Spectroscopic studies on the interaction of Congo Red with bovine serum albumin

Spectrochimica Acta Part A 72 (2009) 907–914

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Spectroscopic studies on the interaction of Congo Red with bovine serum albumin Ye-Zhong Zhang a , Xia Xiang b , Ping Mei a , Jie Dai a , Lin-Lin Zhang a , Yi Liu a,b,∗ a b

Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023, PR China State Key Laboratory of Virology, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, PR China

a r t i c l e

i n f o

Article history: Received 7 August 2008 Received in revised form 4 December 2008 Accepted 9 December 2008 Keywords: Bovine serum albumin Congo Red Fluorescence quenching Binding site Circular dichroism (CD) Three-dimensional fluorescence spectra

a b s t r a c t The binding interaction of Congo Red (CGR) with bovine serum albumin (BSA) was investigated by spectroscopic techniques including fluorescence spectroscopy, UV–vis absorption, and circular dichroism (CD) spectroscopy under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by CGR was the result of the formation of a BSA–CGR complex, and the corresponding binding constants (Ka ) at the four different temperatures (292, 298, 304, and 310 K) were obtained according to the modified Stern–Volmer equation. The thermodynamic parameters H and S were calculated to be −12.67 kJ mol−1 and 58.60 J mol−1 K−1 , respectively, which suggested that both hydrophobic force and hydrogen bond played major roles in stabilizing the BSA–CGR complex. Site marker competitive experiments showed that the binding of CGR to BSA primarily took place in site I of BSA. The distance r between CGR (acceptor) and tryptophan residues of BSA (donor) was calculated to be 3.89 nm based on Förster’s non-radioactive energy transfer theory. The conformational investigation showed that the presence of CGR resulted in the change of BSA secondary structure and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Serum albumin (SA), as the most abundant protein constituent of blood plasma, facilitates the disposition and transportation of various endogenous and exogenous ligands including fatty acids, steroids, metal ions, etc. [1]. It is responsible for the maintenance of blood pH [2] and the contribution of colloid osmotic blood pressure. The absorption, distribution, metabolism, excretion properties as well as the stability and toxicity of fatty acids and dyes can be significantly affected as a result of their binding to serum albumins [3]. Moreover, there is evidence of conformational changes of serum albumin induced by its interaction with low molecular weight dyes and drugs, which appears to affect the secondary and tertiary structure of albumins [4]. Consequently, the investigation on the interaction of dyes and drugs with serum albumin is of great importance. Many industries generate coloured effluents containing various dyes and pigments and discharge the same to natural water bodies [5]. Among these dyes and pigments, many are toxic and

∗ Corresponding author at: Department of Chemistry, College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou, Hubei 434023, PR China. Tel.: +86 27 68756667; fax: +86 27 68754067. E-mail address: [email protected] (Y. Liu). 1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2008.12.007

have carcinogenic and mutagenic effects that affect aquatic biota and also humans [6–8]. In this present work, Congo Red (CGR) [1-naphthalenesulfonic acid, 3,3 -(4,4 -biphenylenebis (azo)) bis(4amino-)disodium salt] is chosen as the typical dye due to its chemical composition and environmental concern. The molecular structure of CGR is illustrated in Fig. 1. Effluent containing CGR is produced from textiles, printing, dyeing, paper, and plastic industries [9,10]. Due to the complex aromatic structure of CGR, it has the property of physicochemical, thermal and optical stability, and resistance to biodegradation and photodegradation. Yet, this anionic dye can be metabolised to benzidine, a known human carcinogen [11]. As the particular interaction mechanism between CGR and protein and the impact of CGR on the conformation of protein are still unknown, efforts are made in this paper to study these aspects in detail. In this work, BSA was selected as our protein because of its medical importance, stability, and unusual ligand-binding properties [12,13]. Studies direct toward the siteand conformation-specific reagents with protein enable chemists to extensively study the ability of these complexes to act as probes and thereby providing some scientific information at life science and clinical medicine fields. Fluorescence spectroscopy is a powerful tool for the study of the reactivity of chemical and biological systems since it allows nonintrusive measurements of substances in low concentration under physiological conditions [14]. Because of its high sensitivity, rapid-

