Determination of Singlet Oxygen and Electron Transfer Mediated

Kazutaka Hirakawa†, Hironobu Umemoto†, Ryo Kikuchi†, Hiroki Yamaguchi†, ... *Tel/Fax: +81-53-478-1287; E-mail: [email protected]...
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Determination of Singlet Oxygen and Electron Transfer Mediated Mechanisms of Photosensitized Protein Damage by Phosphorus(V)porphyrins Kazutaka Hirakawa,*,† Hironobu Umemoto,† Ryo Kikuchi,† Hiroki Yamaguchi,† Yoshinobu Nishimura,‡ Tatsuo Arai,‡ Shigetoshi Okazaki,§ and Hiroshi Segawa∥ †

Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu, Shizuoka 432-8561, Japan ‡ Department of Chemistry, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8571, Japan § Medical Photonics Research Center, Hamamatsu University School of Medicine, Handayama 1-20-1, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan ∥ Research Center for Advanced Science and Technology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan S Supporting Information *

ABSTRACT: The mechanism of photosensitized protein damage by phosphorus(V) tetraphenylporphyrin derivatives (P(V)TPPs) was quantitatively clarified. P(V)TPPs bound to human serum albumin (HSA), a water-soluble protein, and damaged its tryptophan residue during photoirradiation. P(V)TPPs photosensitized singlet oxygen (1O2) generation, and the contribution of 1O2 to HSA damage was confirmed by the inhibitory effect of sodium azide, a 1O2 quencher. However, sodium azide could not completely inhibit HSA damage, suggesting the contribution of an electron transfer mechanism to HSA damage. The decrement in the fluorescence lifetime of P(V)TPPs by HSA supported the electron transfer mechanism. The contribution of these processes could be determined by the kinetic analysis of the effect of sodium azide on the photosensitized protein damage by P(V)TPPs.

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mechanism is hardly observed, whereas 1O2 can be easily generated by visible light with its long wavelength. Therefore, the Type II mechanism is considered to be important for PDT. Porphyrins have been extensively examined as photosensitizers for PDT.1−4 In clinical use, porfimer sodium and talaporfin sodium, an oligomer and a monomer of free-base anionic porphyrin, respectively, are important photosensitizers. Since porphyrins are visible-light photosensitizers (excitation energy ∼ 2 eV), the generation of 1O2 is an important process. However, the phototoxic effect of 1O2 on PDT is restricted because a tumor exists under hypoxic conditions.9 Therefore, the Type I mechanism is also necessary for PDT. The central atoms of porphyrin strongly affect its photochemical property. For example, high-valent porphyrin complexes, such as phosphorus(V)10−13 and antimony(V)14−16 complexes, demonstrate a lower redox potential for one-electron reduction in their photoexcited state than do free-base or low-valent metal complexes. Photosensitized biomolecule damage by these porphyrins through dual mechanisms has been reported.11,13,15,16 However, the contribution of electron transfer

INTRODUCTION

Photosensitized biomolecule damage is closely related to photocytotoxicity and the mechanism of photodynamic therapy (PDT), which is a less invasive treatment for cancer and some nonmalignant conditions.1−4 PDT involves the administration of a photosensitizer followed by exposing the tissue to visible light. The photoexcited photosensitizer induces photochemical damage to biomacromolecules, resulting in cell death. In general, the following two mechanisms are important for photosensitized biomolecule damage: photoinduced electron transfer from biomolecules such as protein and DNA (Type I) and the oxidation of biomolecules through the generation of reactive oxygen species (Type II).5 Singlet oxygen (1O2), which is formed through energy transfer from the photoexcited photosensitizer to molecular oxygen, is a major species of reactive oxygen.6,7 The Type I mechanism requires relatively large oxidation activity for the photosensitizer.8 The oxidation activity of a photoexcited photosensitizer is determined by its redox potential for one-electron oxidation and its excitation energy (singlet energy). Therefore, the photosensitizer, which has excitation energy comparable to that of the ultraviolet photon, induces biomolecule damage through the Type I mechanism. For a visible-light photosensitizer, the Type I © 2015 American Chemical Society

