DNA binding and cleavage activity of quercetin nickel(ii) complex

Article abstract of DOI:10.1039/b901353a

The interaction of a quercetin nickel(ii) complex with DNA was investigated using UV-vis spectra, fluorescence measurements, viscosity measurements, agarose gel electrophoresis and thiobarbituric acid-reactive substances assay. The results indicate that t

Full text of DOI:10.1039/b901353a

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/dalton | Dalton Transactions
DNA binding and cleavage activity of quercetin nickel(II) complex
Jun Tan,*a,b,c Liancai Zhua and Bochu Wang*a
Received 21st January 2009, Accepted 26th March 2009
First published as an Advance Article on the web 27th April 2009
DOI: 10.1039/b901353a
The interaction of a quercetin nickel(II) complex with DNA was investigated using UV-vis spectra,
fluorescence measurements, viscosity measurements, agarose gel electrophoresis and thiobarbituric
acid-reactive substances assay. The results indicate that the quercetin nickel(II) complex can intercalate
into the stacked base pairs of DNA, and compete with the strong intercalator ethidium bromide for the
intercalative binding sites with Stern–Volmer quenching constant Ksq = 1.0. The complex successfully
promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount
of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on
the concentration of the complex, ionic strength and the duration of incubation of the complex with
DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in
the presence of 100 mM of the complex is found to be 0.76 ¥ 10-4 s-1. The hydrolytic cleavage of DNA by
the complex was supported by the evidence from free radical quenching and thiobarbituric
acid-reactive substances assay.
In this study, we investigated the mode of DNA binding and
Introduction
DNA cleavage activity of the quercetin nickel(II) complex. We
demonstrate that the quercetin nickel(II) complex could be bound
to DNA via an intercalation mode and could promote the cleavage
of plasmid DNA via a hydrolytic pathway.
In recent years, many researches1–3 have been focused on the
interaction of small molecules with DNA. DNA is generally the
primary intracellular target of anticancer drugs, so the interaction
between small molecules and DNA can cause DNA damage in
cancer cells, inhibiting the growth of cancer cells and resulting in
cell death or apoptosis.4–5 Small molecules can interact with DNA
through the following three non-covalent modes: intercalation,
groove binding and external static electronic effects. Among
these interactions, intercalation is one of the most important
DNA binding modes that is related to the antitumor activity
of the compound. Recently, there has been a great interest on
the binding of transition metal complexes with DNA, owing to
their possible applications as new cancer therapeutic agents and
their photochemical properties that make them potential probes
of DNA structure and conformation.6–8 Moreover, transition
metal complexes have been widely exploited for metallohydrolases
capable of mimicking the function of these endonucleases.9–13 So it
is essential to develop synthetic, sequence-selective DNA binding
and cleavage agents for DNA itself and for new potential DNA
targeting antitumor drugs.
Experimental
Materials
All chemicals and reagents were purchased from commercial
sources and were used without further purification. The plasmid
pBR322 DNA was purchased from TaKaRa Biotechnology
Co. Ltd. (Japan). Quercetin, calf thymus DNA (CT DNA),
catalase and ethidium bromide (EB) were obtained from Sigma
(Sigma Chemical Co., St. Louis, MO, USA). Nickel(II) chlo-
ride (NiCl2·6H2O) was from Guangfu Fine Chemicals, Tianjing
(China). 2-Thiobarbituric acid (TBA) was from Sinopharm Chem-
ical Reagent Co. Ltd., Shanghai (China). Agarose (molecular
biology grade) was from Oxoid Limited Basingstoke, Hampshire
(England). The Tris-HCl buffer solution was prepared with triple
distilled water.
Quercetin
(Que,
3,5,7,3¢,4¢-pentahydroxyflavone),
a
Preparation of quercetin nickel(II) complexes
bioflavonoid widely distributed in fruits and vegetables, has
been reported to exert multiple biological effects as an antioxidant
and antitumor activity.14–16 Quercetin can chelate metal ions
to form metal complexes that have better antioxidation and
antitumor activity than quercetin alone.17–18 However, little
research has been devoted to DNA-binding properties and DNA
cleavage mechanism of the quercetin nickel(II) complex.
