Using the Decay of Incorporated Photoexcited Triplet Probes to Study

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Using the Decay of Incorporated Photoexcited Triplet Probes to Study Unilamellar Phospholipid Bilayer Membranes Pe´ter Baranyai,† Susanne Gangl,‡ Gottfried Grabner,*,‡ Martin Knapp,‡,§ Gottfried Ko¨hler,‡,§ and Tama´s Vido´czy† Institute of Chemistry, Chemical Research Center of the Hungarian Academy of Sciences, H-1525 Budapest, Pf. 17, Hungary, Institute for Theoretical Chemistry and Radiation Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria, and ASM - Institute for Biophysics, Lustkandlgasse 52/3, A-1090 Vienna, Austria Received April 30, 1999. In Final Form: June 23, 1999 The decay kinetics of triplet excited probes (meso-tetraphenylporphyrin or anthracene) in unilamellar phospholipid bilayer membranes was studied using nanosecond laser-induced transient absorption spectroscopy. Two decay modes, quenching by molecular oxygen and triplet-triplet annihilation, were investigated as a function of temperature. The rates of both processes were sensitive to the phase of the membrane and underwent a significant change at the temperature corresponding to the main phase transition. The analysis of triplet annihilation data obtained in dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine unilamellar vesicles, and mixtures thereof, demonstrates that the phase transition can be analyzed in these systems with an accuracy matching that of other techniques, including calorimetry; this accuracy applies to the determination of critical temperatures as well as to the estimation of transition cooperativity. Triplet quenching by molecular oxygen is shown to be largely determined by diffusion of oxygen from the bulk medium to the membrane. Triplet decay is thus a suitable probe of fluidity as well as of diffusional processes in unilamellar bilayers.

Introduction The complexity of phospholipid bilayer membranes is such that, despite decades of intensive research1,2 their structural and dynamic properties are still not completely elucidated. This is the case even for membranes consisting of a single phospholipid component, but still more so for systems in which steroids (such as cholesterol), peptides, or membrane proteins are mixed with one or more phospholipid components; yet, only these complex structures can claim to serve as models for real biological membranes. Moreover, the latter are always of unilamellar structure, whereas model studies very often are performed on multilamellar composites, the multilamellar large vesicles (MLVs). Although MLVs do have many physical properties that are interesting in their own right, such as rich polymorphism, they are not in all respects good models for unilamellar membranes. As an example, evidence has accumulated over the years indicating that properties of the main (gel-to-liquid crystalline) phase transition of phospholipid vesicles depend strongly on their size and structure.3-6 These measurements, using calorimetric as well as fluorescence probe techniques, concurred to show that, with decreasing vesicle size, both in terms of thickness and diameter, the phase transition tends to broaden, implying that the van’t †

Hungarian Academy of Sciences. University of Vienna. § Institute for Biophysics. ‡

(1) Lipowsky, R.; Sackmann, E. Structure and Dynamics of Membranes; Elsevier: Amsterdam, The Netherlands, 1995. (2) Koynova, R.; Caffrey, M. Biochim. Biophys. Acta 1998, 1376, 91. (3) Suurkuusk, J.; Lentz, B. R.; Barenholtz, Y.; Biltonen, R. L.; Thompson, T. E. Biochemistry 1976, 15, 1393. (4) Takemoto, H.; Inoue, S.; Yasunaga, T.; Sukigara, M.; Toyoshima, Y. J. Phys. Chem. 1981, 85, 1032. (5) Nagano, H.; Nakanishi, T.; Yao, H.; Ema, K. Phys. Rev. E 1995, 52, 4244. (6) Heimburg, T. Biochim. Biophys. Acta 1998, 1415, 147.

Hoff enthalpy, ∆HvH, of the transition decreases with decreasing vesicle size. On the other hand, calorimetry indicated that the transition enthalpy, ∆Hcal, is not significantly size-dependent.6 Consequently, the cooperativity of the transition, which is the ratio ∆HvH/∆Hcal7, also decreases with vesicle size. This size dependence also appears to be the case when the size of unilamellar vesicles is decreased from a diameter of the order of 100 nm or more (large unilamellar vesiclessLUVs) to 50 nm or less (small unilamellar vesiclessSUVs).4 Experimental methods that are able to probe unilamellar vesicles are therefore of great value. Among these, spectroscopic probe techniques are attractive because they can provide both structural and dynamic information about membranes. Fluorescence spectroscopy is by far the most widely used method for this purpose. Compared to it, triplet-state properties of membrane-incorporated probes have received much less attention; this lack of attention appears understandable at first sight because there are very few probe molecules having a suitably high phosphorescence yield in fluid solutions8,9 and because triplet-triplet absorption cannot match emission techniques in sensitivity. The use of triplet probes does, however, present some attractive features. The most significant difference with respect to fluorescence methods is provided by the usually much longer lifetime, which opens a different time scale for dynamic studies, namely, the microsecond-to-millisecond range. Triplet probes should therefore be more suitable than singlet probes for studies of molecular diffusion in and through the membrane, including diffusion of the probe itself and diffusion (7) Blume, A. In: Physical Properties of Biological Membranes and Their Functional Implications; Hidalgo, C., Ed.; Plenum Press: New York, 1988; p 71. (8) Johnson, P.; Garland, P. B. FEBS Lett. 1983, 153, 391. (9) Vanderkooi, J. M.; Wright, W. W.; Erecinska, M. Biochemistry 1990, 29, 5332.

