Accepted Manuscript In vitro studies on the behavior of salmeterol xinafoate and its interaction with calf thymus DNA by multi-spectroscopic techniques S. Tingting Zhao, Shuyun Bi, Yu Wang, Tianjiao Wang, Bo Pang, Tingting Gu PII: DOI: Reference:

S1386-1425(14)00731-8 http://dx.doi.org/10.1016/j.saa.2014.04.158 SAA 12122

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

9 February 2014 18 April 2014 28 April 2014

Please cite this article as: S. Tingting Zhao, S. Bi, Y. Wang, T. Wang, B. Pang, T. Gu, In vitro studies on the behavior of salmeterol xinafoate and its interaction with calf thymus DNA by multi-spectroscopic techniques, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.04.158

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In vitro studies on the behavior of salmeterol xinafoate and its interaction with calf thymus DNA by multi-spectroscopic techniques Tingting Zhaoa, Shuyun Bia,, Yu Wanga ,Tianjiao Wanga, Bo Pangb, Tingting Gub a

College of Chemistry, Changchun Normal University, Changchun 130032, China

Technology Center of Inspection and Quarantine, Jilin Entry-Exit Inspection and Quarantine Bureau,

b

Changchun 130062, China

Abstract The salmeterol xinafoate (SX) binding to calf thymus DNA in vitro was explored by fluorescence, resonance light scattering (RLS), UV-vis absorption, as well as viscometry, ionic strength effect and DNA melting techniques. It was found that SX could bind to DNA weakly, and the binding constants (Ka) were determined as 8.52  103, 8.31  103 and 6.14  103 L mol-1 at 18, 28 and 38 °C respectively. When bound to DNA, SX showed fluorescence quenching in the fluorescence spectra and hyperchromic effect in the absorption spectra. Stern-Volmer plots revealed that the quenching of fluorescence of SX by DNA was a static quenching. Furthermore, the relative viscosity and melting temperature of DNA solution were hardly influenced by SX, while the fluorescence intensity of SX–DNA was observed to decrease with the increasing ionic strength of system. Also, the binding constant between SX and double stranded DNA (dsDNA) was much weaker than that between SX and single stranded DNA (ssDNA). All these results suggested that the binding mode of SX to

 Corresponding author. Tel.: +86431 86168098; Fax: +86431 86168096 E-mail address: [email protected] 1

DNA should be groove binding. The obtained thermodynamic parameters indicated that electrostatic force might play a predominant role in SX binding to DNA. The quantum yield (φ) of SX was measured as 0.13 using comparative method. Based on the Förster resonance energy transfer theory (FRET), the binding distance (r0) between the acceptor and donor was calculated as 4.10 nm. Keywords: Salmeterol xinafoate (SX); DNA; Groove binding; Fluorescence; Resonance light scattering (RLS) Introduction As we know, deoxyribonucleic acid (DNA) is an important hereditary substance in the living organism. In recent years, the studies of the binding of small molecules, such as drug [1-6], coordination compound [7-10] to DNA have been of great interest. It is extremely useful to understand the drug–DNA binding mechanism for predicting the consequences of these interactions in the human body, design the structure of new and efficient drugs, and elucidate the nature of this biologically important complex in vitro[11, 12]. Salmeterol xinafoate (SX) (Fig. 1), a common type of β2-adrenoceptor agonists, it is particularly useful for treatment of bronchial asthma and airways obstruction [13-15]. Meanwhile, SX is often used as a growth promoter in animal feed because of the repartitioning ability of carcass composition to decrease fat deposition and to increase muscle mass [16]. So far, few papers on the study of the interaction of SX with DNA have been reported. It might be the first report on the interaction of SX and DNA to the best of our knowledge. When investigating the interaction between DNA and drugs, more attention was