Quercetin nickel(II) complex was prepared according to literature.9
The solid quercetin·2H2O (2 mM) was dissolved into 60 mL of
ethanol. Then the pH of the solution was adjusted to 6–7 with
EtONa. After 5 min, NiCl2·6H2O (1 mM) was added to the above
mixture. After being stirred and heated to reflux for 5 h at 60 ◦C,
the reaction mixture was cooled to room temperature and poured
into H2O. The pale yellow precipitate, which formed immediately,
was set aside for 48 h, filtered and washed thrice with 1 : 3 EtOH–
H2O. The solid product was dried under vacuum for 48 h at
room temperature. Yield: 72%. Anal. Found: Ni(Que)2(H2O)2:
C, 51.2%; H, 3.7%; Ni, 8.2%. IR (KBr) cm-1: vmax = 3450–3098
(b, m), 1631 (s), 1392 (s), 1263 (s), 657 (w), 623 (w), 399 (m).
aBioengineering College, Chongqing University, Chongqing, 400030, P. R.
China
bDepartment of Life Science & Chemistry, Chongqing Education College,
Chongqing, 400067, P. R. China
cPost-doctoral Research Center of Mechanics, Mechanical Resource and En-
vironment College, Chongqing University, Chongqing, 400030, P. R. China
4722 | Dalton Trans., 2009, 4722–4728
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
UV (EtOH) nm: lmax = 424 (12151), 261 (31983). Attempts to
grow single crystals suitable for crystal structure determination
were unsuccessful. These data indicated that stoichiometric ratio
(Ni : Que) of Ni(Que)2(H2O)2 was 1 : 2 and quercetin could chelate
nickel(II) via 3-OH and 4-oxo groups. The possible structure model
of the complex is shown in Fig. 1.
directly into the EB-DNA system (cEB = 5 mM, cDNA = 100 mM bp,
0.01 M Tris buffer, pH 7.2). Emission spectra were recorded in the
region 520–650 nm using an excitation wavelength of 500 nm. All
measurements were performed at 25 ◦C.
Viscosity measurements were carried out using an Ubbelodhe
viscometer maintained at a constant temperature at 30.0 0.1 ◦C
in a thermostatic bath. Flow time was measured with a digital
stopwatch and each sample was measured three times and an
average flow time was calculated. Data are presented as (h/h0)1/3
versus binding ratio,19 where h is the viscosity of DNA in the
presence of the complex and h0 is the viscosity of DNA in the
absence of the complex. Viscosity values were calculated from
the observed flow time of DNA-containing solutions (t > 100 s)
corrected for the flow time of buffer alone (t0), h = t - t0.
DNA cleavage studies
DNA cleavage was measured by the conversion of supercoiled
pBR322 plasmid DNA to nicked circular and linear DNA forms.
Supercoiled pBR322 plasmid DNA (0.25 mg per reaction) in Tris–
HCl buffer (50 mM) with 50 mM NaCl (pH 7.2) was treated with
the indicated amount of Ni(Que)2(H2O)2 complex, followed by
dilution with the Tris–HCl buffer to a total volume of 10 mL. The
Fig. 1 The possible structure of quercetin nickel(II) complex.
◦
samples were incubated for 1 h at 37 C. After the reaction had
DNA-binding measurements
been stopped by addition of 1/10 volume of the loading buffer
(0.25% bromophenol blue, 40% sucrose, 0.25% xylene cyanole and
200 mM EDTA), the samples were loaded on 1% neutral agarose
gel containing 40 mM Tris/acetate and 1 mM EDTA (TAE buffer,
pH 8.0), and were subjected to electrophoresis in a horizontal slab
gel apparatus and 1 ¥ TAE buffer, which was performed at 75 V
for 1.5 h. The gel was stained with a solution of 0.5 mg mL-1
ethidium bromide for 30 min, followed by destaining in water.
Agarose gel electrophoresis of plasmid DNA was visualized by
photographing the fluorescence of intercalated ethidium bromide
under a UV illuminator. The cleavage efficiency was measured by
determining the ability of the complexes to convert the supercoiled
DNA (SC) to nicked circular form (NC) and linear form (L).
