Journal of Molecular Structure 842 (2007) 60–66

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Spectroscopic study on binding of rutin to human serum albumin

Alexander V. Pastukhov *, Lidiya A. Levchenko, Anatoli P. Sadkov
Institute of Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Moscow Region 142432, Russia

Received 3 September 2006; received in revised form 7 December 2006; accepted 8 December 2006
Available online 16 December 2006

Abstract

Steady-state and time-resolved fluorescence spectroscopy techniques were used to study the interaction of the flavonoid rutin with
human serum albumin (HSA) as well as spectral properties of the protein-bound flavonoid. Both quenching of the intrinsic fluores-
cence of the protein (Trp214) and the ligand fluorescence, appearing upon complexation with HSA, were used to determine binding
parameters. The binding constant determined from the quenching of the Trp214 fluorescence by rutin is equal to
6.87 ± 0.22 · 104 M1 and that obtained from the fluorescence of HSA-bound rutin is 3.8 ± 0.4 · 104 M1. Based on the Job plot
analysis, the 1:1 binding stoichiometry for the HSA-rutin complex was determined. The efficient quenching of the Trp214 fluorescence
by rutin, fluorescence resonance energy transfer from excited Trp214 to rutin, and competitive binding of warfarin indicate that the
binding site for the flavonoid is situated within subdomain IIA of HSA. The presence of the sugar moiety in the flavonoid molecule
reduces affinity of rutin for binding to HSA but does not affect the binding stoichiometry and location of the binding site compared
with aglycone analogues.
 2006 Elsevier B.V. All rights reserved.

Keywords: Rutin; Albumin; Binding; Fluorescence

1. Introduction may result in enhancing their anti-inflammatory and anti-

Flavonoids are a large class of naturally occurring poly-
phenols widely distributed in plants. Being both dietary
and biologically active compounds, flavonoids have
attracted much attention of investigators as potent species
capable of affecting various biological processes in living
organisms. It has been recognized that flavonoids display
anticancer, antiviral, anti-inflammatory, and heart disease
protective activities [1,2]. They are able to modulate vari-
ous enzymes [3,4]. These highly potent biological activities
of flavonoids are thought to result from their antioxidant
and free radical scavenging properties. Flavonoids are also
capable of chelating transition and noble metal ions what
Abbreviation: HSA, human serum albumin.
*
Corresponding author. Present address: Department of Pharmacology
and Toxicology, The University of Texas Medical Branch, 301 University
Blvd., Galveston, TX 77555, USA. Tel.: +1 409 772 9670; fax: +1 409 772
9642.
E-mail address: avpastuk@utmb.edu (A.V. Pastukhov).
oxidant properties [5] or in arising new activities like in
the case of the Au–rutin complex that was shown to oxidize
methane to methanol [6].
In body, bioavailability and bioactivity of flavonoids
depend on how they are distributed and transformed in dif-
ferent tissues. It is of vital importance for such a distribu-
tion and transformation how flavonoids interact with
proteins. It has been demonstrated that a plasma protein,
albumin, is a primary carrier of flavonoids in blood [7,8]
and that binding of flavonoids to albumins can occur both
non-covalently [7] and covalently [9] depending on condi-
tions. Despite extensive investigation, molecular mecha-
nisms of binding are not well understood while that is the
mode of binding of flavonoids to albumin which is primar-
ily responsible for their bioavailability and bioactivity in
body. One of the techniques used to probe interaction of
flavonoids with albumins is fluorescence spectroscopy that
has been exploited to study binding of flavonoids to
human, bovine, and rat albumins [7,10–14].

