Spectroscopic studies on the interaction of morin-Eu(III) complex with calf thymus DNA

Article abstract of DOI:10.1016/j.molstruc.2009.02.011

The interaction between morin-Eu(III) complex and calf thymus DNA in physiological buffer (pH 7.4) was investigated using UV-vis spectrophotometry, fluorescence spectroscopy, viscosity measurements and DNA melting techniques. Hypochromicity and red shift

Full text of DOI:10.1016/j.molstruc.2009.02.011

Journal of Molecular Structure 923 (2009) 114–119
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Journal of Molecular Structure
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Spectroscopic studies on the interaction of morin–Eu(III) complex with
calf thymus DNA
*
Guowen Zhang , Jinbao Guo, Junhui Pan, Xiuxia Chen, Junjie Wang
State Key Laboratory of Food Science and Technology, Nanchang University, 235# Nanjing East Road, Nanchang 330047, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The interaction between morin–Eu(III) complex and calf thymus DNA in physiological buffer (pH 7.4) was
investigated using UV–vis spectrophotometry, fluorescence spectroscopy, viscosity measurements and
DNA melting techniques. Hypochromicity and red shift of the absorption spectra of morin–Eu(III) com-
plex were observed in the presence of DNA, and the fluorescence intensity of morin–Eu(III) complex
was greatly enhanced with the addition of DNA. Moreover, fluorescence quenching and blue shift of
the emission peak were seen in the DNA–ethidium bromide (EB) system when morin–Eu(III) complex
was added. The relative viscosity of DNA increased with the addition of morin–Eu(III) complex, whereas
the value of melting temperature of DNA–EB system decreased in the presence of morin–Eu(III) complex.
All these results indicated that morin–Eu(III) complex can bind to DNA and the major binding mode is
Received 19 November 2008
Received in revised form 6 February 2009
Accepted 13 February 2009
Available online 24 February 2009
Keywords:
DNA
Morin–Eu(III) complex
Spectroscopy
Interaction
intercalative binding. The 3:1 morin:Eu(III) complex (estimated binding constant = 2.36 ꢀ 106 L molꢁ1
)
is stabilized by intercalation into the DNA. The calculated binding constants of morin–Eu(III) complex
Ethidium bromide (EB)
with DNA at 292, 301 and 310 K were 7.47 ꢀ 104, 8.89 ꢀ 104 and 1.13 ꢀ 105 L molꢁ1, respectively. The
thermodynamic parameters were also obtained:
D D
Hh was 20.14 kJ molꢁ1 > 0 and Sh was
161.70 J molꢁ1 Kꢁ1 > 0, suggesting that hydrophobic force plays a major role in the binding of morin–
Eu(III) complex to DNA.
Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction
inflammatory, antioxidant, anticancer and cardiovascular protec-
tion [10–12].
The interaction of drug molecules with DNA has become an
active research area in recent years [1]. Because the intracellular
target for a wide range of anticancer and antibiotic drugs is DNA
[2–4], illustrating the binding between small molecules and DNA
can greatly help understand drug–DNA interactions and design
new and promising drugs for clinical use. Generally, a variety of
small molecules interacts reversibly with DNA, primarily through
three modes: (i) intercalative binding that small molecules interca-
late into the base pairs of nucleic acids [5]; (ii) groove binding in
which the small molecules bound on nucleic acids are located in
the major or minor groove [5]; (iii) long-range assembly on the
molecular surfaces of nucleic acids so that the small molecules
are not related to the groove structure of the nucleic acids [6].
The intercalative binding is stronger than other two binding modes
because the surface of intercalative molecule is sandwiched be-
tween the aromatic, heterocyclic base pairs of DNA [7,8].
Morin is known to complex with many metal ions to form sta-
ble compounds. In recent years, tremendous interest has been
drawn to interactions between transition metal complexes of mor-
in and nucleic acids due to potential applications of the metal com-
plexes as anticancer drugs or as complexes with other biological
functions [13,14]. Some authors reported that morin, Zn(II) and
Cu(II) complexes of morin can bind to DNA, respectively, but the
binding mode is different. The complexes bind to DNA mainly by
intercalating mode, while morin binds in a non-intercalating mode
[15,16]. Moreover, the complexes show higher antitumor activities
than that of morin [17]. Rare earth metals not only have more
physiological activities, but also their toxicities are decreased after
coordinating with a ligand [18]. Zhou et al. found that the antitu-
mour activities of rare earth metal complexes of quercetin with
La(III), Eu(III) and Gd(III) are superior to quercetin, the major bind-
ing mode of quercetin–La(III) complex with DNA is intercalative
binding [19]. Rare earth metals and their complexes as chemical
nucleases are superior to transition metals and their complexes be-
cause they can bind to nucleic acid more efficiently by hydrogen
bonding and hydrolyzed mode [20]. Whereas, so far the interac-
tions between rare earth metal complexes of morin with DNA have
seldom been reported.
