Photoprocesses of merocyanine 540 bound to serum albumin and lysozyme

Journal of Molecular Structure 1011 (2012) 94–98

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Photoprocesses of merocyanine 540 bound to serum albumin and lysozyme Yazhou Zhang 1, Helmut Görner ⇑ Max-Planck-Institut für Bioanorganische Chemie, D-45413 Mülheim an der Ruhr, Germany

a r t i c l e

i n f o

Article history: Received 24 March 2011 Received in revised form 22 July 2011 Accepted 22 July 2011 Available online 6 August 2011 Keywords: Photoprocesses Protein oxidation Merocyanine dye Quantum yield

a b s t r a c t The binding of merocyanine 540 to either lysozyme or bovine serum albumin (BSA) in aqueous solution and the related photodecomposition processes were studied. Absorption, fluorescence and trans ? cis photoisomerization demonstrate a shift from free dimers to monomers upon binding to BSA, in contrast to lysozyme, where the binding appears spectroscopically less pronounced. The quantum yield (Ured) of merocyanine damage is generally small (60.0004). However, Ured is markedly enhanced upon binding and was found to be comparable to the quantum yields of protein oxidation, which are ca. 0.002. The mechanisms of protein oxidation were discussed. The major effect is electron transfer from aromatic amino acid residues of the protein to the triplet state of merocyanine 540. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Merocyanine 540 (MC) is an anionic polymethine dye which has frequently been studied as sensitizer for application in the photodynamic therapy [1–11]. MC is readily soluble in alcohols and lipid membranes, making it attractive for technical applications and biological activity studies of plasma membranes and tumor cells. The major deactivation process is trans ? cis photoisomerization, followed by thermal isomerization to the trans form [2]. The fluorescence of MC is substantial in organic solvents and sensitive to the solvent polarity, for example, the quantum yield of fluorescence in dioxane and ethanol is Uf = 0.52 and 0.15, respectively [2,10]. The quantum yields of intersystem crossing (Uisc) and formation of molecular singlet oxygen are small, e.g. in ethanol 0.04 and 0.002, respectively [7]. A correlation between these quantum yields and the efficiency of cell killing has been reported [7]. Three products of photooxidation mediated by molecular singlet oxygen have been identified [5]. Structural modifications of MC have marked implications on the photophysical properties and biological activity. Harriman and his group studied the photoisomerization of derivatives, e.g. with sulfur and selenium atoms; Uisc of the latter is strongly enhanced, whereas the spin–orbital coupling for sterically hindered merocyanines is unaffected by substitution [12]. The role of a zwitterionic resonance form, where the barbiturate unit acts as electron acceptor, has been discussed [13]. Whitten and his group found for bis-merocyanine type dyes with methylene spacers that exciton and charge-transfer interactions in nonconjugated merocyanine ⇑ Corresponding author. E-mail address: [email protected] (H. Görner). On leave from the Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China. 1

0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.07.042

dye dimers cause a novel solvatochromic behavior for tethered bichromophores and excimers [14]. In liposomes MC is incorporated into the lipid bilayer as monomer and Uisc is smaller than 0.1 [2]. MC has a high recognition potential for leukemia cells [11], but optimized photophysical and photochemical properties does not necessarily go along with therapeutic improvements. The photophysical properties of MC in water [3,15–20] and in the presence of proteins [15,19,21–24] are complex. The absorption spectrum in neat aqueous solution at pH 7.8 has two peaks at 500 and 536 nm which have been attributed to dimer formation [10,18]. The photobehavior of MC bound to human serum albumin (HSA) has recently been studied by Alarcón et al. [24]. Radicals of MC have been characterized by radiolysis [25]. A related class of anionic dyes are meso-thiacarbocyanine dyes which are the subject of various spectroscopic, kinetic and photochemical studies [26–33]. Specific alkylmeso-thiacarbocyanine dyes are able to form J-aggregates. One way to induce J-aggregates is by the addition of ions [26,27,31]. Another possibility is binding to proteins, such as bovine serum albumin (BSA) or HSA. For non-covalently linked complexes of a series of thiacarbocyanine dyes with HSA, the number of binding sites (n) of 0.13–0.75 has been determined [29]. In aqueous solution Uf of MC is 0.04 and increases to 0.27 when bound to 0.36 mM BSA [10]. Various other pigment systems reveal changes in Uf upon binding to proteins [34–39]. Here, we studied the binding of MC to either BSA or lysozyme and the relative photoprocesses in aqueous buffer-free solution by steady-state and time-resolved methods. Serum albumin and lysozyme are two of the most frequently studied proteins showing significant differences when bound to other dyes [29–39]. In the literature, there are not so many contributions about the interactions of the excited merocyanine dye with proteins. A rationale for the work is the lack of quantum yields of both merocyanine damage and protein oxidation. The quantum yields Ured of reduction of MC and Uox of