After electrophoresis, the proportion of DNA in each fraction
was estimated quantitatively from the intensity of the bands using
Glyko BandScan software.
All the experiments involving the interaction of compound with
CT DNA were conducted in Tris buffer (0.01 M Tris–HCl/50 mM
NaCl, pH 7.2). The purity of the DNA was determined by
monitoring the value A260/A280 about 1.8–1.9 : 1, indicating that
the DNA was sufficiently free of protein. The DNA concentration
per nucleotide was determined by absorption spectroscopy using
the molar absorption coefficient (6600 M-1 cm-1) at 260 nm.
UV-vis spectra were measured on a Lambda 900 UV/Vis/NIR
Spectrometer (Perkin–Elmer) in 0.01M Tris buffer. Spectroscopic
titrations were carried out at room temperature to determine
the binding affinity between DNA and quercetin metal com-
plex. Initially, the solutions (2000 mL) of the blank buffer and
Ni(Que)2(H2O)2 complex sample (10 mM) were placed in the
reference and sample cuvettes (1 cm path length), respectively, and
then the first spectrum was recorded in the range of 260–440 nm.
During the titration, an aliquot (20 mL) of buffered DNA solution
(concentration of 1 mM in base pairs) was added to each cuvette to
eliminate the absorption from DNA itself, and the solutions were
mixed by repeated inversion. After the solutions had been mixed
for 10 min, the absorption spectra were recorded. The titration
processes were repeated until there was no change in the spectra for
four titrations at least, indicating that binding saturation had been
achieved. During the spectrophotometric titration, the changes in
the metal complex concentration are negligible.
To study the mechanism of the DNA cleavage reaction
performed by Ni(Que)2(H2O)2 complex, different scavengers or
reactive oxygen intermediates such as dimethyl sulfoxide (DMSO)
(0.4 M), glycerol (0.4 M), mannitol (0.2 M) catalase (15 units),
and oxidant hydrogen peroxide (50 mM) were added to reaction
mixtures, respectively. Samples were treated as described above.
Thiobarbituric acid-reactive substances (TBARS) assay
Fluorescence measurements were made using Perkin–Elmer LS-
50B fluorescence spectrophotometer with a slit width 5 nm for
the excitation and emission beams. Fluorescence titrations were
carried out by adding increasing amounts of CT DNA directly
into the cell containing the solution of Ni(Que)2(H2O)2 complex
(c = 30 mM, 0.01 M Tris buffer, pH 7.2). The concentration range
of the DNA was 0–60 mM bp. Emission spectra were recorded
in the region 330–370 nm using an excitation wavelength of
312 nm. Fluorescence quenching study was conducted by adding
increasing amounts of Ni(Que)2(H2O)2 complex (0–120 mM)
Each sample containing 0.5 mM CT DNA and 100 mM
Ni(Que)2(H2O)2 complex in 50 mM phosphate buffer (pH 7.2)
was incubated at 37 ◦C for 24 h in a total volume of 2 mL. After
incubation, samples were treated with 2 mL of 1% (w/v) solution
of 2-thiobarbituric acid in 50 mM NaOH and 2 mL of glacial acetic
acid, and were incubated at 100 ◦C for 30 min. After cooling,
the absorbance at 532 nm was measured. Blanks contained all
components except the complex. The control group contained
[Fe(EDTA)]2- (100 mM) and hydrogen peroxide (10 mM) instead
of the complex.
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 4722–4728 | 4723
©

View Article Online
Results and discussion
DNA binding properties
The binding of Ni(Que)2(H2O)2 complex with CT DNA was mea-
sured using absorption, fluorescence spectrophotometric titrations
and viscosity measurements.
