Synthesis and luminescent properties of Tb(III) complex with a novel pyrazolone ligand and its interaction with bovine serum albumin

Article abstract of DOI:10.1016/j.molstruc.2012.07.023

A novel aromatic pyrazolone ligand, methyl 6-(3-methyl-1-phenyl-5- pyrazolone-4-carbonyl) pyridine-2-carboxylic acid (L), and its corresponding Tb(III) complexes, Tb(L)₃·2H₂O, was successfully prepared. L and its complex were charact

Full text of DOI:10.1016/j.molstruc.2012.07.023

Journal of Molecular Structure 1030 (2012) 19–25
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis and luminescent properties of Tb(III) complex with a novel
pyrazolone ligand and its interaction with bovine serum albumin
⇑
Zhengfa Yang, Ruiren Tang , Zhenfeng Zhang
School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, PR China
h i g h l i g h t s
"
"
"
"
A novel ligand and its Tb(III) complex were synthesized.
The luminescence properties of Tb(III) complex was studied.
The binding characteristics of Tb(L)3ꢀ2H2O with BSA was investigated.
Circular dichroism spectra indicated the changed conformation of BSA.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 May 2012
Received in revised form 23 June 2012
Accepted 15 July 2012
Available online 22 July 2012
A novel aromatic pyrazolone ligand, methyl 6-(3-methyl-1-phenyl-5-pyrazolone-4-carbonyl) pyridine-2-
carboxylic acid (L), and its corresponding Tb(III) complexes, Tb(L)3ꢀ2H2O, was successfully prepared. L
and its complex were characterized by means of mass spectrum (MS), elemental analysis, infrared spec-
troscopy (IR), 1HNMR and thermogravimetric analysis (TGA). The luminescence spectra showed that L
was an efficient sensitizer for Tb(III) luminescence. The interaction of Tb(L)3ꢀ2H2O with bovine serum
albumin (BSA) has been investigated through fluorescence spectroscopy under physiological conditions.
The Stern–Volmer analysis indicated that the fluorescence quenching of BSA by Tb(L)3ꢀ2H2O was resulted
from static mechanism, and the binding constants (Ka) were 2.36 ꢁ 104, 1.54 ꢁ 104 and 1.12 ꢁ 104 at
288 K, 298 K and 310 K, respectively. The binding sites (n) and the corresponding thermodynamic param-
Keywords:
Synthesis
Dimethyl pyridine-2,6-dicarboxylate
Lanthanide complex
Luminescence
eters
DH, DS, and DG were calculated at different temperatures. According to the theoretical and exper-
imental results, van der Waals interactions and hydrogen bonds were found to play major roles in the
binding reaction. Furthermore, synchronous fluorescence spectra and circular dichroism (CD) spectra
indicated that the conformation of BSA was changed. The results obtained in the work can help under-
stand the action mode between rare earth complexes with BSA, and they are also expected to provide
important information of designs of new inspired drugs based on rare earth compounds.
Ó 2012 Elsevier B.V. All rights reserved.
Fluorescence quenching
Bovine serum albumin
1. Introduction
by the use of high absorbent chelating ligands, which served as
efficient sensitizers [4]. Based on the different ligands and the cen-
The unique physical properties of lanthanide ions are the main
reason lanthanide complexes are of great importance in industrial,
chemical, medical, and sensor applications [1,2]. Several of the lan-
thanide (Ln) ions show very intense luminescence characterized by
very narrow emission bands, a long decay time, and a large differ-
ence between the excitation and emission wavelengths, a property
that can be used to decrease the interference caused by back-
ground and scattering [3]. Nevertheless, the Ln(III) complexes usu-
ally give weak luminescence due to the weak absorption
coefficient of the parity-forbidden f–f transitions which limits their
practical application considerably. However, this can be overcome
tral Ln(III), many novel ligands and complexes have been designed
and synthesized [5]. The main ligands include aromatic carboxylic
acid, 1,10-phenanthroline (phen), pyridine and b-diketone, etc. [6–
8]. Among these ligands, aromatic b-diketone compounds have
been more attractive for their highly efficient intermolecular en-
ergy transfers [9,10].