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oxidation of the proteins were determined in the presence and absence of air. The mechanisms in both air- and argon-saturated aqueous solution can now be discussed. Moreover, the results were compared with those of related pigments, such as eosin, erythrosin, rose bengal, flavins, porphyrins or chlorin e6. 2. Experimental MC (Fluka) was used as received. Lysozyme and BSA were the same as used previously [30,34–37], the lipid content of our BSA preparation was <0.2 mg/g. Water was from a Millipore (milli Q) system. A diode array spectrophotometer (HP 8453) was employed to measure absorption spectra. The dye samples in the concentration range 2–10 lM were freshly prepared in water. The steadystate absorption and fluorescence measurements were used to determine the concentration of free and bound dyes as well as to analyze the concentration and pH dependences. The absorption maxima vary between the value in the absence of macromolecules ðk0a Þ or sufficient protein amounts ðkPa Þ. The molar absorption coefficient of MC is e554 = 1.6  105 M1 cm1 in methanol and e517 = 5.5  104 M1 cm1 in water [7,18]. The pH was varied by addition of HClO4 or NaOH and the samples were without buffer unless indicated otherwise. The fluorescence spectra were recorded on a spectrofluorimeter (Cary, Eclipse). The quantum yield changes between the value in the absence of macromolecules ðU0f Þ and sufficient protein amounts ðUPf Þ. Irradiation was performed with a 250-W Hg lamp and filters, cutting-off the range below 400 nm, or a 1000-W Xe–Hg lamp and a monochromator. The relative quantum yield of decomposition of the dye in argon-saturated aqueous was obtained from the changes in absorption upon continuous irradiation at >400 nm. The changes were measured by (A0  At)/(A0  Ae), where At is the absorption at the given time, A0 at the beginning and Ae is the (end) value, when the changes are negligible. The relative quantum yield of photooxidation of proteins was obtained by the fluorescence intensity (kex = 280 nm) of the tryptophan residues at kf = 350 nm which decreases vs. the irradiation time [40]. As reference we used Ured = 2  103 for chlorin e6 in air-saturated aqueous solution at pH 7 [41]. The flash photolysis was studied with kexc = 530 nm using a Nd-YAG laser [34–37]. The measurements were carried out at 24 °C and refer to air-saturated aqueous (for convenience) unless indicated otherwise.

Fig. 1. Absorption spectra of 10 lM MC in 100% (vol) 80%, 50% glycerol and neat aqueous solution at pH 7.8, 1–4, respectively.

Fig. 2. Plots of A555 of MC (13 lM, buffer-free) in the presence BSA (s) and of A540 for lysozyme (N) as a function of [P]/[D]; insets: absorption spectra of MC (1) on addition of BSA (2, 3) and lysozyme (4, 5).

Table 1 Spectroscopic data of free and bound MC.a

ka (nm)

3. Results

kfex (nm)

3.1. Absorption a

The absorption band of MC in methanol at concentrations of <0.02 mM has a peak at ka = 560 nm originating from the monomer. Upon addition of water, the maximum is blue-shifted and the band in neat aqueous solution at pH 7.8 has two peaks at 500 and 536 nm. The latter species has been attributed to the dimer [10,16,18,19]. The dissociation constant has been reported to be 3.1  104 M and the amount of monomers for 5 lM MC has been calculated to be as low as 5% [16]. The absorption spectra of MC in mixtures of water with glycerol are similar, examples are shown in Fig. 1. The spectral and kinetic features change with increasing the protein concentration [P], keeping the dye concentration [D] constant. The maximum of the absorption band of MC in aqueous solution is red-shifted to 550 nm on addition of BSA, (Fig. 2, inset). The signal at kPa ¼ 555 nm increases with increasing the [BSA]/[D] ratio (Fig. 2). The characteristic protein/dye concentration ratio for 50% change, ð½P=½DÞa1=2 , is close to 1. With lysozyme the long-wavelength curve becomes less steep, but otherwise no significant change was observed. The maxima in buffer-free solution (Table 1) and in the

pH

Free

BSA

Lysozyme

8 4 8 4

536 500, 536 572 572

555 558 582 590

538 500 574 574

In air or argon-saturated aqueous (buffer-free) solution at [P]/[D] = 1.

