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Impact of the alkyl chain length on binding of imidazolium-based ionic liquids to bovine serum albumin
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 196 (2018) 323–333
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Impact of the alkyl chain length on binding of imidazolium-based ionic liquids to bovine serum albumin Mengyue Zhang a, Ying Wang a, Hongmei Zhang a, Jian Cao a, Zhenghao Fei a,b, Yanqing Wang a,b,⁎ a
Institute of Environmental Toxicology and Environmental Ecology, Yancheng Teachers University, Yancheng City, Jiangsu Province 224051, People's Republic of China Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers University, Yancheng City, Jiangsu Province 224051, People's Republic of China b
a r t i c l e
i n f o
Article history: Received 5 November 2017 Received in revised form 8 February 2018 Accepted 12 February 2018 Available online 14 February 2018 Keywords: Imidazole-based ionic liquids Bovine serum albumin Binding mechanism Functional properties Thermal denaturation
a b s t r a c t The effects of six imidazolium-based ionic liquids (ILs) with different alkyl chain length ([CnMim]Cl, n = 2, 4, 6, 8, 10, 12) on the structure and functions of bovine serum albumin (BSA) were studied by multi-spectral methods and molecular docking. ILs with the longer alkyl chain length have the stronger binding interaction with BSA and the greater conformational damage to protein. The effects of ILs on the functional properties of BSA were further studied by the determination of non-enzyme esterase activity, β-fibrosis and other properties of BSA. The thermal stability of BSA was reduced, the rate of the formation of beta sheet structures of BSA was lowered, and the esterase-like activity of BSA were decreased with the increase of ILs concentration. Simultaneous molecular modeling technique revealed the favorable binding sites of ILs on protein. The hydrophobic force and polar interactions were the mainly binding forces of them. The calculated results are in a good agreement with the spectroscopic experiments. These studies on the impact of the alkyl chain length on binding of imidazoliumbased ionic liquids to BSA are of great significance for understanding and developing the application of ionic liquid in life and physiological system. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Ionic Liquids, ILs, are a kind of new materials with unique physical and chemical properties [1], such as low melting points, low or negligible vapor pressure, high thermal stability, and good catalytic properties [2,3]. Therefore, they have become increasingly attractive as green solvents for industrial applications [4]. At present, the application of ionic liquids in the field of biochemistry is also very broad, such as biocatalysis, biomass processing, drug delivery [5–7]. However, from an environmental viewpoint, the concomitant toxicological effects to various environments of them in the process of application should not be neglected if they are released into nature [8]. Accordingly, the negative environmental aspects of ILs should be characterized before their safe applications. Numerous toxicity effects of ILs on the environment have been reported. There are some ionic liquids with low to high hazard potential for human being and the environment [9–14]. The toxicity of some ILs on Vibrio fischeri microorganism was found to be more toxic than toluene [15]. The toxicity of some imidazolium-based ionic liquids toward ⁎ Corresponding author at: Institute of Environmental Toxicology and Environmental Ecology, Yancheng Teachers University, Yancheng City, Jiangsu Province 224051, People's Republic of China. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.saa.2018.02.040 1386-1425/© 2018 Elsevier B.V. All rights reserved.
