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J. Phys. Chem. B 2008, 112, 4276-4282
Interaction of Zinc Tetrasulfonated Phthalocyanine with Cytochrome c in Water and Triton-X 100 Micelles Ce´ sar A. T. Laia* and Sı´lvia M. B. Costa Centro de Quı´mica-Estrutural, Complexo 1, Instituto Superior Te´ cnico, 1049-001 Lisboa, Portugal ReceiVed: July 31, 2007; In Final Form: December 17, 2007
The interaction of a zinc tetrasulfonated phthalocyanine with cytochrome c was studied using steady-state spectroscopic techniques and time-correlated single photon counting in water and Triton-X 100 micelles. The dye forms dimers in water with a high equilibrium constant (70 × 106 M-1). Because of a specific electrostatic interaction, the presence of cytochrome c does not lead to a dissociation of this dimer, but increases its formation, with an equilibrium constant of about 7.9 × 109 M-1. Triton-X 100 micelles dissociate the dimer, creating two populations of dye molecules: one in a hydrophilic media, probably on the surface of the micelles, another on a hydrophobic environment, probably inside the micelles. However, when cytochrome c is added the dye aggregation is again induced leading to a strong fluorescence quenching. This fluorescence quenching may also be caused by a photoinduced electron-transfer due to the formation of a 1:1 complex between the dye and the protein, but the present work does not give direct evidence of such an effect because the fluorescence decays did not show the presence of an extra component. The results presented here are quite different from those reported for aluminum sulfonated phthalocyanines, where aggregation does not occur and the fluorescence quenching is solely due to photoinduced electron-transfer reactions.
Introduction The interaction of synthetic molecules with biomacromolecules leads to a wide range of effects.1 Namely, the active site of enzymes may be blocked, inducing the inhibition of the biological function. Synthetic inhibitors (namely, porphyrin derivatives or calixarenes) can quench biochemical reactions because the complexation of those molecules with proteins prevents the reactants from docking at the active site where those reactions occur.2 Therefore, the understanding of the interaction of small molecules with the protein’s active site is important in order to develop new efficient drugs. Cytochrome c (Cyt c) is not a very large protein with a heme group resting in the globule.3-5 It is water-soluble, with a high positive charge (around +8) at neutral pH. The active site that allows the protein to dock on other proteins like cytochrome c oxidase (Cyt c Ox)3-7 or caspase proteins,8,9 which play important roles in respiration and apoptosis, has a high positive charge density. This allows a strong binding to solutes with a high negative charge that mimics the architecture of the active site of Cyt c Ox and caspases.1,2,6 Anionic porphyrins and phthalocyanines as well as calixarenes and dendrimers designed to interact with Cyt c proved that such molecules could bind with equilibrium constants as high as those found in nature (higher than 105 M-1).1,2,6,10-22 The interaction is driven mainly by electrostatic interaction (although some contribution of hydrophobic interactions is also important) and breaks up as the ionic strength increases.20-22 Alternatively, the effect on the binding of organic solutes in organized media like membranes is still largely unexplored. The interaction of aluminum tetrasulfonated phthalocyanine (AlPcS4) with Cyt c was studied previously.20-22 Strong * Corresponding author. Fax: +351 212948550; tel: +351 212948300; e-mail:
[email protected]. Present adress: CQFB-REQUIMTE, Faculdade de Ciencias e Tecnologia, 2829-516 Monte de Caparica, Portugal.
