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Journal of Photochemistry and Photobiology B: Biology 92 (2008) 98102

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Spectroscopic studies on binding of 1-phenyl-3-(coumarin-6-yl)sulfonylurea

to bovine serum albumin
Xiao-Hui Liu, Pin-Xian Xi, Feng-Juan Chen, Zhi-Hong Xu, Zheng-Zhi Zeng *
College of Chemistry and Chemical Engineering and State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The interaction of 1-phenyl-3-(coumarin-6-yl)sulfonylurea (SU22) with bovine serum albumin (BSA) has
Received 3 August 2007 been investigated by uorescence quenching spectroscopy combined with UV-absorption, circular
Received in revised form 29 January 2008 dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy techniques under simulative physiolog-
Accepted 29 April 2008
ical conditions for the rst time. Fluorescence data and UV-absorption spectra revealed that the quench-
Available online 8 May 2008
ing mechanism of uorescence of BSA by SU22 was a static quenching process and the number of binding
sites was about 0.8858; the thermodynamic parameters (DG = 29.23 kJ mol1, DH = 47.48 kJ mol1,
Keywords:
and DS = 61.24 J mol1 K1) explained that hydrogen bond and Van der Waals interaction were the
1-phenyl-3-(coumarin-6-yl)sulfonylurea
Bovine serum albumin
main binding force stabilizing the complex. The binding average distance between SU22 and BSA was
Fluorescence quenching obtained (3.20 nm) on the basis of the Frsters theory. In addition, The CD spectra and FT-IR spectra have
Circular dichroism proved that BSA secondary structure changed in the presence of SU22 in aqueous solution.
Fourier transform infrared Crown Copyright 2008 Published by Elsevier B.V. All rights reserved.

1. Introduction
As the major soluble protein constituents of the circulatory sys-
tem, serum albumin has many physiological functions and has
been one of the most extensively studied of all proteins. It contrib-
utes to colloid osmotic blood pressure and is chiey responsible for
the maintenance of blood pH [1]. The most remarkable property of
albumin is that it serves as a depot and transport protein for
numerous endogenous and exogenous compounds. It can transport
unesteried fatty acids, but is also capable of binding an extraordi-
narily diverse range of metabolites, drugs and organic compounds.
The remarkable binding properties of albumin accounts for the
central role it can play in both the efcacy and rate of delivery of
drugs. Many drugs, including anti-coagulants, tranquilizers, and
general anaesthetics, are transported in the blood while bound to
albumin (often more than 90% of the drug is bound) [2]. This has
stimulated scientists interest and became an important research
eld in life sciences, chemistry and clinical medicine. The molecu-
lar interactions are often monitored by using optical techniques.
These methods are sensitive and relatively easy to use whereas
uorescence spectroscopy is a valuable technique for study of the
binding of ligands to proteins. For serum albumin, different com-
pounds have been used as probes successfully such as dyes [3],
drugs [4], and metal complexes [5].
The sulfonylureas have a wide range use of in biology, and many
of their ramications have been used as anti-diabetic medicines
* Corresponding author. Tel.: +86 0931 8610877; fax: +86 0931 8912582.
E-mail address: zengzhzh@yahoo.com.cn (Z.-Z. Zeng).
and herbicides [6,7]. Coumarin also has many noticeable bioactiv-
ities and pharmacological properties, for example, hypoglycemic
effect [8]. In the literature [9], a series of new coumarin-6-sulfonyl-
ureas have been synthesized with convergent strategy and were
evaluated for their anti-diabetic properties using rats. It is very sig-
nicant that whether the coumarin-6-sulfonylureas could be
transported in the blood while bound to albumin. In this paper,
the interactions of Bovine serum albumin (BSA) with 1-phenyl-3-
(coumarin-6-yl)sulfonylurea (SU22) have been studied by using
spectroscopy under simulative physiological conditions. The
quenching mechanism of uorescence of BSA by SU22 was ex-
plored by uorescence spectrum at different temperatures and
UVvis spectrum. The numbers of binding sites and main sorts of
binding force in the medium of Tris/HCl buffer solution (pH 7.4)
have been suggested. In addition, according to the mechanism of
Frster theory, the transfer efciency of energy and distance be-
tween the acceptor BSA and the donor SU22 were calculated.
Moreover, the change of BSAs secondary structures induced by
SU22 binding was investigated by CD and FT-IR spectroscopy.
2. Materials and methods
2.1. Materials
All starting materials were of analytical grade and double dis-
tilled water was used throughout the experiment. BSA, purchased
from Sinopharm Group Chemical Reagent Co. Ltd., was dissolved in
the pH 7.40 buffer solution, and BSA stock solution (1.0 mg mL1)
was kept in the dark at 04 C. Tris/HCl buffer solution (pH 7.40)

