Study on the binding of chloroamphenicol with bovine serum albumin by fluorescence and UV–vis spectroscopy

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 74–79

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Study on the binding of chloroamphenicol with bovine serum albumin by fluorescence and UV–vis spectroscopy Jun Zhang a,⇑, Lingnan Chen b, Birong Zeng b,⇑, Qilong Kang b, Lizong Dai b a b

Department of Physics, School of Physics and Information Engineering, Fuzhou University, Fuzhou 350108, China Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" We explored the interaction of BSA

The interaction between chloroamphenicol (CPC) and bovine serum albumin (BSA) was studied by fluorescence and UV–visible spectroscopy. The quenching mechanism, binding constants and binding distance were obtained. The effects of some common metal ions on the binding constant between CPC and BSA were examined. What’s more, we investigated the possible sub-domains on BSA that bind CPC by displacement experiments.

" "

"

200

Fluorescence Intensity

"

and CPC by spectroscopic methods. The fluorescence quenching mechanism is combined quenching. The binding constants and binding sites were calculated. The synchronous fluorescence spectra indicated that the conformation of BSA has been changed. The displacement experiment indicated that CPC does bind at the region of site I.

a

150

100

j

50

0 300

350

400

450

500

Wavelength (nm)

a r t i c l e

i n f o

Article history: Received 24 August 2012 Received in revised form 9 November 2012 Accepted 16 November 2012 Available online 5 December 2012 Keywords: Chloroamphenicol Bovine serum albumin Fluorescence spectroscopy UV–visible spectroscopy Metal ions Displacement experiments

a b s t r a c t The binding of chloroamphenicol (CPC) to bovine serum albumin (BSA) at 296 K, 303 K, and 310 K by fluorescence and UV–visible absorption spectroscopy were investigated under imitated physiological conditions. The experimental results showed that the fluorescence quenching mechanism between CPC and BSA was combined quenching (dynamic and static quenching) procedure at low CPC concentration, or a dynamic quenching procedure at high concentrations. The binding constants, binding sites and the corresponding thermodynamic parameters of the interaction system were calculated. According to Förster non-radiation energy transfer theory, the binding distance between CPC and BSA was calculated to be 3.02 nm. Both synchronous fluorescence and FT-IR spectra confirmed the interaction, and indicated the conformational changes of BSA. The effects of some common metal ions Ca2+, Ni2+, Mg2+, Fe2+, and Cu2+ on the binding constant between CPC and BSA were examined. Furthermore, we investigated the possible sub-domains on BSA that bind CPC by displacement experiments. Ó 2012 Published by Elsevier B.V.

Introduction Chloramphenicol (CPC) (Scheme 1) also known as chlornitromycin, is effective against a wide variety of Gram-positive and ⇑ Corresponding authors. Tel.: +86 592 8387220; fax: +86 592 2185170. E-mail addresses: [email protected] (J. Zhang), [email protected] (B. Zeng). 1386-1425/$ - see front matter Ó 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.saa.2012.11.064

Gram-negative bacteria, including most anaerobic organisms. It is considered a prototypical broad-spectrum antibiotic, alongside the tetracyclines, and as it is both cheap and easy to manufacture. In the West, CPC is still widely used in topical preparations (oint-

J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 74–79

75

Procedures

Scheme 1. Molecular structure of CPC.

