J. Phys. Chem. 1996, 100, 8567-8572
8567
Hole-Burning Spectroscopy of Proteins in External Fields: Human Serum Albumin Complexed with the Hypericinate Ion M. Ko1 hler, J. Gafert, and J. Friedrich* Physikalisches Institut und Bayreuther Institut fu¨ r Makromoleku¨ lforschung, UniVersita¨ t Bayreuth, D-95440 Bayreuth, Germany
H. Falk and J. Meyer Institut fu¨ r Chemie, Johannes Kepler UniVersita¨ t, A-4040 Linz, Austria ReceiVed: October 20, 1995; In Final Form: February 26, 1996X
We investigated human serum albumin complexed with the hypericinate ion by using optical hole-burning techniques. The response of the burned-in holes to external pressure variations and electric fields was measured as a function of burn frequency within the inhomogeneous band. The results for the protein-dye complex were compared with the respective behavior of the dye in a host glass. In addition, the spectral properties of the serum albumin hypericinate complex in an electric field were compared with the respective properties of protoporphyrin substituted myoglobin. On the basis of our results we concluded that there must be two stereoisomers with a relative weight of about 50%. Further, the binding site in human serum albumin seems to be quite shallow. The dye is not shielded from the host glass, contrary to the respective behavior of protoporphyrin-substituted myoglobin.
Introduction Photochemical hole-burning techniques have become an attractive tool in protein spectroscopy.1-5 The reason is its high resolution which can be trimmed to work at the natural linewidth limit. Hence, on the one hand, the technique can be applied to do relaxation spectroscopy in the frequency domain, which is widely used in photosynthesis research.2-4,6-11 On the other hand, it can be applied to study the influence of small perturbations on the protein. Whenever these perturbations cause a shift or a broadening of the hole of the order of the natural line width, it can, in principle, be measured. Thus, the sensitivity is orders of magnitude higher as compared to a situation where the external perturbation is monitored through the change of the inhomogeneous band. We made use of this advantage in studying the influence of pressure,12-17 electric fields,18 and temperature jumps19-21 on frozen proteins. In addition, we used the technique to investigate their long-time (days!) relaxation dynamics.22 In this paper we use hole burning to investigate human serum albumin (HSA) complexed with the dye hypericin.23 HSA is a transport protein in the blood plasma for quite a series of rather different molecules. It consists of six helical subdomains. The polypeptide backbone is formed by 585 amino acids. The complete X-ray structure of HSA was recently published.24 The protein has two major binding sites labeled IIA and IIIA according to the subdomains where they occur. The IIIA binding site is much more active. As to the binding of hypericin, it is the only one.23 Hypericin is a plant pigment which plays an important role as a photodynamic agent, especially in anticancer and antiviral applications.25 Hypericin is also an efficient hole-burning dye.26 Recently, it has been shown to be present in solutions and complexed to HSA as the dissociated species, the hypericinate ion.27 The respective photoreaction of the hole-burning process is supposed X
Abstract published in AdVance ACS Abstracts, April 15, 1996.
S0022-3654(95)03101-7 CCC: $12.00
Figure 1. Phototautomerism of the hypericinate ion.
to be a proton transfer (tautomerization) in the bay region (Figure 1), which differs from that suggested for quinizarin.28 We employ hole-burning Stark and pressure tuning spectroscopy to characterize the HSA-hypericin binding site. There are two major questions which we want to address: The first concerns the depth of the binding site. From the X-ray results it is suggested that this site is rather shallow. As a consequence, the ligand is exposed to the solvent. A comparative Stark effect experiment between glassy solutions of the complex and the ligand on one hand and between another protein-dye complex, namely, protoporphyrin-IX substituted myoglobin (Mb), on the other hand, should add to the qualitative understanding. The second question concerns complex formation itself. Since hypericin can exist in enantiomeric propellerlike conformations, one expects two diastereoisomers of the HSAhypericin complex. Is there spectroscopic evidence for two forms? If yes, what is their relative weight? © 1996 American Chemical Society
8568 J. Phys. Chem., Vol. 100, No. 20, 1996
Ko¨hler et al.