In the past few years, many studies on the molecular structure
of metal complexes and their biological activities aroused much
interest in the field of inorganic chemistry [11,12]. Serum albumin
as one of the most abundant carrier proteins plays an important
role in the transport and disposition of endogenous and exogenous
ligands present in blood [13,14]. Recently, there have been several
studies showed that some components of metal complexes have
high affinity for human serum albumin binding site-II and to
⇑
Corresponding author. Tel.: +86 731 88836961; fax: +86 731 88879616.
E-mail address: trr@mail.csu.edu.cn (R. Tang).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.07.023

20
Z. Yang et al. / Journal of Molecular Structure 1030 (2012) 19–25
exhibit a variety of pharmacological properties [15–17]. Metal
complexes that bind with DNA and proteins under physiological
conditions are of current interests for their varied applications.
The nature and magnitude of drug–albumin interactions signifi-
cantly influence the pharmacokinetics of drugs, and the binding
parameters are useful in studying protein–drug binding as they
greatly influence absorption, distribution, metabolism, and excre-
tion properties of typical drugs [18]. Furthermore, it was found
that rare earth metals possessed much biological activity which
suggests that we ought to pay extensive attention to the biological
activity and pharmacological action of rare earth complexes
in vivo.
Based on the above consideration, we synthesized and charac-
terized a novel pyrazolone ligand, methyl 6-(3-methyl-1-phenyl-
5-pyrazolone-4-carbonyl) pyridine-2-carboxylic acid (L), and its
Tb(III) complex. The fluorescence property of the complex was
investigated in detail which showed that L was an efficient sensi-
tizer for Tb(III) luminescence. Moreover, the interactions of the
complex with BSA have been investigated through fluorescence
quenching under physiological conditions due to its potential
drugs value. The effect of the complex on conformational changes
of BSA was discussed on the basis of the synchronous fluorescence
spectra and CD spectra. Through this investigation, we hope it can
provide a clue for drug design and pharmaceutical research. The
synthetic route of L was expressed in Scheme 1.
Excitation and Emission spectra were recorded on a Hitich F-4500
luminescence spectrophotometer (The width of emission slit and
the voltage of photomultiplier tube was 5 nm and 400 V). Thermal
gravimetric (TG) were performed in the nitrogen atmosphere using
a Netzsch TG 209 thermogravimetric analyzer at a heating rate of
10 °C minꢂ1 from 20 to 800 °C.
2.2. Preparation of ligand (L)
2.2.1. Synthesis of 6-(methoxycarbonyl)pyridine-2-carboxylic acid (2)
A solution of 1 (5.85 g, 30.0 mmol) in methanol (150 mL) was
cooled to 0 °C. After KOH pellets (1.76 g, 31.0 mmol) were added,
the reaction mixture was stirred at 0 °C for 2 h and then at room
temperature for 24 h. The solvent was removed under reduced
pressure, and the residue was suspended in H2O (100 mL) and ex-
tracted with ethyl acetate (3 ꢁ 30 mL). The aqueous layers were
acidified to pH 3 with 1 M diluted HCl solution and extracted with
chloroform (5 ꢁ 30 mL). The collected organic layers were dried
over anhydrous Na2SO4. The chloroform was removed in vacuo to
provide the desired product 2 (2.80 g, 52%) as a white solid: m.p.
144–146 °C. 1HNMR (DMSO-d6): dH 8.12–8.24 (m, Py-3,4,5, 3H)
3.93 (s, ACH3,3H); 13CNMR(DMSO-d6): d = 52.2, 127.4, 127.7,
139.0,147.4, 148.9, 164.9, 165.8 ppm; IR (KBr), v/cmꢂ1: 3073,
2964, 2852, 1725,1581, 1325.
2.2.2. Synthesis of methyl 6-(chlorocarbonyl)pyridine-2-carboxylate
(3)
2. Experimental
2 (0.72 g, 4 mmol) was treated with thionyl chloride (10 mL)
and one drop of N,N-dimethylformamide, then the mixture was re-
fluxed at 70 °C for 4 h. The excess thionyl chloride was removed by
evaporation under reduced pressure. Residual thionyl chloride was
removed by co-evaporation with 30 mL of anhydrous benzene to
afford the corresponding diacid chloride 3. This crude product
was used in the following step without further purification.
