Study of the interaction between 2,5-di-[2-(4-hydroxy-phenyl)ethylene]- terephthalonitril and bovine serum albumin by fluorescence spectroscopy

Article abstract of DOI:10.1016/j.saa.2011.02.013

A new compound, 2,5-di-[2-(4-hydroxy-phenyl)ethylene]-terephthalonitrile (DHPEPN), was synthesized. The interaction between bovine serum albumin (BSA) and DHPEPN in Tris-HCl buffer solution (pH 7.4) was investigated using fluorescence and UV-vis absorption spectroscopy. The mechanism of BSA fluorescence quenched by DHPEPN is discussed according to the Stern-Volmer equation. The binding constant and the thermodynamic parameters ΔH, ΔS, ΔG at different temperatures were calculated. The results indicate that the van der Waals interaction and hydrogen bonding play major roles in the binding process. The distance between BSA and DHPEPN is estimated to be 3.59 nm based on the Fo?rster resonance energy transfer theory. The spectral changes of synchronous fluorescence and three-dimensional fluorescence suggest that both of the microenvironment of DHPEPN and the conformation of BSA are changed during binding between DHPEPN and BSA.

Full text of DOI:10.1016/j.saa.2011.02.013

Spectrochimica Acta Part A 79 (2011) 97–103
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Study of the interaction between
2,5-di-[2-(4-hydroxy-phenyl)ethylene]-terephthalonitril and bovine serum
albumin by fluorescence spectroscopy
Li-Na Zhang, Fang-Ying Wu∗, Ai-Hong Liu
Department of Chemistry/Center of Analysis and Testing, Nanchang University, Nanchang 330031, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new compound, 2,5-di-[2-(4-hydroxy-phenyl)ethylene]-terephthalonitrile (DHPEPN), was synthe-
sized. The interaction between bovine serum albumin (BSA) and DHPEPN in Tris–HCl buffer solution
(pH 7.4) was investigated using fluorescence and UV–vis absorption spectroscopy. The mechanism of
BSA fluorescence quenched by DHPEPN is discussed according to the Stern–Volmer equation. The binding
constant and the thermodynamic parameters ꢀH, ꢀS, ꢀG at different temperatures were calculated. The
results indicate that the van der Waals interaction and hydrogen bonding play major roles in the binding
process. The distance between BSA and DHPEPN is estimated to be 3.59 nm based on the Förster reso-
nance energy transfer theory. The spectral changes of synchronous fluorescence and three-dimensional
fluorescence suggest that both of the microenvironment of DHPEPN and the conformation of BSA are
changed during binding between DHPEPN and BSA.
Received 5 November 2010
Received in revised form 23 January 2011
Accepted 7 February 2011
Keywords:
2,5-Di-[2-(4-hydroxy-phenyl)ethylene]-
terephthalonitrile
Bovine serum albumin
Fluorescence quenching
Thermodynamic parameters
Three-dimensional fluorescence spectrum
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
requires high fluorescence quantum yield and long excitation and
emission wavelengths. Consequently, the construction of highly
Investigations into the interaction between small molecular
drugs and proteins have been attracting much attention because
proteins play a fundamental role in sustaining life and are closely
related to metabolism, the origin and evolution of life [1,2]. Serum
albumin is the most abundant and the main transport protein in
blood plasma. And it plays an important role in transporting and
metabolizing of many endogenous and exogenous compounds in
metabolism [3]. Thus study on the small molecular to serum albu-
min is of great importance. Bovine serum albumin (BSA) is usually
selected as the protein model, because of its abundance, low cost,
ease purification, stability, medical importance and ligand-binding
properties. Moreover all the study results are consistent with
the fact that human and bovine serum albumins are homologous
proteins [4]. Therefore, the investigations of interaction between
proteins and drugs are mostly concentrated on bovine serum albu-
mins.
efficient fluorescent compound at the desired wavelength region
for sensing events is very challenging [9]. Stilbenes derivatives
which were conjugated with different electron donor or accep-
tor groups usually possessed good optical property like longer
emission and applied for several sensing events [10]. Moreover,
stilbenes derivatives have shown some merits such as protection
lipoproteins from oxidative damage and cancer chemopreventive
activity [11,12]. Therefore, synthesis of stilbenes derivatives
shows increasing concern. Wang et al. [13] synthesized a novel
distyrylbenzene derivative containing 1-oxa-4,10-dithia-7-aza-
cyclododecane whose excitation and emission wavelengths were
460 and 600 nm respectively, and it could sense Ag+ and Zn2+
.
