Synthesis and fluorescence properties of Tb(III) complex with a novel β-diketone ligand as well as spectroscopic studies on the interaction between Tb(III) complex and bovine serum albumin

Article abstract of DOI:10.1016/j.molstruc.2011.11.041

A novel aromatic β-diketone ligand, 4-isopropyl-2,6-bisbenzoylactyl pyridine (L), and its corresponding Tb(III) complex Tb₂(L) ₃·5H₂O were synthesised in this paper. The ligand was characterized by FT-IR and ¹H NMR. The complex was characterized with elemental analysis and FT-IR. The investigation of fluorescence property of the complex showed that the Tb(III) ion could be sensitized efficiently by the ligand. Furthermore, the interaction of Tb₂(L)₃· 5H₂O with bovine serum albumin (BSA) has been investigated by fluorescence quenching spectra, UV-vis absorbance and synchronous fluorescence spectra. The fluorescence quenching mechanism of BSA by Tb₂(L) ₃·5H₂O was analyzed. The binding constants, binding site number and the corresponding thermodynamic parameters at different temperatures were calculated. The results indicated that the Van der Waals and hydrogen bond interactions were the predominant intermolecular forces in stabilizing the complex. Moreover, the effect of Tb₂(L) ₃·5H₂O on the conformation of BSA was analyzed according to synchronous fluorescence.

Full text of DOI:10.1016/j.molstruc.2011.11.041

Journal of Molecular Structure 1010 (2012) 116–122
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis and fluorescence properties of Tb(III) complex with a novel
b-diketone ligand as well as spectroscopic studies on the interaction
between Tb(III) complex and bovine serum albumin
⇑
Zhenfeng Zhang, Ruiren Tang
School of Chemistry and Chemical Engineering, Central South University, Hunan 410083, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 October 2011
Received in revised form 24 November 2011
Accepted 24 November 2011
Available online 1 December 2011
A novel aromatic b-diketone ligand, 4-isopropyl-2,6-bisbenzoylactyl pyridine (L), and its corresponding
Tb(III) complex Tb2(L)3ꢀ5H2O were synthesised in this paper. The ligand was characterized by FT-IR
and 1H NMR. The complex was characterized with elemental analysis and FT-IR. The investigation of fluo-
rescence property of the complex showed that the Tb(III) ion could be sensitized efficiently by the ligand.
Furthermore, the interaction of Tb2(L)3ꢀ5H2O with bovine serum albumin (BSA) has been investigated by
fluorescence quenching spectra, UV–vis absorbance and synchronous fluorescence spectra. The fluores-
cence quenching mechanism of BSA by Tb2(L)3ꢀ5H2O was analyzed. The binding constants, binding site
number and the corresponding thermodynamic parameters at different temperatures were calculated.
The results indicated that the Van der Waals and hydrogen bond interactions were the predominant
intermolecular forces in stabilizing the complex. Moreover, the effect of Tb2(L)3ꢀ5H2O on the conforma-
tion of BSA was analyzed according to synchronous fluorescence.
Keywords:
Aromatic b-diketone
Lanthanide complex
Fluorescence property
Bovine serum albumin (BSA)
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
and drugs [11]. Recently, the interactions of serum albumin with
lanthanide complexes have received much interest owing to their
Within recent years, luminescent lanthanide–organic frame-
works have attracted significant attention due to their absorbing
architectures and enormous potential applications in many areas,
such as light emitting diode [1], luminescence probes or sensors
[2,3]. The absorption and emission spectra intensity of the trivalent
rare earth ions (RE(III)) is weak since intra-configuration 4f–4f
transitions in trivalent lanthanide ions are forbidden (Laporte rule).
In order to overcome this drawback and obtain outstanding lumi-
nescent properties, an organic ligand with excellent absorption
coefficient and high efficient ligand-to-RE(III) ions energy trans-
fers, which acts as an ‘‘antenna’’ [4] is very necessary. Based on
the different ligands and the central RE(III), many novel ligands
and complexes have been designed and synthesised [5]. The main
ligands include aromatic carboxylic acid, 1,10-phenanthroline
(phen), pydine and b-diketone, etc. [6–8]. Among these ligands,
aromatic b-diketone compounds have been more attractive for
their highly efficient intermolecular energy transfers [9,10].