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centered on the binding mode. It is well known that small molecules bind to DNA primarily through three modes: electrostatic binding, groove binding, intercalation binding. Electrostatic interaction generally occurs along the exterior of the DNA helix. Groove binding usually interacts in the deep major groove or the minor groove of the helix. While the intercalation binding perpendicularly inserts to the two adjacent base pairs [17]. The binding mode is most convincingly established by high resolution structural studies, using X-ray diffraction or NMR. However, in this study, the binding mode of SX to DNA was explored only by UV-vis spectrophotometer, spectrofluorophotometer and viscometers without the aid of the expensive and intricate equipments. Previously, various probes of DNA were used to study the binding of drug and DNA [18-20] because of two main disadvantages: (1) DNA has much lower fluorescence quantum efficiency, and (2) most of the studied drugs have no fluorescence. However, the using of the various probes made the studies on the interaction of drug and DNA more intricate. In this study, it was found delightedly that SX could emit fluorescence, so the interaction between SX and DNA in vitro could be investigated directly without any probe. Furthermore, SX as a fluorophore, its quantum yield φ has not been reported to our knowledge. The task of obtaining the value of φ of SX was accomplished, and the binding distance according to FRET was determined. The results will provide valuable hints in drug design and synthesis in the future work. Experimental

3

Apparatus The

fluorescence

measurements

were

performed

on

a

RF-5301PC

spectrofluorophotometer (Shimadzu, Japan) with a quartz cell of 1 cm path length and a xenon lamp source. The absorption spectra were recorded on TU-1901 UV-vis spectrophotometer (Beijing Purkinje General Instrument Co. Ltd.). The viscosity determination was carried out using Ubbelohde viscometer. An electronic thermostat water-bath (Shandong Juancheng Instrument Company) was used for controlling the temperature. A pH-3S digital pH-meter (Nanjing Sangli electronic equipment factory, Nanjing, China) was used to measure pH. Materials and reagents The DNA (DNA) from calf thymus was purchased from Sigma Chem. Co. and used without purification. To prepare the stock solution, the solid DNA was directly dissolved in double distilled water overnight and stored at 4 ℃ in the dark. The concentration was determined by absorption technique, using the absorptivity ε260 (25℃) = 6600 L mol-1cm-1 [21, 22]. The stock solution gave a ratio of absorbance at 260 and 280 nm (A260/A280>1.8), indicating that the DNA was sufficiently free of protein contamination [23]. SX purchased from Chinese Drug Biological Products Qualifying Institute was prepared in ethanol, and the concentration was 1.00  10-4 mol L-1. A Tris–HCl buffer solution (0.05 mol L−1, pH 7.4) containing 0.1 mol L−1 NaCl was used in this work. All reagents were of analytical reagent grade in this work and the double distilled water was used throughout the experiments.

4

Procedures RLS spectra measurements The resonance light scattering (RLS) spectra were recorded on a RF-5301PC spectrofluorophotometer at 18 ± 0.5oC, and the wavelength range was from 220 to 700 nm by means of synchronous fluorescence measurements with λem = λex = 220 nm. Both slits width of excitation and emission were 3 nm. Fluorescence measurements The fluorescence spectra of samples were recorded from 280 to 700 nm when it was excited at 249 nm. Both excitation and emission slits were set at 3 nm. The fluorescence measurements were carried out by fixing the concentration of SX and varying the concentration of DNA. The mixture was diluted with pH 7.4 Tris–HCl buffer. All the fluorescence data were corrected for absorption of exciting light and reabsorption of emitted light according to the following formula [24]: Fcorr  Fobs e( Aex  Aem )/2

(1)

Fcorr and Fobs are the corrected and observed fluorescence intensities respectively; Aex and Aem are the absorbance of DNA at the excitation and emission wavelengths respectively. Absorption spectra The UV–vis absorption spectra of the mixture of SX–DNA were measured when the concentration of DNA were increased. At 18 ± 0.5 oC, the absorbance was obtained against the blank solution. The slit width was 3 nm, and the scan wavelengths were from 220 to 600 nm. All measurements were carried out in a pH 7.4

