Photosensitized damage of protein by fluorinated diethoxyphosphorus(V) porphyrin

Article abstract of DOI:10.1142/S1088424612501258

The effect of the axial ligand fluorination of the water-soluble P(V)porphyrin complex on photosensitized protein damage was examined. The activity of singlet oxygen generation by diethoxyP(V) porphyrin was slightly improved by the fluorination of the ethoxy chains. Absorption spectrum measurements demonstrated the binding interaction between the P(V)porphyrins and human serum albumin, a water-soluble protein. Photo-irradiated P(V)porphyrins damaged the amino acid residue of human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of P(V)porphyrin by human serum albumin supported the electron transfer mechanism. The estimated contributions of the electron transfer mechanism are 0.57 and 0.44 for the fluorinated and non-fluorinated P(V)porphyrins, respectively. The total quantum yield of the protein photo-oxidation was slightly enhanced by this axial fluorination. Copyright

Full text of DOI:10.1142/S1088424612501258

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2013; 17: 56–62
DOI: 10.1142/S1088424612501258
Published at http://www.worldscinet.com/jpp/
Photosensitized damage of protein by fluorinated
diethoxyphosphorus(V)porphyrin
Kazutaka Hirakawa*a◊, Keito Azumia, Yoshinobu Nishimurab, Tatsuo Araib,
Yoshio Nosakac and Segetoshi Okazakid
a Department of Basic Engineering (Chemistry), Faculty of Engineering, Shizuoka University, Johoku 3-5-1,
Naka-ku, Hamamatsu, Shizuoka 432-8561, Japan
b Department of Chemistry, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8571, Japan
c Department of Materials Science and Technology, Nagaoka University of Technology, Kamitomioka 1603-1,
Nagaoka, Niigata 940-2188, Japan
d Medical Photonics Research Center, Hamamatsu University School of Medicine, Handayama 1-20-1,
Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan
Received 22 June 2012
Accepted 20 July 2012
ABSTRACT: The effect of the axial ligand fluorination of the water-soluble P(V)porphyrin complex on
photosensitized protein damage was examined. The activity of singlet oxygen generation by diethoxyP(V)
porphyrin was slightly improved by the fluorination of the ethoxy chains. Absorption spectrum
measurements demonstrated the binding interaction between the P(V)porphyrins and human serum
albumin, a water-soluble protein. Photo-irradiated P(V)porphyrins damaged the amino acid residue of
human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue
of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the
damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein
damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of P(V)porphyrin
by human serum albumin supported the electron transfer mechanism. The estimated contributions of the
electron transfer mechanism are 0.57 and 0.44 for the fluorinated and non-fluorinated P(V)porphyrins,
respectively. The total quantum yield of the protein photo-oxidation was slightly enhanced by this axial
fluorination.
KEYWORDS: P(V)porphyrin, fluorination, photosensitizer, singlet oxygen, electron transfer, protein
oxidation.
1
However, the phototoxic effect of O2 on the PDT is
INTRODUCTION
restricted because the oxygen concentration in a cancer
Porphyrins and their analogues are the most commonly
cell is relatively low [4]. Another important mechanism
administered photosensitizers in the photodynamic
therapy (PDT), which is a promising treatment of
of photosensitized biomolecule damage is the oxidation
reaction through electron transfer (ET) (Type
I
cancer and some non-malignant conditions [1–3]. In
general, administered photosensitizers damage cancer
cells by the generation of singlet oxygen (1O2) (Type II
mechansim), which is formed through energy transfer to
molecular oxygen from the photoexcited photosensitizer.
mechanism), which requires absolutely no oxygen [5].
The ET mechanism requires highly oxidative activity (a
lower reduction potential) in the photoexcited state of the
photosensitizer. Larger excitation energy is advantagous
for the lower reduction potential of the photosensitizer
in the photoexcited state. Ultra-violet photosensitizers
mainly induce biomolecule photodamage through the
ET mechanism, whereas a visible-light photosensitizer
is not appropriate for this mechanism. Therefore, it is
SPP full member in good standing
*Correspondence to: Kazutaka Hirakawa, email: tkhirak@ipc.
shizuoka.ac.jp, tel: +81 53-478-1287, fax: +81 53-478-1287
Copyright © 2013 World Scientific Publishing Company

