Accepted Manuscript Investigation on the interactions of clenbuterol to bovine serum albumin and lysozyme by molecular fluorescence technique Shuyun Bi, Bo Pang, Tianjiao Wang, Tingting Zhao, Wang Yu PII: DOI: Reference:

S1386-1425(13)01144-X http://dx.doi.org/10.1016/j.saa.2013.09.137 SAA 11116

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

19 July 2013 13 September 2013 29 September 2013

Please cite this article as: S. Bi, B. Pang, T. Wang, T. Zhao, W. Yu, Investigation on the interactions of clenbuterol to bovine serum albumin and lysozyme by molecular fluorescence technique, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2013), doi: http://dx.doi.org/10.1016/j.saa.2013.09.137

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Investigation on the interactions of clenbuterol to bovine serum albumin and lysozyme by molecular fluorescence technique Shuyun Bi*, Bo Pang, Tianjiao Wang, Tingting Zhao, Wang Yu College of Chemistry, Changchun Normal University, Changchun 130032, PR China

Abstract Clenbuterol interacting with bovine serum albumin (BSA) or lysozyme (LYS) in physiological buffer (pH 7.4) was investigated by the fluorescence spectroscopy and UV–vis absorption spectroscopy. The results indicated that clenbuterol quenched the intrinsic fluorescence of BSA and LYS via a static quenching procedure. The binding constants of clenbuterol with BSA and LYS were 1.16 × 103 and 1.49 × 10 3 L mol−1 at 291 K. The values of ΔH and ΔS implied that hydrophobic and electrostatic interaction played a major role in stabilizing the complex (clenbuterol–BSA or clenbuterol–LYS). In the presence of Fe2+, Fe3+, Cu 2+, Mg2+, Ca2+, or Zn2+, the binding constants of clenbuterol to BSA or LYS had no significant differences. The distances between the donor (BSA or LYS) and acceptor (clenbuterol) were 2.61 and 2.19 nm for clenbuterol–BSA and clenbuterol–LYS respectively. Furthermore, synchronous fluorescence spectrometry was used to analyze the conformational changes of BSA and LYS.

Keywords: Clenbuterol; Bovine serum albumin; Lysozyme; Fluorescence quenching; Synchronous fluorescence 1. Introduction *

Corresponding author. Tel: +86 431 86168098; Fax: +86 431 86168096.

E-mail address: sy_bi @sina.com 1

Clenbuterol

(4-amino-α-[t-butylaminomethyl]-3,

5-dichlorobenzyl

alcohol

hydrochloride) (Fig. 1) is a long-acting beta-2-adrenergic receptors (β2-AR) agonist, which was originally used in non-infectious respiratory diseases [1]. In addition, because the drug initiates fat metabolism resulting in promoting muscle growth and fat breakdown [2], it is illegally used to feed livestock in order to keep the economical benefits. The use of clenbuterol has been forbidden in the world [3, 4]. But human food poisoning caused by the misuse of clenbuterol was still found in some countries [5-7]. Therefore, the continuous surveillance of clenbuterol has become more and more necessary for human health. Proteins are very important biochemical compounds in cell having many diverse functions. The ability of the binding of clenbuterol to proteins can determine the amount of clenbuterol bound to protein and the effectiveness of clenbuterol in body. Therefore it is important to study the interaction of clenbuterol with proteins. Bovine serum albumin (BSA) is the most common model protein. Its intrinsic fluorescence arose from Trp−134 and Trp−212 located subdomains IA and IIA, respectively [8]. Because it has structural homology with human serum albumin (HSA) [8, 9], BSA was chosen for this study. Lysozyme (LYS) was also chosen as the target protein molecule because it has often been applied for the study of biological process, and the property of LYS significantly differs from that of BSA [10]. LYS has six tryptophanes (Trp), and Trp−62 and Trp−108 are the most dominant fluorophores which play important roles [11].We believe, LYS as the study model will provide new insights on the understanding how this protein interacts with clenbuterol. Up to now, various methods have been reported about the determination of clenbuterol [12-16]. However, little research has been conducted on the interaction of clenbuterol with BSA (or LYS) by fluorescence spectroscopy and UV–vis absorption 2

