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Comprehensive spectroscopic probing the interaction and conformation impairment of bovine serum albumin (BSA) by herbicide butachlor
Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 332–339
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Comprehensive spectroscopic probing the interaction and conformation impairment of bovine serum albumin (BSA) by herbicide butachlor Xiaoyi Liu a, Zhaoxing Ling a, Xing Zhou b, Farooq Ahmad a, Ying Zhou a,c,⁎ a b c
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China College of Atmospheric Science, Lanzhou University, Lanzhou, China Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou, China
a r t i c l e
i n f o
Article history: Received 11 January 2016 Received in revised form 4 July 2016 Accepted 7 July 2016 Available online 09 July 2016 Keywords: Interaction Butachlor Herbicide Pesticide Bovine serum albumin (BSA) Spectroscopic method
a b s t r a c t Butachlor is an effective herbicide to deal with undesired weeds selectively and is used at high levels in Asian countries. However, its interaction and impairment effect on BSA was still not clear. In this study, we investigated the interaction between butachlor and bovine serum albumin (BSA) by multi-spectroscopic methods including UV absorption, circular dichroism (CD) spectra, Fourier transform infrared (FTIR) spectra and fluorescence spectra under physiological conditions (pH = 7.4). The results revealed that there was a static quenching of BSA induced by butachlor stemmed from the formation of complex. Based on thermodynamic data, the interaction of butachlor with BSA was due to happen, and van der Waals force as well as hydrogen bond were the major forces contributed to the interaction. The binding constant Kb and number of binding site of butachlor with BSA were 5.158 × 105 and 1.372 at 303 K, respectively. The distance r between donor (BSA) and acceptor (butachlor) was 0.113 nm, obtained according to the Förster theory. The results revealed that butachlor induced conformational changes in BSA but the secondary structure of BSA was still retained. In addition, the microenvironment around chromophore residues of BSA, for example, tryptophan, changed as well, resulting from the formation of more hydrogen bonds. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Herbicides are applied to fields to get rid of undesired weeds selectively without injuring crops [1]. Butachlor, whose chemical name is N-butoxymethyl-2-chloro-2′, 6′-diethyl acetanilide (Fig. 1), is a preemergence herbicide and belongs to the chloroacetanilide herbicides of which the effect is inhibiting protein synthesis in developing plant tissue [2]. Field experiments have confirmed that butachlor can control the weeds population effectively and increase the crop yield [3,4]. Butachlor is used at high levels in Asian countries like China and India [2,5], and the consumption of butachlor is nearly 4.5 × 107 kg per year in Asia alone [6], resulting in the inevitable entrance of butachlor into environment via routes like agricultural run-off, leaching and rainfall. Some herbicides including butachlor have been detected in soil, water and sediment in several countries [6,7]. The environmental concentration of butachlor detected in the Mekong Delta area in Vietnam ranged from 0.01 μg L−1 to 0.59 μg L−1 [8]. Once released into the environment, ⁎ Corresponding author at: Research Center of Analysis and Measurement, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, Zhejiang Province, China. E-mail address: [email protected] (Y. Zhou).
http://dx.doi.org/10.1016/j.jphotobiol.2016.07.005 1011-1344/© 2016 Elsevier B.V. All rights reserved.
butachlor potentially exerts toxic effects on organisms like earthworm [9,10], flea, alga [11,12], frog [13,14], fish [15–17] and even humans [18]. By now, there have been a few reports on the damage of butachlor on DNA [18,19]. Apart from DNA, many functional proteins are also considered as targets or carriers of drugs. However, most of the interaction studies are focused on pharmaceuticals and sophisticated materials while those of pesticides with biological molecules (proteins) are still mostly ignored and the interaction between butachlor and protein has not been seen. Serum albumin (SA), which is synthesized in liver, is one of the most abundant carrier proteins in blood plasma [20]. It plays a vital role in life body as it combines and delivers endogenous and exogenous compounds present in blood. Generally, the weak binding between ligands and serum albumin could cause ligands' poor distribution while the strong binding results in the decreasing concentration of free ligands in plasma [21]. Consequently, the investigation of ligands-serum albumin interaction is a fundamental step to understand the pharmacology of drugs clearly. Bovine serum albumin (BSA) is used in many studies because of its highly stability, lower cost, possibility of being isolated in highly pure form and unusual ligand-binding properties [20,22,23] and what's the most important is that BSA is in structural similarity with human serum albumin (HAS) in 76% [22].
X. Liu et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 332–339
Fig. 1. The structure of butachlor.
In this study, BSA was chosen as a target protein molecule. The interaction between butachlor and BSA was investigated using spectroscopic methods including UV, circular dichroism (CD), FTIR, and fluorescence spectroscopy. The structural changes of BSA under the effect of butachlor were estimated. The binding parameters and main type of binding force were also obtained. This study is of great significance to offer a better understanding of the mechanism of butachlor's toxicity and also experimental basis for the designing of pesticide which is safe not only at the organism level but also at the molecular level. 2. Materials and Methods 2.1. Materials Butachlor (97% purity) was obtained from Hangzhou Qingfeng Agrochemical Limited Company (China). A stock solution of butachlor (5 × 10−3 M) was prepared in analytical-grade ethyl alcohol and stored at 4 °C in darkness. The test solutions for the following experiments were prepared by diluting the stock solution in HBS buffer (HEPES buffer solution, containing 2 × 10−3 M HEPES buffer and 1 × 10−3 M NaCl, pH = 7.4). BSA was purchased from Aladdin (Shanghai, China) and BSA stock solution (5 × 10−5 M) was prepared in HBS buffer as well. BSA solutions were prepared based on its molecular weight of 67,000. Ultrapure water produced by a Milli-Q system was used throughout the study. 2.2. Procedures 2.2.1. UV–Vis Measurement The UV spectra of all BSA solutions in the absence and presence of butachlor were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan) with a 1 cm quartz cell at room temperature. The wavelength ranged from 190 to 350 nm. BSA concentration was fixed at 2 × 10− 6 M while the butachlor concentration varied from 0 to 40 × 10−6 M (0, 4, 6, 10, 20, 40 × 10−6 M respectively). All these solutions were prepared in HBS buffer. The corresponding solutions of butachlor were used as the reference.
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excitation wavelength was at 280 nm. Mixed solutions were prepared by adding appropriate volumes of butachlor and BSA stock solutions to HBS buffer to obtain mixture containing butachlor from 0 to 50 × 10−6 M (0, 5, 10, 15, 20, 30, 40 and 50 × 10−6 M, respectively) and the BSA 2 × 10−6 M. Then 200 μL of these solutions was transferred with a suited pipette to a 96-well plate. Each sample was repeated three times and each spectrum was the average of three scans. However, inner filter effect (IFE), which is caused when the quencher in mixed solution has ultraviolet absorption at excitation and emission wavelength, would reduce the fluorescence intensity [21]. In this work, all fluorescence intensities were corrected for IFE by the following equation: F cor ¼ F obs 10ðA1 þA2 Þ=2
ð1Þ
where Fcor is the corrected fluorescence intensity, Fobs is the fluorescence intensity obtained in experiment, A1 and A2 are the absorption of the fluorescence determination system at 280 nm and at emission wavelength, respectively.
2.2.4. Synchronous and Three-dimensional Fluorescence Measurement The synchronous and three-dimensional fluorescence spectra (SFS) of butachlor-BSA solutions were recorded on a FluoroMax-4 (Horiba Jobin Yvon, France) equipped with a 1 cm quartz cell by scanning two different intervals of Δλ (15 nm and 60 nm) at room temperature. The wavelength ranges were 280–350 nm (Δλ = 15 nm) and 310– 510 nm (Δλ = 60 nm), respectively, characterizing the properties of tyrosine (Tyr) and tryptophan (Trp), respectively [21,24]. Concentration of BSA was 2 × 10−6 M while concentrations of butachlor were 0, 10, 20, 30 and 40 × 10−6 M, respectively. Three-dimensional fluorescence spectroscopy was conducted with setting excitation and emission wavelength range as 250–350 nm and 250–500 nm, respectively.
2.2.5. Fourier Transform Infrared (FTIR) Spectra Measurement At room temperature, the Fourier transform infrared (FTIR) spectra of BSA in the presence and absence of butachlor were measured on Nicolet 6700 FTIR spectrophotometer (Thermo, America) in the range of 1200–2000 cm−1. The spectrum of BSA-butachlor was obtained by subtracting the absorption of corresponding butachlor solution from the spectrum and the spectrum of BSA was obtained by subtracting the absorption of buffer.
3. Results and Discussion 3.1. UV Absorption Spectra
2.2.2. Circular Dichroism (CD) Measurement The CD measurements of BSA solutions in the absence and the presence of butachlor were measured on a J-815 CD spectrometer (Jasco, Japan) with a 0.1 cm quartz cell at room temperature with the wavelength from 200 to 350 nm, under constant nitrogen flush. The butachlor concentrations were 0, 10, 20 and 40 × 10−6 M, respectively, while the concentration of BSA was fixed at 2 × 10−6 M. HBS buffer was used as a baseline. With scanning speed of 100 nm/min and intervals of 1.0 nm, each spectrum was the average of three scans, corrected by HBS buffer blanks. 2.2.3. Fluorescence Measurement The fluorescence spectra measurements of BSA-butachlor mixed solutions at different temperatures (298, 303 and 308 K) were performed on a microplate reader M2 (Molecular devices, USA) with a 96-well plate. The emission wavelength ranged from 290 to 500 nm while the
UV absorption spectroscopy is a simple way to explore the conformational changes and the complex formation. The UV spectra of mixed solutions of BSA (2 × 10−6 M) with different concentrations of butachlor were shown in Fig. 2. From the figure it can be seen that the BSA solution has two absorption bands near 203 nm and 280 nm respectively, though the later one was very weak. The strong absorption peak near 203 nm reflects the framework conformation of BSA and the weak one around 280 nm is due to the π → π⁎ transition of aromatic amino acids such as Trp, Tyr and Phe [25,26]. Moreover, the UV absorption of BSA decreased regularly when the concentration of butachlor increases. Red shift of maximum absorption of BSA at 203 nm (Δλ = 3 nm) was also noticed and the red shift of peak absorption at 280 nm was not obvious. They were triggered possibly due to the complex formation between BSA and butachlor, leading to the conformational change of BSA.