ments and eye drops) for the treatment of bacterial conjunctivitis. In low-income countries, CPC is still widely used because it is inexpensive and readily available [1–4]. Serum albumins (52–60% of the total amount of plasma proteins) are the major soluble protein constituents of the circulatory systems, and they have many physiological functions, such as combined with many endogenous and exogenous compounds. It also plays an important role in storage and transport of energy [5,6]. We have selected bovine serum albumin (BSA), for which association constants and binding sites are known for a number of compounds. BSA and human serum albumin (HSA) display approximately 76% sequence homology. The 3D structure of BSA is believed to be similar to that of HSA. BSA has two tryptophan residues (Trp 134 and Trp 212), located in sub-domains IA and IIA, respectively [7,8]. The ability and the state of drug binding is an ideal model to study the interaction between the biomacromolecules and small drug molecules. It also provides useful clinical information on the compatibility and use of drugs via the reaction mechanism and the molecular level [9,10]. In this article, the interaction between CPC and BSA has been investigated using fluorescence and UV–visible absorption spectroscopy. The quenching mechanism was analyzed by the Stern–Volmer equation. The binding constants, the binding sites, thermodynamic parameters, and binding distance were obtained. The possible sub-domains on BSA that bind CPC were investigated by displacement experiments. Furthermore, the conformational changes of BSA were explored by synchronous fluorescence spectroscopy and the effect of some common metal ions on the binding constant of CPC and BSA were also examined. Experimental Reagents BSA (P99%) was obtained from Jitian Bioengineering Co. (Shanghai, China) and was dissolved in a Tris–HCl (0.10 mol L1, pH = 7.4) buffer to form the BSA solution with a concentration of 1.00  105 mol L1. A Tris–HCl buffer (0.10 mol L1, pH = 7.4) containing 0.10 mol L1 NaCl was selected to keep the pH value constant and to maintain the ionic strength of the solution. CPC was obtained from Cangzhou guangming pharmaceutical Co., LTD. (Cangzhou, China), and the CPC (3.87  103 mol L1) solution was prepared in double-distilled water. All other reagents were of analytical grade and double-distilled water was used in the experiments. Apparatus Fluorescence measurements were conducted on a Shimadzu RF5301 fluorescence spectrophotometer (Tokyo, Japan) with a SB-11 water bath (Eyela) and 1.0 cm quartz cells. The excitation and emission slits were 5 and 10 nm, respectively. The synchronous fluorescence spectra were obtained by setting the excitation and emission wavelength interval (Dk) at 15 and 60 nm respectively. The absorption spectra were obtained from a Cary 5000 spectrophotometer (Varian, USA) and the pH measurement was carried out by a Leici pHS-2 digital pH-meter (Shanghai, China).

Fluorescence spectroscopy A 2.5 mL solution containing 1.00  105 mol L1 BSA was titrated by successive additions of 3.87  103 mol L1 CPC solution and the concentration of CPC varied from 0 to 13.9  105 mol L1. Titrations were done manually by using micro-injector. Fluorescence spectra were measured in the range of 280–600 nm at the excitation wavelength of 280 nm. The fluorescence spectra were performed at three temperatures (296 K, 303 K, and 310 K). The fluorescence spectra of displacement experiments for BSA were measured in the presence of warfarin sodium (the marker of site I) and ibuprofen (the marker of site II) at different concentrations of CPC. The concentrations of BSA, warfarin sodium and ibuprofen were kept at 1.00  105 mol L1, while the concentration of CPC was varied from 0 to 13.9  105 mol L1. The Fluorescence intensity was recorded in the range of 280–600 nm at 310 K. The fluorescence spectra of BSA were also recorded in the presence of some metal ions, which contain Ca2+, Ni2+, Mg2+, Fe2+, and Cu2+ at 310 K in the range of 280–600 nm at excitation wavelength of 280 nm. In the system, the overall concentration of BSA was fixed at 1.00  105 mol L1, and the common metal ion was maintained at 4.00  105 mol L1. UV–vis absorption spectra The absorption spectra of BSA in the presence of different concentrations of CPC were recorded in the range of 200–400 nm at room temperature. The concentration of BSA was kept at 1.00  105 mol L1, while that of CPC was varied from 0 to 3.10  105 mol L1. The UV–visible absorption spectra of CPC solution at 1.00  105 mol L1 concentration were measured in the range of 200–600 nm at 310 K. FT-IR spectra FI-IR measurements were carried out at room temperature on Perkin–Elmer FT-IR spectrometer (America) equipped with a germanium-attenuated total reflection (ATR) accessory, a DTGS KBr detector, and a KBr beam splitter. All spectra were taken via the attenuated total reflection (ATR) method with resolution of 4 cm1 and 60 scans. Spectra processing procedures: spectra of buffer solution were collected under the same conditions. Then, the absorbance of buffer solution from the spectra of sample solution was subtracted to get the FT-IR spectra of proteins. Results and discussion The mechanism of quenching of BSA fluorescence by CPC Fig. 1 shows the fluorescence quenching of Chloramphenicol (CPC) with Bovine serum albumin (BSA) at 310 K. It was observed that the fluorescence intensity of BSA decreased with the increasing concentration of CPC. With the addition of CPC, the fluorescence intensity of BSA shows significant decrease and there is a red shift (from 341 to 346 nm) of the maximum emission wavelength. These data indicates that CPC can interact with BSA and quench its intrinsic fluorescence [11,12]. Fluorescence quenching can proceed via different mechanisms, usually classified as dynamic quenching and static quenching. The dynamic quenching is the fluorescence substance collides with quencher brought on the quantum yield was lowered and the strength of fluorescence was weakened. The static quenching was initiated from the formation of a non-fluorescent compound, it was formed by quencher and fluorescence substance. Dynamic and static quenching can be distinguished by their different depen-

