Photoluminescence studies of a Terbium(III) complex as a fluorescent probe for DNA detection

Journal of Luminescence 143 (2013) 56–62

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Photoluminescence studies of a Terbium(III) complex as a fluorescent probe for DNA detection Mozhgan Khorasani-Motlagh n, Meissam Noroozifar, Sona Niroomand, Asieh Moodi Department of Chemistry, University of Sistan & Baluchestan, Zahedan, P.O. Box 98155-147, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 7 January 2013 Received in revised form 12 March 2013 Accepted 9 April 2013 Available online 25 April 2013

The photoluminescence properties of a Tb(III) complex of the form [Tb(phen)2Cl3
OH2] (phen ¼1,10phenanthroline) in different solvents are presented. It shows the characteristic luminescence of the corresponding Ln3+ ion in the visible region. The emission intensity of this complex in coordinating solvent is higher than non-coordinating one. The suggested mechanism for the energy transfer between the ligand and Tb3+ ion is the intramolecular energy transfer mechanism. The interactions of the Tb(III) complex with fish salmon DNA are studied by fluorescence spectroscopy, circular dichroism study and viscosity measurements. The results of fluorescence titration reveal that DNA strongly quenches the intrinsic fluorescence of the complex through a static quenching procedure. The binding constant (Kb) of the above metal complex at 25 1C is determined by the fluorescence titration method and it is found to be ð8:06 7 0:01Þ  103 M−1. The thermodynamic parameters (ΔH0 40, ΔS0 4 0 and ΔG0 o0) indicate that the hydrophobic interactions play a major role in DNA–Tb complex association. The results support the claim that the title complex bonds to FS-DNA by a groove mode. & 2013 Elsevier B.V. All rights reserved.

Keywords: Fluorescence Terbium(III) complex 1,10-phenantroline DNA binding Thermodynamic parameters Groove binding

1. Introduction Recently, lanthanide ions, Ln(III) with organic ligands have attracted much attention because of their unique luminescence and magnetic properties [1]. Due to these properties, the use of lanthanide-based probes is becoming more common in a wide variety of photonic applications such as luminescence spectroscopy, planar waveguide amplifiers, plastic lasers, and lightemitting diodes [2,3]. Among Ln(III) ions, Eu3+ and Tb3+ have intense, long-lived, and line-like emission in the visible region that enables them to be promising in applications from display devices to biological assays [4]. Unfortunately, the low extinction coefficients of free lanthanide ions result in very inefficient light absorption, which makes direct excitation impossible. However, the luminescence properties of lanthanides can be improved by means of organic ligands containing suitable chromophores coordinated to the lanthanides [5]. These luminescent ligands act as antenna by efficiently absorbing light in the UV region and transforming the absorbed energy to the emitting level of the lanthanide ion. Moreover, the ligands can protect the lanthanide ions from vibrational coupling that may quench the luminescence [6].

n

Corresponding author. Tel.: +98 541 244 6565; fax: +98 541 244 6888. E-mail address: [email protected] (M. Khorasani-Motlagh).

0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.04.011

In addition to the spectroscopic and magnetic properties, the biological activities of the lanthanide complexes such as antitumor, antimicrobial, antivirus, anticoagulant action, prevention from arteriosclerosis, luminescent bioprobes, etc., have been investigated in the last few years [7,8]. The luminescence properties of Tb3+, Eu3+, and Ce3+ make them useful in the analysis of biomolecular structures. The first two of these cations are of special interest because their resonance energy levels overlap the triplet energy states of nucleic acid and protein aromatic residues and increase their natural fluorescence by energy transfer when irradiated with ultraviolet light [9]. Furthermore, lanthanide ions have been used as chemical nucleases and their complexes can efficiently bind to DNA [10]. Since DNA is the primary target molecule for most anticancer and antiviral therapies, the binding studies of small molecules to DNA are basis to development of new classes of pharmaceutical molecules [11]. Nucleic acids can be regarded as ambidentate ligands, with diverse potential binding sites, including hydroxyl groups on the ribose sugar, nitrogen and oxygen donors on the bases and negatively charged oxygen atoms in the phosphate groups which make the Ln(III) ions readily interact with DNA [9]. One of the interested categories of small molecules which interact with DNA are metal bipyridyl complexes and metal phenanthroline complexes. In fact some metal complexes containing polypyridyl ligands such as 1,10-phenanthroline have strong effect of inserting into DNA and display remarkable antibacterial and antitumor activities [12,13]. Also, these ligands act as antenna units and improve the luminescence properties of

