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Journal of Lipid Research, Vol. 48, 1518-1532, July 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology
* Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
Hormel Institute, University of Minnesota, Austin, MN 55912
Published, JLR Papers in Press, April 7, 2007.
1 I. A. Boldyrev and X. Zhai contributed equally to this work. 
2 To whom correspondence should be addressed. e-mail: reb{at}umn.edu (R.E.B.); jgmol{at}ibch.ru (J.G.M.)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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506–515 nm) but also showed the absence of the 620–630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me4-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.
Supplementary key words spectral properties • monolayers • lipid lateral packing • surface compressional modulus • lipid phase state • fluorophore position • fluorescence quenching • iodide • fluorophore location in bilayers • parallax analysis • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
Abbreviations: AV12-PC, 1-acyl-2-[12-(9-anthryl)-11E-dodecenoyl]-sn-glycero-3-phosphocholine; B3-, B5-, B7-, or B9-PC, phosphatidylcholine bearing at the sn-2 position 
-Me4-BODIPY-8-C3-, -C5-, -C7-, or -C9-fatty acid, respectively; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; I7- or I11-PC, phosphatidylcholine bearing 7-iodoheptanoic or 11-iodoundecanoic acid, respectively, at the sn-2 position; Ksv, Stern-Volmer quenching constant; Me2-BODIPY-3, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl; Me4-BODIPY-8, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl; PB-PC, 1-palmitoyl-2-Me2-BODIPY-3-pentanoyl-sn-glycero-3-phosphocholine; Tm, phase transition temperature
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the past decade, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore probes have found a wide range of applications in cell biology and biophysics, even though this zwitterionic fluorophore was first synthesized by Treibs and Kreuzer (8) in 1968. The BODIPY fluorophore family has gained popularity because of its overall excellent spectral properties (9, 10), which include high photostability, high molar absorptivities, high quantum yields, and strong, narrow-wavelength emission maxima in the visible region. Although the great majority of BODIPY fluorophores are nearly insensitive to environmental polarity and this property is advantageous for many applications, a few environmentally responsive varieties of BODIPY have been developed (11). Many BODIPY derivatives, including labeled fatty acids and complex lipid probes, have been synthesized (12, 13) and applied in numerous biological studies (1, 14). These probes are commercially available (Invitrogen) (15) but are relatively expensive, presumably because of their rather complicated syntheses.
Because the BODIPY group carries no net charge, one might expect it to be a more reliable depth-dependent membrane probe than dansyl or NBD. Despite the seemingly favorable properties, studies of the localization of lipid-attached BODIPY in model membranes have led to conflicting conclusions. Based on iodide quenching studies of different BODIPY probes in phospholipid vesicles and in water, Johnson, Kang, and Haugland (9) concluded that lipid-attached BODIPY fluorophores reside mainly in the bilayer at the expected depth. On the other hand, Menger, Keiper, and Caran (16), using spin-labeled lipids as quenchers incorporated into the membrane, found that BODIPY fluorophores linked to various positions of the phosphatidylcholine acyl chain, including the position, reside close to the membrane surface, 17–20 Å from the bilayer center, as determined by parallax analysis. However, Kaiser and London (17), also using spin-labeled lipid quenchers and parallax analysis, concluded that lipid-attached BODIPY in the bilayer distributes between two populations: in one, the fluorophore is embedded into bilayer along the entire length of the acyl chain; in the other population, BODIPY resides closer to the membrane surface by causing bending/looping of the flexible acyl chain. Even though such dual fluorophore localization and related uncertainties do not diminish the effectiveness of BODIPY for many purposes (e.g., monitoring lipid trafficking within a cell), it can be problematic in other cases, such as in resonance energy transfer experiments in which the fluorophore serves as a molecular ruler, thus requiring its membrane position to be accurately known.