the Channel Catfish Ovary cell was chiefly related to the shape and hydrophobicity parameters of cations [16]. Some proteins have been highly denatured when exposed to ILs [17]. Incidentally, the distribution and metabolism of ILs in the body are correlated with their affinities toward serum albumin. The studies about ILs binding with albumin are of imperative and fundamental importance [18,19,20]. Bovine serum albumin (BSA) is selected as a model protein to study the interaction and its conformational damage induced by ILs because of their low cost, ready availability. BSA consisting of a single chain polypeptide chain of 583 amino acid residues is structurally similar to human serum albumin (HSA) [21]. It is indispensable in the transport and disposition of endogenous and exogenous ligands in vivo, and plays an important role in the life activities of organism [22–24].The interactions between ionic liquid and protein have been discussed in some articles [25–27], but the mechanism of action of globular protein and ionic liquid is still limited. In particular, there is a lack of research on the subsequent changes in protein structure and the ionic liquid tail chain length alters the different effects on protein conformation [28]. In this work, firstly, the intrinsic fluorescence quenching, UV–vis, and circular dichroism(CD) spectra have been used to study the effects of six imidazole-based ionic liquids ([CnMim]Cl, n = 2, 4, 6, 8, 10, 12) with different tail length on the conformational changes of protein. CD signal changes of bilirubin-BSA system, 8-Anilino-1-naphthalenesulfonic acid
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Fig. 1. The fluorescence quenching (A) and the maximum emission wavelength (B) of BSA in the presence of different concentration of ILs, c(BSA) = 5.0 μM, pH = 7.40, T = 298 K, λex = 295 nm.
(ANS) fluorescence probe and molecular modeling techniques were used to analysis the binding site of ILs in BSA. In addition, the non-enzyme
esterase activity and fibrillation of BSA in the presence of ILs were also studied. It is hoped that the information obtained from this paper on
Fig. 2. Effects of ILs on the fluorescence intensity of BSA, Δλ = 15 nm, c(BSA) = 5 μM, pH = 7.40; T = 298 K;The concentration of ILs (A–E) (from 1 to 15): 0.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 mM; (F) (from 1 to 15): 0.0, 0.12, 0.24, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8 mM.
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the binding interactions of serum albumin with ILs will be helpful for the scientific community in choosing and designing ILs in biochemical processes and the toxicity effects of ILs on the environment.
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All other chemicals were of analytical grade. Ultrapure water was used throughout the experiments. 2.2. Fluorescence Quenching Measurements
2. Materials and Methods 2.1. Materials BSA (A1933, lyophilized powder, ≥98%) was purchased from Sigma (St, Louis, MO, USA) was prepared at 10 μM in NaH2PO4 -Na2HPO4 (pH = 7.40), then preserved at 0–4 °C, and diluted as required. 1ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-butyl-3methylimidazolium chloride, ([C4mim]Cl), 1-hexyl-3methylimidazolium chloride ([C6mim]Cl), 1-Oct-3-methylimidazolium chloride([C8mim]Cl), 1-decyl-3-methylimidazolium chloride ([C10mim]Cl), and 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl) with purities of 99% were purchased from MonILs Chem. Eng. Sci. & Tech. (Shanghai) co. The ILs stock solutions were prepared at a concentration of 0.1 M in NaH2PO4-Na2HPO4 (pH = 7.40) buffer.
The samples were placed in a fluorescent curette at room temperature and recorded with fluorescence emission spectra and synchronous fluorescence spectra on a LS-50B fluorescence photometer purchased from Perkin-Elmer(USA), and the light source was a pulsed xenon arc lamp. The scanning range is 300–450 nm, the emission and excitation slit width is 10.0 and 2.5 nm, and the scanning speed is 300 nm/min. Fluorescence spectra were obtained by fluorescence scanning at 295 nm. The fluorescence spectra were obtained by fluorescence scanning with fixed excitation wavelength and emission wavelength spacing of 15 and 60 nm in order to analysis the environment around tyrosine(Tyr) and tryptophan(Trp) residues. In the ANS fluorescence probe experiment, the excitation wavelength was 350 nm with the scan emission wavelength range from 400 to 600 nm. In addition, BSA fibrillation was studied by incubating the sample solutions at 338 K in the absence and presence of ILs. Thioflavin T (ThT) was used as
Fig. 3. Effects of ILs on the fluorescence intensity of BSA, Δλ = 60 nm, c(BSA) = 5 μM, pH = 7.40; T = 298 K; The concentration of ILs (A–E) (from 1 to 15): 0.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 mM; (F) (from 1 to 15): 0.0, 0.12, 0.24, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8 mM.