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quenching. Dynamic quenching results from molecular collision between quenchers and excited-state fluorophores. Static quenching, however, is caused by the formation of ground-state complex of fluorophores and quenchers [28]. Dynamic quenching constant and static quenching constant can be distinguished by fluorescent lifetime and their dependence along with the change of temperature [29]. The dynamic quenching constant increases with the increase of temperature as higher temperature lead to a larger diffusion coefficient. On the contrary, the static quenching constant decreases when temperature increases because the higher temperature would cause decreasing stability of complexes [21]. Therefore, fluorescence spectra of BSA-butachlor system at different temperatures were measured to identify the changing trend of quenching constant (K sv). Fluorescence quenching is described by the Stern-Volmer equation [30]: F 0 =F ¼ 1 þ K q τ0 ½Q ¼ 1 þ K sv ½Q Fig. 2. UV absorption of BSA in the absence and the presence of butachlor. Numbers 1–6 indicate concentration of butachlor as 0, 4, 6, 10, 20, and 40 × 10−6 M, respectively. BSA concentration applied was 2 × 10−6 M.
3.2. Fluorescence Spectra 3.2.1. Fluorescence Quenching The fluorescence quenching measurement is a sensitive method for investigating the binding interaction of small molecules with proteins. There are two tryptophan residues in BSA (Trp-213 and Trp-134) [27] have relatively strong fluorescence intensity and are sensitive to the microenvironment changes. The fluorescence spectra of BSA with addition of varied concentrations of butachlor were shown in Fig. 3. As seen, under the excitation wavelength of 280 nm, the emission peak of BSA appeared around the wavelength of 340 nm. And the fluorescence intensity of BSA decreased gradually when the butachlor increased yet without obvious changes of the maximum emission wavelength and the shape of peak. The result of fluorescence quenching indicated that the interaction between BSA and butachlor caused changes in the microenvironment around Trp within BSA.
ð2Þ
where F0 and F represent the fluorescence intensities in the absence and the presence of quencher (butachlor), respectively. Kq is the bimolecular quenching rate constant, Ksv the Stern-Volmer quenching constant, τ0 the average fluorescent lifetime of protein without quencher, which is generally 6 × 10− 9 s for BSA [21], and [Q] the concentration of quencher. The Stern-Volmer plots of F0/F versus [Q] at three temperatures, 298 K, 303 K and 308 K, were shown in Fig. 4, and the values of quenching constant Ksv and Kq at different temperatures were calculated by slope of Fig. 4 and the results were presented in Table 1. The results of the study, where the Ksv decreased with the increase of temperature, suggested that the quenching mechanism of BSA fluorescence by butachlor was a static quenching process. What's more, the values of Kq were higher than that of the maximum scattering collision quenching constant, 2 × 1010 M−1 s−1 [31], indicating that the quenching was initiated by the formation of a complex rather than the dynamic collision. 3.2.3. Binding Constant and Number of Binding Sites When small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Kb) and the number of binding sites (n) fit the double-logarithm equation [32]:
3.2.2. Fluorescence Quenching Mechanism Protein contains endogenous fluorophores. Fluorescence quenching occurs when there are protein-drug interactions such as complex formation, collision and energy transfer, etc. Generally, fluorescence quenching includes dynamic quenching and static
where Kb is the binding constant and n is the number of binding sites. The plots of log[(F0 − F) ∕ F] against log[Q] at three temperatures
Fig. 3. Fluorescence spectra of BSA in the absence and the presence of butachlor. Numbers 1 to 8 indicate concentrations of butachlor: 0, 5, 10, 15, 20, 30, 40 and 50 × 10−6 M, respectively. BSA concentration was 2 × 10−6 M.
Fig. 4. Stern-Volmer linear plot of fluorescence quenching of BSA by butachlor at different temperatures. [BSA] = 2 × 10−6 M, λex = 335 nm.
log½ð F 0 −F Þ=F ¼ logK b þ n∙ log½Q
ð3Þ
X. Liu et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 332–339 Table 1 The Stern-Volmer quenching constants of the butachlor-BSA system at different temperatures. T (K)
Ksv (M−1)
Kq (M−1 s−1)
R2
298 303 308
1.579 × 104 1.311 × 104 1.113 × 104
2.632 × 1012 2.185 × 1012 1.855 × 1012
0.9789 0.9670 0.9695
(298, 303 and 308 K, respectively) were presented in Fig. 5. The values of Kb and n can be obtained from the intercept and slope of the above equation and were shown in Table 2. As results shown, both values of K b and n decreased with increase in temperature. This indicated that temperature effectively decreased the number of binding sites as well as the stability of the complex. Furthermore, these results also demonstrated that static quenching is predominant [33], which is consistent with the results above. 3.2.4. Thermodynamic Parameters and Binding Force Generally, the types of acting force between exogenous small molecular ligands and biological macromolecules are non-covalent forces including hydrogen bonds, van der Waals force, hydrophobic force, electrostatic interactions and so forth. The thermodynamic parameters, enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG) could be used as main evidences to determine the type of major acting forces [34]. The ΔH is considered to be temperature independent in a small temperature range [26,31], thus ΔH, ΔS and ΔG can be obtained from the van't Hoff equation: lnK b ¼ −ΔH=RT þ ΔS=R
ð4Þ
ΔG ¼ ΔH−TΔS
ð5Þ
where ΔH and ΔS are the standard enthalpy and entropy change of the reaction, respectively and ΔG is free energy change. R is the gas constant and T is the temperature. The plot of lnKb versus 1/T at 298 K, 303 K and 308 K was made and ΔH, ΔS and ΔG calculated according to the slope and intercept were shown in Table 2 as well. According to Ross's [34] study, the negative ΔG values of suggested that the binding process of butachlor to BSA is spontaneous, meaning that the interaction between butachlor and BSA was due to happen. Negative values of both ΔH and ΔS suggest that the van
Fig. 5. The plot of log[(F0 − F)/F] versus log[Q] for quenching process of butachlor with BSA at 298, 303 and 308 K. [BSA] = 2 × 10−6 M, λex = 335 nm.
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Table 2 Binding constants Kb, number of binding sites n and the thermodynamic parameters of BSA-butachlor system at different temperatures.
T (K)
Kb (L mol−1)
n
R
298 303 308
7.513 × 105 5.158 × 105 2.043 × 105
1.412 1.372 1.298
0.9803 0.9607 0.9796
ΔS (J mol−1 K−1)
ΔH (kJ mol−1)
ΔG (kJ mol−1)
−219.1
−99.12
−33.83 −32.73 −31.64
der Waals force and hydrogen bonds played the major role in the interaction. 3.3. Conformational Change of BSA Induced by Butachlor 3.3.1. CD Measurement Circular dichroism (CD) is seen as a sensitive and powerful method to investigate the conformational changes in proteins and chiral molecules [35,36]. Therefore, CD spectra were carried out to observe the conformation change of BSA in the presence of butachlor and the results were shown in Fig. 6. From Fig. 6 it can be found that the CD spectra of BSA showed two negative bands in the UV region near 208 nm and 220 nm which are characteristic of α-helical structure of protein [25,37]. The two peaks are contributed by the n → π* transfer for the peptide bond of α-helix [31,38]. From Fig. 6, it can be noticed that the CD spectra of BSA with or without butachlor were similar in shape, indicating that the BSA structure was predominantly α-helix. Besides, the CD signals rose when butachlor concentration increased but without any significant shift. The secondary structures, calculated on the basis of Yang's equation, showed that the original BSA has an α-helix content of 57.6%, beta content of 17.2% as well as turn and random content of 25.2%. With the increase of the butachlor concentration, the αhelix content increased by 8.2% and beta content decreased by 5.7% (Table 3). The increased α-helix and decreased beta content in BSA may suggest the formation of hydrogen bond, and the BSA structure became refolding. In addition, the CD spectra on the wavelength range from 240 nm to 350 nm reflect the information of tertiary structure of protein [21]. As seen, the intensities of this character band increased as well. In conclusion, the higher CD singles has shown that there was an increase in the α-helix content [39], hinting a trend of BSA refolding to adopt an α-helix rich structure. This conformational rearrangement of
Fig. 6. CD spectra of BSA in the absence and the presence of butachlor. BSA concentration applied was 2 × 10−6 M. The number 1 indicates 40 × 10−6 M butachlor solution alone, and numbers 2 to 5 indicate mixture with concentrations of butachlor as 0, 10, 20, and 40 × 10−6 M, respectively.
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between butachlor and BSA induced certain conformational change in BSA.
