Spectroscopic studies on the interaction between Pr(III) complex of an ofloxacin derivative and bovine serum albumin or DNA

Article abstract of DOI:10.1016/j.saa.2010.11.018

The binding properties on [PrL₂(NO₃)](NO ₃)₂ (L = 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1- piperaziny)-7-oxo-7Hpyrido[1,2,3-de]-1,4-benzoxazine-6-carbaldehyde benzoyl hydrazone) to bovine serum albumin (BSA

Full text of DOI:10.1016/j.saa.2010.11.018

Spectrochimica Acta Part A 78 (2011) 503–511
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectroscopic studies on the interaction between Pr(III) complex of an ofloxacin
derivative and bovine serum albumin or DNA
Min Xu, Zhao-Rong Ma, Liang Huang, Feng-Juan Chen, Zheng-zhi Zeng∗
Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province, State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2010
Received in revised form 12 October 2010
Accepted 15 November 2010
The binding properties on [PrL2(NO3)](NO3)2 (L = 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-
piperaziny)-7-oxo-7Hpyrido[1,2,3-de]-1,4-benzoxazine-6-carbaldehyde benzoyl hydrazone) to bovine
serum albumin (BSA) have been studied for the first time using fluorescence spectroscopy in combination
with UV–Vis absorbance spectroscopy. The results showed that [PrL2(NO3)](NO3)2 strongly quenched the
intrinsic fluorescence of BSA through a static quenching procedure, and non-radiation energy transfer
happened within molecules. The number of binding site was about 1, and the efficiency of Förster energy
transfer provided a distance of 4.26 nm between tryptophan and [PrL2(NO3)](NO3)2 binding site. At 288,
298, 310 K, the quenching constants of BSA–[PrL2(NO3)](NO3)2 system were 5.11 × 104, 4.33 × 104 and
3.71 × 104 l M−1. ꢀH, ꢀS and ꢀG were obtained based on the quenching constants and thermodynamic
theory (ꢀH < 0, ꢀS > 0 and ꢀG < 0). These results indicated that hydrophobic and electrostatic interac-
tions are the mainly binding forces in the [PrL2(NO3)](NO3)2–BSA system. In addition, the CD spectra have
proved that BSA secondary structure changed in the presence of [PrL2(NO3)](NO3)2 in aqueous solution.
Moreover, the interaction between [PrL2(NO3)](NO3)2 and calf thymus DNA (CT DNA) was studied by
spectroscopy and viscosity measurements, which showed that the binding mode of the [PrL2(NO3)](NO3)2
with DNA is intercalation. The DNA cleavage results show that in the absence of any reducing agent, the
[PrL2(NO3)](NO3)2 can cleave plasmid pBR322 DNA and its hydrolytic mechanism was demonstrated
with hydroxyl radical scavengers and singlet oxygen quenchers.
Keywords:
Pr(III) complex
Bovine serum albumin
Calf thymus DNA
pBR 322 DNA
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
chemical behavior of some compounds including their interactions
with serum proteins and DNA, which are primary target molecules,
In the past years, many studies on the molecular structure of
metal complexes and their biological activity caused much interest
in the field of inorganic chemistry [1,2]. Serum albumin as one of the
most abundant carrier proteins plays an important role in the trans-
port and disposition of endogenous and exogenous ligands present
in blood [3]. The nature and magnitude of drug–albumin interac-
tions significantly influence the pharmacokinetics of drugs, and the
binding parameters are useful in studying protein–drug binding
as they greatly influence absorption, distribution, metabolism, and
excretion properties of typical drugs [4]. Furthermore, DNA is an
important cellular receptor, many chemicals exert their antitumor
effects through binding to DNA thereby changing the replication
of DNA and inhibiting the growth of the tumor cell, which is the
basis of designing new and more efficient antitumor drugs and their
effectiveness depends on the mode and affinity of the binding [5–7].
Therefore, this inspires considerable interest in the study of the bio-
when metal compound is administered intravenously [8–12].
Ofloxacin (OFLX, 9-fluoro-2,3-dihydro-3-methyl-
(
)
10-(4-methyl-1-piperaziny)-7-oxo-7H-pyrido[1,2,3-de]-1,4-
benzoxazine-6-carboxylic acid, Fig. 1) is one of the most frequently
used fluorinated quinolones antibiotics in the world [13]. It has a
broad spectrum of activity against gram-positive, gram-negative
aerobic, facultatively anaerobic bacteria, chlamydiae, and some
related organisms, such as mycoplasmas or mycobacteria [14].
Several transition metal complexes of quinolones antibiotics have
been synthesized, characterized and evaluated for various biolog-
ical activities [15–17]. However, studies on biological activities
of the rare earth metal complexes with ofloxacin derivative have
been rarely reported.
Based on the above consideration, we synthesized and char-
acterized a novel ligand derivated from ofloxacin and its Pr(III)
complex. The interactions of BSA with Pr(III) complex have been
studied by using spectroscopy under simulative physiological con-
ditions. The quenching mechanism of fluorescence of BSA by Pr(III)
complex was explored by fluorescence spectrum at different tem-
peratures and UV–Vis spectrum. The number of binding site and
∗
Corresponding author. Tel.: +86 931 8912582; fax: +86 931 8912582.
E-mail address: zengzhzh@yahoo.com.cn (Z.-z. Zeng).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.11.018

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M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
6600 M−1 cm−1 at 260 nm. Stock solution of CT-DNA was stored at
277 K and used after no more than 4 days.
2.2. Physical measurements
The melting points of the compounds were measured on a
Beijing XT4-100× microscopic melting point apparatus (the ther-
mometer was not corrected). Elemental analysis was carried
out by using an Elemental Vario EL analyzer. Infrared spectra
(4000–400 cm−1) were recorded with KBr disks by using a Therrno
Mattson FTIR spectrometer. The UV–Vis spectra were recorded on
a Varian Cary 100 Conc spectrophotometer. 1H NMR spectra were
measured on a Varian VR 400-MHz spectrometer by using TMS as
a reference in DMSO-d6. Mass spectra were measured on aVGZAB-
HS (FAB) instrument. The fluorescence spectra were recorded on a
Hitachi RF-4500 spectrofluorophotometer.