Basic Aspects of the Hole-Burning Technique. In disordered systems such as glasses, polymers, and also proteins the absorption transitions of probe molecules are inhomogeneously broadened. Typical numbers for the inhomogeneous width are 100 cm-1. Since the static disorder does not significantly depend on temperature, inhomogeneous broadening is rather independent of it. The homogeneous width, on the other hand, is determined by dynamic disorder (thermal vibrations) which can be frozen out as the temperature goes to zero. At 1.7 K, the temperature of our experiment, typical values for the homogeneous width are several tens to hundreds of megahertz. If the dye probes are photoreactive, laser irradiation into the inhomogeneous band leads to a depletion of the number of absorbers at the laser frequency and to the appearance of a narrow hole whose shape is close to the homogeneous line shape. Since the hole is, in many cases, persistent, it is easy to investigate how it shifts and broadens under external perturbations like pressure and electric fields (for a review, see ref 29). Holes in an Electric Field. The frequency shift of an electronic transition in an external electric field B E0 is determined by
SE ) f∆µ b‚E B0/hc
(1)
with f being the Lorentz correction, and b µ being the difference vector of the dipole moments in the ground and excited state.30,31 In general, ∆µ b is a sum of two terms, namely, ∆µ b0 and ∆µ bi. ∆µ b0 is the difference of the permanent dipole moments, whereas ∆µ bi is the respective difference of the induced dipole moments. From an experimental point of view the polarization direction of the laser with respect to the external field is important. Hole burning with polarized light induces a macroscopic anisotropy in the sample. Since the transition moment and the ∆µ b vector have a fixed orientation in a molecular frame, polarized hole burning induces an anisotropic distribution of ∆µ b vectors, too. The point is that the director of the ∆µ b distribution can be oriented with respect to the external field by choosing the polarization direction in a proper way. If this direction is orthogonal to the external field B E0, the hole just broadens. However, if it is parallel to B E0, the hole splits. The field broadening of the holes has a 2-fold reason, namely, bi distribution. The latter arises the ∆µ b0 distribution and the ∆µ because, in a random matrix, the matrix fields fluctuate in direction and size; hence, the induced dipole moments fluctuate in direction and size. Holes under Pressure. Burned-in holes shift and broaden under pressure. In a hole-burning pressure tuning experiment we study a frequency-selected ensemble of molecules whose solvent shift νs is defined by
νs ) νb - νvac
(2)
with νb being the burn-frequency and νvac the vacuum absorption frequency of the dye probe. The pressure shift Sp can be viewed as a pressure-modulated solvent shift:
Sp )
∂νs ∆p ∂p
(3)
Assuming that the main contributions to the solvent shift are due to dispersive and higher order electrostatic interactions which fall off as R-6, eq 3 leads to
Sp ) 2κ(νb - νvac)
(4)
κ is the isothermal compressiblility of the sample. With eq 4 it is possible to measure κ by a purely optical experiment:12-17
As Sp/∆p is plotted as a function of burn frequency νb, one gets a straight line with slope 2κ.32 As to the broadening of the holes under pressure, it arises from the fact that in disordered solids such as glasses, polymers, and also proteins, the probe lattice configurations are highly degenerate. Pressure lifts this degeneracy, and as a consequence, broadening results. Experimental Section Sample Preparation. Hypericin was prepared according to ref 33. Its potassium salt was complexed with HSA (Sigma) in phosphate buffer (pH 7) and purified by chromatography over a Sephadex G 15 column following the procedure given in ref 23. Using absorption spectroscopy and circular dichroism measurements, it was confirmed that the HSA-hypericinate complex was stable in the solvent mixtures used. The HSA-hypericinate complex and the pure dye were dissolved in a mixture of potassium phosphate buffer (pH 7) with glycerol (1:3, v/v) and dimethylformamide with glycerol (1:3, v/v), respectively. The concentration was chosen in a way that the optical density was about 0.5. The preparation of the protoporphyrin-IX samples is described in ref 15. Spectroscopy. The temperature of the experiments was in all cases 1.7 K. The broad-band spectra were measured with a 1 m Jobin-Yvon spectrometer whose resolution was set to 2 cm-1. Holes were burned and detected with an argon ion laserpumped ring dye laser. The laser bandwidth was a few megahertz, and the scan width 30 GHz. Detection was performed in the transmission mode. Usually quite deep holes were burned in order to get a sufficiently good