5

Tris–HCl buffer. Viscosity measurements The viscosity measurements of DNA solutions were performed at 18 ± 0.5℃ in the absence and presence of SX. The flow times of samples were measured by using a digital stopwatch. The relative specific viscosities (η/η0)1/3 were plotted versus binding ratio r (r = CSX/CDNA), where η0 and η are the viscosities of DNA in the absence and presence of SX, respectively [25]. Melting studies The DNA melting experiments were recorded by monitoring the absorbance of DNA at 260 nm at various temperatures in the absence and presence of SX. The temperature was continuously increased from 20 to 100 oC, and the absorbance was recorded every 2–5 oC. Effect of ionic strength A series of solutions containing various concentrations of NaCl and a fixed amount of SX-DNA were prepared to measure the fluorescence intensity. Results and discussion RLS spectral studies Fig. 2 shows the RLS spectra of DNA, SX, and the mixture of DNA and SX in a pH 7.4 Tris–HCl buffer, respectively. It could be seen that DNA alone had a weak RLS signal. However, when the DNA was mixed with SX, the RLS signal was enhanced remarkably, which indicated that the interaction occurred between SX and DNA. According to the RLS theory, the intensity of RLS is related to the extent of the

6

electronic coupling among chromophores and the particle dimension of the formed aggregate [26]. Therefore, it was inferred that the aggregation of SX molecule on the surface of DNA produced the larger particle in size, thus the enhanced RLS signal for the binding system was observed. Fluorescence spectroscopic studies Quenching mechanism Fig. 3 displays the excitation spectra of SX. It could be observed that the maximum excitation wavelength was 249 nm, so 249 nm was chosen as the excitation wavelength. The fluorescence spectrum of SX (Fig. 4) showed SX had a strong emission peak at 415 nm after being excited at 249 nm. When the increasing concentrations of DNA were added into the solution of SX, the position and shape of the peak did not change, while the fluorescence intensity of SX regularly decreased. The results revealed that SX could interact with DNA and a new non-fluorescent complex of SX-DNA was formed, but the microenvironment of SX was not influenced by DNA. It is well known that the fluorescence quenching may be dynamic, resulting from the diffusive encounter between quencher and fluorophore during the lifetime of the excited state, or resulting from the formation of a non-fluorescent ground-state complex (fluorophore–quencher) [27, 28]. Dynamic and static quenching could be distinguished by their different dependence on temperature and excited-state lifetime. Being restricted by our spectrofluorometer, the excited-state lifetime failed to measure. The following simple method was used to ascertain the quenching type: generally,

7

dynamic quenching of fluorescence was analyzed according to the Stern-Volmer equation [27-29],

F0  1  K q 0 [Q]  1  K sv [Q] F

(1)

where F0 and F are the fluorescence intensities of SX in the absence and in presence of DNA, respectively. Kq is the quenching rate constant of the bimolecules, Ksv is the Stern−Volmer dynamic quenching constant. [Q] is the concentration of quencher (DNA), τ0 is the average lifetime of molecules in the absence of quencher and its value is about 10−8 s [29]. The Stern–Volmer plots of F0 / F versus [Q] at 18, 28, and 38 oC are presented in Fig. 5. The values of Ksv are listed in Table 1. It was found the values of Ksv decreased with the increasing temperatures, showing that the quenching was not a dynamic quenching. Moreover, the order of magnitude of Kq at different temperatures were calculated as 1011, which were much higher than the maximum diffusion collision quenching rate constant of bimolecules, 2.0×1010 L mol–1 s–1 [30]. The results confirmed that the quenching type was a static quenching, not a dynamic quenching. Binding constant and binding site For a static quenching process, the binding constants and binding sites were calculated by following equation [31]: lg

F0 - F 1 1 F0 - F  lg K a  lg([ Bt ][ Dt ]) F n n F0

(2)

Where [Dt] and [Bt] are the total concentration of drug and DNA, respectively. The plots of lg

F0  F 1 F0  F versus lg([ Bt ]  [ Dt ]) are shown in Fig. 6, and the values F n F0 8

of Ka and n at various temperatures are listed in Table 2. Thermodynamic parameters and binding forces The binding force between a biomacromolecule and a small molecule mainly contains van der Waals forces, hydrogen bonds, electrostatic force, and hydrophobic interactions. According to the point of view of Ross and Subramanian, when ΔH < 0 or ΔH ≈ 0, ΔS > 0, the main binding force is electrostatic force; when ΔH < 0, ΔS < 0, the main binding force is van der Waals force or hydrogen bond; when ΔH > 0, ΔS > 0, the main acting force is hydrophobic force [32]. In order to clarify the interaction force of SX with DNA, we calculated the thermodynamic parameters from Eqs. (3) and (4), Ka 2 1 1 H )(  ) K a1 T1 T2 R