spectroscopy in vitro. This research is necessary because of the dangerous effects from the use of clenbuterol. In this paper, the accurate and full data will make a significant contribution to the understanding of the action mechanism and metabolic process of clenbuterol in living organisms. 2. Experimental 2.1. Reagents BSA and LYS (Sigma packaging) were bought from Changchun Dingguo Biotechnology Company and stored in refrigerator at 4 °C. Clenbuterol was got from Chinese Drug Biological Products Qualifying Institute. Tris-HCl buffer solution (pH 7.4) containing 0.1 mol L-1 NaCl was prepared. FeSO4, FeCl3, FeCl2, CuCl2, MgCl2, and CaCl2 were used to study the effects of co-ions on the binding of clenbuterol to BSA and LYS. All other reagents were of analytical reagent grade without further purification. Double distilled water was used throughout experiments. 2.2. Apparatus All fluorescence spectra were recorded on a RF-5301PC spectrofluorometer (Shi madzu, Japan) equipped with a xenon lamp source, a 1.0 cm ×1.0 cm × 4.0 cm quartz cell and a thermostatic controller. The absorption spectra were recorded on TU-1901 UV-Spectrometer (Beijing Purkinje General Instrument Co., Ltd.). 2.3. Procedures A fixed amount of protein and various amounts of clenbuterol were added to each mark tube and the total volume was fixed to 2.5 mL with Tris-HCl buffer of pH 7.4 containing 0.1mol L-1 sodium chloride. All solutions in tubes were mixed well. The excitation wavelength was 280 nm and the fluorescence spectra were recorded in the wavelength range 280–500 nm at different temperatures. The slit widths of excitation/emission were set to 3/3 nm. Synchronous fluorescence spectra of BSA and 3

LYS in the presence of various amounts of clenbuterol were scanned at Δλ = 15 nm and 60 nm, respectively. The measurement of UV/Vis spectrum of clenbuterol was performed under the conditions: the scan rate was 8 nm s-1, and the scan range was 290–500 nm. 3. Results and discussion 3.1. Binding characteristics of clenbuterol to BSA or LYS Fig. 2 shows the effects of clenbuterol on the fluorescence of BSA and LYS. The concentrations of BSA and LYS were all 5.0 × 10−6 mol L-1, and the concentration of clenbuterol was varied from 0.0 to 25.0 × 10−5 mol L-1. It could be seen that when BSA and LYS were excited at 280 nm, both of them emitted strong fluorescence, and the maximum emission wavelengths were at 342 nm and 338 nm respectively, which mainly arose from Trp. In fact, the intrinsic fluorescence of BSA and LYS primarily arises from Trp and Tyr. But the emission wavelength of Tyr was not often found at about 304–310 nm because its fluorescence was almost quenched by Trp owing to the efficient energy transfer from Tyr to Trp, i.e. the internal quenching effect [17, 18]. It was also obvious that the emission intensity of the two proteins dropped regularly with different amounts of clenbuterol, suggesting that the interaction occurred between clenbuterol and the protein and the non-fluorescent complex was formed. Furthermore, a blue shift of the maximum emission peak (from 342 to 339 nm) was found in the emission spectra of BSA when the concentration of clenbuterol was increased continuously, suggesting that Trp was located in a more hydrophobic environment [19, 20]. In the emission spectra of LYS, a blue or red shift of the maximum emission peak was not found. Two quenching processes are known: static and dynamic quenching. Dynamic quenching resulted from the diffusive encounter between quencher and fluorophore 4

during the lifetime of the excited state; static quenching resulted from the formation of a non-fluorescent ground-state complex (fluorophore–quencher) [21]. The fluorescence quenching for the binding of drug (D) and protein (P) are analyzed by the Stern–Volmer equation [21, 22] F0 = 1 + K qτ 0 [ D ] = 1 + K sv [ D ] F

(1)

where F0 and F denote the fluorescence emission intensities in the absence and in presence of quencher respectively. Kq is the bimolecule quenching rate constant, τ0 is the average lifetime of a fluorophore in the absence of quencher and its value is about 10−8s [22], [D] is the concentration of the drug, Ksv is the Stern−Volmer quenching constant. For eliminating the inner filter effects, absorbance of the sample was measured at the excitation and emission wavelengths of the fluorescence measurements. The fluorescence data were corrected by the following equation [23]: Fc = Fm × e

( A1 + A2 )