presence of 10 mM phosphate are similar. For MC in the presence of 0.1 M phosphate buffer saline (PBS) ka = 505 nm and 545 nm when bound to HSA [24]. 3.2. Fluorescence The fluorescence emission maximum of MC ðkfem Þ in aqueous solution at pH 7.8 is centered at 575 nm and the excitation peaks ðkfex Þ at 510 and 540 nm. The small Uf of 0.04 and the fluorescence lifetime sf = 0.1 ns of MC in aqueous solution could indicate a low amount of monomers, taking into account that dimers of cyanine dyes are not fluorescing [10,19]. On the other hand, the small Uf has been ascribed to isomerization [10]. Thus, both effects could be operating. In methanol or glycerol both Uf and sf indicate only monomers, as in other organic solvents. Note that MC in dioxane has a much larger Uf = 0.5 and sf = 1.5 ns [11].

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Fig. 3. Plots of If (kexc = 520 nm) of 5 lM MC in buffer-free argon-saturated aqueous solution at pH 7.8 as a function of [P]/[D] for BSA (s) and lysozyme (N); inset: fluorescence spectra of free MC (1) bound to BSA; [P]/[D] = 0.1, 0.3 and 4, curves 2, 3 and 4, respectively.

Formation of a ground state complex between photosensitizer dye and protein is also indicated by fluorescence spectroscopy. The emission maximum is slightly red-shifted with increasing the BSA concentration, e.g. 15 nm for kfem at [P]/[D] = 1 (Table 1). The excitation maxima in the absence of protein and presence of lysozyme and BSA are at 560, 545 and 565 nm, respectively. Examples of the emission spectra in the absence and presence of BSA are shown in Fig. 3, inset. Uf initially decreases on slightly increasing [P]/[D] to 0.2 and then strongly increases, the UPf =U0f ratio is 10 for [P]/[D] = 4, i.e. becomes 10 times larger UPf than U0f . The characteristic protein/dye concentration ratio for 50% change, ð½BSA=½DÞf1=2 , is similar to that obtained by absorption. In contrast, lysozyme decreases Uf and the intensity If is reduced by 30% at for [P]/ [D] = 2. The spectra and effects are similar at pH 4.5 on the one hand and at pH 7 in the presence of 10 mM phosphate buffer on the other. For MC/PBS when bound to HSA kf = 573 nm and UPf ¼ 0:40 [24].

Fig. 4. Plots of A540 vs. irradiation time of 10 lM MC in buffer-free argon-saturated aqueous solution at pH 7.8 (j) and for BSA (circles) and lysozyme (triangles) at [P]/ [D] = 1 (open) and 2 (full); inset: absorption spectra for [BSA]/[D] = 1 at 0, 120 and 800 s, 1–3, respectively.

Table 2 Quantum yields of reduction of MC and oxidation of proteins.a Solvent

Gas

Urel red Free dye

BSA

Lysozyme

BSA

Lysozyme

Water, pH 8

Air Airb Argon Argonb Argon Argon

0.2 0.1 0.15 0.2 0.1 0.1

0.8 0.2 0.3 0.3 0.2 0.4

1.0c 0.2 0.5 0.9

0.2 0.2 0.04 0.4

0.3 0.4 0.14 0.5

+10% Methanol +10% Glycerol a b c

Urel ox

In argon-saturated buffer-free solution at [P]/[D] = 1. Using 5 mM phosphate buffer. Absolute value Ured = 0.002.