Liu et al. [14] synthesized 2-[(E)-4-(dimethylamino)styryl)]-
5-[(E)-4-aminostyrul)terephthalonitrile] whose excitation and
emission wavelengths were 460 nm and 540 nm respectively,
and it was used for sensing anti-BSA. Ahn and co-workers
[15] synthesized 1-{4-[N,N-bis(2-pyridyl)amino]styryl}-4-[4-
(diphenylamino)stryl]-2,5-dicyanobenzene for sensing metal ions,
which emitted fluorescence of 610 nm excited at 425 nm. Pond
et al. [16] synthesized a serial of donor-acceptor-donor structure
compounds possessing one or two mono-aza-15-crown-5 ether.
These compounds showed selective response to Mg2+ based on
the fluorescence change of 625 nm.
Owing to proteins possessing intrinsic fluorescence and the
advantages of fluorescent technique such as high sensitivity,
convenience, and on-site or in situ analyses [5,6], fluorescent
assay has been extensively used for investigation of proteins
[7,8]. As an ideal fluorescent probe for sensing biomolecular, it
Up to now, stilbenes derivative which used to identify proteins
have reported rarely [17]. Herein a new stilbenes derivative
∗
Corresponding author. Tel.: +86 7913969882.
E-mail address: fywu@ncu.edu.cn (F.-Y. Wu).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.02.013

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L.-N. Zhang et al. / Spectrochimica Acta Part A 79 (2011) 97–103
CN
Br
CN
Br
a
b
c
Br
Br
NC
NC
CN
CN
e
HO
d
(EtO) OP
2
OH
PO(OEt)
2
NC
NC
DHPEPN
Scheme 1. The synthesis route for DHPEPN. Reagents and conditions: (a) Br2, CH2Cl2, 12 ◦C, 18 h; (b) CuCN, DMF, refluxed, 48 h; (c) NBS, AIBN, hꢁ, refluxed, 5 h; (d) P(OEt)3,
160◦C, 18 h; (e) (i) KOtBu, THF, 4-formylphenyl acetate, r.t., 6 h; (ii) H2O, r.t.
solution of 1.0 × 10−4 mol L−1 was prepared by directly dissolving
(2,5-di-[2-(4-hydroxy-phenyl)ethylene]terephthalonitrile,
DHPEPN) was prepared and its wavelengths of emission/excitation
are 505/394 nm in methanol which meet the demands as biomolec-
ular probe. Meanwhile the hydroxy group provides the binding
site with protein. Thus DHPEPN is potential candidate for sensing
protein.
in the 5.0 × 10−2 mol L−1 NaCl solution. Tris (Sigma Chemical Co.)
buffer had a pH of 7.4 containing 5.0 × 10−2 mol L−1 NaCl. DMSO is
of chromatographic grade. Other chemicals are of analytical grade.
Doubly distilled water was used throughout. All testing aqueous
solution contained 1% (v/v) DMSO for fluorescence determination
and 20% (v/v) DMSO for absorption determination.
2. Experimental
2.3. Procedures
2.1. Apparatus
2.3.1. Determination of quantum yield
All fluorescence measurements were carried out on a F-
4600 spectrofluorimeter (Hitachi, Japan) equipped with a xenon
lamp source and a 1.0 cm quartz cell, and the scan speed was
1200 nm min−1. Absorption spectra were recorded on a Shimadzu-
2550 UV–vis spectrophotometer (Shimadzu, Japan) using a 1.0 cm
quartz cell. All pH measurements were measured with a pHS-3C
digital pH-meter (Shanghai REX Instrument Corp., China) with a
combined glass-calomel electrode. 1H NMR spectra were carried
out in DMSO-d6 on a Bruker Avance 600 MHz NMR spectrome-
ter using TMS as the internal standard. ESI mass spectrum was
recorded using a Waters ZQ4000/2695 LC-MS spectrometer (USA).