0022-2860/$ - see front matter  2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2006.12.008
 A.V. Pastukhov et al. / Journal of Molecular Structure 842 (2007) 60–66
5`
6`
8
1 B
HO 7 O 2
1`
2`
A C
6
3
5 4
O cence intensities, respectively; A1 and A2 are the absor-
OH O bances at the excitation and emission wavelengths,
HO O O CH 2
CH 3
O Elmer Lambda EZ-210 spectrophotometer. Concentration
OH
OH OH HO
HO OH
Scheme 1. Structure of rutin.
Rutin is a flavonol glycoside consisting of the flavonol
quercetin and the disaccharide rutinose, Scheme 1. Rutin
is found in many plants and common dietary sources of
rutin are black tea and apple peals. Like other flavonoids,
rutin exhibits many of the above activities and being an
abundant dietary flavonoid it may affect various processes
in a human body. At the same time, little is known about
the way rutin interacts with its main carrier protein in plas-
ma, albumin. Moreover, the presence of a sugar moiety in a
flavonoid molecule can considerably affect interactions
with proteins compared with those of its aglycone [4,7].
Therefore, it would be interesting to obtain more detailed
information on the rutin–albumin interaction at the molec-
ular level.
In this work, we present the results of the study on bind-
ing of rutin to HSA obtained by using steady-state and
time-resolved fluorescence spectroscopy techniques. The
binding parameters, location of the binding site, and spec-
tral characteristics of the protein-bound flavonol are deter-
mined. The effect of the presence of the sugar moiety in the
flavonol molecule on the binding characteristics is
discussed.
2. Materials and methods
Human serum albumin (essentially globulin and fatty
acid free) was purchased from Fluka. Rutin hydrate and
warfarin were from Sigma. All other chemicals used were
reagent grade. 0.01 M Tris–HCl buffer, pH 8.0, was used
in all experiments. All measurements were carried out at
25 C.
Steady-state fluorescence measurements were performed
on a Perkin-Elmer LS-55 spectrofluorometer using a
0.3 · 1 cm quartz cell, with the shorter path parallel to
the excitation light direction. The emission was collected
in the range of 305–500 nm upon excitation at 295 nm
and in the range of 460–620 nm upon excitation at
61
450 nm. The obtained fluorescence data were corrected
OH
for the background fluorescence of buffer and HSA, and
4`
for inner filter effects according to the expression [15]
A þA 
3` OH 1 2
F corr ¼ F obs 10 2 ; ð1Þ
where Fobs and Fcorr are the observed and corrected fluores-
respectively. Every spectrum recorded was an average of
three measurements. All experiments were repeated at least
in triplicate. Absorption spectra were recorded on a Perkin-
of HSA was determined spectrophotometrically based on
the extinction coefficient, e280 = 34,600 M1 cm1 [16].
Fluorescence lifetime decays were recorded using a time-
correlated single-photon counting FluoTime 200 spectrom-
eter. Sub-nanosecond pulsed light emitting diode, kpeak =
295 nm, and picosecond diode laser, kpeak = 405 nm,
(PicoQuant GmbH, Germany) were used to excite the
sample. A 0.5 · 0.5 cm quartz cell was used in all fluores-
cence decay measurements. The lifetime decays were
analyzed with the Global Fluorescence Decay Data Analy-
sis Software (PicoQuant) as a sum of exponentials
X
IðtÞ ¼ ai expðt=si Þ; ð2Þ
i
where I(t) is the intensity decay, si is the decay time of the
ith component, and ai is the weighting factor for the ith
component. The quality of fitting was estimated by the re-
duced v2 value [17]. To analyze the data, the average fluo-
rescence lifetime, <s>
P
ai s i
hsi ¼ Pi ð3Þ
i ai
was used because this parameter is more appropriate for
representing a fluorophore with non-exponential lifetime
decay [18].
Binding of rutin to HSA was studied by both quenching
of the intrinsic fluorescence of the protein (Trp214) by rutin
and the enhancement of the rutin fluorescence in the pres-
ence of HSA. In the quenching experiments, the binding
constant was determined from the Stern–Volmer equation
[15]:
s0 F0
¼ 1 þ k q s0 ½L0  ¼ 1 þ K SV ½L0  ¼ ; ð4Þ
s F
where s0 and s are lifetimes of Trp214 in the absence and
presence of quencher (rutin), respectively; F0 and F are
steady-state fluorescence intensities of the tryptophan resi-
due in the absence and presence of rutin, respectively. KSV
is the Stern–Volmer quenching constant, kq is the bimolec-
ular quenching constant, [L0] is the total concentration of
the quencher in the sample. In experiments based on the ru-
tin fluorescence enhancement accompanying its binding to
HSA, several procedures were used to obtain the binding
parameters. The binding constant and number of binding
sites for the ligand on the protein were determined from
62 A.V. Pastukhov et al. / Journal of Molecular Structure 842 (2007) 60–66

the procedure of Scatchard [19]. For this purpose, a fixed

amount of the ligand was first titrated with increasing
amounts of HSA. The obtained data were plotted as 1/F
versus 1/[P0], where [P0] is a total concentration of the pro-
tein in the sample. The reciprocal of the intercept of the
plot with the y-axis was considered as the fluorescence,
Fmax, of all the ligand totally bound to the protein. This
value was then used to calculate the concentration of the
bound ligand when a fixed amount of HSA was titrated
with increasing amounts of rutin under the same apparatus
settings. The concentration of the bound ligand was deter-
mined from the relationship