3.3. Transients The major transient of MC in argon-saturated methanol and aqueous solution at pH 7.8 at the end of the 530 nm pulse has absorption bands at 400 and 600 nm and a bleaching minimum around 550 nm (not shown). This transient is attributed to a cis isomer. The decay follows first-order kinetics and is not affected by oxygen. This thermal recovery is due to cis ? trans isomerization. The quantum yield Ut–c of trans ? cis isomerization of MC in aqueous solution at pH 7.8 was estimated to be 0.08. For comparison Ut–c = 0.4 in ethanol [2,11]. The other (minor) transient at the pulse end has a band at 640 nm, a lifetime of sT = 20 ls under argon and the decay is quenched by oxygen. It is attributed to the triplet state, the rate constant for quenching by oxygen is kox = 0.2– 0.6  109 M1 s1. Addition of BSA at [P]/[D] = 1 only slightly reduces Ut–c and Uisc but prolongs the decay 5–8-fold. Such an increase, sT = 0.8 ms in the presence of HSA and 0.1 M PBS, has recently been reported [24]. In contrast, the addition of lysozyme has almost no effect. Intersystem crossing from upper triplet levels has been achieved for MC in methanol, where the quantum yield of intersystem crossing to the lowest triplet state is substantial [9], but this effect cannot have a significance under our conditions, where Uisc is smaller than 0.02. 3.4. Photodamage Upon irradiation of free and bound MC at >400 nm, the absorption band becomes lower. A540 as a function of the irradiation time

0

Fig. 5. Plots of I340 (kexc = 280 nm) vs. irradiation time of 7 lM MC in buffer-free argon-saturated aqueous solution at pH 7.8, 5 mM phosphate buffer, [P]/[D] = 2 (s) and 0.5 (d) for BSA and 1 for lysozyme (N); inset: fluorescence spectra at [BSA]/ [D] = 1 at 0, 120, 300 and 600 s, 1–4, respectively.

is initially linear and the slope is taken as Ured. Examples are shown in Fig. 4. The quantum yield of dye damage in argon-saturated aqueous solution at pH 7.8 is Ured = 0.0004 and 4 or 5-fold larger in the presence of either BSA or lysozyme at [P]/[D] = 1 (Table 2). The results are similar in the presence of 10 mM phosphate buffer. For comparison, Ured is also given in air-saturated solution, the values are of the same order of magnitude. For MC in air-saturated ethanol Ured is smaller than 6  104 and in the presence of 0.1 M PBS at pH

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7.4 Ured = 0.004 [24]. The decrease of Amax vs. time is proportional to Ured also for other specific protein/dye systems [38–41]. The change in the fluorescence intensity of the protein/dye system (kex = 280 nm, kf = 350 nm) as a function of the irradiation time is a measure of the protein oxidation due to damage of the tryptophan residues [40]. Examples are shown in Fig. 5. Uox is small for BSA at [P]/[D] = 1 and larger for lysozyme (Table 2). In the presence of 5 mM phosphate buffer Uox increases 3–10-fold. The reason of the strong buffer-enhanced effect in the presence of BSA is open to question. 4. Discussion 4.1. Photophysical properties of merocyanine 540 The solvent polarity on the one hand and the viscosity on the other have significant influences on sf and Uf of MC [6,8]. An intrinsic barrier height of 3 kJ mol1 was determined for isomerization in the excited singlet state in polar solvents based on dynamic solvation effects on the rate constant [6]. The presence of 7% water in glycerol also has an effect on sf, Uf and Ut–c [20]. Environmental effects on radiative and nonradiative transitions were studied for merocyanines in homogeneous and microheterogeneous systems. The triplet in methanol has Uisc = 0.04, a lifetime of sT = 10–20 ls and kox = 3  109 M1 s1 [2,7]. MC in organic solvents is present as monomer, but in aqueous solution at pH 7–8 the dye is also present as dimer. The relative absorbances at 500 and 550 nm is a rough estimate of the dimer/ monomer ratio [24]. The equilibrium between these forms is established [10,18]. An analysis has indicated that at a MC concentration of 5 lM only 5% is present as dimer [10]. This conflicts with the spectra shown in Fig. 1, where the amount of dimer is larger than 50%. The A500/A550 ratio is 0.7–1 for glycerol and [HSA]/[D] = 6 [24]. An analysis of the equilibrium for 3-acetyl-5,12-(3-ethyl-2benzothiaxolydene) rhodanine, a related merocyanine, shows that in water the dye is mainly present as dimer [42]. Note that the inflection point of MC at pH 8.5 is not a pKa value and that the behavior in the alkaline range is not reversible [15]. The effect of pH presented in this work in the range 4–8 was found to be minor. It is worth mentioning that the monomer–dimer equilibrium of MC resembles the case of other cyanine dyes [26–33]. 4.2. Binding of merocyanine 540 to proteins Formation of complexes between the dye and the protein is revealed by UV–vis absorption (Fig. 2) and fluorescence (Fig. 3) spectroscopy. These plots as a function of the [P]/[D] ratio are strikingly different for the two proteins. Note that the isoelectric point of lysozyme is pH 11, whereas that of BSA is much lower, 4.7. On the other hand, BSA has no b-sheets, in contrast to lysozyme with 12% b-sheets. These differences, however, are proposed not to be the reasons of the different spectroscopic effects. Binding of MC to the albumin leads to stronger changes in the ground state