2

(2)

where Fc and Fm are the fluorescence intensities corrected and observed, respectively, and A1 and A2 are the absorption of clenbuterol at excitation and emission wavelength, respectively. The Stern–Volmer plots for the two proteins quenched by clenbuterol at different temperatures are displayed in Fig. 3. All plots had a good linearity. According to Eq. (1), the KSV and Kq values for clenbuterol–BSA and clenbuterol–LYS systems are listed in Table 1. The values of Kq were all larger than 2.0 × 1010 L mol−1 s−1, the maximum diffusion collision quenching rate constant of various quenchers with the biopolymer [21, 22, 24]. If the value of Kq is greater than 2.0 × 1010 L mol−1 s−1, the quenching process will be static, not dynamic. Moreover, the values of Ksv for 5

clenbuterol–LYS decreased with increasing temperature. The values of Ksv for clenbuterol–BSA had little change, showing the temperature had little effect on the quenching process. Higher temperature results in faster diffusion and hence larger amount of dynamic quenching, which will typically result in dissociation of weakly bound complexes, and hence smaller amounts of static quenching [21]. The observations suggested that the fluorescence quenching of BSA or LYS by clenbuterol occurred via a static quenching mechanism. 3.2. Binding constants and binding sites For the static quenching, the binding constant KA and the number of binding sites n were determined from an improved equation developed by us [25]:

lg

F0 − F F −F ) = n lg K A + n lg ([ Dt ] − n [ Pt ] 0 F F0

(3)

F0 and F are the fluorescence intensity of protein when the drug D was present and absent. [Dt] is the total drug concentration and [Pt] is the total protein concentration. Fig. 4 displayed

the plots of lg((F0−F)/F) for BSA or LYS against

lg([Dt]−n[Pt](F0−F)/F0). The values of KA and n at different temperatures were listed in Table 2. The KA values decreased with the increasing temperature implied the complex of clenbuterol–BSA or clenbuterol–LYS became less stable at higher temperature, which further evidenced that the fluorescence quenching was a static quenching process. 3.3. Thermodynamic parameters and nature of the binding forces

In general, acting forces between small molecular and biomacromolecule mainly include hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic forces, etc. By the values of the binding constants KA at 291, 301 and 311 K, the

6

thermodynamic parameters such as ΔH, ΔG and ΔS were estimated according to the following equations: ln

K A2 1 1 1 = ( − ) ΔH K A1 R T1 T2

ΔG = − RT ln K A = ΔH − TΔS

(4) (5)

where the values of ΔH, ΔG and ΔS are enthalpy change, free energy change and entropy change. The calculated results for the interaction between clenbuterol and BSA or LYS are listed in Table 3. The negative ΔG values mean that the interaction process between clenbuterol and BSA or LYS was spontaneous. According to the point of view of Ross and Subramanian [26], when ΔH ﹤0 or ΔH ≈ 0, ΔS ﹥0, the main binding force was electrostatic force; when ΔH ﹤0, ΔS ﹤0, the main binding force was van der Waals force or hydrogen bond and when ΔH ﹥0, ΔS ﹥0, the main binding force was hydrophobic force. So the results indicated that electrostatic force was the main binding force to stabilize the complex of clenbuterol–BSA and clenbuterol–LYS. Moreover, ΔS ﹥0, hydrophobic interaction should not be excluded. 3.4. Effect of the metal ions on the binding constants

The presence of metal ions in plasma may affect the interactions of the drug with proteins. The effects of Fe2+, Fe3+, Cu2+, Mg2+, Ca2+, and Zn2+ on the binding clenbuterol to the two proteins were determined at 291 K, and data are shown in Table 4. When Fe2+, Fe3+, Cu2+, Mg2+, Ca2+, or Zn2+ was added into clenbuterol–BSA or clenbuterol–LYS solution, the ratio of binding constant KA´ to KA ranged from 91.38% to 101.72% for clenbuterol–BSA and 90.61% to 102.01% for clenbuterol–LYS. These results showed that the binding constants had no apparent change as compared to the binding constants without the metal ions. Thus, these metal ions had no significant effects on the binding of clenbuterol to the two proteins.