10.1021/la990532x CCC: $18.00 © 1999 American Chemical Society Published on Web 08/13/1999

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of molecules able to interact with the probe, such as molecular oxygen. The use of triplet-triplet absorption of membraneincorporated probe molecules is by no means new. The ability of a lipid-bound anthracene probe to sense the main phase transition of sonicated SUVs was demonstrated using microsecond flash photolysis.10 The same result was obtained in a later laser flash-photolysis study of SUVs prepared by the ethanol injection technique, using hematoporphyrin and its dimethyl ester as probes.11 Very recently, the deactivation of triplet tetra(N-methyl-4pyridyl)porphyrin was used to study surface diffusion on small dihexadecyl phosphate vesicles.12 In all these studies, an abrupt change of the rate of triplet deactivation was observed in the range of the main phase transition. Despite positive results, these studies have remained isolated, and the potential provided by the application of triplet probes to membrane studies has, to our knowledge, never been systematically investigated. In the present study, we applied nanosecond lasertransient absorption spectroscopy to assess the potential of triplet probes for studies of unilamellar vesicles, with the main emphasis on the sensitivity and accuracy of the detection of the main phase transition in vesicles consisting of the well-studied phospholipids dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). Different vesicle preparation techniques and two kinds of probe molecules, meso-tetraphenylporphyrin (TPP) and anthracene, were used. We will show that the performance of the triplet absorption method in these model systems is satisfactory and that the technique also holds promise in yielding valuable results in more complex membrane systems. Experimental Section

Baranyai et al. Anthracene was either added to the lipid/cholate mixture before dialysis or injected from a concentrated ethanol solution into the dialyzed lipid solution. Incorporation was successful by both procedures. Lipid/cholate ratios of 0.635 were used for the preparations; vesicles with a diameter of about 60 nm are expected to be formed under these conditions.15 The extrusion technique was employed in a few runs as an alternative method to produce LUVs, using an Avestin extruder with a 100 nm pore size.16,17 Although these preparations yielded basically the same experimental results as the dialyzed ones, they were more difficult to handle due to significantly higher light scattering. Furthermore, the probe-to-lipid ratio proved to be difficult to control in the extrusion process. The greater part of the data was therefore obtained with dialyzed samples. Spectroscopic Techniques. The third harmonic of a Nd: YAG laser (Coherent Infinity) and a broadband optical parametric oscillator (Lambda-Physik OPPO) were used as the excitation sources in transient absorption spectroscopy. They delivered pulses with a duration (fwhm) of 3-5 ns; pulse-to-pulse reproducibility was of the order of 5-10%. Time-resolved absorbance was measured in a right-angle geometry as described earlier;18 in this setup, both exciting and analyzing light beams are delimited by rectangular apertures, which define a reaction volume of dimensions 0.17 cm (height), 0.32 cm (width), and 0.13 cm (depth) at the entrance front of the sample cell. The pulse energy delivered to the sample was varied between 0.05 and 7 mJ/pulse by adjusting the pumping level and was measured at the sample cell location using a ballistic calorimeter (RayconWEC 730). The time resolution of the experiments was on the order of 10 ns, and the sensitivity of absorbance measurement was on the order of 0.001. TPP was excited at the maximum of the first Q-band (514 nm) using the OPPO, and anthracene was excited at 355 nm. A thermostatted cell holder was used to measure the temperature dependence of the triplet lifetime. The temperature was measured inside the cell. For oxygen quenching measurements, the solutions were air equilibrated by slow bubbling of air. For the triplettriplet annihilation studies, samples were deoxygenated by bubbling with nitrogen. Fluorescence polarization measurements were performed with a Perkin-Elmer LSB-50 spectrofluorimeter equipped with a thermostatted cell holder.

Chemicals. Anthracene (scintillation grade, Merck), 5,10,15,20-tetraphenylporphyrin (TPP, Aldrich), L-R-dipalmytoylphosphatidyl-choline (DPPC, 99%, Sigma), and L-R-dimiristoylphosphatidyl-choline (DMPC, 99%, Sigma) were used as received. Spectroscopic- or HPLC-grade organic solvents were used. All aqueous solutions were prepared with doubly distilled water buffered with either 0.01 M phosphate to pH 7.4 or Tris to pH 7.2. Liposome Preparation and Probe Incorporation. Two liposome preparation methods were mainly used in the present work, the injection technique and the dialysis technique. The injection method was used mainly for preparing porphyrincontaining SUVs, according to the literature method.13 In this method, 9.6 mM phospholipid was dissolved in ethanol, together with the required probe. Ethanol solution (0.2 mL) was slowly injected into 3 mL phosphate buffered (pH ) 7.4) saline solution, which was magnetically stirred and thermostatted a few degrees above the critical phase transition temperature of the lipid. This method produces SUVs of approximately 50 nm diameter. A higher lipid concentration (40 mM) in the ethanol solution results in LUVs with a diameter of about 100 nm. The freshly prepared liposome solution showed very little opacity and was stable for several hours. The dialysis (detergent solubilization) technique14 was employed to prepare SUVs with incorporated anthracene. Liposomes were typically prepared from 6 × 10-4 or 1.2 × 10-3 M lipid with probe-to-lipid ratios nominally ranging from 1:200 to 1:20.