1011-1344/$ - see front matter Crown Copyright 2008 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2008.04.008
 Author's personal copy

X.-H. Liu et al. / Journal of Photochemistry and Photobiology B: Biology 92 (2008) 98102 99

and NaCl solution (0.5 mol L1) which was used to maintain the ion
strength were prepared. SU22 (shown in Fig. 1) was synthesized 800
according to the literature [10]. Its stock solution (1.00  103 a
mol L1) was prepared in absolute ethanol.

2.2. Apparatus and methods
Fluorescence spectrum scan was measured with a Hitachi F-
4500 uorophotometer, and uorometric titration experiments at
different temperatures were recorded on a LS-55 uorophotometer
equipped with a thermostat bath.
Fluorometric titration experiments: 2.0 mL operating solution
(BSA: 1.50  106 mol L1, NaCl: 0.10 mol L1, pH 7.40) was ti-
trated by successive additions of a 1.00  103 mol L1ethanol
stock solution of SU22. Titrations were done manually by using a
micro injector, and the uorescence intensity was measured (exci-
tation at 280 nm and 5 nm/5 nm slit widths). The experiments
were measured at two temperatures (298 and 310 K). The temper-
ature of sample was kept by recycled water throughout the exper-
iment. Fluorescence spectrum scan was recorded at room
temperature and similar in methods to titration experiments.
A Varian Carry 100 UVvis spectrophotometer equipped with
1.0 cm quartz cells was used for scanning the UV spectrum on
the range of wavelength from 190 to 500 nm. The Tris/HCl buffer
solution was used as a reference solution.
Circular dichroism (CD) measurements were made on an Olis
RSM 1000 CD spectrometer in cell of 1 mm path length at room
temperature. CD spectra (200300 nm) were taken at a BSA con-
centration of 1.50  106 mol L1, and the results were taken as
molar ellipticity ([h]) in deg cm2 dmol1; The a-helical content of
BSA was calculated from the [h] value at 222 nm using the equa-
tion [11]: %helix = {([h]222  2340)/30300}  100.
FT-IR measurements were carried out at room temperature on a
Nicolet Nexus 670 FT-IR spectrometer equipped with a Germanium
attenuated total reection (ATR) accessory, a deuterated triglycine
sulphate (DTGS) detector and a KBr beam splitter. All spectra were
taken via the attenuated total reection (ATR) method with a res-
olution of 4 cm1 and 60 scans. Spectra processing procedures:
spectra of buffer solution were collected at the same condition.
Then, we subtracted the absorbance of buffer solution from the
spectra of sample solution to get the FT-IR spectra of proteins.
The subtraction criterion was that the original spectrum of protein
solution between 2200 and 1800 cm1 was featureless [12]. Fou-
rier self-deconvolution and secondary derivative were applied to
this range respectively to estimate the number, position and width
of component bands. Based on these parameters, curve-tting pro-
cess was carried out by Galactic Peak solve to get the best Gauss-
ian-shaped curves that t the original protein spectrum. After the
identication of the individual bands, the representative structure
of BSA was calculated using the area of their respective component
bands.
Fluorescence intensity
600
g
400
200
h
0
320 360 400 440
Wavelength(nm)
Fig. 2. Fluorescence spectra of BSASU22 system. (a)(g) respectively, 1.50  106
mol L1 BSA in the presence of 0.00, 2.00, 4.00, 6.00, 8.00, 10.0, 12.0  106 mol L1
SU22; (h) 1.00  105 mol L1 SU22 slits: 5 nm/5 nm; kex = 280 nm.
ment condition, the uorescence intensity of SU22 was very weak,
so the effect of the maximum uorescence intensity could be ig-
nored. As can be seen from Fig. 2, BSA has strong uorescence
emission with a peak at 344 nm on excitation at 280 nm and
SU22 causes a concentration-dependent quenching of the intrinsic
uorescence of BSA. These results indicated that there were strong
interactions and energy transfer between SU22 and BSA. Also, this
graph indicated that addition of quencher did not change the max-
imum wavelength of BSA emission spectra, indicating that SU22
bound to BSA without altering the local dielectric environment.
Fig. 3 shows the UV-absorption spectra of BSA in the absence
and presence of SU22. As can be seen in Fig. 3, BSA has strong
absorbance with a peak at 206 nm and the absorbance of BSA de-
creased with the addition of SU22. The absorption of BSA at about
210 nm represents the helix structure of BSA [13]. As we all known,
the dynamic quenching only affected the excited state of uoro-
phore and did not change the absorption spectrum, but the forma-
tion of non-uorescence ground state complex induced the change
of absorption spectrum of uorophore, so the possible quenching
mechanism of BSA by SU22 was a static quenching process which
formed the BSASU22 complex.
2.5
2.4
a
2.0
Absorbance