7

3.5. Synchronous fluorescence spectra

Synchronous fluorescence spectrometry is a very useful method to further investigate the conformations about the microenvironment in the vicinity of the chromophore molecules [27], and it offers many advantages, such as sensitivity, spectral simplification, and avoiding different perturbing effects [28]. According to Miller [29], when Δλ (the difference between excitation wavelength and emission wavelength) is stabilized at 15 or 60 nm, the synchronous fluorescence spectra were obtained to evidence the characteristic information of Tyr or Trp residues, respectively [30]. The synchronous fluorescence spectra of BSA and LYS with a fixed concentration in the presence of various concentrations of clenbuterol were recorded when Δλ = 60 nm and Δλ = 15 nm. The corresponding results are shown in Fig. 5. For the two proteins, the fluorescence intensity of Tyr and Trp was decreased, and the emission wavelength of them had no consistent change. There was no obvious change in the emission wavelength of Tyr (Fig. 5(1) and Fig. 5(2)), suggesting that the polarity and hydrophobicity around Tyr microenvironment was not affected by clenbuterol. Fig. 5(1´) displays that the maximum emission peak have a notable blue shift (from 342 to 338) nm, demonstrating that Trp of BSA exposed to a more hydrophobic microenvironment [19, 30]. However, from Fig. 5(2´), the wavelength change could not be observed, which showed that the microenvironment of Trp of LYS was not changed by clenbuterol. 3.6. Binding distance

According to the Förster non-radiation energy transfer theory [31], the distance r between donor (BSA or LYS) and acceptor (clenbuterol) can be calculated. The efficiency (E) energy transfer is related to the r between the two proteins and clenbuterol by 8

E = 1−

R6 F = 6 0 6 F0 R0 + r0

(6)

here E is the efficiency of transfer between the donor and the acceptor; R 0 is called the Förster distance or critical distance, at which the efficiency of transfer is 50%.

R06 = 8.8 × 10 −25 K 2 N −4φJ

(7)

In Eq. (7) K2 is the spatial orientation factor of the dipole, N is the refractive index of the medium, φ is the fluorescence quantum yield of the donor, J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J can be given by the expression J=

∑

F (λ )ε (λ )λ4 Δλ

∑ F (λ )Δλ

(8)

where F(λ) is the fluorescence intensity of the fluorescent donor at wavelength λ, and ε(λ) is the molar absorptivity of the acceptor at wavelength λ and its unit is L mol−1

cm−1. The overlapping graphs of the fluorescence spectra of BSA and LYS with the absorption spectra of clenbuterol are shown in Fig. 6. In the present case, K2 = 2/3, φ = 0.13, N = 1.33 [32], the values of J, E, R0 and r were calculated and presented in Table 5. Apparently, the value of r0 < 8 nm [33], the academic value of energy transfer distance between the donor and acceptor, suggesting that non-radiative energy transfer occurred between clenbuterol and the protein with high probability. 4. Conclusion

The binding properties of clenbuterol to the two important proteins (BSA and LYS) were studied by fluorescence quenching, synchronous fluorescence, and UV–vis absorption. The results showed that clenbuterol quenched the fluorescence of BSA and LYS through complex formation with the distance between the donor and acceptor less than 8 nm. BSA and LYS could bind with clenbuterol by hydrophobic 9

and electrostatic interactions. The quenching rate constant, binding constant, and number of binding sites were obtained. Fe2+, Fe3+, Cu2+, Mg2+, Ca2+, or Zn2+ had little effect on the binding constants. From synchronous fluorescence spectra, the microenvironment of Tyr of BSA and LYS was not changed, but Trp of them were exposed to a more hydrophobic microenvironment. This work focused on the mechanism of the binding of clenbuterol to protein, we believe the data will give a valuable reference on the relevant subject.

Acknowledgements This paper was supported by Twelfth Five-Year Program of Science and Technology of the Educational Office of Jilin Province and the Science Program of Changchun Normal University (No. 2010-034).

10

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[15] W. Haasnoot, M.E. Ploum, R.J.A. Paulussen, R. Schilt, F.A. Huf, J. Chromatogr. A. 519 (1990) 323–335. [16] A. Posyniak, J. Zmudzki, J. Niedzielska, Anal. Chim. Acta. 483(2003) 61–67. [17] D.Freifelder, Physical Biochemistry: Applications to Biochemistry and Molecular Biology, San Francisco, 1976, pp. 410–430. [18] A. Sułkowska, J. Mol. Struct. 614 (2002) 227–232. [19] T. Yuan, A.M. Weljie, H.J. Vogel, Biochemistry. 37 (1998) 3187–3195. [20] A. Sułkowska, J. Równicka, B. Bojko, J Poźycka, I. Zubik-Skupień, W. Sulkowski, J. Mol. Struct. 704 (2004) 291–295. [21] J.R. Lakowicz, Principles of Fluorescence Spectroscopy (second ed.), Plenum Press, New York, 1999, p.237. [22] T.G. Dewey (Ed.), Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, Plenum Press, New York, 1991. [23] R.F. Steiner, L. Weinryb, Excited States of Protein and Nucleic Acid, Plenum Press, New York, 1971, p. 40. [24] W.R. Ware, J. Phys. Chem. 66 (1962) 455–458. [25] S. Bi, D. Song, Y. Kan, D. Xu, Y. Tian, X. Zhou, H. Zhang, Spectrochim. Acta, Part A. 62 (2005) 203–212. [26] P.D. Ross, S. Sabramanian, Biochemistry. 20 (1981) 3096–3102. [27] B. Klajnert, M. Bryszewska, Bioelectrochemistry. 55 (2002) 33–35. [28] T. Ueno, S. Abe, T. Koshiyama, T. Ohki, T. Hikage, Y. Watanabe, Chem. A Eur. J. 16 (2010) 2730–2740. [29] J.N. Miller, Proc. Anal. Div. Chem. Soc. 16 (1979) 203–208. [30] E.A. Burstein, N.S. Vedenkina, M.N. Ivkova, Photochem. Photobiol. 18 (1973) 12