(3)

G  H  T S  RT ln K a

(4)

ln(

where the values of ΔH, ΔS, and ΔG are the enthalpy change, the entropy change and the free energy change, respectively. Based on the binding constants Ka obtained from the fluorescence results at 18 oC and 28 oC, the values of ΔH, ΔS, and ΔG are listed in Table 3. The negative sign for ΔG indicated that the binding process was spontaneous. The result of ΔH < 0 and ΔS > 0 indicated that the electrostatic force was a major binding force in the binding of SX and DNA. Absorption spectra of interaction between SX and DNA The UV-vis spectrometry is one of the most important methods to investigate the interaction of small molecules with DNA [33]. From Fig. 7, it could be seen that the 9

maximum absorption were at 250 and 260 nm for SX and DNA, respectively. The absorption of SX exhibited red-shift on addition of DNA. It seemed that the red-shift could not reveal the type of the binding mode, because the spectral changes of SX might just overlap with the absorption spectra of DNA. However, the absorbance analysis might give an information on the binding mode: at 250 nm, the absorbance of mixture of DNA (1.78 × 10–5 mol L–1) and SX (5.00 × 10−6 mol L−1) was compared with the sum of absorbance of the SX (5.00 × 10−6 mol L−1) alone and the DNA (1.78 × 10–5 mol L–1) alone. The results were as follows: ADNA + ASX = 0.3268, ADNA–SX = 0.3380. Obviously, ADNA–SX was larger than the sum of ADNA and ASX. It revealed that interaction of SX with DNA did occur. Generally, hypochromic and red-shift are observed in the absorption spectra of small molecules if they intercalate to the base pairs, because of strong stacking interactions between the aromatic chromophore of the small molecules and the base pairs of DNA [34]. However, the hyperchromic effect was found in the UV-vis spectra, revealing that SX binding to DNA probably through groove binding mode. Viscosity studies Viscosity experiment is regarded as the most effective test for the binding mode between DNA and small molecules. A classical intercalation binding leads to an increase of DNA viscosity due to the lengthening of DNA helix as the space of the adjacent base pair are widened enough to accommodate the binding ligands [35]. However, there is little effect on the viscosity of DNA if a partial non-classical intercalation, such as groove binding and electrostatic binding, occurred in the binding

10

process [36, 37]. In the present study, the relative viscosities (η/η0)1/3 of DNA were plotted versus binding ratio r (r = CSX/CDNA). The results shown in Fig. 8 indicated that there was no obvious change in relative viscosity of DNA in the presence and absence of SX. It should be pointed out that the binding mode of SX with DNA was a non-intercalative, and might be a groove binding. Melting studies The double helix structure of DNA can be denatured into single helix by heating at the melting temperature (Tm). Intercalation of small molecules into the double helix could increase the DNA melting temperature about 5 – 8 oC, and stabilized the double helix structure, but the non-intercalation binding caused no obvious increase in Tm [32]. The melting experiments were performed to obtain the melting temperature of DNA. The DNA melting curves in the absence and in the presence of SX are presented in Fig. 9. As we could see, the value of Tm for DNA in the absence of SX is 87  1℃, while the observed melting temperature of DNA in the presence of SX is about 86  1℃. After fixed amount of SX was added into the DNA solution, the Tm of the complex was not obviously increased. The results revealed that the binding mode of DNA with SX was non-intercalative, it was presumably a groove binding mode. Effect of ionic strength on the fluorescence properties In this work, NaCl was used to control the ionic strength of the solutions. Generally, when a small molecule intercalated into the adjacent base pairs of DNA, the relative fluorescence intensity was not susceptible to the surrounding change [38]; for electrostatic binding mode, Na+ ions inclined to bind with the phosphate groups of