Morin(20,3,40,5,7-pentahydroxyflavone, Fig. 1), a flavonoid com-
pound, is present in tea, coffee, cereal grains, fruits, vegetables and
many traditional Chinese herbal medicines [9]. It has been shown
in various biological and pharmacological activities, including anti-
* Corresponding author. Tel.: +86 7918305234.
E-mail address: gwzhang@ncu.edu.cn (G. Zhang).
0022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2009.02.011

G. Zhang et al. / Journal of Molecular Structure 923 (2009) 114–119
115
(EB) stock solution (1.34 ꢀ 10ꢁ3 mol Lꢁ1) was prepared by dissolv-
ing its crystals (Sigma Chem. Co., USA) in doubly distilled water
and stored in a cool and dark place. All the solutions were adjusted
with the Tris–HCl buffer solution (0.05 mol Lꢁ1, pH 7.4), which was
prepared by mixing and diluting 25 mL of Tris solution
(0.2 mol Lꢁ1) with 45 mL of HCl (0.1 mol Lꢁ1) to 100 mL. All chem-
icals were of analytical reagent grade, and doubly distilled water
was used for the preparation of the test solutions.
HO
OH
4'
2'
B
HO
O
C
7
A
3
5
OH
OH
O
2.3. Procedures
Fig. 1. Molecular structure of morin.
2.3.1. Interaction between morin and Eu(III)
A number of techniques have been employed to study the inter-
action of drugs with DNA, including fluorescence spectroscopy
[21], UV-spectrophotometry [22], electrophoresis [23], nuclear
magnetic resonance [24], electrochemical methods [25], etc. UV–
vis absorption and fluorescence spectroscopy are regarded as effec-
tive methods among these techniques because they are sensitive,
rapid and simple [26].
The molar ratio method introduced by Yoe and Jones [29] al-
lows us to deduce the composition of complexes in solution from
the spectrophotometric spectra. For this purpose, solutions con-
taining a constant concentration (4.2 ꢀ 10ꢁ5 mol Lꢁ1) of morin
and variable concentration of Eu(III) (from 1.5 ꢀ 10ꢁ6 mol Lꢁ1 to
8.4 ꢀ 10ꢁ5 mol Lꢁ1) were prepared.
In this work, we used UV–vis absorption, fluorescence spectros-
copy, viscosity measurements and DNA melting techniques to ex-
plore the interaction between morin–Eu(III) complex and calf
thymus DNA. We believe this will be helpful to further understand
the mechanism of interactions between DNA and morin’s rare
earth metal complexes as well as further understand morin’s phar-
macological effects. The knowledge gained from this study should
be useful for the development of potential probes for DNA struc-
ture and new therapeutic reagents for tumours and other diseases.
2.3.2. Binding ofmorin–Eu(III) complex with DNA in the presence of EB
An aliquot from each of the DNA solutions in a concentration
series, was added to a 2.0 mL solution mixture consisting of morin
at a given concentration and Eu(III) in a 3:1 ratio adjusted by the
Tris–HCl buffer (pH 7.4). Each sample mixture was combined in a
10 mL volumetric flask, and diluted to the mark with doubly dis-
tilled water. After these solutions were allowed to stand for
10 min to equilibrate, the UV–vis measurement was then made.
The competitive interaction between EB and morin–Eu(III) com-
plex with DNA was carried out as follows: fixed amounts of the EB
and DNA were titrated with increasing amounts of morin–Eu(III)
complex solution. The changes in fluorescence intensities, as
appropriate, were monitored against a blank after each addition
of the morin–Eu(III) complex.
2. Materials and methods
2.1. Apparatus
UV–vis absorption spectra were measured on a Shimadzu
UV-2450 spectrophotometer using a 1.0 cm cell. Fluorescence
measurements were performed with a Hitachi spectrofluorimeter
Model F-4500 equipped with a 150 W Xenon lamp and a thermo-
stat bath, using a 1.0 cm quartz cell. The widths of both the excita-
tion slit and emission slit were set at 5.0 nm, and the scan rate at
1200 nm minꢁ1. pH measurements were carried out with a pHS-
3C digital pH-meter (Shanghai Exact Sciences Instrument Co. Ltd.,
Shanghai, China) with a combined glass-calomel electrode. The vis-
cosity measurements were carried out using an NDJ-79 viscosity
meter (Yinhua Flowmeter Co. Ltd., Hangzhou, China). An electronic
thermostat water-bath (Shanghai Yuejin Medical Instrument Com-
pany, Shanghai, China) was used for controlling the temperature.
