Photophysics and photochemistry of the UV filter kynurenine covalently attached to amino acids and to a model protein

Article abstract of DOI:10.1021/jp104485k

The photophysics and photochemistry of kynurenine (KN) covalently bound to the amino acids lysine, cysteine, and histidine, the antioxidant glutathione, and the protein lysozyme have been studied by optical spectroscopy with femto- and nanosecond time res

Full text of DOI:10.1021/jp104485k

J. Phys. Chem. B 2010, 114, 11909–11919
11909
Photophysics and Photochemistry of the UV Filter Kynurenine Covalently Attached to
Amino Acids and to a Model Protein
Peter S. Sherin,*,†,‡ Jakob Grilj,‡ Lyudmila V. Kopylova,† Vadim V. Yanshole,†
Yuri P. Tsentalovich,† and Eric Vauthey‡
International Tomography Center SB RAS, Institutskaya str. 3a, 630090 NoVosibirsk, Russia, and Department
of Physical Chemistry, UniVersity of GeneVa, quai Ernest-Ansermet 30, CH-1211 GeneVa, Switzerland
ReceiVed: May 17, 2010; ReVised Manuscript ReceiVed: August 3, 2010
The photophysics and photochemistry of kynurenine (KN) covalently bound to the amino acids lysine, cysteine,
and histidine, the antioxidant glutathione, and the protein lysozyme have been studied by optical spectroscopy
with femto- and nanosecond time resolution. The fluorescence quantum yield of the adducts of KN to amino
acids is approximately 2 times higher than that of the free KN in solution; KN attached to protein exhibits
a 7-fold increase in the fluorescence quantum yield. The S1 state dynamics of KN-modified lysozyme reveals
a multiphasic decay with a broad dispersion of time constants from 1 ps to 2 ns. An increase of the triplet
yield of KN bound to lysozyme is also observed; the triplet state undergoes fast intramolecular decay. The
obtained results reveal an increase of the photochemical activity of KN after its covalent attachment to amino
acids and proteins, which may contribute to the development of oxidative stress in the human lensessthe
main causative factor for the cataract onset.
Introduction
modified by KN and its derivatives has been reported.21,22 The
underlying mechanism, however, remains unclear. Thus, the
Our eyes are protected from UV irradiation by low-molecular-
weight molecules contained in the lens, kynurenine (KN) and
its derivatives.1-4 These natural metabolites of the amino acid
tryptophan absorb UV light in the 300-400 nm region and
exhibit low photochemical activity,5-8 which allows the function
of kynurenines as UV filtering molecules, protecting the eye
tissues from the harmful UV irradiation. The most abundant
UV filters in the lens are (in decreasing order): 3-hydroxy-
kynurenine O-ꢀ-D-glucoside (3OHKG), 4-(2-amino-3-hydroxy-
phenyl)-4-oxobutanoic acid O-ꢀ-D-glucoside (AHBG), kynure-
nine (KN), and 3-hydroxykynurenine (3OHKN).
photochemical reactions of KN-modified lens proteins may
accelerate the aging processes and give a contribution to the
cataract development.
Recently, we reported a detailed study of the photophysics
of KN.23 It has been revealed that the lifetime of the singlet
excited state S1 (τs) is highly sensitive to the nature of the local
environment: in aqueous solutions, τs is about 30 ps, it increases
in alcohols by an order of magnitude, and it exceeds 1 ns in
the aprotic solvents dimethylformamide (DMF) and dimethyl
sulfoxide (DMSO). The change of the medium character from
protic to aprotic also leads to a tremendous increase of the triplet
quantum yield (from 1.7% in aqueous solution to 35% in
DMSO).23 It has been shown that solvent-assisted internal
conversion (IC) is the main deactivation channel of the S1 state
in protic solvents: the rate constant of the fast radiationless S1
f S0 transition is determined by the ability of the solvent to
form hydrogen bonds with KN. This may explain the increased
photoactivity of protein-bound UV filters in comparison to the
free ones: in the hydrophobic environment of a protein, the IC
rate constant decreases, so the lifetime of the singlet excited
state and the yield of the chemically active triplet state increase.
The latter can be readily quenched by oxygen, by the antioxidant
ascorbate, and by the amino acids tryptophan and tyrosine.24
The peroxides and the products of tyrosine oxidation, observed
after UV irradiation of UV filter-modified lens proteins,21,22 may
originate from the reactions of triplet UV filters with molecular
oxygen and tyrosine residues of lens proteins.
Kynurenine-based UV filters (3OHKG, KN, and 3OHKN) are
thermally unstable molecules under physiological conditions:9,10
the spontaneous fragmentation (deamination) leads to the
formation of R,ꢀ-unsaturated compoundsscarboxyketoalkenes
(CKAs)10-12swhich can covalently bind to nucleophilic residues
of the lens proteinsscysteine (Cys), histidine (His), and lysine
(Lys).10,13-16 A covalent attachment of the UV filters to the lens
proteins has been shown to be one of the numerous age-related
post-translational modifications of the lens proteins.9,11,13 CKAs
can also react with the reduced gluthatione (GSH), forming the
GSH-KN, GSH-3OHKN, and GSH-3OHKG adducts.10,14,17 With
aging, the concentration of free UV filters in the lens decreases,
whereas the levels of GSH-bound and protein-bound kynurenines
increase.18 These processes can increase the susceptibility of
the lens tissue to UV irradiation: the KN adducts absorb in the
UV-A wavelength region as well as the free UV filters, but they
are photochemically more active than the parent compound KN:
in a neutral aqueous solution, the adducts are more fluorescent15,19
and exhibit higher triplet yields.20 The generation of reactive
oxygen species (ROS) upon aerobic photolysis of lens proteins
At present, there is no report on the photophysics and
photochemistry of the lens proteins modified by UV filters and
the present work is a first step toward this. The main goal of
this work is to examine the photochemical activity of KN
covalently bound to biologically relevant molecules: (a) to the
amino acids cysteine (Cys-KN), histidine (His-KN), and lysine
(Lys-KN), (b) to the antioxidant glutathione (GSH-KN), which
is present in the human lens,18 and (c) to a protein molecule.