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length was set at 200 nm with an increment of 5 nm, the number of scanning curves was 31, and the other scanning parameters were just the same as those of the fluorescence emission spectra. 2.3. Site marker competitive experiments

Fig. 1. Molecular structure of Congo Red.

ity, and simpleness [15], fluorescence technique has been widely used for drug–protein studies [16–18]. In this paper, the interaction between BSA and CGR was studied by spectroscopy including fluorescence, UV–vis absorption and circular dichroism (CD). Great attempts were made to explore the interaction mechanism, the specific binding site and the effect of CGR on the conformational changes of BSA. We hope this work can not only provide useful information for appropriately understanding the toxicological action of active components in dyes, but also illustrate its binding mechanisms at a molecular level. 2. Materials and methods 2.1. Materials BSA (fatty acid free and electrophoresis grade reagents) and Congo Red were purchased from Sigma Chemical Co. (USA). Stock solution of BSA was prepared by dissolving it in Tris–HCl buffer solution (0.05 mol L−1 Tris base (2-amino-2-(hydroxymethyl)-1,3propanediol), 0.10 mol L−1 NaCl, pH 7.4) with the final concentration of 2.0 × 10−6 mol L−1 and was kept in dark at 277 K. The Tris–base had a purity of no less than 99.5% and NaCl, HCl, etc., were all of analytical purity. The stock solution of CGR was prepared by dissolving it in doubly distilled water. Warfarin and ibuprofen (analytical purity) were obtained from Medicine Co. Ltd., Jiangshu (China), prepared by the Tris–HCl buffer (pH 7.4) to form 1.0 × 10−3 mol L−1 solution. Other chemicals were all of analytical grade and doubly distilled water was used throughout the experiment. 2.2. Fluorescence measurements All fluorescence spectra were measured on an LS-55 fluorophotometer (Perkin–Elmer Co., USA) equipped with a 1.0 cm quartz cell and a thermostat bath. In a typical fluorescence measurement, 2.0 mL BSA solution with the concentration of 2.0 × 10−6 mol L−1 was added accurately into the quartz cell, and then was titrated by successive additions of 4.0 × 10−4 mol L−1 CGR using a 2 ␮L trace syringe to attain a series of final concentrations. Titrations operated manually and mixed moderately. The fluorescence emission spectra were measured at 292, 298, 304, and 310 K with the width of the excitation and emission slit adjusted at 15.0 and 4.0 nm, respectively. An excitation wavelength of 285 nm was chosen and the emission wavelength was recorded from 300 to 450 nm. Very dilute solutions were used in the experiment (BSA 2.0 × 10−6 mol L−1 , CGR in the range of 0–4.0 × 10−6 mol L−1 ) to avoid inner filter effect. The synchronous fluorescence spectra were obtained by simultaneously scanning the excitation and emission monochromators. It was recorded at  = 15 and 60 nm in the absence and presence of various amounts of CGR over a wavelength range of 240–350 nm. The appropriate blanks corresponding to the buffer were subtracted to correct background of fluorescence. The three-dimensional fluorescence spectra were measured under the following conditions: the emission wavelength was recorded between 200 and 500 nm, the initial excitation wave-