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UV–visible absorption measurement is a very simple method and applicable to explore the structural change and formation of a complex [22]. The absorption intensity of BSA increased at 277 nm with the increasing concentration of CPC. Furthermore, the absorption peak at 280 nm had an obvious blue shift (about 1.2 nm). It indicates that the fluorescence quenching of BSA is mainly caused by complex formation between BSA and CPC. In other words, it supports the conclusion that the static quenching exists in the interaction of BSA and CPC [23–25].

Fluorescence Intensity

200

150

a

100

Binding parameters and identification of the binding site between CPC and BSA

j 50

When small molecules are bound independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by the Eq. (3) [26,27]: 0 300

350

400

450

lg

500

Wavelength (nm) Fig. 1. The fluorescence quenching spectra of BSA by CPC at 310 K. kex = 280 nm; c(BSA) = 1.00  105 mol L1; c(CPC) (a–j): 0, 1.55, 3.10, 4.64, 6.19, 7.74, 9.29, 10.8, 12.4, and 13.9 (105 mol L1).

dence on temperature of binding constants and viscosity, or preferably by lifetime measurements. The quenching constants decrease with increasing temperature for static quenching, while the reverse effect is for dynamic quenching. Assuming that the process belongs to dynamic quenching, we analyzed the fluorescence data at different temperatures with the well-known Stern–Volmer Eq. (1) [13–17]:

F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q  F

ð1Þ

where F and F0 are the fluorescence intensity of BSA in the presence of and in the absence of the quencher, respectively; Ksv is the dynamic quenching constant; Kq is the quenching rate constant, Kq = Ksv/s0; s0 is the average lifetime of the molecule without CPC (108 s) [18,19]; [Q] is the concentration of CPC. The Stern–Volmer plots of the quenching of BSA fluorescence by CPC at three different temperatures are displayed in Fig. 2A and B. From Fig. 2, we observed that a combined quenching process (dynamic and static quenching) occurs at low CPC concentration, but a dynamic quenching process at high CPC concentration. The values of KSV and Kq are illustrated in Table 1. All we known that fluorophore can be quenched with the same quencher by both colliding and forming complex (combined quenching process). In this case, F0/F versus [Q] is described by the following modified form of the Stern–Volmer Eq. (2) [20]:

F0 ¼ ð1 þ K D ½Q Þð1 þ K S ½QÞ ¼ 1 þ ðK D þ K S Þ½Q  þ K D K S ½Q 2 F

ð2Þ

where KS and KD are the static and dynamic quenching constants, respectively. It is second order in [Q] and thus leads to upward curvy plots of F0/F versus [Q] at lower [Q] arising from a combined quenching (both dynamic and static) process. Furthermore, Kq between BSA and CPC are all greater than 2.0  1010 L mol1 S1, it suggest that there is a static quenching process between CPC and BSA [21]. In a word, the fluorescence quenching of BSA by CPC is initiated by a combined quenching (dynamic and static quenching). UV–visible absorption studies The absorption spectra of BSA in the presence and absence of CPC were recorded and presented in Fig. 3. As all we known that