* Corresponding author. Phone: +7-383-330-31-36. Fax: +7-383-333-
13-99. E-mail: Sherin@tomo.nsc.ru, Petr.Sherin@unige.ch.
† International Tomography Center SB RAS.
‡ University of Geneva.
10.1021/jp104485k  2010 American Chemical Society
Published on Web 08/19/2010

11910 J. Phys. Chem. B, Vol. 114, No. 36, 2010
Sherin et al.
CHART 1
Hen egg white lysozyme (HEWL) was used as a model protein
for this purpose. The chemical structures of Cys-KN, His-KN,
Lys-KN, and GSH-KN are presented in Chart 1.
water (pH 6.5) for 6 h at 4 °C, and finally was lyophilized and
stored at 4 °C.
For the in-solution tryptic digests, 5 µg of HEWL and
m-HEWL were dissolved in 10 µL of 8 M urea and incubated
for 1 h at 60 °C. Afterward, 5 µL of 20 mM DTT was added
and incubated for 30 min at 60 °C. To protect reduced cysteines
from forming disulfide bonds over again, 5 µL of 50 mM
iodoacetamide was added to the solution and stored at room
temperature in the dark for 30 min. Before introducing trypsin
to samples, 2 µL (0.5 mg/mL) of the enzyme was dissolved in
118 µL of 40 mM ammonium bicarbonate buffer. Then, 60 µL
of the buffered trypsin solution was put into the HEWL and
m-HEWL solutions and incubated overnight at 37 °C.
HPLC. Semipreparative HPLC separations were performed
with the use of an Agilent LC 1100 chromatograph equipped
with a UV-vis detector. Separations were carried out on a 9.4
× 250 mm ZORBAX Eclipse XBD-C18 column using an
acetonitrile/0.05% (v/v) TFA in H2O gradient. The adduct
separations were performed with the gradients 0% (0-5 min),
0-50% (5-25 min), 50-100% (25-26 min), and 100%
(26-30 min); the flow rate was 3.0 mL/min. CKA separations
were performed with the gradients 0% (0-3 min), 0-30% (3-5
min), 30-55% (5-35 min), 55-100% (35-37 min), and 100%
(37-40 min) at a flow rate of 1.0 mL/min.
Mass-Spectrometry Analysis. The mass spectra were ob-
tained with a MALDI-TOF/TOF spectrometer Ultraflex III
(Bruker Daltonics, Germany). In all experiments, a standard
MTP ground steel plate (Bruker Daltonics, Germany) was used.
The mass spectra of HEWL and m-HEWL were recorded in
linear positive mode in the 3000-20000 m/z range; the mixture
of 0.5 µL of concentrated and purified by Zip Tip pipet tips
(Millipore, USA) protein solution and 0.5 µL of sinapinic acid
solution (70% acetonitrile, 0.1% TFA) was dried under air. The
mass spectra of HEWL and m-HEWL tryptic digests were
recorded in reflective positive mode in the 500-4200 m/z range;
the mixture of 0.5 µL of concentrated and purified by Zip Tip
pipet tips protein digests and 0.5 µL of R-cyano-4-hydroxycin-
namic acid (70% acetonitrile, 0.1% TFA) was dried in the air.
MS/MS spectra of the peptides were recorded from the same
spots on the plate as for MS spectra using the LIFT technology
of the spectrometer; briefly, the daughter ions were generated
using the increase of kinetic energy of parent ions with the
subsequent fragmentation. First, the parent ion was chosen from
Experimental Methods
Materials. D,L-Kynurenine (KN), D,L-cysteine (Cys), N-
acetyl-L-histidine (His), D,L-lysine (Lys), reduced L-glutathione
(GSH), trifluoroacetic acid (TFA), and quinine bisulfate were
used as received from Sigma/Aldrich. Sinapinic acid and
R-cyano-4-hydroxycinnamic acid were used as received from
Bruker Daltonics, Germany. Hen egg white lysozyme (HEWL)
and dithiothreitol (DTT) were from Helicon, Russia; sequencing
grade modified trypsin from Promega, USA. H2O was doubly
distilled. Deuterated water (D2O), methanol (MeOH), ethanol
(EtOH), N,N-dimethylformamide (DMF), and dimethylsulfoxide
(DMSO) were used as received from Fluka. Acetonitrile (HPLC
grade) was purchased from Cryochrom, Russia, and used as
received.
The KN adducts of Cys, His, Lys, and GSH were synthesized
according to the method described in ref 25: 33.3 mg of
kynurenine sulfate salt were dissolved in 20 mL of phosphate
buffer at pH 9.5, and the amino acids were added in 10-fold
molar excess. The pH was readjusted to 9.5 with NaOH. The
solution, placed in a glass vial, was bubbled with argon, sealed,
and then incubated at 37 °C for 48 h. After adjusting the pH to
4.5 with hydrochloric acid, the reaction mixture was separated
by semipreparative HPLC. The adduct-containing fractions were
collected, lyophilized, and stored at 4 °C.
KN-modified lysozyme (m-HEWL) was prepared in two
steps: first, CKA was synthesized, and second, HEWL was
modified by CKA. For the CKA synthesis, the unbuffered
aqueous KN solution (5 mM) was adjusted to pH 8.3 by
dropwise addition of NaOH. The solution (volume 20 mL) was
placed in a glass vial, bubbled with argon, capped, sealed with
parafilm, and incubated at 70 °C. The pH of the solution was
controlled with an Orion Research pH-meter with a glass
electrode before and after incubation. CKA was isolated from
the reaction mixture using semipreparative HPLC. For HEWL
modification, 28.3 mg of HEWL was added to 11 mL of CKA-
containing fraction; the pH was again adjusted to 8.6. The
reaction mixture was incubated for 51 h at 37 °C, after which
the protein fraction was isolated via dialysis against distilled

Photophysics and Photochemistry of UV Filter KN
J. Phys. Chem. B, Vol. 114, No. 36, 2010 11911
the mass spectrum of the digest. Second, the value of the chosen
ion was declared as a parent mass in LIFT mode, and the MS
spectrum of the isolated parent ion followed by the MS/MS
spectrum of daughter ions was recorded. The detection of
daughter ions was performed with standard built-in parameters;
the registered m/z range was from 40 Da to the parent mass
+20%.