Received: November 28, 2014 Published: January 23, 2015 262

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and 1O2 generation to these mechanisms has not been welldetermined. In this study, the contribution of these mechanisms to photosensitized protein damage was exactly determined by spectroscopic experiments. As photosensitizers, porphyrin phosphorus(V) complexes bis(ethyleneglycoxy)P(V) tetraphenylporphyrin (EGPP) and bis(3-hydroxypropoxy)P(V) tetraphenylporphyrin (PRPP) (Figure 1)17 were used. The purposes

Article

RESULTS AND DISCUSSION Interaction between P(V)porphyrins and Human Serum Albumin. In the presence of HSA, a water-soluble protein, the bathochromic effect and a red shift were observed in the UV−vis absorption spectra of P(V)porphyrins (Figure 2), indicating static interaction between P(V)porphyrins and

Figure 1. Structures of EGPP (left) and PRPP (right).

of this study are the confirmation of the protein photodamaging activity of these P(V)porphyrins and the development of an analysis method to determine the contribution of the Type I and Type II mechanisms toward this activity. Since a small difference of the axial ligand of P(V)porphyrin affects its photochemical property,13 two kinds of water-soluble P(V)porphyrins, EGPP and PRPP, were used.

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EXPERIMENTAL PROCEDURES

Materials. EGPP and PRPP were synthesized according to previous reports.17−20 Human serum albumin (HSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Spectroscopic grade water (H2O) was from Dojin Chemicals Co. (Kumamoto, Japan) and used as received. Sodium phosphate buffer (pH 7.6) was from Nakalai Tesque Inc. (Kyoto, Japan). Sodium azide (NaN3) was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Measurements of Absorption and Fluorescence Spectra. The absorption spectra of P(V)porphyrins were measured with a UV1650PC UV−vis spectrophotometer (Shimadzu, Kyoto, Japan). The fluorescence spectra of samples were measured with an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The fluorescence quantum yields (Φf) of P(V)porphyrins in sodium phosphate buffer were determined relative to that of EGPP (Φf =0.048 in distilled water).18 These values were corrected by the refractive index of the solvents.21 Experimental details of other spectroscopic measurements (fluorescence lifetime, near-infrared 1O2 emission, and transient absorption) are described in the Supporting Information. Evaluation of HSA Damage by P(V)porphyrins. The sample solution containing P(V)porphyrins and HSA in 10 mM sodium phosphate buffer (pH 7.6) was irradiated with a light-emitting diode (LED) (λmax = 519 nm, 1 mW cm−2, CCS Inc., Kyoto, Japan). The intensity of the LED light source was measured with an 8230E optical power meter (ADC Corporation, Tokyo, Japan). The protein damage by P(V)porphyrins was evaluated by the measurement of fluorescence intensity from amino acid residues as previously reported.22 The amount of damaged HSA was estimated from the results of fluorometry using the following equation damaged HSA =

F0 − F cV F0

Figure 2. Absorption spectra of EGPP (A) and PRPP (B) with or without HSA. Inset: Absorbance at 445 nm vs [HSA]. The sample solution contained 5 μM porphyrins and HSA in 10 mM sodium phosphate buffer (pH 7.6).