Table 3 Secondary structure of butachlor-BSA complex based on CD data. Butachlor con. (×10−6 M) α-Helix (%) Beta (%) Random coil (%)
0 57.6 17.2 25.2
10 61.0 15.4 23.6
20 63.4 13.8 22.8
40 65.8 11.5 22.7
BSA may be affected by the polarity as well, leading to changes of the tertiary structure of BSA. 3.3.2. Synchronous Fluorescence Spectroscopy The synchronous fluorescence spectra (SFS) were obtained by scanning the excitation and emission monochromators simultaneously, providing the characteristic information about the microenvironment in vicinity of Tyr and Trp residues when the wavelength interval (Δλ) is 15 nm and 60 nm, respectively [24, 40]. The spectra of BSA-butachlor system were shown in Fig. 7. As seen in Fig. 7 (A), when butachlor concentrations increased, the fluorescence emission intensity of Trp residues decreased, along with a slight red shift, which was attributed to the exposure of the buried tryptophan moieties to a hydrophilic phase. However, in Fig. 7 (B), the fluorescence emission intensity of Tyr residues decreased with increased butachlor concentration yet no obvious shift was observed. The results suggested that there was slight change of the polarity around the Trp residues due to interaction between butachlor with BSA while the microenvironment of Tyr remained unchanged. 3.3.3. Three-dimensional Fluorescence Spectroscopy Three-dimensional fluorescence spectroscopy is a recent technique that provides information about fluorescence characteristics by simultaneously changing excitation and emission wavelength [41]. Three-dimensional fluorescence spectra of BSA in the absence (Fig. 8 (A)) and presence (Fig. 8 (B)) of butachlor were presented in Fig. 8. According to the figure, peak a is the Rayleigh scattering peak (λem = λ ex ) and peak b is the second-order scattering peak (λem = 2λex). Peak 1 (λex = 280 nm, λem = 340 nm) is the fluorescence peak that mainly represents the spectral behavior of the Trp residues. As shown in Fig. 8, the fluorescence intensity of peak 1 decreased obviously with the addition of butachlor, revealing that the microenvironment of tryptophan residues has been changed. The decrease of fluorescence intensity was consistent with the result of synchronous fluorescence spectra, showing that the interaction
3.3.4. Fourier Transform Infrared (FTIR) Spectra Measurement Fourier transform infrared (FTIR) is a well-defined tool for the determination of protein's secondary structure, which is maintained by the hydrogen bonding between carbonyl group and amide group. There are two unique bands, protein amides I band (1700–1600 cm−1, mainly C_O stretch) and amide II band (1550–1500 cm− 1, C\\N stretch coupled with N\\H bending mode), characterize the spectra of BSA. The FTIR spectra of BSA in the absence and presence of butachlor were accumulated over the range of 1200–2000 cm− 1 and the result was shown in Fig. 9. With the addition of butachlor, no major spectral shifting was observed at protein amide I bond. However, the peak within amide II bond moved slightly from 1540 cm−1 to 1550 cm−1 along with an obvious decrease in the intensity. The result indicated that the secondary structure of BSA was changed after the interaction with butachlor and this result agreed with that of the CD results. 3.4. Red-edge Excitation Shift Red edge excitation shift (REES) is a shift in the wavelength of maximum fluorescence emission toward higher wavelengths with an increase in the excitation wavelength [42]. The effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases [43]. For proteins, Trp residues show REES and the majority of the REES occurs at excitation wavelengths higher than 290 nm [44], avoiding the contribution of Tyr fluorescence. The REES measurements of BSA in the absence and presence with butachlor of two levels were conducted at room temperature and the results are shown in Table 4. As seen, the value of the REES for system in addition of butachlor was 10 nm, while the butachlor-free system didn't show a shift. This indicates that after treatment with butachlor, Trp residues in BSA acquired an environment where its mobility relative to the solvent molecules is restricted. So there were some conformational changes in BSA and more hydrogen bound formed around the Trp residues, which agrees with the results above. 3.5. Energy Transfer Between BSA and Butachlor Fluorescence resonance energy transfer (FRET) is a reliable way for evaluating the donor- acceptor distance as well as for studying the structure, conformation and spatial distribution of complex proteins. It
Fig. 7. Synchronous fluorescence spectrum of BSA in the absence and the presence of butachlor. BSA concentration was 2 × 10−6 M, numbers 1 to 5 indicate concentrations of butachlor: 0, 10, 20, 30 and 40 × 10−6 M respectively. (A) Δλ = 60 nm (B) Δλ = 15 nm.
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Fig. 8. Three-dimensional fluorescence spectra of BSA in the absence (A) and presence (B) of butachlor. The concentrations of BSA and butachlor were 2 × 10−6 M and 50 × 10−6 M, respectively.
is an interaction between the electronic states of two fluorophores in which the excitation energy is transferred from a donor to an acceptor without emission of a photon [41]. The efficiency of FRET depends mainly on the following factors: (1) the enough overlap between the donor (BSA) emission and the acceptor (ligand) absorption; (2) the small (no more than 8 nm) distance between donor and acceptor [21]. The energy transfer efficiency E is defined by the following equation: E ¼ 1− F=F 0 ¼ R60 = R60 þ r 6
ð6Þ
where F0 and F are the fluorescence intensities of BSA in the absence and presence of butachlor, respectively. r is the average distance between the donor (BSA) and the acceptor (butachlor). R0 is the Förster critical distance, at which the excitation energy transferred to the acceptor is 50%. R0 can be calculated from donor emission and acceptor absorption spectra by the Förster formula, Eq. (7): R60 ¼ 8:8 10−25 k n−4 JΦ 2
ð7Þ
where k2 is the spatial orientation factor of the dipole and k2 = 2/3. n is the refractive index of medium, n = 1.36. Φ is the fluorescence quantum yield of the donor, and for BSA, Φ = 0.15. J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor, which can be calculated by the following equation: J ¼ ∑F ðλÞεðλÞλ4
Δλ ∑F ðλÞΔλ
ð8Þ
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. The spectral overlap between the UV absorption spectrum of butachlor (acceptor) and the fluorescence emission spectrum of free BSA (donor) is shown in Fig. 10. The value of J of the BSA-butachlor system is determined by integrating the overlapped portion of the spectra in Fig. 10, which was 4.67 × 10− 23 cm3 M− 1. The value of E, R0 and r is 0.330, 0.100 nm and 0.113 nm, respectively. The average distances between butachlor and BSA is b8 nm and consistent with the rule 0.5R0 b r b 1.5R0 [26]. 4. Conclusions In this study, the interaction between BSA and butachlor was investigated by several spectroscopic methods. The experimental results revealed that butachlor molecules could effectively bind to BSA molecules and triggered static fluorescence quenching of BSA, and the van der Waals force and hydrogen bonds played a major role in the binding reaction. The binding constant K b and binding sites of butachlor with BSA were dependent with temperature and they were 5.158 × 105 and 1.372 at 303 K, respectively. The binding distance r between butachlor and BSA was 0.113 nm according to Table 4 Red edge excitation shift effects at λex = 295 nm and 310 nm, at 298 K. Maximum emission wavelength (nm)
Fig. 9. FTIR spectra of BSA in the absence (1) and the presence (2) of butachlor.
System
λex = 295 nm
λex = 310 nm
REES = Δλ (nm)
BSA BSA-butachlor (1:25)
340 340
340 350
0 10
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Fig. 10. Overlap of UV absorption (a) of butachlor and fluorescence emission spectrum (b) of BSA. The concentrations of butachlor and BSA were 40 × 10−-6 M and 2 × 10−6 M, respectively.
Förster's theory. In addition, the results also suggested that there was a rearrangement of BSA and more hydrogen bound formed around the Trp, resulting in a changed microenvironment of Trp residues. This study contributes to a better understanding of the ligand effects on herbicide-proteins interactions. Furthermore, it provides not only experimental basis on reasonably controlling the use of this kind of herbicide but also pavement for the designing of pesticide which is safe not only at the organism level but also at the molecular level. Acknowledgments The authors appreciate the financial support of the Project of Science and Technology Department of Zhejiang Province (2012C37058) and the Key Innovation Team of Science and Technology in Zhejiang Province (2010R50018) for financial support. References [1] A. Debnath, C.A. Das, D. Mukherjee, Persistence and effect of butachlor and Basalin on the activities of phosphate solubilizing microorganisms in wetland rice soil, Bull. Environ. Contam. Toxicol. 68 (2002) 766–770. [2] R.S. Mohanty, K. Bharati, S.B.T. Moorthy, B. Ramakrishnan, R.V. Rao, N. Sethunathan, K.T. Adhya, Effect of the herbicide butachlor on methane emission and ebullition flux from a direct-seeded flooded rice field, Biol. Fertil. Soils 33 (2001) 175–180. [3] A.K. Gogoi, H. Kalita, Effect of fertilizer and butachlor on weeds and yield of transplanted rice (Oryza sativa), Indian. J. Agron. 41 (1996) 230–232. [4] S. Chander, J. Pandey, Effect of herbicide and nitrogen on yield of scented rice (Oryza sativa) under different rice cultures, Indian. J. Agron. 41 (1996) 209–214. [5] A.R. Abdullah, C.M. Bajet, M.A. Matin, D.D. Nhan, A.H. Sulaiman, Ecotoxicology of pesticides in the tropical paddy field ecosystem, Environ. Toxicol. Chem. 16 (1997) 59–70. [6] M.E.A. Abigail, S.M. Samuel, C. Ramalingam, Addressing the environmental impacts of butachlor and the available remediation strategies: a systematic review, Int. J. Environ. Sci. Technol. 12 (2015) 4025–4036. [7] N.D.G. Chau, Z. Sebesvari, W. Amelung, F.G. Renaud, Pesticide pollution of multiple drinking water sources in the Mekong Delta, Vietnam: evidence from two provinces, Environ. Sci. Pollut. Res. 22 (2015) 9042–9058. [8] P.V. Toan, Z. Sebesvari, M. Bläsing, I. Rosendahl, F.G. Renaud, Pesticide management and their residues in sediments and surface and drinking water in the Mekong Delta, Vietnam, Sci. Total Environ. 452–453 (2013) 28–39. [9] C. Chen, Y. Wang, X. Zhao, Q. Wang, Y. Qian, Comparative and combined acute toxicity of butachlor, imidacloprid and chlorpyrifos on earthworm, Eisenia fetida, Chemosphere 100 (2014) 111–115. [10] G. Muthukaruppan, S. Janardhanan, G. Vijayalakshmi, Sublethal toxicity of the herbicide butachlor on the earthworm Perionyx sansibaricus and its histological changes (5 pp), J. Soils Sediments 5 (2005) 82–86. [11] H. He, G. Chen, J. Yu, J. He, X. Huang, S. Li, Q. Guo, T. Yu, H. Li, Individual and joint toxicity of three chloroacetanilide herbicides to freshwater cladoceran Daphnia carinata, Bull. Environ. Contam. Toxicol. 90 (2013) 344–350. [12] H. He, J. Yu, G. Chen, W. Li, J. He, H. Li, Acute toxicity of butachlor and atrazine to freshwater green alga Scenedesmus obliquus and cladoceran Daphnia carinata, Ecotoxicol. Environ. Saf. 80 (2012) 91–96.