Steady-State UV-vis Measurements. Steady-state absorp-
tion and fluorescence spectra were measured with a Cary 50
(Varian) spectrophotometer and a Cary Eclipse (Varian) fluo-
rometer, respectively. All fluorescence spectra were corrected
for the wavelength-dependent sensitivity of the detection. All
solutions were deoxygenated by argon bubbling for 15 min prior
to measurements, capped, and sealed with parafilm. The
fluorescence quantum yields were determined relatively to a 1.0
N H2SO4 aqueous solution of quinine bisulfate (fluorescence
quantum yield ΦF ) 0.546 at 365 nm excitation26). For all
measurements, a 10 × 10 mm2 quartz cell was used and the
absorbance of the samples was below 0.1 at the absorption
maximum.
Time-Resolved Fluorescence Measurements. The early
fluorescence dynamics was measured using the fluorescence
upconversion setup described in detail elsewhere.27 Briefly, part
of the output of a mode locked Ti:sapphire oscillator (Spectra
Physics “Tsunami”) was frequency doubled and used to excite
the sample at 400 nm. The fluorescence was gated by sum-
frequency mixing with the fundamental of the oscillator output.
The up-converted UV photons were directed into a monochro-
mator and detected by a photomultiplier tube with photon
counting electronics. The sample solutions were kept in a 1.0
mm thick spinning cell. The concentration was adjusted so that
the absorbance of the sample was around 0.15 at the excitation
wavelength. The full width at half-maximum (fwhm) of the
instrument response function was around 210 fs.
Figure 1. Mass spectra of HEWL and m-HEWL.
150 mJ). The spot size of the laser beam at the cell entrance
was 2.5 × 8 mm2. The monitoring system includes a DKSh-
150 xenon short-arc lamp connected to a high current pulser, a
homemade monochromator, a 9794B photomultiplier (Electron
Tubes Ltd.), and a LeCroy 9310A digitizer. The monitoring
light, concentrated in a 2.5 × 1 mm2 rectangle, passed through
the cell along the front (laser irradiated) window. Thus, in all
experiments the excitation optical length was 1 mm and the
monitoring optical length was 8 mm. All solutions were bubbled
with argon or oxygen for 15 min prior to, and during, irradiation.
Results
Mass-Spectrometry Analysis of m-HEWL. The mass
spectra of lysozyme (HEWL) and KN-modified lysozyme (m-
HEWL) are presented in Figure 1. The strongest signal with
m/z 14313 observed in the m-HEWL spectrum reveals the
presence of unmodified protein in solution; the signals separated
from the main peak by n*191 ( 4 Da correspond to proteins
modified by one (n ) 1), two (n ) 2), and three (n ) 3)
deaminated KN molecules.
Time-Correlated Single Photon Counting (TC-SPC) Mea-
surements. Time profiles longer than 1 ns were measured using
the TC-SPC unit described in ref 28. A pulsed laser diode at
395 nm (Picoquant model LDH-P-C-400B) with a repetition
rate of 20 MHz was used for excitation; a MCP-PMT Hamamat-
su R3809U-51 was used as a photomultiplier. The fwhm of the
instrument response function was around 60 ps. A 10 × 10 mm2
quartz cell was used, and the absorbance at 395 nm was about
0.1.
Transient Absorption (TA) Measurements. The experi-
mental setup for pump-probe transient absorption (TA) has
been described in detail elsewhere.29 Excitation was performed
at 400 nm using the frequency-doubled output of a standard 1
kHz amplified Ti:sapphire system (Spectra-Physics). The pump
intensity on the sample was around 3 µJ. Probing was achieved
with a white-light continuum obtained by focusing a small
fraction of the 800 nm pulses into a CaF2 plate. The polarization
of the probe pulses was at magic angle relative to that of the
pump pulses. All spectra were corrected for the chirp of the
white light probe pulses. The fwhm of the instrument response
function was ca. 200 fs. The sample solutions were placed in a
1 mm thick quartz cell where they were continuously stirred
by N2 bubbling. Their absorbance at the excitation wavelength
was around 0.15. All time-resolved measurements were carried
out at room temperature.
In order to determine the sites of the modification, HEWL
and m-HEWL were subjected to the tryptic digestion. The mass
spectrum of the peptides obtained from m-HEWL is presented
in Figure 2a. The analysis of the spectrum reveals three peaks
with m/z 797, 2528, and 2869, which were not present in the
spectrum of the HEWL digest. These signals are separated by
191 Da from the peaks with m/z 606 (KVFGR), 2337
(NLCNIPCSALLSSDITASVNCAK), and 2678 (GYSLGN-
WVCAAKFESNFNTQATNR) observed in both HEWL and
m-HEWL spectra and correspond to KN-modified peptides. It
should be noted that each mentioned peptide contains a lysine
amino acid residue, a possible site for KN attachment.10,15
Figure 2b depicts the MS/MS spectrum of the m/z 797 signal.
The intense peak with m/z 606 corresponds to the peptide
KVFGR, formed by the loss of the KN fragment. Less intense
MS/MS peaks with m/z 175, 232, 379, and 478 correspond to
y-ions of the KVFGR peptide. The signals from b-ions (m/z
129, 228, 375, 432, 588) are much weaker, which probably
indicates the localization of the positive charge on the C-
terminus of the peptide. The attachment of KN to the lysine
residue (K1 of HEWL) is confirmed by the presence of a signal
with m/z 320 corresponding to a b-ion of K-KN; no signals
from b-ions of KV-KN, KVF-KN, and KVFG-KN fragments
were found in the MS/MS spectrum.
