Fluorescence studies, DNA binding properties and antimicrobial activity of a dysprosium(III) complex containing 1,10-phenanthroline

Journal of Photochemistry and Photobiology B: Biology 127 (2013) 192–201

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Fluorescence studies, DNA binding properties and antimicrobial activity of a dysprosium(III) complex containing 1,10-phenanthroline Mozhgan Khorasani-Motlagh ⇑, Meissam Noroozifar, Asieh Moodi, Sona Niroomand Department of Chemistry, University of Sistan & Baluchestan, P.O. Box 98155-147, Zahedan, Iran

a r t i c l e

i n f o

Article history: Received 18 June 2013 Received in revised form 25 July 2013 Accepted 19 August 2013 Available online 29 August 2013 Keywords: Dysprosium complex DNA binding Binding constant Thermodynamic parameters Antibacterial activity

a b s t r a c t Luminescence and binding properties of dysprosium(III) complex containing 1,10-phenanthroline (phen), [Dy(phen)2(OH2)3Cl]Cl2H2O with DNA has been studied by electronic absorption, emission spectroscopy and viscosity measurement. The thermodynamic studies suggest that the interaction process to be endothermic and entropically driven, which indicates that the dysprosium(III) complex might interact with DNA by a non intercalation binding mode. Additionally, the competitive fluorescence study with ethidium bromide and also the effect of iodide ion and salt concentration on fluorescence of the complexDNA system is investigated. Experimental results indicate that the Dy(III) complex strongly binds to DNA, presumably via groove binding mode. Furthermore, the complex shows a potent antibacterial activity and DNA cleavage ability. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction DNA is a genetic material and primary target molecule for most anticancer and antiviral therapies according to cell biologists that acts as a form of memory storage for genetic information. The interaction of small molecules with DNA is important in the design of new pharmaceutical molecules [1–3]. The study on the interaction of small molecules (often termed drugs or ligands) with DNA has been the focus of some recent research works in the scope of life science, chemistry and clinic medicine [4]. Drugs that bind with genomic DNA are effective as anti-tumor, antivirus and anti-bacterial therapeutic agents. Therefore, the interactions of metal complexes with DNA have been the subject of interest in the development of anticancer drugs, sequence-specific cleaving agents and effective chemotherapeutic agents for numerous diseases and provide routes toward rational drug design because possess better bioactivities, such as antioxidant activity, cytotoxic activity and DNA binding affinity [2,5–7]. Study on DNA hybridization or its interactions with various molecules is detected by various methods including fluorescence, surface plasmon resonance, quartz crystal microbalance and electrochemistry [1–3]. In recent years, many lanthanide complexes with ligands such as tetracycline, phenanthroline, adriamycin and pyridine have been synthesized as a probe for DNA [5,7]. Among these heterocyclic ligands, 1,10-phenanthroline and the related derivatives have attracted much more attention because of being applicable to chelating ⇑ Corresponding author. Tel.: +98 541 244 6565; fax: +98 541 244 6888. E-mail address: [email protected] (M. Khorasani-Motlagh). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.08.009

ligands forming stable complexes with lanthanides ions and act as new reagents for biotechnology and medicine [5,8]. In the previous papers, we reported optical spectroscopic studies designed to characterize the interaction of some complexes of lanthanides ions with 1,10-phenanthroline and 2,20 -bipyridine and DNA [9–14]. We have already established in our laboratory to investigate the interaction of the Dy(III) complex with 1,10-phenanthroline ligand (Scheme 1) and fish salmon DNA and explore the DNA binding properties by fluorescence and UV spectroscopic techniques and viscosity experiments. We have also reported the antibacterial activity of the Dy(III) complex against Gram +ve and Gram ve bacteria and good activity was obtained. Also, the DNA cleavage ability of the complex is studied by gel electrophoresis.

2. Experimental 2.1. Apparatus and reagents Electronic spectra were measured on an analytikjena SPECORD S100 spectrometer with photodiode array detector with thermo stated cell compartment, using a 1 cm optical-path quartz cell. Emission spectra were recorded on a PERKIN ELMER, LS-3. Viscosity experiments were carried out using a viscometer (SCHOT AVS 450) maintained at a constant temperature immersed in a thermostated water-bath. Commercially pure chemicals were used as purchased from Merck and Aldrich Chem. Co. Fish salmon DNA purchased from Fluka Biochemika (Switzerland) was stored at 4 °C and used as received. [Dy(phen)2(OH2)3Cl]Cl2H2O was synthesized

M. Khorasani-Motlagh et al. / Journal of Photochemistry and Photobiology B: Biology 127 (2013) 192–201

where F0 and F is fluorescence intensities in the absence and in the presence of quencher, respectively, [Q] the concentration of quenching reagent and KSV is the Stern–Volmer constant [12]. The intrinsic binding constant (Kb) of the Dy(III) complex with DNA was obtained by monitoring the changes in centered charge transfer bands of phenanthroline ligand with increasing concentration of DNA using the following function equation [1,19]:

H2O Cl N

N

Cl2 H2O

Dy N

N

H2O H2O

½DNA

ea  ef

Scheme 1. The structure of complex [Dy(phen)2Cl(OH2)3]Cl2H2O.

by literature method [15]. Other chemicals and solvents used were of analytical reagent grade and without further purification. 2.2. General procedure for DNA-binding studies All the experiments involving the interaction of the Dy(III) complex with DNA were carried out in Tris–HCl buffer solution containing 5 mM Tris–HCl and 50 mM NaCl, and adjusted to pH = 7.2 with hydrochloric acid. Doubly distilled water was used to prepare buffer. The concentration and the purity of DNA was determined by UV–Vis spectroscopy. A solution of DNA gave a ratio of UV absorbance at 260 and 280 nm of about 1.8–1.9, indicating that the DNA was sufficiently free of protein [16]. The DNA concentration per nucleotide was determined spectrophotometrically by employing an extinction coefficient of 6600 M1 cm1 at 260 nm [17]. Stock solutions were stored at 4 °C and used no more than 4 days after preparation. For UV–Vis spectral titrations, 1  105 M concentration of Dy(III) complex solution was used and fish salmon DNA was added in steps from 0.17 lM to 25.2 lM. Centered charge transfer bands included p ? p transitions attribute to phenanthroline ligand were monitored to follow the interaction of the complex with DNA. For fluorescence intensity measurements, the excitation wavelength was fixed at 264 nm and the emission range was adjusted from 270 to 700 nm. The emission peak was observed at 369 nm. All measurements were made in a thermostated cuvette. For emission spectral titrations, 6  107 M concentration of the Dy(III) complex solution was used while fish salmon DNA concentration was varied from 1.0 lM to 21.5 lM. In the competitive binding study of the Dy(III) complex with ethidium bromide, fluorescence quenching experiments were conducted by adding small aliquots of a 2.8  103 M stock solution of Dy(III) complex to the sample containing 8.3 lM ethidium bromide (EB) and 140 lM DNA in Tris–HCl buffer solution ([DNA]/[EB] = 16.9). Samples were excited at 340 nm and emission was observed between 500 and 700 nm. 2.3. Binding data analysis The intrinsic binding constant (Kb) of the complex with DNA was obtained through fluorescence spectra according to the following equation [18]:

log

F0  F ¼ log K b þ n log½Q  F

ð1Þ

where Kb and n are the binding constant and the number of binding sites in base pairs, respectively and [Q] is the concentration of quenching reagent. A constant parameter namely Stern–Volmer constant (KSV) was used to measure the fluorescence quenching efficiency and was achieved from classical Stern–Volmer equation [12]:

F0 ¼ 1 þ K SV ½Q  F

193

ð2Þ

¼

½DNA

eb  ef

þ

1 K b ðeb  ef Þ

ð3Þ

where [DNA] is the concentration of DNA in base pairs, ea, ef, and eb correspond to the apparent absorption coefficient Aobsd/[complex], the extinction coefficient for the observed absorption value at given DNA concentrations, extinction coefficient for the free complex and the extinction coefficient for the complex in the fully bound form, respectively. The following equations were used to calculate the thermodynamic parameters [12,18]:

DG ¼ DH  T DS

ln K b ¼ 

  DG0 DH0 1 DS0 þ ¼ T RT R R

ð4Þ

ð5Þ

where Kb is the binding constant at corresponding temperature, R is the gas constant and T is the temperature in Kelvin, DG°, DS°, DH° are the free energy change, the entropy change and enthalpy change respectively. 2.4. Optimal conditions for fluorescence emission titrations The emission spectra of the Dy(III) complex were recorded in three solvents and have been shown in Fig. 1A. From Fig. 1A is seen that the fluorescence spectra of the Dy(III) complex exhibit a slightly red shift with increasing solvent polarity from 366 nm in acetonitrile to 369 nm in water. The change in the solvent polarity is reflected in the fluorescence emission intensities. The fluorescence emission intensity increases with increasing the solvent polarity from acetonitrile to water where it reached maximum intensity. So, doubly distilled water was used throughout all experiment as a solvent medium. For optimization of concentration, the fluorescence spectra of the Dy(III) complex were recorded in the range of 1  103 to 1  109 M. The experimental results indicated that the maximum intensity could be obtained when the complex concentration was 1  107. The subsequent studies were carried out in the range of 1  107 to 10  107 M (Fig. 1B) and a complex concentration of 6  107 M was selected as optimal. The preliminary investigation indicated that addition of DNA would quench the fluorescence of the Dy(III) complex. So, the effect of pH on the fluorescence intensity of complex was studied without and with of 4.7  106 M DNA at different pH values (from 2 to 11). Results have been shown in Fig. 1C. From this figure, it is clear that DF value reaches to the maximum at the pH 7. Here, DF = F0F, that F0, F are the fluorescence intensity of the Dy(III) complex in absence and presence of DNA, respectively. So pH 7.2 (physiological pH condition) was recommended for DNA binding investigations and the subsequent experiments were done in Tris–HCl buffer (pH 7.2). The effect of incubation time on the DF was investigated in the range of 0–12 h. The maximum DF was obtained immediately after the solutions were mixed. Therefore, the fluorescence spectra were directly recorded after mixing the solutions and an additional incubation time was not needed in our work.

