Binding interaction of quinclorac with bovine serum albumin: A biophysical study

Spectrochimica Acta Part A 74 (2009) 781–787

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

Binding interaction of quinclorac with bovine serum albumin: A biophysical study Xiao-Le Han a , Ping Mei b,∗ , Yi Liu a,b,∗∗ , Qi Xiao a , Feng-Lei Jiang a , Ran Li a a b

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

a r t i c l e

i n f o

Article history: Received 14 January 2009 Received in revised form 14 July 2009 Accepted 7 August 2009 Keywords: Quinclorac Bovine serum albumin Fluorescence quenching Binding site Circular dichroism Three-dimensional fluorescence

a b s t r a c t Quinclorac (QUC) is a new class of highly selective auxin herbicides. The interaction between QUC and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy, synchronous fluorescence, three-dimensional fluorescence, CD spectroscopy and UV–vis absorption spectroscopy under simulative physiological condition. It was proved that the probable quenching mechanism of BSA by quinclorac was dynamic quenching. The Stern–Volmer quenching model has been successfully applied and the activation energy of the interaction as much as 8.03 kJ mol−1 , corresponding thermodynamic parameters H␪ , S␪ and G␪ were calculated. The results indicated that the acting forces between QUC and BSA were mainly hydrogen bonding and van der Waals forces. According to the Förster non-radiation energy transfer theory, the average binding distance between donor (BSA) and acceptor (QUC) was obtained (r = 3.12 nm). The alterations of protein secondary structure in the presence of QUC were confirmed by the evidences from three-dimensional fluorescence, synchronous fluorescence and CD spectroscopy. Furthermore, the site marker competitive experiments indicated that the binding of QUC to BSA primarily took place in Sudlow site I. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Quinclorac (3,7-dichloro-8-quinolinecarboxylic acid; formula: C10 H5 Cl2 NO2 ; molecular weight: 242.06; its structure is shown in Scheme 1) is used in rice (Oryza sativa L.) and turf fields to control the growth of some broad-leaved weeds and major grass weeds such as Echinochloa, Digitaria, and Setaria species [1,2]. In chemical fallow, QUC is used to suppress Convolvulus arvensis L. and Lactuca serriola L. [3]. After being readily absorbed by germinating seeds, roots, and leaves, it could be translocated both acropetally and basipetally. It has very good applications because of its high biological activity and low doses, whereas, as a kind of herbicides, QUC has some toxicity to humans via the oral, dermal and inhalation routes of exposure. It is of low acute toxicity to microorganisms, but its effect on non-target organisms and overall environmental impact are still not fully understood. Studies on the interaction between QUC and BSA are of fundamental importance for providing more information about the potential toxicological effect of herbicides. Serum albumins, as the most abundant proteins in the circulatory system, act as a transporter and disposer of many endogenous and exogenous compounds [4]. They are also capable of modulating

∗ Corresponding author. Tel.: +86 716 8060472; fax: +86 716 8060472. ∗∗ Corresponding author. Tel.: +86 27 8721 8284; fax: +86 27 6875 4067. E-mail addresses: [email protected] (P. Mei), [email protected] (Y. Liu). 1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2009.08.018

their delivery to cells in vivo and in vitro and playing a dominant role in drug or toxicity chemicals disposition and efficacy [5]. BSA is frequently used in biophysical and biochemical studies since it has a well-known primary structure, and it has been associated with the binding of many different categories of small molecules, such as dye, drugs and toxic chemicals [6–9]. In this work, we selected BSA as our protein model, because of its medically important, abundance, low cost, ease of purification, unusual ligand-binding properties, stability, and all the studies are consistent with the fact that human and bovine serum albumins are homologous proteins [10,11]. Binding of herbicide to plasma proteins has toxicological importance, since controls their free, active concentrations and, as a consequence the degree and time of action in the body, affects duration and intensity of their effects. In other words, binding to proteins will significantly affect the distribution, metabolism, and excretion of herbicide. Fluorescence spectroscopy is essentially a probe technique sensing changes in the local environment of the fluorophore. In the present work, we demonstrated the affinity of QUC to BSA and the thermodynamics of their interaction. In order to attain these objectives, we planned to carry out detailed investigation of QUC–BSA association using fluorescence spectroscopy and UV/vis absorption spectroscopy. Through fluorescence resonance energy transfer and synchronous fluorescence, CD and three-dimensional fluorescence spectra, we planned to further investigate the effect of the energy transfer and the effect of QUC on the conformation of BSA.