signal-to-noise ratio. The holes were largely saturated. However, their shape could still be approximated by Lorentzians very well. The external Stark-field B E0 was varied up to 11.7 kV/cm. We stress that all features investigated changed linearly with the field. At all frequencies holes were burned with the polarization parallel as well as perpendicular to B E0. Burning times and burning powers were on the order of 100 s and 100 µW, respectively. For details of the fit procedure see ref 18. For the pressure experiments the samples were sealed in small plastic bags to ensure isotropic pressure conditions. Pressure was transmitted via He gas. It was varied up to 2.4 MPa, the limiting value where He solidifies at 1.5 K. The accuracy of the pressure level was about 0.001 MPa. The fit procedure for the holes under pressure is based on the observation that the pressure kernel has a Gaussian shape. The pressure broadening plotted in the figures is the width of this kernel. The unpressurized hole could be very well represented by a Lorentzian shape. Much attention was paid to the symmetry of the line shape under pressure. As a rule, this shape becomes asymmetric or the hole can even split when two different transitions, which overlap with their inhomogeneous widths, are simultaneously burned.16,17 Results Stark Effect. To learn something on the specific nature of the HSA-IIIA binding site, we compare, on one hand, the HSA-hypericinate complex with the behavior of hypericinate directly dissolved in the host glass, and, on the other hand, the HSA-hypericinate complex with the Mb-protoporphyrin complex.18 Figure 2a shows the inhomogeneous absorption of the HSAhypericinate complex. The arrow indicates the frequency where hole-burning was performed. The insert shows the hole with and without an external field of 11.7 kV/cm. It is clearly discernible that the hole splits in the field but broadens as well.
Hole Burning in Human Serum Albumin
Figure 2. (a) Origin region of the inhomogenously broadened absorption spectrum of the HSA-hypericinate complex in a glycerol/ water (3:1) solvent. The insert shows a hole with and without an electric field of 11.7 kV/cm . The arrow indicates the burning position; temperature 1.7 K. (b) Same as (a) but without HSA. The dye is directly dissolved in a host glass (glycerol/dimethylformamide, 3:1).
The splitting is of the order of 3 GHz. These results are for a laser polarization parallel to the external field. For orthogonal polarization (not shown) a symmetric broadening is observed but no splitting. Figure 2b shows the respective behavior of the hypericinate ion in the glass. Surprisingly, the inhomogeneous line is much narrower, roughly by a factor of 2. However, the pattern of the hole in the external field is very much the same as for the protein dye complex. We stress that comparative measurements on myoglobinprotoporphyrin and protoporphyrin in a glass, respectively, showed a significantly different behavior: In the glass the hole just broadens irrespective of the polarization of the laser field. In the protein sample, however, the hole clearly splits if the polarization of the laser field is parallel to B E0.15 Figures 3 and 4 show the frequency dependence of the Stark effect across the inhomogeneous band for the hypericinate ion in the host glass (Figure 3) and for the HSA-hypericinate complex (Figure 4). We note that the frequency-dependent effects are not small. The splitting and the broadening change by more than a factor of 2 from the blue to the red edge of the band. Frequency-dependent features in the field broadening have been analyzed before.37 For a dye in a random matrix, the frequency dependence in the splitting, however, is quite surprising, because the splitting should be determined solely by ∆µ b0 which is a purely molecular quantity. Comparing the dye in the glass (Figure 3) with the complex (Figure 4) we see quite specific features: On one hand, the variation in the splitting with frequency is of the same magnitude in the frequency range common to both experiments. On the other hand, for the protein complex, the broadening runs through kind of a maximum at a frequency which coincides with the band maximum, whereas for the glass sample the broadening is linear with frequency. We stressed before that the absorption band of the complex is much broader than the respective band of the dye. In Figure 4a we represent the inhomogeneous band as a superposition of
J. Phys. Chem., Vol. 100, No. 20, 1996 8569
Figure 3. Frequency-dependent features of the hole-burning Stark effect for the hypericinate ion in a host glass (glycerol/dimethylformamide, 3:1). (a) Origin region of inhomogeneous absorption. (b) Stark splitting per field as a function of burn frequency. (c) Stark broadening as a function of burn frequency; temperature 1.7 K.