Fig. 1. The structure of ofloxacin.
main sort of binding force in the medium of Tris–HCl buffer solu-
tion (pH 7.4) have been suggested. In addition, according to the
mechanism of Förster’s theory, the transfer efficiency of energy
and distance between the acceptor BSA and the donor Pr(III) com-
plex were calculated. Moreover, to explore the conformational
changes of BSA during the course of binding, we employed CD, a
well-known spectroscopic technique for obtaining information on
the secondary structure of proteins [18,19], together with meth-
ods (SELCON3, CONTIN, CDSSTR) for estimating protein secondary
structures from CD spectra [20–23]. The assignments obtained from
these methods gave six types of secondary structures: regular ␣-
helix, ␣R; distorted ␣-helix, ␣D; regular ␤-strand, ␤R; distorted
␤-strand, ␤D; turns, T; and unordered, U. The results suggest that
the Pr(III) complex influence the secondary structure of BSA. Mean-
while, we described a comparative study of the interaction of Pr(III)
complex and the ligand with CT DNA using spectroscopy and vis-
cosity measurements. Furthermore, this Pr(III) complex was found
to cleave pBR 322 DNA at physiological pH and temperature.
2.3. Procedure
2.3.1. Preparation of the ligand
The ligand (Fig. 2) was prepared according to a modified method
of the literature [25]. Ofloxacin (18.82 g; 50 mmol) was esterified
to 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperaziny)-7-
oxo-7H-pyrido [1,2,3-de]-1,4-benzoxazine-6-carboxylate, treat-
ments of the esters with N2H4·H2O gave the corresponding
hydrazine (18.19 g; (86%)), which was refluxed 3 h mixed with 2,4-
dihydroxybenzaldehyde, the ligand was obtained in 84% yield.
Ester: white solid, m.p. 238–240 ◦C, 1H NMR (CD3OH, 400 MHz)
ı (ppm): 1.432–1.449 (3H, d, J = 6.8 Hz, –CH3), 2.361–2.375(3H,
d, J = 5.6 Hz, –N–CH3), 2.642 (4H, s, 1ꢀ,4ꢀ-H), 3.230–3.238(1H,
m, 2-H), 3.325–3.336(4H, d, 2ꢀ,3ꢀ-H), 4.331–4.535(2H, m, 1-H),
7.360–7.391(1H, d, 3-H), 8.511(1H, s, 7-H).
Hydrazine: yellowish solid, m.p. 200–202 ◦C, 1H NMR (CD3OH,
400 MHz) ı (ppm): 1.462–1.478 (3H, d, J = 6.4 Hz, –CH3), 2.290(3H,
s, –N–CH3), 2.526 (4H, s, 1ꢀ,4ꢀ-H), 3.229–3.236 (1H, m, 2-H),
3.261–3.333 (4H, d, 2ꢀ,3ꢀ-H), 4.289–4.444 (2H, m, 1-H), 7.305–7.336
(1H, d, 3-H), 8.559 (1H, s, 7-H).
2. Experimental
2.1. Materials
All chemicals were purchased from commercial sources, and
used as received without further purification. CT-DNA and pBR322
DNA were obtained from Sigma–Aldrich (USA). Agarose was pur-
chased from Promega (German). EB and other chemicals were local
products of analytical grade. Solutions of CT-DNA in 50 mM NaCl,
5 mM Tris–HCl (tris(hydroxymethyl)aminomethane hydrochlo-
ride) (pH 7.2) gave a ratio of UV–Vis absorbance of 1.8–1.9:1 at 260
and 280 nm, indicating that the DNA was sufficiently free of protein
[24]. The CT-DNA concentration per nucleotide was determined
spectrophotometrically by employing an extinction coefficient of
Ligand: C25H26FN5O5, white solid, m.p. 264–266 ◦C, 1H NMR
(DMSO-d6, 200 MHz) ı (ppm): 1.703–1.733(3H, d, J = 6.0 Hz, –CH3),
2.507–2.781 (7H, m, –N–CH3, 1ꢀ,4ꢀ-H), 3.443–3.628 (5H, m, 2-H, 2ꢀ,
3ꢀ-H), 4.396–4.665 (2H, m, 1-H), 4.811–5.201 (2H, s, –OH, –OH),
7.099–7.138 (2H, d, 1ꢀ, 3ꢀ-H), 7.786 (1H, s, 7-H), 8.550 (1H, s, 4ꢀ-
H), 9.157 (1H, s, 3-H), 10.215 (1H, s, –CH N). FAB-MS: m/z = 496.1
[M+H]+. Anal. Calcd for C25H26FN5O5: C, 60.62; H, 5.32; N, 14.16.
Found: C, 60.60; H, 5.29; N, 14.13. IR vmax (cm−1): v(N–H):3352,
v(hydrazonic)C O: 1658, v(C N): 1608. UV: ꢁmax (nm): 300, 356.
Fig. 2. The synthetic route of the ligand: (i) C2H5OH, reflux, 20 h; (ii) C2H5OH, reflux, 4 h; (iii) CH3OH, reflux, 3 h.

M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
505
2.3.2. Preparation of the complex
sities were recorded in the absence and presence of DNA in the
mixture solution of each compound and NaCl at room temperature.
Viscosity experiments were conducted on an Ubbdlodhe vis-
cometer, immersed in a thermostated water-bath maintained to
25.0 ◦C. Titrations were performed for the complexes (1–5 ␮M), and
each compound was introduced into DNA solution (5 ␮M) present
in the viscometer. Data were presented as (Á/Ác)1/3 versus the ratio
of the concentration of the compound and DNA, where Á is the vis-
cosity of DNA in the presence of compound and Á0 is the viscosity
of DNA alone. Viscosity values were calculated from the observed
flow time of DNA containing solution corrected from the flow time
of buffer alone (t0), Á = t − t0 [27,28].
Ligand (0.119 g, 0.25 mmol) and NaOH (0.01 g, 0.25 mmol) was
mixed in 15 ml CH3OH, which stirred for 0.5 h at 60 ◦C, then
Pr(NO3)3·6H2O (0.1299 g, 0.3 mmol) was added to the yellowish
solution. The solution turned to deep yellow immediately and fur-
ther stirred 3 h at room temperature. A yellow precipitate, the Pr(III)
complex was separated from the solution by suction filtration, puri-
fied by washing several times with methanol, and dried for 24 h
under vacuum. Yield: 65%. Anal. Calcd for C50H52F2N13O19Pr: C,
45.60; H, 3.99; N, 13.84. Found: C, 45.56; H, 3.95; N, 13.82. IR vmax
(cm−1): v(N–H): 3373, v(C O): 1621, v(C N): 1587, v(NO3): 1476,
UV: ꢁmax (nm): 305, 363. ꢁm (S cm2 M−1): 146.8.
2.3.3. BSA binding experiments
2.3.5. DNA cleavage
The protein-binding study was performed by fluorescence
quenching experiments using BSA, 3 ␮M in Tris–HCl buffer (pH
7.4). The quenching of the emission intensity of BSA at 341 nm was
monitored using complexes as quenchers with increasing concen-
tration. Fluorescence spectra were recorded from 300 to 500 nm at
an excitation wavelength of 280 nm. The experiments were mea-
sured at three temperatures (288, 298 and 310 K). The temperature
of sample was kept by recycled water throughout the experiment.