Binding location studies between CGR and BSA in the presence of two site markers (warfarin and ibuprofen) were measured using the fluorescence titration methods. The concentrations of BSA and warfarin/ibuprofen were all stabilized at 2.0 × 10−6 mol L−1 . CGR was then gradually added to the BSA–warfarin or BSA–ibuprofen mixtures. An excitation wavelength of 285 nm was selected and the fluorescence spectra were recorded over a wavelength range of 300–475 nm. 2.4. UV–vis absorption and circular dichroism spectra The UV–vis absorption spectra of BSA in the presence and absence of CGR were carried out on a TU-1901 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells. The spectra were recorded over a wavelength range of 320–200 nm. Circular dichroism spectra were measured on a J–810 Spectropolarimeter (Jasco, Tokyo, Japan) at room temperature under constant nitrogen flush over a wavelength range of 260–200 nm. The instrument was controlled by Jasco’s Spectra ManagerTM software and the scanning speed was set at 200 nm min−1 . A quartz cell having path length of 0.1 cm was used and each spectrum was the average of three successive scans. Appropriate buffer solution running under the same conditions were taken as blank and subtracted from the experimental spectra. The concentration of BSA was kept at 2.0 × 10−6 mol L−1 and the molar ratio of BSA to CGR was varied as 1:0, 1:1, 1:3 and 1:6. The CD results were expressed as mean residue ellipticity (MRE) in deg cm2 dmol−1 which is defined as [19]: MRE =  obs /(10 × n × l × Cp ), where  obs is the CD in millidegree, n is the number of amino acid residues (585), l is the path length of the cell, and Cp is the mole fraction. The ␣-helix content was then calculated from the MRE values at 208 nm using the following equation [20]: ␣-helix (%) = {(−MRE208 –4000)/(33,000–4000)} × 100; where MRE208 is the observed MRE at 208 nm, 4000 is the MRE value of the ␤-form and random coil conformation cross at 208 nm, and 33,000 is the MRE value of a pure ␣-helix at 208 nm. 3. Results and discussion 3.1. Interactions between CGR and BSA Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions, such as excited-state reactions, energy transfer, ground-state complex formation and collisional quenching [14]. By measuring the intrinsic fluorescence intensity of protein before and after the addition of a drug, some changes of molecular microenvironment may be provided in the vicinity of fluorophore. Fig. 2 shows the fluorescence emission spectra of BSA in the presence of various concentrations of CGR at 298 K. When different amount CGR was titrated into a fixed concentration of BSA, the fluorescence intensity of BSA at around 350 nm decreased regularly but the emission maximum did not move to shorter or longer wavelength. These results indicated that CGR could interact with BSA and quench its intrinsic fluorescence, but there was no change in the local dielectric environment of BSA. The inset in Fig. 2 corresponds to the Stern–Volmer plot at 298K. It shows that within the investigated concentrations range, the Stern–Volmer plot exhibits a good linear relationship.

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Fig. 2. Emission spectra of BSA in the presence of various concentrations of CGR (T = 298 K and ex = 285 nm). c(BSA) = 2.0 × 10−6 mol L−1 ; c(CGR) (×10−6 mol L−1 ), A–K: 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, respectively. Curve L shows the emission spectrum of CGR only, c(CGR) = 2.0 × 10−6 mol L−1 . The insert corresponds to the Stern–Volmer plot.

Fluorescence quenching mechanisms are usually classified as either dynamic or static quenching, which can be distinguished by their different dependence on temperature or viscosity, or preferably by lifetime measurements [21,22]. For static quenching, the quenching constants decrease with the increased temperature, while the reverse effect is observed for the case of dynamic quenching [23]. In order to figure out which mechanism play a dominant role in the interaction, the fluorescence quenching process is firstly assumed to be a dynamic mechanism. For dynamic quenching, the decrease in intensity is usually analyzed using the Stern–Volmer equation [22]: F0 = 1 + KSV [Q ] = 1 + kq 0 [Q ] F

(1)

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively; KSV is the Stern–Volmer quenching constant; [Q] is the concentration of the quencher;  0 is the average fluorescence lifetime of bimolecular and equal to 10−8 s [24]; kq , which is equal to KSV / 0 , is the apparent bimolecular quenching rate constant. For dynamic quenching, the maximum scattering collisional quenching constant of various quenchers is 2.0 × 1010 L mol−1 s−1 [25]. Table 1 presents the calculated KSV and kq at each temperature studied. The results show that the Stern–Volmer quenching constants KSV decreased with the increase of temperature and the values of kq were much larger than 2.0 × 1010 L mol−1 s−1 . As a result, the probable quenching mechanism of the intrinsic fluorescence of BSA was not initiated by a dynamic process, but resulted from a complex formation between BSA and CGR. Meanwhile, it was found that the values of KSV were all great; the reason might be that the fluorescent quantum yield of BSA increased or a strong binding existed between BSA and CGR [26].