Infrared spectra were obtained as KBr pellets on a Nicolet 5700 FTIR
spectrometer.
Fluorescence-quantum yield of DHPEPN in different organic sol-
vents is determined by using quinoline sulfate with 0.5 mol L−1
H2SO4 solution (˚ = 0.546) as a standard [21].
2.3.2. Fluorescence titration of DHPEPN against BSA
20 ␮L DHPEPN was added to a quartz cell and diluted to 2.0 mL
with Tris–HCl buffer, different amounts of BSA were added using
sample injector. The fluorescence spectra from 370 to 550 nm of
the above solutions at different temperatures were collected with
the excitation wavelength at 360 nm.
2.3.3. Fluorescence titration of BSA against DHPEPN
20 ␮L BSA was added to a quartz cell and was diluted to 2.0 mL
with Tris–HCl buffer and mixed, different amounts of DHPEPN were
added using sample injector. The fluorescence spectra from 290
to 550 nm of the above solutions at different temperatures were
collected with the excitation wavelength at 280 nm.
2.2. Materials
The compound DHPEPN was prepared according to the Refs.
[18–20] and the route for synthesis is shown in Scheme 1.
Tetraethyl-(2,5-dicyano-␣,␣ꢀ-p-xylylenediphosphonate (676 mg,
1.6 mmol), potassium tert-butoxide (359 mg, 3.2 mmol) and 4-
formylphenyl acetate were dissolved in tetrahydrofuran (15 mL)
in 250 mL flask and kept 6 h at room temperature under nitro-
gen. The distilled water (75 mL) was added to the reaction mixture,
and the mixture was extracted with dichloromethane. The organic
layer was washed three times with water and dried with magne-
sium sulfate. Organic solvent was reduced via rotary-evaporation
and yellow precipitate was formed. Finally, a yellow solid was
obtained and separated by column chromatography on silica gel.
Compound DHPEPN was characterized by 1H NMR, IR and Mass
data. The results were assigned as follows: 1H NMR (600 MHz,
DMSO–d6), ı (ppm): 9.98(s, 2H), 8.46(s, 2H), 7.63(d, 16.2 Hz, 2H),
7.49(d, 8.40 Hz, 4H), 7.09(d, 16.2 Hz, 2H), 6.85(d, 8.40 Hz, 4H). ESI-
MS: m/z calcd for [M − H]− 363.12, found [M − H]+ 362.88. IR:
2.3.4. Synchronous fluorescence spectroscopy
20 ␮L BSA was added to a quartz cell and was diluted to 2.0 mL
with Tris–HCl buffer and mixed, different amounts of DHPEPN were
added using sample injector. The fluorescence spectra were col-
lected with the ꢀꢂ = 15 nm or ꢀꢂ = 60 nm.
2.3.5. Three-dimensional fluorescence spectroscopy
20 ␮L BSA was added to a quartz cell and was diluted to 2.0 mL
with Tris–HCl buffer and mixed, 20 ␮L DHPEPN was added using
sample injector. The three-dimensional fluorescence spectra were
collected with setting excitation wavelength from 200 to 450 nm,
emission wavelength from 200 to 520 nm and increment 5 nm.
2.3.6. Absorption titration of DHPEPN against BSA
3361(ꢁO–H), 2243(ꢁ–CN), 1631(ꢁC C), 1602, 1514, 1478, 1440(ꢁC
Ph-), 1167(ꢁC–O).
Compound DHPEPN was prepared into a concentration of
1.0 × 10−4 mol L−1 in dimethyl sulphoxide (DMSO). Bovine serum
albumin (BSA) was purchased from Beijing Solarbio Co. Ltd. BSA
,
C
600 ␮L DHPEPN was added to a quartz cell and was diluted to
3.0 mL with Tris–HCl buffer and mixed, different amounts of BSA
were added using sample injector. Absorption spectra of the above
solutions were collected.

L.-N. Zhang et al. / Spectrochimica Acta Part A 79 (2011) 97–103
99
Fig. 2. Fluorescence spectra of DHPEPN (1.0 × 10−6 mol L−1) in the presence and
absence of BSA (ꢂex = 360 nm). [BSA] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.5, 5.5, 6.5,
7.5, 19.5 × 10−7 mol L−1. Inset plot shows the linear calibration curve between fluo-
rescent intensity of DHPEPN and the concentration of BSA.