263–279. [31] T. Förster, O. Sinanoglu (Eds.), Modern Quantum Chemistry (3rd ed.), Academic Press, New York, 1996, p. 93. [32] B. Zhang, W. Wang, F. Bai, Chem. J. Chin. U. 15 (1994) 373 –378. [33] G. Chen, X. Huang, J. Xu, Z. Zheng, Z. Wang, Fluorimetry (2nd ed.), Science Press, Beijing, 1990, p.123.

13

Captions of illustrations

Fig. 1 Structure of clenbuterol.

Fig. 2 The effects of clenbuterol on the fluorescence emission spectrum of (1) BSA

and (2) LYS in Tris–HCl buffer of pH 7.4 at 291K. The concentrations of BSA and LYS are all 5.0 × 10−6 mol L−1. The concentration of clenbuterol in BSA and LYS are all: (a) 0.0, (b) 4.0, (c) 8.0, (d) 12.0, (e) 16.0, (f) 20.0 and (g) 24.0 × 10−5 mol L−1.

Fig. 3 Stern–Volmer curves for clenbuterol binding to (1) BSA and (2) LYS at

different temperatures.

Fig. 4 Plots of lg((F0−F)/F) vs. lg([Dt]−n[Pt](F0−F)/F0) for clenbuterol binding to (1)

BSA and (2) LYS at different temperatures.

Fig. 5 Synchronous fluorescence spectra of (1) BSA and (2) LYS in the presence of

clenbuterol at Δλ = 15 nm and those of (1′) BSA and (2′) LYS at Δλ = 60 nm. The concentrations of BSA and LYS are all 5.0 × 10−6 mol L−1. The concentration of clenbuterol in BSA and LYS are all: (a) 0.0, (b) 4.0, (c) 8.0, (d) 12.0, (e) 16.0, (f) 20.0 and (g) 24.0 × 10−5 mol L−1.

Fig. 6 The overlaps of the fluorescence spectra of BSA and LYS with the absorption

spectra of clenbuterol. The concentrations of BSA, LYS and clenbuterol are all 5.0 × 10−6 mol L−1.

14

OH H N

CH3

H3C

CH3

Cl

H2N

. HCl Cl

Fig. 1

15

(1)

600

a

Fluorescence Intensity

500

400

g

300

200

100

0 300

350

400

450

Wavelength (nm)

(2) 140

a

120

Fluorescence Intensity

100

g 80

60

40

20

0 300

350

400

450

Wavelength (nm)

Fig. 2

16

(1) 1.30

BSA 291 K BSA 301 K BSA 311 K

1.25

F0 / F

1.20

1.15

1.10

1.05

1.00

0

5

10

15 -5

20

25

-1

[D] (10 mol L )

(2)

1.5

LYS 291K LYS 301K LYS 311K

1.4

F0 / F

1.3

1.2

1.1

1.0

0

5

10

15 -5

20

25

-1

[D] (10 mol L )

Fig. 3

17

(1) BSA 291 K BSA 301 K BSA 311 K

-0.6

log((F0-F)/F)

-0.8

-1.0

-1.2

-1.4

-1.6 -4.4

-4.2

-4.0

-3.8

-3.6

lg([Dt]-n[Pt](F0-F)/F0)

(2) -0.3

LYS 291 K LYS 301 K LYS 311 K

-0.4 -0.5 -0.6

log((F0-F)/F)