½PL F
¼ ; ð5Þ
½L0  F max

where [PL] is the concentration of the ligand–protein com-

plex observed at the total ligand concentration [L0]. Final-
ly, a plot of n/[L] versus n, where n is the number of binding
sites per protein molecule occupied by the ligand and [L] is
the concentration of the free ligand, provided values for the
binding constant and number of binding sites.
Additionally, the Job plot method [20] was used to eval-
uate the binding stoichiometry of the complex. In this
method, the total concentration of the ligand and HSA
was maintained constant, whereas their molar fractions
were varied. The fluorescence intensity measured at
525 nm and attributed to rutin associated with HSA was
plotted versus the ligand molar fraction. The abscissa of
the maximum of the Job plot provided the value of the
ligand molar fraction for the binding stoichiometry.

3. Results and discussion

Fig. 1 presents the absorption and fluorescence spectra

of rutin in the presence of HSA. Adding the protein to
rutin solution resulted in the shift of the Band I maximum
from approximately 386 to 403 nm in the absorption spec-
trum of the flavonol (Fig. 1a). This red shift indicates the
ground state complex formation between rutin and HSA.
Moreover, the spectral position of the band may mean that
binding of rutin to HSA causes a shift in the protonation
equilibrium of the ligand as it was supposed for binding
of rutin parent compounds (3-hydroxyflavone, quercetin)
to albumins [12,14].
Another argument in favor of the complexation of rutin
with HSA comes from the appearance of the weak fluores-
cence in the range 460–620 nm upon addition of HSA to
buffer solution containing free rutin (Fig. 1b). It is known
that flavonols bearing a 5-OH group usually do not fluo- Fig. 1. (a) Normalized absorption (1, 2) and fluorescence excitation (3)
resce due to the formation of an intramolecular H-bond spectra of rutin: (1) free in buffer (2) in the presence of HSA (conditions:
between C(4)@O and C(5)–OH which facilitates the non- [rutin] = 2 lM, [HSA] = 100 lM); (3) HSA-bound rutin (conditions:
radiative deactivation of the excited flavonols [21]. Indeed, [rutin] = [HSA] = 50 lM, kem = 520 nm). (b) Fluorescence emission spec-
we did not observe any noticeable fluorescence of free rutin trum of HSA-bound rutin. (Conditions: [rutin] = 50 lM, [HSA] = 10 lM,
kex = 450 nm). (c) Normalized fluorescence decay of the HSA-bound
in buffer solution under apparatus settings used in our flavonol (solid line) and instrument response function (dot line). (Condi-
experiments. The fluorescence of rutin arising in the presence tions: [rutin] = [HSA] = 50 lM, kex = 405 nm, kem = 525 nm).
of HSA had the maximum at about 525 nm (excitation at
 A.V. Pastukhov et al. / Journal of Molecular Structure 842 (2007) 60–66 63