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Fig. 3. UV–vis absorption spectra of CGR, BSA, and BSA–CGR solutions; c(BSA) = c(CGR) = 2.0 × 10−6 mol L−1 . (A) The absorption spectrum of BSA only; (B) the absorption spectrum of CGR only; (C) the difference absorption spectrum between CGR–BSA and CGR at the same concentration.

The UV–vis absorption spectra of CGR, BSA, and BSA–CGR (subtracting the corresponding spectrum of CGR in the buffer) solutions were also measured to confirm the quenching mechanism. As shown in Fig. 3, the absorption intensity of BSA decreased obviously at around 224 nm with the addition of CGR. It indicates that the fluorescence quenching of BSA was mainly cased by complex formation between BSA and CGR [27]. Therefore, the fluorescence quenching of BSA by CGR should be analyzed using the modified Stern–Volmer equation [28]: F0 F0 1 1 1 = + = F0 − F F fa Ka [Q ] fa

(2)

In this case, fa is the fraction of accessible fluorescence, and Ka is the effective quenching constant for the accessible fluorophores. F0 /F is linear with the reciprocal value of the quencher concentration [Q], with slope equal to the value of (fa Ka )−1 . Fig. 4 shows the modified Stern–Volmer plots at the four different temperatures, and the corresponding values of Ka are presented in Table 2. The decreasing trend of Ka with increasing temperature is in accordance with KSV ’s dependence on temperature, which coincides with the static type of quenching mechanism [29]. Besides, we can find that the binding constants between CGR and BSA are great and the effect of temperature is small. Thus, CGR can be stored and carried by protein in the body, while it would be harmful to human beings if released.

Table 1 Stern–Volmer quenching constants for the interaction of CGR with BSA at different temperatures. pH

T (K)

KSV (×105 L mol−1 )

kq (×1013 L mol−1 s−1 )

Ra

S.D.b

7.4

292 298 304 310

1.996 1.928 1.831 1.797

1.996 1.928 1.831 1.797

0.9985 0.9994 0.9991 0.9997

0.016 0.010 0.011 0.007

a b

The correlation coefficient. The standard deviation for the KSV values.

Fig. 4. The modified Stern–Volmer plots for the CGR–BSA system at the four different temperatures, pH 7.4.

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Table 2 Modified Stern–Volmer association constants Ka and relative thermodynamic parameters of the CGR–BSA system. T (K)

Ka (×105 L mol−1 )

Ra

292 298 304 310

2.126 1.947 1.717 1.583

0.9995 0.9998 0.9999 0.9998

a b

H␪ (kJ mol−1 )

G␪ (kJ mol−1 ) −29.78 −30.17 −30.46 −30.86

−12.67

S␪ (J mol−1 K−1 )

Rb

58.60

0.9967

The correlation coefficient for the Ka values. The correlation coefficient for the van’t Hoff plot.

3.2. Determination of the force acting between CGR and BSA Essentially, the interaction forces between ligands and biological macromolecules may include hydrophobic force, multiple hydrogen bond, van der Waals force and electrostatic interactions, etc. [30]. The signs and magnitudes of the thermodynamic parameters (H and S) can account for the main forces involved in the binding process. For this reason, the temperature-dependent binding constant was studied. The temperatures chosen were 292, 298, 304 and 310 K, thus BSA would not undergo any structural degradation. If the enthalpy change (H) does not vary significantly in the temperature range studied, both the enthalpy change (H) and entropy change (S) can be evaluated from the van’t Hoff equation: ln K = −

H S + RT R

(3)

where K is analogous to the effective quenching constants Ka at the corresponding temperature and R is the gas constant. It can be seen from Fig. 5 that there was a good linear relationship between ln K and 1/T. The enthalpy change (H) could be calculated from the slope of the van’t Hoff plot. The free energy change (G) was then estimated from the following relationship: G = H − T S = −RT ln K

(4)