Fig. 1. Fluorescence spectra of DHPEPN in different organic solvents.
3. Results and discussion
3.3. Fluorescence spectral change of BSA upon addition of
DHPEPN
3.1. Spectral parameters of DHPEPN in organic solvents
3.3.1. Fluorescence quenching of BSA
The fluorescence spectra ofDHPEPNin different organicsolvents
were collected and are shown in Fig. 1. The spectral parameters of
DHPEPN are summarized and presented in Table 1. As can be seen
from Fig. 1 and Table 1, the emission spectra of DHPEPN were red
shifted from 460 nm (in diethyl ether, DEE) to 505 nm (in methanol,
MeOH) with increasing polarity of solvents and the quantum yield
decreased. It implies that DHPEPN is sensitive to the polarity of
solvent and is expected to act as fluorescent probe for biomolecule
like protein.
It is well known that BSA emits a strong fluorescence peaked
at 342 nm with excitation wavelength of 280 nm. Viewed from
Fig. 3, the presence of DHPEPN led the emission intensity of BSA to
decrease coupled with slight blue shift of the maximum wavelength
from 342 to 338 nm. The quench of BSA’s intrinsic fluorescence and
the enhancement of DHPEPN’s fluorescence ca. 470 nm demon-
strate the efficient energy transfer occurs between DHPEPN and
BSA. The appearance of a clear isoemissive point at 436 nm further
suggests complex formation.
3.3.2. Synchronous fluorescence spectral change of BSA
3.2. Fluorescent titration of DHPEPN against BSA
The fluorescence of BSA comes from the tyrosine and tryp-
tophan residues. When ꢀꢂ between excitation wavelength and
emission wavelength is set as 15 nm, the synchronous fluorescence
The fluorescent titration of DHPEPN with BSA was carried
out in pH 7.4 Tris–HCl aqueous solution and the results are
presented in Fig. 2. With increasing the amount of BSA, the
fluorescence intensity of DHPEPN was enhanced and emission
band slightly blue shifted. The results suggest that the polar-
ity of microenvironment of DHPEPN decrease, which means that
DHPEPN had entered the hydrophobic microenvironment of BSA.
Thus an interaction between DHPEPN and BSA occurred. At the
same time a good linear response of fluorescence intensity as a
function of BSA concentration was obtained. The linear equation is
IF460 nm = 9.96 + 8.91CBSA × 10−7 (n = 7, R = 0.996) while the concen-
tration of BSA ranges from 0 to 3.0 × 10−7 mol L−1 and the detection
limit is 1.07 × 10−8 mol L−1
.
Table 1
Spectral parameter of DHPEPN in different organic solvents.
f
Solvent
ꢂmax (nm)
ꢂem (nm)
˚
DEEa
EAb
395
397
395
393
394
460
470
488
496
505
0.53
0.43
0.39
0.36
0.21
c
CHCl3
CH3CNd
CH3OHe
Fig. 3. Fluorescence spectra of BSA (1.0 × 10−6 mol L−1) at different concentrations
of DHPEPN (T = 298 K, ꢂex = 280 nm). [DHPEPN] = 0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.2, 4.0,
(a) Diethyl ether; (b) ethyl acetate; (c) chloroform; (d) acetonitrile; (e) methanol;
(f) fluorescence quantum yield.
5.4, 7.2 × 10−6 mol L−1
.

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L.-N. Zhang et al. / Spectrochimica Acta Part A 79 (2011) 97–103
Fig. 5. The quenching degree of BSA (1.0 × 10−6 mol L−1) synchronous fluorescence
by DHPEPN. [DHPEPN] = 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.5, 4.5, 6.1, 7.8, 9.5 × 10−6 mol L−1
In a three-dimensional fluorescence spectrum, the excitation wave-
length, the emission wavelength and the fluorescence intensity can
be observed.