It is well known that serum albumin is the most abundant pro-
tein in plasma, playing the most important physiological role in the
maintenance of colloid osmotic blood pressure and in the binding
and transportation of various ligands such as fatty acids, hormones,
application in a great deal of medical and chiroptical systems
[12,13]. The luminescent Eu and Tb complexes are capable of selec-
tively binding to ‘drug site ll’ of serum albumin and playing a sig-
nificant part in drug pharmacokinetics and pharmacodynamics
[12]. In addition, bovine serum albumin (BSA) has been studied
as a model protein due to its structural homology with human ser-
um albumin (HSA) [14].
Accordingly, in this work, a novel aromatic b-diketone ligand of
4-isopropyl-2, 6-bisbenzoylactyl pyridine (L) with large conjugated
plane and rigid structure, was designed and synthesised (Scheme 1).
And its corresponding Tb(III) complex was also prepared. The fluo-
rescence property of the complex was investigated in detail. Fur-
thermore, the information related to the binding mechanisms with
BSA, such as binding modes, binding constants, and quenching rate
constants, was explored by UV–vis and fluorescence measurements.
2. Experimental
2.1. Materials
Isobutylaldehyde was purchased from Jiangsu (China) Medicine
Co. Ltd., BSA, obtained from Sigma Chemical Co. Ltd., was dissolved
in 0.1 M Tris–HCl buffer solution (pH = 7.40, 50 mM NaCl). BSA
stock solution (1.0 ꢁ 10ꢂ5 M) was kept in the fridge at 0–4 °C.
⇑
Corresponding author. Tel.: +86 731 8883691; fax: +86 731 88879616.
E-mail address: trr@mail.csu.edu.cn (R. Tang).
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.11.041

Z. Zhang, R. Tang / Journal of Molecular Structure 1010 (2012) 116–122
117
Scheme 1. Synthetic route of ligand L.
The complex Tb2(L)3ꢀ5H2O was dissolved in ethanol to form 1 mM
stock solution. And other chemicals were of A.R. grade without fur-
ther purification. Doubly distilled and deionized water was used in
the whole experiments.
687 cmꢂ1 1H NMR (400 MHz, CDCl3): d = 1.35 (d, J = 8 Hz, 6 H),
;
3.05(m, J = 8 Hz, 1 H), 7.53–8.10 (m, J = 8 Hz, 10 H), 8.1 (s, 2 H).
16.35 ppm (s, 2 H).
2.4. Synthesis of the Tb(III) complex
2.2. Methods
A solution of TbCl3 (0.4 mmol) in ethanol (5 mL) was added to
the mixture of ligand L (0.6 mmol) in ethanol (5 mL). And the
resulting mixture was treated with aqueous NaOH (0.1 M) to pH
6, and stirred at 60 °C for 24 h. The resulting yellow precipitate
was collected by filtration, washed with the mixture solution of
ethanol and chloroform mixture (v:v, 1:1), and dried in vacuum
to give a yellow solid.
Melting points were determined on a XR-4 apparatus (ther-
mometer uncorrected); elemental analysis was carried out by a
Perkin Elmer 2400 elemental analyzer; infrared spectra were re-
corded on a Nicolet NEXUS 670 FT-IR spectrophotometer using
KBr discs in the 400–4000 cmꢂ1 region; 1H NMR spectra were mea-
sured with a Bruker-400 MHz nuclear magnetic resonance spec-
trometer with CDCl3 as solvent and TMS as internal reference.