[13] R.B. Geng, D. Yao, Q.Q. Xue, Acute toxicity of the pesticide dichlorvos and the herbicide butachlor to tadpoles of four anuran species, Bull. Environ. Contam. Toxicol. 75 (2005) 343–349. [14] W.Y. Liu, C.Y. Wang, T.S. Wang, G.M. Fellers, B.C. Lai, Y.C. Kam, Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan, Ecotoxicology 20 (2011) 377–384. [15] W. Tu, L. Niu, W. Liu, C. Xu, Embryonic exposure to butachlor in zebrafish (Danio rerio): endocrine disruption, developmental toxicity and immunotoxicity, Ecotoxicol. Environ. Saf. 89 (2013) 189–195. [16] S. Anbumani, M.N. Mohankumar, Cytogenotoxicity assessment of monocrotophos and butachlor at single and combined chronic exposures in the fish Catla catla (Hamilton), Environ. Sci. Pollut. Res. 22 (2015) 4964–4976. [17] H.D. Xu, J.S. Wang, M.H. Li, Y. Liu, T. Chen, A.Q. Jia, 1H NMR based metabolomics approach to study the toxic effects of herbicide butachlor on goldfish (Carassius auratus), Aquat. Toxicol. 159 (2015) 69–80. [18] S. Dwivedi, Q. Saquib, A.A. Al-Khedhairy, J. Musarrat, Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells, Toxicology 302 (2012) 77–87. [19] X.H. Yin, S.N. Li, L. Zhang, G.N. Zhu, H.S. Zhuang, Evaluation of DNA damage in Chinese toad (Bufo bufo gargarizans) after in vivo exposure to sublethal concentrations of four herbicides using the comet assay, Ecotoxicology 17 (2008) 280–286. [20] B. Chakraborty, S. Basu, Interaction of BSA with proflavin: a spectroscopic approach, J. Lumin. 129 (2009) 34–39. [21] J.H. Shi, J. Chen, J. Wang, Y.Y. Zhu, Q. Wang, Binding interaction of sorafenib with bovine serum albumin: spectroscopic methodologies and molecular docking, Spectrochim. Acta A 149 (2015) 630–637. [22] J. Gao, Y. Guo, J. Wang, Z. Wang, X. Jin, C. Cheng, Y. Li, K. Li, Spectroscopic analyses on interaction of o-Vanillin-D-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-LLevodopa Schiff Bases with bovine serum albumin (BSA), Spectrochim. Acta A 78 (2011) 1278–1286. [23] S.P. Boulos, T.A. Davis, J.A. Yang, S.E. Lohse, A.M. Alkilany, L.A. Holland, C.J. Murphy, Nanoparticle–protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces, Langmuir 29 (2013) 14984–14996. [24] C. Barakat, D. Patra, Combining time-resolved fluorescence with synchronous fluorescence spectroscopy to study bovine serum albumin-curcumin complex during unfolding and refolding processes, Luminescence 28 (2013) 149–155. [25] Z. Tian, F. Zang, W. Luo, Z. Zhao, Y. Wang, X. Xu, C. Wang, Spectroscopic study on the interaction between mononaphthalimide spermidine (MINS) and bovine serum albumin (BSA), J. Photochem. Photobiol. B 142 (2015) 103–109. [26] N. Shahabadi, M. Maghsudi, S. Rouhani, Study on the interaction of food colourant quinoline yellow with bovine serum albumin by spectroscopic techniques, Food Chem. 135 (2012) 1836–1841. [27] R. Kumaran, P. Ramamurthy, Photophysical studies on the interaction of amides with bovine serum albumin (BSA) in aqueous solution: fluorescence quenching and protein unfolding, J. Lumin. 148 (2014) 277–284. [28] H. Xu, S.-L. Gao, J.-B. Lv, Q.-W. Liu, Y. Zuo, X. Wang, Spectroscopic investigations on the mechanism of interaction of crystal violet with bovine serum albumin, J. Mol. Struct. 919 (2009) 334–338. [29] L. Ding, P. Zhou, H. Zhan, X. Zhao, C. Chen, Z. He, Systematic investigation of the toxicity interaction of ZnSe@ZnS QDs on BSA by spectroscopic and microcalorimetry techniques, Chemosphere 92 (2013) 892–897. [30] F.L. Cui, J. Fan, J.P. Li, Z.D. Hu, Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy, Bioorg. Med. Chem. 12 (2004) 151–157. [31] Q. Wang, X. Zhang, X. Zhou, T. Fang, P. Liu, P. Liu, X. Min, X. Li, Interaction of different thiol-capped CdTe quantum dots with bovine serum albumin, J. Lumin. 132 (2012) 1695–1700. [32] X.L. Wei, J.B. Xiao, Y. Wang, Y. Bai, Which model based on fluorescence quenching is suitable to study the interaction between trans-resveratrol and BSA? Spectrochim. Acta A 75 (2010) 299–304. [33] F. Ahmad, Y. Zhou, Z. Ling, Q. Xiang, X. Zhou, Systematic elucidation of interactive unfolding and corona formation of bovine serum albumin with cobalt ferrite nanoparticles, RSC Adv. 6 (2016) 35719–35730. [34] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [35] G. Siligardi, R. Hussain, S.G. Patching, M.K. Phillips-Jones, Ligand- and drug-binding studies of membrane proteins revealed through circular dichroism spectroscopy, Biochim. Biophys. Acta Biomembr. 1838 (2014) 34–42. [36] C. Bertucci, M. Pistolozzi, A. De Simone, Circular dichroism in drug discovery and development: an abridged review, Anal. Bioanal. Chem. 398 (2010) 155–166. [37] S. Monti, F. Manoli, S. Sortino, R. Morrone, G. Nicolosi, Binding of a chiral drug to a protein: an investigation of the 2-(3-benzoylphenyl)propionic acid/bovine serum albumin system by circular dichroism and fluorescence, Phys. Chem. Chem. Phys. 7 (2005) 4002–4008. [38] B. Ranjbar, P. Gill, Circular dichroism techniques: biomolecular and nanostructural analyses- a review, Chem. Biol. Drug Des. 74 (2009) 101–120. [39] M. Sarkar, S.S. Paul, K.K. Mukherjea, Interaction of bovine serum albumin with a psychotropic drug alprazolam: physicochemical, photophysical and molecular docking studies, J. Lumin. 142 (2013) 220–230. [40] M.D. Meti, K.S. Byadagi, S.T. Nandibewoor, S.A. Chimatadar, Multi-spectral characterization & effect of metal ions on the binding of bovine serum albumin upon interaction with a lincosamide antibiotic drug, clindamycin phosphate, J. Photochem. Photobiol. B 138 (2014) 324–330.
X. Liu et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 332–339 [41] D. Sarkar, Probing the interaction of a globular protein with a small fluorescent probe in the presence of silver nanoparticles: spectroscopic characterization of its domain specific association and dissociation, RSC Adv. 3 (2013) 24389–24399. [42] A. Chattopadhyay, S. Mukherjee, Fluorophore environments in membrane-bound probes: a red edge excitation shift study, Biochemistry 32 (1993) 3804–3811. [43] G. Maglia, A. Jonckheer, M. De Maeyer, J.M. Frère, Y. Engelborghs, An unusual rededge excitation and time-dependent Stokes shift in the single tryptophan mutant
339
protein DD-carboxypeptidase from Streptomyces: the role of dynamics and tryptophan rotamers, Protein Sci. 17 (2008) 352–361. [44] S.K. Patra, M.K. Pal, Red edge excitation shift emission spectroscopic investigation of serum albumins and serum albumin-bilirubin complexes, Spectrochim. Acta A Mol. Biomol. Spectrosc. 53A (1997) 1609–1614.