Figure 4. Same as Figure 4, yet for the HSA-hypericinate complex in glycerol/water (3:1). The inhomogeneous absorption line is decomposed into two components. The shape for the band of an individual component is taken from Figure 3.
twice the band of the dye (Figure 3a) with a relative frequency shift of 178 cm-1. Considering the fact that the complex on one hand and the pure dye on the other hand are quite different systems and considering the fact that there are just three fit parameters involved, namely, the relative spectral weight of the bands and the respective frequencies, the two component representation of the spectrum of the complex becomes quite satisfactory, when the relative weight is about 50%.
8570 J. Phys. Chem., Vol. 100, No. 20, 1996
Ko¨hler et al. by the arrow, as it deforms under pressure. Note that the shapes of the holes are perfectly symmetric. From the perfect linearity with frequency, we conclude that in the frequency range where the pressure experiments were performed there is no additional state which plays a dominant role. Figure 5c shows the broadening per pressure. It is roughly constant with frequency, in agreement with theory.32 Figure 6 shows the respective pressure experiments for the complex. The experimentally determined frequency dependence of the pressure shift is quite complicated. Contrary to the glass case, it is definitely nonlinear indicating that more than one states are involved. There is a frequency range where the holes under pressure become quite asymmetric. An example is shown in the right insert of Figure 6b. The pressure broadening data strongly support this view: There is a significant increase of the broadening in the overlap region with the tendency of running through a maximum. This increase in the width comes from the difference in the pressure shifts of the components which cannot be resolved at the pressure levels of the experiment. Summary of Experimental Results
Figure 5. Frequency-dependent features of pressure on spectral holes for the hypericinate ion in a host glass (glycerol/dimethylformamide, 3:1). (a) Origin region of the inhomogeneous absorption. (b) Shift per pressure as a function of burn frequency. (c) Broadening per pressure as a function of burn frequency; temperature 1.7 K.
We found that (i) there is no significant difference in the qualitative behavior of the Stark effect between the HSAhypericin complex and the hypericin glass system. These findings are quite in contrast to the respective behavior of Mbprotoporphyrin-IX. (ii) The frequency-dependent features of the Stark experiments and the pressure experiments strongly suggest that the inhomogeneous line of the HSA-hypericinate complex is built from two states of about equal population. Discussion
Figure 6. Same as Figure 5, but for the HSA-hypericinate complex in glycerol/water (3:1). Note the nonlinear behavior of the pressure shift (b) and the distinct asymmetry of the holes under pressure in the overlap region of the two components (lower insert right).