Fluorescence spectrum scan was recorded at room temperature and
similar in methods to titration experiments.
The cleavage reactions were performed incubating pBR 322
(12 ␮M base pairs) at 37 ◦C in the presence/absence of increas-
ing amounts of metal complex for 5 h in TBE buffer [0.045 M
tris(hydroxymethyl)aminomethane (tris), 0.045 M boric acid, and
1 mM EDTA, pH 8.3]. And then the samples were analyzed by elec-
trophoresis for 1 h at 100 V on a 0.8% agarose gel in TBE buffer. The
gel was stained with 1 mg/ml ethidium bromide and photographed
on an Alpha Innotech IS-5500 fluorescence chemiluminescence and
visible imaging system.
A Varian Carry 100UV–Vis spectrophotometer equipped with
1.0 cm quartz cells was used for scanning the UV spectrum on the
range of wavelength from 190 to 400 nm. The Tris/HCl buffer solu-
tion was used as a reference solution.
3. Results and discussion
3.1. Structure of the Pr(III) complexes
Circular dichroism (CD) measurements were made on an Olis
RSM 1000 CD spectrometer in cell of 1 mm path length at room
temperature. CD spectra (200–260 nm) were taken at a BSA con-
centration of 1.50 × 10−6 M, and the results were taken as molar
absorbance ([ꢀε]) in cm2 dM−1. In order to obtain the secondary
structure fractions of BSA, the CDPro software package was used.
This consists of the three programs, SELCON3, CONTIN, and CDSSTR,
and a program for determining tertiary structure class (CLUSTER).
One of the major advantages of the CDPro software package is
that the programs have been modified to accept any given set
of reference proteins (CD spectra and secondary structure frac-
tions), and seven such reference sets are provided. Moreover,
input data files for these three programs are identical. More
information about CDPro is available at the following website:
http://lamar.colostate.edu/sreeram/CDPro [26].
The complex is air-stable for extended periods and very solu-
ble in DMSO and DMF; soluble in water, slightly soluble in ethanol
and methanol; insoluble in benzene and diethyl ether. The molar
conductivity of the complex is 146.8 S cm2 M−1 in DMF solution,
showing that this complex is 2:1 electrolytes [29]. The elemental
analyses and molar conductivities show that formulas of the Pr(III)
complex conform to [PrL2(NO3)](NO3)2.
3.1.1. Infrared spectrum study
IR spectra usually provide a lot of valuable information on coor-
dination reactions. All the spectra are characterized by vibrational
bands mainly due to the NH, C O, C N and NO3 groups. The
v(N–H) appears at 3352 cm−1 for the ligand, and this peak shifts
at 3373 cm−1 or so for the Pr(III) complex. The v(C O) vibration of
the free ligand is at 1658 cm−1; for the Pr(III) complex, the peak
shifts to 1621 cm−1, ꢀv(ligand–complexes) is equal to 37 cm−1. In
the complex, the band at 585 cm−1 or so is assigned to v(M–O).
It demonstrates that the oxygen of carbonyl has formed a coor-
dinative bond with the lanthanide ions [30]. The band at 1608−1
cm for the free ligand is assigned to the v (C N) stretch, which
shifts to 1587 cm−1 for its Pr(III) complex, ꢀv (ligand–complexes)
is equal to 21 cm−1. Weak bands at 410 cm−1 or so are assigned to v
(M–N) in the complex. These shifts and new bands further confirm
that the nitrogen of the imino-group bonds to the Pr(III) ions [31].
The absorption bands of the coordinated nitrates were observed
at about 1476 (vas) and 855 (vs) cm−1. The v3 free nitrates appear
at 1384 cm−1 or so in the spectra of the Pr(III) complexes. In addi-
tion, the separation of the two highest frequency bands |v4 − v1|
2.3.4. DNA binding experiments
The interaction of complexes with CT DNA has been studied with
UV spectroscopy in order to investigate the possible binding modes
to CT DNA and to calculate the binding constants to CT DNA (Kb).
In UV titration experiments, the spectra of CT DNA in the pres-
ence of each complex have been recorded for a constant CT DNA
concentration in diverse [complex]/[CT DNA] mixing ratios (r).
The competitive studies of each compound with EB have been
investigated with fluorescence spectroscopy in order to examine
whether it is able to displace EB from its CT DNA–EB complex. The
CT DNA–EB complex was prepared by adding 0.32 ␮M EB and 4 ␮M
CT DNA in Tris–HCl buffer (pH 7.2). The intercalating effect of com-
pounds with the DNA–EB complex was studied by adding a certain
amount of a solution of the compound step by step into the solu-
tion of the DNA–EB complex. The influence of the addition of each
compound to the DNA–EB complex solution has been obtained by
recording the variation of fluorescence emission spectra.
Iodide quenching experiments were used potassium iodide as
the quencher to determine the relative accessibilities of the free
and bound the Pr(III) complex.
is approximately 160 cm−1, and accordingly the coordinated NO3
ion in the complex is a bidentate ligand [32].
−
3.1.2. Thermal studies
The thermogravimetric analysis of the complexes have been
carried out in nitrogen environment over the temperature range
30–1200 ◦C. The complexes begin to decompose at 300 ◦C or so and
there are two exothermic peaks appear around 300–564 ◦C. The
corresponding TG curves show a series of weight loss. Under 200 ◦C,
The effect of the ionic strength on the compounds was also
investigated with fluorescence spectroscopy. Fluorescence inten-

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M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
Fig. 4. Fluorescence spectra of BSA–Pr(III) complex system. 1–11 respectively,
1.5 × 10−6 M BSA in the presence of 0.00, 1.25, 3.75, 6.25, 8.75, 12.5, 18.8, 25.0, 31.3,
37.5, 43.8 × 10−6 M Pr(III) complex.
The Stern–Volmer and Lineweaver–Burk graphs may be used in
order to study the interaction of a quencher in presence of BSA
(see Supporting Information Fig. S1). The curves have fine lin-
ear relationship according to Stern–Volmer quenching equation
(Eq. (1)) [37].
Fig. 3. The suggested structure of [PrL2(NO3)](NO3)2.
F0
there are no endothermic peaks and no weight loss on the corre-
sponding curves. It indicates that there are no crystal or coordinate
solvent molecules. While being heated to 800 ◦C, the curve became
plateau because complexes become their corresponding oxides.