monomer–dimer equilibrium than binding to lysozyme. The proposed mechanism of conversion of free dimers to bound monomers is illustrated in Scheme 1. A minimum in Uf at [P]/[D] = 0.1 indicates non-fluorescent dimers bound to BSA and the increase with the BSA concentration is due to monomer formation. Apparently, the dye is located in an environment, where trans ? cis isomerization is strongly restricted. Otherwise, Ut–c would be larger since it is enhanced at the expense of Uf. This enhancement of Ut–c for BSA at [P]/[D] = 1–3 was found not to take place. The enhancement of Uf has been attributed to incorporation of the dye to a more hydrophobic part of the albumin [19]. The aggregation of MC in water and some disaggregation by HSA has been considered [24]. The features of the dye bound to lysozyme are different since A540 (Fig. 2) and Uf (Fig. 3) decrease in a similar way with the lysozyme concentration. Electron transfer from an aromatic amino acid entity of lysozyme to the singlet state of MC can therefore be excluded since this should only affect the fluorescence. One could argue that no binding takes place, but this has also to be excluded based on the enhanced efficiency of photodamage (Fig. 4 and Table 2). The lysozyme-bound cyanine dyes are present partly as dimer and partly as monomer, whereby the equilibrium is only slightly shifted with respect to the free dye, Scheme 1. The proposed mechanism is binding of the dye as monomer and dimer and a slightly larger dimer contribution causes the observed fluorescence quenching (Fig. 3).

4.3. Photoreduction of merocyanine 540 and protein oxidation The photoproducts of reduction of MC have been reported to absorb only weakly [25]. Photodamage of MC takes place both without air and in the presence of oxygen. A mechanism has been proposed to account for the photooxidation of MC by molecular singlet oxygen [4,22]. The photoproducts of oxidation of related dyes by molecular singlet oxygen have been discussed [35]. The Uox of protein oxidation with and without oxygen are similar to the Ured values (Fig. 5 and Table 2). This could indicate that the molecular singlet oxygen destroys partly the aromatic amino acids of the proteins. On the other hand, electron transfer and radicals rather than energy transfer and molecular singlet oxygen have recently been proposed by Alarcón et al. for MC in air-saturated aqueous PBS (0.1 M), where at [HSA]/[D] = 6 Ured = 0.005 and Uox = 0.02 [24]. The latter is much larger than our Uox = 0.0005 in the absence of buffer or presence of 5 mM phosphate, indicating a significant effect due to PBS. Such a type I radical-induced protein oxidation rather than a type II energy transfer mechanism had been considered [21,23]. From the results under argon a damage of the merocyanine chain can be concluded. A possible reason is self-quenching, ion pair formation and radical-induced damage of a peptide bond, as proposed for other cases [40]. The enhancement of Ured in the presence of macromolecular environment, shown in Table 2 and Fig. 4 for [P]/[D] = 1 or 2, may be ascribed to electron transfer from an aromatic amino acid entity of the protein to the triplet state.

isc(S-T) Mcis

Mcis +Protein Protein-M

M

+M

M-M

+Protein Protein-Dimer

Lysozyme Scheme 1.

Mcis M-Protein-M

BSA

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Table 3 Quantum yields of decomposition of free dyes and oxidation of proteins.a Dye

Ured Free

Uox BSA

Uox Lysozyme

Typeb

Merocyanine 540 Rose bengal Eosin Riboflavin FMN TSPP Chlorin e6

0.0004

0.0016 0.001 0.002 0.09 0.10 0.0003 0.0002

0.002 0.003 0.002 0.10 0.12 0.0005 0.0014

A B B A A C C

0.001 0.02 0.0001 0.003

a

In argon-saturated buffer-free aqueous solution at pH 7.8, see Refs. [34–37] for xanthenes, flavins, TSPP and chlorin e6, respectively. b Electron transfer from aromatic amino acid residues of: (A) BSA or lysozyme to the sensitizer triplet state, (B) proteins to the sensitizer excited singlet and triplet states, and (C) lysozyme to sensitizer excited singlet state.