Pressure Effects. Figures 5 and 6 compare the influence of pressure on spectral holes for hypericin in the host glass (Figure 5) and for the HSA-hypericinate complex (Figure 6). Figure 5b shows the shift per pressure as a function of frequency. The data points perfectly fall on a straight line in agreement with eq 4. The respective slope is determined by the compressibility of the host glass, which is, in this case, 0.104 GPa-1. The insert shows a hole, taken at the frequency marked
Stark Effect and the Depth of the IIIA Binding Site. It is instructive to start this discussion with the behavior of Mbprotoporphyrin. The fact that a splitting occurs in the protein, whereas no splitting can be observed in the dye-glass system leads to important conclusions: The dye has an effective inversion symmetry. The splitting in the protein is due to a dipole moment difference with a well-defined direction induced by the field generated by the protein.18,34-36 So the relevant interaction of the chromophore with its environment cannot significantly exceed the typical dimension of the protein radius which is on the order of 10 Å. If it did, the chromophore would feel the random glass environment and we would not see a splitting but a broadening only. The Mb pocket obviously is deep enough to shield the chromophore from the solvent. The Stark pattern of the HSA-hypericinate complex shows quite a different behavior. First, we observe a rather large splitting in the glass, pointing to the fact that the hypericinate ion has a permanent dipole moment which changes significantly upon excitation. Second, this splitting does not significantly change if the hypericinate ion is complexed with HSA (Figure 2). From the color effect (Figures 3 and 4) we definitely see that there is a significant influence on the effective dipole moment of the hypericinate ion through polarization effects of the solvent. Yet the difference between glass and protein is not significant. In the frequency range common to both samples the measured values for the splitting per field fall almost on top of each other. (The same is true for the field-induced broadening, although the respective frequency dependence is different (Figure 3c and 4c). Where this difference in the frequency dependence comes from is discussed in the next section.) The conclusion from this observation is that the hypericinate ion at the IIIA binding site of HSA is obviously not shielded
Hole Burning in Human Serum Albumin
J. Phys. Chem., Vol. 100, No. 20, 1996 8571
from the surrounding host glass, quite in contrast to protoporphyrin in Mb. The binding site seems to be strongly exposed to the glass, hence, it must be quite shallow. This conclusion is in agreement with the X-ray structure of the protein.24 Frequency-Dependent Hole-Burning Features and the Characterization of the HSA-Hypericinate Complex. Frequency-dependent features in the Stark effect were considered in detail by Kador et al.37 The situation which these authors considered was confined to a centrosymmetric probe in an amorphous material. The underlying physics of the frequency dependence is the following: The matrix induces a dipole moment in the centrosymmetric probe. Since the internal matrix fields are supposed to increase with the solvent shift, the induced dipole moment increases as well. This gives rise to the observed color effect in the field-induced line broadening. What is surprising in our case is the fact that we observe a color effect in the splitting as well, i.e., for a noncentrosymmetric molecule with a permanent dipole moment. This implies that the random matrix fields of the glass would induce a dipole moment whose difference vector adds constructively to the ∆µ b0 vector of the probe, a situation which is hard to imagine. We interpret this finding by assuming that the probe itself induces local order in the solvent through polarizing the respective solvent dipoles. These dipoles, in turn, induce a dipole moment in the probe which reinforces the dipole moment already present.38 Hence
∆µ b ) ∆µ b0 + 1/2∆Rˆ ‚E B
(5)
with ∆Rˆ being the difference of the polarizability tensors of the molecular states involved. B E is the internal field. It increases with the solvent shift thus giving rise to the observed frequency dependence of the splitting. For the characterization of the complex the field-induced broadening is of interest. In the glass sample (Figure 3c) this broadening depends in a rather linear fashion on frequency.37 In the complex, however, it runs through a kind of maximum (Figure 4c). We can understand this maximum by assuming that the inhomogeneous band of the complex is built from two components as indicated in Figure 5a. Each component behaves in the usual way, like in Figure 3c. When hole burning occurs in the overlap region, the high-frequency component is burned on the red side, whereas the low-frequency component is burned on the blue side. Since red-side and