= 1 + Kqꢂ0[Q] = 1 + KSV[Q]
(1)
F
where F0 and F are the fluorescence intensity in the absence
and presence of the quencher, [Q] is the concentration of the
quencher, ꢂ0 is the average bimolecular lifetime in the absence of
quencher and Kq is the bimolecular quenching rate constant which
is expected to be proportional to the diffusion coefficients and so to
be proportional to the solvent temperature. KSV is the Stern–Volmer
quenching constant.
3.1.3. UV spectra
The UV–Vis absorption spectra of the investigated compounds
in the absence and in the presence of the CT-DNA were obtained in
DMF:Tris–HCl buffer (pH 7.20) containing 50 mmol NaCl of 1:100
solutions, respectively. The UV–Vis spectra of ligands have two
types of absorption bands at ꢁmax in the regions of 294–302 nm and
352–359 nm, which can be assigned to ␲–␲* transitions within the
organic molecules, and ␲–␲* of the C N and C O groups, respec-
tively. While the UV–Vis spectra of its Pr(III) complex has two types
of absorption bands at ꢁmax in the regions of 300–308 nm and
356–365 nm, which can be, respectively, assigned to ␲–␲* tran-
sitions of the larger conjugated organic molecules and ␲–␲* of the
charge transfers from ligands to metal ions (L → Pr3+) [33,34]. The
band shifts of ꢁmax and the changes of ε for complex in comparison
with ligand indicate the formations of the complexes.
Since the crystal structures of its Pr(III) complexes have not been
obtained yet, we characterized the complexes and determined its
possible structure by elemental analyses, molar conductivities, IR
spectra, TG/DTA and UV–Vis spectra. The ligand and the Pr(III) ions
can form mononuclear ten-coordination [PrL2(NO3)](NO3)2 com-
plex with 1:2 metal-to-ligand stoichiometry at the Pr(III) centers
(Fig. 3).
Taking as fluorescence lifetime (ꢂ0) of BSA at around 10−8 s [38],
the dynamic quenching constant (KSV, M−1), and subsequently the
approximate quenching constant (Kq, M−1 s−1), can be obtained
by the slope of the diagram. The calculated values of KSV and Kq
for the interaction of the Pr(III) complex with BSA are given in
Table 1. Usually, the maximum quenching rate constant of diffusion
collision of various quenchers for the biomacromolecule is about
2.0 × 1010
M
−1 s−1 [39]. Obviously, the derived quenching rate con-
stants (which were about 10 × 1012
M
−1 s−1) were higher than the
Kq of the diffusion course by two orders of magnitude for com-
plexes. Thus, the fluorescence quenching was the consequence of
the static quenching instead of the dynamic collision quenching.
Meanwhile, in this study the KSV were found to decrease with an
increase in temperature, indicating that static quenching actually
occurred in the quenching process [40,41].
To further reveal the nature of quenching, fluorescence life-
time measurements were carried out by using TCSPC technique.
Fluorescence lifetime serves as a sensitive parameter for explor-
ing the local environment around a fluorophore, and it is sensitive
to excited-state interactions [42]. It also contributes to the under-
standing of the interactions between the probes and the proteins
[43]. The results show that no observed change in the magnitude of
3.2. BSA binding studies
3.2.1. The quenching mechanism of fluorescence of BSA by Pr(III)
complex
Table 1
BSA solutions exhibit a strong fluorescence emission with a peak
at 341 nm when excited at 280 nm [35]. An excitation wavelength
of 280 nm would excite both tyrosine and tryptophan side chains in
proteins, with the overall fluorescence intensity depending, in part,
on their relative abundances in the protein [36]. Under the same
experiment condition, the fluorescence intensity of its Pr(III) com-
plex was very weak. Addition of the Pr(III) complex to BSA causes
a concentration-dependent quenching (Fig. 4).
The quenching and dissociation constants of BSA with [PrL2(NO3)](NO3)2 at different
temperatures.
T (K)
Kq
KSV
R2
(×1012 M−1 s−1
)
(×104 M−1
)
288
298
310
5.11
4.33
3.71
5.11
4.33
3.71
0.998
0.996
0.996

M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
507
Table 2
Binding constants KA, number of binding sites n and thermodynamic parameters for
[PrL2(NO3)](NO3)2–BSA.
T (K) KA
n
R2
ꢀG
(kJ mol−1
ꢀH
(kJ mol−1
ꢀS
(×104 M−1
)
)
)
(J mol−1 K−1
)
288
298
310
5.40
4.21
3.29
0.911
0.884
0.912
0.994 −26.08
0.999 −26.41
0.999 −26.80
−16.72
32.50
the fluorescence lifetime of BSA with the addition of Pr(III) complex
was made [44].
The above results obtained both from the steady state and time
resolved spectroscopic measurements hint to the occurrence of
static-type fluorescence quenching phenomena caused by ground
state complex formations between BSA and Pr(III) complex.
Fig. 5. Overlap of fluorescence spectra of BSA and UV absorption spectrum of Pr(III)
complex. (a) Fluorescence spectra of BSA (1.50 × 10−6 M); (b) UV absorption spec-
trum of Pr(III) complex (1.50 × 10−6 M).
3.2.2. Binding constant and binding sites
The apparent binding constant KA and binding sites n can be
evaluated using the following equation (Eq. (2)) [45].
ꢀ
ꢁ
3.2.4. Energy transfer from BSA to complexes
F0 − F
1
log
= n log KA − n log
(2)
According to Förster nonradiative energy-transfer theory [48],
the efficiency of energy transfer is E, the critical distance for 50%
energy transfer is R0, and the actual distance of separation is r. These
values were calculated by Eqs. (5) and (6).
F
[Dt] − (F0 − F)[Pt]/F0
where F0 and F are the fluorescence intensities before and after
the addition of the quencher, respectively, and [Dt] and [Pt] are
the total quencher concentration and the total protein concen-
tration, respectively. Based on the plot of log(F0 − F)/F versus log
(1/([Dt] − (F0 − F)[Pt]/F0)) (see Supporting Information Fig. S2), the
number of binding sites n and binding constant KA can be obtained,
as presented in Table 2. It was noticed that the binding constant
values decreased with increase in temperature due to reduction of
the stability of Pr(III) complex–BSA system. The value of n is helpful
to know the number of binding sites and to locate the binding site
in BSA for the drug, which was noticed to be almost unity indicat-
ing that there was one independent class of binding site on BSA for
complex.
R06
R06 + r6
F
E = 1 −
=
(5)
F0
R06 = 8.78 × 10−25 Ä2n−4
˚TrpJ
(6)
where Ä2 is the orientation factor, ˚Trp is the quantum yield of
the donor tryptophan in the absence of acceptor, n is the refractive
index of the medium intervening between the donor and acceptor,
and J is the spectral overlap integral defined by Eq. (7).