The three-dimensional fluorescence spectra of BSA and
DHPEPN–BSA complex are shown in Fig. 6, and the characteristic
parameters are summarized in Table 2. From Fig. 6, peak 1 reveals
the spectral characteristic of tryptophan and tyrosine residues,
peak a and peak b are the rayleigh scattering peak (ꢂex = ꢂem) and
the second-ordered scattering peak (ꢂem = 2ꢂex) respectively [24].
After the addition of DHPEPN, the fluorescence intensity of BSA
decreased from 2522 to 2360. This suggests a less polar environ-
ment of both residues and almost all the hydrophobic amino acid
residues of BSA were buried in the hydrophobic pocket. Less polar
environment means that the binding position between DHPEPN
and BSA located within this hydrophobic pocket, the addition of
DHPEPN changed the polarity of this hydrophobic microenviron-
ment and the conformation of BSA [25].
Besides, peak 2 mainly exhibits the fluorescence spectra behav-
ior of polypeptide backbone structures, which is caused by the
transition of ꢃ−ꢃ* of BSA’s characteristic polypeptide backbone
structure C O [25]. After the addition of DHPEPN, peak 2 dra-
matically decreased with the increasing concentration of DHPEPN.
The fluorescence intensity of BSA decreased from 2210 to 453.8.
The result indicates that the interaction between DHPEPN and BSA
induced the unfolding of the polypeptides of BSA and conforma-
tional change of the BSA [26]. These phenomenon and analyses
results reveal that the binding of DHPEPN to BSA induced confor-
mational and microenvironment changes in BSA.
Fig. 4. (a) Synchronous fluorescence spectra of BSA (1.0 × 10−6 mol L−1) in the
present of different amount of DHPEPN with ꢀꢂ = 15 nm. [DHPEPN] = 0, 0.5, 1.0,
1.5, 2.0, 2.5, 3.5, 4.5, 6.1, 7.8, 9.5 × 10−6 mol L−1. (b) Synchronous fluorescence spec-
tra of BSA (1.0 × 10−6 mol L−1) in the present of different amount of DHPEPN with
ꢀꢂ = 60 nm, [DHPEPN] = 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.5, 5.2, 6.1, 7.8,
9.5 × 10−6 mol L−1
.
spectrum gives the characteristic information of tyrosine residues
in BSA. When ꢀꢂ is 60 nm, it gives the characteristic of the trypto-
phan residues [22]. The shift of the maximum emission can prove
the changes of the residues’ surrounding environment in polarity.
Red shift of fluorescence spectrum suggests the polarity of the sur-
rounding environment increasing, the hydrophobicity decreasing
and BSA molecule extending and vice versa [23].
Synchronous fluorescence spectral changes of BSA upon addi-
tion of DHPEPN are presented in Figs. 4 and 5. Viewed from Fig. 4,
the maximum emission peak of tyrosine residues (ꢀꢂ = 15 nm) and
tryptophan residues (ꢀꢂ = 60 nm) had a slight shift. However, from
Fig. 5, we could see obviously that the pitch of quenching at ꢀꢂ
60 nm is higher than that of ꢀꢂ 15 nm. Such result means DHPEPN
is closer to tryptophan residues than tyrosine residues, namely
binding sites mainly are focused on tryptophan moiety.
Table 2
Three-dimensional fluorescence spectra characteristic parameters of the BSA and
DHPEPN–BSA complex.
Systems
BSA
Parameters
Peak 1
Peak 2
Peak position (ꢂex/ꢂem, nm/nm)
Relative intensity
Stokes shift (ꢀꢂ/nm)
278.0/353.5
2522
75.5
226.0/358.5
2210
132.5
DHPEPN–BSA
Peak position (ꢂex/ꢂem, nm/nm)
Relative intensity
Stokes shift (ꢀꢂ/nm)
278.0/353.5
2360
75.5
236.4/358.5
453.8
122.1
3.3.3. Three-dimensional fluorescence spectral change of BSA
The three-dimensional fluorescence spectrum is another pow-
erful method for the studying of conformation change of BSA [24].

L.-N. Zhang et al. / Spectrochimica Acta Part A 79 (2011) 97–103
101
Fig. 7. Stern–volmer plots for the quenching of BSA by DHPEPN at different tem-
peratures.
results show that the values of Kq were much larger than the maxi-
mum scattering collision quenching constant 2.0 × 1010 L mol−1 s−1
[28]. This suggests that the quenching mechanism between
DHPEPN and BSA maybe a static quenching [27].