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2.5. Molecular Modeling
Fig. 4. ANS Fluorescence spectra analysis of BSA in the absence and presence of Ils. c(BSA) = 5.0 μM, c(ANS) = 5.0 μM, c([C2mim]Cl) = c([C4mim]Cl) = c([C6mim]Cl) = c([C8mim] Cl) = c([C10mim]Cl) = c([C12mim]Cl) = 3.0 mM.
fluorescence probe to monitor the BSA fibrillation. The excitation wavelength of λex = 440 nm in the range from 460 to 600 nm. 2.3. UV–vis Spectroscopy Samples were placed at room temperature in a quartz curette and the UV absorption spectra were recorded on a SPECORD-S600 UV–vis spectrophotometer purchased from Jena Analytical Instruments (Germany). The measurement range is 200–350 nm with reference to the same concentration of ionic liquid solution. 2.4. Circular Dichroism Spectroscopy Samples were placed at room temperature in a curette with a 0.1 cm optical path equipped with temperature controlled quantum purchased from the United Kingdom. The scanning range is 180–260 nm with three scans averaged and scanning speed was set at 30 nm/min for each CD spectrum. After the single scan, select three groups of samples for thermal denaturation experiments. The temperature was changed from 20 to 90 °C in 5 °C step with 240 s increments. Global Analysis Software equipped with the spectrometer was used to obtain the melting temperature (Tm). When measuring, the light source system through the nitrogen to remove air, water vapor. In addition, The scanning range of bilirubin-BSA system was 300–550 nm.
Using the Gaussian09 quantum chemical package, the density functional theory (DFT) B3LYP6-311++G(d,p) method of quantum chemistry was chosen to optimize the structural data of six ILs and obtain the stable configuration [29]. The crystal structure of BSA (PDB ID 3V03) is taken from the protein crystal database [30]. The interactions between ILs with different tail length and BSA were simulated by Autodock 4.2.3 molecular docking software [31]. In the blind docking calculations, a grid box of 126 × 126 × 126 grid points with spacing of 0.457 Å was used. The Lamarckian Genetic Algorithm(GA) method was used as the searching algorithm. The number of GA run and the population size were set at 100 and 150, respectively. In addition, the maximum number of evals, generations, top individuals were set at 150, 2,500,000, 27,000, and 1, respectively. At last, all other parameters were default settings parameters. The calculation structure was further analyzed using the Molegro MolecuLar Viewer software [32]. 3. Results and Discussion 3.1. Fluorescence Spectra of BSA in ILs Solution Fig. S1(A–F) shows the steady-state fluorescence of BSA at 295 nm excitation wavelengths in the presence of ILs. The fluorescence emission peak of BSA is near 346 nm originating mainly from the Trp-134 and Trp-213 tryptophan residues [33]. The fluorescence quenching and the maximum emission wavelength of BSA in the presence of different concentration of ILs were shown in Fig. 1(A,B). The changes of the emission intensity of fluorescence and the shift of the emission wavelength in the presence of different ILs were two main characteristics of the fluorescence spectra of BSA. The results showed that [C2mim]Cl, [C4mim]Cl, and [C6mim]Cl with the increase of ILs concentration, both the fluorescence emission peak intensity and the emission peak shape did not change significantly. In contrast, obviously significant fluorescence quenching as the concentration of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl increases. Concomitantly, the emission peak wavelengths of BSA obviously blue shift, indicating the increase of the hydrophobicity around Trp residues of BSA induced by ILs. Especially [C12mim]Cl, the fluorescence intensity decreased by 90% and the emission peak wavelength blue shift about 19 nm when the concentration of ILs was 4 mM. From the slopes of fluorescence quenching and emission wavelength curves, it can be concluded that ILs with the short tail chain are weak against the protein and did not significantly alter the microenvironment of the Trp residues. And ILs with the long tail chain have a strong effect on proteins and can significantly alter the
Fig. 5. CD spectra of BSA in the absence and presence of Ils. c(BSA) = 5.0 μM, c([C2mim]Cl) = c([C4mim]Cl) = c([C6mim]Cl) = c([C8mim]Cl) = c([C10mim]Cl) = 10.0 mM. c([C12mim]Cl) = 0.8 mM.