Triplet-Triplet Absorption. Unilamellar phospholipid vesicles containing probe molecules were subjected to transient absorption spectroscopy. Incorporation of the probe molecules into the phospholipid vesicles was evident from the appearance of their known triplet-triplet absorption bands.19 Because the aqueous solubility of the utilized probes is exceedingly low (2 × 10-7 M in the case of anthracene and below this value in the case of TPP), the majority of the probe molecules must be dissolved in the lipid fraction. In the case of anthracene, membrane incorporation is also shown by a characteristic shift of the triplet-triplet absorption which is displayed in Figure 1, using as an example the more intense band of the characteristic anthracene triplet-triplet spectrum. This transition, which is located near 420 nm in most common organic solvents,19 undergoes a red shift of 7 nm on incorporation into the gel phase (18 °C) of a DMPC vesicle. A red-shift of about 3 nm can be readily observed in homogeneous solutions when longer chain-length alkanes are used as

(10) Razi Naqvi, K.; Behr, J.-P.; Chapman, D. Chem. Phys. Lett. 1974, 26, 440. (11) Ricchelli, F.; Olsen, K.; Lindqvist, L. J. Photochem. Photobiol., B: Biology 1988, 2, 475. (12) Khairutdinov, R. F.; Hurst, J. K. J. Phys. Chem. B 1999, 103, 3682. (13) Kremer, J. M. H.; DeEsker M. W. J.; Pathmamanohoran C.; Wieserma P. H. Biochemistry 1977, 16, 3932. (14) New, R. R. C. In Liposomes. A Practical Approach; New, R. R. C., Ed. IRL Press (Oxford University Press): New York, 1990; p 83.

(15) Zumbu¨hl, O.; Weder, H.-G. Biochim. Biophys. Acta 1981, 640, 252. (16) Nayar, R.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1989, 986, 200. (17) MacDonald, R. C.; MacDonald, R. I.; Menco, B. P. M.; Takeshita, K.; Subbarao, N. K.; Hu, L. R. Biochim. Biophys. Acta 1991, 1061, 297. (18) Grabner, G.; Getoff, N.; Gantchev, T.; Angelov, D.; Shopova, M. Photochem. Photobiol. 1991, 54, 673. (19) Carmichael, I.; Hug, G. L. J. Phys. Chem. Ref. Data 1986, 15, 1.

Results

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Figure 1. Triplet-triplet absorption spectrum of anthracene in hexane (A) and in DMPC SUVs at 25 °C (B) and 18 °C (C). Figure 3. Temperature dependence of the decay rate of TPP triplets in DPPC SUVs in air-equilibrated solutions. The inset shows oscilloscope traces recorded at various temperatures (10, 20, 30, 40, and 60 °C, from upper trace to lower trace).

Figure 2. Laser pulse energy dependence of the initial triplettriplet absorbance of TPP in DMPC SUVs in air-equilibrated solutions (left axis) at 20 °C (9) and 26 °C (0) as well as the dependence of the rate of oxygen quenching (right axis) at 20 °C (b) and at 26 °C (O).

solvents, due to the influence of the solvent refractive index. A further contribution to the shift is expected from the change in molecular polarizability upon excitation, reflecting a strong specific solute-solvent interaction. Crossing the main phase transition, which in LUVs is located at 24.1 °C by calorimetry20 has little influence on the transition, the red-shift with respect to the ethanol solution being reduced by less than 1 nm. The band halfwidths are nearly equal in all conditions, from which it can be deduced that the extinction coefficient is the same as in the organic medium ( ) 6 × 104 M-1 cm-1 at band maximum19). The triplet-triplet spectrum of anthracene incorporated in vesicles prepared from DMPC or DPPC by different techniques showed no sign of an influence of the mode of preparation. The dependence of the laser-induced initial absorbance of the TPP triplet in DMPC on laser pulse energy is shown in Figure 2, along with measured values of the triplet decay rate in air-equilibrated solution (see below). The absorbance data exhibit saturation at high pulse energies, as expected for a triplet-triplet transition, and again show little influence of the membrane phase. Triplet Quenching by Molecular Oxygen. The ability of triplet-triplet absorption to serve as a probe of structural changes in phospholipid membranes was investigated by studying its decay kinetics as a function of temperature. In the presence of O2, triplet decay is dominated by oxygen quenching, provided that the local concentration of triplets is not high enough to favor triplet-triplet annihilation processes. TPP incorporated (20) Jutila, A.; Kinnunen P. K. J. J. Phys. Chem. A 1997, 101, 7635.