PHOTOSENSITIZED DAMAGE OF PROTEIN BY FLUORINATED DIETHOXYPHOSPHORUS(V)PORPHYRIN
57
Cl-
Cl-
N
N
N
N
+
+
F3CH2CO
P
OCH2CF3
H3CH2CO
P
OCH2CH3
N
N
N
N
EtPP
Fig. 1. Structures of FEtPP (left) and EtPP (right)
FEtPP
important to select the appropriate molecular design
to achieve ET-mediated biomolecule damage using a
visible-light photosensitizer. Since high-valent porphyrin
complexes demonstrate a lower reduction potential in
their photoexcited state than free-base or low-valent
metal complexes, these porphyrins are advantageous for
the oxidative ET reaction [5–13]. Indeed, derivatives of
high-valent porphyrin complexes, such as P(V) [5, 9]
and Sb(V) [13] complexes, photosensitize DNA damage
through two mechanisms, i.e. 1O2 generation and the ET
reaction. In this study, photosensitized protein oxidation
by a porphyrin P(V) complex (Fig. 1), diethoxyP(V)
tetraphenylporphyrin (EtPP) and its axial fluorinated
compound (FEtPP), was examined. The specific
characteristics of the porphyrin P(V) complexes are the
variety of the substituted axial ligand and the relatively
low redox potential of the one-electron reduction in the
photoexcited state. In addition, P(V)porphyrin is cationic
and water-soluble. The purpose of this study is the
evaluation of a fluorination effect of the axial ligand on
the photosensitized reaction. As a target protein model,
human serum albumin (HSA), a water-soluble protein,
was used, because its structure and property were
elucidated.
a pure product with 78% yield. 1H NMR (CDCl3, TMS):
d, ppm -2.40 ~ -2.29 (4H, m, P-OCH2CH3), -1.74 (6H, td,
JH-H = 6.0 Hz, JP-H = 2.1 Hz, P-OCH2CH3), 7.78 ~ 7.81
(12H, m, meta- and para-H of phenyl group), 7.94 ~ 8.01
(8H, m, ortho-H of phenyl group), 9.07 (8H, d, JH-H
=
2.7 Hz, H). MS (FAB): m/z 733 (calcd. for [M]+ 733).
UV-vis (ethanol): lmax, nm 423.5, 555.0, 594.0.
FEtPP was synthesized by the similar procedure with
that of EtPP. 20 mg of Cl2PP was dissolved in 2 mL of
trifluoroethanol to reflux at 80 °C for 2 h. Solvent was
removed under vacuum. The residue was purified by
column chromatography on silica gel with an eluent of
chloroform-methanol (4/1, vol/vol), resulting in a pure
product with 91% yield. 1H NMR (CDCl3, TMS): d, ppm
-2.05 ~ -1.94 (4H, m, P-OCH2CF3), 7.79 (4H, t, JH-H = 1.8
Hz, para-H of phenyl group), 7.81 (8H, d, JH-H = 1.8 Hz,
meta-H of phenyl group), 7.96 ~ 8.00 (8H, m, ortho-H
of phenyl group), 9.19 (8H, d, JH-H = 3.0 Hz, H). MS
(FAB): m/z 841 (calcd. for [M]+ 841). UV-vis (ethanol):
l
max, nm 428.5, 559.0, 600.0.
HSA was from Sigma-Aldrich Co. LLC. (St. Louis,
MO, USA). Ethanol and sodium azide were from Wako
Chemicals Co.. Deuterium oxide (D2O) was fromAcrross
Organics (New Jersey, USA). Sodium phosphate buffer
(pH 7.6) was from Nacalai Tesque Inc. (Kyoto, Japan).
EXPERIMENTAL
Spectroscopic measurements
The absorption spectrum of P(V)porphyrins and
HSA was measured with a UV-vis spectrophotometer
UV-1650PC (Shimadzu, Kyoto, Japan). The fluorescence
spectra of P(V)porphyrins and HSA were measured with
an F-4500 fluorescence spectrophotometer (Hitachi,
Tokyo, Japan).
Materials
DichloroP(V)tetraphenylporphyrin chloride (Cl2PP)
was obtained by the phosphorus incorporation into
commercially available tetraphenylporphyrin (Wako
Chemicals Co., Osaka, Japan) according to the previous
report [14].
EtPP was synthesized according to the previous
report [6] using the following procedure. 20 mg of Cl2PP
was dissolved in 2 mL of ethanol to reflux at 80 °C for 2
h. Solvent was removed under vacuum. The residue was
purified by column chromatography on silica gel with an
eluent of chloroform-methanol (4/1, vol/vol), resulting in
Detection of damage to HSA photosensitized by P(V)
porphyrins
As a target biomacromolecule, HSA, a water-
soluble protein, was used. The interaction between P(V)
porphyrins and HSA was examined by a UV-vis absorption
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 57–62