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Fig. 6. CD spectra of bilirubin-BSA system (A) and the radio of millidegrees θ/θ0(426 nm) value of bilirubin-BSA system (B) in the absence and presence of ILs, c(BSA) = c (bilirubin) = 5.0 μM, pH = 7.40, T = 298 K.
microenvironment of Trp residues [34]. In a word, the binding ability of ILs with BSA became stronger with increasing the length of alkyl-chain of ILs. In addition, ILs with longer alkyl-chain could pose more of a health threat than those with shorter alkyl-chain [35]. To make the trends more clear of the effects of ILs on the aromatic amino acids of BSA, synchronous fluorescence spectral method was used in this work [36]. The change of the fluorescence emission peak position of the protein and the change of the peak reflect the change of the microenvironment of the amino acid residue, and the change of the protein conformation can be judged by changing the emission wavelength [37]. The fluorescence spectra of Tyr and Trp residues were separated by synchronous fluorescence with wavelength difference of 15 and 60 nm, respectively [38,39]. The synchronous fluorescence spectra of Tyr and Trp residues of BSA in the presence of ILs were shown in Fig. 2(A–F) and Fig. 3(A–F), respectively. As can be seen from Fig. 2 and Fig. 3 that the fluorescence emission peak intensity did not change significantly and the peak shape of the emission peak did not change obviously in the presence of [C2mim]Cl, [C4mim]Cl, and [C6mim]Cl, indicating that the effect of the short-tailed ionic liquid on the microenvironment of Tyr and Trp residues in BSA is weak. In contrast, with the increase of the concentration, the fluorescence intensity changed significantly and the maximum emission wavelength blue shifted, implying that the hydrophobicity of the microenvironment of Tyr and Trp residues are enhanced. These results prove that the presence of long tailed ionic liquids changes the tertiary structure of BSA. In addition, Fig. 2(E,F) showed that the intensity of Tyr residues increased first and then decreased with the increase of concentration in the synchronous fluorescence of Δλ = 15 nm in the presence of [C10Mim]Cl, [C12Mim]Cl, confirming that different binding ability of ILs with the different length of alkyl-chain with protein lead to different effects on the tertiary structure of protein [40]. The results from the synchronous fluorescence spectra showed good agreement with the steady-state fluorescence spectra data. The longer alkyl-tail ILs have, the stronger binding ability with BSA ILs have.
reduced the fluorescence intensity of ANS by 38.9% and 78.5%, which implied that [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl changed the structure and the original properties of the proteins. This shows that the hydrophobic patches of BSA for ANS binding was gradual unfolding by the presence of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl. BSA becomes loose in the presence of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl. At the same concentration of ILs, other ILs caused less fluorescence intensity decrease than that of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl, implying that the existence of some intermediate state which is not completely unfolded of BSA in the presence of [C2mim]Cl, [C4mim]Cl, and [C6mim] Cl. The results are in agreement with the results of the previous fluorescence spectral results. 3.3. CD Spectra of BSA in ILs Solution The polypeptide backbone structure of BSA in the presence of ILs was investaged by using CD spectroscopy, which is a powerful technique to analysis the secondary structural changes of proteins. Fig. 5 (A) showed CD spectra of BSA in the absence and presence of ILs. BSA shows two characteristic negative peaks in the UV region at 208 and 222 nm, which are characteristic of α-helical structure [44]. As shown in Fig. 5(A), the presence of ILs significantly changes the CD spectra of BSA. The quantitatively measured secondary structure variation of BSA is recorded in Fig. 5(B). BSA in native form exhibits 67.4% α-helical
3.2. ANS Fluorescence Spectra of BSA in ILs Solution ANS fluorescence probe is often used to explore the characterization of intermediate states and aggregates, unfolding state, tertiary structure changes of protein [41]. When ANS bound to the hydrophobicity cavity of protein and other substances, its the fluorescence intensity will significantly increase, which can be used to characterize the protein surface hydrophobicity [42,43]. Fig. 4 showed the fluorescence spectra of ANS of BSA in the absence and presence of ILs. The presence of different ILs induced varying degree reduction of the ANS fluorescence intensity. Especially, the presence of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl
Fig. 7. Variation in Tm values of BSA in buffer (green) and in various ILs which is obtained from CD analysis with 10 mM (red), and 20 mM (blue). The values of [C12mim]Cl are 0.8 mM (red), and 2.4 mM (blue).
