Anal. Chem. 1995, 67,2659-2665
Phospholipid Vesicles as Organized Media in Spectroluminescence Analysis Sharon L. Neal* and Michele M. Villegast
Department of Chemistry, University of Califomia, Riverside, Califomia 92521
The membrane properties of pure dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylserine (DMPS), and dimyristoyl phosphatidylglycerol (DMPG) vesicles were compared using the fluorescence of the probe pyrene in an assessment of the potential utility of phospholipids as organized media in spectroluminescence analysis. The partition coefficients, excimer stability constants, vibronic band ratios, triethylamine quenching constants, and fluorescence lifetime distributionwidths of pyrene were used as measures of the solute capacity, solute aggregation promotion, polarity, sequestering efficiency, and heterogeneity of the three media. The results indicate that the head group hydrophobicity and membrane fluidity required to reduce the incidence of excimer formation and to promote solute sequestering in the most hydrophobic part of the aggregate precludes efficient sequestering from cosolubilized quenchers. This implies that significant improvements in dynamic range would be expeded in spectroluminescence analyses utilizing DMPG, but improvements of detection limits would be moderate compared to those observed in bile salts, cyclodextrins, or surfactant aggregates. Organized media (OM)' are amphiphilic polymers and selfassembled aggregates that form macromolecular structures in solution. These structures constitute microheterogeneous phases, which often exhibit dramatic polarity and viscosity gradients within the structure as well as across the structure/solvent boundary. Consequently, many OM are capable of sequestering solutes in inhospitable solvents. Moreover, solutes are sequestered in microenvironments that have considerably more structure than isotropic solvents. Increasingly, OM are being incorporated into spectroluminescence analyses because sequestering by the medium can improve analyte detection and discrimination. For example, bile salt and detergent micelles have been used in energy transfer2 and room temperature phosphorescence measurements3 to reduce the distance between fluorophores and reagents, increasing the analytical signal and reducing the detection limits of the analysis. Cyclodextrin~~ and bile salts5have been observed to increase the quantum yield of a fluorophore by shielding it from nonradiative deactivation by quenchers and polar solvent mol' Present address: Allergan, Inc., 2525 Dupont Dr., P.O. Box 19534, Imine. CA 927139534, (1) Fendler, J. H. Membrane Mimetic Cbemistly; John Wiley and Sons: New York, 1982. (2) Nithipatikom, IC; McGown. L. B. Anal. Cbem. 1988,60,1043-1045. (3) Cline Love, L. J.: Skrilec, M.: Habarta, J. G. Anal. Cbem. 1980,52, 754759. (4) Nelson, G. A; Patonay, G.: Warner, I. M. Anal. Cbem. 1988,60,274-279. (5) Ritenour Hertz, P. M.: McGown, L. B. Anal. Cbem. 1992,64, 2920-2928.
0003-270019510367-2659$9.00/0 0 1995 American Chemical Society
ecules. Quantitative analyses utilizing OM can be less susceptible to matrix effects if the medium reduces intermolecular interactions by effective analyte c~mpartmentalization.~ Additionally, sample preparation procedures can be simplified by the use of OM when samples can be prepared by simple dissolution or extraction into aqueous OM dispersion^.^ To some degree, all these benefits derive from the ability of the subphases and interfaces to act as solute scaffolds, facilitating association with reagents while shielding the solute from interferents. Phospholipid bilayer vesicles, depicted in Figure 1with typical examples of the aggregates listed above, have not been utilized in spectroluminescence analysis but are promising molecular scaffolds due to the high degree of order in alkyl chains near the polar head groups and the ability of the interior aqueous vacuole to serve as a reagent reservoir. In this paper, the measurement of the membrane properties of three types of phospholipid vesicles are described. These properties are then used to assess the potential utility of phospholipid vesicles in spectroluminescence analysis. Phospholipid bilayer vesicles have several properties that make them potentially useful as organized media and may offer advantages over other types of organizing media in some applications. Phospholipid bilayers are large, and above their liquid crystal phase transition temperature, solubilization depends on the oil/water partition coefficient of the solute and the lipid headgroup packing of the membrane.6 Consequently, vesicles are capable of solubilizing a wide range of solute sizes. Above the phase transition, the alkyl chains near the bilayer midplane, where lamellae of assembled lipids meet, are relatively fluid,' making the hydrocarbon interior accommodating of nonpolar solutes. Lipid dynamics are not likely to affect the microenvironment of solubilized molecules on the time scale of luminescence measurements. Free lipid monomer concentrations in aggregate solutions can be as low as 1O-Io M, and monomer dissociation occurs on the order of hours.* Moreover, aggregation numbers are very large, 2500-3000 for small unilamellar vesicles? further reducing the impact of lipid dynamics on individual solutes. The combination of vesicle size, membrane accessibility, and the ordered structural diversity produced by polar headgroups, moderately polar glycerol fatty acid ester linkages, and apolar hydrocarbon chains could promote discrimination of cosolubilized solutes in the vesicle. Finally, the presence of the interior aqueous vacuole, whose contents can be different from the bulk aqueous phase,I0 (6) Walter, A.; Gutknecht, J. J. Membr. Biol. 1986,90,201-217.