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(A)

500

a

Intensity/a.u.

400 300

b 200 100

c

0 300

350

400

450

500

550

Wavelength/nm

(B) 500

(C) 450

450

400 350

350

Intensity/a.u.

Intensity/a.u.

400 300 250 200 150

300 250 200 150

100

100

50

50

0

0 1

2

3

4

5

6

7

8

9

10

[Complex]×107/M

2

3

4

5

6

7

8

9

10

11

pH

Fig. 1. Optimum conditions for fluorescence emission titrations. (A) The emission spectra of Dy(III) complex in different solvents; [complex] = 6  107 M; (a) water; (b) methanol; (c) acetonitril. (B) The effect of Dy(III) complex concentration on fluorescence intensity; (C) The influence of pH on the fluorescence spectra; [complex] = 6  107 M; [DNA] = 47  107 M.

2.5. Antimicrobial experiment The in vitro evaluation of antimicrobial activity was carried out to provide antimicrobial efficiency of the Dy(III) complex. The antimicrobial effect of the prepared compound was tested against several bacterial species and the minimum inhibitory concentration (MIC) for the complex was measured. MIC is the lowest concentration of solution to inhibit the growth of a test organism. Antibacterial activity of the Dy(III) complex was tested against the Gram +ve bacteria namely Micrococcus luteus, Enterococcus, Bacillus and Bacillus cereus and Gram ve bacteria namely Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Shigella, Acinetobacter baumannii, and Salmonella paratyphi B, C cultured on Mueller–Hinton agar and performed by the disk diffusion method [20]. The stock solution (1  103) of the complex was prepared in H2O. Overnight grown bacteria cultures were adjusted to 106CFU = mL. Then, 100 lL cell suspensions were spread on the surfaces of Mueller–Hinton agar (Oxoid). The disks (6 mm in diameter) were impregnated with 10 lL of the extracts or fractions as 30 lg per disk and placed on the inoculated media. The Petri dishes were stood for 2 h at 4 °C for diffusion of the metabolites and then incubated at 37 °C for 24 h. Antimicrobial activity was determined by measuring the radius of the clear inhibition zone around each disk (in mm). Each test was carried out in triplicate in individual experiments and the average was reported. 2.6. DNA cleavage experiment The cleavage of fish salmon DNA was determined by agarose gel electrophoresis. The gel-electrophoresis experiments were carried by incubation of the samples containing1.4  103 M fish salmon

DNA, 2.4  102 and 3.1  102 M dysprosium complex and in the absence and presence of 20  103 hydrogen peroxide (H2O2) as oxidant in Tris–HCl/NaCl buffer (pH 7.2) for 60 min at 25 °C and loaded on 0.8% agarose gel after mixing 5 ll of loading buffer (25% bromophenol blue + 0.25% xylene cyanol + 30% glycerol (3 ll) + sterilized distilled water. The samples were analyzed by electrophoresis for 80 min at a constant voltage, 50 V, using Tris– acetic acid–EDTA buffer (pH 7.2), until the bromophenol blue reached to three-fourth of the gel. The gel was then stained for 20 min by immersing it in 1 lg/cm3 ethidium bromide solution. It was then de-stained by keeping in sterile distilled water for 20 min. The bands were visualized by viewing the gel under UV light and photograph. 3. Results and discussion In general, there is a variety of binding sites and binding modes in the DNA molecule and small molecules interact with double helix DNA in covalent or non-covalent way. Non-covalent interaction is included three distinct modes: electrostatic interaction, major or minor groove binding and intercalation into the base pairs. In order to investigate the mode interaction of small molecules with DNA, a number of techniques have been used such as UV–Vis and fluorescence spectroscopy, circular dichroism, cyclic voltammetry measurements and mass spectrometry. Among these techniques, simple but sensitive methods are UV–Vis and fluorescence spectroscopy [18]. The binding properties of the Dy(III) complex with DNA were investigated by means of spectroscopic studies including UV–Vis and fluorescence. Furthermore, other tests namely ethidium bromide displacement experiments, iodide quenching, salt effect, thermodynamic studies and viscosity measurements also were carried out to determine binding mode.