The goal of this study was to develop a set of lipid probes, bearing a BODIPY fluorophore at the end of the acyl chain, in which the fluorophore location within the bilayer corresponded to the maximal depth allowed by the acyl chain. Our strategy was to modify the BODIPY structure to counteract the polarity originating from its compensating positive (on nitrogen atom) and negative (on boron atom) internal charges to optimize its capacity for localization in the apolar interior of the bilayer. As a rule, insertion of substituents at positions 1, 3, 5, 7, and 8 of the BODIPY ring (Fig. 1 ), but not at positions 2 and 6, is known to be well tolerated without negatively affecting emission quantum yield (9). It should be noted that the previously described studies of lipid-bonded BODIPY localization in bilayers (9, 16, 17) were performed using fluorophore containing methyl substituents at positions 5 and 7 and linked to acyl chains via position 3. We reasoned that BODIPY could be optimized for deep bilayer localization by maximizing the number and symmetry of apolar substituents. Our considerations led to the hypothesis that the desired fluorophore should have the BODIPY ring, with identical apolar alkyls at positions 1, 3, 5, and 7, and be linked to acyl chains via position 8. To evaluate this hypothesis, we analyzed the spectral and membrane properties of BODIPY probes with the aforementioned structural features, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8). For this purpose, a novel set of phosphatidylcholines was synthesized with the Me4-BODIPY-8 fluorophore attached to the end of sn-2 fatty acids consisting of three, five, seven, or nine carbon atoms. The location and insertion depth of the Me4-BODIPY-8 fluorophore in unilamellar bilayer vesicles have been assessed by quenching with free iodide (Stern-Volmer analysis) as well as with iodolabeled phosphatidylcholines (parallax analysis) (17). The physical and spectral features of these new phosphatidylcholine probes, in their pure states and in mixtures with other lipids, have been characterized in both bilayer and monolayer model membrane systems to assess the occurrence of lipid-packing distortions caused by the fluorophore as well as concentration-dependent dimerization associated with the BODIPY family of fluorophores (18, 19).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vesicle preparation
An aliquot of lipid solution in chloroform was rotary-evaporated in a round-bottomed flask and kept in vacuo (20 Pa) for 2 h. The resulting lipid film was suspended in PBS at the desired lipid concentration (400 µg/ml) and subjected to 10 cycles of freezing (liquid nitrogen) and thawing (water bath at 40°C for POPC and at 50°C for DMPC or DMPC/cholesterol). Then, the lipid suspension was extruded through polycarbonate membranes (Nucleopore) with 100 nm pores 20 times using the mini-extruder (Avanti%20Polar%20Lipids">Avanti Polar Lipids). The resulting vesicles, as shown previously (22), are unilamellar with mean diameters of 120 nm.
Spectral measurements
The UV spectra were recorded on an Ultrospec II spectrophotometer (Pharmacia LKB, Bromma, Sweden). Steady-state fluorescence measurements were performed on an F-4000 fluorimeter (Hitachi, Ltd., Tokyo, Japan) using thermostatted 10 x 10 mm cuvettes. Me4-BODIPY-8 probes were excited at 497 nm, unless specified otherwise, and the emission was recorded at 505 nm using excitation and emission band passes of 5 nm. Anthrylvinyl probe was excited at 373 nm, and the emission was recorded at 434 nm (excitation and emission band passes of 5 and 10 nm, respectively). In the liposome experiments, a cross-oriented configuration of the polarizers (Expol = 0° and Empol = 90°) was used for maximal suppression of liposome-scattering side effects (23). Fluorescence of high-concentration probe solution (5% in chloroform) was measured in three sealed glass capillaries (inner diameter, 0.5 mm) placed cornerwise in a quartz cuvette filled with toluene to minimize light reflection at the external surface of the capillaries.
In iodide quenching experiments involving Me4-BODIPY-8 probes (0.1 mol% total lipid) and AV12-PC (0.5 mol% total lipid) in vesicles (0.4 mg lipid/ml), the data were collected by sequential addition of the stock quencher solution (1.5 M sodium iodide containing 10 mM Na2S2O3 in PBS) to vesicles containing the probe and subsequent measurement of fluorescence intensity after system equilibration (up to 60 min) similar to our previous studies (24, 25). During the quenching experiments, osmolarity changes caused by the addition of iodide salts to the suspension, apart from dilution, can bring about vesicle swelling and change the relative refraction index at the solution/vesicle boundary. Because both effects can influence vesicle turbidity (26, 27), steps were taken to correct for their contributions to the emission quenching data by performing parallel control measurements in which equal aliquots of 1.5 M sodium chloride solution in PBS were added to vesicle samples instead of quencher solution.