ꢂ
F(ꢃ)ε(ꢃ)ꢃ−4ꢀꢃ
ꢂ
J =
(7)
F(ꢃ)ꢀꢃ
3.2.3. Main binding force between complexes and BSA
where F(ꢃ) is the fluorescence intensity of the donor, ε(ꢃ) is the
molar extinction coefficient of the acceptor in units of M−1 cm−1
,
The interaction force between compounds and proteins pertains
to weak interaction, including hydrogen bond, hydrophobic force,
electrostatic force, and Van der Waals’ interaction. If ꢀH ≈ 0, ꢀS > 0,
the main force is hydrophobic interaction; if ꢀH < 0, ꢀS > 0, the
main force is electrostatic effect; if ꢀH < 0, ꢀS < 0, Van der Waals
and hydrogen bond interactions play major role in the reaction
[46]. The enthalpy change ꢀH and entropy change ꢀS for a binding
reaction can be derived from the vant’t Hoff equations (Eq. (3)).
and ꢃ is the frequency in cm−1. The fluorescence emission spec-
trum of BSA and the UV-absorption spectrum of the Pr(III) complex
was shown in Fig. 5, which reveals that they have some overlap.
The value of J was 1.98 × 1014 cm3 l M−1 for the complex. The ori-
entation factor, Ä2 taken as 2/3 and the refractive index, n, was
taken as 1.36 [48]. The quantum yield, ˚Trp, was determined in the
study as 0.15 [49]. With use of the values of J, Ä2, n and ˚Trp, R0
value was calculated as 2.86 nm for the Pr(III) complex. The E was
0.08357 for the complex, so the energy transfer efficiency from BSA
to the Pr(III) complex is very weak. The actual distance r, between
the binding site of complexes binding in BSA molecule and Trp212
in BSA polypeptide chain was 4.26 nm for its Pr(III) complex. The
average distance of 2–8 nm between a donor and acceptor indicated
that the energy transfer from BSA to complexes occurred with high
probability [50].
ꢀH
RT
ꢀS
R
ln KA = −
+
(3)
where KA is analogous to the binding constant at the correspond-
ing temperature, R is gas constant. The free energy change can be
obtained from the following relationship (Eq. (4)).
ꢀG = ꢀH − TꢀS
(4)
Using the above two equations the values of ꢀG, ꢀH, ꢀS were
obtained and shown in Table 2. The binding process was always
spontaneous as demonstrated by the negative value of ꢀG and
the formation of the Pr(III) complex–BSA is an exothermic process,
accompanied by negative enthalpy and positive entropy changes.
Positive ꢀH and ꢀS values are frequently taken as typical evidence
of hydrophobic interactions, while negative enthalpy and entropy
changes arise from van der Waals and hydrogen bonding formation
[47,46]. Therefore from these results, the binding of Pr(III) complex
to BAS appears to involve hydrophobic interactions as shown by the
positive value of ꢀS although a lower component of electrostatic
interactions cannot be excluded.
3.2.5. Changes of BSA’s secondary structures induced by
complexes binding
Further experiments were carried out with CD technique to ver-
ify the binding of complexes to BSA. As Fig. 6 shows, a CD spectrum
of BSA gives double negative peaks, and the peak at 208 nm is higher
than the peak at 222 nm. When the complex added in BSA, the
intensity of the two bands increased, clearly indicating the increase
of the a-helical content in protein, respectively [51]. The average
fractions of secondary structure estimated from the CD data are
given in Table 3. The regular ␣-helix content was 42.27% and the
distorted ␣-helix content was 14.57% in the BSA, while the reg-

508
M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
where [DNA] is the concentration of DNA in base pairs, εa cor-
responds to the extinction coefficient observed (Aobsd/[M]), εf
corresponds to the extinction coefficient of the free compound, εb
is the extinction coefficient of the compound when fully bound to
DNA, and Kb is the intrinsic binding constant. The ratio of slope to
intercept in the plot of [DNA]/(εa − εf) versus [DNA] gives the values
of Kb [54].
The values of Kb are 5.17 × 103 M−1 and 1.01 × 104 M−1 for the
ligand and its Pr(III)complex. The Kb values of the complex are
higher than that of ligand suggesting that ligand presents higher
affinity to CT DNA, when it is coordinated to Pr(III). The higher
binding affinity of the Pr(III) complex is probably attributed to the
extension of the ␲ system of the intercalated ligand, which leads to
a planar area greater than that of the free ligand, so the coordinated
ligand penetrates more deeply into and stacks more strongly with
DNA base pairs.
Fig. 6. CD spectra of the BSA–Pr(III) complex system. (a) 1.50 × 10−6 M BSA; (b)
1.50 × 10−6 M BSA + 1.50 × 10−6 M Pr(III) complex.
3.3.2. EB–DNA quenching assay
The DNA binding modes of the compounds were also monitored
by a fluorescent EB displacement assay. EB is a phenanthridine flu-
orescence dye and is a typical indicator of intercalation, forming
soluble complexes with nucleic acids and emitting intense fluores-
cence in the presence of CT DNA due to the intercalation of the
planar phenanthridinium ring between adjacent base pairs on the
double helix [55].
The emission spectra of EB bound to CT DNA in the absence and
presence of each compound have been recorded for [EB] = 0.32 ␮M,
[DNA] = 4 ␮M for increasing amounts of each compound. The addi-
tion of the Pr(III) complex to the DNA-bound EB solutions caused
marked reduction in emission intensities, however, the addition
of the ligand arouse little reduction in intensities (see Supporting
Information Fig. S4). The observed quenching of DNA–EB flu-
orescence from Pr(III) complex suggests that the complex can
moderately displace EB from the DNA–EB complex and it can prob-
ably interact with CT DNA by the intercalative mode [56,57], but
for the ligand, the external contact may not excluded.
ular ␣-helix structure were reduced to 37.20% and the distorted
␣-helix structure increase to 23.50%, when the complex bind to BSA.
Moreover, the binding of this complex to BSA arouse the increase of
the regular ␤-strand content and turn, the decrease of distorted ␤-
strand content and unordered structure. The calculated results still
exhibited a reduction of ␣-helix structure from 56.84% to 50.2% for
Pr(III) complex, as described in Fig. 6. All the results indicate that the
binding of Pr(III) complex to BSA induces a conformational change
in BSA.
3.3. DNA binding studies
3.3.1. Electronic absorption titration
The UV–Vis absorption spectra of the investigated compounds
in the absence and in the presence of the CT DNA were obtained in
DMF:Tris–HCl buffer (5 mmol, pH 7.20) containing 50 mmol NaCl
of 1:100 solutions, respectively (see Supporting Information Fig.