58
K. HIRAKAWA ET AL.
measurement. The sample solution containing 8 μM P(V)
porphyrins and 10 μM HSA in a sodium phosphate buffer
(pH 7.6) was irradiated with a light-emitting diode (LED)
(lmax = 519 nm, 1 mW.cm-2, CCS Inc., Kyoto, Japan).
The intensity of the LED light source was measured with
a light meter (LM-331, AS ONE, Osaka, Japan). Protein
damage by P(V)porphyrins was evaluated by measurement
of the fluorescence intensity from the amino acid residues
as previously reported [15]. The excitation and detection
wavelengths were 298 and 350 nm, respectively.
excitation at 410 nm was achieved by using a diode
laser (LDH-P-C-410, PicoQuant, Berlin, Germany)
with a power control unit (PDL 800-B, PicoQuant) in
a repetition rate of 2.5 MHz. The temporal profiles of
fluorescence decay were detected by using a micro-
channel plate photomultiplier (R3809U, Hamamatsu
Photonics) equipped with a TCSPC computer board
module (SPC630, Becker and Hickl Gmbh, Berlin,
Germany). The full-width at half-maximum (FWHM) of
the instrument response function was 51 ps. The values
of c2 and the Durbin-Watson parameters were used to
determine the quality of the fit obtained by nonlinear
regression.
Detection of singlet oxygen
1
The O2 generation was directly measured by near-
1
infrared luminescence at around 1270 nm from O2,
1
which corresponds to the O2(1Dg)–3O2(3Sg-) transition.
RESULTS AND DISCUSSION
The procedure is the same as that described in an earlier
report [16]. The quantum yield of 1O2 generation (FD) was
estimated from the comparison of the emission intensity
with that of the reference photosensitizer, methylene blue
(FD = 0.52 in H2O) [17].
Interaction between P(V)porphyrins and HSA
In the presence of HSA, the hyperchromic effect and
red-shift were observed in the UV-vis absorption spectra
of FEtPP (Fig. 2), indicating the static interaction between
FEtPP and the protein. The analysis of the absorption
spectrum suggests the 4:1 complex formation between
both P(V)porphyrins and HSA (inset of Fig. 2). Similar
results were observed in the case of EtPP. Job’s plot of the
absorption change showed the intersection points at ca.
0.2 in the both cases of FEtPP and EtPP, supporting the
0.8:0.2 (= 4:1) complex formation (Fig. 3). The apparent
association constant (Kap) between P(V)poprhyrins and
HSA was evaluated under an assumption of the following
equation:
1
The kinetics of O2 generation and its decay were
examined by the time-resolved near-infrared emission
measurement. The sample solutions of 2 mL contained
8 μM P(V)porphyrins with or without HSA in a sodium
phosphate buffer (pH 7.6). The excitation light was the
second harmonic (532 nm) of a pulsed Nd:YAG laser (5
ns, 10 Hz, Minilite-II, Continuum, CA, USA). The beam
was passed through a set of dielectric multilayer film
mirrors to eliminate stray light and irradiate from the 45°
direction of the surface of a 1 cm × 1 cm × 4.5 cm quartz
cell. The emission from the front surface of the sample cell
was collected with a set of quartz lenses, passed through
a cold mirror (CLDM-50S, Sigma Koki, Tokyo, Japan),
separated by a Bosch-Lomb Shimadzu monochromator,
and then introduced into a photomultiplier (R5509–41,
Hamamatsu Photonics, Hamamatsu, Japan), which was
cooled to 200 K with liquid nitrogen. The signal from the
photomultiplier was amplified by 75 with an amplifier
(SR-455, Stanford Research, CA, USA) and then counted
with a scaler/averager (SR430, Stanford Research). By
changing the wavelength, the luminescence intensity
showed a maximum at 1270 nm, confirming the detection
of the phosphorescence of 1O2. To analyze the time profile
[P(V)porphyrin-HSA]
Kap
=
(1)
[P(V)porphyrin][HSA]b
1
of O2 emission, the signal obtained at 1270 nm was
accumulated for 20,000 scans with a bin width of 40 ns.
Electrochemical measurements
The redox potentials of P(V)porphrins were measured
with a differential pulse voltammometry (Hokuto Denko,
Tokyo, Japan) using a platinum working electrode, a
platinum counter electrode, and a saturated calomel
reference electrode (SCE) in acetonitrile.
Fig. 2. Absorption spectra of FEtPP in the presence of HSA.
The sample solution contained 8 μM FEtPP and HSA (1, 2, 5,
10, or 20 μM) in a 10 mM sodium phosphate buffer (pH 7.6).
The inset indicates the relationship between the absorbance of
P(V)porphyrin at 440 nm and the concentration of HSA. The
intersection point of two asymptotes indicates almost 2 μM
of HSA, suggesting the 4:1 complex formation between P(V)
porphyrin and HSA
Fluorescence lifetime measurements
Fluorescence decay was measured using a time-
correlated single-photon counting method [18]. Laser
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 58–62