Table 2 shows the values of H and S obtained from the slopes and ordinates at the origin of the fitted lines. As can be found from the values of H (−12.67 kJ mol−1 ) and S (58.60 J mol−1 K−1 ) that the binding process was mainly driven by the entropy change, the enthalpy change has little contribution. The negative value of G revealed the binding process was spontaneous. Ross and Subramanian [31] have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interaction that may take place in protein association process. From the point of view of water structure, a positive S value is frequently taken as evidence for a hydrophobic interaction because the water molecules are arranged in an orderly fashion

Fig. 5. Van’t Hoff plot for the interaction between BSA and CGR in Tris–buffer, pH 7.4.

around the drug and protein acquires a more random configuration [32]. A negative H value is frequently taken as evidence for hydrogen bond in the binding interaction [33], and from the structure of CGR, H-bond can be formed in the nitrogen atom. And the negative value of H (–12.67 kJ mol−1 ) observed in this experiment cannot be attributed to electrostatic interactions, since the value of H is very small [33]. Thus, from the thermodynamic characteristics summarized above, hydrophobic forces and hydrogen bond played major roles in the CGR–BSA binding reaction and contributed to the stability of the complex. 3.3. Number of binding sites and identification of the binding location of CGR on BSA As discussed above, CGR-induced fluorescence quenching of BSA was a static quenching process. Florescence quenching data of BSA were analyzed to obtain various binding parameters. The binding constant (Kb ) and the number of binding sites (n) can be calculated according to the equation [34]: log

F0 − F = log Kb + n log[Q ] F

(5)

where F0 and F are the fluorescence intensity without and with the ligand, respectively. A plot of log [(F0 − F)/F] vs. log [Q] gave a straight line using least squares analysis whose slope was equal to n (binding sites) and the intercept on Y-axis to log Kb (Kb equal to the binding constant). From Eq. (5), the values of Kb and n at 298 K were obtained to be 2.098 × 105 L mol−1 and 1.008 respectively, which implied that CGR bound strongly to BSA and there was one independent class of binding sites for CGR towards BSA. The linear coefficient R (0.9996) indicated that the assumptions underlying the derivation of Eq. (5) were satisfactory. Crystal structure of BSA shows that BSA is a heart-shaped helical monomer composed of three homologous domains named I–III, and each domain includes two sub-domains called A and B to form a cylinder [35]. There are two tryptophan residues (Trp134 and Trp212) in BSA: the former was embedded in the first sub-domain IB and was more exposed to a hydrophilic environment, while the latter was embedded in sub-domain IIA and deeply buried in the hydrophobic loop [36,37]. According to Refs. [38,39], the principal regions of ligands bound to BSA are usually located in hydrophobic cavities in sub-domains IIA and IIIA, and the binging cavities associated with sub-domains IIA and IIIA are also referred to as sites I and II. As the data in the preceding discussion did not allow us to give the precise binding location of CGR on BSA, the site marker competitive experiments were then carried out, using drugs which specifically bind to a known site or region on BSA. As described in the literature, warfarin has been demonstrated to bind to the sub-domain IIA while ibuprofen is considered as sub-domain IIIA binder [40]. By monitoring the changes in the fluorescence intensity of CGR bound BSA that brought about by site I (warfarin) and site II (ibuprofen) markers, information about the specific binding site of CGR in BSA can be gained (Fig. 6). During the experiment, CGR was gradually added to the solution of BSA with site markers held in equimolar concentrations (2.0 × 10−6 mol L−1 ). As shown in Fig. 6(A), with addition of warfarin

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Fig. 7. Modified Stern–Volmer plots for the CGR–BSA system in the absence and presence of site markers (T = 298 K and pH 7.4).

3.4. Energy transfer between CGR and BSA The Förster’s non-radioactive energy transfer theory is often used to determine the drug binding site distance between the site and the amino acid residues [41]. The extent of energy transfer depends upon the extent of overlap and the distance between these molecules [40]. The efficiency of energy transfer between the donor and acceptor, E, could be calculated using the equation [42]: R6 F = 6 0 F0 R0 + r 6

E =1−

Fig. 6. Effect of selected site markets on the fluorescence of CGR bound BSA (T = 298 K and ex = 285 nm). (A) c(BSA) = c(ibuprofen) = 2.0 × 10−6 mol L−1 ; (B) c(BSA) = c(warfarin) = 2.0 × 10−6 mol L−1 ; c(CGR) (×10−6 mol L−1 ), A–K: 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, respectively. The inserts correspond to the molecular structures of site markets.