Laser Flash Photolysis (LFP) Measurements. A detailed
description of the LFP equipment has been published earlier.30,31
The solutions, placed in a square cell (inner dimensions 10 mm
×10 mm), were irradiated with a Quanta-Ray LAB-130-10 Nd:
YAG laser (pulse duration 8 ns; 355 nm; pulse energy up to
A similar MS/MS analysis was also performed for the m/z
2869 ion (data not shown). The MS/MS spectrum contains an

11912 J. Phys. Chem. B, Vol. 114, No. 36, 2010
Sherin et al.
Figure 3. UV-vis absorption and fluorescence spectra of (a) Lys-
KN, (b) Cys-KN, (c) His-KN, (d) GSH-KN, and (e) HEWL (dashed
line) and m-HEWL (solid line) in neutral aqueous solution at room
temperature. Red lines, compounds under study; black lines, KN in
neutral aqueous solution.
Figure 2. (a) Mass spectrum of m-HEWL after tryptic digestion. (b)
MS/MS spectrum of the 797 m/z ion of hydrolyzed m-HEWL.
intense signal with m/z 2678 corresponding to the GYSLGN-
WVCAAKFESNFNTQATNR peptide formed by the loss of the
KN fragment. This sequence contains a K-F bond (lysine-
phenylalanine), which was not hydrolyzed by trypsin likely due
to the presence of the KN modification on the lysine residue
(K33 of lysozyme). Furthermore, the signals from y-ions are
present in the spectrum only until m/z 1428, which corresponds
to the fragments of the FESNFNTQATNR peptide up to the
lysine amino acid. Heavier y-ions containing lysine with
probable modification are not detected.
The MS/MS spectrum of the m/z 2528 ion has not been
recorded due to the low intensity of this signal. Summarizing
the mass-spectrometric analysis, at least three positions in
m-HEWL are modified by KN, and at least two of them are
proved to be the lysine residues, namely, K1 and K33.
UV-vis Absorption Spectra and Fluorescence Quantum
Yields. Figure 3a-d shows the UV-vis absorption and
fluorescence spectra of Lys-KN, Cys-KN, His-KN, and GSH-
KN (red lines) and KN (black lines) in neutral aqueous solution,
pH 6.6. The differences in peak positions and spectral shapes
between KN and its adducts are negligible. The spectra obtained
with these compounds in organic solvents (MeOH, EtOH, DMF,
and DMSO) are also very similar to each other (data not shown).
The solvent-induced changes in the optical spectra of the adducts
follow those observed with KN: a weak blue shift of the
absorption band and a relatively large red shift of the fluores-
cence band when passing from aprotic to protic solvents.23 These
observations show a minor influence of the substituent on the
spectral properties of KN.
The fluorescence quantum yields ΦF of Lys-KN, Cys-KN,
His-KN, and GSH-KN in various solvents are summarized in
Table 1. In aqueous solutions, the ΦF values of the adducts
increase in the following order: Lys-KN, Cys-KN, His-KN,
GSH-KN. For the Lys-KN adduct, the ΦF value is similar to
that for KN, whereas for GSH-KN it is twice as high. The
increase of ΦF from Lys-KN to GSH-KN can be observed in
all protic solvents (D2O, MeOH, and EtOH). In the aprotic
solvents DMF and DMSO, the ΦF values of the adducts are
close to those of KN.
Figure 3e shows the UV-vis absorption and fluorescence
spectra of m-HEWL in neutral aqueous solution (pH 5.8). Both
the absorption and fluorescence bands of the protein-bound KN
are shifted with respect to the spectra of KN aqueous solution,
which may be attributed to the less protic and less polar local
environment of the protein in comparison with the bulk solution.
In fact, the m-HEWL fluorescence spectrum in H2O is similar
to the fluorescence spectrum of KN in MeOH,23 which confirms
this suggestion. Besides, the m-HEWL absorption band is
broader than that of KN in MeOH; that agrees with the presence
of several sites of KN attachment to HEWL. The average
number of KNs attached to a protein molecule has been
evaluated in the following way: the concentration of the protein
dissolved in the sample was determined with use of the Bradford
protein assay.32 The concentration of KN in the sample was
estimated by the absorption at 370 nm, assuming the
extinction coefficient of KN attached to the protein to be
the same as that of free KN, ε370 ) 4500 M-1 cm-1.8 The

Photophysics and Photochemistry of UV Filter KN
J. Phys. Chem. B, Vol. 114, No. 36, 2010 11913
TABLE 1: Photophysical Properties of KN and Its Adducts to Lysine (Lys-KN), Cysteine (Cys-KN), Histidine (His-KN), and
Glutathione (GSH-KN) in Various Solvents at Room Temperature*
solvent
compound
KN
ΦF, %
τ1, ps
τ2, ps
τ3, ps
kF, s-1
kNR, s-1
H2O (R ) 1.17)
0.08
0.9a
1.0b
1.1a
1.1b
0.9a
1.3b
1.2a
1.5b
1.3a
1.1b
1.2a
1.4a
1.3b
1.2a
1.8b
1.5a
2.3b
1.7a
1.7b
4.5a
5.4b
4.7a
4.2b
8.5a
9.5b
6.4a
8.5b
7.2a
8.8b
6.6a
8.1a
6.1b
11a
27a
3.0 × 107
3.7 × 1010
26b
Lys-KN
Cys-KN
His-KN
GSH-KN
0.07
0.12
0.15
0.15
28a
2.6 × 107
2.7 × 107
3.1 × 107
3.3 × 107
3.6 × 1010
2.3 × 1010
2.1 × 1010
2.1 × 1010
29b
44a
41b
48a
49b
47a
46b
D2O
KN
Lys-KN
0.14
0.11
43a
3.1 × 107
2.3 × 1010
45a
2.4 × 107
2.2 × 1010
46b
Cys-KN
His-KN