PHOTOSENSITIZED DAMAGE OF PROTEIN BY FLUORINATED DIETHOXYPHOSPHORUS(V)PORPHYRIN
59
Fig. 4. Time course of HSA damage photosensitized by FEtPP
and EtPP. The sample solution contained 8 μM P(V)porphyrins
and 10 μM HSA in a 10 mM sodium phosphate buffer (pH 7.6).
The vertical axis “[HSA]” indicates the relative concentration
of non-damaged HSA
Fig. 3. Job’s plots of the Soret band peak of P(V)porphyrins
with HSA. The sample solution contained 0 ~ 10 μM FEtPP or
EtPP and 0 ~ 10 μM HSA in a 10 mM sodium phosphate buffer
(pH 7.6). The total concentration of P(V)porphyrin and HSA
was 10 μM
where [P(V)porphyrin] is the concentration of the non-
binding photosensitizer, FEtPP or EtPP, [HSA]b is the
concentration of the binding sites of HSA without a
binding porphyrin (four times the actual concentration of
freeHSA), and[P(V)porphyrin-HSA]istheconcentration
of the HSA-binding photosensitizer. The estimated values
of Kap were 4.6 × 104 M-1 and 2.6 × 104 M-1 for FEtPP
and EtPP, respectively. The affinity between FEtPP and
HSA is slightly larger than that of EtPP.
Fig. 5. Effect of sodium azide (NaN3) on HSA photo-oxidation
by FEtPP. The sample solution contained 8 μM FEtPP, 10 μM
HSA and indicated concentration of NaN3 in a 10 mM sodium
phosphate buffer (pH 7.6). The vertical axis “[HSA]” indicates
the relative concentration of non-damaged HSA
Photosensitized damage of HSA by P(V)porphyrins
The intensity of HSA fluorescence around 350 nm,
assigned to the tryptophan residue, was decreased by
photo-irradiation in the presence of these P(V)porphyrins.
The fluorescence decrement of HSA can be explained by
the amino acid oxidation through the photosensitized
reaction [15]. The observed extent of this HSA damage
by the fluorinated P(V)porphyrin, FEtPP, was almost the
same as that of the EtPP (Fig. 4). The quantum yields
of tryptophan degradation photosensitized by P(V)
porphyrins for 120 min irradiation were estimated from
the decrease of the tryptophan fluorescence and the
absorbed photon number by the porphyrins.The estimated
yields were 2.9 × 10-5 and 2.2 × 10-5 for FEtPP and EtPP,
respectively. The quantum yield of HSA photodamage by
FEtPP was slightly larger than that of EtPP.
suggest that the ET mechanism is partly responsible for
1
HSA photodamage, as is the O2 mechanism. Because
1
the almost all O2 can be quenched by 10 mM sodium
azide, the damage of HSA photosensitized by P(V)
porphyrins with 10 mM sodium azide should be due to
the ET mechanism. The quenching rate coefficient of 1O2
by sodium azide is almost diffusion control limit (kdif),
which is calculated as follows:
8000RT
kdif
=
(2)
3η
where R is the gas constant, T is the absolute temperature,
and h is the viscosity of water (8.91 × 10-4 kg.m-1.s-1). The
quenching efficiency of 1O2 by sodium azide (Efq) can be
calculated from the following equation using the lifetime
of 1O2 (tD = 3.5 μs , described in latter):
This HSA damage was partially inhibited by
1
sodium azide, a physical quencher of O2 [19] (Fig. 5).
Furthermore, HSA damage was enhanced in D2O (data
1
not shown), in which the lifetime of O2 is markedly
elongated (about 2 ~ 4 μs in H2O to 70 μs in D2O) [20].
These findings suggest HSA oxidation by 1O2. However,
HSA damage was not completely inhibited by an excess
amount of sodium azide (~10 mM). These results
kq[NaN3 ]
Efq =
(3)
kq[NaN3 ]+1/ τD
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 59–62