All experiments, unless specified otherwise, were carried out at
room temperature (25 1 °C).
2.3.3. Viscosity measurements
Viscosity measurements were performed using a viscometer,
which was immersed in a thermostat water-bath at 25 0.1 °C.
Typically, 10.0 mL of buffer and 10.0 mL of DNA solution were
transferred into the viscometer separately. An appropriate amount
of morin–Eu(III) complex was then added into the viscometer to
give a certain r(r = [drug]/[DNA]) value while keeping the DNA con-
centration constant. After a thermal equilibrium was achieved
(15 min), the flow times of the samples were repeatedly measured
with an accuracy of 0.2 s by using a digital stopwatch. Flow times
were above 200 s, and each point measured was the average of at
1/3
least five readings. The data were presented as (
where
g
/g0)
versus r,
g
and g0 are the viscosity of DNA in the presence and ab-
sence of the complex, respectively.
2.2. Materials
2.3.4. DNA melting studies
DNA melting experiments were carried out by monitoring the
fluorescence intensities of the sample at different temperatures
in the absence and presence of the morin–Eu(III) complex. The
temperature of the sample was continuously monitored with a
thermocouple attached to the sample holder. The fluorescence
intensities were then plotted as a function of temperature ranging
from 20 to 100 °C. The melting temperature (Tm) of DNA was deter-
mined as the transition midpoint.
A stock solution (3.0 ꢀ 10ꢁ3 mol Lꢁ1) of morin (Sigma Chem.
Co., USA) was dissolved in anhydrous methanol. Calf thymus
DNA (Sino-American Biotechnology Company, Beijing, China) was
used without further purification, and its stock solution was pre-
pared by dissolving appropriate solid DNA into doubly distilled
water and stored at 4 °C. The concentration of DNA in stock solu-
tion was determined by UV absorption at 260 nm using a molar
absorption coefficient e
260 = 6600 L molꢁ1 cmꢁ1 [27]. Purity of the
DNA was checked by monitoring the ratio of the absorbance at
260 nm to that at 280 nm. The solution gave a ratio of >1.8 at
A260/A280, indicating that DNA was sufficiently free from protein
[28]. The stock solution of Eu(III) (1.0 ꢀ 10ꢁ3 mol Lꢁ1) was pre-
pared by dissolving its oxide, Eu2O3 (99.95%, Fourth Reagent Fac-
tory, Shanghai, China) in a minimum amount of hydrochloric
acid and diluted by doubly distilled water. Ethidium bromide
3. Results and discussion
3.1. Interaction between morin and Eu(III)
The changes in UV–vis absorption of morin in the presence of
Eu(III) (with increasing concentration) were examined in the

116
G. Zhang et al. / Journal of Molecular Structure 923 (2009) 114–119
level decreasing and absorbance bond red shifting. The effect of
hauling electrons caused by the bond of Eu(III) with morin results
in a lower absorbance at 265 nm. This rationale is also in accor-
dance with previous work, which demonstrated that the 3-hydro-
xyl and 4-carbonyl structure in ring C of morin is important for rare
earth metal chelating [19,35]. The curves of absorbance versus
[Eu(III)]/[morin] molar ratio plotted at 390 and 431 nm allow us
to find a stoichiometry of 1:3 for the complex (Fig. 3). A threefold
excess of Eu(III) is necessary to obtain a full complexation of morin,
more important excess of Eu(III) has no effect on the absorbance
value. According to Job’s method [36], the binding constant of
morin–Eu(III) complex is calculated to be 2.36 ꢀ 106 L molꢁ1
3.2. Binding characteristics of morin–Eu(III) complex with DNA
3.2.1. UV–vis absorption spectroscopy
.
Fig. 2. Absorption spectra of morin in the presence of Eu(III) at pH 7.4. cEu(III) = 0,
0.3, 0.6, 0.9, 1.2 and 2.1 ꢀ 10ꢁ5 mol Lꢁ1 for curves 1–6; cmorin = 4.2 ꢀ 10ꢁ5 mol Lꢁ1
.