The UV–vis spectra were recorded on an UV-2450 spectrophotom-
eter. Luminescence measurements were made on a Hitich F-4600
spectrophotometer, the widths of both the excitation and emission
slit were set to 5 nm with the photomultiplier tube voltage at
700 V.
2.5. Interaction of complex with BSA
2.5.1. Fluorescence spectroscopy
To 2 mL of BSA stock solution in a 1 cm quartz cell, the complex
Tb2(L)3ꢀ5H2O working solution was gradually added using a pip-
ette. The accumulated volumes were 0, 10, 15, 20, 30, 35, 40, 50,
2.3. Synthesis of the ligand
75 and 100 lL. The quenching of the emission intensity of BSA at
around 340 nm was observed with the increased concentration of
the complex. Fluorescence spectra were recorded from 300 to
460 nm at an excitation wavelength of 280 nm at three tempera-
tures (291 K, 298 K, and 310 K). Both of the excitation and emission
slit widths were 5 nm.
2.3.1. Synthesis of dimethyl 4-isopropyl-2,6-pyridinedicarboxylate
The synthesis of ligand referred to the previously reported
method [15]. Briefly, to a mixture of dimethyl 2,6-pyridinedicar-
boxylate (4.9 g, 25 mmol), isobutylaldehyde (5.4 g, 75 mmol) and
H2SO4 (w/w, 30% aqueous solution, 36 mL), aqueous solution of
FeSO4 (4.9 g, 30 mmol) and H2O2 (w/w, 30% aqueous solution,
40 mL) were added dropwise under vigorous stirring over 15 min
at 0 °C. The reaction mixture was stirred for an additional
15 min, treated with K2CO3 powder to pH 2, and extracted with
chloroform (3 ꢁ 35 mL). The combined extracts were dried, evapo-
rated, and purified by flash chromatography to give white needles
of dimethyl 4-isopropyl-2,6-pyridinedicarboxylate (3.9 g, yield:
2.5.2. UV–vis absorption spectroscopy
Absorption spectra (UV) measurements were made by using a
Shimadzu 2450 UV–Vis spectrophotometer at 298 K in the region
of 260–300 nm. The 2.0 mL of BSA stock solution was titrated with
successive additions of the complex Tb2(L)3ꢀ5H2O working solu-
tion. The Tris–HCl buffer solution was used as a reference solution.
66%) m.p. 58–60 °C; IR:
v
max = 3081, 2954, 2878, 1719, 1600,
1558, 1443, 1358, 1331, 1216 cmꢂ1
;
1H NMR (400 MHz, CDCl3):
3. Results and discussion
d = 1.35 (d, J = 8 Hz, 6 H), 3.07 (m, J = 8 Hz, 1 H), 4.02 (s, 6 H),
8.17 ppm (s, 2 H).
3.1. Composition and properties of the complex
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
be Tb2(L)3ꢀ5H2O. The complex was yellowish powder, stable under
atmospheric condition and soluble in H2O, ethanol, DMF, DMSO
and acetone.
2.3.2. Synthesis of the ligand (4-isopropyl-2,6-bisbenzoylactyl
pyridine)
Sodium metal (0.92 g, 40 mmol) was added to a solution of di-
methyl 4-isopropyl-2,6-pyridinedicarboxylate (2.37 g, 10 mmol) in
20 mL dry toluene, and then a solution of acetophenone (4.8 g,
40 mmol) in 10 mL dry toluene was added dropwise under stirring
at 60 °C for 4 h. The sodium salt was collected by filtration, washed
with petroleum ether. The dried solid was added to dilute hydro-
chloric acid (2.0 M) and the resulting precipitate was collected by
filtration. The crude products were recrystallized over methanol
to give the title compound (L) (3.06 g, yield: 74%). m.p. 178–
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 3459 cmꢂ1
180 °C; IR:
vmax = 3459, 3054, 2965, 1611, 1571, 1487, 1342,

118
Z. Zhang, R. Tang / Journal of Molecular Structure 1010 (2012) 116–122
Table 1
Composition analytical data for the complex/%.
Complex
C found (calc.)
56.18 (56.22)
H found (calc.)
4.83 (4.75)
N found (calc.)
Tb found (calc.)
20.07 (20.18)
Tb2(L)3ꢀ5H2O
2.58 (2.52)
Scheme 2. Chemical structure of Tb2(L)3ꢀ5H2O.
Table 3
Fluorescence spectra date for the complex Tb2(L)3ꢀ5H2O.