60
K. HIRAKAWA ET AL.
where [NaN3] is the concentration of sodium azide. In the
presence of 10 mM sodium azide, the Efq becomes 0.996.
The roughly estimated contributions of the HSA damage
through the ET mechanism for 120 min irradiation were
0.57 and 0.44 for FEtPP and EtPP, respectively. Therefore,
the contributions of the 1O2 mechanism are 0.43 and 0.56
for FEtPP and EtPP, respectively. The ET mechanism
was slightly enhanced in the case of fluorinated P(V)
porphyrin.
The HSA damage was not observed under anaerobic
conditions. The formed radical cation of the amino acid
residue through the ET should undergo a reaction with the
surrounding elements, such as molecular oxygen or water.
Furthermore, re-oxidation of the reduced photosensitizer,
which is formed thorugh ET from the amino acid residue
to the photoexcited photosensitizer, is important. In vivo,
oxidative agents, such as metal ions, might oxidize the
reduced photosentizer. In in vitro experiments, molecular
oxygen is an important oxidative agent to remove the
electron from the reduced photosensitizer. The rapid
reverse-ET should inhibit the following reactions in
simple aqueous solution without oxygen. These results
showed that the following reaction with molecular
oxygen is necessary for protein oxidation through ET in
this experimental condition. Formed superoxide through
re-oxidation of the reduced photosensitizer should be
dismutated into hydrogen peroxide and decomposed
into water and molecular oxygen. The electron, which
is removed from the photosensitizer, should be used
to form hydroxide ion or final product of decomposed
amino acid.
1
Fig. 6. Near-infrared emission spectra of O2 generated by the
photosensitization of FEtPP and EtPP. The sample solution
contained 8 μM FEtPP or EtPP in a 10 mM sodium phosphate
buffer (pH 7.6)
Table 1. Singlet oxygen quantum
yields and the related time constants
Porphyrin
FD
tD (μs) tT (μs)
FEtPP
EtPP
0.68
0.59
3.5
3.5
1.8
1.9
The sample solution contained 8 μM
P(V)porphyrins in a 10 mM sodium
phosphate buffer (pH 7.6).
Singlet oxygen generation by the photosensitized
reaction of P(V)porphyrins
where I0 is the pre-exponential factor, td is the decay
time constant of the emission, and tr is the rise time
constant of this emission. When the tD is longer than tr, td
corresponds to tD. In general, tr equals to tT because the
T1 is dominantly quenched by O2 molecules. In contrast,
tr indicates tD, if tT is longer than tD. The analysis of the
1
The photosensitized O2 generation by these P(V)
porphyrinswasconfirmedbythedetectionofnear-infrared
emission around 1270 nm (Fig. 6), which is assigned to
-
the 1O2(1Dg)–3O2(3Sg ) transition. The estimated FD values
for FEtPP and EtPP were 0.68 and 0.59, respectively.
The fluorination of this porphyrin slightly improved the
1O2 generating ability. These relatively large values of
FD indicate that the 1O2 mechanism is also important for
photosensitized biomolecule damage in the presence of a
sufficient concentration of molecular oxygen.
1
time-resolved O2 emission gave the kinetic parameters,
as shown in Table 1. The observed value of td, around 3.5
μs, almost coincided with the typical lifetime of 1O2 (tD)
in H2O (2 ~ 4 μs) [20]. The values of tr should correspond
to the tT in H2O. The estimated tT values indicate that 1O2
is generated within about two μs in the photochemical
process of P(V)porphyrin in a phosphate buffer.
Time profile of 1O2 emission and estimated lifetime of
the triplet excited state
Redox potentials of P(V)porphyrins
The lifetime of the 1O2 (tD) and the triplet excited state
(T1) of these photosensitizers (tT) was estimated from
the time-resolved emission of 1O2 (data not shown). The
emission intensity of O2 as a function of time, I(t), can
be expressed with the following equation [21]:
The reversible reduction peak was observed for
both P(V)porphyrins (FEtPP: -0.40 V vs SCE; EtPP:
-0.30 V vs SCE) (Table 2). The oxidation potentials of
P(V)porphyrin (Eox) were roughly estimated from the
reduction potential (Ered) and the energy of the singlet
excited state (S1) of P(V)porphyrins (ES1). The ES1 was
calculated from the fluorescence maximum of P(V)
porphyrins. The redox potential of EtPP was smaller
1