HSA. Analysis of the absorption spectrum showed a 2:1 complex formation between P(V)porphyrins and HSA. A Job’s plot also supported this binding ratio (Figure S1). These findings suggest that porphyrins bind into two hydrophobic pockets23 of HSA. Photosensitized Damage of Human Serum Albumin by P(V)porphyrins. The typical fluorescence spectrum assigned to the tryptophan residue of HSA is shown in Figure 3. The intensity was decreased by the photosensitized reaction

(1) Figure 3. Fluorescence spectra of HSA before and after photoirradiation with EGPP. The sample containing 5 μM EGPP and 10 μM HSA in 10 mM sodium phosphate buffer (pH 7.6) was irradiated with an LED (519 nm, 1 mW cm−2, 30 min). The excitation wavelength is 298 nm.

where F0 is the initial fluorescence intensity of HSA, F is the fluorescence intensity of HSA photosensitized by P(V)porphyrins, c is the initial concentration of HSA (10 μM), and V is the volume of the sample solution (1.2 cm3). 263

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Chemical Research in Toxicology of P(V)porphyrins. HSA has one tryptophan residue,23 which is easily oxidized by Type I and II mechanisms, leading to the extinguishment of fluorescence.22 This fluorescence decrement of HSA can be explained by tryptophan oxidation by photoexcited P(V)porphyrins. Although other amino acids of HSA should be damaged by photoexcited P(V)porphyrins, tryptophan damage can be selectively analyzed by fluorometry. Therefore, tryptophan is a convenient indicator of the oxidative damage of HSA. Analysis with high-performance liquid chromatography and fluorometry also demonstrated photosensitized damage to isolated tryptophan by these P(V)porphyrins (Supporting Information). HSA damage, determined from the fluorescence intensity of HSA, increased in a dose-dependent manner with photoirradiation (Figure 4). The

Figure 5. Effect of NaN3 on HSA damage photosensitized by P(V)porphyrins. The curves were calculated by eq 7.

porphyrins was confirmed using near-infrared luminescence measurements (Supporting Information). Typical phosphorescence spectra of 1O2 at around 1270 nm were observed during the photoexcitation of EGPP and PRPP. (Figure 6). The

Figure 4. Time course of HSA damage photosensitized by P(V)porphyrins. The sample solution containing 5 μM porphyrins and 10 μM HSA in 10 mM sodium phosphate buffer (pH 7.6) was irradiated with an LED (519 nm, 1 mW cm−2). The excitation wavelength is 298 nm.

Figure 6. Near-infrared emission spectra of 1O2 generated by the photosensitization of EGPP (A) and PRPP (B). The sample solution contained 5 μM EGPP or PRPP with or without 10 μM HSA in 10 mM sodium phosphate buffer (pH 7.6).

quantum yield of this HSA damage (tryptophan damage) (Φdamage) was estimated from the initial decomposition rate within 30 min of the results in Figure 4 and the absorbed photon number by these P(V)porphyrins. The resulting value is 2.5 × 10−2 for both EGPP and PRPP. HSA damage was enhanced in D2O (data not shown), in which the lifetime of 1 O2 (τΔ) is elongated (approximately 3.5 μs in H2O24 to 70 μs in D2O25). These findings suggest the contribution of 1O2 to HSA photooxidation by P(V)porphyrins. The time course of HSA damage (Figure 4) demonstrated that the photodamage of the protein by these P(V)porphyrins proceeds linearly within 30 min. Thus, the effect of NaN3, a physical quencher of 1O2,26 on the photosensitized HSA damage by P(V)porphyrins for 30 min was examined (Figure 5). In this figure, the relative HSA damage (DHSA), which is determined to be 100% in the absence of NaN3, is presented. HSA damage was effectively inhibited by NaN3, supporting the contribution of 1O2. However, the damage to HSA was not completely inhibited by the excess of NaN3, suggesting the contribution of another mechanism (Type I). In addition, the excess of NaN3 did not completely inhibit the damage of isolated tryptophan photosensitized by EGPP and PRPP (Figure S2), supporting the contribution of the Type I mechanism. The results in Figure 5 were kinetically analyzed to determine the contribution of the Type I and Type II mechanisms (described later). Singlet Oxygen Generation Photosensitized by P(V)porphyrins. Photosensitized 1O2 generation by these P(V)-

quantum yields of photosensitized 1O2 generation (ΦΔ) were calculated from the comparison of the 1O2 phosphorescence intensities by P(V)porphyrins in sodium phosphate buffer (pH 7.6) and methylene blue (ΦΔ = 0.52).27 The estimated values of ΦΔ were relatively large (Table 1). The apparent value of ΦΔ decreased in the presence of HSA, suggesting the chemical quenching of 1O2 through the oxidation of HSA. Table 1. Photophysical Properties of P(V)porphyrins with or without HSA sensitizer