2.1. Materials and methods
The stock solution of BSA (purity P 99%, purchased from Sino-
Biotechnology Company, Shanghai, China) was prepared to be the
concentration of 1.0 ꢁ 10ꢂ5 mol Lꢂ1 and kept it in the dark at
0–4 °C [19]. After Tb(L)3ꢀ2H2O was dissolved in water, and diluted
to the desired concentration, the working solutions of Tb(L)3ꢀ2H2O
was obtained. The buffer Tris was purchased from Acros (Geel,Bel-
gium). Still, Tris-HCl buffer solution (PH = 7.4) was given via the
dropwise addition of HCl (0.1 mol Lꢂ1) to Tris solution (0.1 mol Lꢂ1).
0.9% NaCl solution was used to maintain the ionic strength.
Dimethyl pyridine-2,6-dicarboxylate 1 was prepared by literature
[20]. 3-Methyl-1-phenyl-5-pyrazolone was prepared by literature
[21]. Other chemicals were of A.R. grade and used without further
purification. Water used in this study was doubly deionized.
Melting points were determined on a XR-4 apparatus (thermom-
eter uncorrected). Elemental analysis was carried out by a PerkinEl-
2.2.3. Synthesis of methyl 6-(3-methyl-1-phenyl-5-pyrazolone-4-
carbonyl)pyridine-2-carboxylate (4)
To
a mixture of 3-methyl-1-phenyl-5-pyrazolone (1.15 g,
6.6 mmol) and Ca(OH)2 (0.73 g, 9.9 mmol) in dioxane (50 mL,
stored over 4 A molsieve) was added the appropriate carboxylic
acid chloride 3 (6.6 mmol, 1.32 g) in dioxane (10 mL) within
5 min. The resulting mixture was heated to reflux for 4 h and then
allowed to cool to room temperature. After addition of 2 N HCl
(15 mL) the mixture was stirred for 1 h, then poured onto H2O
(100 mL). The precipitated product was filtered off, washed several
times with H2O and recrystallized from methyl alcohol to give
4(1.44 g, 65%) as a yellow crystals. m.p. 188–191 °C. Anal. Calcd
for C18H15N3O4 (%): C, 64.09; H, 4.48; N, 12.46; Found: C, 63.15;
H, 4.42; N, 12.40; 1HNMR (CDCl3): d 8.25–8.66 (m, Py-3,4,5, 3H),
7.29–7.88 (m, Ph, 5H), 2.61 (s, Pyrazole-CH3, 3H), 4.09 (s, ACH3,
3H); 13CNMR(CDCl3): d = 21.8, 51.6, 53.4, 124.6, 126.6, 127.5,
mer 2400 elemental analyzer. Infrared spectra (4000–400 cmꢂ1
)
were recorded with samples as KBr discs using a Nicolet NEXUS
670 FT-IR spectrophotometer. 1HNMR was measured with a Bru-
ker-400 MHz nuclear magnetic resonance spectrometer with CDCl3,
DMSO and D2O as solvents and TMS as internal reference. The
EI-mass spectra were recorded on a Finnigan MAT 90 instrument.
KOH
0
SOCl2
CH3OH
CH3COCl
HOOC
N
COOH
COCl
H3COOC
N
COOCH3
H3COOC
N
COOH
1
2
Ca(OH)2
KOH
COOCH3
N
N
N
N
1,4-Dioxane
N
N
COOH
H3COOC
N
O
O
O
O
3
4
L
Scheme 1. The synthetic route of L.

Z. Yang et al. / Journal of Molecular Structure 1030 (2012) 19–25
21
Table 1
128.0, 128.9, 138.9, 139.2, 148.0, 151.8, 155.6, 165.4, 169.1, 196.9;
IR (KBr), v/cmꢂ1: 3104, 2954, 2923, 1743, 1719, 1621, 1556, 1491,
1236; EI-MS: m/z: 337.1[M]+.
Composition analytical data for the complex.
Complex
C (%) Found (calc.) H (%) Found (calc.) N (%) Found (calc.)