ðF 0  FÞ ¼ lgK a þ nlg½Q  F

ð3Þ

where Ka is the binding constant of CPC with BSA, and n is the number of binding sites per BSA molecule. Thus, Ka and n can be determined from the slope and the intercept of the regression curve (Fig. 4 and Table 2). The values of Ka for the association of CPC with BSA decreased with the rising temperature, it indicates the formation of an unstable compound, while partly decomposition at relatively higher temperatures [28]. What’s more, the value of n roughly equal to 1 indicates just one association site exist between CPC and BSA. Competitive displacement experiments were carried out using site-specific probes, warfarin sodium and ibuprofen by fluorescence quenching method. It has been found that warfarin sodium mostly located in site I (subdomain IIA) on the BSA, ibuprofen possesses one high affinity sites in site II (subdomain IIIA) [29]. In order to ensure the binding site of CPC to BSA, we used warfarin sodium and ibuprofen as two probes by adding them to BSA–CPC system, respectively. Table 3 displays the Ka calculated from the probe fluorescence spectrometry in the presence of competition drugs (warfarin sodium and ibuprofen). As we can see, Ka was reduced by 36.3% in the presence of warfarin sodium, and reduced by 8.86% in the presence of ibuprofen. It is evident that CPC does bind at the region of site I (subdomain IIA) and is buried in this hydrophobic pocket [30,31]. Thermodynamic parameters and nature of the binding forces As all we known that the acting forces between a small molecule and macromolecule mainly include hydrogen bond, electrostatic force, van der Waals force and hydrophobic interaction force. The binding studies are carried out at three different temperatures (296 K, 303 K, and 310 K). The thermodynamic parameters, free energy (DG), enthalpy (DH) and entropy (DS) of interaction are important to interpret the binding mode. The binding parameters of BSA–CPC complex were calculated from the Van’t Hoff Eqs. (4) and (5) as follows [32]:

ln K a ¼

DH DS þ RT T

DG ¼ DH  T DS ¼ RT ln K a

ð4Þ ð5Þ

where Ka is the binding constant at the corresponding temperature, R is the gas constant, T is the experimental temperature. The thermodynamic parameters are listed in Table 4. From Table 4, The values ofDG , DH, and DS indicate that hydrogen bonds and van der Waals forces played a major role in the reaction between CPC and BSA. While the negative sign for DG indicates the spontaneity of the binding for CPC with BSA [33,34].

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A

B 6.0

296K 303K 310K

296K 303K 310K

5.6

4.5

F0/F

F0/F

4.8

4.0

3.0

3.2 1.5

2.4 0

3

6

CPC

9

12

15

8

-5

×10 mol/L

10

CPC

12

14

-5

×10 mol/L

Fig. 2. Stern–Volmer plots of BSA (1.00  105 mol L1) quenched CPC at three different temperatures (w, 296 K;d, 303 K;N, 310 K); (A) c(CPC): 1.55, 3.10, 4.64, 6.19, 7.74, 9.29, 10.8, 12.4, and 13.9 (105 mol L1); and (B) c(CPC): 7.74, 9.29, 10.8, 12.4, and 13.9 (105 mol L1).

Table 1 The quenching constants of BSA by CPC at different temperatures.

0.8

KSV (Lmol1)

Kq (Lmol1 s1)

R

296 303 310

2.07  104 2.19  104 2.34  104

2.07  1012 2.19  1012 2.34  1012

0.9988 0.9987 0.9951

1.2

0.4

Absorbance

0.3

e 0.9

Absorbance

310K 303K 296K

lg F0-F /F

T (K)

0.0

f

0.2

-0.4 0.1

0.0 210

245

280

315

350

-0.8

Wavelength (nm)

0.6

-4.75

-4.50

-4.25

-4.00

-3.75

lg CPC

a

Fig. 4. A plot of lg[(F0  F)/F] versus lg[CPC] at three different temperatures.