The UV–vis spectral measurement can distinguish static
quenching and dynamic quenching. Dynamic quenching only
affects the excited states of the fluorophores, and there are no
changes in the absorption spectra. However, ground-state complex
formation will frequently result in perturbation of the absorp-
tion spectrum of the fluorophore [4,27]. From Fig. 8, in the range
of 300–500 nm, the absorbance of DHPEPN was increased upon
addition of BSA. The result implies that an interaction between
DHPEPN and BSA occurs. In other words, the fluorescence quench-
ing between DHPEPN and BSA is mainly ascribed to be static
quenching.
3.4.2. Energy transfer between DHPEPN and BSA
According to Förster’s resonance energy transfer theory, the
effective energy transfer from donor to acceptor can happen under
the following conditions: (1) the donor can produce fluorescence
light; (2) the fluorescence emission spectrum of the donor and the
Fig. 6. Three-dimensional fluorescence spectra of BSA(A) and BSA-DHPEPN(B). Both
concentrations of BSA and DHPEPN are 1.0 × 10−6 mol L−1
.
3.4. Mechanism of interaction between DHPEPN and BSA
3.4.1. Mechanism of fluorescence quenching
Generally speaking, fluorescence quenching can classified as
static quenching, dynamic quenching, and static and dynamic
quenching participating in simultaneously. In order to prove the
fluorescence quenching mechanism of BSA by DHPEPN, the pro-
cedure of the fluorescence quenching is assumed to be a dynamic
quenching process first. For the dynamic quenching, the mecha-
nism is usually analyzed via the classical Stern–Volmer equation
[27]:
F0
= 1 + Kqꢄ0[Q] = 1 + Ksv[Q]
F
where F0 and F are the fluorescence intensities of BSA before and
after addition of quencher, respectively, KSV is the Stern–Volmer
quenching constant; and [Q] is the concentration of the quencher;
ꢄ0 is the average fluorescence lifetime of biomolecular without
addition of quencher which is considered as 10−8 s [27].
The Stern–Volmer plots at different temperatures are shown in
Fig. 7. The values of Kq at different temperatures (298, 303 and
308 K) are estimated as 1.80, 2.41 and 2.62 × 1013 L mol−1 s−1. These
Fig. 8. UV absorption spectra of DHPEPN (2.0 × 10−5 mol L−1) upon addition of BSA.
Curve A shows the absorption spectrum of BSA (2.0 × 10−5 mol L−1). Curves from B
to G show the absorption spectra of DHPEPN in the presence and absence of BSA, and
the concentrations of BSA are 0, 1.0, 2.0, 3.0, 4.0, and 5.0 × 10−5 mol L−1 respectively.

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L.-N. Zhang et al. / Spectrochimica Acta Part A 79 (2011) 97–103
where Kb is the binding constant, n is the number of binding sites
per BSA.
According to the plot of lg(F0 − F)/F vs. lg[DHPEPN] at different
temperatures, the values of binding constants Kb are estimated as
11.5, 0.46, and 0.068 × 105 L mol−1and the binding sites n are 1.14,
0.87, and 0.71 respectively in different temperatures (298, 303 and
308 K). The values of Kb decreased with increasing temperature
which was assumed to decrease stability of DHPEPN–BSA complex.
And the values of n decreased with increasing temperature, which
might be due to changes in the protein structure with temperature
[23].
3.4.4. Thermodynamic parameters and nature of the binding
forces
The interaction forces between proteins and ligands may involve
hydrophobic force, electrostatic interaction, Van der Waals inter-
action, hydrogen bonds, etc. [32]. According to the views of Ross
and Subramanian [33], the relationship between acting force and
thermodynamic parameters can describe as follows: (1) ꢀH > 0 and
ꢀS > 0, hydrophobic forces, (2) ꢀH < 0 and ꢀS < 0, Van der Waals
interaction and hydrogen bond, (3) ꢀH < 0 and ꢀS > 0, electro-
static interactions. The values of the enthalpy change (ꢀH) and the
entropy change (ꢀS) can be estimated from the following relation-
ship:
Fig. 9. Spectral overlap between the fluorescence spectrum of BSA
(a) (1.0 × 10−6 mol L−1
)
and the absorption spectrum of DHPEPN (b)
(1.0 × 10−6 mol L−1).