electrostatic interactions leads to the formation of a 1:1 complex between the dye and Cyt c, which causes a strong fluorescence quenching of the dye.20 This is caused by a photoinduced electron-transfer reaction from AlPcS4 to Cyt c that occurs with a high rate constant both in the singlet (above 109 s-1) and triplet (above 107 s-1) excited state. This behavior is also observed in micellar systems.21 The present work deals with zinc tetrasulfonated phthalocyanines (ZnPcS4), which aggregate very easily in water, leading to the formation of a non-emissive dimer at room temperature.23-25 Micelles and proteins induce the dissociation of dye aggregates frequently. Thus, it was considered interesting to investigate the balance of specific electrostatic interactions and nonspecific hydrophobic interactions that could occur, respectively, with Cytc and Triton X-100 micelles. The manuscript is organized in the following manner: (i) spectroscopic evidence of both monomer and dimer forms of ZnPcS4; (ii) interaction of both forms with Cyt c in water with evaluation of the induced rate constant of dimerization; (iii) interaction of ZnPcS4 with TX100 micelles; (iv) analysis of the overall system in micelles with the addition of Cyt c. It will be shown herein that in the balance of two types of interactions the electrostatic factor prevails, leading to a strong dimerization (and fluorescence quenching) of the dye in presence of the protein. Therefore, even in the presence of micelles, dye aggregates prevail on the system with Cyt c, preventing the dye from undergoing photoinduced electron-transfer reactions with the protein. Materials and Methods ZnPcS4 was purchased from Porphyrin Products (99% purity) and used as received. Cyt c was purchased from Aldrich (97% purity) and used without further purification. Bidistilled water
10.1021/jp076100+ CCC: $40.75 © 2008 American Chemical Society Published on Web 03/19/2008
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was used. Triton X-100 surfactant (critical micellar concentration (cmc) ) 2.4 × 10-4 M, aggregation number (Nagg) ) 105) was purchased from Sigma and used as received. Absorption spectra were recorded at room temperature with a JASCO V-560 UV/vis absorption spectrophotometer. Steadystate emission measurements were carried out with a PerkinElmer LS 50B Spectrofluorimeter with the sample holder thermostated at 22 °C. The instrument response at each wavelength was corrected by means of a curve provided with the instrument. Time-correlated single-photon counting was used to obtain the fluorescence decays in a MicroTime 200 fluorescence lifetime microscope system from Picoquant. The excitation source consisted of a pulsed red diode laser (PDL 800, PicoQuant Berlin) with 635 nm wavelength, providing output pulses of 10 µM as well. These results show a two-stage behavior: for [Cyt c] < 10 µM, ZnPcS4 dimerization is again induced by the presence of the protein; for [Cyt c] > 10 µM higher-order aggregates with Cyt c are formed, a behavior that is not observed in aqueous solutions. The emission spectra intensity decreases as Cyt c increases (see Figure 9). This result does not constitute a surprise because either the formation of dimers or complexes 1:1 with Cyt c would imply a fluorescence quenching. The extinction is
Figure 9. ZnPcS4 fluorescence spectra (λex ) 640 nm) in water/TX100 micellar system ([TX100] ) 1.6 mM) as a function of Cyt c concentration (from 0 M to 54.2 µM).
Figure 10. ZnPcS4 fluorescence intensity (λex ) 640 nm, O) in water/ TX100 micellar system ([TX100] ) 1.6 mM) as a function of Cyt c concentration (from 0 M to 54.2 µM) and the maximum fluorescent intensity wavelength (4, right axis). Line is fitting of the fluorescence intensity data with eq 6.
accompanied by a solvatochromic shift of the emission spectra (see Figure 10), which denounces a more polar environment for ZnPcS4 as Cyt c is added. The analysis of the data presented is not a simple task because many processes are involved (see Scheme 4): Therefore, severe assumptions would be necessary (like neglecting some processes completely), or else too many adjustable variables would be required, and therefore the fitting would not give reliable results because of the errors involved.