Electronic absorption spectral studies. The binding of the
complex to DNA was characterized classically through absorption
titration. A complex bound to DNA through intercalation is
characterized by the change in absorbance (hypochromism) and
red shift in wavelength, due to the intercalative mode involv-
ing a stacking interaction between the aromatic chromophore
and the DNA base pairs. The electronic absorption spectra of
Ni(Que)2(H2O)2 complex exhibited broad absorption bands in
the region 260–440 nm, typical for transitions between the p-
electronic energy levels of the quercetin skeleton. The electronic
spectra of the complex in the presence and absence of DNA were
monitored over the wavelength range of 260–440 nm, as shown in
Fig. 2. With increasing concentration of CT DNA (0–40 mM), a
considerable drop in the absorptivity (64% hypochromicity) at
about 270 nm and a substantial red-shift (Dl = 6.6 nm) was
observed. The hypochromicity suggests the complex may bind to
DNA by intercalation mode, due to a strong interaction between
the electronic states of the intercalating chromophore and those
of the DNA bases.
Fig. 3 The change of emission fluorescence spectra of quercetin nickel(II)
complex (30 mM) in 0.01 M Tris-HCl buffer at pH 7.2, in the absence and
presence of increasing amounts of CT DNA (from bottom to top, 0–60 mM
bp), lex = 312 nm. The arrow shows the intensity increased with increasing
amounts of DNA.
To understand the interaction pattern of the complex with DNA
more clearly, a fluorometric competitive binding experiment was
carried out using ethidium bromide (EB) as a probe that shows no
apparent emission intensity in buffer solution because of solvent
quenching. However, ethidium bromide emits intense fluorescent
light in the presence of DNA due to its strong intercalation
between the adjacent DNA base pairs. A competitive binding of
a small molecule to CT DNA could result in the displacement of
EB or quenching of the bound EB by the compound decreasing its
emission intensity. It was previously reported that the enhanced
fluorescence could be quenched by the addition of another
molecules.1,13 The emission spectra of EB bound to DNA in
the absence and in the presence of Ni(Que)2(H2O)2 complex are
given in Fig. 4. The emission band at 587 nm of the DNA-EB
system decreased in intensity with the increasing concentration
of the complex, and an equal absorption point appeared at
543.2 nm (data not shown). It has been reported that some small
organic molecules interact with DNA by intercalation when the
concentration ratio of them to DNA (cM/cDNA) is less than 100
and the fluorescence intensity of EB-DNA system is decreased by
50%.1,20 The inset in Fig. 4 shows the quenching extent has reached
up to 52% when ccomplex/cDNA = 1.2. The changes observed here are
Fig. 2 Absorption spectra of quercetin nickel(II) complex (10 mM) in
0.01 M Tris-HCl buffer at pH 7.2, in the absence and presence of increasing
amounts of CT DNA (from top to bottom, 0–40 mM bp). The arrow shows
the intensity decreased with increasing amounts of DNA.
Fluorescence spectral studies. The additional evidence for in-
tercalation into the DNA was obtained from fluorescence spectral
studies. The fluorescence spectra of Ni(Que)2(H2O)2 complex,
exhibiting a broad emission band in the range 340–360 nm,
were monitored at a fixed concentration of 30 mM. Enhanced
fluorescence without wavelength shift was observed (Fig. 3), when
CT DNA was added into the complex solution. The result suggests
that the stronger enhancement of fluorescence intensity for the
complex may be largely due to the interaction between adjacent
base pairs of CT DNA and the complex. Furthermore, the binding
of the complex to the DNA helix could decrease the collisional
frequency of solvent molecules with the complex, which usually
leads to emission enhancement of the complex. The result also
agrees with those observed for other intercalators.1,7–8 So the
complex may intercalate into the adjacent base pairs of CT DNA.
Fig. 4 Fluorescence spectra of the binding of EB to DNA in 0.01 M
Tris-HCl buffer at pH 7.2, in the absence and presence of increasing
amounts of quercetin nickel(II) complex (from top to bottom, 0–120 mM),
lex = 500 nm, cEB = 5 mM, cDNA = 100 mM bp. The arrow shows the
intensity decreased with increasing concentration of the complex. The inset
is Stern–Volmer quenching plots of DNA–EB system by the complex. r =
c
complex/cDNA
.