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which is comparable with reported literature values [45]. It has been observed that BSA undergoes a consecutive unfolding process in the presence of ILs. There are decreases of 54.0%, 53.7%, 56.5%, 60.8%, and 70.3% α-helical content in the presence of 10 mM [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, and [C10mim]Cl, respectively. Especially [C12mim]Cl, there is a decreases of 89.6% in the presence of 0.8 mM. Since most of the α-helices of BSA are present in subdomains, the decrease of α-helical indicating that BSA undergoes a consecutive unfolding process and the presence of ILs opens up the structure of BSA and exposes the helices sequentially. In addition, [C12mim]Cl shows the strong destruction of the secondary structure of BSA, which implied that the hydrophobic chain of ILs played an important role in the binding of ILs with BSA. This behavior corroborates to the blueshift in fluorescence wavelength of BSA with added ILs with the longer alkyl-tail [46]. 3.4. CD Spectra of Bilirubin-BSA System in ILs Solution The unfolding of BSA may indicate the decrease of the binding capacity of BSA with small molecules. Bilirubin is bound to BSA causing it to
Fig. 9. The esterase-like activity of BSA in the absence and presence of ILs. c(BSA) = 5 μM, pH = 7.40, T = 310 K.
Fig. 8. (A–G)ThT fluorescence spectra of BSA in the absence and presence of ILs(A, BSA; B, BSA + [C2mim]Cl; C, BSA + [C4mim]Cl; D, BSA + [C6mim]Cl; E, BSA + [C8mim]Cl; F, BSA + [C10mim]Cl; G, BSA + [C12mim]Cl) after different incubation time at 338 K, (H) Change in ThT fluorescence intensity at 485 nm of BSA at 338 K with time in the absence and presence of ILs(10 mM), pH = 7.40, λex = 440 nm.
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become optically active. CD spectroscopy is a method inherently sensitive to the structure of bilirubin binding with BSA [47]. The CD spectra of bilirubin-BSA system in the absence and presence of ILs were shown in Fig. 6(A). There was a negative CD couplet with maxima at 475(−)/426 (+)nm, which is characteristic for M-helical conformer of bilirubin binding with BSA [47]. The presence of ILs in bilirubin-BSA system change the CD signal, especially ILs with the hydrophobic chain, [C10mim]Cl and [C12mim]Cl. We suggested that the presence of ILs led to variation in the angle between two dipyrrinone chromophores of bilirubin, or destroy the binding domain of BSA with bilirubin. Fig. 6 (B) presents the change of the CD signal at 426 nm of bilirubin-BSA system with increase of ILs concentration. As evident from Fig. 6(B), there are appreciable decrease in the radio of millidegrees θ/θ0(426 nm) value of bilirubin-BSA in the presence of ILs. In the same concentration range of ILs, the value of θ/θ0(426 nm) decrease sharply for [C10mim] Cl and [C12mim]Cl. When the concentration is b0.4 mM, the value of θ/ θ0(426 nm) change little in the presence of [C2mim]Cl, [C4mim]Cl, and [C6mim]Cl. The decrease in the θ/θ0(426 nm) values with increase
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cation chain length indicated the order of unfolding BSA to be [C12mim]Cl N [C10mim]Cl N [C8mim]Cl N [C6mim]Cl N [C4mim]Cl N [C2mim]Cl. 3.5. Thermal Stability of BSA in ILs Solution Fig. 7 shows the transition temperature (Tm) of BSA in ILs obtained by using Global Analysis Software. The value of Tm of BSA in tuffer was found to be 64.9 ± 0.2 °C. However, the value of Tm in the presence of ILs was different from that of BSA in buffer. [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, and [C8mim]Cl shows a similar pattern. The desorption temperature of BSA increased first in the presence of 10 mM [Cnmim]Cl (n = 2, 4, 6, 8) and then decreased in the presence of 20 mM ILs. It was found that [Cnmim]Cl (n = 2, 4, 6, 8) stabilized BSA at low concentration and destroyed at high concentration. As for [C10mim]Cl and [C12mim]Cl, the value of Tm in the presence of ILs decrease sharply. [Cnmim]Cl (n = 10, 12) acted as the destabilizer of BSA. The results show that the decrease in Tm was more pronounced by ILs with the longer hydrophobic