into DMPC or DPPC vesicles was used for these measurements. Experiments performed with anthracene yielded similar results, but TPP was a more suitable probe due to its somewhat slower quenching by O2 and a more favorable ratio of O2 quenching versus annihilation. Furthermore, TTP was highly photostable when experiments were carried out in the presence of oxygen. This stability is in contrast to anthracene, where photodegradation was evident as a gradual decrease of triplettriplet absorption, necessitating frequent solution exchange. The photostability of TTP is confirmed by literature data21 showing that oxidation of this molecule by 1O2, which is the main product of the triplet quenching reaction, is a hardly detectable, totally negligible process. Triplet decay was studied in an air-equilibrated solution; first-order kinetics were observed in all cases. Figure 3 shows the temperature dependence of the triplet decay rate in DPPC SUVs prepared by the injection method. The data allow the distinction of three well-separated temperature intervals, characterized by an almost constant decay rate at high temperatures (T > 40 °C), a much stronger temperature dependence in the low-temperature range (T < 36 °C), and an intermediate region in which the rate abruptly increases by a factor of about 1.3 over an interval of only 4 °C. The upper limit of this “jump” interval is near the phase transition temperature of DPPC, as determined by calorimetry in extruded or sonicated LUVs (Tc ) 41 °C).6 In Figure 4, the rate constants for TPP triplet quenching by O2 are plotted as Arrhenius diagrams for SUVs of DMPC and DPPC. The rate constants were calculated by using the O2 concentration in air-equilibrated water at the same temperature; as will be discussed below, this approach is appropriate because the larger part of the quenching reaction involves O2 molecules diffusing into the membrane from the surrounding bulk aqueous medium. On the logarithmic scale chosen here, the “jump” region is less apparent, but the abrupt change in slope occurring at its upper boundary is still easily visible. The slopes are of comparable magnitude for both systems, yielding apparent activation energies in the range of 42-58 kJ/ mol at T < Tc and of 9-19 kJ/mol at T > Tc. Figure 5 shows a comparison of two measurements on DPPC vesicles of different average size (average diameters of 50 and 100 nm), prepared by the injection method using (21) Krasnovsky, A. A., Jr. Photochem. Photobiol. 1979, 29, 29.

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Figure 4. Arrhenius diagram for the oxygen quenching rate constant of TPP triplets in DMPC (O) and DPPC (b) SUVs.

Figure 5. Arrhenius diagram for the oxygen quenching rate constant of TPP triplets in DPPC vesicles of different sizes: average diameter 50 nm (O) and 100 nm (b). The inset shows fluorescence depolarization measurements performed on the same samples.

different concentrations of phospholipid.13 These two vesicle sizes lie in the range usually assigned to SUVs and LUVs, respectively. The TPP triplet decay rate data are distinct for the two systems at T < Tc; the decay is faster in the smaller vesicles at a given temperature, whereas the activation energy remains the same. The insert in Figure 5 shows results of a fluorescence depolarization measurement carried out on the same system. This technique, too, is sensitive to the size difference in vesicle preparations, although to a lesser extent than the triplet quenching method. In addition, comparison of the techniques shows that the phase transition is reflected more clearly in the triplet quenching data than in the fluorescence depolarization data, which exhibits a continuous change in the degree of polarization over a considerably larger temperature interval (>10 °C) than the “jump” interval seen in the triplet decay. Triplet-Triplet Annihilation. In solutions of phospholipid vesicles deoxygenated by bubbling with nitrogen, probe triplet decay was considerably retarded and showed a very marked temperature dependence. Again comparing TPP and anthracene as probe molecules, it was apparent that in the case of TPP, the decay always involved firstand second-order components with a sizable contribution from the former, particularly in the gel phase of the membranes. In contrast, anthracene excitation produced faster decays of almost pure second order, reflecting easier diffusivity of the probe in the membrane and therefore less impact of residual O2 or other quenchers on the decay. Anthracene was therefore chosen as the more suitable

Baranyai et al.

Figure 6. Arrhenius diagram for the triplet-triplet annihilation rate of anthracene (probe-to-lipid ratio 1:125) in SUVs of DPPC (triangles), DMPC (squares) and of a 1:1 mixture of both (circles). The inset shows two typical measurements of the decay of triplet-triplet absorption at the indicated temperatures; the dotted lines represent the calculated second-order decay.

probe for these measurements. Probe degradation by photochemistry was noticeable after prolonged experiments on the same solution; it has been shown previously that triplet-triplet annihilation leads to anthracene photodimerization.22 A series of measurements covering about 10 temperature settings could usually be performed before the solution had to be changed. Experimental results on temperature-dependent annihilation kinetics of anthracene triplets in SUVs of 60nm diameter, prepared by the dialysis method from DMPC, DPPC, and a 1:1 mixture of DMPC and DPPC are shown in Figure 6. The insert displays typical oscilloscope traces obtained in the DMPC/DPPC mixture at two temperatures immediately below and above the expected phase transition; the calorimetrically determined solidus and liquidus points for LMVs of this composition lie at 29.5 and 33.5 °C, respectively.23 Fits to second-order decay are indicated by dotted lines. The corresponding decay rates are 2k/l ) 6.1 × 106 s-1 at 29.0 °C and 2k/l ) 1.84 × 107 s-1 at 34.4 °C; second-order decay is thus accelerated by a factor of 3 over this temperature interval. The effect of the phase transition is thus much more important for triplet-triplet annihilation than for O2 quenching and results in characteristic three-part Arrhenius diagrams as shown in Figure 6. A transition interval covering 3-4 °C and characterized by a distinctly steeper slope can be discerned for each system. The liquid crystalline phase data indicate that the activation energy is similar for the three systems above the phase transition; its value is 27.6 kJ/mol. At temperatures far below the phase transition, the Arrhenius plots tend to depart from linearity, possibly because of increasing competition from other decay channels when triplet lifetimes get longer. The intersection points defined by the liquid-crystalline phase activation energy and the slopes in the phase transition region allow the determination of characteristic temperatures: 24.6 °C for DMPC, 33.5 °C for the 1:1 mixture of DMPC and DPPC, and 41.5 °C for DPPC. These values are very close to the literature values of the main phase transition temperatures of these systems.2 A more quantitative treatment of these data will be discussed below. (22) Charlton, J. L.; Dabestani, R.; Saltiel, J. J. Am. Chem. Soc. 1983, 105, 3473. (23) Mabrey, S.; Sturtevant, J. M. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 3862.