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57

3.2. Iodide quenching experiment

lanthanide ions. These attractive properties of lanthanide complexes containing polypyridyl ligands persuaded us to investigate their spectroscopic and DNA binding properties in our last works [14–18]. In the present work, luminescence and DNA binding properties of Tb(III) complex with 1,10-phenanthroline, [Tb(phen)2Cl3
OH2] have been studied by fluorescence, circular dichroism (CD) and viscosity measurements.

Iodide quenching experiments were conducted by adding small amount (40 mL) of the potassium iodide stock solution (1.2 M) to the Tb(III) complex and the complex–DNA mixture.

2. Material and methods

The effect of salt concentration was studied by adding different aliquots (0.03–0.6 M) of the NaCl stock solution to the complex– DNA mixture.

All chemicals and reagents were obtained commercially and used without further purification. Also, deionized double distilled water was used. Fish salmon DNA and ethidium bromide was obtained from Sigma. A solution of fish salmon DNA in buffer gave a ratio of 1.8–1.9 UV absorbance at 260 and 280 nm indicating that DNA was sufficiently free of protein [14]. The DNA concentration per nucleotide was obtained by using a molar absorption coefficient (6600 M−1 cm−1) at 260 nm. All the measurements about interaction of the complex with FS DNA were conducted using solutions of DNA and the complex in Tris–HCl buffer (pH 7.2) containing 5 mM Tris–HCl and 50 mM NaCl and at ambient temperature 25 1C. [Tb(Phen)3Cl3
OH2] was synthesized by the literature method [19]. IR spectrum of this complex showed the broad peaks at 1637 cm−1 and in the 3050–3330 cm−1 region which may be assigned to the bending and stretching vibrations of coordinated water. Moreover, the resulted elemental analysis confirms the proposed formulation. Fluorescence spectra were recorded using a PERKIN ELMER, LS-3 with a 1 cm quartz cell. Circular dichroism (CD) measurements were recorded on an AVIV (Model-215) spectropolarimeter.

3. Theory and calculation 3.1. Fluorescence study Florescence spectra of complex were obtained in several solvents and different excitation wavelengths. Concentration and pH were optimized by considering maximum intensities. In the binding studies fixed Tb(III) complex concentration (1:0  10−4 M) was taken and to this varying concentration (0–30 μM) of DNA was added. The excitation wavelength was fixed at 270 nm and the emission was scanned in the range of 300–500 nm. The quenching process type was determined by the Stern– Volmer quenching method (Eq. (1)) [20]: F0 ¼ 1 þ K sv ½Q  F

ð1Þ

where F0 and F have the same meaning mentioned above, [Q] is the concentration of the quenching reagent and KSV is the Stern– Volmer constant. The intrinsic binding constant Kb was obtained by the fluorescence titration methods and Eq. (2) [21]:   F 0 −F ¼ logK b þ n log½Q  ð2Þ log F where Kb is the intrinsic binding constant and n is the exclusion parameter in DNA base pairs and [Q] is the concentration of quenching reagent (DNA). F0 and F are the fluorescence intensities of the complex alone and in the presence of DNA, respectively. For EB quenching experiments small amounts of complex stock solution were added to DNA–EB solution and the change of fluorescence intensity was recorded. The excitation wavelength was 525 nm and the emission wavelength was 589 nm.