11

DNA ,which resulted in the binding of small molecule with DNA being much weaker; nevertheless, for groove binding mode, when the increasing concentrations of NaCl were added, the minor groove of DNA was narrower and deeper, and the double stranded helix of DNA converged longitudinally, it was beneficial for the interaction between the small molecule and DNA, thus the relative fluorescence intensity would be decreased [36, 39]. As we can see from Fig. 10, the relative fluorescence intensity of SX did not change while the relative fluorescence intensity of SX-DNA decreased when the concentrations of NaCl ranged from 0 to 0.5 mol L−1. It indicated that ionic strength was irrelevant to SX, and the fluorescence change of SX-DNA with the increasing ionic strength was obviously concerned with DNA. The results just proved that SX bound DNA was in the groove binding mode. Comparison of the interactions of SX with single-stranded and double-stranded DNA The binding of SX to single stranded DNA (ssDNA) was further investigated. The ssDNA was obtained by dsDNA in a boiling water bath for 10 min and immediately cooling in an ice–water bath for 10 min. If the binding mode of SX with DNA was a groove binding, the ssDNA would have less opportunity to bind SX than dsDNA would do [36]. As shown in Table 4, the Ka (SX–ssDNA) < Ka (SX–dsDNA), which further confirmed that the binding mode between SX and DNA was groove binding. Binding distance The theory of FRET has been extensively used to measure the binding distance [40, 41], it refers to the process that the energy is transferred from donor to acceptor.

12

The donor of fluorescence here was SX and the acceptor was DNA base pairs. In order to calculate the binding distance, the fluorescence quantum yield of SX was required. Up to now, there has not been any report on the quantum yield of SX to the best of our knowledge. In this paper, fluorescence quantum yield () of SX was obtained by comparing fluorescence intensity of the sample with that of standard solution (having known quantum yield) under the selected conditions. The quantum yield was calculated as follows [42]:

  st  x

Fx Ast  Fst Ax

(5)

In this equation, the subscripts ‘‘x’’ and ‘‘st’’ denote sample and standard. φx and φst are the fluorescence quantum yield. Ax and Ast are the absorption of sample and standard at the excitation wavelength of standard. Fx and Fst are the integrals of the fluorescence intensity. In this study, L-Trptophan was selected as a standard, for which the fluorescence quantum yield of 0.14 at 25 oC was reported [43]. The measurements were carried out under the selected experimental conditions. According to Eq. (5), the quantum yield φ of SX was measured as 0.13. Based on FRET theory, the rate of dipole–dipole energy transfer depends on the relative orientation of the donor and acceptor transition dipoles, the extent of overlapping of the acceptor absorption spectrum with the donor emission spectrum, and the distance between the donor and acceptor [44, 45]. The energy transfer efficiency (E) is inversely proportional to the sixth power of the distance between donor and acceptor, it can be obtained by the following equations. 13

E  1

R6 F  6 0 6 F0 R0  r0

(6)

F and F0 are the fluorescence intensity of the donor SX in the presence and absence of the acceptor DNA, respectively. r0 is the acting distance between acceptor and donor. The critical distance R0 is the distance at 50% of the energy transfer efficiency, and is given as:

R0  8.8 1025  K 2 n 4 J 

1

6

(7)

Where φ is the fluorescence quantum yield, and  =0.13 for SX as above, K2 is the spatial orientation factor of the dipole, and its value is 2/3, n is the refraction index of the medium and equal to 1.336 [46], J is the overlap integral between the donor fluorescence emission spectrum and the acceptor absorption spectrum. The value of J can be given by the following expression.