0.3 Table 2 The binding constants and the number of binding sites of BSA with CPC.

240

270

300

330

Wavelength (nm) Fig. 3. UV–visible absorption spectra of BSA in the presence of different concentrations of CPC. (a–e): c(BSA) = 1.00  105 mol L1; c(CPC) = 0.00, 6.20, 12.4, 18.6, and 24.8 (105 mol L1); and (f): c(BSA) = 0, c(CPC) = 1.00  105 mol L1.

T (K) 296 303 310

Ka (L mol1) 5

5.26  10 4.14  105 3.50  105

n

R

1.34 1.30 1.29

0.9925 0.9905 0.9972

Energy transfer between CPC and BSA FRET has been used as a ‘‘spectroscopic ruler’’ for measuring molecular distances in biological and macromolecular systems. It takes place when the fluorescence emission band of one molecule (donor) overlaps with an excitation band of a second (acceptor) that is within 2–8 nm [35,36]. The overlap of the fluorescence emission spectra of BSA and the absorption spectra of CPC at

Table 3 Effects of site I and site II ligands on the interaction of BSA and CPC at 310 K. System

Ka (L mol1)

n

R

CPC CPC + warfarin sodium CPC + ibuprofen

3.50  105 4.77  105 3.19  105

1.29 1.32 1.28

0.9972 0.9974 0.9964

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Table 4 The thermodynamic parameters of the interaction between BSA and CPC.

E¼1

T (K)

DH (kJ mol1)

DG (kJ mol1)

DS (J mol1 K1)

296 303 310

195.5

32.42 32.59 32.91

550.9 537.6 524.4

ð7Þ

F0 are F mean the fluorescence intensity of BSA in the absence and in the presence of quencher, respectively; where r is the distance from the ligand to the tryptophan residue of the protein, and R0 is the Förster critical distance at which 50% of the excitation energy is transferred to the acceptor. 0.60

200

  F R6 ¼ 6 0 F0 R0 þ r6

R60 ¼ 8:8  1025 K 2 N4 uJ

ð8Þ

2

K is a factor describing the relative orientation in space of the transition dipoles of donor and acceptor, N is the refractive index of the medium, u is the fluorescence quantum of the donor in the absence of acceptor, J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. K2 is 2/3 for two albumin solutions and N is 1.336 for HSA and 1.360 for BSA, u is 0.118 and 0.150 for HSA and BSA, respectively [40]. Combining the data with the equations, we calculated J = 7.8947  1015 cm3 L mol1, E = 0.18, R0 = 2.36 nm, and r = 3.02 nm. The value of r is within 2–8 nm, and 0.5 R0 < r < 1.5 R0, which indicates that the non-radiative energy transfer exists between CPC and BSA [41–43].

150

0.45

100

0.30

50

Absorbance

Fluorescence Intensity

a

0.15

b Conformation investigation 0 300

350

400

The synchronous fluorescence spectra were measured, shown in Fig. 6. In the synchronous spectra, the sensitivity associated with fluorescence is maintained while offering several advantages: spectral simplification, spectral bandwidth reduction and avoidance of different perturbing effects. It shows the tyrosine residues (Tyr) and tryptophan residues (Trp) of BSA when the wavelength interval Dk is 15 nm and Dk is 60 nm, respectively [44,45]. It is obvious that the emission strengths of both tyrosine and tryptophan decreased. The shift of the maximum of emission wavelength from 304 to 309 nm when Dk = 15 nm, which is consistent with the fact that the conformation of BSA was changed and the polarity around the tyrosine residue increases and the hydrophobicity decreases, while the tryptophan residues does not show significant shift. The conclusion agrees with the result of conformational changes by UV–visible spectra. What’s more, it can be seen from Fig. 6 that a stronger fluorescence quenching effect of tyrosine res-

0.00 500

450

Wavelength (nm) Fig. 5. The overlap of fluorescence spectrum of BSA (a) and absorption spectrum of CPC (b). c(BSA) = c(CPC) = 1.00  105 mol L1; T = 310 K.