4.4. Comparison with other dyes For the MC case in argon-saturated aqueous solution we propose mechanism (A) of electron transfer from aromatic amino acid residues, see Table 3. It appears attractive to compare the protein/ chlorin system with those of xanthene dyes, such as eosin Y, erythrosin B and rose bengal [34,43], flavins [35], porphyrins, such as meso-tetra-4-sulfonato-phenylphorphyrin (TSPP) and meso-tetra4-hydroxy-phenylphorphyrin (THPP) [36] and chlorin e6 [37]. Selective quantum yields to illustrate the damage in the absence and presence of proteins at [P]/[D] = 1 are compiled in Table 3. The pigments differ markedly by their binding and aggregation potentials in aqueous solution. Ured is largest for flavin mononucleotide (FMN), where Uox is even larger. In the cases of flavins, only the sensitizer triplet state is involved. The high Ured for free flavin is suggested to be the result of efficient self-quenching [35]. On the other hand, Ured is smallest for TSPP. The yield for TSPP/BSA is Ured = 0.0003 and that for lysozyme larger. For bound TSPP, THPP and chlorin e6 electron transfer from tyrosyl/tryptophan residues of lysozyme to the excited singlet state has been proposed [36,37]. For binding of eosin and rose bengal to either lysozyme or BSA the triplet yield is significantly reduced, UPisc =U0isc ¼ 0:2  0:3 for [P]/[D] = 1. For rose bengal or eosin-sensitized photooxidation of lysozyme or BSA in argon-saturated aqueous solution at pH 7–10 Ured = 0.001–0.004. The major mechanism for xanthene dyes is electron transfer from aromatic amino acid residues of proteins to both excited singlet and triplet states of the sensitizer. 5. Conclusions The photophysical properties and photoisomerization process of MC, the frequently used anionic polymethine dye, and related structural modifications have been studied by many groups due to its application in the photodynamic therapy. This article reports on the results of the photoprocesses studies of MC with proteins such as serum albumin and lysozyme. The presence of proteins shifts the equilibrium between free MC monomers and dimers from the free to the bound dye. BSA does not markedly affect the low efficiency of trans ? cis photoisomerization but causes strong changes in absorption and fluorescence due to bound monomers. In contrast, MC bound to lysozyme shows only small spectral changes. The quantum yield of dye damage Ured in aqueous buffer-free solution in the presence or absence of air is strongly enhanced upon binding