UV–vis absorbance spectrum of the acceptor have overlap; (3) the
distance between the donor and the acceptor approaches is within
2–8 nm [29].
ꢀH
RT
ꢀS
R
ln Kb = −
+
R06
R06 + r6
F
E = 1 −
=
where Kb is binding constant at the different temperatures, R is the
gas constant, T is the experimental temperature.
F0
From the van’t Hoff plots between log K and 1/T, the values
of enthalpy change ꢀH and entropy change ꢀS were obtained as
−392.5 kJ mol−1and −1.20 kJ mol−1 K, respectively. The free energy
changes ꢀG in different temperature are estimated as −34.35
(298 K), −28.34 (303 K), −22.33 (308 K). The values of ꢀG were
negative at all experimental temperature, indicating that the reac-
tion is spontaneous. The values of ꢀH and ꢀS were negative at the
experimental temperature, which suggests that the Van der Waals
interaction and hydrogen bond play major roles in the binding pro-
cess.
where F0 is the fluorescent intensity of BSA; F is the fluorescent
intensity of BSA upon addition of DHPEPN when the concentration
of which is equal to BSA; R0 is the distance at which 50% of the
energy is transferred and is a function of the spectral overlap of the
donor emission and acceptor absorption. And R0 is calculated using
the following equation:
R06 = 8.79 × 10−25 K2
N
−4 ꢅJ
where K2 is the spatial orientation factor related to the geometry
of the donor and acceptor of dipoles and K2 = 2/3; N is the aver-
age refractive index of medium and N = 1.336; ϕ is the fluorescence
quantum yield of the donor and ϕ = 0.15 [30]; J is the effect of spec-
tra overlap between the fluorescence emission spectrum of the
donor and the UV–vis absorbance spectrum of the acceptor, and
J can calculated by the equation:
4. Conclusions
In this paper,
a new compound, 2,5-di-[2-(4-hydroxy-
phenyl)ethylene]-terephthalonitrile (DHPEPN) is synthesized
and used as fluorescent probe for protein. Upon addition of
DHPEPN into BSA solution, the fluorescence of DHPEPN enhanced
whereas the emission of BSA decreased, which was attributed
to energy transfer between them. The quenching mechanism of
BSA is mainly belonged to static quench, meanwhile that dynamic
quenching can not be ignored. Thermodynamic parameters sug-
gest that the interaction forces between BSA and DHPEPN are the
Van der Waals interaction and hydrogen bond.
ꢀ ∞
F(ꢂ)ε(ꢂ)ꢂ4dꢂ
0
J =
ꢀ
∞ F(ꢂ)dꢂ
0
where F(ꢂ) is the corrected fluorescence intensity of the donor in
the wavelength range from ꢂ to ꢂ + ꢀꢂ, ε(ꢂ) is the molar absorption
coefficient of the acceptor at wavelength ꢂ. The spectral over-
lap between the fluorescence emission spectrum of BSA and the
UV–vis absorbance spectrum of DHPEPN are shown in Fig. 9. From
the equations above, we could calculate the values of following
parameters, J = 1.44 × 10−14 cm3 L mol−1, R0 = 2.57 nm, r = 3.59 nm
(2 < r < 8 nm) [29]. The results suggest that effective energy transfer
between BSA and DHPEPN happened.
Acknowledgement
This work is supported by Natural Science Foundation of China
(no. 20965006).
3.4.3. Binding constant and binding sites
References
When a small quencher binds independently to a set of equiva-
lent sites on a fluorescent macromolecule, the equilibrium between
binding constant (Kb) and the number of binding sites (n) is given
by the following equation [31]:
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approach, J. Lumin. 129 (2009) 34–39.
[2] E. Chambers, E.M. Hoey, A. Trudgett, I. Fairweather, D.J. Timson, Binding of
serum albumin to the anthelmintic drugs albendazole, triclabendazole and
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