Fig. 10. Cluster analyses of the AutoDock docking runs of ILs in the binding sites of BSA with ILs (a-1, [C2mim]Cl; b-1, [C4mim]Cl; c-1, [C6mim]Cl; d-1, [C8mim]Cl; e-1, [C10mim]Cl; f-1, [C12mim]Cl); the binding sites for the five BSA–ILs most stable complexes(a-2, [C2mim]Cl; b-2, [C4mim]Cl; c-2, [C6mim]Cl; d-2, [C8mim]Cl; e-2, [C10mim]Cl; f-2, [C12mim]Cl).
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Fig. 10 (continued).
chain. The drop of the value of Tm implied that the presence of high concentration of ILs could reduce the thermal stability of BSA by accelerating the rate of thermal denaturation process [48]. 3.6. The Fibrillation Process of BSA in ILs As discussed earlier, the presence of surfactants may play a significant role in the protein fibrillation pathway [49]. The formation of fibrils by BSA in the absence and presence of ILs was verified using ThT fluorescence probe [50]. As indicated in Fig. 8(A), the fluorescence intensity of ThT upon binding to BSA was increased significantly with increase of incubation time, indicating the fibrillar aggregates of BSA. The increase shift at about 485 nm peak indicated the increase in beta sheet assemblies. As indicated in Fig. 8(B–G), the presence of [Cnmim]Cl with the different length hydrophobic chain did cause varying degrees of influence on the fibrillation process of BSA. Fig. 8(H) showed the kinetic of amyloid aggregation for BSA-ILs solutions by curving the fluorescence emission of ThT at 485 nm versus incubation time. [Cnmim]Cl (n = 2,
4, 6) (10 mM) had little effect on the slope and intensity of ThT fluorescence emission kinetics. However, [Cnmim]Cl (n = 8, 10, 12) (10 mM) decreased significantly the slope and intensity of ThT fluorescence kinetic, indicating that the rate of the formation of beta sheet structures of BSA was lover in the presence of ILs with the longer hydrophobic chain, especially [C10mim]Cl and [C12mim]Cl. These results implied that the nature of functionalization of alkyl chain of ILs has a significant effect on their interactions with BSA and a variety of structurally distinct complexes between ILSs and BSA formed [51]. Ils with the longer hydrophobic chain have led to the formation of unordered large selfassembled structures of BSA. 3.7. The Esterase Activity of BSA in ILs Solution The retention of BSA activity after ligand binding is of particular importance for any biological application [52]. The esterase-like activity is an interesting enzymatic property of BSA, which is evaluated by monitoring the hydrolysis of p-nitrophenyl acetate to p-nitrophenol by the
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action of BSA [46]. In order to study the possibility of the ILs binding affecting on the enzymatic activity of BSA, the evaluation of esterase-like activity of BSA in the absence and presence of ILs are depicted in Fig. 9. The results showed that the esterase-like activity of BSA were decreased with the increase of ILs concentration. Especially, the binding interactions with [Cnmim]Cl (n = 8, 10, 12) is associated with a discernible reduction in the esterase-like activity of BSA. Such loss of BSA activity in the presence of [Cnmim]Cl (n = 8, 10, 12) is consistent with the breakdown of the native protein structure. The presence of ILs opens up the structure of BSA and exposes the helices sequentially, which affected the activity pocket of protein. In addition, ILs maybe bind into the activity pocket of protein. The