In general, if a small molecule interacts with DNA, changes in
absorbance (hypochromism) and in the position of the band (red
shift) should occur. This phenomenon indicates that the small
molecule has intercalated into DNA base pairs, and is involved in
a strong interaction in the molecular stack between the aromatic
chromophore and the base pairs. The spectral effects have been
Tris–HCl buffer solution (pH 7.4) (Fig. 2). The UV–vis spectra of
morin showed an intense absorbance at 390 nm (band I) and
265 nm (band II) (curve 1). Band I located in the wavelength range
of 300–400 nm is related to conjugated system between ring B and
carbonyl of ring C, and band II located in the wavelength range of
240–300 nm is related to conjugated system between ring A and
carbonyl of ring C (Fig. 1) [30,31]. When the solution of Eu(III)
was added, band I gradually shifted to longer wavelengths, accom-
panied with decrease in absorption. Simultaneously, a new stron-
ger absorbance peak appeared at 431 nm (curve 6). The results
indicated formation of a complex between morin and Eu(III). The
observation of an isobestic point at 422 nm suggested the exis-
tence of a fairly simple equilibrium in solution that involved only
two dominant species: the free and complexed molecules. There
are two possible chelating sites on morin that can interact with
Eu(III): the 3- or 5-hydroxyls, and the 4-carbonyl. Presence of the
peak at 265 nm demonstrated that ring A remained unchanged.
The appeared new peak at 431 nm suggested that Eu(III) had
bonded to 3-hydroxyl and 4-carbonyl of ring C [32]. In deed, a great
number of proofs clearly indicated that this complex involves the
3-hydroxyl-4-keto group: (i) among 3-hydroxyl and 5-hydroxyl
molecules, 3-hydroxyl has greater chelation power; (ii) the delo-
calization of the oxygen electrons of 3-hydroxyl group is higher
rationalized as follows [37,38]: the empty
molecule couples with the
*-orbital of the DNA base pairs, which
causes an energy decrease, and a decrease of the
p* transition
energy. Therefore, the absorption of the small molecule should ex-
hibit a red shift. At the same time, the empty
*-orbital is partially
p
*-orbital of the small
p
p–
p
filled with electrons to reduce the transition probability, which
leads to hypochromism.
In this work, UV–vis absorption spectra were obtained by titra-
tion of 3.5 ꢀ 10ꢁ5 mol Lꢁ1 morin–Eu(III) complex solution with
increasing concentration of DNA (Fig. 4). In the absence of DNA,
the spectra of the morin–Eu(III) complex (curve 1) was character-
ized by a peak at 431 nm. With the increase of DNA concentration,
the absorption spectra showed clear hypochromicity at the maxi-
mum of 431 nm together with a red shift of
Dk = 8 nm. The hypo-
chromicity and red shift in the absorption spectra of morin–Eu(III)
complex indicated the intercalative binding of the morin–Eu(III)
complex to DNA bases.
3.2.2. Fluorescence studies
than 5-hydroxyl, which facilitates the delocalization of the
p elec-
The fluorescence spectra of morin–Eu(III) complex is shown in
Fig. 5. As can be seen from Fig. 5, the fluorescence intensity of mor-
in–Eu(III) complex was dramatically enhanced when the DNA was
added, which indicated morin–Eu(III) complex could bind to DNA.
The observation reflected that the stronger enhancement for
morin–Eu(III) complex may be largely due to the increase of the
trons [33,34]. Band I bathochromic shift can be explained by the
interaction of Eu(III) with the 3-hydroxyl group of morin resulting
in electronic redistribution between the morin molecule and Eu(III)
to become a big extended
p
bond system. Electron distribution in
morin changed from n– transition to p–
p
p* transition and the en-
ergy of this electron transition is decreased. Morin can attract p
bond electron cloud to orientate by itself and make p bond electron
cloud excurse and polarize, which lead to difference of p–p
* energy
Fig. 4. Absorption spectra of morin–Eu(III) complex in the presence of DNA at pH
7.4. cDNA = 0, 0.71, 1.42, 2.13, 2.84, 3.55, 4.26 and 4.97 ꢀ 10ꢁ5 mol Lꢁ1 for curves 1–
Fig. 3. Absorbance versus [Eu(III)]/[morin] molar ratio plot at 390 and 431 nm.
8; cmorin–Eu(III) = 3.5 ꢀ 10ꢁ5 mol Lꢁ1
.