(7) Seek. A; Seelig, J. Biochemistry 1974,13, 4839-4845. (8) Gennis, R B. Biomembranes: Molecular Structure and Function: Springer Verlag: New York, 1989 Chapter 2. (9) Watts, A.; Marsh, D.; Knowles, P. F. Biocbemisty 1978,17, 1792-1801. (10) Redelmeier, T. E.; Hope: M. J.; Cullis. P. R Biochemistry 1990,29, 30463053.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995 2659
y-Cyclodextrin
Dodecyl Sulfate Micelle
Taurocholate Micelle
Dimyristoyl Phoshatidylcholine Vesicle
Organized media used in spectroluminescent analysis and a phospholipid bilayer vesicle. These schematics are intended to show hydrophobic regions and do not depict aggregation numbers, conformational states, or relative sizes.
Figure 1.
facilitates the design of analyses requiring controlled interaction between reagents. These properties contrast many of the limitations of traditional OM. Cyclodextrin encapsulation places stringent constraints on the relative dimensions of the solute and the cyclodextrin cavity." Since these molecules are only available in a limited number of sizes, the scope of applications based on these media is limited. Surfactant micelles are capable of solubilizing species of various sizes and polarities but are limited by the consequences of their small size. At high solute concentration, cosolubilization of multiple solutes in micelles can promote molecular interactions that compete with fluorescence and lower analysis detection limits.5 More importantly, micelles participate in a rapid dynamic (-W s) equilibrium with millimolar concentrations of surfactant monomers in the bulk aqueous phase.'* These free monomers can form premicellar aggregates with nonelectrolytes in the aqueous phase,13 reducing the driving force for micelle solubilization. There is also evidence of substantial contact between water and the alkyl chains in surfactant micelles,14no doubt increased by the monomer dynamics described above, which reduces the efficiency with which solutes are sequestered. Since micelle aggregation numbers are small, from -50 to 2008, dissociation of even a single monomer from an aggregate could have a profound effect on the solute microenvironment. Bile salts form small aggregates that compartmentalize solutes very effe~tively,~ but they lack the organized structural diversity of bilayers and the resultant capacity to controlled reagent solute interactions. (11) De la pefla, A. M.; Ndou, T. T.; Zung, J. B.; Greene. K. L.; Warner, I. M. j. Am. Chem. Sot. 1991,113, 1572-1577. (12) Fendler, J. H. Membrane Mimetic Chemistry: John Wiley and Sons: New
York, 1982; Chapter 2. (13) Bertolotti, S. G.;Zimerman, 0. E.; Cosa, J. J.; Previtali, C. M. j. Lumin. 1993,55, 105-113. (14) Menger. F. M.Acc. Chem. Res. 1979,12,111-117.
2660 Analytical Chemistry, Vol. 67, No. 75,August 1, 1995
Several variables influence the viability of phospholipid membranes as OM: individual amphiphile structure; membrane composition; and solution properties, e.g., pH, ionic strength, temperature, and pressure. In the work reported here, the medium properties of pure dimyristoyl phosphatidylcholine (DMPC) , pure dimyristoyl phosphatidylglycerol (DMPG) , and pure dimyristoyl phosphatidylserine (DMPS) vesicles in unbuffered aqueous solution were determined and compared. DMPC, DMPS, and DMPG (depicted in Figure 2) are 14-carbon diacylglycerol phospholipids, which differ in size and charge of the phosphate ester headgroup. Five properties characterizing solute/ medium interactions were measured using pyrene as a probe to maximize the validity of the comparisons by avoiding spurious differences induced by probe chemistry. The change in the fluorescence intensity with monomer concentration is used to measure the partition coefficient,15 i.e., solute capacity of the medium. The change in the monomer/excimer ratio with pyrene concentration is used to measure the tendency of the medium to promote solute aggregation and self-quenching, Le., the medium fluidity.lfi Solute capacity and aggregation reflect the impact of a medium on the linear dynamic range of luminescence analyses since they reveal the ability of the medium to minimize interactions between analyte molecules. The ratio of vibronic band intensities of pyrene solubilized by the medium is used as a measure of medium p01arity.l~Triethylamine quenching studies indicate the ability of the medium to sequester analytes from molecules in the aqueous phase that partition substantially in the medium.l8 The polarity and sequestering efficiency are correlated (15) Huang, 2.; Haughland, R. P. Biochem. Biophys. Res. Commun. 1991,181, 166-171. (16) Macdonald, A. G.; Wahle, K. W. J.; Cossins. A. R.: Behan. M. K. Biochim. BiOPhyS. A h 1988,938, 231-242. (17) Dong. D. C.; Winnik. M. A. Can. j. Chem. 1984,62, 2560-2565.