310 K is shown in Fig. 5. The efficiency of energy transfer E and the distance between the acceptor and donor r can be defined as the following Eqs. (6)–(8) [37–39]:

FðkÞeðkÞk4 Dk J¼ P FðkÞDk

ð6Þ

F(k) is the fluorescence intensity of the fluorescence donor in the wavelength range k to k + Dk, and e(k) is the molar absorbance of the acceptor at wavelength k.

B 30

200

24

160

Fluorescence Intensity

Fluorescence Intensity

A

a 18

12

j 6

a 120

80

j

40

0 280

300

320

Wavelength (nm)

340

360

0 300

325

350

375

400

Wavelength (nm)

Fig. 6. The synchronous fluorescence spectra of BSA. (A) Dk = 15 nm, and (B) Dk = 60 nm; T = 310 K; c(BSA) = 1.00  105 mol/L; c(CPC) (a–j): 0, 1.55, 3.10, 4.64, 6.19, 7.74, 9.29, 10.8 ,12.4, and 13.9 (105 mol L1).

J. Zhang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 74–79 Table 5 Effects of common metal ions on the binding constants of BSA and CPC at 310 K. System

Ka (105 L mol1)

n

R

BSA + CPC BSA + CPC + Ca2+ BSA + CPC + Ni2+ BSA + CPC + Mg2+ BSA + CPC + Fe2+ BSA + CPC + Cu2+

3.50 6.12 4.74 4.13 3.43 1.55

1.29 1.34 1.32 1.31 1.28 1.20

0.9972 0.9977 0.9960 0.9918 0.9962 0.9902

idues compared with the tryptophan residues after CPC are added. It indicates that the binding site of CPC is nearer to tyrosine than that of the tryptophan residues [46–48]. Fourier Translation Infrared absorption spectroscopy (FT-IR) is helpful in the characterization in a general way of the structure of proteins. It can provide valuable information on their conformational changes in aqueous solution. The protein amide I band (1600–1700 cm1, mainly C@O stretch) and amide II band (1548 cm1, CAN stretch coupled with NAH bending mode) both have a relationship with the secondary structure of the protein [49–51]. In this case, BSA in the absence of CPC shows the amide II peak at 1535.5 cm1 and in the presence of CPC the amide II peak was shifted to 1546.6 cm1, while amide I peak at 1654.7 cm1 and show no significant shift (from 1654.7 cm1 to 1654.9 cm1). From the shift in peak position we reconfirmed that the conformation of BSA has been affected by the addition of CPC [52]. The effect of other ions the binding constant In plasma, there are some inorganic ions, which can affect the reactions of drugs with the serum albumins [53,54]. The previous studies demonstrated that CPC can interact with some metal ions. The effects of some common metal ions (Ca2+, Ni2+, Mg2+, Fe2+, and Cu2+) on the binding were investigated at 310 K. The results are shown in Table 5. The CPC–BSA binding constant decreases in the presence of Fe2+ and Cu2+, while increases in the presence of Ca2+, Ni2+, and Mg2+. In a word, the presence of common ions may shorten or prolong the storage time of CPC in blood plasma and enhance or weaken its maximum effects [55]. Conclusions In this paper, the fluorescence and UV–visible spectrometry were applied to investigate the interaction of CPC and BSA. The results show that the fluorescence quenching mechanism a combined quenching process (dynamic and static quenching) occurs at low CPC concentration, but a dynamic quenching process at high CPC concentration. The values of thermodynamic parameters indicate that CPC binds to BSA mainly by hydrogen bonds and van der Waals interactions. The binding distance (r = 3.02 nm) suggests that there is a non-radioactive energy transfer between BSA and CPC. The synchronous fluorescence spectroscopy and FT-IR indicates that the conformation of BSA is changed in the presence of CPC. In addition, the binding constant between CPC and BSA increases in the presence of Ca2+, Ni2+, and Mg2+, decreases in the presence of Fe2+ and Cu2+. Competitive binding experiments with warfarin sodium and ibuprofen indicated the binding site was located in a hydrophobic cavity near Drug sites I (subdomain IIA). This work has provided insight that will guide development of future pharmacological applications of chloroamphenicol. Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No. 51103122), Natural Science Foundation

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