Tb(L)3ꢀ2H2O 52.54(52.58)
3.72(3.69)
10.79(10.82)
2.2.4. Synthesis of methyl 6-(3-methyl-1-phenyl-5-pyrazolone-4-
carbonyl) pyridine-2- carboxylic acid (L)
4 (0.5 g, 1.48 mmol) was dissolved in 50 mL anhydrous metha-
nol with stirring in an ice bath. 2.5 g KOH solution (30%) were
added, and the reaction mixture was stirred for 24 h. Then the sol-
vent was evaporated to gave a yellow oily residue, 10 mL H2O was
added and change pH = 1–2 to give L (0.42 g, 87.5%) as a white so-
lid. m.p. 270–272 °C. Anal. Calcd for C17H13N3O4 (%): C, 63.16; H,
4.05; N, 13.00; Found: C, 63.12; H, 4.01; N, 13.03; 1HNMR (DMSO):
d 8.43–8.45 (m, Py-3,4,5, 3H), 7.35–7.79 (m, Ph, 5H), 2.51 (s, Pyra-
zole-CH3, 3H); 13CNMR(DMSO): d = 21.9, 53.6, 124.6, 126.9, 127.5,
128.0, 129.0, 138.6, 138.9, 148.2, 151.6, 165.4, 168.3, 169.0, 196.5;
IR (KBr), v/cmꢂ1: 3061, 2505, 1720, 1625, 1551, 1497, 1229; EI-MS:
m/z: 323.3[M]+.
be Tb(L)3ꢀ2H2O. The complex was yellowish powder, stable under
atmospheric condition and soluble in ethanol, DMF, DMSO and
acetone.
3.2. FT-IR spectra
The FT-IR spectra and characteristic absorption bands of the free
ligand L and its Tb(III) complex were shown in Fig. 1 and Table 2,
respectively. In the free ligand L, the weak band at 3454 cmꢂ1
can be attributed to the
m
(OAH) and the enol-isomer
m
(C@O) vibra-
tion is at 1625 cmꢂ1. However, the enol-isomer
m(C@O) vibration at
1625 cmꢂ1 was not observed in the complex, indicating that six-
membered ring was constructed by the intramolecular hydrogen
bond generated from the carbonyl groups of the ligand [23]. In
2.3. Preparation of the complex
the complex of Tb(L)3ꢀ2H2O, the
m(C@O) of carboxyl peak disap-
A mixture of L (0.9 mmol) and a solution of LnCl3ꢀ6H2O
(0.3 mmol) (Ln = Tb, Eu) in methanol (6 mL) was stirred for
10 min at room temperature, following which the aqueous NaOH
(0.01 M) was added dropwise to pH 6.0. Then the mixture was stir-
red at 60 °C for 12 h. The resulting precipitate was collected by fil-
tration, washed three times with methanol, dried and purified by
recrystallization from a methanol–acetone mixture.
peared and new bands appeared at 1608 cmꢂ1 and 1402 cmꢂ1
which are assigned to the two characteristic bands of asymmetric
(ca. 1600 cmꢂ1) and symmetric (ca. 1400 cmꢂ1) stretching vibra-
tion modes of the carboxylate group [24]. The m(C@N) peak has
slightly shifts (1583 cmꢂ1 for the ligand L, 1589 cmꢂ1 for the com-
plex Tb(L)3ꢀ2H2O), which suggests that pyridine nitrogen atom
does not participate in coordination reaction. The absorption band
assigned to the coordinated TbAO was found at around 429 cmꢂ1
.
2.4. Interactions with BSA
The band m
(OAH) vibration at 3373 cmꢂ1 is relatively intense and
can be assigned to solvated water. However, the qc(H2O) and
qx(H2O) bands at approximately 850 cmꢂ1 and 650 cmꢂ1 were
not observed in the spectra of the complex, indicating that there
was no coordinated water molecules [25].
The results of elemental analysis and IR spectroscopy indicated
that the enol-forms of the ligand L and carbonyl group of carbox-
ylic acid were involved with the coordination, and we suspected
that the chemical structure of complex may be as shown in Scheme
2.
2.4.1. Fluorescence spectrum
A 2.5 mL solution containing 1.0 ꢁ 10ꢂ5 mol Lꢂ1 BSA was ti-
trated by successive additions of 1.0 ꢁ 10ꢂ3 mol Lꢂ1 stock solution
of Tb(L)3ꢀ2H2O and the concentration of it varied from 0 to
1.0 ꢁ 10ꢂ4 mol Lꢂ1. Titrations were done manually by using mi-
cro-injector. An excitation wavelength (Ex) of 280 nm was chosen
in the experiment. The excitation and emission slit widths (5 nm
each) and scan rate (240 nm/min) were constantly maintained
for all experiments. The fluorescence spectra were recorded at
300, 310 and 320 K in the range of 300–440 nm, respectively.