dimyristoy 1 phosphatidylcholine
dimyristoyl phosphatidylserine
dimyristoyl phosphatidylglycerol Figure 2. Structures of dimyristoyl phospholipids: dimyristoyl phosphatidylcholine(DMPC), dimyristoyl phosphatidylserine(DMPS), and dimyristoyl phosphatidylglycerol (DMPG).
to the medium impact on analysis detection limits because they reflect the ability of the medium to minimize nonradiative deactivation of the excited state. Finally, the width of the pyrene lifetime distribution in the medium is used as a measure of medium heter~geneity.’~ Medium heterogeneity or better structural heterogeneity in the probe solubilization site would indicate poor control of solute localization by the medium and reduced usefulness as a molecular scaffold. MATERIALS AND METHODS
Materials. The following reagents were used as received: pyrene (99%purity, Aldrich Chemical Co., Milwaukee, WI); p-bis[2-(5phenyloxzoyl)]benzene (POPOP) and rhodamine 610 chloride (Exciton Chemical Co., Inc., Dayton, OH); triethylamine (99% purity, Sigma Chemical Co., St. Louis, MO); dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and dimyristoyl phosphatidylserine (9% purity, Avanti Polar Lipids, Alabaster, AL). Methylene chloride, methanol, 1-propanol, 2-propanol, and l-butanol were obtained from Burdick and Jackson (Muskegon, MI) or Fisher (FairLawn, NJ) at the highest purity available. Absolute ethanol was purchased from Quantum Chemical Co. fhscola, IL). 1-Pentanolwas purchased from Wiley Organics (Coshocton, OH) at 99% purity. All aqueous solutions were prepared using doubly distilled-deionized water. Sample Preparation. Fresh lipid solutions were used for all measurements. Pyrene/lipid solutions were prepared using various modifications of the method of Huang.20 Generally, aliquots of pyrene stock solution in methylene chloride and lipid (18) Gratzel, M.; Thomas, J. K. J. Am. Chem. SOC.1973,95, 6885-6889. (19) Siemiarczuk, A; Ware, W. R. Chem. Phys. Left. 1990, 167,263-268. (20) Huang, C.-H. Biochemistry 1969,8, 344-352,
stock solution in chloroform were mixed in a round bottom flask, the solvent was removed under vacuum at approximately 50 “C for at least 10 h, and then the solution was made by adding the appropriate volume of hot water (-50 “C) to the dried sample. Vesicles were formed by sonicating the aqueous dispersion for 2 h. The size distribution was reduced by filtering it through a 0.22 pm pore filter after the solution had cooled to room temperature. The concentration of the lipid in the stock solution was determined using a modification of the spectrometric molybdate assay of Chen et aLZ1 Unless otherwise stated, the lipid concentration was approximately 0.3 mM (-0.105 pM vesicles), and pyrene concentration was 1pM. These concentrations correspond to a lipid/ pyrene ratio of 300:l and a probe/vesicle ratio of approximately 101. All samples were purged with agitation for at least 1h with argon. All fluorescence measurements were made at 5 “C above the lipid gel phase transition temperature. The procedure described above was used without variation to prepare samples for the vibronic band ratio and lifetime measurements. The partition coefficient, excimer/monomer ratio, and quenching measurements are different in that they record more dynamic aspects of vesicle/solute interactions; therefore, the lipid vesicles were formed before the solute was introduced to the sample. Aqueous lipid dispersions were prepared using the method described above, omitting the pyrene. In the case of samples for partition coefficient and excimer formation measurements, the vesicle solutions were added to dried aliquots of pyrene stock solutions and then sonicated briefly (-30 s) after the filtration step. In the quenching measurements, the quencher is the solute whose interaction with the vesicle is being measured, but adding different amounts triethylamine solution to the vesicle dispersions would have effected the pyrene and lipid concentrations. Therefore, these samples were prepared by dispersing the dried pyrene and lipid in half the volume of water used in the procedure above and by diluting the sonicated dispersion using water and increasingly concentrated aliquots of triethylamine solution in order to maintain the pyrene and lipid concentrations in the samples. These solutions were not buffered to avoid the potential impact of salt effects on solute partitioning and membrane organization. Aqueous salts would be expected to increase the partition coefficient of pyrene in the potentially damping the differences in pyrene association that we hoped to measure. Salts also screen the electrostatic effects of charged headgroups, reducing the surface p0tential.2~Since membrane order is clearly influenced by the surface polarity and hydration,24salts can also modulate membrane permeability. The literature on the pH dependence of vesicle structure indicates that signiicant changes do not occur unless the pH is less than 6 and greater 9 than for DMPSZ5or less than 4 for DMPCSz6Analogous studies for DMPG have not been reported, but structural changes would not be expected at pH levels above 4 since the pKa of the phosphate group is approximately 2. The pH of the solutions used in the (21) Chen, P. S.; Toribara. T. Y.; Warner, H. Anal. Chem. 1956,28,1756-1758. (22) Almgren, M.; Grieser, F.; Thomas, J. IC]. Am. Chem. SOC.1979,101,279291. (23) Gennis, R B. Biomembranes: Molecular Structure and Function; Springer Verlag: New York, 1989 Chapter 7. (24) Cevc, G. Biochemistry 1987,26. 6305-6310. (25) de Kroon, A. I. P. M.; Timmermans, J. W.; Killian, J. A,: de Krujff, B. Chem. Phys. Lipids 1990,54, 33-42. (26) Massari, S.; Folena. E.: Ambrosin, V.; Schiavo, G.; Colonna. R. Biochim. Biophys. Acta 1991, 1067, 131-138.