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3.1. Binding constant and number of binding sites

interactions, such as excited-state reactions, molecular rearrangements, energy transfer and ground-state complex formation [9,10]. In our experiments, the Stern–Volmer constant, KSV, has been determined from the slope of the plot of F0/F versus [DNA] and shown in Fig. 2C. According to Eq. (2), KSV was calculated to be about (6.58 ± 0.1)  104 M1 at 298 K. The value is too large to be due to the collisional quenching. It proves that the quenching process is being static [12,21].

The fluorescence intensity data can be used to obtain the binding constant (Kb) and the number of binding sites (n) for a small molecules bound to a macromolecule [18]. The spectral results of the emission titrations for the Dy(III) complex with DNA are illustrated in the titration curves (Fig. 2). The intensity of emission bands at 369 nm of the complex decreased with increasing concentrations of DNA but the shape of the bands remained unchanged. Based on Eq. (1) and the plot of log(F0/F)/F versus log [DNA] (Fig. 2B), n and binding constant (Kb) were obtained. The calculated values of n and Kb were 1.24 and (8.51 ± 0.12)  105 M1 at 298 K, respectively. The binding constant for some typical intercalators such as ethidium bromide and acridine orange has been reported 2.6  106 M1 and 4  105 M1, respectively [18]. In this work, the binding constant value of the DNA–Dy(III) complex is comparable to that of acridine orange and the observed value for [Nd(bpy)2Cl3OH2] (2.4  105 M1) that the major interaction mode between Nd(III) complex with DNA is groove binding [9].

3.3. Competitive binding studies by EB–DNA quenching assay To further clarify the interaction of the Dy(III) complex with DNA, the competitive binding experiment was carried out by a fluorescent EB displacement assay. EB is one of the most sensitive and a common fluorescent probe for DNA structure and can bind to DNA by the strong intercalation mode of the planar phenanthridinium ring between adjacent base pairs on the double helix, so fluorescence emission enhances due to intercalation of EB to DNA [22,23]. Mode and process of metal complexes binding to DNA is examined by measuring of quenching extent of fluorescence of DNA–EB system. Because the binding sites of DNA that available for EB are decreased by complex binding [22]. In this work, the emission intensity of EB bound to DNA before and after adding the Dy(III) complex into the solution of DNA–EB complex, was measured by fluorescence spectroscopy. The fluorescence intensity

3.2. The fluorescence quenching mechanism Decrease in fluorescence intensity of compound that is called fluorescence quenching occurs by a variety of molecular

(B)

-1.8

(C)

y = 1.2444x + 5.9299 R2 = 0.9945

-1.6

y = 0.0658x + 0.89 R2 = 0.9839

2.1

-1.2 -1

F0/F

log(F0-F)/F

-1.4

2.5

-0.8 -0.6

1.7 1.3

-0.4 -0.2

0.9

0 0.2

0.5 -4.7

-4.9

-5.1

-5.3

-5.5

-5.7

-5.9

0

-6.1

4

8

(A)

12

16

20

24

[DNA]×106/M

log[DNA] 500

a 450 400

Intensity/a.u.

350 300 250

l

200 150 100 50 0 300

320

340

360

380

400

420

440

460

480

500

Wavelength/nm Fig. 2. (A) The emission diminishment spectra of Dy(III) complex (6  107 M) in the presence of different concentrations of DNA: a = 0, b = 1.0, c = 2.8, d = 3.7, e = 5.6, f = 7.5, g = 9.3, h = 11.2, i = 13.1, j = 15.9, k = 18.7, l = 21.5 lM (pH = 7.2, T = 298 K, kex = 264 nm). (B) The plot of log (F0F)/F versus log [DNA] at 298 K. (C) The Stern–Volmer plot for the quenching of Dy(III) complex by DNA.

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at 587 nm (kex = 340 nm) of DNA–EB was examined against the Dy(III) complex concentration. Fig. 3 shows the emission spectra of the DNA–EB system with increasing amount of the Dy(III) complex. The addition of the Dy(III) complex to the DNA-bound EB solution caused an obvious reduction in emission intensity, indicating that the complex competes with EB in binding to DNA. The decrease in the fluorescence of the system is due to the quenching of DNA–EB complex by the bound Dy(III) complex. Generally, if the interaction mode between a compound and DNA is being intercalation mode, the emission intensity of the DNA–EB system will be decrease more than 50% and the ratio of the concentration of drug to DNA will be less than 100 [4]. From the inset of Fig. 3, it is obvious that by adding the Dy(III) complex to the DNA– EB system, the intensity of DNA–EB system decreased by 44% which is less than 50%. The result indicates that the Dy(III) complex do not intercalate between the base pairs but releases the EB molecules and decreases intensity. Therefore, the binding mode of the complex with DNA may be groove than intercalation binding.