2.1

d
Absorbance

1.5
3. Results and discussion
1.8
3.1. The quenching mechanism of uorescence of BSA by SU22
1.0
200 204 208 212

To investigate whether SU22 binds to BSA, uorescence mea- Wavelength(nm)

surements were carried out. Fig. 2 shows the emission spectra of

0.5
BSA in the absence and presence of SU22. Under the same experi-

e
O O 0.0
200 220 240 260 280 300
O
SO2NH C NH
Wavelength(nm)

Fig. 3. UVvis absorbance spectra. (a)(d) respectively, 1.50  106 mol L1 BSA in
Fig. 1. The structure of 1-phenyl-3-(coumarin-6-yl)sulfonylurea (SU22). the presence of 0, 1.6, 4.8, 8.0  106 mol L1 SU22; (e) 1.5  106 mol L1 SU22.
 Author's personal copy
100 X.-H. Liu et al. / Journal of Photochemistry and Photobiology B: Biology 92 (2008) 98102
The uorescence quenching mechanism can be understood by
SternVolmer equation [14],
F 0 =F 1 K q s0 Q  1 K sv Q 
where F0 and F are the uorescence intensity in the absence and
presence of the quencher, [Q] is the concentration of the quencher,
s0 is the uorescence lifetime in the absence of quencher, and Kq is
the bimolecular quenching rate constant which is expected to be
proportional to the diffusion coefcients and so to be proportional
to the solvent temperature. Ksv is the SternVolmer quenching
constant.
Fluorescence intensity data were analyzed according to plotting
F0/F versus [Q] at 298 and 310 K (Fig. 4), and Ksv of the two systems
was calculated (Table 1). The value of Kq was also obtained (the
uorescence lifetime of the biopolymer is 108 s [15]).
Because the maximum scatter collision quenching constant of
various quenchers with the biopolymer is usually 2.0  1010 L
mol1 s1, the values of Kq at 298 and 310 K are far greater than
it [16]. Furthermore, the values of Ksv were lowered with increas-
ing solvent temperature. As was indicated again, the quenching
mechanism of the reaction between BSA and SU22 was not dy-
namic quenching but static quenching due to the formation of a
non-uorescence ground state complex.
3.2. Main binding force between SU22 and BSA
The interaction force between drugs and proteins pertains to
weak interaction, including hydrogen bond, hydrophobic force,
electrostatic force, and Van der Waals interaction. If DH  0,
DS > 0, the main force is hydrophobic interaction; if DH < 0,
DS > 0, the main force is electrostatic effect; if DH < 0, DS < 0, Van
der Waals and hydrogen bond interactions play major role in the
reaction [17]. When the change of temperature is not obvious,
DH can be considered a constant.
In order to obtain the values of association constant b, uores-
cence intensity data were also analyzed according to static quench-
ing formula [18].
1=F 0  F 1=F 0 1=F 0 bQ 
The result was shown in Table 1 and it indicated that there was a
kind of strong binding force between SU22 and BSA, and the BSA
SU22 complex was steady. The decreasing trend of b with increas-
ing temperature was in accordance with Ksvs dependence on
temperature as mentioned above.
0.8
0.6 a
b tum yield, uTrp, was determined in the study as 0.15. With use of
0.4
F0/F-1
0.2
0.0
0 2 4 6 8 10
[Q] ( 10-6molL-1)
Fig. 4. The SternVolmer curve of BSASU22 system (a) 298 K; (b) 310 K.