Analytical Chemistry, Vol. 67,No. 15, August 1, 1995
2661
measurements described here was monitored to ensure that these extremes were not exceeded in the results reported here. Instrumentation. Dynamic Light Scattering. The diameters (hydrodynamic radius) of the lipid vesicles studied in the work reported here were measured using a dynamic light scattering photometer (Nicomp 370, Particle Sizing Systems, Santa Barbara, CAI. The photometer correlates fluctuations in the intensity of the light scattered by the sample to particle diameter through the decay constant of the intensity autocorrelation function. The principles of this method have been thoroughly described elsewhere.27 Fluorescence. Fluorescence data were acquired using the K2 multifrequency cross-correlation phase and modulation spectrofluorometer OSS, Champaign, IL). Excitation at 335 nm was provided by a 300 W Xe arc lamp. The fluorescence emission was monitored at 90" to the excitation by a photomultiplier tube. For steady-state measurements, a quantum counter consisting of 25 mg/L rhodamine 610 chloride in ethanol was used to correct the fluorescence intensity for excitation fluctuations. Pyrene monomer emission was monitored at 386 nm unless otherwise stated. Pyrene excimer emission was monitored at 475 nm. Slits of 2 mm 16 nm bandpass, were used on the excitation channel, and 1 mm slits, 4 nm bandpass, were used on the emission channel. Pyrene spectra were dispersed by a spectrograph equipped with a 150 groove/" grating (Model HF320, Instruments SA, Metuchen, NJ) and measured between 290 and 800 nm using a 1024 element-intensitied photodiode array (Model IRY1024, Princeton Instruments, Trenton, NJ) , For liietime measurements, the excitation was sinusoidally modulated at 15 logarithmically spaced frequencies from 0.30 to 30 MHz via a Pockels cell. Slits of 2 mm were used on both the excitation and emission channels, and the fluorescence was observed through a 380 nm long pass filter (Schott type, Oriel Corp., Stratford, 0. The liietime data were acquired using POPOP in ethanol as the reference liietime standard. The reference lifetime was assigned the widely reported value of 1.35 nsZa Data Acquisition and Analysis. Vibronic Band Ratio. Polar solvents enhance the symmetry forbidden 0-0 band via symmetry reducing a~sociation,~~ making the intensity of the vibronic bands of pyrene dependent on solvent Many efforts have been made to relate the magnitude of this enhancement, usually reported as the ratio of steady-state intensities near 373 and 384 nm O/III), to macroscopic solvent properties such as the dielectric constant or dipole moment. In general, these efforts have been unsuccessful, but I/III can be related to solvent polarity when the interactions producing polarity differences are captured within a chemical class.17 In this work, a linear calibration curve relating I/III to the solvent dielectric constant, e, was constructed using the fluorescence intensities of alcohol solutions containing 1mM pyrene at 373 and 384 nm. We found that
measured, and the apparent polarity of the pyrene microenvironment was calculated from eq 1. Partition Coefficient. The partition coefficient Kp is the ratio of probe concentration in the aggregate to that in the aqueous phase. In this work, the value of Kpis determined by measuring the probe fluorescence as a function of increasing aggregate concentration.16 As the aggregate concentration increases, an increasing number of probe molecules can partition into the nonpolar phase of the aggregate. The intensity of the probe fluorescence increases because the quantum yield of the probe in the medium is larger than it is in the aqueous phase. The rate of the fluorescence intensity growth with amphiphile concentration depends on how many molecules were encapsulated per added aggregate, i.e., the partition coefficient. Huang and Haughland16 show that the partition coefficient can be calculated from the slope and yintercept of the regression between the inverse fluorescence intensity and inverse medium concentration according to
1/F = (55.6/F&J (l/[mediuml)
+ l/Fo
(2)
where F is the fluorescence intensity in the presence of vesicles, FOis the fluorescence intensity in pure water, and [medium] is the vesicle concentration. Excimer/Monomer Ratio. The formation rate of pyrene excimer and the excimer to monomer intensity ratio have been shown to obey reversible two-state kinetics and Einstein-Smoluckowski diffusion in isotropic solvent.31 However, Blackwell et al.32have investigated the behavior of pyrene in a variety of model membranes and found that the criteria for diffusion-controlled (collisional) excimer formation defined by Birks are not satisfied in these anisotropic media. They attribute excimer formation in membranes to a static process between aggregated pyrene molecules. In more recent work, some authors33have used a three-state model in which the excimer is formed by a combination of static and diffusion-controlled mechanisms to describe pyrene excimer kinetics, but these models, their sophistication notwithstanding, fit pyrene emission in membranes rather poorly.33 Since these are steady-state measurements, they have no value in determining the mechanism of excimer formation. For convenience, we use the static model to describe excimer formation as a simple reaction between molecules in an aggregate, and the excimer/monomer intensity ratio is related to the equilibrium constant of that reaction, KE:
ZM,,
- X*
(34
The intensity ratio of probes solubilized by the lipid vesicles were
where Mag, represents the aggregated monomer and X* represents the excimer. The value of KE is determined by measuring the intensity ratio of excimer fluorescence, ZX, to monomer fluorescence, IM,as a function of increasing pyrene concentration. The slope of the least-squares lines is taken as a measure of the tendency of a medium to promote solute aggregation.
(27)PhiIlies, G. D. J. Anal. Chem. 1990, 62,1049A-1057A (28)Lakowicz, J. R.: Cherek, H.; Baker, A.J. Biochem. Biophys. Methods 1981, 5. 131-146. (29)Lianos. P.; Georghiou, S. Photochem. Photobiol. 1979,30, 355-362. (30) Nakajima, A. Bull. Chem. Soc. Jpn. 1971,44,3272-3277.
(311 Birks, J. B.; Dyson, D. J.: Munro, I. H. Proc. R. Soc. London,A 1963,275, 575-588. (32)Blackwell, M. F.: Gounaris. IC; Barber, J. Biochim. Biophys. Acta 1986, 858, 221-234. (33)Sugar, I. P.; Zeng. J.; Chong, P. L. G.J. Phys. Chem. 1991.95,7524-7534.
I/III = 0.216
+ 0.60
2662 Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
(1)
Fluorescence Quenching. Fluorescence quenching processes are distinguished by their effect on the excited state.34 Static quenchers form fluorophore-quencher complexes, which reduce the number of excited states produced. Dynamic quenchers have no effect on fluorophore excitation, rather they increase the rate of excited state deactivation, generally via collision. Dynamic quenching is related to the rate of collision via the Stem-Volmer equation:
(4) where to is the lifetime of the fluorophore in the absence of quencher and k, is the bimolecular quenching constant. In micelles and bilayers, quenching is also influenced by the partitioning of the quencher and fluorophore between the aggregate and the aqueous phase.35 In the case of a probe that is completely bound to the medium and a partitioning quencher, the quenching constant becomes an apparent quenching constant, k,,, that combines the rate of collision and the entrance rate of the quencher into the medium. The quencher concentration becomes the effective local quencher concentration, [Qllod,which is related to the analytical quencher concentration, [Qltod,through the quencher partition coefficient, [Qllocal= [QItod(KJq[MI/@+(&),[MI) where [MI is the concentration of the medium in solution. In the studies reported here, an apparent quenching constant was determined using eq 4, by measuring the relative lifetime of pyrene solubilized in vesicles as a function of quencher concentration. The apparent quenching constant can be considered a measure of the probe accessibility to water-soluble molecules that partition substantially in the membrane phase. Fluorescence Lifetime Distributions. The fluorescence lifetime, t,is the reciprocal of the excited state deactivation rate. Processes other than photon emission including vibrational relaxation, intersystem crossing, collisional deactivation, and photodecomposition contribute to excited state dea~tivation.~~ In organized media, structural diversity in the solubilization site can lead to local variations in the rates of these processes. In some cases, these local variations are best described by distributions rather than discrete fluorophore lifetime values.'g The lifetime distribution can be measured in either the time or frequency domain. In this work, the frequency domain method was used. The phase, 4, and modulation, m, of sinusoidally modulated emissions are measured relative to the phase and modulation of sinusoidally modulated excitation at several frequencies. The fluorescence lifetime can be estimated from these measured parameters using procedures that have been described elsewhere." In this work, lifetime estimation was performed using globals lifetime analysis software (Laboratory for Fluorescence Dynamics, Champaign, IL) running on a 33 MHz 486 computer. The program minimizes the x 2 function
comparing the experimental data to data simulated from the (34) Blatt, E.; Sawyer, W. H. Biochim. Biophys. Acta 1985,882, 43-62. (35) Yetka, A; Aikawa, M.; Turro, N. J. Chem. Phys. Lett. 1979,63, 543-548. (36) Lakowicz, J. R. Principles ofFhorescence Spectroscopy; Plenum Press: New York, 1983; Chapter 3. (37) Beecham, J. M.; Knutson, J. R; Ross, J. B. A; Turner, B. W.; Brand, L. Biochemisty 1983,22, 6054-6058.