4724 | Dalton Trans., 2009, 4722–4728
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
often characteristic of intercalation. Moreover, this phenomenon
indicates that Ni(Que)2(H2O)2 complex competes with EB in
binding to DNA.
supercoiled form will relax to produce a nicked circular form. If
both strands are cleaved, a linear form will be produced. Relatively
fast migration is observed for supercoiled form when the plasmid
DNA is subjected to electrophoresis. The nicked circular form
migrates slowly and the linear form migrates between SC and
NC.26 Hence, DNA strand breaks were quantified by measuring
the transformation of the supercoiled form into nicked circular
and linear forms.
According to the classical Stern–Volmer equation:21
I0/I = 1 + Ksqr
(1)
where I0 and I represent the fluorescence intensities in the
absence and presence of the complex, respectively, and r is the
concentration ratio of the complex to DNA. Ksq is a linear Stern–
Volmer quenching constant dependent on the ratio of the bound
concentration of EB to the concentration of DNA (Fig. 4, inset).
The Ksq value is obtained as the slope of I0/I versus r linear plot.
From the inset in Fig. 4, the Ksq value for Ni(Que)2(H2O)2 complex
is 1.0, which suggests that the interaction of the complex with
DNA is strong.22 It may be due to the complex interacting with
DNA through intercalation binding, so releasing some free EB
from the DNA–EB complex,23 which is consistent with the above
absorption spectral results.
Effects of the concentration of the complex and ionic strength
on DNA cleavage. The ability of the Ni(Que)2(H2O)2 complexes
to induce DNA cleavage was studied by gel electrophoresis using
supercoiled pBR 322 DNA in 50 mM Tris-HCl/50 mM NaCl
buffer (pH 7.2). Both quercetin (data not shown) and Ni2+ (Fig. 6,
lane 2) alone induced little DNA cleavage. Fig. 6 shows the results
of the gel electrophoresis experiment carried out with supercoiled
DNA cleavage induced by various concentrations of the complex.
The complex scarcely catalyzed the cleavage of plasmid DNA both
at 10 mM and at 25 mM. When the concentration of the complex
was up to 50 mM, the cleavage of plasmid DNA was observed
obviously. And the increase in the amounts of nicked DNA was
observed associated with the increase of the concentration of the
complex.
Viscometric studies. To investigate further the DNA-binding
mode of Ni(Que)2(H2O)2 complex, viscosity measurements on
solutions of CT DNA incubated with the complex were performed.
It is well known that intercalative DNA binding would cause
elongation of DNA polymer by effecting separation of DNA base
pairs, resulting in an increase in viscosity. In contrast, a partial or
non-classical intercalation of the ligand could bend or kink DNA
resulting in a decrease in its effective length with a concomitant
decrease in its viscosity.24–25
The relative specific viscosity (h/h0) of DNA generally reflects
the increase in contour length associated with separation of DNA
base pairs caused by intercalation. Fig. 5 shows the increase in the
relative specific viscosity of DNA when Ni(Que)2(H2O)2 complex
is added into CT DNA solution. So the results demonstrate that
the complex binds to DNA by the intercalation mode, which is
consistent with the above absorption and fluorescence spectral
results.
Fig. 6 Agarose gel (1%) of pBR322 (0.25 mg) incubated for 1.5 h at 37 ◦C
and pH 7.2 (50 mM Tris-HCl) with increasing complex concentrations.
Lane 1: DNA control; lane 2: Ni2+ 100 mM; lane 3–8: Ni(Que)2(H2O)2
complex 10, 25, 50, 100, 200, 400 mM, respectively.
The effects of ionic strength on the double-strand DNA cleavage
by adding NaCl were studied. Fig. 7 shows that the process of
cleavage was sensitive to the change of ionic strength. When
the concentration of NaCl was not more than 50 mM, the
ionic strength could promote DNA cleavage. However, the extent
of DNA cleavage became obviously weakened when the ionic
strength from 50 to 800 mM. The results suggest that electrostatic
interactions contribute to the DNA cleavage.
Fig. 5 Effect of increasing amounts of quercetin nickel(II) complex on
the relative viscosity of CT DNA in 0.01 M Tris-HCl buffer (pH 7.2) at
30 0.1 ◦C. cDNA = 50 mM, r = ccomplex/cDNA
.