Fig. 1. IR spectra of ligand L and complex Tb2(L)3ꢀ5H2O.
complex
kex (nm)
kem (nm)
FIa
Assignment
Tb2(L)3ꢀ5H2O
274
491
546
587
623
2241
3773
1045
485
5D4
5D4
5D4
5D4
?
?
?
?
7F6
7F5
7F4
7F3
Table 2
Characteristic IR bands (cmꢂ1) of the ligand L and the complex Tb2(L)3ꢀ5H2O.
Sample
v
(O–H)
v
(C@O)
v
(C@C)
v
(C@N)
v(C–O)
a
Fluorescence intensity.
L
3459
3422
1611
1597
1533
1517
1571
1573
1298 1263
1266
Tb2(L)3ꢀ5H2O
can be attributed to the
v
(O–H) and the enol-isomer
v
(C@O) vibra-
tion is at 1611 cmꢂ1. However, the keto-isomer
v(C@O) vibration at
1640 cmꢂ1 was not be observed, indicating that six-membered ring
was constructed by the intramolecular hydrogen bond generated
from the two carbonyl groups of the ligand [16]. In the complex
of Tb2(L)3ꢀ5H2O, the
v(C@O) peak of enol-isomer is shifted down-
field to 1597 cmꢂ1, which suggests that the carbonyl group may
participate in coordination reaction. In addition, high intensity
sharp bands are observed at 1517 cmꢂ1 and 1266 cmꢂ1 which are
attributed to
v(C@C) and v(C–O), respectively, indicating that the
enol-form anion of ligand is coordinated to Tb(III) ion [17]. The
v
(C@N) peak is slightly shifted (1571 cmꢂ1 for the ligand L,
1573 cmꢂ1 for the complex Tb2(L)3ꢀ5H2O), which suggests that pyr-
idine nitrogen atom doesn’t participate in coordination reaction.
The absorption band assigned to the coordinated Tb–O was found
at around 436 cmꢂ1. The band (O–H) vibration at 3422 cmꢂ1 is
v
Fig. 2. The excitation and emission spectra of Tb2(L)3ꢀ5H2O.
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, indicat-
ing that there was no coordinated water molecules [18].
The results of elemental analysis and IR spectroscopy indicated
that the enol-forms of the ligand L was only involved with the
coordination, and we suspected that the chemical structure of
complex may be as shown in Scheme 2.
The maximum excitation wavelength of the complex
Tb2(L)3ꢀ5H2O is 274 nm, due to the
pꢃ transition centered at
p–
the ligand L. The complex displays the characteristic line emission
of 4f–4f transition of Tb(III) ion, consisting of four main lines at
491 nm (5D4 ? 7F6), 546 nm (5D4 ? 7F5), 587 nm (5D4 ? 7F4), and
623 nm (5D4 ? 7F3) (Fig. 2). There is no trace emission from ligand
L (about 450 nm) in the complex, which shows that an efficient en-
ergy transfer in the complex system occurs from the singlet excited
state of the ligand L to the singlet excited state of b-diketone unit,
and followed by intersystem crossing to the excited triplet state of
b-diketone unit, where it is finally transferred to the central Tb(III)
ion. Therefore, Tb(III) ion emits characteristic luminescence. Due
3.3. Luminescence properties
The fluorescence characteristics data of the solid complex
Tb2(L)3ꢀ5H2O at room temperature are listed in Table 3. The corre-
sponding excitation and emission spectra are shown in Fig. 2.

Z. Zhang, R. Tang / Journal of Molecular Structure 1010 (2012) 116–122
119
to the presence of a scattering signal at 491 nm, and the 5D4 ? 7F5
emission is a magnetic dipole transition, which is less affected by
the ligand field, so the peak height at 546 nm for terbium was used
to measure the fluorescence intensities. The fluorescence emission
maximum at 546 nm is observed. The emission band (5D4 ? 7F5) is
obviously stronger than the other emission bands (5D4 ? 7F6,
5D4 ? 7F4, 5D4 ? 7F3) (Fig. 2). The typical narrow emission bands
of Tb(III) ion can be detected upon excitation of the ligand-cen-
tered absorption band, indicating that the ligand is a comparative
good organic chelator to sensitize fluorescence of Tb(III) ion.
interactions and radiationless energy transfer between BSA and the
complex Tb2(L)3ꢀ5H2O [23].