For Stern-Volmer plots, F0/F values were calculated as follows:
 | (Eq. 1) |
 | (Eq. 2) |
 | (Eq. 3) |
For quenching experiments with iodolabeled phosphatidylcholines, POPC vesicles containing 5 mol% I7- or I11-PC and a Me4-BODIPY-8 probe (0.1 mol%) were prepared as described above. To avoid inner filter effects, total absorbance was kept low (0.02 at 498 nm). The distance of the fluorophore from the center of the bilayer was calculated using the parallax equation (17, 28):
 | (Eq. 4) |
Monolayer conditions
The monolayer properties of the lipid probes, in pure form and mixed with other lipids, were characterized using a computer-controlled, Langmuir-type film balance designed to measure the surface pressure (
) and dipole potential (
V) as a function of cross-sectional molecular area (A) while simultaneously acquiring the fluorescence emission intensity as a function of wavelength at the desired surface pressures. Surface pressure and area calibration of the film balance were performed as detailed previously (30, 31). Fluorescence emission measurements were performed using a slightly modified set-up compared with that described by Dahim et al. (32). Briefly, BODIPY lipid films were excited at a 90° incident angle using 488 nm unpolarized light from an argon-ion laser (model 2122-45L; JDS Uniphase, San Jose, CA) equipped with a model-3 light-intensity controller and a fiber-optic coupler (model HPUC-23-488-S-3, FAC-2BL; Oz Optics, Nepean, Ontario, Canada). Fluorescence emission was collected perpendicular to the interface at a distance of 1 cm using a fiber-optic spectrometer (model PC2000-ISA; Ocean Optics, Dunedin, FL) equipped with an L2 lens and a 200 µm slit. A 500 nm long-pass filter (500EFLP; Omega Optical, Brattleboro, VT) was mounted between the emission collimator and the detector to reduce scattered excitation light. Fluorescence emission spectral intensities were collected each second. Although monolayer compression was continuous during the spectral data acquisition cycle, the fractional change in lipid concentration during each acquisition cycle was 
0.0073. Emission spectra were not affected by gas phase (i.e., air or argon) or by 0.01% sodium azide in the subphase buffer.
Lipid monolayers were formed by spreading (51.67 µl aliquots) mixtures made from stock solutions dissolved in toluene-ethanol (5:6) or hexane-isopropanol-water (70:30:2.5). Solvent purity was verified by dipole potential measurements using a 210Po ionizing electrode (31). After spreading on the subphase surface, lipid films were compressed at a rate of 4 Å2/molecule/min after a delay period of 4 min. Subphase buffer was maintained at 24°C via a thermostatted, circulating water bath and was produced using water previously purified by reverse osmosis, activated charcoal adsorption, and mixed-bed deionization, then passed through a Milli-Q UV Plus system (Millipore Corp., Bedford, MA) and filtered through a 0.22 µm Millipak 40 membrane. Subphase buffer contained 10 mM potassium phosphate (pH 6.6), 100 mM NaCl, and 0.2% NaN3 and was kept stored under argon, which was cleaned by passage through a seven stage series filtration set-up consisting of an Alltech activated charcoal gas purifier, a LabClean filter, and a series of Balston disposable filters consisting of two adsorption (carbon) units and three filter units (93% and 99.99% efficiency at 0.1 µm). The film balance was housed in an isolated laboratory supplied with clean air by a Bioclean air filtration system equipped with charcoal and High Efficiency Particulate Air (HEPA) filters and was kept under humidified argon in a separate enclosure. Other features contributing to isotherm reproducibility include automated lipid spreading via a modified HPLC autoinjector, automated surface cleaning by multiple barrier sweeps between runs, and highly accurate, reproducible setting of the subphase level by an automated aspirator. Glassware was acid-cleaned and rinsed with purified water and then with hexane-ethanol (95:5) before use.
Analyses of Monolayer Isotherms
The dipole potential versus cross-sectional molecular area (V – A) behavior of lipid monolayers can be described by
 | (Eq. 5) |
V is the potential measured in millivolts and µ
is the dipole moment (in milliDebyes) perpendicular to the lipid-water interface and can be determined from the slope of V versus 1/A plots (30). The intercept term, V0, is lipid-specific and appears to arise from an epitaxial ordering of interfacial water molecules (30, 33). V versus 1/A plots of various liquid-expanded lipids are linear from 1 mN/m up to surface pressures at which the second derivative of the -A isotherms (d2/dA2) goes from positive to negative values (d) (33, 34). The linearity indicates a lack of significant dipole reorientation over the range of surface pressures leading up to d and typically encompasses 80–90% of the – A data for liquid-expanded films.
Monolayer compressibilities at the indicated experimental mixing ratios were obtained from – A data using:
 | (Eq. 6) |
). To facilitate comparison with elastic moduli of area compressibility values in bilayer systems, we expressed our data as the reciprocal of CS, originally defined as the surface compressional modulus (CS–1) by Davies and Rideal (35). High CS–1 values correspond to low lateral elasticity among packed lipids forming the monolayer. Comprehensive descriptions of the mechanoelastic properties of model membranes have been reviewed by Needham (36), Behroozi (37), and Brown and Brockman (38). Whenever possible, we used a 100 point sliding window that used every fourth point to calculate a CS–1 value before advancing the window one point. Reducing the window size by 5-fold did not significantly affect the observed CS–1 values. Each plot of CS–1 versus average molecular area consisted of 200 CS–1 values obtained at equally spaced molecular areas along the – A isotherms. The standard errors of our CS–1 values are 2%. CS–1 values, which use data available in the slopes of the isotherms, respond to changes in surface pressure and phase state. CS–1 values are known to be especially sensitive to phosphatidylcholine acyl structure during mixing with cholesterol at high surface pressures that mimic the biomembrane environment (39–41). CS–1 data complement area condensation data, which are inherently less reliable at high surface pressures because of the small changes that occur in average area.