The temperatures of the samples were kept by recycled water
throughout the experiment.
3.3. Thermal gravimetric analyses
The results of TGA of the complex Tb(L)3ꢀ2H2O is given in Fig. 2.
We can find that it have the relatively high thermal stability. There
are two main successive mass loss stages in the TG curve. The first
stage of the decomposition appears within 60–120 °C correspond-
ing to the loss of the solvated water (Weight loss: 3.62%, Calcd:
2.4.2. CD spectra
CD measurements were carried out on a J-810 spectropolarim-
eter (Jasco, Tokyo, Japan) in a cell of path length 1.0 mm at room
temperature. The induced ellipticity was obtained by the ellipticity
of the Tb(L)3ꢀ2H2O–BSA mixture subtracting the ellipticity of
Tb(L)3ꢀ2H2O at the same wavelength and is expressed in degrees.
The results are expressed as mean residue ellipticity (MRE) in
deg cm2/dmol, which is defined as [MRE hobs (m deg)/10nl Cp].
The hobs represents the CD in millidegree, n is the number of amino
acid residues (583), l is the path length of the cell and Cp is the
mole fraction. The a-helical content of HSA was calculated
from the MRE value at 208 nm using the equation
a%
helix = [(ꢂMRE208 ꢂ 4000)/33000 ꢂ 4000] ꢁ 100 [22].
3. Results and discussion
L
Tb (L)
2H O
2
3
3.1. Composition and properties of the complex
4000 3500 3000 2500 2000 1500 1000
Wavenumbers (cm-1)
500
The elemental analytical data for the complex was presented in
Table 1, which was in good agreement with the values calculated,
indicating that the composition of the complex was confirmed to
Fig. 1. IR Spectrum of ligand L and complex Tb(L)3ꢀ2H2O.

22
Z. Yang et al. / Journal of Molecular Structure 1030 (2012) 19–25
Table 2
Characteristic IR bands (cmꢂ1) of the ligand and its complexes.
3000
2500
2000
1500
1000
500
EX
EM
Sample
mOH
m
(C@O) of carboxyl
m(C@O)
m
py(C@N)
mReAO
L
3454
3373
1720
1625
1583
1589
Tb(L)3ꢀ2H2O
429
-
0
200
300
400
500
600
700
· 2H2O
Wavelength (nm)
Fig. 3. Excitation (left) and emission (right) spectra of Tb(L)3ꢀ2H2O.
Table 4
Luminescence data for the complex.
Complex
kex (nm)
kem (nm)
LI
Assignment
Scheme 2. The chemical structure of Tb(L)3ꢀ2H2O.
Tb(L)3ꢀ2H2O
278
488
542
581
618
1746
2596
505
5D4-7F6
5D4-7F5
5D4-7F4
5D4-7F3
248
100
80
0.000
300 nm which was attributable to the p-
pꢃ transition of the coor-
-0.001
-0.002
-0.003
-0.004
dinated ligands. The emission spectrum of the Tb(III) complex, ob-
tained with the excitation wavelength of 278 nm, is composed of
the typical emission lines for Tb(III), which are assigned to the
transitions between the first excited state (5D4) and the ground
multiplet 7FJ (J = 6–3). The emission spectrum of the Tb(III) com-
plex consisted of four main bands at approximate 488 nm
(5D4 ? 7F6), 544 nm (5D4 ? 7F5), 581 nm (5D4 ? 7F4) and 618 nm
(5D4 ? 7F3), and we can see that the emission band at 544 nm is
obviously stronger than the other emission bands [26]. Still, each
emission band is very narrow and the widths of half bands are
about several nanometers, indicating that the complex have high
color purity and the ligand is a comparative good organic chelator
to sensitize fluorescence of Tb(III) ion [17].
60
40
TG
20
-------DTA
100 200 300 400 500 600 700 800
Tempareture (0C)
Fig. 2. TGA diagrams of Tb(L)3ꢀ2H2O.