ΦΔa

Φf

τf/ns

τT/μs

τTN2b/μs

EGPP +HSA PRPP +HSA

0.88 0.63 0.80 0.57

0.048 0.036 0.043 0.033

4.23 4.03 4.34 4.23

2.6 27 2.4 26

125 166 139 145

a Reference, methylene blue (ΦΔ = 0.52 in water).27 bTriplet excited state lifetime under nitrogen bubbling.

Possibility of Protein Damage through Electron Transfer Mechanism. The fluorescence intensity of these P(V)porphyrins was slightly decreased with an increase in the HSA concentration, suggesting electron transfer quenching by HSA (Figure 7). The Φf values of the P(V)porphyrins are shown in Table 1. The fluorescence intensity time profile of 264

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Chemical Research in Toxicology Table 2. Reaction Parameters Determined from the Experimental Results sensitizer EGPP PRPP

ΦETa 0.051 0.026

Φdcb 0.088 0.140

Φreactc

kqf/s−1 M−1

kqΔ/s−1 M−1

0.032 0.037

2.1 × 10 2.1 × 109

1.2 × 108 1.1 × 108

9

ΦET = kET τf. bΦdc was calculated by eq 5. cΦreact was calculated by eq 6.

a

concentration of P(V)porphyrin T1, was decayed with a single-exponential function (Figure S3). The evaluated lifetime (τT) is shown in Table 1. The values of τT increased in the presence of HSA, suggesting that interaction with HSA suppresses the vibrational deactivation of P(V)porphyrins T1 and protects T1 from being quenched by molecular oxygen. Furthermore, this result suggests that the quenching of T1 through electron transfer is negligible. In general, the oxidative activity of the porphyrin T1 is smaller than that of S1. This result does not support a contribution of T1 for electron transfer mediated HSA damage. The values of τT strongly depend on the oxygen concentration and markedly elongate under nitrogen. The quenching of the T1 by oxygen can be explained by the energy transfer to molecular oxygen that is responsible for 1O2 generation (Figure S4). Mechanisms of Photosensitized Protein Damage by P(V)porphyrins. The mechanisms of photosensitized damage of HSA by EGPP and PRPP are summarized in Scheme 1.

Figure 7. Fluorescence quantum yields of P(V)porphyrins in the presence of HSA. The sample solution contained 5 μM EGPP or PRPP and the indicated concentration of HSA in 10 mM sodium phosphate buffer (pH 7.6). Inset: Fluorescence spectra of EGPP with or without HSA. The excitation wavelength is 550 nm.

these P(V)porphyrins without HSA could be fitted by a singleexponential function. The determined fluorescence lifetime (τf) is also shown in Table 1. In the presence of 10 μM HSA (the same as the experimental conditions for protein damage), the time profiles could also be fitted by a single-exponential function, and the obtained values of τf were slightly smaller than those without HSA (Table 1), supporting the quenching of the singlet excited state (S1) of these P(V)porphyrins by HSA. From these results, the quenching of P(V)porphyrins S1 through electron transfer from HSA was suggested. The Gibbs energy (−ΔG) of the electron transfer was calculated using the following equation −ΔG = E0 − 0 − e(E + − E−)

Scheme 1. Proposed Mechanisms of Protein Damage Photosensitized by P(V)porphyrins