The area-condensing effect of cholesterol on different phosphatidylcholine species was determined from plots of average molecular area versus composition (39, 41–43). Experimentally observed areas of mixtures were compared with areas calculated by summing the molecular areas of the pure components (apportioned by mole fraction in the mixture). The calculated average molecular area (A) of two component mixtures was determined at a given surface pressure () using the following equation:
 | (Eq. 7) |
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl)-propionic (XIII), -pentanoic (XIV), -heptanoic (XV), and -nonanoic (XVI) acid. It should be mentioned that acids XIII and XVI are manufactured by Invitrogen, but their high cost restricts their availability. Phosphatidylcholines, XVII (B3-PC), XVIII (B5-PC), XIX (B7-PC), and XX (B9-PC), bearing the above acids were prepared by acylation of lysophosphatidylcholine (derived from egg yolk phosphatidylcholine) with the corresponding acid in the presence of dicyclohexylcarbodiimide (5).
Probe spectral properties in solution and in bilayers
The absorption and fluorescence spectra of probes XVII–XXII and their corresponding acids XIII–XVI in ethanol are nearly identical, sharing similar characteristics with other Me4-BODIPY-8 derivatives (15), with absorption maximum at 498 nm (
8–10 x 104 M–1 cm–1; in ethanol). The fluorescence spectra show excitation maxima at 497–498 nm and emission maxima at 506–508 nm, typical for BODIPY derivatives (15). Representative excitation and emission spectra, for phosphatidylcholine probe B7-PC in DMPC vesicles, are shown in Fig. 2A
. The fluorescence spectra measured in solvents of different polarity were similar (data not shown), in agreement with previous reports indicating low environmental sensitivity for BODIPY fluorescence (9, 10). The quantum yields of the Me4-BODIPY-8 derivatives described here were not determined absolutely, but comparative measurements of relative fluorescence intensities in ethanol of acids XIII–XVI and of known BODIPY probes [D-2190 and D-3834 products of Invitrogen; quantum yields 
0.9 (13)] revealed no noticeable differences.
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0.13 M) solution in chloroform are shown in Fig. 2B. To reduce the side effects of strong absorption at high concentration (i.e., inner filter effects), measurements were carried out in thin glass capillaries to provide short light paths for both excitation and emission (45). Under these conditions, the excitation spectrum showed a red-shifted wavelength maximum (4 nm) compared with dilute fluorophore in DMPC (Fig. 2A). In the emission spectrum, dramatic changes were evident at high Me4-BODIPY-8 solution concentrations. Apart from a red shift of the wavelength maximum from 506–508 to 518 nm, a new peak appeared at 540 nm (Fig. 2B, peak 1), which seemed to result from the high probe concentrations in the capillaries, in which calculated peak absorbance can exceed 500 even at path lengths as short as 0.05 cm. However, there was no evidence of the longer wavelength emission peak (620–630 nm region) that has been associated previously with emission by BODIPY dimers (18, 19).
The effects of high Me4-BODIPY-8 concentrations on fluorescence spectra of POPC vesicles containing 0.1, 5, and 10 mol% B7-PC probe were evaluated using total lipid concentrations that yielded nearly equal optical absorbances of 0.08 in the mixtures (Fig. 2B, peaks 2–4). The spectra registered at 0.001 (peak 2) and 0.05 (peak 3) probe mole fractions coincided with each other and with the spectrum of the 0.001 B7-PC mole fraction in DMPC (Fig. 2A). Although negligible change was observed in the excitation spectrum at 10 mol% B7-PC probe, broadening of the emission spectrum to the red side began to show (Fig. 2B, peak 4). Again, no sign of a peak in the 620–630 nm region was visible.