450 nm). An analogous fluorescence band with a maximum

in the range of 510–530 nm was observed in experiments on
binding of flavonoids to albumins and was attributed to the
emission of tautomer species formed by the intramolecular
proton transfer process from either 3-OH or 4 0 -OH group
to C(4)@O [10,12,22]. Because rutin bears the rutinose moi-
ety at C(3), proton transfer from the 4 0 -OH group to 4-OH
group is only possible. We suppose that binding of rutin to
HSA may result in the formation of an intermolecular H-
bond between the 5-OH group of rutin and some amino
acid residue in the binding site of HSA. In particular, a
molecular modelling study of the interaction of the flavone
wogonin with HSA demonstrated the formation of the H-
bond between the 5-OH group of the flavone and Arg257
in HSA, with the 5-OH group donating a proton to the car-
bonyl group of Arg257 [23]. Such an interaction of rutin
with the protein microenvironment would prevent the
non-radiative deactivation of excited rutin. On the other
hand, 4 0 -OH to 4-OH proton transfer could result in the
appearance of the low intensity fluorescence band with
the peak at 525 nm. The fluorescence decay of HSA-bound
rutin was also recorded (Fig. 1c). The decay curve was non-
exponential and was satisfactory described as a sum of two
exponentials with <s1> = 0.39 ns (fraction – 0.76), <s2> =
4.86 ns (fraction – 0.24), and average <s> = 1.45 ns.
The excitation spectrum of HSA-bound rutin is different
from its absorption spectrum, with the maximum being
30 nm more shifted to the red (Fig. 1a). Similar behavior
was observed for the excitation spectrum of albumin-
bound quercetin and was interpreted as the manifestation
of the fact that the protein bound ligand is a mixture of
species with different fluorescence properties [14]. More-
over, another band with the maximum at about 285 nm
(attributable to tryptophan absorption) is observed in the
excitation spectrum. This implies efficient Forster type
energy transfer from excited Trp214 to rutin.
Binding of rutin to HSA was accompanied by efficient
quenching of the intrinsic fluorescence of the protein
(Trp214) excited at 295 nm. Simultaneously, a weak emis-
sion with a peak at about 525 nm appeared (Fig. 2a) what Fig. 2. (a) Fluorescence emission spectrum of the HSA-rutin complex.
is again indicative of fluorescence resonance energy transfer (Conditions: [HSA] = 4 lM, [rutin] = 12 lM, kex = 295 nm). (b) Stern–
from the excited tryptophan to the protein-bound flavonol. Volmer plot for quenching of the Trp214 fluorescence by rutin. Steady-
Quenching of the Trp214 fluorescence by various ligands is state (dark squares) and time-resolved (open circles) experimental data
often used, in conjunction with the Stern–Volmer model, to points were fitted to Eq. (4) (solid lines). (Inset) Fluorescence spectra of
Trp214 in the presence of increasing amounts of rutin. (Conditions:
assess the binding parameters [24,25]. The prerequsite for [HSA] = 4 lM, concentration of rutin varied from 2 to 80 lM,
using the Stern–Volmer analysis in such studies is the static kex = 295 nm, kem = 350 nm).
nature of the quenching process, i.e., the quenching occurs
within a protein–ligand complex, in which case the Stern–
Volmer constant, KSV is equal to the association constant, on the calculation procedure, the binding constant values
KA, [15]. The Stern–Volmer plot for quenching of the for complexation of rutin with HSA were 5.48 · 104,
Trp214 fluorescence by rutin showed good linearity in the 6.74 · 104, and 12.8 · 104 M1, with the latter value being
range of quencher concentrations used in our experiments less correct [24]. However, the linearity of the Stern–Vol-
(Fig. 2b) and provided the binding constant for the HSA- mer plot does not answer the question about the quenching
rutin association, KA = 6.87 ± 0.22 · 104 M1. This value mechanism. The quenching process may be both dynamic
is in a good accordance with values obtained by Bi et al. and static by its nature. Dynamic quenching is a diffusion
[24] who used quenching of the Trp214 fluorescence to controlled process, whereas static quenching is due to the
study binding of various flavonoids to HSA. Depending complexation of a quencher with a fluorophore.
64 A.V. Pastukhov et al. / Journal of Molecular Structure 842 (2007) 60–66
Fig. 3. (a) Normalized fluorescence decays of Trp214 in the absence (1)
and presence (2) of rutin, and instrument response function (3). (Condi-
tions: [rutin] = [HSA] = 50 lM, kex = 405 nm). (b) Job plot for the
complexation of rutin with HSA. (Conditions: total concentration of
rutin and HSA in a sample was 100 lM, kex = 450 nm, kem = 525 nm).
The absorption and fluorescence emission and excitation
spectra of rutin in the presence of HSA provide evidence
for the ground state complex formation and thus support
the static model for the quenching mechanism. The fluores-
cence lifetime measurement can directly distinguish
between these two mechanisms because in the case of the
static quenching the lifetime does not depend on the
quencher concentration, i.e., <s0> = <s> [15]. Fig. 3a pre-
sents the lifetime decays of Trp214 in the absence and pres-
ence of rutin. The fluorescence decay of unquenched
Trp214 was biexponential with s1 = 0.85 ns (fraction –
0.04) and s2 = 5.97 ns (fraction – 0.96), and with <s> =
5.76 ns. These parameters are in good accordance with
Table 1
Average fluorescence lifetime of Trp214 in the presence of rutin
Rutin (lM) <s> (ns) v2
0 5.76 1.05
5 5.67 0.78
10 5.50 0.86
15 5.43 0.79
25 5.27 0.87
40 5.19 0.98
60 5.10 1.11
90 4.93 1.08
Conditions: [HSA] = 3 lM, kex = 295 nm, kem = 345 nm.
those obtained for Trp214 by Helms et al. ([26] and refer-
ences therein). Adding HSA solution with increasing
amounts of rutin caused a small decrease in the average
fluorescence lifetime of Trp214, Table 1. The Stern–Volmer
plot gave the bimolecular quenching constant kq =
4 · 1011 L M1 s1. This value is order of magnitude higher
than the upper limit for the diffusion controlled quenching
constant in solutions of biopolymers, kq = 2 · 1010
L M1 s1 [27]. Moreover, the plot of <s0>/<s> versus
[L0] lies lower than that for F0/F (Fig. 2b) what is also
indicative of the static mechanism of the quenching
because for dynamic quenching I0/I = <s0>/<s>. The
following relationship can be used to estimate the contribu-
tion of each mechanism to overall quenching [18]
I 0 < s0 > 1
¼ ; ð6Þ
I <s> f
where f is the fraction of excited species that are not
quenched statically. In the case of the HSA-rutin complex
it was found that approximately 95% of the observed
quenching occurs by the static mechanism.
From crystallographic data, it is known that HSA con-
tains single tryptophan residue (Trp214) situated in subdo-
main IIA [28]. The efficient quenching of the Trp214
emission by rutin and fluorescence resonance energy trans-
fer from excited tryptophan to rutin indicate that the most
probable binding site for the flavonol is located within sub-
domain IIA. In the literature there are data that HSA may
have one or two binding sites for flavonoids situated in sub-
domains IIA and IIIA [10,11] as well as in the interdomain
loop region [29]. To clarify this issue, two types of experi-
ments were performed: (i) the binding stoichiometry for
the HSA-rutin complex using the method of continuous
variations (the Job plot) [20] was determined; (ii) competi-
tive binding of warfarin to HSA was carried out. Fig. 3b
presents the results of the Job plot experiment. As seen from
Fig. 3b, the prominent increase in the fluorescence intensity
of HSA-bound rutin is observed at the equimolar concen-
trations of the protein and ligand. The 1:1 binding stoichi-
ometry means that HSA has only one binding site for rutin.
It is well known that warfarin has a high affinity binding
site with KA = 2 to 5 · 105 M1 [30] in subdomain IIA of
HSA. Competitive binding of warfarin to HSA is often used
to demonstrate that binding of a ligand under study occurs
 A.V. Pastukhov et al. / Journal of Molecular Structure 842 (2007) 60–66 65