In order to shed light on the fluorescence quenching mecha-
nism, the fluorescence quenching data were analyzed by the
Stern–Volmer equation (Eq. (1)) [20]. The graphs plotted for
F0/F against [Q] from Eq. (1) at different temperatures are shown
in Fig. 4.
F0=F ¼ 1 þ kqs0½Qꢄ ¼ 1 þ Ks
v
½Qꢄ
ð1Þ
where kq, Ksv, s0 and [Q] are the biomolecular quenching rate con-
stant, the dynamic Stern–Volmer quenching constant, the average
biomolecular lifetime in the absence of quencher at about 10ꢂ8
s
3.4. Analysis of the interaction of complex with BSA
[24], and the concentration of quencher, respectively. F0 and F are
the fluorescence intensities in the absence and presence of quench-
er, respectively. The biomolecular quenching rate constant kq and
the dynamic Stern–Volmer quenching constant Ksv at pH 7.40 can
be obtained by the slope of the diagram at different temperatures,
which are given in Table 4. In general, for the dynamic quenching,
the quenching constant Ksv of the fluorescent complex increases
with the temperature due to faster diffusion and larger amounts
of collision quenching in the case of higher temperature. Further-
more, the maximum quenching rate constant kq of various kinds
of bimolecular is 2.0 ꢁ 1010 Mꢂ1 sꢂ1 [25]. The quenching constant
Ksv in Table 4 is inversely correlated with the temperature, and
the quenching rate constant kq is much larger than 2.0 ꢁ
1010 Mꢂ1 sꢂ1. This indicates that the quenching mechanism of fluo-
rescence of BSA by the complex is not initiated by dynamic collision
but the formation of a complex instead.
BSA has been studied as a model protein due to its structural
homology with human serum albumin [14]. There are three types
of fluorophores in BSA, namely tryptophan residues (Trp), tyrosine
residues (Tyr), and phenylalanine residues. Although both Trp and
Tyr residues can be excited at 280 nm, energy transfer effect at
280 nm mostly comes from Trp residues [19]. At the excitation
wavelength of 280 nm, BSA exhibits a strong fluorescence emission
with a peak at around 340 nm [20].
3.4.1. Analysis of fluorescence quenching of BSA by the complex
Fluorescence quenching is the decrease of the quantum yield of
fluorescence from a fluorophore induced by a variety of molecular
interactions with quencher molecules. Under the conditions of
fixed pH, temperature and ionic strength, fluorescence quenching
may result from exciting-state reaction, molecular rearrange-
ments, energy transfer, ground complex formation and collisional
quenching processes [21]. Fluorescence quenching can be dynamic
which results from collisional encounters between the fluorophore
and quencher, or static, owing to the formation of a ground state
complex between the fluorophore and quencher [22]. In general,
static and dynamic quenching can be distinguished from their dif-
ferent binding constants dependent on temperature and viscosity,
or preferably by lifetime measurements.
It is well known that UV–vis absorption measurement is a very
simple method and applicable to explore the structural change and
study the complex formation [26]. Therefore, to further confirm
the quenching mechanism induced by the complex, we studied
the difference of UV–vis absorption spectra of BSA in the absence
and presence of the complex.
Fig. 5 shows the effect of increasing concentrations of the com-
plex on the UV–vis absorption spectra of BSA. We can clearly see
The fluorescence spectra of BSA with increasing concentrations
of the complex at 298 K when excited at 280 nm are illustrated
in Fig. 3.
As shown in Fig. 3, the natural fluorescence of BSA at around
340 nm is gradually quenched by the increasing concentration of
the complex, while the complex Tb2(L)3ꢀ5H2O has no intrinsic fluo-
rescence in this range. Results above indicate that there are strong
Fig. 4. The Stern–Volmer curves of BSA quenched by Tb2(L)3ꢀ5H2O.
Table 4
The quenching and dissociation constants of BSA with Tb2(L)3ꢀ5H2O at different
temperatures.