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SCHEME 4
Rather then using such a route, an analysis of the fluorescence intensity was done with the following empirical equation:35
I)
I[Cytc])0M + ICytcKe[Cytc]γ 1 + Ke[Cytc]γ
(6)
This equation has two empirical parameters: Ke and γ. Equation 6 has two limits that are exact solutions for the formation of 1:1 complexes (when γ ) 1) and 2:1 complexes (γ ) 0.5). Therefore, the intermediate γ value gives insight into the system, even though eq 6 is empirical. Of course, the parameter Ke is not an equilibrium constant, unless γ ) 1 or γ ) 0.5. For intermediate γ values, Ke is merely empirical and cannot be translated into meaningful thermodynamic parameters. The fitting (see Figure 10) gives these results: ICytc ) 0, γ ) 0.81 and Ke ) 1.74 × 104 M-γ. This system displays an intermediate situation in which both mechanisms for the fluorescence quenching by complex formation may be operative. Fluorescence decays were expected to give further insight into the system. Namely, a new component that could be assigned to a 1:1 complex should appear on the decays, as was observed on the AlPcS4 system.20-22 However, such a new component does not appear, even at high Cyt c concentrations. Therefore, it is not possible to obtain direct evidence of a 1:1 complex in these systems from the fluorescence decays. Nevertheless, the fluorescence lifetimes indeed change with Cyt c concentration, as is shown in Figure 11. A Stern-Volmer behavior is found for both components
1 1 ) + kq[Cytc] τ τ0
(7)
where τ0 is the fluorescence lifetime in the absence of Cyt c and kq is the quenching rate constant of the bimolecular reaction. It turns out that kq is different for each component. For the 3 ns component (the one attributed to a hydrophilic environment) kq ) 2.9 × 1011 M-1s-1 is found, which is remarkably similar to that found for the AlPcS4 dye in water.20 The 4.5 ns component hardly changes; probably it reflects ZnPcS4 in a more hydrophobic region, less accessible to interact with Cyt c. The preexponential factors also change with [Cyt c] (see Figure 11B). The longest component preexponential factor increases, although
Figure 11. ZnPcS4 in water/TX100 micellar system ([TX100] ) 1.6 mM) fluorescence lifetimes (a) and preexponential factors (b) vs concentration of Cyt c (analysis with eq 5).
slightly, a result that is in an apparent paradox with the solvatochromic result (Figure 10). The fluorescence decays point to a fluorescence quenching by Cyt c, which might be an indication of a 1:1 complex. The bimolecular quenching seen on Figure 11 is most likely to originate a 1:1 complex on the singlet excited state, which might not be seen directly on the fluorescence decays because its fluorescence is too weak. So the indications are that the Cyt c effect here is twofold: it induces the ZnPcS4 dimerization on the ground state, but it also forms 1:1 complex with ZnPcS4 on the excited state. This might explain why γ ) 0.81. The interactions of ZnPcS4 with Cyt c in water and TX100 micelles display a rich set of phenomena. Dimerization of the dye is strongly induced by this protein because of very strong and specific electrostatic interactions at the protein active site. Such an effect is not found with other proteins, such as human serum albumin where the interaction is caused mainly by nonspecific hydrophobic interactions, which promote the dissociation of the dye aggregates. Like this protein, TX100 micelles promote