Triplet Probes in Phospholipid Bilayer Membranes

To test the possible influence of probe concentration on the results, probe-to-lipid ratios were varied over a wide range in dialyzed samples of DMPC. The boundaries delimiting the sections of the Arrhenius plots in the main phase transition region were reproducible within a few tenths of a degree although absolute values of the annihilation rate constants tended to vary in some runs in a way that did not always show a clear trend. More work will be necessary to discern the factors that determine the magnitude of the rate constants and the possible significance of these values in understanding probe distribution and the influence of the mode of vesicle preparation. Discussion Probe Location and Diffusion. Two kinds of probe molecules, meso-tetraphenylporphyrin and anthracene, were used in the present work in order to gauge the ability of triplet-triplet absorption measurements to yield information on membrane structure and dynamics. Both probes are of small size compared to the phospholipid molecules constituting the membrane and are therefore expected to be able to diffuse relatively freely through it. Moreover, because the probe molecules chosen are hydrophobic, their preferred location can be expected to lie in the lipid chain region. These assumptions are in line with recent literature results; using fluorescence quenching by nitroxide labels bound to different positions of the alkyl chain of the lipid, substituted 9-methylanthracenes were found to be distributed over various depths within the hydrophobic core of a bilayer membrane.24 Anthracene molecules were determined to be oriented with their long axis parallel to the membrane normal by flow dichroism measurements.25 Simple order-of-magnitude calculations allow estimates of the local concentrations of incorporated probes and photoexcited triplet states derived from them under the conditions used in this work. In the experimental decay curves shown in Figure 6, 258 anthracene molecules will be present, on average, in each vesicle, assuming a diameter of 60 nm, a bilayer thickness of 4 nm, and a probe/lipid ratio of 1:125. The overall triplet concentrations can be determined from the measured transient absorbances using values of  ) 6 × 104 M-1 cm-1 for the extinction coefficient of the triplet-triplet transition and l ) 0.32 cm for the optical path length. Assuming total confinement of the probe molecules in the vesicles, the overall concentrations can be translated into number densities, yielding 25 triplets per vesicle for the excitation conditions used in the experiment. About 10% of the probe molecules are excited to the triplet state, creating a situation still far removed from saturation. At the same time, the number of triplets per vesicle is just high enough to fulfill the conditions required for a second-order treatment of annihilation kinetics in a confined volume.26 Triplet-triplet annihilation is known to be a diffusioncontrolled process,27 and its kinetics should therefore reflect the microviscosity of the medium. In view of the unknown parameters involved, a quantitative treatment of the annihilation data in terms of diffusion-controlled reaction kinetics was not attempted in the present work. Based on the Stokes-Einstein relation with an interaction radius of 0.7 nm, and taking into account a spin statistical (24) Asuncion-Punzalan, E.; London, E. Biochemistry 1995, 34, 11460. (25) Ardhammar, M.; Mikati, N.; Norden, B. J. Am. Chem. Soc. 1998, 120, 9957. (26) Rothenberger, G.; Moser, J.; Gra¨tzel, M.; Serpone, N.; Sharma, D. K. J. Am. Chem. Soc. 1985, 107, 8054. (27) Saltiel, J.; Atwater, B. W. Adv. Photochem. 1988, 14, 1.