DNA cleavage activity
Fig. 7 Agarose gel (1%) of pBR322 DNA incubated for 1.5 h at 37 ◦C and
pH 7.2 with 100 mM quercetin nickel(II) complexes for increasing NaCl
concentrations. Lane 1: DNA control; lane 2–8: NaCl concentration was
10, 25, 50, 100, 200, 400, 800 mM, respectively.
The double-stranded plasmid pBR322 exists in a compact super-
coiled conformation (SC). Upon formation of strand breaks, the
supercoiled form of DNA is disrupted into the nicked circular
form (NC) and the linear form. If one strand is cleaved, the
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 4722–4728 | 4725
©

View Article Online
Effects of time on DNA cleavage. The time dependence of the
reaction was carried out in the presence and the absence of the
complex, and pBR322 DNA was incubated with 100 mM complex
◦
in 50 mM Tris-HCl buffer at 37 C for 15–240 min, respectively
(Fig. 8). The increase in the amounts of nicked DNA was observed
to be associated with the increase of reaction time. The amounts
of nicked DNA were 57.5% and 65.3% when the reaction time was
3 h and 4 h, respectively. Increasing the incubation beyond 8 h led
to the appearance of linear DNA (data not shown).
Fig. 10 Agarose gel (1%) of pBR322 (0.25 mg) incubated for 1.5 h at 37 ◦C
and pH 7.2 (50 mM Tris-HCl) with 100 mM quercetin nickel(II) complex
and different scavengers or H2O2. Lane 1: DNA control; lane 2: complex
control lane 3: DMSO (0.4 M); lane 4: glycerol (0.4 M); lane 5: hydrogen
peroxide (50 mM); lane 6: catalase (15 units).
hydroxyl radical and hydrogen peroxide might not occur in the
reaction. And 50 mM hydrogen peroxide could not promote
the cleavage of DNA induced by the complex. Therefore, DNA
cleavage promoted by the complex might not occur via an oxidative
pathway but takes place probably via a hydrolytic pathway.
In order to determine whether the presence of the complex
may increase the formation of reactive oxygen species (ROS), we
analyzed the influence of the complex on the oxidative damage
of CT DNA by measuring the formation of 2-thiobarbituric
acid reacting species (TBARS). It can be seen from Fig. 11 that
the absorbance of a sample treated by complexes is very small
compared with that of a sample treated by [Fe(EDTA)]2-/H2O2.
It suggested that the deoxyribose ring in DNA skeleton was not
cut by ROS and oxidative cleavage of DNA did not occur.
Fig. 8 Agarose gel (1%) of pBR322 (0.25 mg) at 37 ◦C and pH 7.2 (50 mM
Tris-HCl) with 100 mM quercetin nickel(II) complex for increasing reaction
time. Lane 1: DNA control; lane 2–9: 15, 30, 60, 90, 120, 150, 180, 240 min,
respectively.
Fig. 9 shows the extent of DNA cleavage by Ni(Que)2(H2O)2
complex with reaction time. The decrease in SC and the formation
of NC of DNA with time shows the expected exponential nature of
the curves. The plot of ln (%SC-DNA) versus time is linear, which
confirms the process to be pseudo-first-order. The rate constant
k1 is obtained (0.76 ¥ 10-4 s-1), using a complex concentration of
100 mM (Fig. 9, inset).
Fig. 11 The formation of 2-thiobarbituric acid reacting species (TBARS).
Control: [Fe(EDTA)]2-/H2O2. Each value represents the mean SD of
three experiments.
Fig. 9 Disappearance of supercoiled (SC) and formation of nicked
circular (NC) forms of pBR322 DNA in the presence of quercetin nickel(II)
complex (100 mM) with incubation time (pH 7.2, Temperature 37 ◦C).
Inset: Plot of ln (%SC-DNA) versus time for the complex concentration of
100 mM.
