentially restricted to the lipid portion of the monolayer and the monolayer headgroupaqueous interface (see Figure 9). Given that the concentration of oxygen in air-saturated water is 2.9 X 1 V M a t 20 OC$5 and a rate constant of 2.1 X 1OloM-1 s-l for the quenching of pyrene by 0 2 in ~ a t e r , ~the 6 contribution to the total quenching of p y r e n e DPPE by 02,from the aqueous phase, can be calculated. The magnitude of this quenching is small, which suggests that the dominant quenching role is played by the oxygen contained within the monolayer. The diminished quenching of pyrene-DPPE by 0 2 with monolayer compression can be explained by either expulsion of O2 from the monolayer with increasing molecular packing densities or a decrease in the diffusion coefficient of 0 2 contained within the monolayer with compression. It is, of course, plausible that both of these mechanisms are operating simultaneously. The quenching by oxygen of pyrene-DPPE in a DMPC monolayer follows normal Stern-Volmer behavior for a homogeneous environment, as exemplified by the single-exponential kinetics (Figure 4). Thus, by assuming that the quenching of pyreneDPPE by 0 2 in the monolayer can be approximated by that in ethanol solution, a quenching rate constant (k,) of 1.6 X 1010 M-1 s-I and a natural lifetime of 33 ns for pyreneDPPE in the monolayer can be used to calculate the concentration of 02 in the monolayer. Using the 0 2 quenched lifetime of 6.4ns a t 5 m N m-1 (Table I), a local concentration of ca. 8 mM at 5 m N m-1 under air-saturated conditions is calculated. This value is significantly higher than an air-saturated alcohol environment (ca. 2 mM, see Table 11) and that of a pure alkane environment (hexane, 3.15 mM at 20 O C 4 9 . This 0 2 concentration is more like the 0 2 concentration in air (ca. 8.7 mM at 20 "C). The implication of this result is that the monolayer is not tightly packed, with the O2 in the gas phase readily penetrating the lipid hydrocarbon chains at 5 m N m-l. Compression of the monolayer will afford tighter packing of the hydrocarbon chains, and it can be expected that the local concentration of 0 2 in the monolayer will decline. Assuming k, is unchanged with compression, a local concentration of ca. 3 mM is calculated at 35 mN m-1. This concentration of 0 2 in the monolayer is close to that observed in hexane (3.15 mM at 20 OC45). The simplistic treatment of keeping k, constant with compression will lead to an underestimated local concentration
7370 The Journal of Physical Chemistry, Vol. 97,No. 28, 1993 of 02 in the monolayer. If, however, the value of k, is taken to decrease by a factor of 2 from 5 to 35 mN m-I, as reported by Bohorquez and Pattersod for similar phospholipid matrices, the local concentration of 02 in the monolayer is ca. 6 mM. This suggests that, even though located in the headgroup region of the monolayer, the pyrene moiety experiences a gaseous environment and that treating a monolayer as a separate phase may not be strictly appropriate. It is worth noting that 02diffusion from the gaseous phase into the aqueous phase is hardly affected by the presence of phospholipid monolayer^.^^ This would suggest that the dominant reason for the increase in lifetime for pyrene-DPPE is due to expulsion of 02 from the monolayer with compression. We are, however, unable to unequivocally decide on which of the two mechanisms (expulsion of 02 with compression or a decrease in k, with compression) dominates the quenching of pyreneDPPE by 02 within the monolayer. Suffice it to say that, upon compression of the monolayer, the interaction between 02 and the pyrene moiety is reduced.
Conclusions The liquid-expanded nature of the surface pressurearea isotherm of pyreneDPPE embedded in a DMPC monolayer, the monomer emission only observed from steady-state spectra, and single-exponential decays from time-resolved fluorescence measurements all suggest that pyreneDPPE is homogeneously distributed in the monolayer film. The subphase iodide quenching results show pyrene-DPPE is strongly quenched with the quenching independent of surface pressure, suggesting pyreneDPPE is located in the headgroup region of the monolayer at all levels of monolayer compression. The nonlinear increase in steady-state fluorescence intensity with pyreneDPPE concentration can be explained by the diminished interaction of pyreneDPPE and oxygen upon compression. This is observed as an increase in lifetime of p y r e n e DPPE with increasing molecular packing density. Acknowledgment. We are most grateful to Dr. R. C. Ahuja and Dr. D. MiSbius from the Max-Planck-Institut fur Biophysikalische Chemie, Gbttingen, Germany, for the use of equipment in their laboratory. F.C. acknowledges the receipt of a CSIRO Institute of Industrial Technologies Research Postgraduate Scholarship for this work. Financial assistance from the Australian Research Council and the Advanced Mineral Products Research Centre (University of Melbourne) is also gratefully acknowledged. References and Notes (1) Ralston, G. B. Structure and Properties of Cell Membranes; Benga, G., Ed.; CRC: Boca Raton, FL, 1985; Vol. 1, Chapter 2. (2) Singer, S.J.; Nicolson, G. L. Science (Washington,D.C.) 1972,175, 720. (3) Carum, F.; Grieser, F.; Murphy, A.; Thistlethwaite, P. J.; Urquhart, R.; Almgren, M.; Wistus, E. J. Am. Chem. SOC.1991, 113, 4838.