Probe lateral packing and spectral properties in monolayers
To investigate the lateral packing features of B7-PC in monolayers, the surface pressure versus average molecular area behavior was measured using an automated Langmuir-type film balance capable of collecting fluorescence emission spectra during monolayer compression (see Materials and Methods). Figure 3A
shows the force-area isotherms of B7-PC (Me4-BODIPY-8) compared with several phosphatidylcholines containing acyl chains with different cis-unsaturation and commercial BODIPY-PC (PB-PC) containing Me2-BODIPY-3 (Fig. 1). The attachment of Me4-BODIPY-8 fluorophore to phosphatidylcholine resulted in its molecular cross-sectional area being slightly larger than that of the PB-PC fluorophore or naturally occurring, highly unsaturated acyl chains. The cross-sectional molecular area of phosphatidylcholine containing Me4-BODIPY-8 (B7-PC) at 30 mN/m, a surface pressure mimicking biomembrane conditions, was consistent with fluid-like lateral packing of the hydrocarbon region, in which the phosphatidylcholine acyl chain containing the Me4-BODIPY-8 embeds in the hydrophobic interior of the lipid monolayer. Also evident under membrane-like packing conditions was the slightly lower CS–1, indicating higher lateral packing elasticity in the B9-PC films compared with phosphatidylcholines with unsaturated acyl chains (Table 1
).
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350 and 450 mV lower than for unsaturated phosphatidylcholines and for PB-PC, respectively. Table 1 summarizes the values of µ and V0, obtained from plots of the dipole potential versus inverse cross-sectional molecular area (V – A–1). V0 values for B7-PC were 551 mV, compared with 24 mV for PB-PC (26) and 110–140 mV for the fluid nonfluorescent phosphatidylcholines lacking docosahexenoyl acyl chains (30, 33, 34). However, the dipole moment, µ, of B7-PC was found to be –850 milliDebyes, compared with 874 milliDebyes for PB-PC (26) and 480–550 milliDebyes for phosphatidylcholines lacking docosahexenoyl acyl chains (30, 33, 34).
The fluorescence emission response of pure B7-PC monolayers during compression is shown in Fig. 3C. The emission spectra show a peak (S0
S1) in the vicinity of 520 nm with a shoulder at 540 nm that is characteristic of BODIPY fluorophores (10) but no evidence of the 620–630 nm "signature" emission peak characteristic of Me2-BODIPY-3 dimers (32). Even so, there was considerable broadening of the B7-PC emission spectrum, caused by an apparent increase in intensity near 570 nm as well as an overall increase in emission intensity with increasing surface pressure (Fig. 3C).
Mixing of B7-PC with POPC had little effect on the force-area isotherm of POPC at low B7-PC mole fractions (e.g., 0.01) (Fig. 4A
). The small effect of B7-PC on the POPC isotherm, at probe mole fractions of 0.1 and 0.2, suggested a slight ordering effect on POPC by the higher probe concentrations. Consistent with this conclusion was the slight negative deviation from the calculated ideal additivity in average molecular area versus composition plots (data not shown). Figure 4B–E show the fluorescence emission response for the various B7-PC/POPC mixtures measured at different surface pressures. The broadness of the peaks increases at elevated B7-PC mole fractions and surface pressures. Concurrent with the changes in emission broadness are incremental shifts (515–528 nm range) in the wavelength maximum associated with monomer emission (S0S1). The magnitude of the shift in wavelength maximum was found to depend upon the surface concentration of the B7-PC probe (Fig. 4F).
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Figure 7
shows Stern-Volmer plots obtained during quenching of the probes in DMPC vesicles at 17°C (Fig. 7A) and 37°C (Fig. 7B) as well as in DMPC/cholesterol (7:3) vesicles at 37°C (Fig. 7C). The latter composition was chosen because DMPC/cholesterol mixtures with a sterol content of 25–30 mol% form liquid-ordered phase (49) and the phase homogeneity can be expected to simplify the interpretation of results. Similar quenching analyses also were performed with the probes in POPC vesicles at 17°C (Fig. 8A
) and 37°C (Fig. 8B). The quenching in the POPC vesicles at 37°C was compared with data obtained using AV12-PC (Fig. 8B), a fluorophore known to embed deeply in the bilayer (5). Ksv values for the data in Figs. 7 and 8A, B are listed in Table 2
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0.98). The lack of curvature toward the x axis is consistent with a single fluorophore population (50). It is reasonable to expect this single population of fluorophores to be embedded in the bilayer at the maximal depth allowed by the acyl bearer. In all cases, B3-PC, which bears fluorophore at the shortest distance from the polar head, is quenched most efficiently, with Ksv being largest compared with the other probes (Table 2). This finding is consistent with a shallow position in bilayer by the B3-PC probe fluorophore. At the same time, the Me4-BODIPY-8 fluorophore of B3-PC in vesicles is not freely accessible to water molecules. This conclusion follows from the fact that, in vesicle preparations labeled with B3-PC and acid XIII in which the latter is located mainly in water phase (spectrophotometric assessment; data not shown), the Me4-BODIPY-8 fluorophore is quenched much more efficiently (Fig. 8C, upper trace), with initial apparent Ksv = 10.3. In this case, the Stern-Volmer plot clearly bends toward the x axis compared with quenching of phosphatidylcholine B3-PC (Fig. 8C, lower trace), confirming the presence of two fluorophore populations in this system.