1
t

(4)
I(t) = I exp -
-exp -


0 



τ
τr
d 

Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 60–62

PHOTOSENSITIZED DAMAGE OF PROTEIN BY FLUORINATED DIETHOXYPHOSPHORUS(V)PORPHYRIN
61
Table 2. Redox potential and the parameter
Table 3. Fluorescence lifetime of P(V)porphyrins with
about the electron transfer oxidation
or without HSA
Porphyrin
Ered (V)
Eox (V)
-DG (eV)
Porphyrin
FEtPP
HSA
tf (ns) [fraction]
4.43 [1.000]
4.08 [0.449]
FEtPP
EtPP
-0.40
-0.30
1.63
1.73
0.98
1.05
without
+ 5 μM
0.81 [0.551]
0.90 [0.586]
0.94 [0.608]
2.37 [0.215]
1.43 [0.366]
1.39 [0.421]
1.33 [0.444]
+ 10 μM 4.07 [0.414]
+ 20 μM 4.02 [0.392]
The Eox was calculated from the value of Ered and
the wavelength of fluorescence maximum. The
-DG was estimated from the excitation energy of
P(V)porphyrins and the redox potentials of P(V)
porphyrins and tryptophan.
EtPP
without
+ 5 μM
4.95 [0.785]
4.68 [0.634]
+ 10 μM 4.61 [0.579]
+ 20 μM 4.48 [0.556]
than that of FEtPP, suggesting that the axial ligand
fluorination does not increase the electron affinity of the
P(V)porphyrin ring.
The free energy change (-DG) for the ET oxidation
of the tryptophan residue by the photoexcited P(V)
porphyrins was roughly calculated from the following
equation [22]:
The sample solution contained 8 μM P(V)porphyrins
with or without HSA in a 10 mM sodium phosphate
buffer (pH 7.6). Ex = 410 nm. Em = 630 nm.
respectively. These results indicated that a part of P(V)
porphyrin itself is decomposed through photosensitized
reaction.
-DG = ES1 - e(Eox′ - Ered)
(5)
where e is the electronic charge and Eox′ is the oxidation
potential of the amino acid. The oxidation potential of
tryptophan is almost 0.65 V vs. SCE under the similar
conditions of this study [23]. Because the charge of
the P(V) porphyrin is neutralized by the ET, the factor
of the distance between the ET donor and acceptor is
negligible [8]. The estimated values of -DG (Table 2)
suggest that the oxidation of the tryptophan residue of
HSA through the ET by the photoexcited FEtPP and
EtPP is possible.
CONCLUSION
In conclusion, P(V)porphyrins, FEtPP and EtPP, could
induce protein photodamage through 1O2 generation
and the ET mechanism. O2 generation is a well-known
1
mechanism for porphyrin photosensitization [24, 25].
The ET mechanism is hardly observed in the case of
protein or DNA damage by a visible-light photosensitizer
[5]. The time-resolved fluorescence study suggests that
the electron abstraction from the tyrptophan residue
to the S1 of P(V)porphyrins contributes to the ET
mechanism of HSA photodamage. The radical cation
of the tryptophan residue formed through ET should