influence of ILs on the activity of BSA can prove useful for explaining the previously observed physiological phenomena such as the bioaccumulation potential and toxicity of ILs. 3.8. Molecular Modeling Study Molecular docking was often used to analyze the putative binding sites and the intermolecular interactions of some toxicity molecule with serum albumin [53–56]. To determine the binding sites of BSA with ILs, the possible conformations of BSA-ILs system were modeled by using Autodock method. Firstly, the cluster analyses were performed for the possible binding sites of ILs on BSA. The statistical results selected from the minimum energy conformers from 100 runs were shown in Fig. 10(a-1, b-1, c-1, d-1, e-1, and f-1). The total of 16, 24, 40, 46, 51, and 52 multimember conformational clusters for BSA binding with [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl, respectively, indicating that BSA has multi possible binding sites for ILs. In addition, the number of the possible binding sites were increased from 16 to 52 implied that ILs with the longer hydrophobic chain are prone to binding with BSA (Fig. 11), which was in good agreement with the binding energy shown in Fig. 11. The lowest binding energy of [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl with BSA were −3.92, −4.34, −4.47, −4.72, −5.07, and −5.12 kcal/mol, respectively, implying that the binding ability of ILs with BSA increased sequentially with the increase of the number of the carbon atom of the hydrophobic chain. The experimental results are supported by the calculation results. In order to deep insight into the binding amino acids of BSA with ILs, the binding sites for the five BSA-Ils most stable complexes were analyzed and the results were shown in Fig. 10(a-2, b-2, c-2, d-2, e-2, and f-2). As shown in Fig. 10, it was found that site A located the joint gap between subdomain IA and subdomain IIA, site B was close to subdomain IIIA, which is known as “indole-benzodiazepine binding site” [57]. Site C was located in subdomain IIIA and B. In addition, both site D and site E was near subdomain IB. Fig. 12(A–E) presented a sketch of the five binding sites A–E of [C2mim]Cl with BSA. In site A, [C2mim]Cl entered into the binding cavity of BSA formed by Leu-22, Val-23, Ala-26, Leu-46, Leu-66, His-67, Phe70, Gly-247, Asp-248, Leu-249, Leu-250 amino acid residues. Among them, Leu, Val and Phe residues were hydrophobic amino residues, especially, five Leu residues were involved in the binding interaction of BSA with [C2mim]Cl, implying that the hydrophobic force was the main binding force between [C2mim]Cl and BSA, and the hydrophobic core cavity is able to host the hydrophobic carbon chain of ILs. In site B, [C2mim]Cl entered into subdomain IIIA, thirteen amino acid residues including Leu-197, Ser-343, Arg-347, Glu-449, Leu-452, Ser-453, Leu456, Asn-456, Asn-457, Leu-480, Val-481, Arg-483, Arg-484, and Pro485 took part in the binding interaction of BSA with [C2mim]Cl. Besides of Leu, Val, Pro, and other hydrophobic amino residues taking part in the binding interaction, the polar head of imidazole ring with positive charge interacted with Ser-453, Asn-457, Arg-483, and Arg-484. In site C, Thr-411, Arg-412, Pro-415, Pro-492, Asp-493, Glu-494, Tyr-496, Leu-532, Lys-533, Pro-536, and Lys-537 took part in the BSA-[C2mim] Cl complex formation. In sits D, Asp-108, His-145, Pro-146, Tyr-147, Phe-148, Ser-192, Ala-193, Glu-195, Arg-196, and Arg-458 interacted