3.3. Effect of ionic strength

3.4. Viscosity measurements Viscosity experiments were performed on an Ubbelodhe viscometer, immersed in a thermostatic water bath maintained at 25.0 70.1 1C. DNA concentration was kept constant (3.2  10−4 M) and the concentration of the tested compound was gradually increased. The flow time was measured with a digital stopwatch and each sample was measured three times and an average flow time was calculated. The relative viscosity of DNA in the presence and absence of complex was calculated from the following equation: η¼

t−t 0 t0

ð3Þ

where t0 and t are the observed flow times in the absence and presence of the complex. Data were presented as ðη=η∘ Þ1=3 versus binding ratio, where η is the viscosity of DNA in the presence of complex, and η0 is the viscosity of DNA alone [22]. 3.5. Circular dichroism (CD) study The circular dichroism spectra of FS DNA in absence and presence of Tb(III) complex were obtained in 0.5 cm (1 mL) quartz cuvette. The spectra were recorded in the range of 400–200 nm. Each CD spectrum was collected after averaging over at least three accumulations from which the buffer background had been subtracted. The concentration of DNA was 5.84 mM. All measurements were performed at 25 1C. 3.6. Thermodynamic parameters The thermodynamic parameters, enthalpy change (ΔH0), entropy change (ΔS0) and free energy change (ΔG0) of reaction were calculated for confirming binding mode, using the following equations [23]:   ΔG0 ΔH 0 1 ΔS0 þ ¼− ð4Þ lnK b ¼ T RT R R ΔG0 ¼ ΔH 0 −TΔS0

ð5Þ

4. Results and discussion 4.1. Luminescence properties of Tb(III)complex The fluorescent intensity of [Tb(phen)2Cl3
OH2] in solution is sensitive to several factors such as solvent and concentration. The Tb complex in methanol (coordinating solvent) exhibited luminescence characteristic of Tb(III) with obvious bands in the range of 400–700 nm as well as a broad band at 368 nm attributed to the ligand center emission. However, the acetonitrile solution of the Tb(III) complex (non-coordinating solvent) displayed the luminescence of Tb(III) complex with lower intensity at above region. This

M. Khorasani-Motlagh et al. / Journal of Luminescence 143 (2013) 56–62

observation showed that the emission intensity in the coordinating solvents was higher than the non-coordinating solvents, as reported in the literature [1]. The emission intensity increased with decrease of concentration of the complex in the range of 10−2–10−4 M and then decreased with decrease of the complex concentration to 10−9 M. A 1:0  10−4 M solution of [Tb(phen)2Cl3
OH2] was chosen as the optimum concentration for the fluorescent study. The emission spectrum of the [Tb(phen)2Cl3
OH2] complex in methanol is shown in Fig. 1. The emission spectrum of the complex excited at 300 nm displayed four sharp luminescence bands at 491, 545, 586, and 616 nm corresponding to the 5D4-7F6, 5 D4-7F5, 5D4-7F4, and 5D4-7F3 transitions, respectively [24]. This characteristic emission spectrum of the Tb(III) ion declared that the ligand is a good chelating organic chromophore and can be used to absorb and transfer energy to the Ln(III) ions. On the other hand, exhibition of the ligand centered emission band at 368 nm indicated that the efficient energy transfer from phen to the Tb(III) center did not take place and back transfer of energy from Tb(III) ion prevailed. This may be due to the imperfect matches between the lowest triplet state energy level (TL) of the phen (22,200 cm−1) and the lowest resonance energy level of Tb (III) (5D4, 205,000 cm−1) ion. It is important to note that the small energy gap could result in a back-energy transfer process from the excited resonance levels of the Tb(III) to the TL of the ligand, thus leading to a diminution in the luminescence output of the Tb(III) compared with the other reported Ln(III) complexes [25]. In addition, a very weak band between 350 and 400 nm resulting from excitations from the 7F6 state to the 5D3, 5G6, 5L10, 5G5, 5D2, 5 G4 and 5L9 levels of Tb(III) must be observed but these bands overlapped with ligand emission band [26]. The emission spectra of the Tb(III) complex and 1,10-phenanthroline excited at 285 nm are shown in Fig. 2. The Tb(III) complex exhibited only three weak bands at 491, 545, 586 nm as well as ligand emission band; this observation revealed that this excitation band was not the effective energy sensitizer for the luminescence of the Tb(III) ion. The slight red-shift in the fluorescence band of the ligand in Tb(III) complex (368 nm) compared with the free ligand (365 nm) can be attributed to the coordination of Tb(III) ion to the phen. This phenomenon increased the delocalization of electrons and decreased the energy gaps between the π-πn molecular orbitals of the ligand [24]. Several ways have been suggested for the energy transfer from TL of the ligand to the resonance state of Ln(III) ions in lanthanide complexes. In the preference mechanism the ligand is excited to the singlet state (SL) by the strong absorption of UV energy,