G. Zhang et al. / Journal of Molecular Structure 923 (2009) 114–119
117
against 1/[L] confirmed a one-to-one interaction between the two
partners (Fig. 6). According to the above equation, the values of
KDFmax and DFmax are obtained from the slope and intercept of
the linear plot, and the value of K can be calculated by dividing
intercept to slope. The values of K at three temperatures (292,
301, 310 K) are shown in Table 1.
3.2.3. Competitive interaction of morin–Eu(III) complex and EB with
DNA
Further support for the mode of binding between morin–Eu(III)
complex and DNA is given through the competitive experiment.
Here ethidium bromide (EB) has been employed in the examina-
tion of the reaction, as EB presumably binds initially to DNA by
intercalation [13,42]. The experiment was carried out in 3 mL of
6.0 ꢀ 10ꢁ6 mol Lꢁ1 EB and 7.0 ꢀ 10ꢁ5 mol Lꢁ1 DNA (at saturating
binding levels [43]) titrated with 6.0 ꢀ 10ꢁ4 mol Lꢁ1 morin–Eu(III)
complex solution. Fig. 7 shows the emission spectra of the DNA–EB
system in the absence and presence of morin–Eu(III) complex.
When the concentration of morin–Eu(III) complex was added, a
remarkable fluorescence decrease of DNA–EB system was observed
Fig. 5. Fluorescence spectra of morin–Eu(III) complex in the presence of DNA (pH
7.4, T = 292 K, kex = 385 nm, kem = 509 nm). cDNA = 0, 0.71, 1.42, 2.13, 2.84, 3.55, 4.26,
4.97, 5.68, 6.39, 7.10 and 7.81 ꢀ 10ꢁ5 mol Lꢁ1 for curves 1–12; cmorin–Eu(III)
=
3.5 ꢀ 10ꢁ5 mol Lꢁ1
.
molecular planarity of the complex and the decrease of the colli-
sion frequency of the solvent molecules with the complex which
caused by the planar aromatic group of the complex stacks be-
tween adjacent base pairs of DNA. The increase in the molecular
planarity and the decrease of the collision frequency between sol-
vent molecules with the complexes usually lead to emission
enhancement [39]. The binding of morin–Eu(III) complex to DNA
leading to marked increase in emission intensity also agrees with
those observed for other intercalators [40].
at the maximum of 598 nm and blue shift of
Dk = 5 nm. This phe-
nomenon suggested that morin–Eu(III) complex substituted for EB
in the DNA–EB system which led to a large decrease in the emis-
sion intensity of the DNA–EB system. These changes in the state
of intercalation have been observed in several instances [44,45].
3.2.4. Viscosity measurements
Viscosity experiment is an effective tool to determine the bind-
ing mode of small molecules and DNA. A classical intercalation
binding demands the space of adjacent base pairs to be large en-
In order to see the interaction between morin–Eu(III) complex
and DNA, the binding constant was determined from the fluores-
cence intensity based on the following equation [41]:
1=D
F ¼ 1=
DFmax þ ð1=K½LꢂÞð1=
DFmax
Þ
ð1Þ
where
D
F = Fx ꢁ F0 and
D
Fmax = F1 ꢁ F0. F0, Fx and F1 are the fluores-
cence intensities of morin–Eu(III) complex in the absence of DNA, at
an intermediate concentration of DNA, and at the saturation of
interaction, respectively; K is the binding constant and [L] refers
to the DNA concentration. The linearity in the plot of 1/(Fx ꢁ F0)
Fig. 7. Fluorescence spectra of DNA–EB system in the presence of morin–Eu(III) (pH
7.4, T = 292 K, kex = 528 nm, kem = 598 nm). cmorin–Eu(III) = 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0,
7.0, 8.0, 9.0 and 10.0 ꢀ 10ꢁ6 mol Lꢁ1 for curves 1–11; cEB = 6.0 ꢀ 10ꢁ6 mol Lꢁ1, and
cDNA = 7.0 ꢀ 10ꢁ5 mol Lꢁ1
.
Fig. 6. The plots of 1/(Fx ꢁ F0) vs 1/[L]. cmorin–Eu(III) = 3.5 ꢀ 10ꢁ5 mol Lꢁ1
.
Table 1
Binding constants and thermodynamic parameters of the interaction of morin–Eu(III)
complex with DNA at different temperatures.
T (K)
K
Ra
SDb
D
Hh
D
Sh
D
Gh
(L molꢁ1
)
(kJ molꢁ1
)
(J molꢁ1 Kꢁ1
)
(kJ molꢁ1
)
292
301
310
7.47 ꢀ 104
8.89 ꢀ 104
1.13 ꢀ 105
0.9955
0.9975
0.9981
0.024
0.028
0.027
ꢁ27.08
ꢁ28.53
ꢁ29.99
20.14
161.70
a
R is correlation coefficient.