to the BSA solution, the maximum emission wavelength of BSA had a slight red shift (from 352 to 354 nm) and the fluorescence intensity was significantly lower than that of without warfarin. Then, with the continuing addition of CGR into the above system, the fluorescence intensity of the BSA solution with warfarin held in equimolar decreased gradually, and the intensity was much lower than that of without warfarin (Fig. 2), indicating that the binding of CGR to BSA was affected by adding warfarin. By contrast, in the presence of ibuprofen, the fluorescence intensity of the CGR–BSA system almost had no difference from that of without ibuprofen in the same condition (Figs. 2 and 6B), indicating that the site II marker ibuprofen did not prevent the binding of CGR in its usual binding location. In order to compare the effect of warfarin and ibuprofen on the binding of CGR to BSA, the fluorescence quenching data of the Co(phen)3 2+ –BSA system with the presence of site markers were also analyzed using the modified Stern–Volmer equation, as shown in Fig. 7. The binding constants of the systems, which can be calculated from the slope values of the plots, were listed in Table 3. Obviously, the Ka values of the system with warfarin were almost 66% of that without warfarin, while the constants of the systems with and without ibuprofen had only a small difference. It indicated that there was a significant competition between CGR and warfarin, while ibuprofen had only a small influence on the binding of CGR to BSA. The above experimental results and analysis demonstrated that the binding of CGR to BSA mainly located within site I (sub-domain IIA).

(6)

where r represents the distance between the donor and the acceptor, R0 is the critical distance at which transfer efficiency equals to 50%. The value of R0 is calculated using the following equation [43]: R06 = 8.79 × 10−25 K 2 n−4 J

(7)

where K2 is the orientation factor related to the geometry of the donor–acceptor dipole, n is the refractive index of medium,  is the fluorescence quantum yield of the donor, and J expresses the degree of spectral overlap between the donor emission and the acceptor absorption (Fig. 8), which could be calculated by the following equation [43]:

∞

J=

0

F()ε()4 d

∞ 0

(8)

F() d

where F() is the fluorescence intensity of the donor at wavelength range , ε() is the molar absorption coefficient of the acceptor at wavelength . The overlap integral, J, can be evaluated by integrating the spectra in Fig. 8 according to Eq. (8). In the present case, K2 = 2/3, n = 1.36 and  = 0.15 [44]. According to Eqs. (6)–(8), we could calculate that J = 4.582 × 10−14 cm3 L mol−1 , R0 = 3.298 nm, E = 0.270, and r = 3.89 nm. The donor-to-acceptor distance r on the 2–8 nm scale indicated that the energy transfer took place between BSA and CGR with great possibility [45,46]. Table 3 The binding constants of competitive experiments of CGR–BSA system. Site market

Ka (×105 L mol−1 )

Ra

S.D.b

Blank Ibuprofen Warfarin

1.947 1.879 1.280

0.9998 0.9998 0.9998

0.065 0.078 0.097

a b

The correlation coefficient. The standard deviation.

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Fig. 8. Spectral overlap of UV–vis absorption spectrum of CGR (A) with the fluorescence emission spectrum of BSA (B), c(BSA) = c(CGR) = 2.0 × 10−6 mol L−1 , T = 298 K.

3.5. Conformation investigations 3.5.1. Synchronous fluorescence spectroscopy studies Synchronous fluorescence spectroscopy is a kind of simple and effective means to measure the fluorescence quenching and can provide some information about the change of the molecular micro-environment. As the possible shift of the maximum emission wavelength max is related to the alteration of the polarity around the chromophore micro-environment [47], , representing the value of difference between excitation and emis-

Fig. 10. The CD spectra of the CGR–BSA system obtained at room temperature and pH 7.4; c(BSA) = 2.0 × 10−6 mol L−1 ; c(CGR) (×10−6 mol L−1 ), A–D: 0, 2.0, 6.0, 12.0, respectively.