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factor of 0.28,27 the decay traces shown in Figure 6 allow an estimation of the diffusion constant of anthracene in the bilayer, with D ) 3.5 × 10-7 cm2 s-1 at 29.0 °C and D ) 1.0 × 10-6 cm2 s-1 at 34.4 °C. Although these values can be regarded as rough estimates only, they are in agreement with related values for the diffusion of probe molecules such as pyrene.28 An important consequence of these results for the application of the triplet probes can be inferred from a consideration of the distance over which the probes will be able to diffuse during their lifetime: a molecule with a diffusion coefficient on the order of 10-7 cm2 s-1 will diffuse over a distance of approximately the circumference of a 100 nm diameter vesicle in a time interval of about 1 ms. The diffusing triplet molecule will thus be able to probe sizable portions of the bilayer before disappearing by a quenching or annihilation reaction. In particular, it can be surmised that it will sense the presence of fluctuating coexisting phases or domains29-31 in the phase transition region in a “global” way as opposed to shorter-lived probes, such as fluorescence-based ones, which are only able to sense their immediate surroundings. This distinction appears further sharpened if one considers that the characteristic times assumed for fast domain fluctuations29 are of the same order of magnitude as typical fluorophore lifetimes, whereas triplet lifetimes are longer by several orders of magnitude. Finally, it should be noted that in the case of anthracene, the kinetic treatment of the triplet annihilation data using the usual second-order kinetics formalism yielded acceptable fits, although such measurements may require a specific formulation of diffusion-controlled kinetics.10,32 In particular, there was no need to introduce timedependent rate constants to account for the data, as was necessary earlier in a study of pyrene excimer formation.28 This may be due to the fact that the diffusion of anthracene is fast enough to cancel out local inhomogeneities created in the annihilation process. Different results were obtained when studying the annihilation kinetics of TPP, which indeed necessitated a time-dependent treatment; the results of these investigations will be published at a later time. Phase Transition Behavior. The ability of a probe molecule to properly sense the main phase transition of a phospholipid membrane may be regarded as an important criterion of its adequacy. In this respect, the performance of the triplet probes described here is certainly satisfactory; both the oxygen-quenching and the triplet-annihilation data allow a clear experimental determination of a temperature corresponding to the phase transition that is in excellent agreement with literature data. This is an encouraging result because the measurements are carried out in situ in the solution of unilamellar vesicles, without the need for manipulations that might alter their structure. Clearly, the phase transition data obtained by an optical probe technique will not easily match the sensitivity and accuracy available in microcalorimetry. Still, the performance of the triplet annihilation measurements, shown in Figure 6, is similar to the performance of typical calorimetric data obtained on LUVs. To show this, we make use of the argument stated above that diffusing triplet states are “global” membrane probes. To the extent (28) Vanderkooi, J. M.; Callis, J. Biochemistry 1974, 13, 4000. (29) Ruggiero, A.; Hudson, B. Biophys. J. 1989, 55, 1111. (30) Pedersen, S.; Jorgensen, K.; Baekmark, T. R.; Mouritsen, O. G. Biophys. J. 1996, 71, 554. (31) Loura, L. M. S.; Fedorov, A.; Prieto, M. Biophys. J. 1996, 71, 1823. (32) Razi Naqvi, K. Chem. Phys. Lett. 1974, 28, 280.

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Table 1. Results of Fits of Eq 1 to the Data of Figure 6 system DMPC, Eg free DMPC, Eg fixed DMPC/DPPC (1:1), Eg free DMPC/DPPC (1:1), Eg fixed DPPC, Eg free DPPC, Eg fixed

Tc (°C)

∆HvH (kJ/mol)

23.48 ( 0.32 1000 ( 350 23.42 ( 0.22 909 ( 203 32.00 ( 0.20 680 ( 80 32.08 ( 0.10 695 ( 50 40.43 ( 0.18 1500 ( 390 40.44 ( 0.13 1510 ( 290

Eg (kJ/mol) 73.7 ( 35.1 63.9 55.0 ( 36.2 63.9 63.0 ( 17.0 63.9

Figure 7. Fits of eq 1 to the data of Figure 6. The solid lines represent the calculated rate constants with the parameter set given in the second line in Table 1 for each type of vesicle.

that triplet deactivation reactions are controlled by diffusion, the temperature dependence of the triplet decay rate constant, k, can be expressed by the sum of the specific rate constants, kg and kl, for the gel and liquid crystalline phases, respectively, weighted by the mole fractions of these phases; the latter, in turn, are governed by the van’t Hoff equation. The rate constant then becomes33

k ) kg exp(-Eg/RT) + [kl exp(-El/RT) - kg exp(-Eg/RT)] exp[-(∆HvH/RT - ∆HvH/RTc)]/ {1 + exp[-(∆HvH/RT - ∆HvH/RTc)]} (1) Here, ∆HvH is the van’t Hoff enthalpy of the transition, Tc the transition temperature, and El and Eg the activation energies of triplet decay in the liquid-crystalline and gel phases, respectively. Fits of this equation to the data of Figure 6 in the main phase transition range of each system were carried out by first fixing El at 27.6 kJ/mol (see Figure 6) and varying all other parameters. The results are shown in Table 1. It is noteworthy that, despite the limited number of data, values of Tc have a very small statistical error, reflecting the accuracy with which the probe senses the phase transition. The other parameters, including the van’t Hoff enthalpy, have a much larger error because of the limited data set. The values found for Eg are equal within error limits; fixing this parameter at a mean value of 63.9 kJ/ mol allows some further, although rather unimportant, error reduction. The results of this fit are displayed in Figure 7. The transition temperatures found by applying this fitting procedure lie between 1 and 1.5 °C below the intersection points defined in Figure 6. These latter temperatures are reminiscent of the plateau values obtained in fluorescence recovery after photobleaching (FRAP) experiments, which have been interpreted as a (33) Shafirovich, V. Ya.; Batova, E. E.; Levin, P. P. J. Am. Chem. Soc. 1995, 117, 6093. The subscripts referring to the liquid-crystalline (fluid) and gel (solid) phases have to be interchanged in the equation given in this reference.