(2)

where E0−0 is the S1 energy determined from the fluorescence maximum (611 nm: EGPP and PRPP), e is the charge of the electron, E+ is the oxidation potential of tryptophan (0.65 V vs saturated calomel electrode (SCE)),28 and E− is the reduction potential of EGPP and PRPP (−0.51 V vs SCE for both photosensitizers). The calculated value of −ΔG for EGPP, which is the same value as that for PRPP, is 0.52 eV, indicating that the electron transfer from tryptophan to the S1 of these P(V)porphyrins is thermodynamically possible. The Φf and τf of these P(V)porphyrins are decreased by HSA, supporting electron transfer in the S1 state. The electron transfer rate constant (kET) was roughly calculated from the values of τf of P(V)porphyrins with or without HSA using the following relationship 1 1 kET = − 0 τf ′ τf (3)

Photoexcited P(V)porphyrins (S1) oxidize the tryptophan residue of HSA through electron transfer within the S1 lifetime (∼4 ns). Formation of the charge transfer state (CT) possibly reacts further with water or oxygen molecules, resulting in the formation of decomposed products such as N-formylkynulenine.29,30 However, a large part of the CT is considered to return to the ground state through a reverse electron transfer. The quantum yield of the process of decomposition of the CT state is determined as Φdc. The estimation processes for the quantum yields related to the protein damage are described in a later section. The estimated value of Φdc for PRPP (Table 2) was larger than that for EGPP, suggesting a relatively small efficiency of the reverse electron transfer. The longer axial chain might slightly inhibit the electron transfer. The electron transfer mechanism competes with the following process. The alternate process is intersystem crossing from S1 to T1. In the presence of sufficient molecular oxygen, 1O2 can be generated through energy transfer from T1 of these P(V)porphyrins within the T1 lifetime. The energy transfer rate coefficient from the porphyrin T1 to molecular oxygen decreased during interaction with HSA (Supporting Information). Generated 1O2 oxidizes amino acids

where τf′ and τf0 are the value of τf with HSA and without HSA, respectively. The estimated values of kET were 1.2 × 107 s−1 (EGPP) and 6.0 × 106 s−1 (PRPP). The quantum yield of the electron transfer (ΦET) could be calculated by these values (Table 2). The relatively small value for PRPP suggests that the geometry between the binding PRPP and the tryptophan residue is not favorable compared with that of EGPP. The relatively long chain of PRPP, a propionglycoxyl group, might affect the electron transfer. The lifetime of the triplet excited state (T1) of P(V)porphyrin was determined by a transient absorption technique. The transient absorbance, which is proportional to the 265

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Chemical Research in Toxicology within the lifetime (3.5 μs). In this process, a large part of 1O2 should be deactivated through the physical process. The quantum yield of the reaction between 1O2 and tryptophan is determined as Φreact. Consequently, the above-mentioned Φdamage can be expressed as follows Φdamage = ΦETΦdc + ΦΔΦreact

photosensitized reaction of EGPP and PRPP is physically deactivated under these experimental conditions.

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CONCLUSIONS In summary, P(V)porphyrins bound to HSA and photosensitized damage of its tryptophan residue through electron transfer and 1O2 generation. This study clearly determined the contribution of Type I and Type II mechanisms on the photosensitized protein damage by P(V)porphyrins. This method may be applied to the design of photosensitizers with dual process mechanisms and the clarification of photosensitized reactions. The axial ligand of P(V)porphyrins slightly affected the photodamage of the protein, possibly due to the difference in the binding interaction.

(4)

where ΦETΦdc and ΦΔΦreact indicate absolute quantum yields of HSA damage through electron transfer and that of 1O2, respectively. Therefore, the contribution of protein damage through electron transfer (DET) and 1O2 generation (DSO) (DET + DSO = 100%) can be expressed as follows DET =

ΦETΦdc × 100% Φdamage

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(5)

and DSO =

* Supporting Information

ΦΔΦreact × 100% Φdamage

Experimental details, Job’s plots, photosensitized damage of isolated amino acids, time profile of transient absorption of P(V)porphyrins, single oxygen generation rate coefficient, fluorescence quenching of P(V)porphyrins by NaN3, and singlet oxygen quenching by NaN3. This material is available free of charge via the Internet at http://pubs.acs.org.