sion wavelength, is an importance operating parameter. When the values of  are stabilized at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine and tryptophan residues, respectively [48]. The synchronous fluorescence spectra of BSA–CGR system are shown in Fig. 9. It can be seen from Fig. 9(A) that the maximum emission wavelength has a slight blue shift (from 296.15 to 291.5 nm) at the investigated concentrations range when  equals to 15 nm. The blue shift effect expressed that the conformation of BSA was changed and it suggested a less polar (or more hydrophobic) environment of tyrosine residue [49]. While in Fig. 9(B), the maximum emission wavelength almost has no shift at the investigated concentrations range when  equals to 60 nm. This revealed that there was no change of the micro-environment of the tryptophan residue. Moreover, the fluorescence intensity decreased regularly with the addition of CGR in both figures, which further demonstrated the occurrence of fluorescence quenching in the binding process. 3.5.2. Circular dichroism spectroscopy Circular dichroism spectroscopy is a sensitive technique to monitor the secondary structural change of protein upon interaction with ligands [50]. The CD spectra of BSA with various concentrations of CGR at pH 7.4 and room temperature are shown in Fig. 10. As can be seen from Fig. 10, BSA exhibits two negative bands at 208 and 222 nm in the ultraviolet region, characteristic of typical ␣-helix structure of protein [51]. The reasonable explanation is that the negative peaks of 208 and 222 nm both contribute to the n → ␲* transfer for the peptide bond of ␣-helix [52]. With increasing addition of CGR, the band intensity of curves B–D decreased regularly (Fig. 10, curves B–D). The quantitative analysis of the ␣-helix contents of BSA was calculated from the formulae described in Section 2 and the values were listed in Fig. 10. It differed from 62.5% in free BSA to 52.7% when the molar ratio of CGR to BSA was up to 6:1, which was indicative of the loss of ␣-helix content and a secondary structural change of the BSA molecule. The CD spectra of BSA in the Table 4 Fractions of different secondary structures determined by SELCON3a .

Fig. 9. Synchronous fluorescence spectrum of BSA: (A)  = 15 nm; (B)  = 60 nm; c(BSA) = 2.0 × 10−6 mol L−1 , c(CGR) (×10−6 mol L−1 ), A–K: 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, respectively.

Molar ratio [CGR]:[BSA]

H(r) (%)

H(d) (%)

S(r) (%)

S(d) (%)

Trn (%)

Unrd (%)

0:1 1:1 3:1 6:1

42.0 39.1 36.2 33.0

20.5 20.2 20.0 19.7

2.2 2.8 3.6 4.3

3.0 3.8 4.4 5.2

13.2 14.3 15.2 16.8

19.1 19.8 20.6 21.0

a H(r): regular ␣-helix; H(d): distorted ␣-helix; S(r): regular ␤-strand; S(d): distorted ˇ-strand; Trn: turns; Unrd: unordered structure.

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Fig. 11. Three-dimensional fluorescence spectra of BSA (A) and BSA-CGR system (B), c(BSA) (×10−6 mol L−1 ): (A) 2.000, (B) 2.000; c(CGR) (×10−6 mol L−1 ) (A) 0, (B) 2.000. Table 5 Three-dimensional fluorescence spectral characteristic parameters of BSA and CGR–BSA system. Peaks

Rayleigh scattering peaks Fluorescence peak 1 Fluorescence peak 2

BSA

BSA–CGR

Peak position

Stokes shift

Peak position

Stokes shift

ex /em (nm/nm)

 (nm)

Intensity, F

ex /em (nm/nm)

 (nm)