D4→7F5

5

D4→7F6

Intensity/a.u

5

D4 →7F4

5

π→π‫٭‬

320

D4→7F3

5

420

520 Wavelength/nm

620

720

Fig. 1. Emission spectrum of [Tb(phen)2Cl3
OH2] complex (1.0  10−4 M), λexc ¼ 300 nm, in methanol (solid line) and acetonitrile (dotted line) solution at room temperature.

260

195 Intensity/a.u

58

130

65

0 320

430

540 Wavelength/nm

650

760

Fig. 2. Emission spectra of [Tb(phen)2Cl3
OH2] complex (solid line) and 1,10phenanthroline (dashed line), λexc ¼285 nm, in methanol solution (1.0  10−4 M) at room temperature.

SL

TL 5

D4

Absorption Ligand Fluorescence Ligand phosphorescence

Metal emission

Excitation

Ground State of Ligand

Ground State of Tb3+

Scheme 1. The probable mechanism of intramolecular energy transfer between the ligand (L) and the Tb(III).

followed by an energy migration via nonradiative intersystem crossing to the TL. Then the energy is intramolecularly transferred from the TL to a resonance state of the Ln(III) ion and the luminescence occurs in the visible region [24]. We proposed that the luminescence mechanism of [Tb(phen)2Cl3
OH2] was consistent with the intramolecular energy transfer mechanism (Scheme 1). 4.2. DNA binding studies 4.2.1. Fluorescence studies The Tb(III) complex exhibited the luminescence with a maxima at 365 nm in Tris-buffer at ambient temperature when excited at λex ¼ 270 nm. This excitation band was the effective energy sensitizer for the ligand centered luminescence of the Tb(III) complex in Tris-buffer as solvent. The Tb(III) ion characteristic fluorescence in this solvent was not observed; therefore the DNA binding studies were investigated for the ligand center luminescence region. As mentioned above a 1:0  10−4 M solution of [Tb(phen)2Cl3
OH2] was chosen as the optimum concentration for DNA binding study by fluorescence spectroscopy. The effect of the pH on the fluorescence of [Tb(Phen)2Cl3
OH2] was studied and a pH of 7.2 was recommended in the subsequent investigations. Solution behavior of Tb(III) complex in Tris–HCl buffer solution was monitored at room temperature by UV–vis spectroscopy for 24 h before the complex reacts with FS DNA. Liberation of the ligand and its complex was not observed under these conditions. These results suggest that the complex is stable under the condition studies. Fig. 3 displays a well-behaved titration of the [Tb(phen)2 Cl3
OH2] with FS DNA. The luminescent properties of the complex were perturbed when DNA was added to the complex solution,

M. Khorasani-Motlagh et al. / Journal of Luminescence 143 (2013) 56–62

500

59

1.12

a

450

l

400

F0 /F

Emisssion Intensity/a.u

1.08

y = 0.0053x + 1.0037 R2 = 0.9952

1.04

350 300

1 0

5

250

10

15

20

25

[DNA]×106

200 150 350

360

370

380

390

400

Wavelength/nm Fig. 3. The fluorescent spectral characteristics of Tb(III) complex–DNA. Tb(III) complex concentration: 1.0  10−4 M in pH 7.2 Tris-buffer. DNA concentrations: a¼ 0, b¼ 1.3, c ¼2.6, d ¼3.9, e¼ 5.2, f¼ 7.8, g ¼10.5, h ¼12.9, i¼ 15.8, j¼ 16.9, k ¼ 19.5, and l ¼ 23.4 mM. Inset—the Stern–Volmer quenching plot from the fluorescence titration data at 298 K.