 F ( ) ( )  J  F ( ) 4

(8)

F (λ) is the fluorescence intensity of the fluorescence donor at wavelength λ, and ε (λ) is the molar absorption coefficient of the acceptor at wavelength λ. Fig. 11 shows the overlapping of the UV absorption spectrum of DNA with the fluorescence emission spectrum of SX at a 1:1 molar ratio. The values of the parameters were calculated as J = 5.48 × 10-15 cm3 L mol-1, R0 = 2.25 nm, E = 0.027 and r0 = 4.10 nm. Here r0 < 8 nm (the academic value) [47] suggested that non-radiation energy transfer between SX and DNA occurred. Conclusion In this work, the binding mode of SX to calf thymus DNA was probed by various

14

simple and practicable techniques (fluorescence, UV absorption, RLS, viscometry, ionic strength experiment, melting temperature, and ssDNA binding experiment). All the evidences showed that the binding mode between SX and DNA should be groove binding. From the fluorescence quenching spectra, the quenching mechanism, binding constant and binding force were obtained. Comparative method was used to obtain the quantum yield of SX as 0.13. Based on the theory of FRET, the binding distance between SX and DNA was calculated, which implied that the non-radiation energy transfer occurred. These results indicated that DNA might serve as the target of SX. The investigations based on drug–DNA interactions not only help to understand the action mechanisms of drug, but also to design new DNA-targeted drugs and screen drugs in vitro.

Acknowledgements This paper was supported by Twelfth Five-Year Program of Science and Technology of the Educational Office of Jilin Province (NO. 20140257)and Natural Science Foundation of Jilin Province (20140101023JC)and Academic Innovation Foundation of Changchun Normal University (cscxy2013008).

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18

Captions of illustrations

Fig. 1. Structure of salmeterol xinafoate (SX). Fig. 2. RLS spectra of DNA-SX system at pH 7.4 and 18 oC. The RLS spectrum of DNA (1.78 × 10−5 mol L-1) alone is curve a. The RLS spectrum of SX (5.00 × 10−6 mol L-1) alone is curve b. The RLS spectra of DNA–SX are curves c – g, where all the concentrations of SX are identical as 5.00 × 10-6 mol L-1 and the concentrations of DNA are (c) 1.78, (d) 3.56, (e) 5.34, (f) 7.12, and (g) 8.90 × 10 −5 mol L-1 respectively. Fig. 3. Excitation spectra of SX in Tris-HCl buffer of pH 7.4 at 18 oC. The concentration of SX is 5.00 × 10−6 mol L-1. Fig. 4. Fluorescence spectra of SX in the presence of DNA in Tris-HCl buffer of pH 7.4 at 18 oC. The concentration of SX is 5.00 × 10−6 mol L-1 .The concentrations of DNA are (a) 0.00, (b) 1.69, (c) 3.38, (d) 5.06, (e) 6.75, (f) 8.44, (g) 10.13 × 10−5 mol L-1. Fig. 5. Stern–Volmer plots of the system for SX-DNA at various temperatures. Fig. 6. Plots of lg ((F0−F)/F) versus lg ((Bt)−(1/n)(Dt)(F0−F)/F0) for the system of SX–DNA at various temperatures. Fig. 7. The absorption spectra of SX with DNA. The concentration of SX (a) is 5.00 × 10−6 mol L-1, and the concentrations of DNA are 0.00, 1.78, 3.56, 5.34, 7.12, 8.90 × 10−5 mol L-1 for curves b – g, respectively. Fig. 8. Effect of SX on the relative viscosity of DNA. Binding ratio r is equal to CSX/CDNA. Concentration of DNA is 1.80 × 10−5 mol L−1 and the concentrations of SX are 0.0, 3.60, 7.20, 10.80, 14.40, 18.00 × 10−6 mol L−1. Fig. 9. Melting curves of DNA (3.60 × 10−4 mol L−1) (a) in the absence and (b) in the presence of SX (1.00 × 10−4 mol L−1). Fig. 10. Effect of ionic strength on fluorescence intensity of SX (a) and (b) SX–DNA. For SX–DNA system, the concentrations of SX and DNA are 1.00 × 10−4 mol L−1 and 1.19 × 10−5 mol L−1, respectively. Relative fluorescence intensity is equal to F/F0, where F0 and F are fluorescence intensities in the absence and in the presence of NaCl. Fig. 11. Overlapping of (a) the absorption spectrum of DNA with (b) the fluorescence spectrum of SX. Both the concentrations of SX and DNA are all 5.00 × 10–6 mol L–1. 19