3.4. Electronic absorption spectral studies Electronic spectroscopy is commonly used to study the interaction of a metal complex with DNA. When the complex binds with DNA by intercalation mode, usually hypochromism with red shift is seen, because the intercalative mode involving the strong stacking interaction between a complex chromophore and the base pairs of DNA [24]. The electronic absorption spectra of Dy(III) complex in the absence and presence of DNA are given in Fig. 4. From Eq. (3), Kb is given by the ratio of the slope to the intercept. The intrinsic binding constant Kb of the complex was calculated to be (8.41 ± 0.14)  105 M1, from the decay of the absorbance using

the Eq. (3) by plotting of [DNA]/(ea–ef) versus [DNA] (inset of Fig. 4). This value is comparable to that of [Cu(LL)]2+ (1.4  105) (LL = the template condensate of orthophenylene diamine and benzilidene diacetyl curcumin (ben-diacecur)) that this complex interacts with DNA by groove binding [25].

3.5. Thermodynamic studies for assignment of binding mode There are several acting force between small molecular and bio-macromolecule. Intermolecular interacting forces include hydrogen bond, van der Waals force, electrostatic and hydrophobic interactions, etc. Consequently, a free energy change (DG°) for a binding interaction at different temperatures and enthalpy (DH°) and entropy (DS°) can be evaluated by the van’t Hoff equation [18,26]. In the present study, we calculated the thermodynamic parameters from the relationship between ln Kb and the reciprocal absolute temperature (inset of Fig. 5). At 20, 25 and 33 °C, the binding constants of the Dy(III) complex with DNA were calculated by emission spectra and the thermodynamic parameters were determined from linear van’t Hoff plot and the results have been represented in Fig. 5 and Table 1. The enthalpy change (DH°) was calculated from the slope of the van’t Hoff relationship and y-intercept gave DS° (Eq. (5), inset of Fig. 5). The free energy change (DG°) was estimated from Eq. (4). The binding forces of the Dy(III) complex with DNA (DH°, DG° and DS°) were calculated. DH° and DS° for the binding reaction between Dy(III) complex and DNA were found to be +89.03 kJ mol1and +412.31 J/mol K. At 293, 298 and 306 K, DG° calculated were 31.69, 33.75 and 37.04 kJ mol1, respectively. By calculating of binding constants at different temperatures (Fig. 5), it is found that the binding constant increases with the temperature and suggests that the binding reaction of

F/F0

1

0.5

0 0.2

0.6

1

1.4

1.8

2.2

[Complex]/[DNA] 400

a

350

Intensity/a.u.

300 250

o 200 150 100 50 0 545

565

585

605

625

645

Wavelength/nm Fig. 3. The emission spectra of DNA–EB system, in the presence of a = 0, b = 28, c = 56, d = 74.7, e = 93.3, f = 112, g = 140, h = 158.7, i = 177.3, j = 186.7, k = 205.3, l = 224, m = 233.3, n = 252, o = 280 lM of Dy(III) complex (pH = 7.2, kex = 340 nm, kem = 587 nm). [EB] = 8.3 lM; [DNA] = 140 lM. Inset: The plot of F/F0 versus [complex]/[DNA].

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y = 42.136x + 50.074 R2 = 0.9971

[DNA]/(εa-εf)×1012M2cm

1200 1000 800 600 400 200 0 0

3

6

9

12

15

18

21

24

27

[DNA]×106/M 0.8

a

0.7

Absorbance

0.6 0.5

g

0.4 0.3 0.2 0.1 0 240

260

280

300

320

340

Wavelength/nm Fig. 4. Absorption spectra of the Dy(III) complex on the addition of DNA in a Tris buffer medium (pH = 7.2, T = 298 K) in the absence (top spectrum; a = 0) and presence of increasing amounts of DNA (b = 0.17, c = 1.4, d = 2.24, e = 5.6, f = 12.9, g = 25.2 lM; subsequent spectra). [complex] = 1  105 M. Inset: The plot of [DNA]/(ea–ef) versus [DNA].

the Dy(III) complex with DNA is endothermic. This point is further proved by the value of DH°. The binding reaction is spontaneous (DG° < 0) accompanied by positive DS° value. The calorimetric studies and analysis of their thermodynamic data reveal that all groove-binders have positive binding entropies and in no case is the TDS term positive and binding of groove binders was predominantly entropically driven and binding is stabilized by hydrophobic interactions, as well as van der Waals interactions and hydrogen bonding whereas intercalation binding is driven by large, favorable enthalpy contributions [27–29]. For groove-binders, the average binding free energy (9.5 ± 1.6 kcal mol1) is higher than that for monointercalatore (7.3 ± 0.8 kcal mol1). For intercalators, the ratio DH/DG ranges from 0.83 to 1.97 [29]. Our results indicate that the average binding free energy is 8.2 kcal mol1 and ratio DH°/DG° ranges from 2.40 to 2.81. Thus we conclude that the Dy(III) complex interacts to DNA with groove binding mode [29]. 3.6. Absorption spectrum and binding mode investigation The electronic spectrum of Dy(III) complex was recorded with and without DNA and the results have been shown in Fig. 6. The intercalative binders commonly results in hypochromic and bathochromic effect [30]. Fig. 6 was shown that at wavelength of 264 nm, no hypochromic or bathochromic effect was observed with increasing amount of DNA to the Dy(III) complex solution and the absorption of both the Dy(III) complex and DNA have partly overlapped. The following equation was explained their absorption values [30]:

AðDNA þ DyðIIIÞcomplexÞ  AðDNAÞ þ AðDyðIIIÞcomplexÞ

ð6Þ

According to Eq. (6), the absorbance of both the Dy(III) complex and DNA have not been affected by each other obviously [30]. With these results, it is suggested that there existed a slightly interaction

between the complex and DNA and the interaction mode between Dy(III) complex and DNA was not intercalation binding [30]. 3.7. Effect of ionic strength on the binding of Dy(III) complex–DNA system DNA is an anionic polyelectrolyte with phosphate groups. To distinguish the binding modes between molecules and DNA, it is an efficient method to monitor the spectral change with different ionic strength [18]. NaCl is a common salt for control the ionic strength of the solutions. Due to the competition for phosphate groups, by adding the Na+, the electrostatic interaction between molecules and DNA is weaken [18]. In this work, the effect of various concentrations of NaCl on the fluorescence intensity of the Dy(III) complex with DNA was examined. The Na+ concentration was increase from 0.03 to 0.6 mol L1. The results indicated that adding NaCl has been no obvious effect on the fluorescence intensity of the DNA–Dy(III) complex system. These results prove that the interaction between Dy(III) complex and DNA is not electrostatic interaction or surface-binding [31]. 3.8. Iodide quenching studies DNA is an anionic polyelectrolyte and the negatively charged phosphate backbone of DNA repels a highly negatively charged quencher. Therefore when an intercalative bound drug molecule exposes to an anionic quencher, should be protected from being quenched, but the free aqueous complexes or groove binding drugs are quenched readily by anionic quenchers [18]. For above reasons, negatively charged I was selected for determination of DNA binding affinity and KI was used to the iodide quenching experiments. When small molecule intercalates to DNA base pairs, between iodide anions and DNA phosphate backbone electrostatic repelling

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14.7

y = -10.709x + 49.592 R2 = 0.9943 lnKb

14.1

13.5

12.9 3.26

3.3

3.34

3.38

3.42

1/T×1000(K-1) -1.8

293 298 306

log(F0-F)/F

-1.4

-1

-0.6

-0.2

0.2 -4.7

-4.9

-5.1

-5.3

-5.5

-5.7

-5.9

-6.1

log[DNA] Fig. 5. Plots of log (F0F)/F versus log [DNA] at different temperatures. Inset: Van’t Hoff plot of 1/T versus ln Kb in the temperature range of 293–306 K. (pH = 7.2, kex = 264 nm, kem = 369 nm).

Table 1 Binding constants, number of binding sites and thermodynamic parameters of [Dy(phen)2(OH2)3Cl]Cl2H2O at different temperatures by emission measurements. T (K)

Kb  105 (M1)

n

DG° (kJ mol1)

DH° (kJ mol1)

DS° (J mol1 K1)

293 298 306

4.94 8.51 21.82

1.22 1.24 1.34

31.69 33.75 37.04

+89.03

+412.31

2.5

c

Absorbance

2

b 1.5 1

a

occur and it is difficult for iodide anions to collide with the small molecules, which lead to a decrease in fluorescent quenching [30,32]. If this interaction belongs to the outside binding, the Dy(III) complex molecule will absolutely expose in the solution and the quenching extent of fluorescence by I will equal to that of the solution without containing DNA. In the groove binding interaction, the Dy(III) complex molecule will be partly protected by DNA, and I can partly quench its fluorescence [30,32]. Here, to further deduce the binding mode of the Dy(III) complex with DNA, the iodide quenching experiment was carried out. The quenching behavior of KI in the Dy(III) complex–DNA system has been shown in Fig. 7. In this figure (a–d) are fluorescence emission intensity Dy(III) complex, Dy(III) complex + KI, Dy(III) complex + DNA and Dy(III) complex + DNA + KI respectively. Results indicate that F(a)  F(b) > F(c)  F(d) and the fluorescence of Dy(III) complex–DNA system partly has been quenched by iodide anions compared with the quenching of the solution that only containing Dy(III) complex [32]. These observations indicate that the rate of KI quenching effect of the Dy(III) complex is slower in the presence of DNA than in the absence of it. Thus, the Dy(III) complex interacts with DNA with groove binding mode [30,32].