The thermodynamic parameters, enthalpy (DH) and entropy
(DS) of reaction are important for conrming the acting force.
The values of DG, DH and DS were calculated according to the data
1
of association constant b at 298 and 310 K and Eqs. (3)(5).
DG RT ln b 3
lnb2 =b1 1=T 1  1=T 2 DH=R 4
DG DH  T DS 5
The values of thermodynamic parameters were DG = 29.23 kJ mol1,
DH = 47.48 kJ mol1, and DS = 61.24 J mol1 K1(in Table 1).
They indicated that the main binding force between SU22 and
BSA was hydrogen bond and Van der Waals interaction.
3.3. The number of binding sites n
For the static quenching interaction, the relationship between
the uorescence intensity and the quenching medium can be de-
duced from the formula [19]
F 0  F
lg lg K 0 n lgQ  6
F
where F0, F and [Q] is the same as Eq. (1), K0 is equilibrium constant,
and n is the number of binding sites. According to the formula, we
could obtain that the number of binding sites n is about 0.8858. Fur-
thermore, the molar ratio of BSA to SU22 was studied by Jobs curve
of ultraviolet photometric titration (Fig. 5). The result n  0.8640
was approximatively consistent with uorescence quenching
spectrum.
3.4. Interaction distance (r) between BSA and SU22
According to Frster type dipoledipole nonradiative energy
transfer theory [20,21], the efciency of energy transfer, E, the crit-
ical distance for 50% energy transfer, R0, and the actual distance of
separation, r, were calculated by the following Eqs. (7)(9)
E 1 r=R0 6 1 1  F=F 0 7
2 R60 8:78  1025 j2 /Trp n4 J 8
where j2 is the orientation factor, uTrp is the quantum yield of the
donor tryptophan in the absence of acceptor, and n is the refractive
index of the medium intervening between the donor and acceptor. J
is the spectral overlap integral dened by Eq. (9),
X X
J Fmemm4 Dm= FmDm 9
where F(v) is the uorescence intensity of the donor, e(v) is the mo-
lar extinction coefcient of the acceptor in units of M1 cm1, and v
is the frequency in cm1.
The uorescence emission spectrum of BSA and the UVvis
absorption spectrum of SU22 were shown in Fig. 6 which revealed
that they had some overlapping. The value of J was
2.8541  1015 cm3 L mol1. The orientation factor, j2, was taken
as 2/3 and the refractive index, n, was taken as 1.36 [20]. The quan-
the values of J, j2, n and uTrp, R0 value was calculated as 2.04 nm.
The efciency of energy transfer E was 0.06313. The actual dis-
tance, r, between the binding site of SU22 molecule binding in
BSA molecule and Trp212 in BSA polypeptide chain was 3.20 nm.
3.5. Changes of BSAs secondary structures induced by SU22 binding
Further experiments were carried out with CD technique to ver-
ify the binding of SU22 to BSA. As Fig. 7 showed, CD spectra of BSA
exhibited two negative bands in the ultraviolet region at 208 and
222 nm, characteristic of a-helical structure of protein [11]. The
 Author's personal copy

X.-H. Liu et al. / Journal of Photochemistry and Photobiology B: Biology 92 (2008) 98102 101

Table 1
Binding parameters and thermodynamic parameters of BSASU22

T (K) Kq  1012 L mol1 s1 Ksv  104 L mol1 b  105 L mol1 DG kJ mol1 DH kJ mol1 DS J mol1 K1
298 7.078 7.078 1.319 29.23 47.48 61.24
310 6.309 6.309 0.6286