Fig. 1

20

240

g 210

f 180

RLS Intensity

e 150

d

120

c

90

b

60 30

a 0 200

300

400

500

600

700

Wavelength (nm)

Fig. 2

21

700

Fluorescence Intensity

600

500

400

300

200

100

0 200

240

280

320

360

400

Wavelength (nm)

Fig. 3

22

300

a

Fluorescence Intensity

250

200

g 150

100

50

0 350

400

450

500

550

Wavelength (nm)

Fig. 4 23

1.45 1.40

o

18 C o 28 C o 38 C

1.35

F0 / F

1.30 1.25 1.20 1.15 1.10 1.05 1.00 2

4

6

8 -5

10

-1

Concentration (10 mol L )

Fig. 5 24

-0.2

o

18 C o 28 C o 38 C

-0.4

lg((F0-F)F)

-0.6

-0.8

-1.0

-1.2

-1.4 -4.8

-4.6

-4.4

-4.2

-4.0

lg([Bt]-(1/n)[Dt](F0-F)/F0)

Fig. 6 25

1.4

1.2

g

Absorbance

1.0

f

0.8

e 0.6

d 0.4

c b

0.2

0.0 220

240

a

260

280

300

320

340

Wavelength (nm)

Fig. 7

26

1.6

1.4

( / 0)

1/3

1.2

1.0

0.8

0.6

0.4 0.0

0.2

0.4

0.6

0.8

1.0

r

Fig. 8

27

a

2.4 2.2

b 2.0

A20 /A

1.8 1.6 1.4 1.2 1.0 0.8 20

40

60

80

100

o

Temperature ( C)

Fig. 9

28

a

Relative fluorescence intensity

1.00

0.98

0.96

0.94

b

0.92

0.90

0.88

0.86 0.0

0.1

0.2

0.3

0.4

0.5

-1

cNaCl ( mol L )

Fig. 10 29

0.045

250

0.040

0.035

200

0.030 150 0.025 100

Absorbance

Fluorescence intensity

300

0.020 50 0.015 0 300

400

500

600

Wavelength (nm)

Fig. 11 30

Table 1 The Stern-Volmer quenching constant Ksv and the quenching rate constants Kq at different temperatures.

a

Temperature (oC)

Ksv (103 L mol-1 )

Kq (1011 L mol-1 s-1 )

ra

18

4.49±0.02

4.49±0.02

0.9922

28

4.17±0.07

4.17±0.07

0.9973

38

3.73±0.11

3.73±0.11

0.9944

The regression coefficient.

21

Table 2 The binding constants (Ka) and the number of binding sites (n) of SX with DNA at various temperatures.

a

Temperature (oC)

Ka (103 L mol-1 )

n

ra

18

8.52±0.08

0.9177

0.9935

28

8.31±0.04

0.9249

0.9958

38

6.14±0.08

0.9580

0.9882

The regression coefficient.

22

Table 3 Thermodynamic parameters for the interaction of SX with DNA at 291K. ΔH ( kJ mol-1 )

ΔG ( kJ mol-1 )

ΔS ( J mol-1K-1 )

-1.73

-21.89

69.28

c

23

Table 4 Binding constants and binding sites of SX with double-stranded (dsDNA) and single-stranded DNA (ssDNA).

a

Ka (103 L mol-1)

n

ra

dsDNA

8.52±0.08

0.9177

0.9935

ssDNA

4.20±0.04

1.0556

0.9968

The regression coefficient.

24

Highlights > Salmeterol xinafoate interacted with DNA by groove binding mode. > Various simple and practicable techniques to explore the binding mode. > DNA quenched the fluorescence of salmeterol xinafoate. > Binding parameters were determined. > Energy transfer occurred between salmeterol xinafoate and DNA.

300

0 mM 250

Fluorescence Intensity

DNA 200

0.1013mM 150

100

50

0 350

400

450

Wavelength (nm)

500

550

In vitro studies on the behavior of salmeterol xinafoate and its interaction with calf thymus DNA by multi-spectroscopic techniques.

The salmeterol xinafoate (SX) binding to calf thymus DNA in vitro was explored by fluorescence, resonance light scattering (RLS), UV-vis absorption, a...
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