undergo further reaction with the surrounding elements,
such as water and oxygen. An oxidized product, such
as N-formylkynurenine, should be finally formed [26].
Since HSA damage was not observed under anaerobic
conditions, the final protein damage depends on
the oxygen under this experimental condition. The
fluorination of the axial ligand of P(V)porphyrin slightly
improved the FD value. The total quantum yield of the
protein photodamage was slightly enhanced through
this fluorination of the axial ligand of P(V)porphyrin.
FEtPP and EtPP should have ability to induce DNA
Fluorescence quenching of P(V)porphyrins by HSA
The fluorescence lifetime (tf) of P(V)porphyrins
with or without HSA was summarized in Table 3. The
time-resolved fluorescence intensity could be fitted by
a single exponential function in the FEtPP case. The
double exponential function was well-fitted in the case
of EtPP, suggesting the conformation difference. In the
presence of HSA, the decay curves could be fitted by the
double exponential function for the cases of both P(V)
porphyrins, indicating that the microenvironment of
porphyrins is affected through the interaction of HSA.
The value of tf was decreased by the interaction with
HSA, supporting the ET reaction between amino acid
and the S1 of P(V)porphyrins.
1
photodamage. The ET and O2 generation selectively
cause guanine-specific oxidation. Indeed, we have
previously reported the guanine-specific damage by other
Photostability of P(V)porphyrins
P(V)porphyrin,
dihydroxoP(V)tetraphenylporphyrin
The stability of these P(V)porphyrins during the
photosensitized reaction was checked by UV-vis
absorption measurements. The Soret band absorbance of
P(V)porphyrins was decreased by photo-irradiation (lmax
= 519 nm, 1 mW.cm-2, 120 min) in the presence of 10
μM HSA as follows: 5% and 8% for FEtPP and EtPP,
(OHPP) [9]. The values of FD of FEtPP and EtPP are lager
than that of OHPP (0.28). Furthermore, the values of Ered
of FEtPP and EtPP are higher than that of OHPP (-0.5 V).
Therefore, we can speculate that FEtPP and EtPP induce
severe DNA photodamage at guanine residues compared
with the previous reported P(V)porphyrin.
Copyright © 2013 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2013; 17: 61–62

62
K. HIRAKAWA ET AL.
Acknowledgements
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The authors thank Dr. Jotaro Nakazaki and Professor
Hiroshi Segawa (The University of Tokyo) for
measurement of the redox potential. This work was
supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of the Japanese Government.
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