T (K)
Ksv (L molꢂ1
)
kq (L molꢂ1 sꢂ1
)
R2
291
298
310
1.150 ꢁ 105
0.951 ꢁ 105
0.665 ꢁ 105
1.150 ꢁ 1013
0.951 ꢁ 1013
0.665 ꢁ 1013
0.99741
0.99745
0.99712
Fig. 3. Effect of Tb2(L)3ꢀ5H2O on fluorescence spectrum of BSA. T = 298 K, pH = 7.4,
kex = 280 nm. c(BSA) = 1.0 ꢁ 10ꢂ5 mol/L, c(Tb2(L)3ꢀ5H2O)/(10ꢂ5 mol/L), a–j: 0, 0.10,
0.15, 0.20, 0.30, 0.35, 0.40, 0.50, 0.75, 1.0.

120
Z. Zhang, R. Tang / Journal of Molecular Structure 1010 (2012) 116–122
Fig. 6. Double-log plots of Tb2(L)3ꢀ5H2O quenching effect on BSA fluorescence at
Fig. 5. Effect of Tb2(L)3ꢀ5H2O on UV–vis absorption spectra of BSA. T = 298 K,
pH = 7.4. c(BSA) = 1.0 ꢁ 10ꢂ5 mol/L, c(Tb2(L)3ꢀ5H2O)/(10ꢂ5 mol/L), a–f: 0, 0.1, 0.15,
0.2, 0.25.
different temperatures.
Table 5
that the absorption of BSA is increased regularly around 280 nm
with the addition of the complex, indicating that the complex
Tb2(L)3ꢀ5H2O has penetrated into the hydrophobic pocket of the
Trp residues, which inevitably result in the gradual exposure of
the Trp residues inside the hydrophobic cavities. It is responsible
for the gradual increase of absorption [27]. Research consistently
suggests the dynamic quenching only affects the excited state of
fluorophore and does not change the absorption spectrum. How-
ever, the formation of non-fluorescence ground state complex in-
duced the change of absorption spectrum of fluorophore [28]. It
is clear that the mechanism of fluorescence quenching was a static
quenching procedure.
The binding constants Ka, binding sites n, and the thermodynamic parameters at
different temperatures for Tb2(L)3ꢀ5H2O–BSA system.
T (K) Ka
(L molꢂ1
n
R2
D
H
D
S
DG
)
(kJ molꢂ1
)
(J molꢂ1 Kꢂ1
)
(kJ molꢂ1
)
291
298
310
66.9 ꢁ 105 1.38 0.99807 ꢂ147.03
20.6 ꢁ 105 1.26 0.99214 ꢂ147.03
1.5 ꢁ 105 1.02 0.99338 ꢂ147.03
ꢂ374.36
ꢂ374.36
ꢂ374.36
ꢂ38.09
ꢂ35.74
ꢂ31.25
3.4.2. Calculation of binding constants and binding sites
If the non-fluorescence ground state complex is formed
between BSA and quencher, the apparent binding constant Ka
(L molꢂ1) and binding sites n can be expressed by the following
equation [29]:
F
1
Ka
1
½Qꢄ
log
¼ log
þ n log
ð2Þ
F0 ꢂ F
where F0 and F are the fluorescence intensities before and after the
addition of the quencher and [Q] is the concentration of quencher.
Based on the measured fluorescence data at different temperatures,
the plots of log F/(F0 ꢂ F) versus log1/[Q] were obtained (Fig. 6). And
then binding constants Ka and binding sites n of the complex with
BSA obtained from the intercept and slope of the linear dependence
of logF/(F0 ꢂ F) on log1/[Q] were listed in Table 5. The correlation
coefficient was about 0.99, which proves that the interaction be-
tween the complex and BSA is in accordance with the site-binding
model underlined in the above equation. The binding constants Ka
were in the range of 105–106 L molꢂ1, which means that the com-
plex quenches the fluorescence emission of BSA with higher binding
affinity and experiences a static quenching procedure. The corre-
sponding binding sites n at the temperatures of 291 K, 298 K and
310 K were 1.38, 1.26 and 1.02, respectively, which roughly equal
to 1, indicating the interaction of the complex with BSA has only
a single binding site.