GSH-KN
0.24
0.26
0.24
71a
3.4 × 107
3.1 × 107
3.1 × 107
1.4 × 1010
1.2 × 1010
1.3 × 1010
14b
73b
10a
85a
15b
98b
15a
87a
12b
78b
MeOH (R ) 0.93)
KN
Lys-KN
0.79
0.65
240c
170b
170c
280b
290c
300b
310c
380b
380c
480c
360c
670c
590c
790c
1400c
1350c
1350c
1450c
1050c
2300a
2400b
1850b
1800c
1850b
1850c
1700b
1750c
1500b
1500c
3.3 × 107
4.1 × 109
4.7b
4.6b
5.1b
4.0b
21b
26b
26b
34b
3.8 × 107
5.8 × 109
Cys-KN
His-KN
GSH-KN
1.23
1.10
1.38
4.3 × 107
3.6 × 107
3.6 × 107
4.8 × 107
3.6 × 107
3.7 × 107
3.7 × 107
4.7 × 107
3.8 × 107
3.7 × 107
4.4 × 107
3.9 × 107
5.2 × 107
4.0 × 107
5.1 × 107
5.6 × 107
5.6 × 107
5.3 × 107
3.4 × 109
3.2 × 109
2.6 × 109
2.0 × 109
2.7 × 109
1.5 × 109
1.7 × 109
1.2 × 109
6.8 × 108
7.1 × 108
7.0 × 108
6.6 × 108
9.0 × 108
4.0 × 108
5.0 × 108
4.9 × 108
5.2 × 108
6.2 × 108
EtOH (R ) 0.83)
DMF (R ) 0)
KN
2.3
1.3
2.5
2.2
3.7
5.3
5.0
5.9
5.5
5.5
9.0
Lys-KN
Cys-KN
His-KN
GSH-KN
KN
Lys-KN
Cys-KN
His-KN
GSH-KN
KN
not measured
not measured
not measured
not measured
DMSO (R ) 0)
3.3a
4.3b
5.6b
41a
52b
Lys-KN
Cys-KN
His-KN
GSH-KN
9.3
10.3
9.8
100b
4.3b
8.7b
6.6b
66b
120b
72b
7.8
* R, Kamlet-Taft’s solvatochromic parameter characterizing the hydrogen-bonding ability of the solvent;35,36 τi, time constants obtained from
the fits of (a) fluorescence up-conversion, λex ) 400 nm, (b) TA, λex ) 400 nm, or (c) TC-SPC, λex ) 395 nm, measurements (see the
superscript letters in the table); ΦF, fluorescence quantum yield; kF and kNR: radiative and non-radiative rate constant. Standard error: 10%.
TABLE 2: Fluorescence Properties of m-HEWL in Aqueous Solvents at Room Temperature*
solvent
ΦF, %
τ1, ps
τ2, ps
τ3, ps
τ4, ns
τ5, ns
H2O (pH 5.7)
0.54
0.9 (0.20)a
34 (0.36)a
360 (0.44)a
1.0b
52b
640b
380 (0.72)c
460 (0.57)c
2.0 (0.25)c
2.7 (0.36)c
12 (0.03)c
9.5 (0.07)c
D2O
1.00
* ΦF, fluorescence quantum yield; τi, time constants obtained from fits of (a) fluorescence up-conversion, λex ) 400 nm, (b) TA, λex ) 400
nm, or (c) TC-SPC, λex ) 395 nm, measurements. (See the superscript letters in the table. Data in parentheses show the amplitude factors of
the temporal components.) Standard error: 10%.
concentration ratio [KN]/[m-HEWL] gives a value of 1.6.
Thus, the investigated m-HEWL represents a mixture of
unmodified proteins and proteins mainly modified by one and
two KN molecules, that is in agreement with the mass-
spectrometry data (see Figure 1).
The fluorescence quantum yields of m-HEWL were measured
in nonbuffered H2O and D2O; the obtained values are listed in
Table 2. In aqueous solutions, m-HEWL exhibits a 7-fold higher
fluorescence quantum yield than KN; the measurements in D2O
clearly indicate the presence of an isotopic effect. No differences

11914 J. Phys. Chem. B, Vol. 114, No. 36, 2010
Sherin et al.
Figure 4. (a-c) Intensity normalized time-resolved fluorescence spectra of (a) KN, (b) His-KN, and (c) GSH-KN observed after 400 nm excitation.
(d-f) Decay-associated amplitude spectra obtained from the global analysis of the fluorescence time profiles measured with (d) KN, (e) His-KN,
and (f) GSH-KN. Gray lines: steady-state fluorescence spectra.
in the spectral properties or fluorescence quantum yield of
m-HEWL were observed between buffered and nonbuffered
solutions.
Ultrafast Excited State Dynamics of Lys-KN, Cys-KN,
His-KN, and GSH-KN. The temporal evolution of Lys-KN,
Cys-KN, His-KN, and GSH-KN fluorescence in H2O (pH 6.6)
has been measured at eight equidistant wavelengths from 420
to 580 nm throughout the emission band over different time
windows up to 300 ps. The dynamics for all adducts
qualitatively reproduce the one found for KN: an initial red
shift of the emission band, induced by solvent relaxation (so-
called dynamic Stokes shift), is followed by a monotonic
decay.23 This spectral dynamics can be clearly seen in Figure
4a-c. The main difference in the emission dynamics between
KN and its adducts is a faster signal evolution in the case of
KN (Figure 5a).
The measured fluorescence data were treated by global
analysis using a sum of exponential functions convolved with
a Gaussian-like instrument response function, as described in
detail in refs 33 and 34. For all adducts, a good agreement
between experimental and calculated data was obtained with
three exponentials. The time constants obtained from such
analysis are listed in Table 1. Figure 4d-f shows the wavelength
dependence of the amplitude factors associated with the various
time constants, A1(λ), A2(λ), and A3(λ), obtained from the global
analysis with KN, His-KN, and GSH-KN. Positive values
correspond to a decay, negative ones to a rise. The spectra
associated with the fast components, A1(λ) and A2(λ), exhibit a
similar shape, indicating a decay on the high-energy side and a
rise on the low-energy side. These components can thus be
Figure 5. (a) Fluorescence and (b) TA time profiles recorded with
KN, His-KN, and GSH-KN at the emission maximum, 500 nm, and at
the TA band maximum, 570 nm, correspondingly.