(6)

As mentioned above, the electron transfer mechanism competes with the 1O2 mediated oxidation process. The contribution of these mechanisms was estimated from the effect of NaN3 on HSA photodamage (Figure 5). Sodium azide acts as a quencher for photoexcited photosensitizers and as a 1O2 quencher. The fluorescence of these P(V)porphyrins was effectively quenched by NaN3. The quenching rate coefficient (kqf) was estimated from Stern−Volmer plots of fluorescence quenching (Figure S5) and is listed in Table 2. On the other hand, the T1 state of these P(V)porphyrins was hardly quenched by NaN3. Thus, the above-mentioned value of DHSA (Figure 5) could be expressed as follows DHSA = DET ×

1 1 + τf kqf [NaN3]

+ DSO

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81-53-478-1287; E-mail: hirakawa.kazutaka@ shizuoka.ac.jp. Funding

This research was funded by JSPS KAKENHI Grant Number 23750186.

1

Notes

The authors declare no competing financial interest.

1 + τf kqf [NaN3]

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1 1 + τΔkqΔ[NaN3]

ASSOCIATED CONTENT

S

ABBREVIATIONS CT, charge transfer state; DET, protein damage through electron transfer; DHSA, relative HSA damage; DSO, protein damage through 1O2 generation; E0−0, S1 energy; EGPP, bis(ethyleneglycoxy)P(V) tetraphenylporphyrin; E+, oxidation potential; E−, reduction potential; ΔG, Gibbs energy; HSA, human serum albumin; kET, electron transfer rate constant; kqf, quenching rate coefficient of fluorescence by NaN3; kqΔ, quenching rate coefficient of 1O2 by NaN3; LED, light-emitting diode; 1 O 2 , singlet oxygen; P(V)TPP, phosphorus(V) tetraphenylporphyrin; PDT, photodynamic therapy; PRPP, bis(3-hydroxypropoxy)P(V) tetraphenylporphyrin; S1, singlet excited state; SCE, saturated calomel electrode; T1, triplet excited state; Φdamage, quantum yield of HSA damage; Φdc, quantum yield of the decomposition of the CT; ΦET, quantum yield of the electron transfer; Φf, fluorescence quantum yield; Φreact, quantum yield of the reaction between 1O2 and tryptophan; ΦΔ, quantum yield of 1O2 generation; τf, fluorescence lifetime; τT, lifetime of triplet excited state; τΔ, lifetime of singlet oxygen

(7)

where kqΔ is the quenching rate coefficient of 1O2 by NaN3 (Figure S6) (Table 2). The values of DET and DSO could be estimated from analysis of the results of Figure 5 by the leastsquares method. The estimated values of DET were 18% (EGPP) and 15% (PRPP). Similarly, the values of DSO were 82% (EGPP) and 85% (PRPP). Since some 1O2 might be in close proximity to the tryptophan residue and rapidly consumed before quenching by NaN3, the estimated values of DET (18% and 15%) should be the upper limit of the contribution of the electron transfer mechanism under these experimental conditions. These results suggest that 1O2 generation is an important mechanism for the photosensitized reaction of these porphyrins under aerobic conditions. However, the electron transfer reaction should be conserved under hypoxic conditions. The ratio of the contribution of electron transfer mediated protein damage should be changed by the oxygen concentration. In addition, reaction parameters Φdc and Φreact could be also estimated from the above-mentioned values (Table 2). The values of Φdc (0.088 for EGPP and 0.14 for PRPP) suggest that the quantum yields of the reverse electron transfer from the CT to P(V)porphyrins are 0.91 and 0.86 for EGPP and PRPP, respectively. The estimated values of Φreact by EGPP and PRPP are 0.032 and 0.037, respectively. Consequently, these values show that more than 96% of 1 O 2 generated by the

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DOI: 10.1021/tx500492w Chem. Res. Toxicol. 2015, 28, 262−267