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entropy change (S␪ ) can be calculated from the van’t Hoff equation: ln Kb = −

2. Experimental

G␪ = H ␪ − T S ␪

2.1. Materials QUC was obtained from Hubei Sanonda Foreign Trading Co. Ltd. BSA, (hydroxymethyl) aminomethane (Tris) was obtained from Sigma (St. Louis, MO). The samples were dissolved in Tris–HCl buffer solution (0.05 mol L−1 of Tris, 0.10 mol L−1 of NaCl, and pH 7.4 ± 0.1). The QUC solution was prepared in ethanol. Warfarin and ibuprofen were obtained from Jiangsu Medicine Co. Ltd. and Hubei Biocause Heilen Pharmaceutical Co. Ltd. in China, respectively. All other reagents were of analytical reagent grade and doubly distilled water was used throughout. The mass of the samples was accurately weighted using a microbalance (Sartorius, ME215S) with a resolution of 0.1 mg. 2.2. Fluorescence spectral measurements All fluorescence spectra were recorded with a LS-55 Spectrofluorimeter (Perkin-Elmer corporate, UK) equipped with quartz cells (1.0 cm) and a thermostat bath. The fluorescence measurements were performed at different temperatures (292, 298, 304, and 310 K). Excitation wavelength was 280 nm. The slit widths for excitation and emission were set to 15.0 and 4.0 nm, respectively. All data and each spectrum were the average of three scans. Titrations were performed manually by using trace syringes. In each titration, the fluorescence spectrum was collected with the concentration of BSA at 2.0 × 10−6 mol L−1 . Appropriate blanks corresponding to the buffer were subtracted to correct background fluorescence. The quenching effect of ethanol was evaluated and the result indicated that there was almost no effect of ethanol on the QUC–BSA interaction. The data were analyzed by the Stern–Volmer equation [12]: (1)

where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher (QUC), respectively, kq the bimolecular quenching rate constant,  0 the life time of fluorescence in absence of quencher, KSV the Stern–Volmer quenching constant, and [Q] the concentration of quencher. Hence the above equation could be applied to determine KSV by linear regression of a plot of F0 /F against [Q].

When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by the equation [13]:

F − F  0 F

= log Kb + n log[Q]

(2)

where in the present case, Kb is the observed binding constant to a site and n is the number of binding sites per BSA. The dependence of log(F0 /F − 1) on the value of log[Q] is linear with the slope equal to the value of n and log Kb is fixed on the ordinate. If the enthalpy change (H␪ ) does not vary significantly in the temperature range studied, both the enthalpy change (H␪ ) and

(4)

The three-dimensional fluorescence spectra were performed under the following conditions: the emission spectrum was recorded from 200 to 500 nm with the initial excitation wavelength at 200 nm followed by increment of 5 nm. Totally, the number of scanning curves was 31. Other scanning parameters were just the same to those of the fluorescence quenching experiments. 2.4. UV–vis absorption spectral measurements TU-1901 spectrophotometer (Puxi Ltd. of Beijing, China) was used to scan UV–vis spectra; UV–vis absorption spectra of 2 ␮mol L−1 of free QUC in buffer solution as well as the UV–vis absorption spectra of QUC/BSA varieties of molar ratio complexes were recorded from 200 to 500 nm at room temperature. 2.5. CD measurements The CD spectra were recorded on Jasco (J-810-150S) automatic recording spectropolarimeter, using a cylindrical cuvette with 0.1 cm of path-length. CD experiments were conducted at pH 7.40. The spectra were recorded in the absence and presence of QUC with the QUC/BSA ratio 1:1, 5:1 and 30:1. The CD profiles were obtained by employing a scan speed of 500 nm min−1 and a response time of 0.5 s. Each spectrum was the average of three successive scans and was corrected by Tris–HCl buffer solution (containing 0.10 mol L−1 of NaCl, pH 7.4 ± 0.1) at 25 ◦ C. Appropriate baseline corrections in the CD spectra were made. For the CD experiment, the concentration of BSA was kept at 2.0 × 10−6 mol L−1 and the spectra were recorded from 200 to 240 nm. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol−1 according to the following equation: MRE =

observed CD (mdeg) Cp nl × 10

(5)

where Cp is the molar concentration of the protein, n the number of amino acid residues (583 for BSA) and l the path-length (0.1 cm). The ␣-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the equation: ␣-helix (%) =

2.3. Calculation of binding parameters

log

(3)

In Eq. (3), Kb is the binding constant at corresponding temperature and R is the gas constant. The enthalpy change (H␪ ) and entropy change (S␪ ) are calculated from the slope and ordinate of the van’t Hoff relationship. The free energy change (G␪ ) is estimated from the following relationship:

Scheme 1. Molecular structure of QUC.