binding of SU22 to BSA caused a decrease in band intensity at the
far-UV CD, clearly indicating the decrease of the a-helical content
in protein. From the above results, it is apparent that the binding of
SU22 to BSA may induce some conformational changes. The calcu-
lating results exhibited a reduction of a-helix structures from
55.3% to 51.6% at molar ratio BSA/SU22 of 1:1.
Additional evidence regarding the changes of BSAs secondary
structures induced by SU22 binding comes from FT-IR spectros-
copy results. Since infrared spectra of proteins exhibit a number
of amide bands, which represent different vibrations of the peptide
moiety. Of all the amide modes of the peptide group, the single
most widely used one in studies of protein secondary structure is
amide I. This vibration mode originates from the C@O stretching
vibration of the amide group (coupled to the in-phase bending of
the NH bond and the stretching of the CN bond) and gives rise
to infrared bands in the region between approximately 1600 and
1700 cm1 [22]. In Fig. 8, the FT-IR spectra of the SU22-free and
SU22 bound form of BSA with its difference absorption spectrum
were obtained in order to monitor the intensity variations of these
vibrations. From Fig. 8, we concluded that the secondary structure
of BSA is changed because the peak position of amide I band
(1647 cm1) and amide II band (1543 cm1) in the BSA infrared
spectrum has evident shifts and their peak shapes are also chan-
ged. Fig. 9 showed a quantitative analysis of the protein secondary
structure of BSA before and after the interaction with SU22 in Tris
buffer. Based on the literature [23] in which the component bands
of amide I were attributed according to the well-established
assignment criterion, the data obtained from Fig. 9 suggested that
upon SU22BSA complexes, the a-helix structures were reduced
from 41.7% to 37.1%, b-sheet decreased from 27.2% to 21.0%, and
b-turn structures increased slightly from 31.1% to 32.5%, and ran-
dom coil structures increased from an inconspicuous percentage
to 9.40%. The reduction of a-helices and b-sheet in favor b-turn
0

2.21 -5000
[ ] (deg cm dmol )
-1

-10000
2.20
2
Absorbance

-15000
2.19

b
-20000
2.18
a

-25000
2.17 200 210 220 230 240 250 260
Wavelength(nm)

Fig. 7. CD spectra of the BSASU22 system. (a) 1.50  106 mol L1BSA; (b) 1.50 -
2.16
0.0 0.3 0.6 0.9 1.2 1.5 1.8 106 mol L1 BSA + 1.50  106 mol L1 SU22.
cSU22/cBSA
Fig. 5. Jobs curve of ultraviolet photometric titration. 1647

900 0.03

1543
a
a
600 0.02
Fluorescence

Absorbance

1651

300 0.01
b 1541
b

0 0.00 1500 1600 1700 1800

300 350 400 450

Wavelength(nm) Wavenumbers(cm-1)

Fig. 6. Overlap of uorescence spectrum of BSA and absorption spectrum of SU22; Fig. 8. FT-IR spectra and difference spectra [(BSA solution + SU22 solution)-(SU22
(a) Fluorescence spectrum of BSA (1.50  106 mol L1); (b)Absorption spectrum of solution)] of free BSA (a) and BSASU22 complexes (b) in buffer solution in the
SU22 (1.50  106 mol L1). region of 18001500 cm1 (1.50  106 mol L1 BSA; 1.50  106 mol L1 SU22).
 Author's personal copy
102 X.-H. Liu et al. / Journal of Photochemistry and Photobiology B: Biology 92 (2008) 98102
Fig. 9. The curve-t amide I region with secondary structure determination of the
free BSA (a) and BSASU22 complexes (b) in buffer solution in the region of 1700
1600 cm1 (1.50  106 mol L1 BSA; 1.50  106 mol L1 SU22).
and random coil structures is indicative of a partial unfolding of the
protein in the presence of SU22. Similar conformational transitions
were observed for the protein unfolding upon protonation and heat
denaturation [24].
The minor differences between the amounts of a-helices ob-
tained by CD and FT-IR are due to our sample preparation (the
CD spectra were performed in aqueous solution, but the IR spectra
were recorded on hydrated lms) [24].
4. Conclusions
In this paper, the interaction between SU22 and BSA has been
investigated by uorescence method combined with UVvis, CD,
View publication stats
and FT-IR spectroscopy techniques under simulative physiological
conditions. This study showed that the intrinsic uorescence of
BSA was quenched through static quenching mechanism and
SU22 bound to BSA with high afnity which is predominantly ow-
ing to hydrogen bond and Van der Waals interaction. SU22 can be
deposited and transported by albumin. Experimental results also
showed that the binding of SU22 to BSA induced a conformational
change of BSA, which was further proved by the quantitative anal-
ysis data of CD and FT-IR spectrum.
From this paper, accurate measurements of SU22s albumin
binding properties is also expected to open the door to new ave-
nues in the screening and design of appropriate sulfonylurea-based
drugs that will be of important in modern medical research.
Acknowledgements
Authors wish to thank the National Natural Science Foundation
of China (No. 20171019) for nancial support of this work.
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