3.09%). And the second loss stage ranging from 330 to 685 °C is
attributed to the loss of L (Weight loss: 81.25%, Calcd: 83.24%),
which was confirmed by comparing the observed/estimated and
the calculated mass of the complexes. The detailed data are listed
in Table 3. The above results proves that the water in the com-
plexes was the solvated water and did not participate in coordina-
tion, since for the coordinated water of the analogous complexes is
usually released over 200 °C [25].
3.5. BSA binding studies
3.5.1. Fluorescence quenching of BSA by Tb(L)3ꢀ2H2O
Fluorescence quenching mechanisms are usually classified into
dynamic quenching and static quenching. For dynamic quenching,
as the temperature of the system rises, the effective collision times
between molecules, the energy transfer efficiency, and the fluores-
cence quenching constants of substances will all increase. For sta-
tic quenching, increased temperature reduces the stability of the
complex formed, resulting in a reduced quenching constant.
The effect of Tb(L)3ꢀ2H2O on the fluorescence emission spectra
of BSA at 288 K is shown in Fig. 4, and the corresponding calculated
results are given in Table 5. The fluorescence intensity of BSA con-
sistently decreased with the addition of the Tb(L)3ꢀ2H2O. At the
3.4. Fluorescence properties of complex
The emission and excitation spectra measured in the solid state
at room temperature are displayed in Fig. 3, respectively. The lumi-
nescence data for the complex is listed in Table 4. The excitation
spectra of the complex exhibited a broad band between 200 and
same time,
a slight blue shift at the maximum emission
Table 3
TG data of Tb(L)3ꢀ2H2O.
Complex
Stage
Temperature range (°C)
Mass loss (%) found (calc.)
Probable lost molecules
Tb(L)3ꢀ2H2O
I
II
60-120
330-685
3.62(3.09)
81.25(83.24)
2H2O
3L

Z. Yang et al. / Journal of Molecular Structure 1030 (2012) 19–25
23
1800
1500
1200
900
2.2
a
2.0
1.8
i
1.6
1.4
1.2
1.0
600
300
0
2
4
6
8
10
300
320
340
360
380
400
420
440
105c(L)/(mol/L)
Wavelength (nm)
Fig. 5. Stern–Volmer plots of fluorescence quenching of BSA by Tb(L)3ꢀ2H2O at
three different temperatures (j288 K; d298 K; N310 K).
Fig. 4. The quenching effect of Tb(L)3ꢀ2H2O on fluorescence intensity of BSA at
288 K; kex = 280 nm; c(BSA) = 1.0 ꢁ 10ꢂ5 mol Lꢂ1; c(L)/(10ꢂ5 mol Lꢂ1). (a–i): 0, 0.8,
1.6, 2.4, 3.2, 4, 6, 8 and 10, respectively.
3.5.2. Binding constant and binding sites
The relationship between fluorescence intensity and the con-
centration of quenchers can be described by the following Eq. (2)
for static quenching:
Table 5
Stern–Volmer quenching constants Ksv and kq for binding of BSA with Tb(L)3ꢀ2H2O at
different temperatures.
R2a
F0 ꢂ F
Complex
T (K)
Ksv
kq
log
¼ log Ka þ n log½Qꢄ
ð2Þ
(ꢁ104 L molꢂ1
)
(ꢁ1012 L molꢂ1 Sꢂ1
)
F
Tb(L)3ꢀ2H2O
288
298
310
1.2
0.98
0.81
1.2
0.98
0.81
0.99256
0.99561
0.99812
where Ka is the binding constant, and n is the number of binding
sites per BSA [24]. Fig. 6 shows the double-logarithm algorithm
curve and the results of Ka and n are listed in Table 6. It was noticed
that the binding constant values were equal to 104 and decreased
with increase in temperature due to reduction of the stability of
Tb(L)3ꢀ2H2O–BSA system. The above results clearly indicated that
Tb(L)3ꢀ2H2O quenches the fluorescence emission of BSA with higher
binding affinity and experiences a static quenching process. The
binding capacity (n) varies in the range from 1.03 to 1.07, therefore
we can deduce that the number of binding sites approximates 1.0
and the interaction of Tb(L)3ꢀ2H2O with BSA has only a single bind-
ing site.