-0.62 -0.9

log (F0 -F/F)

and binding of the complex to DNA was found to decrease the fluorescence intensity. This obvious decrease in the emission spectra showed that the interaction of Tb(III) complex with DNA was not intercalation [17]. The quenching of luminescence of investigated complex by DNA was in agreement with a photoelectron transfer from the guanine base of DNA to the excited state of the metal complex [27]. The red-shift in the emission spectra occurred because, in the presence of DNA, the metal complex is bound in a relatively non-polar environment compared to water [23]. The results indicated that DNA could quench the intrinsic fluorescence of the Tb(III) complex; different mechanisms are responsible for this fluorescence quenching, which are usually classified as static quenching and dynamic quenching. When the fluorophore and the quencher produce a ground state complex, the static quenching occurs, whereas the dynamic quenching results from the association of the fluorophore and the quencher during the transient existence of the excited state [28]. Inset of Fig. 3 displays the Stern–Volmer plot of the quenching of [Tb (phen)2Cl3
OH2] fluorescence by DNA. As can be seen, the plot of F0/F versus [DNA] ranging from 0 to 24 mM−1 is linear. This observation suggested that a single quenching mechanism, either static or dynamic, occurred at these concentrations [15]. The Stern–Volmer quenching constant calculated from the slope of the curve F0/F versus [DNA] was 5.28  103 M−1, and the values of kq (8.71 70.01  1011, when τ ¼6.2 ns) was greater than the maximum collision diffuse quenching constant of the biomolecules (2.0  1010 L mol−1 s−1) [29], indicating that the fluorescence quenching had mainly arisen from static quenching by complex formation instead of dynamic quenching. To compare quantitatively the affinities of this compound bound to DNA, the intrinsic binding constants Kb can be obtained according to the method described by Chipman et al. (Eq. (2)). A plot of log(F0–F)/F versus log[DNA] gave the binding constant (Kb) ð8:06 7 0:01Þ  103 M−1 and binding site number (n) 1.04 from the fluorescence data for the Tb(III) complex (see Fig. 4). The Kb value obtained here was smaller than the values of typical intercalators, ethidium bromide (2.6  106 L mol−1) and acridine orange (4  105 L mol−1), but corresponds to some groove binders, as reported in literature [14–18,30]. The molecular fluorophore EB (ethidium bromide) has a conjugate planar structure and very weak fluorescence intensity, but it emits high fluorescence light at about 600 nm in the presence of

-1.18 -1.46 -1.74 -2.02 -2.3 -6

-5.74

-5.48 -5.22 log [DNA]

-4.96

-4.7

Fig. 4. Plot of log(F0−F)/F versus log[DNA] at different temperatures (cubic—295 K, triangle—298 K, circle—301 K).

DNA due to its strong intercalation between the adjacent DNA base pairs [31]. It was previously reported that the addition of a competing metal complex could have quenched the enhanced fluorescence. In fact, the degree of the fluorescence quenching for EB bound to DNA is used to determine the extent of binding between metal complex and DNA [32]. If the complex bound to DNA by the intercalation mode, while the ratio of the concentration of the complex to DNA was not more than 100, the emission intensity of the DNA–EB system would decrease more than 50% [33]. In this work, the intensity of DNA–EB system decreased only by 17%, as Tb(III) complex was added to the system and did not decrease anymore (Fig. 5). This observation indicated that Tb(III) complex bound to DNA in a different mode from EB [18] and the competitive inhibition occurred. Probably in this phenomenon the complex interacted with the grooves of DNA and blocked the potential intercalation sites of EB [32].

4.2.2. Iodide quenching experiment To further demonstrate the interaction pattern of the Tb(III) complex with DNA, the iodide quenching experiment was performed. The negatively charged phosphate backbone of DNA can repel a highly negatively charged quencher; therefore intercalative bound molecules should be protected from quenching by anionic

60

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1

a

250

F/F0

Intensity/a.u

200

k

150

100

0.5

0 0

0.04 0.08 [complex]/[DNA]

0.12

50

0 550

570

590

610

630

650

670

Wavelength/nm Fig. 5. Fluorescence quenching of EB–DNA by increasing Tb(III) complex in Tris–HCl buffer. [EB] ¼0.067 mg/mL, [DNA] ¼7.8  10−5 mol L−1, [complex]: a ¼0, b ¼0.83, c¼ 1.66, d ¼ 2.48, e¼ 3.29, f¼ 4.10, g ¼4.98, h ¼ 5.70, i¼ 6.49, j¼ 7.28 and k ¼ 8.06 mM. Inset—effect of complex concentration on the fluorescence intensity for EB–DNA system.

a

500

Intensity/a.u

b

Intensity/a.u

c d

350

200 0

50 340

360

380

400

420

0.1

0.2

0.3 [NaCl]/M

0.4

0.5

0.6

Fig. 7. Effect of salt concentration, [Tb(phen)2Cl3
OH2]: 1.0  10−4 M, [DNA]: 6.4  106 M.