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Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Comprehensive spectroscopic probing the interaction and conformation impairment of bovine serum albumin (BSA) by herbicide butachlor Xiaoyi Liu a, Zhaoxing Ling a, Xing Zhou b, Farooq Ahmad a, Ying Zhou a,c,⁎ a b c
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China College of Atmospheric Science, Lanzhou University, Lanzhou, China Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou, China
a r t i c l e
i n f o
Article history: Received 11 January 2016 Received in revised form 4 July 2016 Accepted 7 July 2016 Available online 09 July 2016 Keywords: Interaction Butachlor Herbicide Pesticide Bovine serum albumin (BSA) Spectroscopic method
a b s t r a c t Butachlor is an effective herbicide to deal with undesired weeds selectively and is used at high levels in Asian countries. However, its interaction and impairment effect on BSA was still not clear. In this study, we investigated the interaction between butachlor and bovine serum albumin (BSA) by multi-spectroscopic methods including UV absorption, circular dichroism (CD) spectra, Fourier transform infrared (FTIR) spectra and fluorescence spectra under physiological conditions (pH = 7.4). The results revealed that there was a static quenching of BSA induced by butachlor stemmed from the formation of complex. Based on thermodynamic data, the interaction of butachlor with BSA was due to happen, and van der Waals force as well as hydrogen bond were the major forces contributed to the interaction. The binding constant Kb and number of binding site of butachlor with BSA were 5.158 × 105 and 1.372 at 303 K, respectively. The distance r between donor (BSA) and acceptor (butachlor) was 0.113 nm, obtained according to the Förster theory. The results revealed that butachlor induced conformational changes in BSA but the secondary structure of BSA was still retained. In addition, the microenvironment around chromophore residues of BSA, for example, tryptophan, changed as well, resulting from the formation of more hydrogen bonds. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Herbicides are applied to fields to get rid of undesired weeds selectively without injuring crops [1]. Butachlor, whose chemical name is N-butoxymethyl-2-chloro-2′, 6′-diethyl acetanilide (Fig. 1), is a preemergence herbicide and belongs to the chloroacetanilide herbicides of which the effect is inhibiting protein synthesis in developing plant tissue [2]. Field experiments have confirmed that butachlor can control the weeds population effectively and increase the crop yield [3,4]. Butachlor is used at high levels in Asian countries like China and India [2,5], and the consumption of butachlor is nearly 4.5 × 107 kg per year in Asia alone [6], resulting in the inevitable entrance of butachlor into environment via routes like agricultural run-off, leaching and rainfall. Some herbicides including butachlor have been detected in soil, water and sediment in several countries [6,7]. The environmental concentration of butachlor detected in the Mekong Delta area in Vietnam ranged from 0.01 μg L−1 to 0.59 μg L−1 [8]. Once released into the environment, ⁎ Corresponding author at: Research Center of Analysis and Measurement, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou 310014, Zhejiang Province, China. E-mail address: [email protected] (Y. Zhou).
http://dx.doi.org/10.1016/j.jphotobiol.2016.07.005 1011-1344/© 2016 Elsevier B.V. All rights reserved.
butachlor potentially exerts toxic effects on organisms like earthworm [9,10], flea, alga [11,12], frog [13,14], fish [15–17] and even humans [18]. By now, there have been a few reports on the damage of butachlor on DNA [18,19]. Apart from DNA, many functional proteins are also considered as targets or carriers of drugs. However, most of the interaction studies are focused on pharmaceuticals and sophisticated materials while those of pesticides with biological molecules (proteins) are still mostly ignored and the interaction between butachlor and protein has not been seen. Serum albumin (SA), which is synthesized in liver, is one of the most abundant carrier proteins in blood plasma [20]. It plays a vital role in life body as it combines and delivers endogenous and exogenous compounds present in blood. Generally, the weak binding between ligands and serum albumin could cause ligands' poor distribution while the strong binding results in the decreasing concentration of free ligands in plasma [21]. Consequently, the investigation of ligands-serum albumin interaction is a fundamental step to understand the pharmacology of drugs clearly. Bovine serum albumin (BSA) is used in many studies because of its highly stability, lower cost, possibility of being isolated in highly pure form and unusual ligand-binding properties [20,22,23] and what's the most important is that BSA is in structural similarity with human serum albumin (HAS) in 76% [22].
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Fig. 1. The structure of butachlor.
In this study, BSA was chosen as a target protein molecule. The interaction between butachlor and BSA was investigated using spectroscopic methods including UV, circular dichroism (CD), FTIR, and fluorescence spectroscopy. The structural changes of BSA under the effect of butachlor were estimated. The binding parameters and main type of binding force were also obtained. This study is of great significance to offer a better understanding of the mechanism of butachlor's toxicity and also experimental basis for the designing of pesticide which is safe not only at the organism level but also at the molecular level. 2. Materials and Methods 2.1. Materials Butachlor (97% purity) was obtained from Hangzhou Qingfeng Agrochemical Limited Company (China). A stock solution of butachlor (5 × 10−3 M) was prepared in analytical-grade ethyl alcohol and stored at 4 °C in darkness. The test solutions for the following experiments were prepared by diluting the stock solution in HBS buffer (HEPES buffer solution, containing 2 × 10−3 M HEPES buffer and 1 × 10−3 M NaCl, pH = 7.4). BSA was purchased from Aladdin (Shanghai, China) and BSA stock solution (5 × 10−5 M) was prepared in HBS buffer as well. BSA solutions were prepared based on its molecular weight of 67,000. Ultrapure water produced by a Milli-Q system was used throughout the study. 2.2. Procedures 2.2.1. UV–Vis Measurement The UV spectra of all BSA solutions in the absence and presence of butachlor were recorded on a UV-2450 spectrophotometer (Shimadzu, Japan) with a 1 cm quartz cell at room temperature. The wavelength ranged from 190 to 350 nm. BSA concentration was fixed at 2 × 10− 6 M while the butachlor concentration varied from 0 to 40 × 10−6 M (0, 4, 6, 10, 20, 40 × 10−6 M respectively). All these solutions were prepared in HBS buffer. The corresponding solutions of butachlor were used as the reference.
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excitation wavelength was at 280 nm. Mixed solutions were prepared by adding appropriate volumes of butachlor and BSA stock solutions to HBS buffer to obtain mixture containing butachlor from 0 to 50 × 10−6 M (0, 5, 10, 15, 20, 30, 40 and 50 × 10−6 M, respectively) and the BSA 2 × 10−6 M. Then 200 μL of these solutions was transferred with a suited pipette to a 96-well plate. Each sample was repeated three times and each spectrum was the average of three scans. However, inner filter effect (IFE), which is caused when the quencher in mixed solution has ultraviolet absorption at excitation and emission wavelength, would reduce the fluorescence intensity [21]. In this work, all fluorescence intensities were corrected for IFE by the following equation: F cor ¼ F obs 10ðA1 þA2 Þ=2
ð1Þ
where Fcor is the corrected fluorescence intensity, Fobs is the fluorescence intensity obtained in experiment, A1 and A2 are the absorption of the fluorescence determination system at 280 nm and at emission wavelength, respectively.
2.2.4. Synchronous and Three-dimensional Fluorescence Measurement The synchronous and three-dimensional fluorescence spectra (SFS) of butachlor-BSA solutions were recorded on a FluoroMax-4 (Horiba Jobin Yvon, France) equipped with a 1 cm quartz cell by scanning two different intervals of Δλ (15 nm and 60 nm) at room temperature. The wavelength ranges were 280–350 nm (Δλ = 15 nm) and 310– 510 nm (Δλ = 60 nm), respectively, characterizing the properties of tyrosine (Tyr) and tryptophan (Trp), respectively [21,24]. Concentration of BSA was 2 × 10−6 M while concentrations of butachlor were 0, 10, 20, 30 and 40 × 10−6 M, respectively. Three-dimensional fluorescence spectroscopy was conducted with setting excitation and emission wavelength range as 250–350 nm and 250–500 nm, respectively.
2.2.5. Fourier Transform Infrared (FTIR) Spectra Measurement At room temperature, the Fourier transform infrared (FTIR) spectra of BSA in the presence and absence of butachlor were measured on Nicolet 6700 FTIR spectrophotometer (Thermo, America) in the range of 1200–2000 cm−1. The spectrum of BSA-butachlor was obtained by subtracting the absorption of corresponding butachlor solution from the spectrum and the spectrum of BSA was obtained by subtracting the absorption of buffer.
3. Results and Discussion 3.1. UV Absorption Spectra
2.2.2. Circular Dichroism (CD) Measurement The CD measurements of BSA solutions in the absence and the presence of butachlor were measured on a J-815 CD spectrometer (Jasco, Japan) with a 0.1 cm quartz cell at room temperature with the wavelength from 200 to 350 nm, under constant nitrogen flush. The butachlor concentrations were 0, 10, 20 and 40 × 10−6 M, respectively, while the concentration of BSA was fixed at 2 × 10−6 M. HBS buffer was used as a baseline. With scanning speed of 100 nm/min and intervals of 1.0 nm, each spectrum was the average of three scans, corrected by HBS buffer blanks. 2.2.3. Fluorescence Measurement The fluorescence spectra measurements of BSA-butachlor mixed solutions at different temperatures (298, 303 and 308 K) were performed on a microplate reader M2 (Molecular devices, USA) with a 96-well plate. The emission wavelength ranged from 290 to 500 nm while the
UV absorption spectroscopy is a simple way to explore the conformational changes and the complex formation. The UV spectra of mixed solutions of BSA (2 × 10−6 M) with different concentrations of butachlor were shown in Fig. 2. From the figure it can be seen that the BSA solution has two absorption bands near 203 nm and 280 nm respectively, though the later one was very weak. The strong absorption peak near 203 nm reflects the framework conformation of BSA and the weak one around 280 nm is due to the π → π⁎ transition of aromatic amino acids such as Trp, Tyr and Phe [25,26]. Moreover, the UV absorption of BSA decreased regularly when the concentration of butachlor increases. Red shift of maximum absorption of BSA at 203 nm (Δλ = 3 nm) was also noticed and the red shift of peak absorption at 280 nm was not obvious. They were triggered possibly due to the complex formation between BSA and butachlor, leading to the conformational change of BSA.