to both proteins. The quantum yield of protein oxidation Uox at a protein to dye molecular ratio of 1 is also low and comparable to Ured. Despite isc of MC is low, the mechanism of damage is attributed to electron transfer from aromatic amino acid residues of BSA or lysozyme to this sensitizer triplet state. Acknowledgments We thank Professor Wolfgang Lubitz for his support and Mr. Leslie J. Currell for technical assistance. References [1] T.G. Easton, J.E. Valinsky, E. Reich, Cell 13 (1978) 475. [2] P.F. Aramendía, M. Krieg, C. Nitsch, E. Bittersmann, S.E. Braslavsky, Photochem. Photobiol. 48 (1988) 187. [3] S. Seret, M. Hoebeke, A. Vandevorst, Photochem. Photobiol. 52 (1990) 601. [4] J. Davilla, A. Harriman, K.S. Gulliya, Photochem. Photobiol. 53 (1991) 1. [5] B. Franck, U. Schneider, Photochem. Photobiol. 56 (1992) 271. [6] Y. Onganer, M. Yin, D.R. Bessire, E.L. Quitevis, J. Phys. Chem. 97 (1993) 2344. [7] R.W. Redmond, M.B. Srichai, J.M. Bilitz, D.D. Schlomer, M. Krieg, Photochem. Photobiol. 60 (1994) 348. [8] D.R. Bessire, E.L. Quitevis, J. Phys. Chem. 98 (1994) 13083. [9] R.W. Redmond, I.E. Kochevar, M. Krieg, G. Smith, W.G. McGimpsey, J. Phys. Chem. A 101 (1997) 2773. [10] D. Mandal, S.K. Pal, D. Sukul, K. Bhattacharyya, J. Phys. Chem. A 103 (1999) 8156. [11] A.C. Benniston, K.S. Gulliya, A. Harriman, J. Chem. Soc., Faraday Trans. 93 (1997) 2491. [12] A.C. Benniston, A. Harriman, C. McAvoy, J. Chem. Soc., Faraday Trans. 93 (1997) 3653. [13] A.C. Benniston, A. Harriman, C. McAvoy, J. Chem. Soc., Faraday Trans. 94 (1998) 519. [14] L. Lu, R.J. Lachicotte, T.L. Penner, J. Perlstein, D.G. Whitten, J. Am. Chem. Soc. 121 (1999) 8146. [15] L. Sikurova, B. Cunderlikova, Spectrochim. Acta A 53 (1997) 293. [16] C. Sato, J. Nakamura, Y. Nakamaru, J. Biochem. 127 (2000) 603. [17] B. Cˇunderlíková, L. Sikurová, Chem. Phys. 263 (2001) 415. [18] A. Adenier, J.J. Aaron, Spectrochim. Acta A 58 (2002) 543. [19] B. Cˇunderlíková, L. Sikurová, J. Moan, Bioelectrochemistry 59 (2003) 1. [20] K.S. Mali, G.B. Dutt, T. Mukherjee, J. Chem. Phys. 128 (2008) 124515. [21] S. Pervaiz, A. Harriman, K.S. Gulliya, Free Radical Biol. Med. 12 (1992) 389. [22] G.S. Anderson, W.H.H. Günther, R. Searle, J.M. Bilitz, M. Krieg, F. Sieber, Photochem. Photobiol. 64 (1996) 683. [23] F. Sieber, Trends Photochem. Photobiol. 10 (2003) 1. [24] E. Alarcón, A. Aspée, M. González-Béjar, A.M. Edwards, E. Lissi, J.C. Scaiano, Photochem. Photobiol. Sci. 9 (2010) 861. [25] A. Harriman, L.C.T. Shoute, P. Neta, J. Phys. Chem. 95 (1991) 2415. [26] V.I. Yuzhakov, Russ. Chem. Rev. 61 (1992) 613. [27] B.I. Shapiro, Russ. Chem. Rev. 63 (1994) 231. [28] I.G. Panova, N.P. Sharova, S.B. Dmitrieva, R.A. Poltavtseva, G.T. Sukhikh, A.S. Tatikolov, Anal. Biochem. 361 (2004) 183. [29] A.S. Tatikolov, S.M.B. Costa, Biophys. Chem. 107 (2004) 33. [30] T.D. Slavnova, H. Görner, A.K. Chibisov, J. Phys. Chem. B 111 (2007) 10023. [31] A.K. Chibisov, T.D. Slavnova, H. Görner, Nanotechn. Russ. 3 (2008) 19. [32] A.K. Chibisov, T.D. Slavnova, G.V. Zakharova, H. Görner, High Energy Chem. 41 (2007) 344. [33] Y. Zhang, J. Xiang, Y. Tang, G. Xu, W. Yan, ChemPhysChem 8 (2007) 224. [34] Y. Zhang, H. Görner, Photochem. Photobiol. 85 (2009) 677. [35] Y. Zhang, H. Görner, Photochem. Photobiol. 85 (2009) 943. [36] Y. Zhang, H. Görner, Dyes. Pigm. 85 (2011) 163. [37] Y. Zhang, H. Görner, Dyes Pigm. 83 (2009) 174. [38] C.V. Kumar, A. Buranaprapuk, G.J. Opiteck, M.B. Moyer, S. Jockusch, N.J. Turro, Proc. Natl. Acad. Sci. 95 (1998) 10361. [39] C.V. Kumar, A. Buranaprapuk, H.C. Sze, S. Jockusch, N.J. Turro, Proc. Natl. Acad. Sci. 99 (2002) 5810. [40] T.R. Hopkins, J.D. Spikes, Photochem. Photobiol. 12 (1970) 175. [41] R. Bonnett, G. Martínez, Tetrahedron 57 (2001) 9513. [42] F. Nüesch, M. Grätzel, Chem. Phys. 193 (1995) 1. [43] E. Alarcón, A.M. Edwards, A. Aspée, C.D. Borsarelli, E.A. Lissi, Photochem. Photobiol. Sci. 8 (2009) 933.