Caruso et al. (4) Urquhart, R. S.;Hall, R. A.; Thistlethwaite, P. J.; Grieser, F. J. Phys. Chem. 1990,94,4173. (5) Subramanian, R.; Patterson, L. K. J . Am. Chem. Soc. 1985. 107, 5820. (6) Subramanian, R.; Patterson, L. K. J . Phys. Chem. 1985,89, 1202. (7) Bohorquez, M.; Patterson, L. K. J. Phys. Chem. 1988, 92, 1835. (8) Bohorquez, M.; Patterson, L. K, Lungmuir 1990,6, 1739. (9) Ahuja, R. C.; MBbius, D. Lungmuir 1992, 8, 1136. (10) Zhu, D. G.; Petty, M. C.; Ancelin, H.; Yarwood, J. hngmuir 1992, 8. 619. (11) Nakahara, H.; Fukuda, K.; Akutsu, H.; Kyogoku, Y. J. Colloid Interface Sci. 1978, 63, 5 17. (12) Smabv, J. M.; Brockman. H. L. Lanmnuir 1991, 7, 1031. (13) Bittman, R.; Blau, L. Biochemistry f972, 11, 4831. (14) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. Soc. 1979, 101, 279. (15) Grieser, F.; Tausch-Treml, R. J . Am. Chem. Soc. 1980,102,7258. (16) Roelants, E.; De Schryver, F. C. Langmuir 1987, 3, 209. (17) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. Soc. 1980, 102, 3188. (18) Atik, S. S.;Thomas, J. K. J . Am. Chem. Soc. 1982,104, 5868. (19) Zachariase, K. A.; Kozankiewicz, B.; Kiihnle, W. Surjacrants in Solution; Mittal, K. L., Ed.; Plenum: New York, 1983. (20) Wistus, E.; Mukhtar, E.; Almgren, M.; Lindquist, S.-E. Langmuir 1992,8, 1366. (21) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. SOC.1977,99, 2039. (22) Dong, D. C.; Winnik, M. A. Photochem. Phorobiol. 1982, 35, 17. (23) Birks, J. B.; Lumb, M. D.; Munro, I. H.Proc. R . Soc. London, A 1964, 280, 289. (24) Galla, H. J.; Sackmann, E. Biochim. Biophys. Acta 1974,339,103. (25) Vanderkooi, J. M.;Callis, J. B. Biochemistry 1974, 13, 4OOO. (26) Dembo, M.; Glushko, V.; Aberlin, M. E.;Sonenberg, M. Biochim. Biophys. Acta 1979, 522, 201. (27) Galla, H.-J.; Hartmann, W. Chem. Phys. Lipids 1980,27, 199. (28) Loughran, T.; Hatlee, M. D.; Patterson, L. K.; Kozak, J. J. J . Chem. Phys. 1980, 72, 5791. (29) Somerharju, P. J.; Virtanen, J. A.; Eklund, K. K.; Vainio, P.; Kinnunen, P. K. J. Biochemistw 1985. 24. 2773. (30) Kinnunen, P. K. J.’; Virtanen, J. A.; Tulkki, A. P.; Ahuja, R. C.; MBbius, D. Thin Solid Films 1985, 132, 193. (31) Hiemenz, P. C. Principles of Colloid andsurface Chemistry; Marcel Dekker: New York, 1977; p 210. (32) Grilniger, H.; Mobius, D.; Meyer, H. J . Chem. Phys. 1983,79,3701. (33) O’Connor, D. V.; Phillips, D. Time-Correlated Single Photon Counting; Academic: London, 1984. (34) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (35) Van Deenen, L. L. M.; Houtsmuller, U. M. T.; De Haas, G. H.; Mulder, E. J. Pharm. Pharmacol. 1962, 14, 429. (36) DBrfler, H.-D. Adv. Colloid Interface Sci. 1990, 31, 1. (37) Urquhart, R.; Grieser, F.; Thistlethwaite, P. J.; Wistus, E.; Almgren, M.; Mukhtar, E. J. Phys. Chem. 1992, 96,7808. (38) Tanaka, F.; Kaneda, N.; Mataga, N. J. Phys. Chem. 1986,90,3167. (39) Birks, J. B. Phorophysics of Aromatic Molecules; Wiley-Interscience: New York, 1970; p 91. (40) Bohorquez, M.; Patterson, L. K. Thin Solid Films 1988, 159, 133. (41) Bohorquez, M.; Patterson, L. K.; Brault, D. Photochem. Photobiol. 1987, 46, 953. (42) Blumen, A.; Klafter, J.; Zumofen, G. Transport and Relaxation in Random Materials; Klafter, J., Rubin, R. J., Shlesinger, M. F., Eds.; World Scientific: Philadelphia, PA, 1986; pp 163-175. (43) Turro, N. J. Modern Molecular Photochemistry; Beniamin-Cumminns: Menlo Park, CA. 1978: ChaDters 6. 8. (44) Kido, N.; T-anaka, F.; Kanuda, N.;Yagi, K. Biochim. Biophys. Acta 1980.603. 255. (45) Battino, R.; Rettich, T. R.;Tominaga, T. J . Phys. Chem. Re/. Data 1983, 12, 163. (46) Thomas. J. K. Chem. Rev. 1980.80. 283. (47) Notter, H. R.; Berg, J. C. J. Colloid Interface Sci. 1973, 45, 421. ~
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