Photophysics and Photochemistry of UV Filter KN
J. Phys. Chem. B, Vol. 114, No. 36, 2010 11915
Figure 6. (a-c) TA spectra recorded at different time delays after 400 nm excitation of (a) KN, (b) His-KN, and (c) GSH-KN. (d-f) Decay-
associated amplitude spectra obtained from the global analysis of the TA spectra of (d) KN, (e) His-KN, and (f) GSH-KN.
attributed predominantly to a dynamic Stokes shift. The
amplitude factors A3(λ) related to the largest time constant
almost coincide with the steady-state emission spectrum (thin
gray line). Thus, the time constant τ3 can be assigned to the
lifetime of the lowest singlet excited state.
TA spectra of KN adducts in aqueous solutions (pH 6.6) were
recorded upon 400 nm excitation, some of them being shown
in Figure 6a-c. As in the case of fluorescence, the TA dynamics
of all adducts are similar to that of KN: a solvent-induced red
shift of the stimulated emission band leads to a blue shift of
the resulting TA band. The disappearance and appearance of
the negative bands around 470 nm and at λ > 600 nm,
respectively, should also be related to the dynamic Stokes shift
of the stimulated emission band. The decay dynamics of the
TA signal at 570 nm are slower for all adducts compared with
KN, and they reproduce well the fluorescence data (Figure 5b).
The same fluorescence upconversion and TA measurements
were carried out in D2O, and TA measurements were also
performed in MeOH and DMSO. The characteristic time
constants obtained from the analysis of the fluorescence and
absorption data as described above are listed in Table 1. In
deuterated water, the excited-state dynamics of all adducts is
slowed down by a factor of approximately 1.5 compared to H2O,
whereas the shapes of the fluorescence and TA spectra remain
the same. These observations indicate a substantial participation
of intermolecular hydrogen bonds in the ultrafast deactivation
of the excited states of the adducts. The spectral evolution of
the TA signals of all adducts in MeOH and DMSO qualitatively
repeats that of KN.23 As in the case of KN, the observed
dynamics with all adducts includes three stages and the
corresponding time constants increase from MeOH to DMSO
(Table 1).
The fluorescence lifetimes of the adducts in MeOH, EtOH,
DMF, and DMSO were obtained using the TC-SPC technique.
The temporal evolution of the emission measured at 500 nm
exhibits an exponential decay in all solvents (data not shown).
The decay times were obtained using the numerical convolution
of the experimental instrument response function with a
monoexponential function. The resulting fluorescence lifetimes
of the adducts in different solvents are assigned to the S1 state
lifetimes (τ3) and are summarized in Table 1. The obtained
lifetimes agree well with the results obtained from the TA
experiments.
A global analysis of the TA spectra was performed with the
sum of three exponential functions and gave the characteristic
times presented in Table 1, which are similar to those obtained
from the fluorescence measurements. The decay associated
spectra, B1(λ), B2(λ), and B3(λ), are shown in Figure 6d-f. The
spectra associated with the fast components, B1(λ) and B2(λ),
are similar to those of the A1(λ) and A2(λ) amplitudes in the
fluorescence measurements but have opposite signs (Figure
4d-f). B3(λ) corresponds to the superposition of the S1 f Sn
absorption and S1 f S0 stimulated emission bands. The negative
feature above 600 nm is due to the red shift of the stimulated
emission. Thus, the TA results are completely consistent with
the time-resolved fluorescence data, and τ3 in Table 1 can be
assigned to the excited state lifetime of the KN adducts.
The obtained ΦF and τ3 values (Table 1) were used to
calculate the rate constants of radiative and nonradiative decay
of the excited state. The kF values amount to about (3-5) ×

11916 J. Phys. Chem. B, Vol. 114, No. 36, 2010
Sherin et al.
Figure 8. TA dynamics of m-HEWL aqueous solution (pH 5.7)
observed after 400 nm excitation. (a) TA spectra recorded at different
time delays after excitation. (b) TA time profiles measured at 510, 570,
and 630 nm; solid lines show best fit; inset shows early dynamics at
the same wavelengths. (c) Decay-associated amplitude spectra obtained
from the global analysis of the TA time profiles.
Figure 7. Fluorescence time profiles of m-HEWL aqueous solutions
(pH 5.7) recorded on (a) picosecond (at the maximum of emission,
480 nm) and (b) nanosecond time scales (at 540 nm). Black lines in
part a: the time profile of KN aqueous solution (pH 6.6) recorded at
the maximum of emission, 500 nm. Insets: the early dynamics at the
same wavelengths.
The measured time profiles were analyzed using the sum of
three exponential functions convolved with a Gaussian-like
instrument response function as described earlier. The best fit
presented by a smooth line in Figure 7a was obtained with the
following time constants: τ1 ) 0.9 ps (amplitude factor 0.20),
τ2 ) 34 ps (0.36), and τ3 ) 360 ps (0.44). Similar time constants
were obtained for the time profiles recorded at 500 and 520 nm
(data not shown). The assignment of the time constants will be
discussed below.
Figure 7b displays the m-HEWL fluorescence dynamics in
H2O (pH 5.7) on a larger time scale recorded at 500 nm using
TC-SPC, whereas the inset presents the early dynamics. The
time profile was analyzed by the convolution of the sum of
exponential functions with the experimental instrument response
function. A good agreement between experimental and calcu-
lated data was obtained with three exponentials. The best fit,
presented by smooth black lines in Figure 7b, was obtained with
the time constants listed in Table 2. These time constants are
designated as τ3, τ4, and τ5, since the fastest component of the
TC-SPC time profile and the slowest component of the
upconversion time profile practically coincide. The fluorescence
time profile of m-HEWL in D2O was obtained in a similar way;
the same fitting procedure gave the time constants summarized
in Table 2.
107 s-1 and depend weakly on the solvent (see Table 1), whereas
the kNR values for KN and adducts increase by 2 orders of
magnitude from DMSO to H2O. The drastic increase of kNR in
protic solvents correlates with the increase of the hydrogen-
bonding ability of these solvents, which can be quantified by
the Kamlet-Taft’s solvatochromic parameter R35,36 shown in
Table 1.