manifestation of liquid-crystalline phase interconnectivity.34,35 This similarity is not unexpected because both FRAP and triplet annihilation reflect probe diffusion. The treatment based on eq 1, on the other hand, yields a value of the transition temperature that corresponds to the points of steepest change in plots such as those of Figure 6 and that is similar to a calorimetric analysis. It is noteworthy that both approaches yield values that lie well within the error limits stated in a recent literature compilation (Tc ) 23.6 ( 1.5 °C for DMPC and 41.3 ( 1.8 °C for DPPC).2 This result is comparable to that obtained in earlier fluorimetric4,36 and recent calorimetric studies,6,20 which found that the transition temperatures of unilamellar vesicles are very near to those of MLVs, as long as the vesicle diameter exceeds about 50 nm. The transition enthalpy, ∆Hcal, was also found to be similar in unilamellar and multilamellar vesicles. On the other hand, there is a very significant difference between MLVs and LUVs in regard to the van’t Hoff enthalpy, that is, the cooperativity of the transition. In differential calorimetry,6,20 a marked decrease in peak height and concomitant broadening is noticed for LUVs as compared to MLVs. Values for cooperative unit sizes for LUVs are not explicitly given in these references;6,20 the results given by Heimburg 6 allow estimates of van’t Hoff enthalpies of H ) 5180 kJ/mol for extruded DPPC LUVs and H ) 1900 kJ/mol for sonicated DPPC LUVs, using the approximate formula given by Mabrey and Sturtevant.37 Using a value of 39.2 kJ/mol for the transition enthalpy,6 the cooperative size unit is 132 in the extruded LUVs and 48 in the sonicated ones. This is contrasted with the value of 38 deduced from our data for DPPC SUVs, using the same value of ∆Hcal (Table 1). Thus, the cooperativity sensed by the triplet probe is somewhat smaller than that deduced from calorimetry; on the other hand, cooperative size units deducible from “local” optical probes using fluorescence4,38 or intramolecular radical ion pair recombination33 yield values that are smaller yet by a factor of up to 4 compared to ours. Thus, it seems that, although cooperativity clearly depends on membrane size and structure, the role of the experimental technique used to study phase transition behavior may also have to be taken into account in the interpretation of the data. One possible reason for this is the influence exerted by the probe itself in terms of the creation of defects in the membrane structure that might reduce cooperativity. Another aspect is that of the different time scales involved; a local probe must be expected to be inherently less sensitive to cooperative motions than a “global” one. In this perspective, using triplet-state diffusion through the membrane turns out to be intermediate in sensitivity between fluorescence and calorimetric techniques. The results of the data treatment (Table 1) demonstrate, moreover, that the triplet probe reports the phase transition in the lipid mixture with the same sensitivity as in the pure lipid vesicles. The trend obtained, in which the van’t Hoff enthalpy in the mixture is markedly lowered, is in full agreement with literature data;7,23 this trend reflects the decreased cooperativity prevailing in the composite bilayer. The drop in cooperativity seems, (34) Vaz, W. L. C.; Melo, E. C. C.; Thompson, T. E. Biophys J. 1989, 56, 869. (35) Vaz, W. L. C.; Melo, E. C. C.; Thompson, T. E. Biophys J. 1990, 58, 273. (36) Lichtenberg, D.; Freire, E.; Schmidt, C. F.; Barenholtz, Y.; Felgner, P. L.; Thompson, T. E. Biochemistry 1981, 20, 3462. (37) Mabrey, S.; Sturtevant, J. M. Methods Membr. Biol. 1978, 9, 237. (38) Kurihara, K.; Onuki, K.; Toyoshima, Y.; Sukigara, M. Mol. Cryst. Liq. Cryst. 1981, 68, 69.

Triplet Probes in Phospholipid Bilayer Membranes

however, much less pronounced than that usually observed in calorimetric measurements performed on MLVs.7,23 Explicit data on values of ∆HvH obtained with unilamellar vesicles composed of phospholipid mixtures do not seem to be available. The triplet probe method may therefore prove useful in studying phase transition behavior in unilamellar vesicles of greater compositional complexity. Oxygen Diffusion. The diffusion of oxygen in and through membranes is a problem of obvious biological interest but is experimentally difficult to access because most techniques used in membrane studies are insensitive to the presence of O2. Pyrene fluorescence quenching by O2 was used earlier to assess oxygen diffusion in membranes.39 In this study, the O2 diffusion coefficient was found to increase by a factor 3-4 at the phase transition of DMPC or DPPC SUVs. Subsequently, a technique based on the measurement of spin-lattice relaxation times of lipid-bound nitroxide spin probes was used to investigate oxygen concentration and transport in membranes.40-43 The use of a triplet state probe appears as a straightforward addition to the list of these methods in view of the usually diffusion-controlled reactivity of triplet states of organic molecules with O2. The results of the present work (Figures 3, 4, and 5), indeed, demonstrate the sensitivity of the oxygen quenching of a porphyrin triplet state to the thermotropic behavior of DMPC and DPPC membranes. Although the sensitivity is not as high as with the triplet annihilation measurement discussed above, the main phase transition is sensed accurately, and the measurement even detects a small influence of vesicle size (Figure 5). Since the diffusion coefficient of O2 is about 2 orders of magnitude higher than that of a large aromatic molecule in a membrane,28 the triplet decay rate is largely determined by oxygen diffusion. It seems appealing, at first glance to discuss the experimental results in the same way as the triplet annihilation, that is, based on a consideration of the motion of O2 in the membrane. A closer look at the measurement conditions, however, shows that this perspective fails for two reasons. One problem arises from the sudden change of oxygen solubility in the membrane the phase transition.44 The second reason is that, even using low excitation pulse energy for the transient absorption measurement, the average number of absorbed photons per vesicle will easily be higher than the average number of O2 molecules dissolved in the vesicle. A model calculation using SUVs of 60 nm diameter (lipid concentration 6 × 10-4 M), incorporating 1.8 × 10-6 M TPP in an air-equilibrated solution (corresponding to the conditions of the experiment of Figure 2) yields an average of 96 probe molecules and 6 O2 molecules per vesicle, assuming an oxygen partition coefficient of 1 between the aqueous and lipid medium and complete incorporation of the probe. Exciting this system with 355 nm pulses in the geometry used for this experiment will result in an average of 17 absorbed photons for 0.1 mJ of pulse energy, the majority of which will yield TPP triplets because the intersystem crossing quantum yield of tetraarylporphyrins in a nonpolar medium exceeds 0.6.45 This (39) Fischkoff, S.; Vanderkooi, J. M. J. Gen. Physiol. 1975, 65, 663. (40) Kusumi, A.; Subczynski, W. K.; Hyde, J. S. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 1854. (41) Subczynski, W. K.; Hyde, J. S. Biophys. J. 1983, 41, 283. (42) Subczynski, W. K.; Hyde, J. S.; Kusumi, A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4474. (43) Subczynski, W. K.; Hyde, J. S.; Kusumi, A. Biochemistry 1991, 30, 8578. (44) Smotkin, E. S.; Moy, F. T.; Plachy, W. Z. Biochim. Biophys. Acta 1991, 1061, 33. (45) Katona Z.; Grofcsik, A.; Baranyai, P.; Bitter, I.; Grabner G.; Kubinyi M.; Vidoczy, T. J. Mol. Struct. 1998, 450, 41.