-0.7 -0.8 -0.9 -1.0 -1.1 -1.2 -1.3 -4.4

-4.2

-4.0

-3.8

-3.6

lg([Dt]-n[Pt](F0-F)/F0)

Fig. 4

18

(1)

(2) 120 140

a

120

80

Fluorescence Intensity

Fluorescence Intensity

100

60

g 40

Δλ=15 nm

20

a

100

80

g

60

Δλ=15 nm

40

20

0 0

300

320

340

360

380

400

300

320

340

Wavelength (nm)

360

380

400

420

440

Wavelength (nm)

(1') 700

(2')

70

600 60

a

a

50

Fluorescence Intensity

Fluorescence Intensity

500

400

300

g

200

Δλ=60 nm

100

g

40

30

Δλ=60 nm

20

10

0

0

300

320

340

360

380

400

Wavelength (nm)

300

320

340

360

380

400

420

440

Wavelength (nm)

Fig. 5

19

0.05

650

BSA

600 550

0.04

450 400 0.03

350 300 250 200

0.02

clenbuterol LYS

150 100

Absorbance

Fluorescence Intensity

500

0.01

50 0 0.00

-50 300

350

400

450

λ (nm)

Fig. 6

20

Table 1 The Stern−Volmer quenching constants Ksv and the quenching rate constants

Kq of clenbuterol binding to BSA and LYS at various temperatures.

Protei n

Temperature (K)

291 BSA

LYS

a

Kq

Ksv 3

（×10 L mol

−1

1.06

）

（×1011 L mol−1 s−1)

ra

1.06

0.9958

301

1.06

1.06

0.9958

311

1.09

1.01

0.9900

1.73

0.9975

291

1.73

301

1.59

1.59

0.9975

311

1.37

1.37

0.9985

The regression coefficient.

21

Table 2 The binding constants KA and binding sites n of clenbuterol to BSA and LYS at different temperatures.

Protein

BSA

LYS

a

Temperature (K)

KA (L mol −1)

n

ra

291

1.16×103

1.09

0.9983

301

1.01×103

0.99

0.9969

311

0.83×103

0.80

0.9920

291

1.49×103

0.86

0.9973

301

1.39×103

0.96

0.9936

311

1.38×103

1.02

0.9984

The regression coefficient.

Table 3 22

Thermodynamic parameters for clenbuterol binding to BSA and LYS at different temperatures.

Protein

BSA

LYS

Temperatu re (K)

ΔH (kJ mol−1)

ΔG (kJ mol−1)

ΔS (J mol−1 K−1)

291

-10.08

-17.07

24.03

301

-15.28

-17.31

6.74

311

-12.59

-17.38

15.40

291

-5.06

-17.46

59.05

301

-0.56

-18.11

58.31

311

-2.89

-18.69

50.80

Table 4 23

Effect of metal ions on the binding of clenbuterol to BSA and LYS at 291K.

Metal ions

Protein BSA KA´ −1

r

(L mol )

LYS KA´/ KA

K A´ −1

(%)

(L mol )

r

KA´/ KA

(%)

Fe2+

1.18×103

0.9926

101.72

1.37×103

0.9920

91.95

Fe3+

1.08×103

0.9976

93.10

1.52×103

0.9900

102.01

Cu2+

1.13×103

0.9942

97.41

1.64×103

0.9996

110.07.

Mg2+

1.17×103

0.9918

100.86

1.36×103

0.9933

91.28

Ca2+

1.06×103

0.9932

91.38

1.35×103

0.9901

90.61

Zn2+

1.09×103

0.9908

93.97

1.37×103

0.9974

91.95

KA and KA´ are the values of binding constant in the absence and presence of ions respectively, n is binding site and r is regression coefficient.

Table 5

24

The parameters of Förster non-radiation energy transfer occurred in the binding process.

Protein

J (L mol−1 cm3)

E (%)

R0 (nm)

r0 (nm)

BSA

4.19×10-16

3.08

1.47

2.61

LYS

3.77×10-14

7.84

1.42

2.19

25

Research Highlights

> Interactions of clenbuterol with BSA and LYS were studied by fluorescence quenching. > Hydrophobic and electrostatic forces were the major forces in the two systems. > Synchronous fluorescence was performed to analyze the conformational changes. > Energy transfer occurred between clenbuterol and the two proteins.

26

Graphical abstract

600

a

Fluorescence Intensity

500

400

g

300

200

100

0 300

350

400

450

Wavelength (nm)

27