blue-side holes have different frequency shifts (Figure 3b), an additional broadening results, if these different shifts cannot be resolved. As holes are burned further to the red, the long-wavelength component increases quickly in relative weight. The additional broadening vanishes and one jumps back onto the usual broadening curve of the red component. The conclusion is that the frequencydependent broadening strongly supports the presence of two components of roughly equal weight in the inhomogeneous band of the complex. As a matter of fact the inhomogeneous band of the complex is twice as broad as the corresponding one in the glass. In Figure 4a we assumed that the complex band consists of two, about equally strong “glass bands” shifted in energy. As can be seen, the fit (three parameters: central frequencies and spectral weight) is satisfactory. The pressure experiments support this view. Again the pressure shift in the glass sample is perfectly linear with frequency in agreement with the model (eq 4). There is no indication of hidden states below the inhomogeneous envelope. The insert of Figure 5b shows the behavior of a hole under pressure. The shape of the hole remains perfectly symmetric. The broadening per pressure as a function of frequency is roughly constant. There are variations on the order of 20%, only. Again the complex has quite different features. First,
the pressure shift is significantly nonlinear with frequency, indicating that different states with different parameters (νvac, κ) contribute to the observed pressure shift. It is quite interesting to note that in the overlap region the holes definitely attain an asymmetric line shape under pressure. An example (hole 2) is shown in the lower right insert of Figure 6b. On the other hand, holes outside the overlap region retain their symmetric shape (hole 1). The asymmetry is a strong indication for the presence of at least two components with slightly different pressure shifts. The difference in the shift is obviously too small compared to the simultaneously occurring pressure broadening to be directly resolved. This implies that the associated parameters (νvac, κ) are rather similar. As in the Stark broadening, we also get an additional pressure broadening in the overlap region resulting from the different shifts of the components. There is a steplike rise of the broadening in the overlap region with an indication of a maximum. Again it is clear, that the two components must roughly be of equal population. How can we understand the occurrence of two components? It has been shown by quite a series of experiments that hypericin and also the hypericinate ion has a propellerlike conformation.39,40 Hence, there are two enantiomers. As a consequence, there are two diasteromers of the HSA-hypericinate complex. It is clear that the presence of the protein must lead to a slight change of the respective absorption frequencies. As our experiments show the respective frequencies differ by about 178 cm-1. Moreover, it seems that the relative population is close to the racemic limit. Summary We performed hole-burning Stark and pressure experiments on the human serum albumin hypericinate complex. From the comparative Stark experiments with Mb-protoporphyrin we concluded that the IIIA binding site has to be quite shallow. The dye binds to the surface of the protein and seems to be in close contact with the solvent molecules. This finding is in line with the X-ray structure.24 From the frequency-dependent features of the Stark and pressure experiments it follows that the inhomogeneous line of the complex is built from two components of roughly equal population. These two components are associated with the two enantiomeric forms of the hypericinate ion which give rise to two diastereomeric forms of the HSA-hypericinate complex. Acknowledgment. We acknowledge support from the DFG (SFB 279, Graduiertenkolleg “Nichtlineare Spektroskopie und Dynamik”) and the “Fonds der Chemischen Industrie”. J.F. would like to acknowledge the discussions with L. Kador. References and Notes (1) Friedrich, J.; Scheer, H.; Zickendraht-Wendelstadt, B.; Haarer, D. J. Am. Chem. Soc. 1981, 103, 1030; J. Chem. Phys. 1981, 74, 2260. (2) Reddy, N. R. S.; Lyle, P. A.; Small, G. J. Photosynth. Res. 1992, 31, 167. (3) Jankowiak, R.; Hayes, J. M.; Small, G. J. Chem. ReV. 1993, 93, 1471. (4) Reddy, N. R. S.; Kolaczkowski, S. V.; Small, G. J. Science 1993, 260, 68. (5) Friedrich, J. Hole burning spectroscopy and physics of proteins. In Methods in EnzymologysBiochemical Spectroscopy; Sauer, K., Ed.; Academic Press: San Diego, 1995; p 226. (6) Johnson, S. G.; Lee, I.-J.; Small, G. J. Solid state spectral linenarrowing spectroscopies. In Chlorophylls; Scheer, H., Ed.; CRC Press: London, 1991; p 739. (7) Reddy, N. R. S.; Picorel, R.; Small, G. J. J. Phys.Chem. 1992, 96, 6458. (8) Reddy, N. R. S.; Cogdell, R. J.; Zhao, L.; Small, G. J. Photochem. Photobiol. 1993, 57, 35.