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quenching. Dynamic quenching results from molecular collision between quenchers and excited-state fluorophores. Static quenching, however, is caused by the formation of ground-state complex of fluorophores and quenchers [28]. Dynamic quenching constant and static quenching constant can be distinguished by fluorescent lifetime and their dependence along with the change of temperature [29]. The dynamic quenching constant increases with the increase of temperature as higher temperature lead to a larger diffusion coefficient. On the contrary, the static quenching constant decreases when temperature increases because the higher temperature would cause decreasing stability of complexes [21]. Therefore, fluorescence spectra of BSA-butachlor system at different temperatures were measured to identify the changing trend of quenching constant (K sv). Fluorescence quenching is described by the Stern-Volmer equation [30]: F 0 =F ¼ 1 þ K q τ0 ½Q ¼ 1 þ K sv ½Q Fig. 2. UV absorption of BSA in the absence and the presence of butachlor. Numbers 1–6 indicate concentration of butachlor as 0, 4, 6, 10, 20, and 40 × 10−6 M, respectively. BSA concentration applied was 2 × 10−6 M.
3.2. Fluorescence Spectra 3.2.1. Fluorescence Quenching The fluorescence quenching measurement is a sensitive method for investigating the binding interaction of small molecules with proteins. There are two tryptophan residues in BSA (Trp-213 and Trp-134) [27] have relatively strong fluorescence intensity and are sensitive to the microenvironment changes. The fluorescence spectra of BSA with addition of varied concentrations of butachlor were shown in Fig. 3. As seen, under the excitation wavelength of 280 nm, the emission peak of BSA appeared around the wavelength of 340 nm. And the fluorescence intensity of BSA decreased gradually when the butachlor increased yet without obvious changes of the maximum emission wavelength and the shape of peak. The result of fluorescence quenching indicated that the interaction between BSA and butachlor caused changes in the microenvironment around Trp within BSA.
ð2Þ
where F0 and F represent the fluorescence intensities in the absence and the presence of quencher (butachlor), respectively. Kq is the bimolecular quenching rate constant, Ksv the Stern-Volmer quenching constant, τ0 the average fluorescent lifetime of protein without quencher, which is generally 6 × 10− 9 s for BSA [21], and [Q] the concentration of quencher. The Stern-Volmer plots of F0/F versus [Q] at three temperatures, 298 K, 303 K and 308 K, were shown in Fig. 4, and the values of quenching constant Ksv and Kq at different temperatures were calculated by slope of Fig. 4 and the results were presented in Table 1. The results of the study, where the Ksv decreased with the increase of temperature, suggested that the quenching mechanism of BSA fluorescence by butachlor was a static quenching process. What's more, the values of Kq were higher than that of the maximum scattering collision quenching constant, 2 × 1010 M−1 s−1 [31], indicating that the quenching was initiated by the formation of a complex rather than the dynamic collision. 3.2.3. Binding Constant and Number of Binding Sites When small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Kb) and the number of binding sites (n) fit the double-logarithm equation [32]:
3.2.2. Fluorescence Quenching Mechanism Protein contains endogenous fluorophores. Fluorescence quenching occurs when there are protein-drug interactions such as complex formation, collision and energy transfer, etc. Generally, fluorescence quenching includes dynamic quenching and static
where Kb is the binding constant and n is the number of binding sites. The plots of log[(F0 − F) ∕ F] against log[Q] at three temperatures
Fig. 3. Fluorescence spectra of BSA in the absence and the presence of butachlor. Numbers 1 to 8 indicate concentrations of butachlor: 0, 5, 10, 15, 20, 30, 40 and 50 × 10−6 M, respectively. BSA concentration was 2 × 10−6 M.
Fig. 4. Stern-Volmer linear plot of fluorescence quenching of BSA by butachlor at different temperatures. [BSA] = 2 × 10−6 M, λex = 335 nm.
log½ð F 0 −F Þ=F ¼ logK b þ n∙ log½Q
ð3Þ
X. Liu et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 332–339 Table 1 The Stern-Volmer quenching constants of the butachlor-BSA system at different temperatures. T (K)
Ksv (M−1)
Kq (M−1 s−1)
R2
298 303 308
1.579 × 104 1.311 × 104 1.113 × 104
2.632 × 1012 2.185 × 1012 1.855 × 1012
0.9789 0.9670 0.9695
(298, 303 and 308 K, respectively) were presented in Fig. 5. The values of Kb and n can be obtained from the intercept and slope of the above equation and were shown in Table 2. As results shown, both values of K b and n decreased with increase in temperature. This indicated that temperature effectively decreased the number of binding sites as well as the stability of the complex. Furthermore, these results also demonstrated that static quenching is predominant [33], which is consistent with the results above. 3.2.4. Thermodynamic Parameters and Binding Force Generally, the types of acting force between exogenous small molecular ligands and biological macromolecules are non-covalent forces including hydrogen bonds, van der Waals force, hydrophobic force, electrostatic interactions and so forth. The thermodynamic parameters, enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG) could be used as main evidences to determine the type of major acting forces [34]. The ΔH is considered to be temperature independent in a small temperature range [26,31], thus ΔH, ΔS and ΔG can be obtained from the van't Hoff equation: lnK b ¼ −ΔH=RT þ ΔS=R
ð4Þ
ΔG ¼ ΔH−TΔS
ð5Þ
where ΔH and ΔS are the standard enthalpy and entropy change of the reaction, respectively and ΔG is free energy change. R is the gas constant and T is the temperature. The plot of lnKb versus 1/T at 298 K, 303 K and 308 K was made and ΔH, ΔS and ΔG calculated according to the slope and intercept were shown in Table 2 as well. According to Ross's [34] study, the negative ΔG values of suggested that the binding process of butachlor to BSA is spontaneous, meaning that the interaction between butachlor and BSA was due to happen. Negative values of both ΔH and ΔS suggest that the van
Fig. 5. The plot of log[(F0 − F)/F] versus log[Q] for quenching process of butachlor with BSA at 298, 303 and 308 K. [BSA] = 2 × 10−6 M, λex = 335 nm.
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Table 2 Binding constants Kb, number of binding sites n and the thermodynamic parameters of BSA-butachlor system at different temperatures.
T (K)
Kb (L mol−1)
n
R
298 303 308
7.513 × 105 5.158 × 105 2.043 × 105
1.412 1.372 1.298
0.9803 0.9607 0.9796
ΔS (J mol−1 K−1)
ΔH (kJ mol−1)
ΔG (kJ mol−1)
−219.1
−99.12
−33.83 −32.73 −31.64
der Waals force and hydrogen bonds played the major role in the interaction. 3.3. Conformational Change of BSA Induced by Butachlor 3.3.1. CD Measurement Circular dichroism (CD) is seen as a sensitive and powerful method to investigate the conformational changes in proteins and chiral molecules [35,36]. Therefore, CD spectra were carried out to observe the conformation change of BSA in the presence of butachlor and the results were shown in Fig. 6. From Fig. 6 it can be found that the CD spectra of BSA showed two negative bands in the UV region near 208 nm and 220 nm which are characteristic of α-helical structure of protein [25,37]. The two peaks are contributed by the n → π* transfer for the peptide bond of α-helix [31,38]. From Fig. 6, it can be noticed that the CD spectra of BSA with or without butachlor were similar in shape, indicating that the BSA structure was predominantly α-helix. Besides, the CD signals rose when butachlor concentration increased but without any significant shift. The secondary structures, calculated on the basis of Yang's equation, showed that the original BSA has an α-helix content of 57.6%, beta content of 17.2% as well as turn and random content of 25.2%. With the increase of the butachlor concentration, the αhelix content increased by 8.2% and beta content decreased by 5.7% (Table 3). The increased α-helix and decreased beta content in BSA may suggest the formation of hydrogen bond, and the BSA structure became refolding. In addition, the CD spectra on the wavelength range from 240 nm to 350 nm reflect the information of tertiary structure of protein [21]. As seen, the intensities of this character band increased as well. In conclusion, the higher CD singles has shown that there was an increase in the α-helix content [39], hinting a trend of BSA refolding to adopt an α-helix rich structure. This conformational rearrangement of
Fig. 6. CD spectra of BSA in the absence and the presence of butachlor. BSA concentration applied was 2 × 10−6 M. The number 1 indicates 40 × 10−6 M butachlor solution alone, and numbers 2 to 5 indicate mixture with concentrations of butachlor as 0, 10, 20, and 40 × 10−6 M, respectively.
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between butachlor and BSA induced certain conformational change in BSA.