According to the experimental results above, we suppose simply
that the mechanism of DNA cleavage induced by Ni(Que)2(H2O)2
complex should be a cooperative process of the nickel cation and
the ligand molecules (quercetin or H2O), involved in the binding of
the complex with DNA. The sketch of the hydrolysis mechanism
is depicted in Fig. 12. The complex intercalating into DNA base
pairs could promote the direct coordination between nickel cation
and the negatively charged oxygen in the phosphodiester backbone
of DNA. The newly formed metal–oxygen bonds possibly include
Mechanism of DNA cleavage. To investigate the mechanism
of DNA cleavage promoted by Ni(Que)2(H2O)2 complex, hydroxyl
radical scavengers (0.4 M DMSO and glycerol), catalase (15 units),
and oxidant (50 mM H2O2) were introduced to the system. As
shown in Fig. 10, no evident inhibition of DNA cleavage was
observed in the presence of scavengers, which suggested that
4726 | Dalton Trans., 2009, 4722–4728
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Fig. 12 Proposed mechanism of hydrolytic cleavage of DNA by quercetin nickel(II) complex.
electrostatic interactions which enhance the electrophilicity of the
phosphorus, so the phosphorus becomes active enough to be target
of nucleophilic attack. There are two possible nucleophilic species
in the complex–DNA system. One is a water molecule linked
to the proximate hydroxyl oxygen of the quercetin ligand. The
hydroxyl oxygen of the quercetin ligand, acting as a Lewis base,
pulls a proton from the water molecule to promote its attack
the phosphorus atom. The other is coordination water linked
to the nickel cation in the complex. The aquo-hydroxo form of
the complex is formed when a proton is removed from one of
the metal-coordinated water molecules in the complex. Then, the
activated phosphorus atom could be nucleophilically attacked by
these two active nucleophilic species, resulting in the formation
of a pentacoordinated intermediate. Finally, one of the P–O ester
bonds of the phosphodiester in the DNA backbone is broken by
the intramolecular charge delivery, resulting in the DNA cleavage.
However, the real hydrolysis mechanism needs to be confirmed
through further detailed research.
of 0.76 ¥ 10-4 s-1, which shows a 107-fold rate acceleration over
that of uncatalyzed supercoiled DNA. DNA cleavage activity of
Ni(Que)2(H2O)2 complex may be explained by the mechanism
proposed for DNA hydrolysis that a pentacoordinated interme-
diate could be formed due to the active nucleophilic species
nucleophilically attacking phosphorus atom in the phosphodiester
backbone of DNA.
In conclusion, we have determined that the intercalation binding
modes of Ni(Que)2(H2O)2 complex with DNA and the hydrolytic
cleavage of DNA by the complex. Bioflavonoid, derived from
a variety of natural resources, can basically chelate metal ions
to form metal complexes, which could be a new source of
artificial metallonucleases, even antitumor agents. The selective
DNA binding mechanism, the potential of the complexes as
artificial nucleases and cytotoxicity activity of the complex are
under investigation.
Acknowledgements
This research was financially supported by the Science and
Technology Project of Education Commission of Chongqing
(Grant No. KJ051503). We are grateful to Chongqing Education
College Science Research Project for support of this research.
Conclusion
In the present study, the DNA-binding properties of
Ni(Que)2(H2O)2 complex have been examined by absorption
spectroscopy, fluorescence spectroscopy and viscosity measure-
ments. Evidence is presented that Ni(Que)2(H2O)2 complex could
interact with DNA via intercalation mode. Furthermore, in the
EB competition fluorescence assay, the Stern–Volmer quenching
constant for this complex, Ksq, is 1.0, illustrating that this complex
could strongly bind to DNA as an intercalator competing with
EB. DNA cleavage of the complex has been studied by agarose
gel electrophoresis and TBARS assay. It was demonstrated that
the complex can effectively promote the cleavage of plasmid DNA
at physiological pH and temperature via a hydrolytic pathway.