SD is standard deviation.
Fig. 8. Effect of increasing amount of bound ligand on the relatively viscosity of
DNA.
b

118
G. Zhang et al. / Journal of Molecular Structure 923 (2009) 114–119
ough to accommodate the bound ligand and to elongate the double
helix, resulting in an increase of DNA viscosity [46]. A series of
solutions was made which contained a fixed concentration of
DNA and various concentrations of morin–Eu(III) complex. Then,
the viscosity measurements were conducted at room temperature
(avoiding from light after a 48 h reaction). The changes in relative
viscosity of DNA with increasing concentrations of morin–Eu(III)
complex are shown in Fig. 8. It can be seen that the relative viscos-
ity of DNA increased steadily with increasing the amounts of the
morin–Eu(III) complex. Such behavior further suggested that an
intercalation binding should be the interaction mode of the
morin–Eu(III) complex with DNA.
3.2.5. Melting studies
Fig. 10. The van’t Hoff plot for the interaction of morin–Eu(III) complex with DNA.
UV–vis absorption spectroscopy is a general method for deter-
mining the melting temperatures (Tm) [47,48]. Considering the
higher sensitivity, the fluorescence technique is used to determine
Tm in this experiment. With the increasing temperature, DNA
denatures and the double helixes unfold. The interaction of classi-
cal intercalators with DNA, such as EB, can increase the stability of
helix of DNA, and cause the Tm of DNA to increase [49,50].
The Tm of the complexes of DNA-EB and morin–Eu(III)–DNA–EB
system were determined, respectively, by monitoring the maxi-
mum fluorescence of the system as a function of temperature rang-
ing from 20 to 100 °C. For each monitored transition, the Tm of
tested solution was determined as the transition midpoint of the
melting curve. The melting curves are shown in Fig. 9. It can be
seen that the Tm of DNA–EB system in the absence of morin–Eu(III)
complex is 88 1 °C under the experimental conditions. The ob-
served melting temperature of DNA–EB system in the presence of
morin–Eu(III) complex is 82 1 °C. Heat and alkali can destroy
the double helix structure of DNA and change it into single helix
at the melting temperature (Tm). Interaction of small molecules
with double stranded DNA can influence Tm. The interaction of
morin–Eu(III) complex with DNA may cause the Tm to be de-
creased. The change in Tm of DNA–EB complex after the addition
of morin–Eu(III) complex reveals that morin–Eu(III) complex
substituted for EB in the DNA–EB system to affect the binding of
EB to DNA and to make the stability of DNA decrease [16]. The re-
sults reconfirmed that the binding mode of the morin–Eu(III) com-
plex with DNA is intercalative binding.
Eu(III) complex and DNA. The interaction forces between a small
molecule and macromolecule mainly include hydrogen bonds,
van der Waals force, electrostatic force and hydrophobic interac-
tion force. The thermodynamic parameters of binding reaction
are the main evidence for confirming the binding force.
If the enthalpy change (
temperature range studied, then its value and that of entropy
change (
Sh) can be determined from the van’t Hoff equation:
D
Hh) does not vary significantly over the
D
D
Hh Sh
2:303RT 2:303R
D
log K ¼ ꢁ
þ
ð2Þ
where K and R are binding constant and gas constant, respectively.
The temperatures used were 292, 301 and 310 K. The enthalpy
change (D D
Hh) and entropy change ( Sh) were obtained from the
slope and intercept of the linear van’t Hoff plot based on logK ver-
sus 1/T (Fig. 10). The free energy change (
D
Gh) is estimated from the
following relationship:
D
Gh ¼
D
Hh ꢁ T
D
Sh
Hh,
ð3Þ
The values of
can be seen that the negative value of
process is spontaneous, while the positive
D
D
Sh and
D
Gh are listed in Table 1. From Table 1, it
Gh revealed the interaction
Hh and Sh values asso-
D
D
D
ciated with the interaction of morin–Eu(III) complex with DNA indi-
cated that the binding is mainly entropy driven and the enthalpy is
unfavorable for it. In other words, the hydrophobic interaction plays
a major role in the binding [51].