Wavelength/nm Fig. 6. The fluorescence spectra of KI quenching for complex–DNA system, [complex]: 1.0  10−4 M; [DNA]: 8.90  10−6 M; [KI]: 0.02 M. (a) Complex; (b) complex+KI; (c) complex+DNA; and (d) complex+DNA+KI.

1.05

(η/η0)1/3

quencher [28], but the groove binding molecules should be partly quenched by anionic quenchers [17]. The quenching behaviors of KI in the [Tb(phen)2Cl3
OH2]–DNA system are shown in Fig. 6. It was apparent that a little change in iodide quenching interactions can be observed when complex bound to DNA (F(a)−F(b)4 F(c)−F (d)), which indicated that the bound molecules had not intercalated into the base pairs of DNA. Iodide quenching results provide direct evidence for the groove binding of [Tb(phen)2Cl3
OH2] with DNA [21].

1.1

1

0.95

0.9 0

4.2.3. Effect of ionic strength The effect of the ionic strength on the compounds fluorescence was tested by the addition of a strong electrolyte, NaCl. DNA is an anionic polyelectrolyte with negatively charged phosphate groups and cations of the salts can neutralize them [28]. If the complex 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. Therefore approaching the DNA is difficult for the complex and the strength of interaction with DNA decreases, and the extent of fluorescence quenching also descends [34]. Addition of NaCl to the Tb(III) complex in the absence and presence of FS DNA had little influence on the fluorescence intensity, showing that the interaction of the Tb(III) complex with FS DNA was not an electrostatic interaction (Fig. 7).

0.1

0.2 0.3 Ccomplex/CDNA

0.4

0.5

Fig. 8. Effect of complex on the viscosity of DNA solution, Tb(III) complex, 1  10−3 M, (10–100 mL) was titrated into a 3.9  10−4 M DNA solution at 298 K.

4.2.4. Viscosity measurements As a means for further clarifying the binding of Tb(III) complex with DNA, viscosity of DNA solutions containing differing amounts of investigated complex was measured. The results indicated that the addition of [Tb(phen)2Cl3
OH2] causes no significant viscosity change, as illustrated in Fig. 8. This observation suggested that the binding of Tb(III) complex with DNA belongs to groove binding model because the electrostatic or groove surface binding has little effect on the viscosity of DNA in the binding process. It is worth to note that a classical intercalation binding is accompanied by an increase of DNA viscosity due to the accommodation of the bound

M. Khorasani-Motlagh et al. / Journal of Luminescence 143 (2013) 56–62

ligands in the space of adjacent base pairs and elongation of the double helix [28].

4.2.6. Thermodynamic parameters of DNA binding Studies of the thermodynamics of complex–DNA interactions can provide insight into the factors that drive bimolecular complex formation. In order to elucidate the interaction of complex with DNA, the thermodynamic parameters were calculated. The first step in most investigations is to experimentally determine the equilibrium binding constant (Kb) at various temperatures (Table 1). This approach supplied a good means to determine indirectly the thermodynamic parameters of DNA binding of the Tb(III) complex by using Van't Hoff plots of 1/T versus ln Kb in the temperature range 22–28 1C (Fig. 10). The standard enthalpy (ΔH0), standard entropy (ΔS0) and standard free energy change (ΔG0) were calculated by Eqs. (4) and (5) [37]. According to the thermodynamic profile, the favorable binding free energy (−4.99 kcal mol−1) resulted from the difference between the unfavorable enthalpic contribution (58.19 kcal mol−1) and the favorable entropic contribution (TΔS0 ¼63.18 kcal mol−1) at 25 1C. Negative binding free energy revealed that the binding of Tb (III) complex and DNA was favorable at 25 1C because the energy of free complex and DNA was higher than the adduct free energy. The binding of the investigated complex and DNA was endothermic 20 15

CD/mdeg

10 5 0 200

250

-5

300 Wavelength/nm

350

400

-10 Fig. 9. Circular dichroism spectra of FS DNA in the absence (dashed line) and presence of the complex (solid line), [DNA] ¼5.84  10−3 M, [complex] ¼ 2.1  10−5.