Table 1. Physlcal Properties of Phospholipid Vesicles
no. of head group charges net head group charge
DMPS
DMPC
3 -1
2 0
aggregation no.
gel-phase transition ("C) waters of hydration head group area (Az) alkyl chain tilt (deg)
vesicle diameter (nm)
38 9-10 0 151
DMPG 1
-1
285014 23 11-12 39 12 131
23 16 44 29 120
proposed fluorescence distribution. The values used for the constants a, and upwere 0.004 and 0.2, respectively; the values frequently used in fitting photomultiplier tube data.38 Lifetime distributions were described as a combination of Dirac (discrete), square wave (uniform), Gaussian, and Lorentzian profiles. The amplitude, mean lifetime, and distribution width were varied in order to simulate data approximating the experimental phases and modulation ratios. RESULTS AND DISCUSSION Several physical properties of DMPC, DMPG, and DMPS that were not measured in these experiments are important in the interpretation of the experimental results and are listed in Table 1. On phosphocholine lipids, the quaternary amine on the ethyl ester makes the headgroup a neutral zwitterion that occupies a maximum surface area equal to 52 Az in lysophosphocholine (single alkyl chain) lipids crystal structure^.^^ In hydrated membranes, choline headgroups occupy a surface area closer to 70 A2.40DMPC bilayers undergo an alkyl chain melting phase transition at 23 0C,41indicating comparatively weak association between alkyl chains. On the phosphoserine lipids, charges on the carboxylate and amine groups cancel each other, imparting an overall negative I charge to the lipid. A crystal structure for DMPS has not been reported, but the gel phase transition temperature is higher than that of DMPC (38 OC),ll and the number of associated water molecules is lower despite the additional charge,42implying that the serine headgroup occupies a smaller surface area. This is corroborated by the observation that the alkyl chains are normal to the membrane surface in DMPS bilayers43whereas they are tilted in DMPC39and DMPG" bilayers to increase close packing of the alkyl chains. In DMPG, the phosphate is esterifled by glycerol, which has neutral hydroxyl groups on carbons 2 and 3, making the lipid negatively charged. The alkyl chain tilt, surface area of the crystal structure headgroup," and number of water molecules hydrating the glycerol h e a d g r o ~ pindicate ~ ~ that the hydrated area is slightly larger than that of DMPC. The DMPG phase transition temperatures are the same,4l which implies that the d ~ e r e n c eis small. However, the glycerol headgroup is more hydrophobic than the choline and (38)Fiorini, R.; Valentino, M.; Wang, S.; Glaser, M.; Gratton, E. Biochemistry 1987,26,3864-3870. (39) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981,650, 21-51. (40) King, G. I.: White, S. H. Biophys. j. 1986,49, 1047-1054. (41) Small, D.M. The Physical Chemisty of Lipids; Plenum Press: New York, 1986. (42) Cevc, G.; Watts, A.; Marsh, D. Biochemistry 1981,20, 4955-4965. (43) Hauser, H.; Paltauf, F.; Shipley, G. G. Biochemisty 1982,21, 1061-1067. (44) Pascher, I.; Sundell, S.; Harlos, IC; Eibl, H. Biochim. Biophys. Acta 1987, 896,77-88. (45) Borle, F.; S e e k J. Biochim. Biophys. Acta 1983,735, 131-136.