F0 = 1 + kq 0 [Q] = 1 + KSV [Q] F

H ␪ S ␪ + RT R

−MRE208 − 4000 33, 000 − 4000

(6)

As described by Lu et al. [14], the observed MRE value at 208 nm, MRE208 , of ␤-form and random coil conformation cross in total and a pure ␣-helix were 4000 and 33000, respectively. From the above equation, the ␣-helicity in the secondary structure of BSA was determined. 3. Results and discussion 3.1. Fluorescence characteristics of bovine serum albumin Quenching may be caused by a variety of molecular interactions including excited-state reactions, molecular rearrangements,

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Fig. 1. Emission spectra of BSA in the presence of various concentrations of QUC (T = 298 K; ex = 280 nm). c(BSA) = 2.0 × 10−6 mol L−1 ; c(QUC)/(10−6 mol L−1 ): a–h: 0; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0, respectively.

Fig. 2. Stern–Volmer plots for the quenching of BSA by QUC at the four different temperatures, pH = 7.4.

energy transfer, ground-state complex formation and collisional quenching. When the concentration of BSA was stabilized at 2 × 10−6 mol L−1 , the content of QUC varied from 0 to 7.0 × 10−6 mol L−1 at an interval of 1.0 × 10−6 mol L−1 . The effect of QUC on BSA fluorescence intensity is shown in Fig. 1. It clearly indicates that the fluorescence intensity of BSA decreases regularly but the emission maximum did not move to shorter or longer wavelength. We can conclude that QUC could interact with BSA and quench its intrinsic fluorescence, but there was no alteration in the local dielectric environment of BSA. The quantitative analysis of binding of QUC to BSA was employed by using the fluorescence quenching at various temperatures as shown in Fig. 2. The value of KSV from Stern–Volmer plots revealed that varying temperature has a moderate effect on fluorescence quenching by QUC. The calculations of KSV at different temperatures (292, 298, 304, and 310 K) are shown in Table 1. The data demonstrated that the Stern–Volmer quenching constant

783

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

KSV is directly correlated with temperatures. This phenomenon indicates that the probable quenching mechanism of a QUC–BSA binding reaction is dynamic collision rather than formation of a complex. Meanwhile, we found that the values of KSV were big enough. The first reason is that the fluorescent quantum yield of BSA increased [15]. The second one is that there may be a strong binding between BSA and QUC in excited state. UV–vis absorption measurement is a very simple method and applicable to explore the structural change [16] and formation of a complex [17]. Dynamic quenching only affects the excited states of the fluorophores, and thus no changes in the absorption spectra are expected. For reconfirming the quenching mechanism, the UV absorption spectrum of BSA (Fig. 3, curve A) in the near ultraviolet band (250–400 nm) and the absorption spectrum (Fig. 3, curve D) by subtracting the absorption spectrum of QUC from that of BSA–QUC at the same concentration could be overlapped within experimental error. This result reconfirms that the probable quenching mechanism of fluorescence of BSA by QUC is a dynamic quenching procedure. 3.2. Binding constant and binding sites Fig. 4 is the plots of log(F0 /F − 1) versus log[Q] for the QUC–BSA system at different temperatures. The binding constants Kb and binding sites n were listed in Table 2. The results showed that the binding constants Kb were decreased with the temperature, which may indicate forming an unstable compound [18]. The unstable compound would be partly decomposed with the rising temperature, therefore, the values of Kb decreased. The values of n approximately equal to 1 indicated the existence of just a single binding site in BSA for QUC. Because higher temperatures will result in faster diffusion and hence lead to larger amounts of collision quenching, one explanation in this case is that the quenching is initiated by dynamic quenching.

Table 1 Stern–Volmer quenching constants for the interaction of QUC with BSA at different temperatures and relative activation energy. pH

T (K)

KSV (×104 M−1 )

Kq (×1012 M−1 s−1 )

Ra

S.D.b

Ea (kJ mol−1 )

7.4

292 298 304 310

6.552 7.070 7.444 7.972

6.552 7.070 7.444 7.972

0.999 0.998 0.996 0.995

0.006 0.009 0.017 0.019

8.25

a b

Linear correlated coefficient. Standard deviation for the KSV values.