the dissociation due to hydrophobic interactions as well, although here another effect kicks in: at some concentrations, these micelles also induce the formation of higher-order dye aggregates. This might be caused by the increase of the ZnPcS4 local concentration around the micelles at low TX100 concentration, which would promote the interactions between dimers and therefore give rise to trimers, tetramers, or even higher-order aggregates. Although the absorption spectra give much qualitative information about all of these aspects, it fails to provide an insightful quantitative description of the systems studied for two reasons: the spectra of monomer and dimer overlap considerably making it difficult to separate clearly the concentration depend-
4282 J. Phys. Chem. B, Vol. 112, No. 14, 2008 ences, and there are several equilibria involved. The fluorescence allows more quantitative insight because it is caused solely by the monomer emission and therefore can be directly connected with ZnPcS4 monomer concentration. The ZnPcS4 fluorescence in water in the presence of Cyt c could be quantitatively unraveled, showing a very high Kb for the dimer, as predicted from previous results recognizing that electrostatic forces play a major role on this system. Alternatively, the ZnPcS4 fluorescence in the presence of TX100 micelles gives rise to a strong emission increase. The quality of the fit was not so good here because higher-order aggregates had to be ignored from the set of equations. Still a good agreement was obtained for Kdimmic, using fluorescence or absorption spectroscopy. The fluorescence decays here show the presence of two ZnPcS4 populations within the micelles: one in a hydrophilic region with a fluorescence lifetime of about 3 ns (probably on the micelle surface) and another in a hydrophobic region with a 4.5 ns fluorescence lifetime (most likely inside the micelle). Such results point out another degree of complexity of the system, which is a heterogeneous distribution of the dye molecules on the micelles. The implications of such an effect on the fluorescence intensity profile shown in Figure 6 are unknown. They might also play a role especially at high TX100 concentration, where the preexponential factor of the hydrophobic component is very important. Final Comments The interaction of sulfonated phthalocyanines with Cyt c gives rise to different phenomenologies. In the planar Zn sulfonated phthalocyanine derivatives studied, which already form dimers in aqueous solutions at low concentrations, the dimer formation is enhanced in the presence of cytochrome c, leading to a significant monomer fluorescence quenching. In Triton-X 100 micelles the dimer is dissociated and a significant monomer emission is found, but the addition of cytochrome c in this system induces fluorescence quenching again, associated with dimer formation, and probably some photoinduced electrontransfer. Acknowledgment. This work was supported by CQE IV and the project POCTI/QUI/57387/2004. C.A.T.L. acknowledges a postdoctoral fellowship SFRH/BPD/11567/2002. References and Notes (1) Peczuh, M. W.; Hamilton, A. D. Chem. ReV. 2000, 100, 24792494. (2) Wei, Y.; McLendon, G. L.; Hamilton, A. D.; Case, M. A.; Purring, C. B.; Lin, Q.; Park, H. S.; Lee, C.-S.; Yu, T. Chem. Commun. 2001, 17, 1580-1581. (3) Pelletier, H.; Kraut, J. Science 1992, 258, 1748-1755. (4) Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1982, 257, 44264437.