S3). In the UV spectrum of ligand, the intensity of the bands cen-
tered at 300 nm and 356 nm decreased in the presence of increasing
amounts of CT DNA. For its Pr(III) complex, the absorption bands at
305 nm and 363 nm exhibited hypochromism of about 46.82 and
48.21%, with no shifts in the region of 305–363 nm. The observed
hypochromism could be attributed to stacking interaction between
the aromatic chromophore of the complexes and DNA base pairs
consistent with the intercalative binding mode [52].
The results derived from the UV titration experiments suggest
that all compounds bind to DNA although the exact mode of binding
cannot be merely proposed by UV spectroscopic titration stud-
ies. Nevertheless, the ofloxacin ligand provides an aromatic/planar
moiety so that the binding of the complex involving intercalation
between the base pairs of CT DNA cannot be ruled out.
The quenching plots illustrate that the quenching of EB bound
to DNA by the compounds which are in good agreement with the
linear Stern–Volmer equation (Eq. (1)). In the plots of F0/F versus
[Q], KSV is given by the ratio of the slope to the intercept. The data
show that the interaction of the Pr(III) complexes (2.78 × 104 M−1
)
with DNA is stronger than that of the free ligand(1.11 × 103 M−1),
which is consistent with the electronic absorption spectral results.
3.3.3. KI quenching
KI can effectively quench the fluorescence of a small molecule,
and the interaction mode of the small molecule with DNA can be
deduced from the variation of the fluorescence in the absence and
presence of DNA. When small molecule intercalates to DNA base
pairs, owing to the electrostatic repelling between DNA phosphate
backbone and the iodide anions, the iodide anions are difficult to
collide with the small molecules, which lead to a decrease in flu-
orescent quenching [58]. On the contrary, surface-binding mode
between small molecule and DNA cannot afford a protection sur-
rounding for the small molecule, the collision probability between
small molecule and iodide anions with the presence and absence of
DNA will be equal. When the interaction mode belongs to groove
binding, the small molecules will be partly protected by DNA, and
iodide anions can partly quench its fluorescence [59,60].
The binding constant Kb is a useful tool in order to calculate the
magnitude of the binding strength of compounds with CT DNA and
represents the binding constant per DNA base pair. It was usually
determined using the following equation (Eq. (8)) [53].
[DNA]
(εa − εf)
[DNA]
(εb − εf + 1/Kb(εb − εf))
=
(8)
Table 3
Average estimates of the secondary structure fractions of BSA solutions obtained
with the CDPro software package after reaction with [PrL2(NO3)](NO3)2.
From the results of the emission titrations for the Pr(III) complex
and potassium iodide with absence and presence of CT DNA, we
can found that the iodide anions could quench the fluorescence of
complex–DNA system and the solution that only contains complex.
The quenching data were plotted according to the Stern–Volmer
equation (Eq. (1)) and the slopes were calculated by the linear least-
squares method. The observed quenching constants were 5.02 M−1
Compound
Fraction of secondary structure (%)
␣
␣
␤
R
␤
D
T
U
R
D
BSA
42.27
26.70
14.57
23.50
3.13
7.40
12.23
11.60
7.00
15.27
21.13
11.40
BSA + [PrL2(NO3)](NO3)2

M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
509
Fig. 9. Effect of increasing amounts of the ligand and Pr(III) complex on the relative
Fig. 7. Stern–Volmer plot of the fluorescence titration data of Pr(III) complex. Effect
viscosity of CT DNA at 25.0 ◦C.
of KI concentration (1: Pr(III) complex + KI; 2: Pr(III) complex + KI + CT DNA).
and 2.21 M−1 with and without CT DNA for the Pr(III) complex
(Fig. 7). The quenching of the complex was in fact enhanced by
a factor of approach 2 when the complex was bound to the DNA
helix. We can conclude that the complexes are intercalated into the
DNA helix and they should be protected from the anionic quencher,
owning to the base pairs above and below the intercalators [61].
binding in solution in the absence of crystallographic structural
data [63,64]. A classical intercalation mode demands that the DNA
helix must lengthen as base pairs are separated to accommodate
the binding complexes, leading to the increase of DNA viscosity, as
for the behaviors of the known DNA intercalators [65]. In contrast,
a partial and/or non-classical intercalation of the complex could
bend (or kink) the DNA helix, reducing its viscosity concomitantly
[66]. The effects of all the compounds on the viscosity of CT DNA are
shown in Fig. 9. The viscosities of the DNA increase steadily with
increasing concentrations of ligand and the Pr(III) complex, and
the extent of the increase observed for the ligand is smaller than
that for the Pr(III) complexes. Viscosity measurements clearly show
that all the compounds can intercalate between adjacent DNA base
pairs, causing an extension in the helix and thus increase the vis-
cosity of DNA, and that the complex can intercalate more strongly
and deeply than the free ligand. The results obtained from the vis-
cosity experiments validate those obtained from the spectroscopic
studies.
3.3.4. Salt effect
The effect of the ionic strength on the complexes fluorescence
intensity was tested by the addition of a strong electrolyte, such
as NaCl, instead of potassium iodide. Cations of the salts can neu-
tralize the negatively charged phosphate. If the compound binds
to DNA through an electrostatic interaction mode, the surface of
DNA will be surrounded by the sodium ions with the increase of
ionic strength. Then the compound is difficult to approach DNA
and the strength of interaction with DNA decreases, the degree of
fluorescence quenching also falls [62]. As seen from Fig. 8, addition
of NaCl to the complexes in the absence and presence of CT DNA
has little or no influence on the fluorescence intensity, showing that
the interaction of the complexes with CT DNA is not electrostatic
interaction.
3.4. Cleavage plasmid pBR322 DNA
3.3.5. Viscosity titration measurements
3.4.1. Chemical nuclease activity
To further clarify the interactions between the studied com-
pounds and CT DNA, viscosity measurements were carried out.
Measurements of DNA viscosity that is sensitive to DNA length
are regarded as the least ambiguous and the most critical tests of
In order to assess the chemical nuclease activities of the Pr(III)
complexes for DNA strand scission, pBR322 DNA was incubated
with the Pr(III) complex under the reaction conditions. The cleav-
age reaction can be monitored by gel electrophoresis. When circular
pBR322 DNA is subjected to electrophoresis, relatively fast migra-
tion will be observed for the intact supercoiled form (Form I). If
scission occurs on one strand (nicking), the supercoiled form will
relax to generate a slower-moving nicked form (Form II). If both
strands are cleaved, a linear form (Form III) that migrates between
Form I and Form II will be generated [67,68].