The other probes tested, B5-, B7-, and B9-PC, are quenched less efficiently by iodide than B3-PC, consistent with the I– concentration diminishing gradually toward the bilayer center and resulting in decreased quenching efficiency and Ksv values, in accordance with the fluorophore distance from the polar head. This finding holds true for the quenching in DMPC vesicles at 17°C and in POPC vesicles at 37°C (Figs. 7A, 8B). Table 2 shows that their Ksv values differ by only 10–15%. In the DMPC bilayer at 17°C [i.e., below the phase transition temperature (Tm) of 24°C], the Ksv values are lowest. In contrast, they are highest in POPC vesicles at 37°C, a temperature well above the Tm of –2°C (Table 2). In liquid-crystalline bilayers at temperatures not far from Tm [DMPC at 37°C (Fig. 7B), POPC at 17°C (Fig. 8A)], deviations from such order occurred, reflecting the complicated profile of the iodide distribution.
When the DMPC vesicles contained cholesterol (0.3 mol fraction), the I– quenching responses of B5-, B7-, and B9-PC were similar to each other, within experimental error (Fig. 7C). The Ksv values (Table 2) were intermediate between those observed for the same probes in pure DMPC at 17°C (gel phase) and 37°C (liquid-crystalline phase), perhaps reflecting the liquid-ordered nature of the system.
Compared with all Me4-BODIPY-8 probes, iodide quenching of anthrylvinyl probe AV12-PC is least efficient (Fig. 8B), consistent with a deep localization of anthrylvinyl near the bilayer center. The linear nature of the Stern-Volmer quenching plot for this anthrylvinyl probe (r = 0.91) provides evidence for a single probe population. These findings are not surprising given the length of the spacer hydrocarbon (12 carbon atoms) and the apolar nature of the anthrylvinyl fluorophore in AV12-PC. The middle-of-the-bilayer localization for the anthrylvinyl fluorophore in AV12-PC revealed by the quenching data is consistent with earlier 1H-NMR data (5).
Fluorophore depth in the bilayer determined by parallax analysis
To confirm the bilayer location of the Me4-BODIPY-8 fluorophore and more precisely estimate the fluorophore depth in the bilayer, we used the parallax quenching approach, involving fluorescence quenching by two quenchers with different, fixed positions in the bilayer (17, 28). Large unilamellar POPC vesicles, doped with B3-, B5-, B7-, or B9-PC probe, were quenched with phosphatidylcholines bearing either 7-iodoheptanoyl (I7-PC) or 11-iodoundecanoyl (I11-PC) sn-2 acyl chains (Fig. 1). Use of the iodine atom as a quencher minimally altered the polarity of the aliphatic chain, as was evident by the nearly identical thin-layer chromatographic mobilities of 11-iodoundecanoic acid and dodecanoic acid. Thus, the behavior of 7-iodoheptanoyl and 11-iodoundecanoyl chains in bilayers is similar to that of bromoacyls (50), and the iodine atom can be expected to locate at the maximal allowed depth, with deviations induced by thermal mobility of chains. In our case, the quencher concentration was limited to 5 mol% to minimize the distortion of the bilayer and avoid separation of the quencher into a separate phase.
Based on the assumption that the liquid-crystalline POPC bilayer is 35 Å thick at 20°C, the distance of the iodine atom from the bilayer center is 8.8 Å for I7-PC and 5.8 Å for I11-PC (see Materials and Methods). Calculation of the Me4-BODIPY-8 fluorophore location from the bilayer center was performed for all four probes by parallax analysis (Table 3
). The distances from the bilayer center to the fluorophore increased in the order B9-PC < B7-PC < B5-PC < B3-PC (Table 3), in agreement with the pattern obtained by soluble iodide quenching (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S1 transition dipoles aligned at 55°, resulting in absorption near 570 nm and emission near 630 nm. Energy transfer to the ground-state DII dimers from excited-state monomers is typically responsible for the DII emission peak observed near 630 nm. A second type of ground-state dimer, denoted DI, characterized by a sandwich-like stacking of the BODIPY rings resulting in parallel alignment of the transition dipoles and absorption near 477 nm, produced no fluorescence emission after excitation (19, 54). Even though an emission peak near 630 nm is not observed at high concentrations for Me4-BODIPY-8 phosphatidylcholine derivatives, substantial broadening of the emission spectrum does occur because of increased fluorescence near 570 nm relative to the 510–520 nm region. The persistence of emission broadening when the total absorbance is low (<0.1) in the bilayers (Fig. 2B) and monolayers (Figs. 2, 3, 5) suggests the involvement of excimer or dimer fluorescence rather than an inner filter effect. However, unequivocal determination among these possibilities will require the application of comprehensive photophysical approaches. Regardless, because the changes in emission broadness and wavelength maximum depend upon the surface concentration of B7-PC, the Me4-BODIPY-8 can be expected to be a useful probe for detecting lateral heterogeneity when attached to other lipids (i.e., sphingolipids).