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estimation does not take saturation into account, whereby the number of absorbed photons will be reduced (see Figure 2), but nevertheless shows that, on the time scale of the measurement, O2 molecules present in the membrane will be depleted by the quenching reaction, even at low pulse energy; the quenching reaction converts O2 into 1O2, which live significantly longer under these conditions than the TPP triplets. The triplet decay kinetics will therefore be determined dominantly by O2 diffusion into the membrane from the surrounding bulk aqueous phase. This assumption was tested by studying the influence of both O2 concentration and laser pulse energy on the TPP triplet-decay kinetics. The effect to be noticed was an increase of decay rate at low pulse energy and/or high O2 concentration because this is the situation in which the number density of O2 in a vesicle might be higher than that of triplet probe molecules; moreover, such an increase of the decay rate might only arise in the liquid-crystalline state because the lipid/water partition coefficient for oxygen is expected to be significantly higher than unity in this phase only.39,41,44 Equilibrating the vesicles with 1 atm O2 did not induce such an effect. The data describing the influence of laser-pulse energy on triplet-decay kinetics are shown in Figure 2. It is apparent that the decay rate is independent of pulse energy over the whole range studied at both temperatures chosen, lying immediately below and above the main phase transition temperature of DMPC. This result demonstrates that a condition in which the triplet decay probes O2 diffusion within the membrane only is probably not attained, even at low excitation levels. This conclusion would be invalid if the lipid/water partition coefficient for oxygen were near unity in both phases, but this is highly unlikely in view of the available evidence, which places its value near 3-4.39,41,44 The fact that the triplet decay rate increases only by a factor of 1.3 within the phase transition region is in agreement with this view. Based on this discussion, the fact that the oxygen quenching experiments show a very marked change of activation energy at the phase transition (Figures 3 and 4) is remarkable. Diffusion of O2 toward the membrane should be characterized by an activation energy on the order of 20 kJ/mol.46-48 The values found above the transition temperature are, in fact, somewhat lower than this value. Below the phase transition, the activation energies are, however, significantly higher. This result can be interpreted to show that a unilamellar bilayer in the gel phase constitutes a significant barrier for oxygen permeation. A similar result has been obtained earlier for MLVs using the spin probe technique.42 Conclusions It has been shown that triplet probes can be successfully applied to the study of the phase transition behavior of unilamellar bilayer membranes, as well as to diffusional motions of the probes in the membranes and of oxygen from the surrounding bulk water toward the membranes. The main purpose of the present work was the assessment of the performance of the triplet-probe technique; analysis of triplet annihilation kinetics in the phase transition region demonstrated that values of critical temperatures, as well as of van’t Hoff enthalpies, can be readily determined. It can, therefore, be concluded that this method represents a valuable addition to the list of techniques (46) Gertz, J. H.; Lo¨schke, H. H. Z. Naturforsch., B 1954, 9B, 1. (47) Gertz, J. H.; Lo¨schke, H. H. Z. Naturforsch., B 1956, 11B, 60. (48) Eisenberg, D.; Kauzmann, W. The Structure and Properties of Water; Clarendon Press: Oxford, 1969; p 205.

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available for the study of unilamellar membrane systems. Studies using a wider variety of probes and more complex bilayers are underway. Acknowledgment. Partial financial support from the Hungarian National Scientific Research Foundation (OTKA Grant No. T 015836) is gratefully acknowledged. Cooperation between the two laboratories was made

Baranyai et al.

possible by the Scientific-Technical Cooperation Program between Austria and Hungary (Grant No. A-45/96). Financial support by the Bundesministerium fu¨r Wissenschaft und Verkehr (Austria), the Jubila¨umsfonds der O ¨ sterreichischen Nationalbank, and the Hochschuljubila¨umsstiftung der Stadt Wien is gratefully acknowledged. LA990532X