Kinetic data of DNA cleavage promoted by the complex (100 mM)
under physiological conditions give an observed rate constant k1
Notes and references
1 Y. M. Song, Q. Wu, P. J. Yang, N. N. Luan, L. F. Wang and Y. M. Liu,
J. Inorg. Biochem., 2006, 100, 1685.
ˇ
2 M. Kozˇurkova´, D. Sabolova´, L. Janovec, J. Mikesˇ, J. Koval, J.
Ungvarsky´, M. Stefanisinova´, P. Fedorocko, P. Kristian and J. Imrich,
Bioorg. Med. Chem., 2008, 16, 3976.
3 C.-P. Tan, J. Liu, L.-M. Chen, S. Shi and L.-N. Ji, J. Inorg. Biochem.,
2008, 102, 1644–1653.
4 G. Zuber, J.C. Jr. Quada and S. M. Hecht, J. Am. Chem. Soc., 1998,
120, 9368.
5 S. M. Hecht, J. Nat. Prod., 2000, 63, 158.
6 C. Metcalfe and J. A. Thomas, Chem. Soc. Rev., 2003, 32, 215.
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 4722–4728 | 4727
©

View Article Online
7 A. Silvestri, G. Barone, G. Ruisi, M. T. Lo Giudice and S. Tumminello,
J. Inorg. Biochem., 2004, 98, 589.
8 M. Navarro, E. J. Cisneros-Fajardo, A. Sierralta, M. Ferna´ndez-Mestre,
P. Silva, D. Arrieche and E. Marcha´n, JBIC, J. Biol. Inorg. Chem., 2003,
8, 401.
17 J. Zhou, L.-F. Wang, J.-Y. Wang and N. Tang, Transition Met. Chem.,
2001, 26, 57.
18 J. Tan, B.-C. Wang and L.-C. Zhu, Bioorg. Med. Chem., 2009, 17,
614.
19 G. Cohen and H. Eisenberg, Biopolymers, 1969, 8, 45.
20 W.-Y. Li, S.-T. Zhu and X.-W. He, Acta Chim. Sinica, 2002, 60,
105.
9 J. Tan, B.-C. Wang and L.-C. Zhu, Bioorg. Med. Chem. Lett., 2007, 17,
1197.
10 P. Nordell and P. Lincoln, J. Am. Chem. Soc., 2005, 127, 9670.
11 J. Tan, B.-C. Wang and L.-C. Zhu, Colloids Surf., B, 2007, 55, 149–152.
12 A. N. R. Pattubala, N. Munirathinam and R. C. Akhil, Eur. J. Inorg.
Chem., 2004, 2007, 1440.
13 N. Ademir, T. Herna´n, H. Rosmari, H. Adolfo, Jr., S. Bruno and S.
Juliet, Inorg. Chem. Commun., 2001, 4, 388.
14 A. Ne`gre-Salvayre and R. Salvayre, Free Radical Biol. Med., 1992, 12,
101.
15 I. B. Afanasev, A. I. Dorozhko, A. V. Brodskii, V. A. Kostyuk and A. I.
Potapovitch, Biochem. Pharmacol., 1989, 38, 1763.
16 I. Morel, G. Lescout, P. Cogrel, O. Sergent, N. Posdeloup, P. Brissot, P.
Cillard and J. Cillard, Biochem. Pharmacol., 1993, 45, 13.
21 J. R. Lakowicz and G. Weber, Biochemistry, 1973, 12, 4161.
22 J. Liu, T.-X. Zhang, T.-B. Lu, L.-H. Qu, H. Zhou, Q.-L. Zhang and
L.-N. Ji, J. Inorg. Biochem., 2002, 91, 269.
23 D. L. Boger, B. E. Fink, S. R. Brunette, W. C. Tse and M. P. Hedrick,
J. Am. Chem. Soc., 2001, 123, 5878.
24 S. Satyanarayana, J. C. Dabrowiak and J. B. Chaires, Biochemistry,
1992, 31, 9319.
25 T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M.
Chikira, P. T. Selvi and M. Palaniandavar, J. Inorg. Biochem., 2005, 99,
1205.
26 Y. Masataka, H. Miyako, N. Makoto and M. Keiko, Mol. Genet.
Metab., 1999, 68, 468.
4728 | Dalton Trans., 2009, 4722–4728
This journal is
The Royal Society of Chemistry 2009
©