R2 is the correlation coefficient of Stern–Volmer equation.
a
wavelength of BSA was observed, which indicated that strong
interaction existed between Tb(L)3ꢀ2H2O and BSA, and the micro-
environment around the chromophore of BSA was also changed
[27]. The quenching can be mathematically expressed by the
Stern–Volmer Eq. (1), which allows for calculating of quenching
constants [28]:
3.5.3. Thermodynamic parameters and binding mode
F0
F
¼ 1 þ Ks ½Qꢄ ¼ 1 þ
s0kq½Qꢄ
ð1Þ
v
The interaction forces between proteins and ligands may in-
clude hydrophobic, hydrogen bonds, van der Waals, electrostatic
interactions, etc. In order to elucidate the interaction between
Tb(L)3ꢀ2H2O and BSA, the thermodynamic parameters were
where kq, Ksv, s0 and [Q] are the quenching rate constant of BSA
(Mꢂ1 sꢂ1), the dynamic Stern–Volmer quenching constant (Mꢂ1),
the average lifetime of BSA and the concentration of quencher
(M), respectively. F0 and F are the fluorescence intensities in the ab-
sence and presence of quencher, respectively. In fact, fluorescence
lifetime of biomolecular s0 is about 10ꢂ8 s [29]; the biomolecular
quenching constant kq at pH 7.40 can be obtained by linear Stern–
Volmer plots (Fig. 5) of F0/F against [Q] from Eq. (1) at different tem-
peratures. In general, maximum collision quenching constant (kq) of
various kinds of biomolecular is 2.0 ꢁ 1010 L molꢂ1 Sꢂ1 [29]. The
rate constants of BSA quenched by Tb(L)3ꢀ2H2O are greater than
2.0 ꢁ 1010 L molꢂ1 Sꢂ1, which suggests that the quenching is initi-
ated by the complex formation rather than by dynamic quenching.
Thus, dynamic collision could be negligible in quantitative analysis
[30]. Meanwhile the results showed that the values of Stern–Volmer
quenching constants Ksv and kq decreased with increasing tempera-
ture, indicating that static quenching actually occurred in the
quenching process [31]. Based on the above discussion, it is clear
that the mechanism of fluorescence quenching was a static quench-
ing procedure and resulted from the formation of ground state com-
plex between Tb(L)3ꢀ2H2O and BSA. Thus the binding constant (Ka)
was evaluated by the method given in the following section.
0.2
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-5.2
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
log [Q]
Fig. 6. Double-log plot of Tb(L)3ꢀ2H2O quenching effect on BSA fluorescence at
three different temperatures (j288 K; d298 K; N310 K).

24
Z. Yang et al. / Journal of Molecular Structure 1030 (2012) 19–25
Table 6
The binding constant (Ka), binding sites (n) and relative thermodynamic parameters for the interactions of Tb(L)3ꢀ2H2O with BSA at different temperatures.
L or complex
Tb(L)3ꢀ2H2O
T (K)
Ka (L molꢂ1
)
n
R2b
D
H (kJ molꢂ1
)
D
S (J molꢂ1 Kꢂ1
)
D
G (kJ molꢂ1
)
R2c
288
298
310
2.36 ꢁ 104
1.54 ꢁ 104
1.12 ꢁ 104
1.07
1.04
1.03
0.99017
0.99732
0.99642
ꢂ24.94
ꢂ24.94
ꢂ24.94
ꢂ2.91
ꢂ2.91
ꢂ2.91
ꢂ24.10
ꢂ24.07
ꢂ24.03
0.99559
0.99559
0.99559
b
c
R2 is the correlation coefficient of double-logarithm equation.
R2 is the correlation coefficient of the value of
DH, DS.
main acting force is electrostatic force; when
D
H < 0,
D
S < 0, the
10.1
10.0
9.9
9.8
9.7
9.6
9.5
9.4
9.3
main acting force is van der Waals or hydrogen bond and when
H > 0, S > 0, the main force is hydrophobic [33]. The negative
enthalpy ( H) and entropy ( S) values of the interaction between
D
D
D
D
Tb(L)3ꢀ2H2O and BSA indicate that van der Waals interactions and
hydrogen bonds played major roles in the binding reaction. Fur-
thermore, the negative values of
DG and DH mean that the inter-
action process is spontaneous and exothermic.