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2663
should present a lower free energy barrier to permeation by nonionic molecules. The particle diameters of the vesicles used here were measured by dynamic light scattering and are also reported in Table 1. Apparent Medium Polarity. The apparent polarities of the microenvironments experienced by pyrene were determined using the calibration curve between the vibronic band ratios and dielectric constants of a homologous series of straight chain alcohols described by eq 1. Apparent dielectric constants equal to 23 1 for DMPS, 19 f 1 for DMPC, and 19 f 1 for DMPG were determined. The vibronic band ratios of the mediumassociated probes indicate that, in DMPC and DMPG, pyrenes experience an environment that is slightly more polar than propanol. The apparent dielectric constant of the DMPS solubilization site is somewhat higher (23), which is closer to the dielectric constant of ethanol. The compactness of the alkyl chains of DMPS as indicated by the data in Table 1 would promote increased solubilization in the more polar regions of the vesicle. In all three cases, the data indicate very polar solubilization sites for pyrene. Thermodynamically, solubilization of hydrophobic molecules at the bilayer midplane would be favorable.46 However, other studies of bilayer polarity using pyrene fluorescence have also been interpreted as indicating a solubilization site in the polar regions of the bilayer.47 Since the alkyl chains of the membrane (-40 A thick) are more ordered near the headgroups, the size and shape of the pyrene molecule (a 10 A oval disk) facilitate its intercalation in the ordered regions of the membrane.I6 Membrane Capacity. The partition coefficients of pyrene in DMPS, DMPC, and DMPG were calculated from the increase in the fluorescence intensity data as a function of the vesicle concentration. Using eq 2, the data yield Kpvalues equal to (1.06 f 0.06) x lo9, (7.3 f 0.6) x 108 and (6.8 f 0.3) x 108,respectively. As packing considerations imply, the partition coefficients for DMPC and DMPG are very similar. The higher capacity of DMPS (44%) does not follow the trend observed in the gel phase transition temperatures and membrane chain tilts, but if the probe has a sterically driven preference for more ordered regions of the membrane, as discussed above, the more rigid membrane would have a higher capacity for that probe. Alternatively, the large DMPS value could be the result of the higher temperature of the DMPS dispersions. All the samples were studied at 5 "C above the gel phase transition temperature: 28 'C for DMPC and DMPG and 43 "C for DMPS, in an attempt to put the fluidity of the hydrophobic phases on the same scale. The higher temperature may also have affected the ability of the solute to permeate the membrane surface. Solute Aggregation. The dependence of the excimer/ monomer ratio on increasing pyrene concentration was used to assess the tendency of the medium to promote solute aggregation. Excimer formation constants, KE, were calculated from the increase in the ratio of excimer to monomer emission intensities @/M) as a function of pyrene concentration. Using eq 3b, formation constants equal to (3.1 f 0.2) x lo4 in DMPS, (3.5 f 0.4) x lo4 in DMPC, and (2.2 f 0.2) x lo4 in DMPG were determined. In this case, DMPC and DMPS provide similar media, while excimer emission increases -30% more slowly with increasing pyrene concentration in DMPG. This is consistent with (46) Marqusee, J. A.; Dill, IC A. J. Phys. Chem. 1986,85, 434.
(47) Limos, P.; Mukhopadhyay, A IC;Georghiou, s.Photochem. ~ h ~ t ~ b1980, i~i. 32, 415-419.
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Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
Table 2. Pyrene Quenching by Triethylamine in Phospholipid Vesicles
DMPS
alkyl chain tilt (deg) waters of hydration hydrated head group area (A2) pyrene lifetime (ns) Stem-Volmer constant (M-I) quenching constant (M-I ns-')
DMPC
DMPG
0 9- 10
12 11-12 70
29 16
209 f 4 81 i 2
272 f 17 119 f 4
280 & 12 169 & 3
0.39 f 0.02
0.45 f 0.03
0.61 i 0.28
Table 3. Pyrene Lifetimes in Phospholipid Vesicles'
fi
71 (ns) 671 (ns) 72 (ns)
DMPS
DMPC
DMPG
0.971 i 0.004 209 f 4 30 f 6 1-5
0.985 f 0.001 272 f 17 0.2 i. 0.0 1-5
0.983 & 0.002 280 f 12 40 f 19 1-5
a Abbreviations: fi, fractional intensity of first component; 71,mean lietime of first component; 671, distribution width of first lifetime component; 72,mean lifetime of second component.