Ultrafast Excited State Dynamics of m-HEWL. The
temporal evolution of m-HEWL fluorescence in aqueous solu-
tion (pH 5.7) has been measured at three wavelengths (480,
500, and 520 nm) over different time windows up to 600 ps.
Figure 7a shows the fluorescence time profiles of m-HEWL and
KN at the maxima of their emission bands, 480 and 500 nm,
respectively. The fluorescence dynamics of m-HEWL is sig-
nificantly slower than that of KN. The early dynamics, displayed
in the inset in Figure 7a, shows that m-HEWL fluorescence
intensity exhibits only a decay, while KN shows a growing
signal due to solvent relaxation. It should be noted that no rising
component was detected for m-HEWL at any wavelength.
Taking into account that the measurements were performed at
wavelengths corresponding to the low-energy side of the
emission band, it might be concluded that the solvent relaxation
dynamics is hidden by a fast process leading to a rapid decay
of a part of the singlet excited state population. Additionally,
solvent relaxation of m-HEWL can be expected to significantly
differ from that of KN in aqueous solution due to the protein
environment.
Figure 8a shows TA spectra of m-HEWL in aqueous solution
(pH 5.7) recorded upon 400 nm excitation. As in the case of
KN and of the adducts, an absorption band with a maximum at
570 nm is immediately present after excitation. The early

Photophysics and Photochemistry of UV Filter KN
J. Phys. Chem. B, Vol. 114, No. 36, 2010 11917
triplet state absorption, which has absorption maxima at 280,
340, and 430 nm,8,37 and of the depletion of the starting material,
which has an absorption maximum at 370 nm (see Figure 3e).
The triplet yield of KN bound to protein was determined
relatively to the free KN in solution. The initial TA values at
430 nm, ∆A430, were measured for aqueous KN and m-HEWL
solutions with the same optical densities at 355 nm (excitation
wavelength) at different laser energies. The triplet yield of
m-HEWL, ΦT ) 3.4 ( 0.5%, was calculated from the ratio of
the slopes of the linear fit of ∆A430 laser pulse energy
dependencies (see the Supporting Information) and using KN
triplet yield as a standard (ΦT ) 1.8%).8
T
The intramolecular character of KN quenching stimulated
the search for probable quenchers among the aromatic amino
acid residues present in lysozyme. The most probable quenchers
of triplet KN are tryptophan and tyrosine residues, both of which
being able to quench TKN via electron transfer reaction.24 As a
consequence, the absorption band with a maximum at 330 nm,
observed 4 µs after laser pulse excitation of m-HEWL in
aqueous solution (Figure 9), can be attributed to the absorption
of Trp• radical (absorption bands at 330 and 510 nm)38-40 formed
upon the deprotonation of the TrpH•+ radical (pKa ) 4.3,38
absorption bands at 330 and 570 nm38,39,41). However, we could
not observe any growing transient absorption signals corre-
sponding to the radical formation at 510 nm (the maximum of
Trp• radical) or at 410 nm (the maximum of Tyr• radical),42,43
probably because of the overlap of radical absorption with the
Figure 9. Transient absorption spectra detected 80 ns (filled squares)
and 4 µs (open circles) after the laser pulse irradiation (355 nm, 70
mJ) of m-HEWL in argon saturated aqueous solution (pH 5.8) in the
presence of 0.01 M acetone. Insets show the time profiles at 340 and
430 nm.
dynamics includes a fast decay and a minor blue shift of the
absorption band (Figure 8a,b). The subsequent dynamics exhibits
a monotonic decay with negligible changes in the spectral shape.
The absence of negative features over the whole spectral window
should be noted.
T
Global analysis of the TA dynamics was performed using
the sum of three exponential functions, the best fit being obtained
with time constants: τ1 ) 1.0 ps, τ2 ) 52 ps, and τ3 ) 640 ps.
The decay associated spectra, B1(λ), B2(λ), and B3(λ), are
displayed in Figure 8c. All amplitudes show positive values over
the whole spectral region; a blue shift of B3(λ) relative to B1(λ)
and B2(λ) can be observed. Interestingly, the fastest component,
B1(λ), gives the largest contribution to the observed dynamics
(Figure 8c). The τ1 values obtained from fluorescence and TA
measurements coincide, but the τ2 and τ3 values exhibit a
significant difference. Probably, this difference can be attributed
to the weakness of the TA signal in the time range t > 100 ps,
where the low signal/noise ratio results in a lower accuracy of
the time constant.
Laser Flash Photolysis of m-HEWL. Figure 9 shows TA
spectra obtained upon 355 nm photolysis of aqueous m-HEWL
solution (1.3 × 10-4 M, pH 6.4) under argon 80 ns and 4 µs
after the laser flash. The TA dynamics was recorded with a laser
pulse energy of 70 mJ in order to minimize a contribution from
the KN biphotonic ionization.37 To exclude a contribution from
the solvated electron, the well-known electron scavenger acetone
was added to the solution. Immediately after the laser pulse, a
positive TA band with the maximum at 430 nm and a negative
TA band with the maximum at 360 nm are observed. The
subsequent dynamics consists of a monotonic decay of the TA
signal below 320 nm and above 380 nm, and of the build-up of
a positive TA band with a maximum at 340 nm. The TA signal
evolution over the whole wavelength region occurs with a rate
constant of (5 ( 1) × 105 s-1, and shows no dependence on
the laser energy and concentration of m-HEWL, indicating an
intramolecular character of the reaction. In the presence of
oxygen, the decay of the TA signal is significantly accelerated
and, after the completion of the signal decay, no absorption was
observed over the whole wavelength region (data not shown).
These observations allow an assignment of the positive absorp-
tion observed immediately after the laser flash to the triplet state
of KN bound to the protein. Thus, the TA spectrum observed
immediately after the laser pulse is a superposition of the KN
broad absorption band of KN.