To assess the DNA cleavage ability of the complexes, super-
coiled pBR322 DNA (20 ␮M) was incubated with 20–100 ␮M of
the Pr(III) complex in TBE buffer for 5 h without the addition of a
reductant [69]. Control experiments show that the free ligand and
Pr(NO3)3·6H2O are cleavage inactive (Fig. 10). The SC DNA (form
I) was cleaved by the complex, especially when the concentration
of the complex is 60 ␮M (Fig. 11). At the concentration of 60 ␮M,
the Pr(III) complex can almost promote the complete conversion
of plasmid pBR 322 from Form I to Form II (lane 4). However, the
cleavage activity decreased when the concentration of the complex
went on increasing. The result indicates that the cleavage efficiency
depends on the concentration of complex.
Fig. 8. Effect of NaCl concentration (Pr(III) complex + NaCl + CT DNA).

510
M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
interactions of the small molecule compounds binding to BSA and
DNA, and helpful in the development of their potential biological,
pharmaceutical and physiological implications in the future.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2010.11.018.
Fig. 10. Agarose gel electrophoresis patterns for the cleavage reaction of the pBR
322 DNA with 60 ␮M of free ligand and free Pr(NO3)3·6H2O for 5 h at 37 ◦C in a
TBE buffer at pH 8.3. Lane 1: pure DNA, without any additives; lane 2: DNA + 60 ␮M
of Pr(NO3)3·6H2O; lane 3: DNA + 60 ␮M of ligand; lane 4: DNA + 60 ␮M of Pr(III)
complex.
References
[1] A. Datta, N.K. Karan, S. Mitra, V.J. Gramlich, J. Chem. Crystallogr. 33 (2003)
579–583.
[2] B. Gillon, C. Mathoniere, E. Ruiz, S. Alvarez, A. Cousson, T.M. Kahn, J. Am. Chem.
Soc. 124 (2002) 14433–14441.
[3] D.C. Carter, J.X. Ho, Adv. Protein Chem. 45 (1994) 153–203.
[4] F. Zsila, Z. Bikadi, M. Simonyi, Biochem. Pharmacol. 65 (2003) 447–456.
[5] Y.B. Zeng, N. Yang, W.S. Liu, N. Tang, J. Inorg. Biochem. 97 (2003) 258–264.
[6] A.E. Friedman, C.V. Kumar, N.J. Turro, J.K. Barton, Nucleic Acids Res. 19 (1991)
2595–2602.
[7] A.M. Pyle, T. Morii, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 9432–9434.
[8] D.C. Cater, J.X. Ho, Adv. Protein Chem. 45 (1994) 153–203.
[9] S. Curry, P. Brick, N.P. Frank, Biochim. Biophys. A 1141 (1991) 131–140.
[10] Y.M. Wang, Y. Song, D.L. Kong, Chin. Sci. Bull. 50 (2005) 1839–1844.
[11] R.K. Mehra, K. Tran, G.W. Scott, P. Mulchandani, S.S. Saini, J. Inorg. Biochem. 61
(1996) 125–142.
Fig. 11. Agarose gel electrophoresis of the cleavage of the pBR 322 DNA in the
absence (lane 1) and in the presence of Pr(III) complex at various concentra-
tions for 5 h at 37 ◦C in a TBE buffer at pH 8.3. Lane 1: DNA control; lanes 2–6:
DNA + [complex] = 20, 40, 60, 80, 100 ␮M, respectively.
[12] Y.B.
Battal,
M.
Topuzogullari,
Z.
Mustafaeva,
J.
Fluoresc.,
doi:10.1007/s10895-009-0484-9.
[13] A.R.A. Abd-Allah, B.B. Gannam, F.M.A. Hamada, Pharm. Res. 42 (2000) 145–150.
[14] W.E. Sanders Jr., Clin. Infect. Dis 14 (1992) 539–554.
[15] K.C. Skyrianou, E.K. Efthimiadou, V. Psycharis, A. Terzis, D.P. Kessissoglou, G.
Psomas, J. Inorg. Biochem. 103 (2009) 1617–1625.
[16] K.C. Skyrianou, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg. Biochem.
104 (2010) 161–170.
[17] A. Tarushi, C.P. Raptopoulou, V. Psycharis, A. Terzis, G. Psomas, D.P. Kessis-
soglou, Bioorg. Med. Chem. 18 (2010) 2678–2685.
[18] R.T. Mostafa, H.M. Seyed, R. Bijan, Biochem. Mol. Biol. Int. 39 (2006) 530–536.
[19] S. Zahra, H. Saman, R. Bijan, N.G. Mohsen, Biochem. Mol. Biol. Int. 39 (2006)
636–641.
[20] N. Sreerama, R.W. Woody, Anal. Biochem. 287 (2000) 252–260.
[21] N.J. Greenfield, Anal. Biochem. 235 (1995) 1–10.
[22] N. Sreerama, R.W. Woody, Anal. Biochem. 209 (1993) 32–44.
[23] J.Y. Zheng, A.C. Celeste, K.R. Vipin, T.C. Cheng, J.D. Joseph, M.L. Roger, J. Phys.
Chem. B 108 (2004) 17238–17242.
[24] A.V. Milyutin, L.R. Amirova, V.E. Kolla, F.Y. Nazmetdinov, L.P. Drovosekova, Y.S.
Andreichikov, Pharm. Chem. J. 32 (1998) 422–424.
[25] P.X. Xi, Z.H. Xu, F.J. Chen, Z.Z. Zeng, X.W. Zhang, J. Inorg. Biochem. 103 (2009)
210–218.
Fig. 12. Agarose gel showing cleavage of pBR322 DNA (0.1 ␮g/␮L) incubated with
0.1 mM Pr(III) complexes for 5 h. at 37 ◦C in TBE buffer at pH 8.3. Lane 1: DNA control;
lane 2: DNA + Pr(III) complex; lane 3: DNA + Pr(III) complex + 100 mM NaN3; lane
4: DNA + Pr(III) complex + 100 mM l-Histidine; lane 5: DNA + Pr(III) complex + 2 ␮L
DMSO; lane 6: DNA + Pr(III) complex + 2 ␮L EtOH.
3.4.2. Mechanistic studies
The DNA cleavage activity of the complexes has been studied
in the presence of several additives to understand the mechanis-
tic pathway involved in the DNA cleavage reaction (Fig. 12). From
Fig. 12, we can see that no obvious inhibitions are observed for the
Pr(III) complex in the presence of NaN3 (lane 3) and l-Histidine
(lane 4), the results rule out the possibility of DNA cleavage by the
singlet oxygen or a singlet oxygen-like entity. The addition of DMSO
(lane 5), EtOH (lane 6) partly diminishes the nuclease activity of the
Pr(III) complex which is indicative of the involvement of hydroxyl
radical in the cleavage process.
[26] F.J. Chen, G.Q. Liu, Z.H. Xu, Z.Z. Zeng, Biochem. Mol. Biol. Int. 41 (2008) 305–309.