the picture of the more fluid, permeable membrane indicated by the chain tilt and hydration data: fewer of the pyrene molecules are solubilized near pyrene neighbors in DMPG. This result is also consistent with the quenching data that will be discussed below. It contrasts the results of Macdonald et al.,I6 who observed a decrease in E/M with increasing membrane rigidity, but the pyrene concentrations used in those measurements were 2 orders of magnitude larger than those used here making direct comparison ill-advised. Sequestering Efficiency. The dynamic quenching of lipidassociated pyrene by the amphiphilic quencher triethylamine was used as a measure of the capacity of the medium to protect the fluorophore from molecules partitioning into the vesicle from the aqueous phase. The Stem-Volmer constants were calculated from the relative lifetime of pyrene in the presence of increasing amounts of triethylamine. The mean lifetimes of the predominant lifetime components were then used to calculate apparent quenching constants. The results of these analyses are listed in Table 2. The alkyl chain tilt and water of hydration data from Table 1 are repeated to facilitate comparison. These data indicate that the permeability of the membrane is dependent on the alkyl chain packing. There is a clear dependence of the apparent quenching constants on the observed chain tilt though there are too few points to warrant a quantitative correlation analysis. Medium Heterogeneity. The results of the pyrene lifetime determinations in the three lipid vesicles are reported in Table 3. In all three cases, two lifetime components were used to fit the decay data: a large, long-lived Lorentzian distribution and a small, short discreet component. In replicate analyses, the value of the short lifetime value varied substantially over the range indicated. Presumably, the long-lived component is sequestered, while the quenched component is either adsorbed to the vesicle surface, relaxed by self-association, scattered light transmitted by the emission filter, or an artifact generated by the data analysis to compensate for an unusual shape in the liietime distribution. The small amplitude of this component precludes more definitive characterization. The widths of the sequestered components
indicate that while pyrene either d f i s e s rapidly between various microenvironments or is solubilized in an essentially homogeneous environment in DMPC, very heterogeneous environments are experienced by the probe in DMPS and DMPG. This is not surprising in DMPS where the strong interlipid interactions would be expected to reduce probe mobility. The width of the DMPG distribution is even wider than that observed in DMPS, an unexpected result given the similarity of the DMPG transition temperature and mean pyrene liietime to those observed for DMPC. If the slightly longer mean lifetime of pyrene in DMPG is attributed to a slightly deeper penetration by pyrene into the more hydrophobic regions of DMPG, the broad distribution derives from the increase in the structural diversity in and/or distance between the sites accessible to pyrene in DMPG. CONCLUSIONS
The goal of this study was to measure the membrane properties of pure phospholipid vesicles using a single probe, pyrene, to investigate the utility of this class of self-assembled aggregates as organizing media in spectroluminescence analysis. A secondary outcome of the study was the exploration of the role of lipid headgroup structure on nonelectrolyte solubilization in pure phospholipid vesicles. The apparent medium polarity, medium capacity, tendency to promote solute aggregation, sequestering efficiency, and medium heterogeneity were measured and compared. The results reported here are consistent with the theory that membrane fluidity depends strongly on the size and degree of hydration of the lipid headgroups. Close packing restricts conformation changes in the alkyl chains, making the membrane less fluid and reducing the mobility of nonelectrolytes. Close packing also increases the tendency of the medium to promote solute self-aggregation since fewer probes are independently solubilized in rigid systems. These data imply that loose headgroup packing (and nonelectrolyte solubilization) is facilitated by more hydrophobic headgroups that experience fewer interlipid electrostatic interactions. The heterogeneity of the solubilization site depends on the combination of membrane fluidity (chain ordering), probe penetration depth, and the size of the polarity/ viscosity gradients near the solubilization site. (48) Villegas. M. M.; Neal, S. L. Anal. Chim. Acta 1995, 307,419-430.
An optimal medium for luminescence analysis would have a large solute capacity, high sequestering efficiency with no tendency to promote solute aggregation, and very nonpolar, homogeneous solubilization site. Unfortunately, the studies reported here indicate that there is a trade-off between several of these properties: high membrane fluidity increases the solute capacity and solubilization site hydrophobicity and decreases solute aggregation, yet reduces the sequestering efficiency as was observed in the case of DMPG. The results also imply that there is a dependence in the site heterogeneity on the penetration depth. Since the polarity drops precipitously and rises again across the membrane, the site heterogeneity must be very probe dependent. Take together, the results indicate that PG lipids would have the greatest potential impact on spectroluminescent analyses of nonpolar fluorophores. Significant improvements in dynamic range would be expected, but emission intensity enhancements would be moderate at best, not nearly as dramatic as those associated with cyclodextrins, surfactant aggregates, and bile salts. In contrast to DMPG, DMPC and DMPS indicate higher sequestering efficiencies but higher incidence of solute aggregation and more polar solubilization sites as well. This prediction is s u b stantiated by the results of a similar comparison of the medium properties of DMPC and sodium dodecyl sulfate micelles48which indicates that the smaller aggregate has a superior sequestering efficiency but lower medium capacity. None of these comparisons explored the potential role of the aqueous reservoir or the structural organization of the bilayer interior. Both of these features could be the source of selectivity enhancements that would compensate for the limited improvements phospholipids provide in intensity enhancements. ACKNOWLEDGMENT
This work was supported by the National Science Foundation Grant CHE-9314935. S.L.N. acknowledges the support of the Beckman Foundation. The authors also wish to thank Prof. Menachem Elimelech for the use of the particle sizing equipment. Received for review November 8, 1994. Accepted March 20, 1995.@ AC941092B @Abstractpublished in Advance ACS Abstracts, May 1. 1995.
Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
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