0.5

3.9. Viscosity measurements 0 240

260

280

300

320

340

Wavelength/nm Fig. 6. The absorption spectra of complex in the presence and absence of DNA (a) 0.05 ml: 1.0  103 M complex + 4.95 ml Tris–HCl buffer, (b) 3.0 ml 2.8  104 M DNA + 2.0 ml Tris–HCl buffer, (c) 0.05 ml: 1.0  103 M complex + 3.0 ml 2.8  104 M DNA + 1.95 ml Tris–HCl buffer.

Viscosity measurement was also carried out to further clarify the interaction nature between the Dy(III) complex and DNA. Hydrodynamic measurements i.e. viscosity and sedimentation that are sensitive to changes in the length and can provide a necessary but not sufficient evidence for determine the binding mode to DNA especially when crystallographic structural data is absence [7]. In

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400

Intensity/a.u.

350 300

a b

18

c d

Zone of Inhibition (mm)

450

250 200 150 100 50 0 320

199

370

420

470

intercalation binding mode, a planar molecule inserts between DNA base pairs and results in a decrease in the DNA helical twist and lengthening of the DNA but when a groove binding agent bounds to DNA, the relative viscosity of DNA do not alter. In electrostatic interaction, binding to DNA causes bending or kinking of the DNA helix and its effective length and its viscosity concomitantly will reduce [33]. The effect of the Dy(III) complex on the viscosity of DNA was examined. On increasing the concentration of complex no obvious change in viscosity was observed. This result indicate that there exists groove binding between Dy(III) complex with DNA helix [30,32]. 3.10. Antibacterial activity All living organisms (prokaryotic and Eukaryotic) replicate DNA, transcript to produce mRNA and translate to synthesis proteins [2]. If inhibition of DNA transcription and replication is occurred, protein synthesis or replication will restricted and induce cell death [2]. In recent years, synthesis of complexes that can behave as target to inhibit the pathogenic gene from replicate and transcript has been developed. Antimicrobial activity of the Dy(III) complex against different of Gram +ve and Gram ve bacteria was explored by determining the values of radius of the inhibition zone around each disk and the results have been shown in Fig. 8. For all bacteria, the MIC value of complex was 7.0 mg/10 ml. It is evident from this figure that the Dy(III) complex shows a efficient antibacterial activity against different bacteria. Such increased activity of the metal chelates can be explained on the basis of chelation theory. Chelation decreases the polarity of the metal ion because of the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups which further leads to increasing the delocalization of p-electrons over the whole chelate ring and the complex’s lipophilicity is enhanced. Since the microorganism cell is surrounded by a lipid membrane which favors the passage of lipid soluble materials, the penetration of complex into lipid membranes and blocking of the metal binding sites in the enzymes of microorganisms is allowed by the increased lipophilic character of chelate and results in restriction of growth of the organism [34,35].

14 12 10 8 6 4 2 0

Wavelength/nm Fig. 7. The KI quenching effect of Dy(III) complex with the presence and absence of DNA: [complex] = 6.0  107 mol L1; [DNA] = 47  107 mol L1; [KI] = 0.004 mol L1; (a) Dy(III) complex; (b) Dy(III) complex + KI; (c) Dy(III) complex + DNA; (d) Dy(III) complex + DNA + KI.

16

e B coli C ns us us us lla nii lus hi nia hi cil ute ere cesce cocc ige man yp mo atyp ichia Ba cus l lus c r ro a au ret neu r e r l t e a b c e i m En p o p c p ch oc ria lle ella Ba ratia lle Es icr cte ne ne r si M ba Se mo leb almo o l t a e K S S cin Sh

A

Fig. 8. Antibacterial activity of [Dy(phen)2(OH2)3Cl]Cl2H2O against different bacteria.

influence of electrical field. This movement is retarded when DNA is bound to dysprosium complex. The DNA gel electrophoresis experiment was conducted at 25 °C using the synthesized complex in the absence and presence of H2O2 as oxidant. Fig. 9 shows the gel electrophoretic results of the interaction of the compound with fish salmon DNA. From control experiment (lane 1) is suggested that untreated DNA did not show any cleavage, while with increasing concentration of the Dy(III) complex (lanes 3–6) did. The complex can cleave DNA in the absence and presence of H2O2 (lane 3–6). The cleavage is more efficient in the presence of H2O2 which may be due to the reaction of hydroxyl radical with DNA (hydroxyl radical scavenger) (lane 4 and lane 6) [36,37]. Even in the absence of oxidant, the complex shows nuclease activity (lane 3 and lane 5). This may be due to the presence of a coordinated water molecule in the structure of the complex may suggest a hydrolytic

3.11. DNA cleavage studies Since the Dy(III) complex showed maximum binding propensity with fish salmon DNA, the DNA cleavage ability of this complex was studied by agarose gel electrophoresis using fish salmon DNA as a substrate. This DNA moves on agarose gel under the

Fig. 9. Gel electrophoresis diagram showing the cleavage of fish salmon DNA (1.4  103 M) by the dysprosium complex; lane 1: DNA control; lane 2: DNA + H2O2 (20  103 M); lane 3: complex (2.4  102 M) + DNA; lane 4: complex (2.4  102 M) + DNA + H2O2; lane 5: complex (3.1  102 M) + DNA; lane 6: complex (3.1  102 M) + DNA + H2O2.