3.2.6. Determination of the thermodynamic parameters
Considering the dependence of binding constant on tempera-
ture, a thermodynamic process was considered to be responsible
for the formation of a complex. Therefore, the thermodynamic
parameters dependent on temperatures were analyzed in order
to further characterize the interaction forces between morin–
4. Conclusion
The binding interactions of morin–Eu(III) complex with DNA in
physiological buffer were illustrated with UV–vis and fluorescence
spectroscopic techniques. The binding constants of morin–Eu(III)
complex with DNA were measured at different temperatures and
the thermodynamic parameters were calculated as well. The inter-
calative binding of morin–Eu(III) complex with DNA was deduced
by taking account of relevant UV–vis absorption spectra, fluores-
cence spectra, viscosity measurements and melting temperature
determinations. It was found that hydrophobic force plays a major
role in the binding of morin–Eu(III) complex to DNA. We believe
that the binding mode of morin–Eu(III) complex with DNA studied
here will provide useful information on the mechanism of antican-
cer drugs binding to DNA and thus will be beneficial to new drug
design.
Acknowledgements
The authors gratefully acknowledge the financial support of this
study by the Jiangxi Province Natural Science Foundation
(2007GZH1924), the Foundation of Jiangxi Provincial Office of
Fig. 9. Melting curves of DNA–EB in the absence (1) and presence (2) of morin–
Eu(III) complex at pH 7.4. cDNA = 8.04 ꢀ 10ꢁ5 mol Lꢁ1, cEB = 6.70 ꢀ 10ꢁ6 mol Lꢁ1 and
cmorin–Eu(III) = 6.70 ꢀ 10ꢁ6 mol Lꢁ1
.

G. Zhang et al. / Journal of Molecular Structure 923 (2009) 114–119
119
[23] F. Araya, G. Huchet, I. McGroarty, G.G. Skellern, R.D. Waigh, Methods 42 (2007)
141–149.
[24] K. Sandstrom, S. Warmlander, M. Leijon, A. Graslund, Biochem. Biophys. Res.
Commun. 304 (2003) 55–59.
[25] L.P. Wang, L. Lin, B.X. Ye, J. Pharm. Biomed. Anal. 42 (2006) 625–629.
[26] S.Y. Bi, H.Q. Zhang, C.Y. Qiao, Y. Sun, C.M. Liu, Spectrochim. Acta A 69 (2008)
23–129.
[27] C.Z. Huang, Y.F. Li, P. Feng, Talanta 55 (2001) 321–328.
[28] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
Education (GJJ08025), the Program for State Key Laboratory of Food
Science and Technology of Nanchang University (SKLF-MB-
200807), the Program for Changjiang Scholars and Innovative Re-
search Team of Ministry of Education of China in Universities
(IRT0540) and the Analysis and Test Foundation of Nanchang Uni-
versity (No. 2008006).
[29] J.H. Yoe, A.L. Joens, Ind. Eng. Chem. (Anal. Ed.) 16 (1944) 111–115.
[30] J. Kang, Y. Liu, M.X. Xie, S. Li, M. Jiang, Y.D. Wang, Biochim. Biophys. Acta 1674
(2004) 205–214.
[31] L. Huang, D.Q. Yu, Application of Ultraviolet Spectroscopy in Organic
Chemistry, Science Press, Beijing, 2000. p. 281.
[32] J.W. Kang, L. Zhuo, X.Q. Lu, H.D. Liu, M. Zhang, H.X. Wu, J. Inorg. Biochem. 98
(2004) 79–86.
[33] J.P. Cornard, J.C. Merlin, J. Inorg. Biochem. 92 (2002) 19–27.
[34] A.C. Boudet, J.P. Cornard, J.C. Merlin, Spectrochim. Acta A 56 (2000) 829–839.
References
[1] S. Rauf, J.J. Gooding, K. Akhtar, M.A. Ghauri, M. Rahman, M.A. Anwar, A.M.
Khalid, J. Pharm. Biomed. Anal. 37 (2005) 205–217.
[2] L.H. Hurley, Nat. Rev. Cancer 2 (2002) 188–200.
[3] H. Mansouri-Torshizi, M. Islami-Moghaddam, A. Divsalar, A.A. Saboury, Bioorg.
Med. Chem. 16 (2008) 9616–9625.
[4] B.K. Sahoo, K.S. Ghosh, R. Bera, S. Dasgupta, Chem. Phys. 351 (2008) 163–169.
[5] R.F. Pasternack, E.J. Gibbs, J.J. Villafranca, Biochemistry 22 (1983) 2406–2414.
[6] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777–2795.
[7] L.S. Ling, Z.K. He, F. Chen, Y.E. Zeng, Talanta 59 (2003) 269–275.