Intensity, F

280/280 → 350/350 280.0/350.0 225.0/350.0

0 70.0 125.0

41.9 → 365.9 539.3 541.1

280/280 → 350/350 280.0/350.0 230.0/350.0

0 70.0 119.0

44.0 → 391.5 387.3 362.8

presence and absence of CGR were similar in shape, indicating that the structure of BSA was still predominantly of ␣-helix. In order to quantify the different types of secondary structure content, the CD spectra have been analyzed by the algorithm SELCON3, with 43 mode proteins with known precise secondary structures used as the reference set [53,54]. The fraction contents of different secondary structures for BSA in the absence and presence of CGR are presented in Table 4. The ␣-helix contents (the sum of the regular and distorted ␣-helix) calculated by this recent model and the above classic model has only a small difference, which means that the results obtained in this paper were credible. A decreasing tendency of the ␣-helix content and an increasing tendency of ␤strands, turn, and unordered structure contents were observed with the increasing concentration of CGR (Table 4). As known, the secondary structure contents are related close to the biological activity of protein, thus the secondary structural changes here meant the loss of the biological activity of BSA upon interaction with high concentration of CGR. The conformational changes here meant that CGR bound with the amino acid residues of the main polypeptide chain of BSA and destroyed their hydrogen bonding networks, making the serum albumin adopt a more incompact conformation state [55]. 3.5.3. Three-dimensional fluorescence spectroscopy To further investigate the conformational changes of BSA by the addition of CGR, three-dimensional fluorescence spectroscopy was employed. Not only can it comprehensively exhibits the fluorescence information of the sample, but also makes the investigation of the characteristic conformational change of protein be more scientific and credible. The three-dimensional fluorescence spectra of BSA and BSA–CGR system are shown in Fig. 11, and the corresponding characteristic parameters are presented in Table 5. By comparing the three-dimensional fluorescence spectral changes of BSA in the absence and presence of CGR, the conformational and micro-environmental changes of BSA can be obtained. As shown in Fig. 11, peak a is the Rayleigh scattering peak (ex = em ) [56] and with the addition of CGR, the fluorescence

intensity of peak a increased. The possible reason may be that a BSA–CGR complex came into being after the addition of CGR, making the diameter of the macromolecule increased, which in turn resulted in the scattering effect enhanced [57]. Peak b is the secondordered scattering peak (em = 2ex ) [56]. Peak 1 (ex = 280.0 nm and em = 350.0 nm), which mainly reveals the spectral behavior of tryptophan and tyrosine residues, is the primary fluorescence peak we studied. Besides peak 1, there is another strong fluorescence peak 2 (ex = 225.0 nm and em = 350.0 nm) that mainly exhibits the fluorescence spectral behavior of polypeptide backbone structures, and the fluorescence intensity of this peak is correlated with the secondary structure of protein [58]. As can be seen from Fig. 11, the fluorescence intensity of peak 2 decreased a lot after the addition of CGR, which means that the peptide strands structure of BSA has been changed. This result was in accordance with that we got from the CD spectra. Analyzing from the fluorescence intensity changes of peaks 1 and 2 (the intensity values were listed in Table 5), they both decreased obviously but to different degrees: the fluorescence intensity of peak 1 has been quenched of 28.06% while peak 2 of 32.89%. The decrease of the fluorescence intensity of the two peaks in combination with the synchronous fluorescence and CD spectra results indicated that the interaction of CGR with BSA induced the slight unfolding of the polypeptides of protein, which resulted in a conformational change of the protein to increase the exposure of some hydrophobic regions that had been buried [59]. All the above phenomenon and analysis of the fluorescence characteristic of the peaks revealed that the binding of CGR to BSA induced some micro-environmental and conformational changes in BSA. 4. Conclusions This paper provided an approach for studying the binding of CGR to BSA by employing different optical techniques. The studies presented here demonstrated that the fluorescence quenching of BSA was resulted mainly from static mechanism and hydrophobic force and hydrogen bonds played major roles in stabilizing the CGR–BSA complex. Site marker competitive experiments suggested

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that MG bound to the sub-domain IIA of BSA, the site of which was the same as that of warfarin binding to BSA (site I). Synchronous fluorescence spectra, CD and three-dimensional fluorescence spectra revealed that the conformation and micro-environment of BSA were changed by the binding of CGR. Acknowledgements We gratefully acknowledge the financial support of Chinese 863 Program (2007AA06Z407); National Natural Science Foundation of China (Grant Nos. 30570015, 20621502 and 20873096); Research Program of Hubei Province Department of Education, China (No. Q20081207); and the Research Foundation of Chinese Ministry of Education ([2006]8-IRT0543). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

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