Table 3 Secondary structure of butachlor-BSA complex based on CD data. Butachlor con. (×10−6 M) α-Helix (%) Beta (%) Random coil (%)
0 57.6 17.2 25.2
10 61.0 15.4 23.6
20 63.4 13.8 22.8
40 65.8 11.5 22.7
BSA may be affected by the polarity as well, leading to changes of the tertiary structure of BSA. 3.3.2. Synchronous Fluorescence Spectroscopy The synchronous fluorescence spectra (SFS) were obtained by scanning the excitation and emission monochromators simultaneously, providing the characteristic information about the microenvironment in vicinity of Tyr and Trp residues when the wavelength interval (Δλ) is 15 nm and 60 nm, respectively [24, 40]. The spectra of BSA-butachlor system were shown in Fig. 7. As seen in Fig. 7 (A), when butachlor concentrations increased, the fluorescence emission intensity of Trp residues decreased, along with a slight red shift, which was attributed to the exposure of the buried tryptophan moieties to a hydrophilic phase. However, in Fig. 7 (B), the fluorescence emission intensity of Tyr residues decreased with increased butachlor concentration yet no obvious shift was observed. The results suggested that there was slight change of the polarity around the Trp residues due to interaction between butachlor with BSA while the microenvironment of Tyr remained unchanged. 3.3.3. Three-dimensional Fluorescence Spectroscopy Three-dimensional fluorescence spectroscopy is a recent technique that provides information about fluorescence characteristics by simultaneously changing excitation and emission wavelength [41]. Three-dimensional fluorescence spectra of BSA in the absence (Fig. 8 (A)) and presence (Fig. 8 (B)) of butachlor were presented in Fig. 8. According to the figure, peak a is the Rayleigh scattering peak (λem = λ ex ) and peak b is the second-order scattering peak (λem = 2λex). Peak 1 (λex = 280 nm, λem = 340 nm) is the fluorescence peak that mainly represents the spectral behavior of the Trp residues. As shown in Fig. 8, the fluorescence intensity of peak 1 decreased obviously with the addition of butachlor, revealing that the microenvironment of tryptophan residues has been changed. The decrease of fluorescence intensity was consistent with the result of synchronous fluorescence spectra, showing that the interaction
3.3.4. Fourier Transform Infrared (FTIR) Spectra Measurement Fourier transform infrared (FTIR) is a well-defined tool for the determination of protein's secondary structure, which is maintained by the hydrogen bonding between carbonyl group and amide group. There are two unique bands, protein amides I band (1700–1600 cm−1, mainly C_O stretch) and amide II band (1550–1500 cm− 1, C\\N stretch coupled with N\\H bending mode), characterize the spectra of BSA. The FTIR spectra of BSA in the absence and presence of butachlor were accumulated over the range of 1200–2000 cm− 1 and the result was shown in Fig. 9. With the addition of butachlor, no major spectral shifting was observed at protein amide I bond. However, the peak within amide II bond moved slightly from 1540 cm−1 to 1550 cm−1 along with an obvious decrease in the intensity. The result indicated that the secondary structure of BSA was changed after the interaction with butachlor and this result agreed with that of the CD results. 3.4. Red-edge Excitation Shift Red edge excitation shift (REES) is a shift in the wavelength of maximum fluorescence emission toward higher wavelengths with an increase in the excitation wavelength [42]. The effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases [43]. For proteins, Trp residues show REES and the majority of the REES occurs at excitation wavelengths higher than 290 nm [44], avoiding the contribution of Tyr fluorescence. The REES measurements of BSA in the absence and presence with butachlor of two levels were conducted at room temperature and the results are shown in Table 4. As seen, the value of the REES for system in addition of butachlor was 10 nm, while the butachlor-free system didn't show a shift. This indicates that after treatment with butachlor, Trp residues in BSA acquired an environment where its mobility relative to the solvent molecules is restricted. So there were some conformational changes in BSA and more hydrogen bound formed around the Trp residues, which agrees with the results above. 3.5. Energy Transfer Between BSA and Butachlor Fluorescence resonance energy transfer (FRET) is a reliable way for evaluating the donor- acceptor distance as well as for studying the structure, conformation and spatial distribution of complex proteins. It
Fig. 7. Synchronous fluorescence spectrum of BSA in the absence and the presence of butachlor. BSA concentration was 2 × 10−6 M, numbers 1 to 5 indicate concentrations of butachlor: 0, 10, 20, 30 and 40 × 10−6 M respectively. (A) Δλ = 60 nm (B) Δλ = 15 nm.
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337
Fig. 8. Three-dimensional fluorescence spectra of BSA in the absence (A) and presence (B) of butachlor. The concentrations of BSA and butachlor were 2 × 10−6 M and 50 × 10−6 M, respectively.
is an interaction between the electronic states of two fluorophores in which the excitation energy is transferred from a donor to an acceptor without emission of a photon [41]. The efficiency of FRET depends mainly on the following factors: (1) the enough overlap between the donor (BSA) emission and the acceptor (ligand) absorption; (2) the small (no more than 8 nm) distance between donor and acceptor [21]. The energy transfer efficiency E is defined by the following equation: E ¼ 1− F=F 0 ¼ R60 = R60 þ r 6
ð6Þ
where F0 and F are the fluorescence intensities of BSA in the absence and presence of butachlor, respectively. r is the average distance between the donor (BSA) and the acceptor (butachlor). R0 is the Förster critical distance, at which the excitation energy transferred to the acceptor is 50%. R0 can be calculated from donor emission and acceptor absorption spectra by the Förster formula, Eq. (7): R60 ¼ 8:8 10−25 k n−4 JΦ 2
ð7Þ
where k2 is the spatial orientation factor of the dipole and k2 = 2/3. n is the refractive index of medium, n = 1.36. Φ is the fluorescence quantum yield of the donor, and for BSA, Φ = 0.15. J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor, which can be calculated by the following equation: J ¼ ∑F ðλÞεðλÞλ4
Δλ ∑F ðλÞΔλ
ð8Þ
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. The spectral overlap between the UV absorption spectrum of butachlor (acceptor) and the fluorescence emission spectrum of free BSA (donor) is shown in Fig. 10. The value of J of the BSA-butachlor system is determined by integrating the overlapped portion of the spectra in Fig. 10, which was 4.67 × 10− 23 cm3 M− 1. The value of E, R0 and r is 0.330, 0.100 nm and 0.113 nm, respectively. The average distances between butachlor and BSA is b8 nm and consistent with the rule 0.5R0 b r b 1.5R0 [26]. 4. Conclusions In this study, the interaction between BSA and butachlor was investigated by several spectroscopic methods. The experimental results revealed that butachlor molecules could effectively bind to BSA molecules and triggered static fluorescence quenching of BSA, and the van der Waals force and hydrogen bonds played a major role in the binding reaction. The binding constant K b and binding sites of butachlor with BSA were dependent with temperature and they were 5.158 × 105 and 1.372 at 303 K, respectively. The binding distance r between butachlor and BSA was 0.113 nm according to Table 4 Red edge excitation shift effects at λex = 295 nm and 310 nm, at 298 K. Maximum emission wavelength (nm)
Fig. 9. FTIR spectra of BSA in the absence (1) and the presence (2) of butachlor.
System
λex = 295 nm
λex = 310 nm
REES = Δλ (nm)
BSA BSA-butachlor (1:25)
340 340
340 350
0 10
338
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Fig. 10. Overlap of UV absorption (a) of butachlor and fluorescence emission spectrum (b) of BSA. The concentrations of butachlor and BSA were 40 × 10−-6 M and 2 × 10−6 M, respectively.
Förster's theory. In addition, the results also suggested that there was a rearrangement of BSA and more hydrogen bound formed around the Trp, resulting in a changed microenvironment of Trp residues. This study contributes to a better understanding of the ligand effects on herbicide-proteins interactions. Furthermore, it provides not only experimental basis on reasonably controlling the use of this kind of herbicide but also pavement for the designing of pesticide which is safe not only at the organism level but also at the molecular level. Acknowledgments The authors appreciate the financial support of the Project of Science and Technology Department of Zhejiang Province (2012C37058) and the Key Innovation Team of Science and Technology in Zhejiang Province (2010R50018) for financial support. References [1] A. Debnath, C.A. Das, D. Mukherjee, Persistence and effect of butachlor and Basalin on the activities of phosphate solubilizing microorganisms in wetland rice soil, Bull. Environ. Contam. Toxicol. 68 (2002) 766–770. [2] R.S. Mohanty, K. Bharati, S.B.T. Moorthy, B. Ramakrishnan, R.V. Rao, N. Sethunathan, K.T. Adhya, Effect of the herbicide butachlor on methane emission and ebullition flux from a direct-seeded flooded rice field, Biol. Fertil. Soils 33 (2001) 175–180. [3] A.K. Gogoi, H. Kalita, Effect of fertilizer and butachlor on weeds and yield of transplanted rice (Oryza sativa), Indian. J. Agron. 41 (1996) 230–232. [4] S. Chander, J. Pandey, Effect of herbicide and nitrogen on yield of scented rice (Oryza sativa) under different rice cultures, Indian. J. Agron. 41 (1996) 209–214. [5] A.R. Abdullah, C.M. Bajet, M.A. Matin, D.D. Nhan, A.H. Sulaiman, Ecotoxicology of pesticides in the tropical paddy field ecosystem, Environ. Toxicol. Chem. 16 (1997) 59–70. [6] M.E.A. Abigail, S.M. Samuel, C. Ramalingam, Addressing the environmental impacts of butachlor and the available remediation strategies: a systematic review, Int. J. Environ. Sci. Technol. 12 (2015) 4025–4036. [7] N.D.G. Chau, Z. Sebesvari, W. Amelung, F.G. Renaud, Pesticide pollution of multiple drinking water sources in the Mekong Delta, Vietnam: evidence from two provinces, Environ. Sci. Pollut. Res. 22 (2015) 9042–9058. [8] P.V. Toan, Z. Sebesvari, M. Bläsing, I. Rosendahl, F.G. Renaud, Pesticide management and their residues in sediments and surface and drinking water in the Mekong Delta, Vietnam, Sci. Total Environ. 452–453 (2013) 28–39. [9] C. Chen, Y. Wang, X. Zhao, Q. Wang, Y. Qian, Comparative and combined acute toxicity of butachlor, imidacloprid and chlorpyrifos on earthworm, Eisenia fetida, Chemosphere 100 (2014) 111–115. [10] G. Muthukaruppan, S. Janardhanan, G. Vijayalakshmi, Sublethal toxicity of the herbicide butachlor on the earthworm Perionyx sansibaricus and its histological changes (5 pp), J. Soils Sediments 5 (2005) 82–86. [11] H. He, G. Chen, J. Yu, J. He, X. Huang, S. Li, Q. Guo, T. Yu, H. Li, Individual and joint toxicity of three chloroacetanilide herbicides to freshwater cladoceran Daphnia carinata, Bull. Environ. Contam. Toxicol. 90 (2013) 344–350. [12] H. He, J. Yu, G. Chen, W. Li, J. He, H. Li, Acute toxicity of butachlor and atrazine to freshwater green alga Scenedesmus obliquus and cladoceran Daphnia carinata, Ecotoxicol. Environ. Saf. 80 (2012) 91–96.