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for confirming binding mode. In this regard, the temperature dependence of binding constant was studied. The thermodynamic parameters were calculated from Eqs. (3) and (4) and summarized in Table 2. The negative H␪ value (−44.22 kJ mol−1 ) observed cannot be mainly attributed to electrostatic interactions since for electrostatic interactions H␪ is very small, almost zero [21]. Negative H␪ value is observed whenever there is hydrogen bonding in the binding. We can see that the binding reaction of QUC to BSA is exothermic (H␪ < 0). This means that higher temperatures should weaken the binding, which is also shown by the decreasing values of Kb in Table 2. The negative H␪ and S␪ values, therefore, showed that both hydrogen bond and van der Waals forces play a role in the binding of QUC to BSA [21]. The negative value of G␪ reveals that the interaction process is spontaneous. Fig. 4. Double-log plots of QUC quenching effect on BSA fluorescence at different temperatures.

Fig. 5. Arrhenius plot for interaction between BSA and QUC in Tris-buffer, pH = 7.4.

3.3. Thermodynamic parameters and nature of the binding forces The interaction forces between a ligand and protein may include hydrophobic force, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc. [19]. The Stern–Volmer quenching constants of BSA were measured at four different temperatures. The slope of a plot of the bimolecular quenching constant vs. 1/T (T, absolute temperature) is linear within experimental error (Fig. 5), which allows one to calculate the activation energy for the quenching process [20]. The activation energy for the quenching process could be determined according to the Arrhenius equation: ln kq = −

Ea + ln A RT

(7)

where kq is the bimolecular quenching rate constant at the corresponding temperature, Ea the activation energy for the quenching process, A the frequency factor (or pre-exponential factor) and R the gas constant. The obtained activation energy for QUC quenching is 8.25 kJ mol−1 (Table 1). The thermodynamic parameters, enthalpy (H␪ ) and entropy (S␪ ) of QUC–BSA interaction are important

3.4. Confirming the binding sites on BSA According to the Refs. [22,23], the principal regions of ligands bound to BSA are usually located in hydrophobic cavities in subdomains IIA and IIIA, and the binging cavities associated with sub-domains IIA and IIIA are also referred to as site I and site II. Most studies of the two probes found that warfarin was mostly bound to site I on the BSA, and that ibuprofen possesses one highaffinity sites in site II and several low-affinity binding sites in site I [24,25]. In order to identify the QUC binding site on BSA, site marker competitive experiments are carried out, using drugs (warfarin and ibuprofen) which specifically bind to a known site or region on BSA. During the experiment, BSA samples containing appropriate concentration of warfarin or ibuprofen (the concentrations of warfarin or ibuprofen were fixed at 2.0 × 10−6 mol L−1 ) were titrated with the continuing addition of QUC [26,27]. The fluorescence spectra were recorded upon excitation at 280 nm. As shown in Fig. 6A, with addition of warfarin to the BSA solution, the maximum emission wavelength of BSA had a slight red shift (from 354 to 356 nm) and the fluorescence intensity was significantly lower than that of without warfarin. Then, with the continuing addition of QUC 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. 1), manifesting that the binding of QUC to BSA was affected by adding warfarin. By contrast, in the presence of ibuprofen, there is no difference between the QUC–BSA system and that of without ibuprofen in the same condition (Fig. 6B), demonstrating that the site II marker ibuprofen did not prevent the binding of QUC in its usual binding location. The quenching data (Fig. 7) were analyzed according to the Stern–Volmer equation (Eq. (1)) and shown in Table 3. Obviously, the KSV values of the system with warfarin were almost 74% of that without warfarin, while the constants of the systems with and without ibuprofen had only a small difference. It revealed that there was a significant competition between QUC and warfarin, while ibuprofen had only a small influence on the binding of QUC to BSA. The above experimental results and analysis demonstrated that the binding of QUC to BSA mainly located within site I (sub-domain IIA).

Table 2 Apparent binding constants Kb at different temperatures and relative thermodynamic parameters of the QUC–BSA system. pH

T (K)

Kb (×104 M−1 )

n

Ra

S.D.b

H (kJ mol−1 )

G (kJ mol−1 )

S (J mol−1 K−1 )

7.4

292 298 304 310

1.962 1.539 1.009 0.696

0.8990 0.8710 0.8293 0.7957

0.999 0.999 0.999 0.998

0.010 0.008 0.011 0.013

−44.22

−23.995 −23.887 −23.301 −22.803

−68.87

a b

Linear correlated coefficient for the Kb values. Standard deviation for the Kb values.