3.5.4. Synchronous fluorescence spectra and CD spectra
The synchronous fluorescence spectra can provide much valu-
able information about the microenvironment in the vicinity of
the chromophore molecules [34]. In the synchronous fluorescence
of BSA, the shift in the position of the maximum emission wave-
length corresponds to the changes of the polarity around the fluo-
3.20
3.25
3.30
3.35
1000/T (K-1)
3.40
3.45
3.50
rophore of amino acid residues. When
Dk values are stabilized at
15 or 60 nm, the synchronous fluorescence of BSA is characteristic
of tyrosine residues (Tyr) and tryptophan residues (Trp), respec-
tively [35]. The effect of Tb(L)3ꢀ2H2O on BSA synchronous fluores-
cence spectroscopy is shown in Fig. 8. As seen from it, the
fluorescence of Tyr was weak and the position of maximum emis-
Fig. 7. Van’t Hoff plots of BSA–Tb(L)3ꢀ2H2O system.
calculated from the van’t Hoff plots. If the enthalpy change (
does not vary significantly in the temperature range studied, both
DH)
sion wavelength had no change when
fluorescence of Trp was strong and the maximum emission wave-
length moderately shifted toward short wave when k was 60 nm.
Dk was 15 nm. However, the
the enthalpy change (
D
H) and entropy change (
D
S) can be evalu-
ated from the van’t Hoff equation [32]:
D
This reflects that the microenvironment of the Trp was signifi-
cantly affected by Tb(L)3ꢀ2H2O binding. The slight blue shift of
Trp fluorescence indicates that the polarity around the tryptophan
residue decreases and the hydrophobicity increases [36]. In a word,
the synchronous fluorescence spectra indicated that the conforma-
tion of BSA has been changed [31,37].
To ascertain the possible influence of Tb(L)3ꢀ2H2O binding on
the secondary structure of BSA, we have performed CD studies
in the presence of same concentrations of Tb(L)3ꢀ2H2O. Fig. 9
shows the CD spectra of BSA, which exhibited characteristic fea-
D
RT
H
DS
ln Ka ¼ ꢂ
þ
R
ð3Þ
where Ka is analogous to the associative binding constants at the
corresponding temperature and R is the gas constant. The calcula-
tion was carried out at three different temperatures, which are
288, 298, and 310 K. The enthalpy change (
the slope of the van’t Hoff relationship. The free energy change
G) is then estimated from the following relationship:
DH) is calculated from
(D
D
G ¼ H ꢂ T
D
D
S
ð4Þ
tures of the typical (a + b) helix structure of the free BSA and its
Fig. 7, by plotting the data in Table 6, shows the values of
D
H
Tb(L)3ꢀ2H2O complex with negative bands at 208 and 222 nm.
The binding of Tb(L)3ꢀ2H2O to BSA caused a decrease in band
intensity without any significant shift of the peaks, indicating a
and S obtained for the binding site from the slopes and ordinates
D
at the origin of the fitted lines. When
D
H < 0 or
D
H ꢅ 0,
D
S > 0, the
100
800
(a)
(b)
a
a
f
80
60
40
20
0
600
f
400
200
0
260
280
300
320
260
270
280
290
300
310
320
Wavelength (nm)
Wavelength (nm)
Fig. 8. Effect of Tb(L)3ꢀ2H2O on the synchronous fluorescence spectra of BSA. (a)
(10ꢂ5 mol Lꢂ1), a ? f: 0, 0.2, 0.4, 0.6, 0.8, 1.0, respectively.
D
k = 15 nm, (b)
D
k = 60 nm; T = 298 K; c(BSA) = 1.0 ꢁ 10ꢂ5 mol Lꢂ1; c (Tb(L)3ꢀ2H2O)/

Z. Yang et al. / Journal of Molecular Structure 1030 (2012) 19–25
25
Acknowledgements
0
-20
-40
-60
-80
Financial support from the National Natural Science Foundation
of China (No. 21071152) is gratefully acknowledged.
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Tb(III) complex were synthesized and characterized. The FT-IR
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that the coordination of the metal ions to the ligand occurred at
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