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mechanism by providing the OH nucleophile [36]. The influence of the concentration of the complex can be observed in lanes (3–6) of Fig. 9. A smear pattern was observed for DNA treated with Dy(III) complex which is consistent with prior observation for [Yb(phen)2(OH2)Cl3](H2O)2 [12]. The increase of smearing is seen for bands with raising of the complex concentration. It results from the generation of smaller fragments of DNA. By increase of the complex concentration, the amount of DNA damage increases. The presence of a smear in the gel diagram also indicates the presence of radical cleavage [38]. 4. Conclusion A novel fluorescent reagent, Dy(III) complex was used to be a probe for DNA. Detailed analyses of the binding of the Dy(III) complex with DNA by fluorescence, UV–Vis spectroscopic techniques and viscosity measurement was carried out under simulative physiological conditions. DNA binding behavior was determined and indicated that the Dy(III) complex can bind to DNA by large binding constant (Kb = (8.51 ± 0.12)  105 L/mol by emission measurements). The effects of temperature, iodide ion and ionic strength on the binding of Dy(III) complex with DNA was discussed. The competitive binding studies by DNA–EB complex in the absence and the presence of the Dy(III) complex also was carried out. All the evidences showed that the Dy(III) complex can bind to DNA with high affinity mainly by groove interaction, not by intercalation interaction. The activity of complex against the some pathogenic bacteria was tested and antimicrobial screening tests shown good efficiency of the Dy(III) complex. Results obtained from our present work would be useful to understand the mechanism of interactions of the small molecule compounds binding to DNA and helpful in the development of their potential biological, pharmaceutical and physiological implications in the future. Acknowledgment We thank the University of Sistan and Baluchestan (USB) for financial support. References [1] S. Sujatha, S. Balasubramanian, B. Varghese, M. Jayaprakashvel, N. Mathivanan, Synthesis, characterization and DNA interaction of hexaaza macrotricyclic copper (II) complexes, Inorg. Chim. Acta 386 (2012) 109–115. [2] B.H.M. Hussein, H.A. Azab, M.F. El-Azab, A.I. El-Falouji, A novel anti-tumor agent, Ln(III) 2-thioacetate benzothiazole induces anti-angiogenic effect and cell death in cancer cell lines, Euro. J. Med. Chem. 51 (2012) 99–109. [3] F. Li, W. Chen, C. Tang, S. Zhang, Recent development of interaction of transition metal complexes with DNA based on biosensor and its applications, Talanta 77 (2008) 1–8. [4] X. Ling, W. Zhong, Q. Huang, K. Ni, Spectroscopic studies on the interaction of pazufloxacin with calf thymus DNA, J. Photochem. Photobiol. B: Biol. 93 (2008) 172–176. [5] J. Lhoste, N. Henry, T. Loiseau, F. Abraham, Molecular assemblies of trichloride neodymium and europium complexes chelated by 1,10-phenanthroline, Polyhedron 30 (2011) 1289–1294. [6] T.R. Li, Z.Y. Yang, B.D. Wang, D.D. Qin, Synthesis, characterization, antioxidant activity and DNA-binding studies of two rare earth (III) complexes with naringenin-2-hydroxy benzoyl hydrazone ligand, Euro. J. Med. Chem. 43 (2008) 1688–1695. [7] Y. Li, Z.Y. Yang, M.F. Wang, Synthesis, characterization, DNA binding properties and antioxidant activity of Ln(III) complexes with hesperetin-4-one-(benzoyl) hydrazone, Euro. J. Med. Chem. 44 (2009) 4585–4595. [8] G. Zhao, F. Li, H. Lin, H. Lin, Synthesis, characterization and biological activity of complexes of lanthanum (III) with 2-(10 -phenyl-20 -carboxyl-30 -aza-n-butyl)1,10-phenanthroline and 2-(10 -p-phenol-20 -carboxyl-30 -aza-n-butyl)-1,10phenanthroline, Bioorg. Med. Chem. 15 (2007) 533–540. [9] M. Khorasani-Motlagh, M. Noroozifar, S. Mirkazehi-Rigi, Fluorescence and DNA-binding spectral studies of neodymium (III) complex containing 2,2´bipyridine, [Nd(bpy)2Cl3OH2], Spectrochim. Acta Part A 75 (2010) 598–603. [10] M. Khorasani-Motlagh, M. Noroozifar, S. Khmmarnia, Study on fluorescence and DNA-binding of praseodymium (III) complex containing 2,2´-bipyridine, Spectrochim. Acta Part A 78 (2011) 389–395.

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