Fig. 7. Van’t Hoff plot for the interaction of BSA and Tb2(L)3ꢀ5H2O.
D
H and entropy change
D
S) were calculated from the Van’t Hoff
H ꢅ 0 and
H < 0 and
S < 0, Van
equation (Eq. (3)) at three different temperatures. If
S > 0, the main force is hydrophobic interaction; if
S > 0, the main force is electrostatic; if H < 0 and D
D
D
D
D
D
der Waals and hydrogen bond interactions play major role in the
reaction [31].
InKa ¼ ꢂ
D
H=RT þ
D
S=R
ð3Þ
where Ka is the effective binding constant at the corresponding
temperature and R is the universal gas constant. If the enthalpy
3.4.3. Thermodynamic parameters and nature of the binding forces
Research shows that the intermolecular forces between the
quencher and biomolecular mainly include hydrophobic forces,
electrostatic interactions, Van der Waals interactions and hydrogen
bonds, etc. [30]. The thermodynamic parameters (enthalpy change
change
studied,
1/T is shown in Fig. 7 The enthalpy change
S were obtained from the slope and intercept of the fitting curve,
D
D
H does not vary significantly over the temperature range
H can be regarded as a constant. The plot of lnKa versus
H and entropy change
D
D

Z. Zhang, R. Tang / Journal of Molecular Structure 1010 (2012) 116–122
121
Fig. 8. Effect of Tb2(L)3ꢀ5H2O on the synchronous fluorescence spectra of BSA. T = 298 K, pH = 7.4. c(BSA) = 1.0 ꢁ 10ꢂ5 mol/L, c(Tb2(L)3ꢀ5H2O)/(10ꢂ5 mol/L), a–j: 0, 0.10, 0.15,
0.20, 0.30, 0.35, 0.40, 0.50, 0.75, 1.0.
respectively. The free energy change can be obtained from the fol-
lowing relationship:
cence method and supplemented by UV–vis under simulative
physiological pH conditions. The fluorescence quenching results
showed that the intrinsic fluorescence of BSA was quenched
through static quenching mechanism. The binding constants and
the number of binding sites at different temperatures were also
obtained, which revealed the interaction of the complex with
BSA has only a single binding site. The values of thermodynamic
parameters and the synchronous fluorescence spectra confirmed
that the hydrophobic and hydrogen bond interactions played a
significant role in the formation of complex Tb2(L)3ꢀ5H2O–BSA
coordination compound. This study is expected to be useful to
understand the mechanism of interactions of the rare earth
complex binding to BSA and provide important information for
theoretical research and application in drug pharmacokinetic and
pharmacodynamic fields.
D
G ¼
D
H ꢂ T
D
S
ð4Þ
The values of thermodynamic parameters are presented in Ta-
ble 5. The H and S values are in accordance with the reported
literature [32]. The negative free energy change G suggests that
the binding process is spontaneous. The negative values of enthal-
py H and entropy S suggest that the binding is mainly enthalpy
D
D
D
D
D
driven and the entropy is unfavorable for it, hydrogen bonds and
van der Waals interactions played major roles in reaction [31].
3.4.4. Synchronous fluorescence spectra
The synchronous fluorescence spectra can provide much valu-
able information about the microenvironment in the vicinity of
the chromophore molecules [33]. According to Miller [34], distinc-
tion of the difference between excitation wavelength and emission
Acknowledgment
wavelength (
ure of chromophores. When
D
k = kemi ꢂ kexc) reflects the spectra of a different nat-
D
k values are stabilized at 15 or
This work was financially supported by the National Natural
Science Foundation of China (No. 21071152).
60 nm, the synchronous fluorescence of BSA is characteristic of
tyrosine or tryptophan residue [35]. The effect of Tb2(L)3ꢀ5H2O on
BSA synchronous fluorescence spectroscopy is shown in Fig. 8 It
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Dk = 15 nm and
D
k = 60 nm are both decreased more and more seriously with
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