[13] R.B. Geng, D. Yao, Q.Q. Xue, Acute toxicity of the pesticide dichlorvos and the herbicide butachlor to tadpoles of four anuran species, Bull. Environ. Contam. Toxicol. 75 (2005) 343–349. [14] W.Y. Liu, C.Y. Wang, T.S. Wang, G.M. Fellers, B.C. Lai, Y.C. Kam, Impacts of the herbicide butachlor on the larvae of a paddy field breeding frog (Fejervarya limnocharis) in subtropical Taiwan, Ecotoxicology 20 (2011) 377–384. [15] W. Tu, L. Niu, W. Liu, C. Xu, Embryonic exposure to butachlor in zebrafish (Danio rerio): endocrine disruption, developmental toxicity and immunotoxicity, Ecotoxicol. Environ. Saf. 89 (2013) 189–195. [16] S. Anbumani, M.N. Mohankumar, Cytogenotoxicity assessment of monocrotophos and butachlor at single and combined chronic exposures in the fish Catla catla (Hamilton), Environ. Sci. Pollut. Res. 22 (2015) 4964–4976. [17] H.D. Xu, J.S. Wang, M.H. Li, Y. Liu, T. Chen, A.Q. Jia, 1H NMR based metabolomics approach to study the toxic effects of herbicide butachlor on goldfish (Carassius auratus), Aquat. Toxicol. 159 (2015) 69–80. [18] S. Dwivedi, Q. Saquib, A.A. Al-Khedhairy, J. Musarrat, Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells, Toxicology 302 (2012) 77–87. [19] X.H. Yin, S.N. Li, L. Zhang, G.N. Zhu, H.S. Zhuang, Evaluation of DNA damage in Chinese toad (Bufo bufo gargarizans) after in vivo exposure to sublethal concentrations of four herbicides using the comet assay, Ecotoxicology 17 (2008) 280–286. [20] B. Chakraborty, S. Basu, Interaction of BSA with proflavin: a spectroscopic approach, J. Lumin. 129 (2009) 34–39. [21] J.H. Shi, J. Chen, J. Wang, Y.Y. Zhu, Q. Wang, Binding interaction of sorafenib with bovine serum albumin: spectroscopic methodologies and molecular docking, Spectrochim. Acta A 149 (2015) 630–637. [22] J. Gao, Y. Guo, J. Wang, Z. Wang, X. Jin, C. Cheng, Y. Li, K. Li, Spectroscopic analyses on interaction of o-Vanillin-D-Phenylalanine, o-Vanillin-L-Tyrosine and o-Vanillin-LLevodopa Schiff Bases with bovine serum albumin (BSA), Spectrochim. Acta A 78 (2011) 1278–1286. [23] S.P. Boulos, T.A. Davis, J.A. Yang, S.E. Lohse, A.M. Alkilany, L.A. Holland, C.J. Murphy, Nanoparticle–protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces, Langmuir 29 (2013) 14984–14996. [24] C. Barakat, D. Patra, Combining time-resolved fluorescence with synchronous fluorescence spectroscopy to study bovine serum albumin-curcumin complex during unfolding and refolding processes, Luminescence 28 (2013) 149–155. [25] Z. Tian, F. Zang, W. Luo, Z. Zhao, Y. Wang, X. Xu, C. Wang, Spectroscopic study on the interaction between mononaphthalimide spermidine (MINS) and bovine serum albumin (BSA), J. Photochem. Photobiol. B 142 (2015) 103–109. [26] N. Shahabadi, M. Maghsudi, S. Rouhani, Study on the interaction of food colourant quinoline yellow with bovine serum albumin by spectroscopic techniques, Food Chem. 135 (2012) 1836–1841. [27] R. Kumaran, P. Ramamurthy, Photophysical studies on the interaction of amides with bovine serum albumin (BSA) in aqueous solution: fluorescence quenching and protein unfolding, J. Lumin. 148 (2014) 277–284. [28] H. Xu, S.-L. Gao, J.-B. Lv, Q.-W. Liu, Y. Zuo, X. Wang, Spectroscopic investigations on the mechanism of interaction of crystal violet with bovine serum albumin, J. Mol. Struct. 919 (2009) 334–338. [29] L. Ding, P. Zhou, H. Zhan, X. Zhao, C. Chen, Z. He, Systematic investigation of the toxicity interaction of ZnSe@ZnS QDs on BSA by spectroscopic and microcalorimetry techniques, Chemosphere 92 (2013) 892–897. [30] F.L. Cui, J. Fan, J.P. Li, Z.D. Hu, Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy, Bioorg. Med. Chem. 12 (2004) 151–157. [31] Q. Wang, X. Zhang, X. Zhou, T. Fang, P. Liu, P. Liu, X. Min, X. Li, Interaction of different thiol-capped CdTe quantum dots with bovine serum albumin, J. Lumin. 132 (2012) 1695–1700. [32] X.L. Wei, J.B. Xiao, Y. Wang, Y. Bai, Which model based on fluorescence quenching is suitable to study the interaction between trans-resveratrol and BSA? Spectrochim. Acta A 75 (2010) 299–304. [33] F. Ahmad, Y. Zhou, Z. Ling, Q. Xiang, X. Zhou, Systematic elucidation of interactive unfolding and corona formation of bovine serum albumin with cobalt ferrite nanoparticles, RSC Adv. 6 (2016) 35719–35730. [34] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [35] G. Siligardi, R. Hussain, S.G. Patching, M.K. Phillips-Jones, Ligand- and drug-binding studies of membrane proteins revealed through circular dichroism spectroscopy, Biochim. Biophys. Acta Biomembr. 1838 (2014) 34–42. [36] C. Bertucci, M. Pistolozzi, A. De Simone, Circular dichroism in drug discovery and development: an abridged review, Anal. Bioanal. Chem. 398 (2010) 155–166. [37] S. Monti, F. Manoli, S. Sortino, R. Morrone, G. Nicolosi, Binding of a chiral drug to a protein: an investigation of the 2-(3-benzoylphenyl)propionic acid/bovine serum albumin system by circular dichroism and fluorescence, Phys. Chem. Chem. Phys. 7 (2005) 4002–4008. [38] B. Ranjbar, P. Gill, Circular dichroism techniques: biomolecular and nanostructural analyses- a review, Chem. Biol. Drug Des. 74 (2009) 101–120. [39] M. Sarkar, S.S. Paul, K.K. Mukherjea, Interaction of bovine serum albumin with a psychotropic drug alprazolam: physicochemical, photophysical and molecular docking studies, J. Lumin. 142 (2013) 220–230. [40] M.D. Meti, K.S. Byadagi, S.T. Nandibewoor, S.A. Chimatadar, Multi-spectral characterization & effect of metal ions on the binding of bovine serum albumin upon interaction with a lincosamide antibiotic drug, clindamycin phosphate, J. Photochem. Photobiol. B 138 (2014) 324–330.
X. Liu et al. / Journal of Photochemistry & Photobiology, B: Biology 162 (2016) 332–339 [41] D. Sarkar, Probing the interaction of a globular protein with a small fluorescent probe in the presence of silver nanoparticles: spectroscopic characterization of its domain specific association and dissociation, RSC Adv. 3 (2013) 24389–24399. [42] A. Chattopadhyay, S. Mukherjee, Fluorophore environments in membrane-bound probes: a red edge excitation shift study, Biochemistry 32 (1993) 3804–3811. [43] G. Maglia, A. Jonckheer, M. De Maeyer, J.M. Frère, Y. Engelborghs, An unusual rededge excitation and time-dependent Stokes shift in the single tryptophan mutant
339
protein DD-carboxypeptidase from Streptomyces: the role of dynamics and tryptophan rotamers, Protein Sci. 17 (2008) 352–361. [44] S.K. Patra, M.K. Pal, Red edge excitation shift emission spectroscopic investigation of serum albumins and serum albumin-bilirubin complexes, Spectrochim. Acta A Mol. Biomol. Spectrosc. 53A (1997) 1609–1614.