[27] M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Gradsland, Biochemistry 33 (1994)
5031–5040.
[28] Y. Xiong, X.F. He, X.H. Zou, J.Z. Wu, X.M. Chen, L.N. Ji, R.H. Li, J.Y. Zhou, R.B. Yu,
J. Chem. Soc., Dalton Trans. 1 (1999) 19–24.
[29] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81–122.
[30] F.D. Lewis, S.V. Barancyk, J. Am. Chem. Soc. 111 (1989) 8653–8661.
[31] N. Raman, A. Kulandaisamy, K. Jeyasubramanian, J. Ind. Chem. 41A (2002)
942–949.
4. Conclusions
[32] B.D. Wang, Z.Y. Yang, D.W. Zhang, Y. Wang, Spectrochim. Acta Part A 63 (2006)
213–219.
[33] M.M. Moawad, W.G. Hanna, J. Coord. Chem. 55 (2002) 439–457.
[34] T.M.A. Ismail, J. Coord. Chem. 58 (2005) 141–151.
[35] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed. Plenum, New
York, 1999.
[36] I. Sjoholm, B. Ekman, A. Kober, I. Ljungstedt-Pahlman, B. Seiving, T. Sjodin, Mol.
Pharmacol. 16 (1979) 767–777.
[37] S.S. Lehrer, Biochemistry 10 (1971) 3254–3263.
[38] J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161–4170.
[39] C.Q. Jiang, M.X. Gao, J.X. He, Anal. Chim. Acta 452 (2002) 185–189.
[40] Y.M. Huang, Z.J. Zhang, D.J. Zhang, Talanta 53 (2001) 835–841.
[41] L.H. Qian, X.L. Wang, Z.H. Tu, Acta Pharmacol. Sin. 22 (2001) 847–850.
[42] P. Das, A. Mallick, B. Haldar, A. Chakrabarty, N. Chattopadhyay, J. Chem. Phys.
125 (2006), 044516/1–6.
The Pr(III) complex are prepared from Pr(NO3)3·6H2O and
acylhydrazone ligands derived from ofloxacin. Firstly, the interac-
tion between the Pr(III) complex and BSA has been investigated
by fluorescence method combined with UV–Vis and CD spec-
troscopy techniques under simulative physiological conditions.
The results showed that the intrinsic fluorescence of BSA was
quenched through static quenching mechanism and the Pr(III) com-
plex bound to BSA with high affinity which is predominantly owing
to hydrophobic and electrostatic effect. The Pr(III) complex can be
deposited and transported by albumin. Experimental results also
showed that the binding of the Pr(III) complex to BSA induced a
conformational change of BSA, which was further proved by the
quantitative analysis data of CD spectrum. Secondly, the DNA bind-
ing mode of Pr(III) complex and ligand with CT DNA were also
studied via spectra and viscosity measurement. The results indi-
cate that the Pr(III) complex bind to DNA via an intercalation mode
and the Pr(III) complex can bind to DNA more strongly than the
free ligand. Noticeably, the Pr(III) complex has been found to pro-
mote cleavage of plasmid pBR 322 DNA. Results obtained from our
present work would be useful to understand the mechanism of
[43] A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem.
14683–14690.
B 109 (2005)
[44] M. Bardhan, G. Mandal, T. Ganguly, J. Appl. Phys. 106 (2009) 034701–034705.
[45] S.Y. Bi, L. Ding, Y. Tian, D.Q. Song, X. Zhou, H.Q. Zhang, J. Mol. Struct. 703 (2004)
37–45.
[46] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102.
[47] S.N. Timaseff, in: H. Peeters (Ed.), Proteins of Biological Fluids, Pergamon Press,
Oxford, 1972, pp. 511–519.
[48] W. De, W. Horrocks, W.E. Collier, J. Am. Chem. Soc. 103 (1981) 2856–2862.
[49] X.H. Liu, X.P. Xian, F.J. Chen, Z.H. Xu, Z.Z. Zeng, J. Photochem. Photobiol. B 92
(2008) 98–102.

M. Xu et al. / Spectrochimica Acta Part A 78 (2011) 503–511
511
[50] Y.J. Hu, Y. Liu, L.X. Zhang, J. Mol. Struct. 750 (2005) 174–178.
[51] Y.H. Chen, J.T. Yang, H.M. Martinez, Biochemistry 11 (1972) 4120–4131.
[52] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.
Chem. Soc. 111 (1989) 3053–3063.
[53] A. Wolf, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392–6396.
[54] S. Bhattacharya, G. Mandal, T. Ganguly, J. Photochem. Photobiol. B: Biol. 101
(2010) 89–96.
[55] G. Zhao, H. Lin, S. Zhu, H. Sun, Y. Chen, J. Inorg. Biochem. 70 (1998) 219–226.
[56] G. Psomas, A. Tarushi, E.K. Efthimiadou, Polyhedron 27 (2008) 133–138.
[57] O. Novakova, H. Chen, O. Vrana, A. Rodger, P.J. Sadler, V. Brabec, Biochemistry
42 (2003) 11544–11554.
[60] C.V. Kumar, R.S. Turner, E.H. Asuncion, J. Photochem. Photobiol. A: Chem. 74
(1993) 231–238.
[61] L.S. Lerman, J. Mol. Biol. 3 (1961) 18–30.
[62] G.M. Howe, K.C. Wu, W.R. Bauer, Biochemistry 15 (1976) 4339–4346.
[63] S. Mahadevan, M. Palaniandavar, Inorg. Chem. 37 (1998) 693–700.
[64] A.B. Tossi, J.M. Kelly, Photochem. Photobiol. 9 (1989) 545–556.
[65] J.G. Liu, Q.L. Zhang, X.F. Shi, L.N. Ji, Inorg. Chem. 40 (2001) 5045–5050.
[66] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993)
2573–2584.
[67] X.W. Liu, J. Li, H. Li, K.C. Zheng, H. Chao, L.N. Ji, J. Inorg. Biochem. 100 (2006)
385–395.
[58] X. Yang, W.H. Liu, W.J. Jin, G.L. Shen, R.Q. Yu, Spectrochim. Acta Part A 55 (1999)
2719–2727.
[59] C.V. Kumar, E.H. Asuncion, J. Chem. Soc. Chem. Commun. 6 (1992) 470–472.
[68] H. Chao, W.J. Mei, Q.W. Huang, L.N. Ji, J. Inorg. Biochem. 92 (2002) 165–170.
[69] M.S. Deshpande, A.A. Kumbhar, A.S. Kumbhar, M. Kumbhakar, H. Pal, U.B. Son-
awane, R.R. Joshi, Bioconjugate Chem. 20 (2009) 447–459.