Laia and Costa (5) Do¨pner, S.; Hildebrandt, P.; Rosell, F. I.; Mauk, A. G.; von Walter, M.; Buse, G.; Soulimane, T. Eur. J. Biochem. 1999, 261, 379-391. (6) Clark-Ferris, K. K.; Fisher, J. J. Am. Chem. Soc. 1985, 107, 50075008. (7) Zhou, J. S.; Rodgers, M. A. J. J. Am. Chem. Soc. 1991, 113, 77287734. (8) Green, D. R.; Reed, J. C. Science 1998, 281, 1309-1312. (9) Reiners, J. J., Jr.; Caruso, J. A.; Mathieu, P.; Chelladurai, B.; Yin, X.-M.; Kessel, D. Cell Death Differ. 2002, 9, 934-944. (10) Zhou, J. S.; Granada, E. S. V.; Leontis, N. B.; Rodgers, M. A. J. J. Am. Chem. Soc. 1990, 112, 5074-5080. (11) Speh, S.; Elias, H. J. Biol. Chem. 1994, 269, 6370-6375. (12) Lahiri, J.; Fate, G. D.; Ungashe, S. B.; Groves, J. T. J. Am. Chem. Soc. 1996, 118, 2347-2358. (13) (a) Larsen, R. W.; Omdal, D. H.; Jasuja, R.; Niu, S. L.; Jameson, D. M. J. Phys. Chem. B 1997, 101, 8012-8020. (b) Croney, J. C.; Helms, M. K.; Jameson, D. M.; Larsen, R. W. J. Phys. Chem. B 2000, 104, 973977. (c) Croney, J. C.; Helms, M. K.; Jameson, D. M.; Larsen, R. W. Biophys. J. 2003, 84, 4135-4143. (14) (a) Lin, Q.; Park, H. S.; Hamuro, Y.; Lee, C. S.; Hamilton, A. D. Biopolymers 1998, 47, 285-297. (b) Jain, R. K.; Hamilton, A. D. Org. Lett. 2000, 2, 1721-1723. (c) Jain, R. K.; Hamilton, A. D. Angew. Chem., Int. Ed. 2002, 41, 641-643. (d) Wilson, A. J.; Groves, K.; Jain, R. K.; Park, H. S.; Hamilton, A. D. J. Am. Chem. Soc. 2003, 125, 4420-4421. (e) Ernst, J. T.; Becerril, J.; Park, H. S.; Yin, H.; Hamilton, A. D. Angew. Chem., Int. Ed. 2003, 115, 553-557. (f) Aya, T.; Hamilton, A. D. Bioorg. Med. Chem. Lett. 2003, 13, 2651-2654. (g) Baldini, L.; Wilson, A. J.; Hong, J.; Hamilton, A. D. J. Am. Chem. Soc. 2004, 126, 5656-5657. (15) Sedla´k, E.; Antalı´k, M. Biopolymers 1998, 46, 145-154. (16) Takashima, H.; Shinkai, S.; Hamachi, I. Chem. Commun. 1999, 23, 2345-2346. (17) Oshima, T.; Goto, M.; Furusaki, S. Biomacromolecules 2002, 3, 438-444. (18) Gong, J.; Yao, P.; Duan, H.; Jiang, M.; Gu, S.; Chunyu, L. Biomacromolecules 2003, 4, 1293-1300. (19) Braun, M.; Atalick, S.; Guldi, D. M.; Lanig, H.; Brettreich, M.; Burghardt, S.; Hatzimarinaki, M.; Ravanelli, E.; Prato, M., van Eldik, R.; Hirsch, A. Chem.sEur. J. 2003, 9, 3867-3875. (20) Laia, C. A. T.; Costa, S. M. B.; Phillips, D.; Beeby, A. J. Phys. Chem. B 2004, 108, 7506-7514. (21) Laia, C. A. T.; Costa, S. M. B. J. Phys. Chem. B 2004, 108, 1718817197. (22) Laia, C. A. T.; Costa, S. M. B.; Ferreira, L. F. V. Biophys. Chem. 2006, 122, 143-155. (23) Farren, C.; Fitzgerald, S.; Beeby, A.; Bryce, M. R. Chem. Commun. 2002, 572-573. (24) Petra´sˇek, Z.; Phillips, D. Photochem. Photobiol. Sci. 2003, 2, 236244. (25) Phillips, D. Prog. React. Kinet. 1997, 22, 176-300. (26) Laia, C. A. T.; Costa, S. M. B.; Phillips, D.; Parker, A. W. Photochem. Photobiol. Sci. 2003, 3, 555-562. (27) Tran-Thi, T.-H. Coord. Chem. ReV. 1997, 160, 53-91. (28) Lipskier, J. F.; Tran-Thi, T.-H. Inorg. Chem. 1993, 32, 722-731 (29) Prayer, C.; Tran-Thi, T.-H.; Pommeret, S.; d’Oliveira, P.; Meynadier, P. Chem. Phys. Lett. 2000, 323, 467-473 (30) Clarke, S. E.; Wamser, C. C.; Bell, H. E. J. Phys. Chem. A 2002, 106, 3235-3242 (31) Gusev, A. V.; Rodgers, M. A. J. J. Phys. Chem. A 2002, 106, 19851992 (32) Streletzky, K.; Phillies, G. D. J. Langmuir 1995, 11, 42-47. (33) Phillies, G. D. J.; Yambert, J. E. Langmuir 1996, 12, 3431-3436. (34) Dutt, G. B. J. Phys. Chem. B 2003, 107, 3131-3136. (35) Costa, S. M. B.; Velazquez, M. M.; Tamai, N.; Yamazaki, I. J. Luminescence 1991, 48-49, 341-351.