binding sites for rutin per a HSA molecule, n = 1.43. The

binding constant determined from the quenching of the
Trp214 fluorescence by rutin is 6.87 ± 0.22 · 104 M1.
We consider the value of the former constant as less accu-
rate since flavonoids bound to albumin are usually a mix-
ture of species with very different fluorescence properties
[14]. The reason for such a variety is the existence of the
differently ionized forms of a protein-bound flavonoid
what originates from the distribution of the configurations
of the binding site microenvironments. Therefore, the bind-
ing constant derived from the fluorescence of rutin bound
to HSA (Scatchard analysis) is likely underestimated
because not every flavonol molecule bound to the protein
may fluoresce in the same range or with the same quantum
yield, if any, to make a proper contribution to the emission
profile of the proton transfer tautomer species which was
used for calculating the binding parameters by the Scat-
chard procedure. At the same time, the efficiency of
quenching of the Trp214 fluorescence by rutin depends
on the absorption properties of the bound ligand which
are less sensitive to the differences in the binding site micro-
environments compared with the emission properties of
HSA-bound rutin. This means that the binding constant
value determined from the Stern–Volmer equation should
be more accurate.
It is interesting to compare our data on binding of rutin
to HSA with those obtained for related aglycone forms.
Table 2 summarizes such data found in the literature. As
seen from Table 2, the absence of the sugar moiety results
in 3- to 15-fold tighter binding of the related aglycones to
HSA (except for the data from Ref. [14] that may be under-
evaluated due to the procedures used to calculate the bind-
ing constant value) compared to that of rutin. The presence
of the sugar moiety in rutin may reduce affinity of the
ligand to HSA in two ways:

(i) The size of rutin approximately doubles that of

quercetin. Moreover, rutin contains two more cyclic
fragments that are less flexible to undertake confor-
mational changes. These reasons may put structural
Fig. 4. (a) Comparative binding of warfarin to HSA in the presence of
constraints on favorable accommodation of rutin
rutin. (Conditions: [rutin] = [HSA] = 50 lM, kex = 450 nm, kem =
540 nm). (b) Scatchard plot for binding of rutin to HSA. (Conditions: within the binding site.
kex = 450 nm, kem = 525 nm).
Table 2
Binding parameters for the complexation of rutin and its aglycone parent
within subdomain IIA. In the case of the HSA-rutin compounds with human serum albumin
complex, adding the solution with increasing amounts of Flavonoid KA · 104 (M1) n (subdomain) Reference
warfarin caused the gradual decrease in the fluorescence Quercetin 26.7 1 (IIA) [8]
of HSA-bound rutin (Fig. 4a). Taken together (the efficient Quercetin 19 1 [11]
quenching of the Trp214 fluorescence, fluorescence Quercetin 5 <1 [14]
resonance energy transfer, 1:1 binding stoichiometry, and Quercetin 110 1.5 [24]
Quercetin 91 1.3 [24]
competitive binding of warfarin) these facts indicate that
Quercetin 52.6 1 [32]
the binding site of rutin is located within subdomain IIA 3-Hydroxyflavone 72 1 (IIA) [10]
of HSA. 3-Hydroxyflavone 25 1 (IIA) [10]
Scatchard analysis [19] of the fluorescence of HSA- Rutina 6.87 1 (IIA)
bound rutin excited at 450 nm (Fig. 4b) gave the binding a
As determined from measurement of the quenching of the Trp214
constant, KA = 3.8 ± 0.4 · 104 M1, and the number of fluorescence by rutin.
66 A.V. Pastukhov et al. / Journal of Molecular Structure 842 (2007) 60–66
(ii) The sugar moiety of rutin bears additionally several
polar hydroxyl groups that may perturb the relatively
hydrophobic environment of the binding site. Such a
perturbation in the secondary and tertiary structure
of a protein was observed upon binding of rutin to
whey proteins [31]. Combining this fact with the sup-
posed structural constraints for the proper accommo-
dation of rutin at the binding site, it is possible to
assume that favorable adjustment of the interactions
of rutin with the protein microenvironment is less
achievable compared to that of quercetin or 3-
hydroxyflavone.
On the other hand, the binding stoichiometry for the
studied aglycones was 1:1 with the binding site situated in
subdomain IIA. Only in the case of 3-hydroxyflavone
another higher affinity binding site, subdomain IIIA, was
reported [10]. These facts indicate that the presence of the
sugar moiety in rutin does not affect the stoichiometry
and location of its binding with HSA.
4. Conclusions
In this work, steady-state and time-resolved fluorescence
spectroscopy techniques were used to study the interaction
of rutin with HSA as well as spectral properties of the pro-
tein-bound flavonol. Both the intrinsic fluorescence of HSA
(Trp214) and fluorescence of HSA-bound rutin were used
to quantify binding of the flavonol to the protein. Fluores-
cence lifetime measurements demonstrated that static
quenching, due to the ground state complex formation, is
the operative mechanism for the decrease in the Trp214
fluorescence in the presence of rutin. Changes in spectral
properties of both Trp214 and HSA-bound rutin, and com-
petitive binding of warfarin allowed us to conclude that the
binding site for rutin is located within subdomain IIA. The
presence of the sugar moiety in the flavonoid molecule
reduces affinity of rutin for binding to HSA but does not
affect the binding stoichiometry and location of the binding
site compared with aglycone analogues.
Acknowledgement
This work was supported by the Russian Foundation
for Basic Research (Grant 06-03-32252).
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