What is clear from the monolayer analyses of Me4-BODIPY-8 phosphatidylcholine is that the probe mimics the behavior of fluid-phase phosphatidylcholines with unsaturated acyl chains reasonably well and does not perturb lipid packing so long as the fluorophore concentration is kept low (e.g., 1 mol%). At higher mole fractions (e.g., 10 or 20 mol%), the probe begins to exert it own influence on the system rather than serving as an "impartial" reporter. Such findings are not surprising and are commonly observed for virtually all probe molecules.
Compared with lipid probes carrying the Me2-BODIPY-3 fluorophore, the Me4-BODIPY-8 fluorophore shows an enhanced tendency for localizing within the apolar region of the bilayer, with the penetration depth dictated by the length of the connecting hydrocarbon chain. This conclusion is supported by the iodide quenching analyses of phosphatidylcholines with different length connecting chains to the Me4-BODIPY-8 fluorophore. Iodide is known to be an effective quencher of BODIPY fluorescence (9), and this small, modestly hydrated anion does not change lipid bilayers when used in concentrations up to 0.5 M (46). It is well known that iodide anions can penetrate into phospholipid bilayers (50), although the details of the transbilayer distribution of iodide remain unclear. Among other quenchers, nitroxide-labeled lipids have been used to determine the immersion depth of different BODIPY probes in bilayers (17). However, we did not use doxyl quenchers because of reported distortions of membrane structure (55, 56) that can occur at doxyl concentrations needed for efficient quenching [15 mol% doxyl (17)]. Also, doxyl labels attached to lipid hydrocarbon chains are broadly distributed in the membrane (57), complicating their use for precise determination of the fluorophore depth. It also should be noted that, among free quenchers tested for this study, iodide was the most effective. Quenchers such as free cesium cation and acrylamide did not quench Me4-BODIPY-8 fluorescence, either in bilayers or in homogeneous water solution (data not shown).
As for the iodide distribution across the bilayer, this issue has been considered by several research groups and the general consensus is that a concentration gradient forms during equilibration with iodide. Cranney et al. (58) noted that the quenching efficiency of diphenylhexatriene and its derivatives by iodide correlated with the extent of the fluorophore immersion and considered strong quenching as evidence of shallow fluorophore location. Such findings imply a transbilayer iodide gradient that is low near the bilayer center. Although such a conclusion was not explicitly stated by the authors, Langner and Hui (59) interpreted these data as evidence of an iodide concentration gradient existing across the bilayer. Barenholz and coauthors (60) investigated the iodide quenching of pyrenyl-labeled lipids in bilayers using fluorophore embedded at different depths. They concluded that I– concentration is somewhat lower in the internal bilayer regions, where the aromatic moieties of 10-(1-pyrenyl)decanoate and 16-(1-pyrenyl)hexadecanoate are located, relative to the fluorophore, 5-(1-pyrenyl)valerate.
Based on these previous findings, it is reasonable to assume that an iodide gradient does exist across the bilayer and that it is not necessarily linear. The situation for iodide can be considered analogous to that of water, which forms a nonlinear gradient across the bilayer with a profile that depends strongly on membrane composition and structure (61). Iodide, being a polar hydrated anion, can be expected to locate in the polar region of the bilayer predominantly together with water entities. However, iodide quenching, as with any bimolecular process, is sensitive to medium fluidity, which increases toward the center in the majority of bilayers (62), together with increased disorder (63). Thus, the higher efficiency of iodide quenching in the egg yolk phosphatidylcholine vesicles of 16-(1-pyrenyl)hexadecanoate fluorophore compared with the shallower immersed fluorophore of 10-(1-pyrenyl)decanoate (60) could reflect a contribution of the increased fluidity near the bilayer center.