Analysis of a m-HEWL solution after irradiation revealed
the presence of 4-hydroxyquinoline (4HQN) and KN yellow,
the products of KN photochemical and thermal degradation.11,12,44
Thus, one of the possible channels of m-HEWL photodecom-
position is the detachment of KN followed by cyclization with
the formation of 4HQN and KN yellow. The detailed mechanism
of these processes is unclear and can include both thermal and
photochemical stages, including biphotonic reactions.
Discussion
Photophysics of the KN Adducts. The fluorescence and
transient absorption dynamics of all adducts in various solvents
are qualitatively similar to those of KN. Therefore, the same
interpretation can be proposed for the time constants: the time
constant τ1 can be assigned to the diffusive motion of the solvent
around the excited molecule, τ2 corresponds to conformational
changes of the adduct in the excited state, and τ3 can be
attributed to the lifetime of the lowest singlet excited state.23
The obtained τ1 values agree with the data reported in literature
for the same solvents;45-47 the τ2 and τ3 values exhibit a
monotonic increase from Lys-KN to GSH-KN and from protic
to aprotic solvents. The increase of the time constant τ2 from
KN to GSH-KN correlates with the increase of the molecular
size, which agrees with its assignment to conformational
relaxation.
The observed isotopic effect and the strong solvent depen-
dence of τ3 point to a solvent-assisted mechanism of the excited
state deactivation for all the adducts under study. The increase
of τ3 from Lys-KN to GSH-KN in protic media indicates a
decrease of the efficiency of the deactivation mechanism. It
seems likely that the presence of a substituent comparable in
size with KN leads to a weakening and/or blocking of
intermolecular hydrogen bonds between the chromophoric
moiety of the adduct and the solvent molecules. As a result, a
slowing down of the radiationless S1 f S0 transition occurs. In
aprotic media, where intermolecular hydrogen bonds are absent,

11918 J. Phys. Chem. B, Vol. 114, No. 36, 2010
Sherin et al.
the lifetimes and quantum yields of fluorescence are similar for
KN and all the adducts. A small decrease of the fluorescence
lifetime and quantum yield from Cys-KN to GSH-KN in DMSO
(Table 1) should be noticed: it might be due to intramolecular
hydrogen bonding between the aminoacetophenone moiety and
the Cys, His, and GSH substituents.
demonstrate even higher photochemical activity. This will be
the topic of our further investigations.
Conclusions
The results obtained in the present work demonstrate that
the covalent attachment of the UV filter kynurenine to amino
acids and proteins results in a significant increase of its
photoactivity. The main reason of this effect is the decrease of
the rate of solvent-assisted internal conversion, the major
deactivation channel of the S1 state. With aging, the concentra-
tions of major UV filters in the human lenss3OHKN, AHBG,
and KNssignificantly decrease,18 whereas the levels of GSH-
3OHKN adduct and kynurenines attached to the lens proteins
increase.18 Thus, the susceptibility to UV light of the aged lens
is much higher than that of the young one, which may give a
significant contribution to the age-related cataract development.
Photophysics and Photochemistry of m-HEWL. The
fluorescence and absorption dynamics of m-HEWL reveal a
multiphasic decay of the singlet excited state population of KNs
covalently attached to HEWL. This dynamics is most probably
due to KNs bound to different sites of the protein. The fastest
component exhibits a time constant close to a characteristic time
for solvent relaxation in aqueous solution;45-47 however, the
dynamic Stokes shift is strongly suppressed by the fast decay
(Figures 7 and 8). A reaction between the S1 state of the bound
KN and neighboring amino acid residues via an electron transfer
can be suggested. In this case, transient bands from kynurenine
radicals with an absorption maximum at 430 nm24 should be
observed. The absence of any long-lived signal in this region
(Figure 8a) points to a fast charge recombination. Considering
the structure of HEWL,48 the possible quenchers of the
photoexcited KNs bound to m-HEWL might be F3 (phenyla-
lanine) and F38 residues located in the immediate proximity of
K1 and F3, F34, F38, and W123 (tryptophan) which are close
to K33. Unfortunately, our data do not allow the mechanism of
this ultrafast reaction, which represents a very efficient deactiva-
tion channel of the KN S1 state, to be fully established.
Acknowledgment. This work was supported by the following
agencies: FASI state contracts 02.512.11.2278, 02.740.11.0262,
and P708, Russian Foundation for Basic Research (projects 08-
03-00539 and 09-04-12135), Grant of the President of Russian
Federation (grant NSh-7643.2010.3), the Division of Chemistry
and Material Science, Russian Academy of Sciences, the SNF
(Project Nr. 200020-124393), and the University of Geneva.
P.S.S. thanks the European Scientific Foundation (DYNA
Exchange Grant #1929).
Supporting Information Available: Laser pulse energy
dependence of the initial absorption at 430 nm of m-HEWL
and KN in neutral aqueous solutions. This material is available
free of charge via the Internet at http://pubs.acs.org.
The second, third, and fourth components of the excited-state
dynamics could be assigned to excited KNs that do not have a
suitable electron donor in a nearest environment. The obtained
τ2 value is close to the S1 state lifetime of KN adducts in water;
the τ3 time constant is similar to the fluorescence lifetime of
the adducts in EtOH; the τ4 value to the KN fluorescence lifetime
in aprotic solvents (see Table 1). Apparently, these time
constants could be associated with KNs attached to different
amino acid residues and having different access to solvent
molecules: the higher τ values correspond to KNs with weaker
exposure to water molecules. Thus, τ2 might be attributed to
KNs located at the outer surface of m-HEWL, τ3 to KNs
partially buried in the protein globule, and τ4 to KNs deeply
inside the protein globule. The largest time constant (τ5)
observed in the TC-SPC experiment is at least 4 times higher
than the fluorescence lifetime of KN in aprotic DMSO. Most
likely, this component corresponds to impurities present in the
solution. Thus, the excited state dynamics of m-HEWL reveals
at least four components of KN excited state decay. Some of
these components could also be attributed to the presence of
several m-HEWL conformations where the same KN-modified
residues are included in protein domains of different proticity.
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