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785

Fig. 8. Spectral overlap of UV–vis absorption spectrum of QUC (a) with fluorescence emission spectrum of BSA (b): c(BSA) = c(QUC) = 2.0 × 10−6 mol L−1 ; T = 298 K.

3.5. Energy transfer between QUC and bovine serum albumin According to Förster theory of molecular resonance energy transfer, the distance r of binding between QUC and BSA, the efficiency E of energy transfer between the donor and acceptor, could be calculated by the equation [28,29]: E=

R06

(8)

R06 + r 6

where E is the efficiency of transfer between the donor and the acceptor and R0 is the critical distance when the efficiency of transfer is 50%. R06 = 8.79 × 10−25 K 2 n−4 J Fig. 6. Effect of selected site markets on the fluorescence of QUC bound BSA (T = 298 K; ex = 285 nm). (a) c(BSA) = c(warfarin) = 2.0 × 10−6 mol L−1 ; (b) c(BSA) = c(ibuprofen) = 2.0 × 10−6 mol L−1 ; c(QUC)/(10−6 mol L−1 ): a–j: 0; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0, respectively.

(9)

K2

is the space factor of orientation, n the refracted In Eq. (9), index of medium,  the fluorescence quantum yield of the donor and J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (Fig. 8), which could be calculated by the equation: J=

∞ F()ε()4 d 0 ∞ 0

F() d

(10)

where F() is the corrected fluorescence intensity of the donor in the wavelength range  to  +  and ε() is the extinction coefficient of the acceptor at . The efficiency of transfer (E) could be obtained by the equation: E =1−

F F0

(11)

In the present case, K2 = 2/3, N = 1.36,  = 0.15 [21], according to Eqs. (9)–(11), we could calculate that J = 1.845 × 10−14 cm3 L mol−1 , R0 = 2.28 nm, E = 0.13 and r = 3.12 nm. Both the average distance between a donor fluorophore and acceptor fluorophore on the 2–8 nm [30] and 0.5R0 < r < 1.5R0 [31], converge to indicate that the energy transfer from BSA to QUC occurs with high probability. Fig. 7. Stern–Volmer plots for the QUC–BSA system in the absence and presence of site markers (T = 298 K; pH = 7.4).

Table 3 The binding constants of competitive experiments of QUC–BSA system. Site maker

KSV (×10−4 M−1 )

Ra

S.D.b

Blank Warfarin Ibuprofen

6.6821 4.9834 6.2295

0.997 0.998 0.998

0.013 0.006 0.009

a b

Linear correlated coefficient. Standard deviation.

3.6. Conformation investigation Synchronous fluorescence spectroscopy is a very useful method to study the microenvironment of amino acid residues by measuring the emission wavelength shift [32,33]. The shift position at maximum emission wavelength corresponds to changes of polarity around the chromospheres molecule. When the D-value () between excitation wavelength and emission wavelength are set as 60 nm, the synchronous fluorescence gives the characteristic information of tryptophan residues [34]. A slightly red shift of tryptophan residues (Fig. 9) is observed, which indicates that the

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Fig. 9. Synchronous fluorescence spectrum of BSA:  = 60 nm, c(BSA) = 2.0 × 10−6 mol L−1 ; c(QUC)/(10−6 mol L−1 ): a–h: 0; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0, respectively.

tryptophan residues are placed in a less hydrophobic environment but more exposed to the solvent molecules during the binding process. CD is widely used in studying peptide and protein conformations. One of the most successful applications of CD, the structural characterization of proteins, depends largely upon the remarkable sensitivity of CD in far-UV region to the backbone conformation of proteins. CD spectra of BSA exhibit two negative bands at 208 and 222 nm that is the characteristic of ␣-helix in the advanced structure of protein [35]. If the ␣-helicity changed, the spectra will change accordingly. A reasonable explanation is that the negative peaks between 208–209 nm and 222–223 nm are both contributed to n → ␲* transition for the peptide bond of ␣-helicity. As shown in Fig. 10, the binding of QUC to BSA caused only a decrease in negative ellipticity at all wavelengths of the far-UV CD without any significant shift of the peaks, clearly indicating the considerable changes in the protein secondary structure, and it may be the result of the formation of complex. In addition, the CD spectra of BSA in the presence and absence of QUC are similar in shape, indicating that structures of BSA are predominantly ␣helicity in two conditions. From the above result, it can be deduced that QUC caused a conformational change on BSA with the loss of