Consideration of the preceding information together with our quenching data led us to conclude that the Me4-BODIPY-8 fluorophore attached to phosphatidylcholine probes embed in the bilayer to the limit allowed by full extension of the acyl chain. In DMPC vesicles at 17°C, Stern-Volmer plots for all Me4-BODIPY-8 probes demonstrate the lowest quenching efficiency (Fig. 7A), which is expected for a gel-state bilayer with relatively low water permeability (64), which, in turn, also reflects the permeability for iodide anions. In POPC vesicles consisting of fluid bilayers at 17°C, the water permeability is higher (64), providing increased iodide quenching efficiency (Fig. 8A, Table 2), although absolute correlation between the Ksv value and the expected fluorophore position in the bilayer is not observed in this case. In DMPC vesicles at 37°C (greater than Tm), the quenching efficiency increases for all probes (Fig. 7B, Table 2), also with deviations from the assumed order: the B5-PC fluorophore is quenched somewhat less efficiently than B7-PC and B9-PC. These discrepancies may result from uneven distribution of iodide within the bilayer at temperatures not far from the Tm. Although the existence of a decreasing I– gradient toward the bilayer center seems reasonable (see above), the character of its fluctuations in particular cases remains unknown. We hypothesize that the apparent discrepancies can be explained by the opposing influences of the I– concentration gradient and the local bilayer disorder. Although the concentration of iodide ions diminishes toward the bilayer center, the secondary effect of increased quenching rate and efficiency in the more disordered medium at the bilayer center (63) may partially override the lower local I– concentration.
It is also noteworthy that iodide quenching efficiency in the DMPC/cholesterol vesicles decreases in comparison with that in DMPC vesicles (Fig. 7B, C), in accordance with decreased water permeability (64, 65). Apart of the shallow labeled B3-PC, all other probes are quenched with nearly equal efficiencies (Table 2). Although cholesterol condenses fluid-phase DMPC (39), thus decreasing iodide quenching efficiencies and Ksv value (Fig. 7B, C), the Stern-Volmer plots remain straight, confirming a single-population distribution of Me4-BODIPY-8 fluorophore in membrane and showing that the cholesterol-induced condensation does not expel fluorophore from the bilayer interior and create dual fluorophore populations.
Verification of the Me4-BODIPY-8 fluorophore position in the bilayer was also achieved from quenching data using parallax analysis (17, 28). However, instead of using doxyl-labeled lipids as quenchers, we used iodolabeled phosphatidylcholines, I7-PC and I11-PC, for this purpose. We also intended to use another iodolabeled phosphatidylcholine, I3-PC, but were unsuccessful because of the high instability of the 3-propanoyl iodoacid. Our strategy of using iodoacids or their derivatives as fluorescence quenchers was based on consideration of the fact that iodide anion is generally a more effective quencher than bromide (50). Indeed, for indole quenching in water, quenching by iodide proceeds with a rate constant 30 times higher than that of bromide (66). In our hands, 5 mol% I7-PC or I11-PC in the POPC bilayer gave 7–24% quenching of the Me4-BODIPY-8 emission in POPC vesicles, enabling the determination of the fluorophore positions within the bilayer with reasonable accuracy (Table 3). As expected, the B9-PC fluorophore was positioned closest to the bilayer center, and B3-PC was positioned nearest to the more polar interfacial zone. Although the fluorophore positions of the B7-PC and B5-PC probes are rather close to one another, this proximity and other small discrepancies, obtained by parallax analysis compared with calculated values of zCF, appear to result from the complicated nature of the lateral pressure within the membrane leaflet, especially when the phosphoglyceride chains contain cis double bonds (67). Overall, however, the parallax analysis confirms that the Me4-BODIPY-8 fluorophore is immersed in the apolar bilayer zone at a depth determined by the linking acyl chain and agrees with the conclusion obtained on the basis of free iodide quenching.
In summary, we conclude that Me4-BODIPY-8-labeled lipids possess attractive photophysical features as membrane probes: longwave emission, high sensitivity, and stability, supplemented with improved residence in the apolar membrane region. Such features endow the Me4-BODIPY-8 fluorophore with obvious advantages over other fluorophores, such as unsymmetrical, dimethylated BODIPYs, NBD (1), or perylene (5). By embedding in the bilayer to the maximal depth allowed by the linking acyl chain, the Me4-BODIPY-8 fluorophore is expected to be particularly useful for studies in which fluorophore localization in the membrane is needed (e.g., studies of membrane topography involving protein-lipid interactions). In such capacity, Me4-BODIPY-8 probes could be used as fluorescence resonance energy transfer acceptors in combination with pyrenyl-, DPH-, or anthroyloxy-labeled lipids (1). Me4-BODIPY-8-labeled lipids also are expected to complement existing BODIPY probes used to monitor lipid traffic between cell membrane processes by fluorescence imaging microscopy, because they offer the same advantages of narrow-wavelength emission maximum and high photostability.
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ACKNOWLEDGMENTS |
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Manuscript received October 18, 2006 and in revised form March 19, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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em = 510 nm) and emission (