[8] X.Q. He, Q.Y. Lin, R.D. Hu, X.H. Lu, Spectrochim. Acta A 68 (2007) 84–190.
[9] J. Kuhnau, World Rev. Nutr. Diet 24 (1976) 117–191.
´
[35] E. Woznicka, M. Kopacz, M. Umbreit, J. Kłos, J. Inorg. Biochem. 101 (2007) 774–
782.
[36] P. Job, Ann. Chim. Phys. 9 (1928) 113–203.
[37] E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271–273.
[38] S.A. Tysoe, R.J. Morgan, A.D. Baker, T.C. Strekas, J. Phys. Chem. 97 (1993) 1707–
1711.
[39] X. Wu, C.Y. Guo, F. Wang, J.H. Yang, D.H. Ran, J.H. Zheng, J.B. Wu, Spectrochim.
Acta A 65 (2006) 543–548.
[40] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 31 (1992) 9319–
9324.
[41] J. Bhattacharya, M. Bhattacharya, A. Chakraborty, U. Chowdhury, R.K. Podder,
Biochem. Pharmacol. 47 (1994) 2049–2052.
[42] S. Nafisi, A.A. Saboury, N. Keramat, J.F. Neault, H.A. Tajmir-Riahi, J. Mol. Struct.
827 (2007) 35–43.
[43] M. Selim, S.R. Chowdhury, K.K. Mukherjea, Int. J. Biol. Macromol. 41 (2007)
579–583.
[44] J.P. Rehman, J.K. Barton, Biochemistry 29 (1990) 1701–1710.
[45] J. Lan, Y. Pin, L.Q. Shan, Chem. J. Chin. Univ. 17 (1996) 1345–1348.
[46] L.S. Lerman, J. Mol. Biol. 3 (1961) 8–30.
[47] P. Nagababu, S. Satyanarayana, Polyhedron 26 (2007) 1686–1692.
[48] T.R. Li, Z.Y. Yang, B.D. Wang, D.D. Qin, Eur. J. Med. Chem. 10 (2007) 1–8.
[49] J.B. Chaires, Biochemistry 25 (1986) 8436–8439.
[50] C.V. Kumar, E.H. Asuncion, J. Am. Chem. Soc. 115 (1993) 8547–8553.
[51] D.P. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102.
[10] S.H. Fang, Y.C. Hou, W.C. Chang, S.L. Hsiu, P.D. Chao, B.L. Chiang, Life Sci. 74
(2003) 743–756.
[11] D.P. Makris, J.T. Rossiter, J. Food Compos. Anal. 15 (2002) 103–113.
[12] Y. Iwase, Y. Takemura, M. Ju-ichi, T. Mukainaka, E. Ichiishi, C. Ito, H. Furukawa,
M. Yano, H. Tokuda, H. Nishino, Cancer Lett. 173 (2001) 105–109.
[13] M.M. Hassanien, K.S. Abou-EI-Sherbini, Talanta 68 (2006) 1550–1559.
[14] Z.H. Xu, F.J. Chen, P.X. Xi, X.H. Liu, Z.Z. Zeng, J. Photochem. Photobiol. A: Chem.
196 (2008) 77–83.
[15] Y.M. Song, J.W. Kang, J. Zhou, Z.H. Wang, X.Q. Lu, L.F. Wang, J.Z. Gao,
Spectrochim. Acta A 56 (2000) 2491–2497.
[16] Y.M. Song, J.W. Kang, Z.H. Wang, X.Q. Lu, J.Z. Gao, L.F. Wang, J. Inorg. Biochem.
91 (2002) 470–474.
[17] Z. Qi, W.L. Fang, L. Xiang, J. Transit. Met. Chim. 21 (1996) 23–27.
[18] X.A. Chen, Q.C. He, T. Guan, Y.E. Cheng, H.F. Chen, B.k. Xiong, Chin. J. Rare
Earths 1 (1995) 70–73.
[19] J. Zhou, L.F. Wang, J.Y. Wang, N. Tang, J. Inorg. Biochem. 83 (2001) 41–48.
[20] R.D. Hu, Q.Y. Lin, W. Huang, Q. Yu, J. Rare Earths 23 (2005) 372–376.
[21] J. Li, Y.L. Wei, L.M. Guo, C.H. Zhang, Y. Jiao, S.M. Shuang, C. Dong, Talanta 76
(2008) 34–39.
[22] Y.N. Ni, D.Q. Lin, S. Kokot, Anal. Chim. Acta 606 (2008) 9–25.