Fig. 10. The far-UV CD spectra of the QUC–BSA system obtained in 0.05 mol L−1 Tris–HCl buffer of pH 7.4 at room temperature. c(BSA) = 2.0 × 10−6 mol L−1 ; c(QUC)/(10−6 mol L−1 ): A–D: 0; 2.0; 10.0; 60.0, respectively.

helical stability [36]. The calculated results exhibited a reduction of ␣-helicity structures (from 61.2% to 59.1%), while the content of ␤-strands (from 5.3% to 6.5%), turn (from 12.5% to 13.5%), and unordered structures (from 21.5% to 23.7%) increased at molar ratio QUC/BSA of 30:1, respectively. The percentage of protein ␣-helicity structure indicated that the herbicide, QUC was bound with the amino acid residue of the main polypeptide chain of protein and destroyed their hydrogen bonding networks [37]. It also revealed that QUC may affect physiological functions of BSA through altering its conformation. The three-dimensional fluorescence spectra for both BSA and QUC–BSA system are shown in Fig. 11. Corresponding characteristic parameters are presented in Table 4. As known to all, normal fluorescence peaks are usually located in the lower right of the Rayleigh scattering regions. It can be seen that the resonance light-scattering spectrum (ex = em ) appears to match with the “chine” pattern in Fig. 11. At the same time, there are two “humps” in the threedimensional fluorescence spectra for BSA and QUC–BSA, in which the peaks are marked peak 1 and peak 2. Fig. 11B shows that both of fluorescence peaks in the three-dimensional fluorescence spectra of BSA have been quenched with the ratio of 0.761 and 0.668, respectively. The results indicate that QUC has formed a complex

Table 4 The characteristic parameters of three-dimensional fluorescence spectra. Systems and parameters

Rayleigh scattering peaks

Fluorescence peak one

Fluorescence peak two

BSA

Peak position (ex /em , nm/nm) Relative intensity, F

350/351.5 → 250/498.5 283.8 → 233.4

285.0/350.0 580.321

230.0/351.0 638.380

BSA + QUC

Peak position (ex /em , nm/nm) Relative intensity, F

350/351.5 → 250/498.5 458.4 → 217.0

285.0/351.0 441.781

230.0/349.5 426.366

Fig. 11. Three-dimensional fluorescence spectra of BSA (A) and QUC–BSA system (B). c(BSA)/(10−6 mol L−1 ): A: 2.0; B: 2.0; c(QUC)/(10−6 mol L−1 ): A: 0; B: 2.0.

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with BSA and thereafter changed its conformation. The maximum excitation and emission wavelengths are 285.0 and 350.0 nm for peak 1 and 285.0 and 351.0 nm for peak 2. In comparison, the intensity of peak 2 is lower than that of peak 1 in the three-dimensional fluorescence spectra for QUC–BSA complex (Fig. 11B) with a ratio of 0.668. It means that the fluorescence quenching of BSA by QUC on peak 2 is greater than on peak 1. According to Ref. [38], the peak 2 may mainly exhibit the fluorescence characteristic of polypeptide backbone structures. The decrease of the fluorescence intensity of peak 2 in combination with the fluorescence quenching and CD spectra results, can conclude that the interaction of QUC with BSA induced the slight unfolding of the polypeptides of protein, which resulted in a conformational change of the protein that increased the exposure of some hydrophobic regions which were previously buried. 4. Conclusions The interaction between QUC and BSA has been investigated in this work using different optical techniques. The data obtained gave preliminary information on the binding of QUC to BSA. The results suggested that QUC could bind to BSA through a dynamic quenching procedure. The interaction is mainly enthalpy-driven, and hydrogen bonding and van der Waals forces played a major role in the reaction. For competition displacement, it appeared that the binding site of QUC on the protein is around site I. Experimental results also showed that the binding of QUC to BSA induced a conformational change of BSA, which was further proved by the analysis data of CD, synchronous and three-dimensional fluorescence spectra. The binding study of QUC with proteins has toxicological importance. This study is expected to provide important insight into the interactions of the protein BSA with auxin herbicides. Acknowledgments We gratefully acknowledge the financial support of Chinese 863 program (2007AA06Z407), National Natural Science Foundation of China (Grant Nos. 20621502, 20873096), Natural Science Foundation of Hubei Province (2005ABC002), and Research Foundation of Chinese Ministry of Education ([2006]8-IRT0543). References [1] K. Grossmann, Weed Sci. 46 (1998) 707–716. [2] K. Grossmann, J. Kwiatkowski, Pestic. Biochem. Physiol. 66 (2000) 83–91.

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