Table 1 Binding constants (Kb), number of binding sites (n) and relative thermodynamic parameters of DNA–Tb(III) complex system. T (K)

Kb  104 (M−1)

n

295 298 301

0.21 0.81 1.69

0.934 1.037 1.038

ΔG0 (kcal mol−1)

ΔH0 (kcal mol−1)

ΔS0 (cal mol−1 K−1)

−4.35 −4.99 −5.62

– 58.19 –

– 212 –

10

9

ln Kb

4.2.5. Circular dichroism spectroscopy CD spectroscopy is a suitable way to monitor the variation in DNA morphology during complex–DNA interactions, as the positive band due to base stacking and the negative one due to polynucleotide helicity are absolutely sensitive to the mode of DNA interaction [35]. As can be seen in Fig. 9 the CD spectrum of FS DNA displayed a decrease in the intensity of the negative and positive bands (shifting to zero level) after binding with the complex [18]. Electrostatic interaction and groove binding of the complexes with DNA reveal slight changes in the intensity of both negative and positive bands of DNA [36], while intercalation interaction enhances the intensity of both the bands, stabilizing the right-handed B conformation of FS DNA. Therefore, CD spectral changes suggest that the Tb(III) complex binds to FS DNA preferentially in a non-covalent groove binding mode.

61

8

y = -29243x + 106.96 2

R = 0.9392 7 0.0033

0.00335

0.0034

1/T Fig. 10. Inset is Van't Hoff plot of 1/T versus ln Kb in the temperature range of 295–301 K.

according to the positive enthalpy. It was reported that when ΔHo0 or ΔH≈0 and ΔS40, the electrostatic force dominates the interaction; when ΔHo0 and ΔSo0, van der Waals interactions or hydrogen bonds dominate the reaction; and when ΔH40 and ΔS40, hydrophobic interactions dominate the binding process [17]. On the basis of the thermodynamical data it can be concluded that the binding process was entropically driven and the hydrophobic interactions may play a main role in the binding of the terbium complex to DNA. In the groove binding, the binding is promoted in large part by the hydrophobic effect and favorable entropy is derived from the release of bound water from the DNA and compound upon complex formation [38].

5. Conclusion Photoluminescence properties and DNA binding studies of [Tb(phen)2Cl3
OH2] were investigated by fluorescence spectroscopy, CD analysis and viscosity measurements. The visible emission spectrum of the terbium complex mainly showed two sharp emission bands at 491 and 545 nm as well as two weak bands at 586 and 616 nm. The characteristic emission spectrum of the Tb (III) ion showed that the phenanthroline acted as an antenna ligand and can be used to absorb and transfer energy to the Ln(III) ions but existence of the ligand centered emission band indicated that the energy transfer from phen to the Tb(III) center did not efficiently take place and back transfer of energy from Tb(III) ion prevailed. DNA binding studies confirm that the Tb(III) complex binds to FS DNA. The binding constant of complex has been determined by the spectrofluorometric method. The binding constant (Kb) of investigated complex was determined asð8:067 0:01Þ 103 M−1. Viscosity measurements and CD studies declared that the binding mode of complex with FS DNA was groove binding mode. Thermodynamic parameters reveal that the binding process was entropically driven and affirmed the groove binding nature of complex–DNA interaction.

Acknowledgment We thank the University of Sistan and Baluchestan (USB) for financial support. References [1] M. Irfanullah, K. Iftikhar, J. Lumin. 130 (2010) 1983. [2] S.-G. Roh, N.S. Baek, K.S. Hong, H.K. Kim, Bull. Korean Chem. Soc. 25 (2004) 343.

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