8572 J. Phys. Chem., Vol. 100, No. 20, 1996 (9) Van der Laan, H.; DeCaro, C.; Schmidt, Th.; Visschers, R. W.; van Grondelle, R.; Fowler, G. J. S.; Hunter, C. N.; Vo¨lker, S. Chem. Phys. Lett. 1993, 212, 569. (10) DeCaro, C.; Visshers, R. W.; van Grondelle, R.; Vo¨lker, S. J. Phys. Chem. 1994, 98, 10584. (11) DeCaro, C.; Visshers, R. W.; van Grondelle, R; Vo¨lker, S. J. Lumin. 1994, 58, 149. (12) Zollfrank, J.; Friedrich, J.; Fidy, J.; Vanderkooi, J. M. J. Chem. Phys. 1991, 94, 8600. (13) Zollfrank, J.; Friedrich, J.; Parak, F. Biophys. J. 1992, 61, 716. (14) Fidy, J.; Vanderkooi, J. M.; Zollfrank, J.; Friedrich, J. Biophys. J. 1992, 63, 1605. (15) Gafert, J.; Friedrich, J.; Parak, F. J. Chem. Phys. 1993, 99, 2478. (16) Gafert, J.; Friedrich, J.; Vanderkooi, J. M.; Fidy, J. J. Phys. Chem. 1994, 98, 2210. (17) Friedrich, J.; Gafert, J.; Zollfrank, J.; Vanderkooi, J. M.; Fidy, J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1029. (18) Gafert, J.; Friedrich, J.; Parak, F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2116. (19) Ko¨hler, W., Friedrich, J. J. Chem. Phys. 1988, 90, 1270. (20) Zollfrank, J.; Friedrich, J.; Vanderkooi, J. M.; Fidy, J. J. Chem. Phys. 1991, 95, 3134. (21) Zollfrank, J.; Friedrich, J.; Vanderkooi, J. M.; Fidy, J. Biophys. J. 1991, 59, 305. (22) Gafert, J.; Pschierer, H.; Friedrich, J. Phys. ReV. Lett. 1995, 74, 3704. (23) Falk, H.; Meyer, J. Monatsh. Chem. 1994, 125, 753. (24) He, X. M., Carter, D. J. Nature 1992, 358, 209. (25) Diwu, Z. Photochem. Photobiol. 1995, 61, 529.
Ko¨hler et al. (26) Pschierer, H.; Friedrich, J.; Falk, H.; Schmitzberger, W. J. Phys. Chem. 1993, 97, 6902. (27) Falk, H.; Mayr, E. Monatsh. Chem., in press. (28) Friedrich, J.; Haarer, D. Ang. Chem. Int. Ed. Engl. 1984, 23, 113. (29) Schellenberg, P.; Friedrich, J. Optical spectroscopy and disorder phenomena in polymers, proteins and glasses. In Disorder effects on relaxation processes; Richert, R., Blumen, A., Eds.; Springer-Verlag: Berlin, 1994; p 407. (30) Maier, M. Appl. Phys. 1986, B41, 73. (31) Scha¨tz, P.; Maier, M. J. Chem. Phys. 1987, 87, 809. (32) Laird, B. B.; Skinner, J. L. J. Chem. Phys. 1989, 90, 3274. (33) Falk, H.; Meyer, J.; Oberreiter, M. Monatsh. Chem. 1993, 124, 339. (34) Schmidt, S.; Reich, R. Ber. Bunsenges. 1972, 76, 1202. (35) Grewer, G.; Lo¨sche, M. Makromol. Chem. Symp. 1991, 46, 79. (36) Kohler, B. E.; Personov, R. I.; Woehl, J. C. Electric field effects in molecular systems studied via persistent hole burning. In Laser Techniques in Chemistry; Rizzo, Th., Myers, A., Eds.; J. Wiley & Sons: New York, 1994. (37) Kador, L.; Jahn, S.; Haarer, D.; Silbey, R. Phys. ReV. 1990, B41, 12215. (38) Liptay, W. Dipole moments and polarizabilities of molecules in excited electronic states. In Excited States; Vol. I, Lim, E. C., Ed.; Academic Press: New York, 1974; p 129. (39) Etzlstorfer, C.; Falk, H.; Oberreiter, M. Monatsh. Chem. 1993, 124, 923. (40) Etzlstorfer, C.; Falk, H.; Mu¨ller, N.; Schmitzberger, W.; Wagner, U. G. Monatsh. Chem. 1993, 124, 751.
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