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with [C2mim]Cl. In site E, Tyr-137, Ile-141, Tyr-160, Ile-181, Met-184, Arg-185, Val-188 were involved the binding interaction of BSA with [C2mim]Cl. In summary, the hydrophobic amino residues are main Leu, Pro, Phe, and Val residues, and the polar amino residues are main Arg, Ser, and Lys residues. The hydrophobic forces and polar interaction are the mainly binding forces of BSA with [C2mim]Cl. The binding sites for the five most stable complexes of the other four Ils with BSA were shown in Fig. 10(b-2, c-2, d-2, e-2, and f-2), and their sketch of the five binding sites A-E of [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, [C10mim]Cl, [C12mim]Cl with BSA were presented in Fig. S2–6. It was found that there were similar binding sites of ILs in BSA. The subdomain IIIA and subdomain IB are the common sites of BSA binding with ILs. With the increase of the hydrophobic carbon chain, [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl entered into the hydrophobic cavity of subdomain IIA. Trp 213 residue was involved in the binding interaction of BSA with [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl, which favorable supported for the view that there were obviously significant fluorescence quenching as the concentration of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl increases. For BSA-[C12mim]Cl system, Trp-134 was involved in their binding interaction each other (Fig. S6 E), therefore, the fluorescence intensity decreased by 90% and the emission peak wavelength blue shift about 19 nm when the concentration of ILs was 4 mM in [C12mim]Cl-BSA system. In addition, with the increase of the hydrophobic carbon chain of ILs, their chain flexibility are enhanced. It can seen from Fig. S4–6 that there are different degree of blending of the hydrophobic carbon chain of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl in order to move into the active site. This conformational modifications of carbon chain indicated the importance of the hydrophobic chain of ILs during the binding interactions of them with BSA [58]. 4. Conclusion In this work, the impact of the alkyl chain length on binding of imidazolium-based ionic liquids to BSA was studied through three spectroscopic techniques including fluorescence, UV–vis, and CD and molecular modeling. On the basis of the results, it was found from fluorescence spectral data that ILs with the longer alkyl chain length have the stronger binding interaction with BSA and the greater conformational damage to protein. The different binding ability of ILs with the different length of alkyl-chain with protein leads to different effects on the tertiary structure of protein. The CD spectral results indicated that the order of unfolding BSA was to be [C12mim]Cl N [C10mim]Cl N [C8mim]Cl N [C6mim]Cl N [C4mim]Cl N [C2mim]Cl. [C12mim]Cl shows the strong destruction of the secondary structure of BSA. The drop of the value of Tm implied that the presence of high concentration of ILs
Fig. 11. The plots of the lowest binding energy (red line) and the conformational clusters (blue line) as a function of the number of carbon atom of the hydrophobic chain of ILs.
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Fig. 12. Details of the binding sites for the five BSA–[C2mim]Cl most stable complexes.
could reduce the thermal stability of BSA by accelerating the rate of thermal denaturation process. ThT fluorescence probe data showed that rate of the formation of beta sheet structures of BSA was lower in the presence of ILs with the longer hydrophobic chain, especially [C10mim]Cl and [C12mim]Cl. The nature of functionalization of alkyl chain of ILs has a significant effect on their interactions with protein. The esterase-like activity of BSA were decreased with the increase of ILs concentration. In addition, molecular modeling information indicated that ILs with the longer hydrophobic chain are prone to binding with BSA by the hydrophobic forces and polar interaction. This study not only provides important insights into the mechanism of interaction between ILs and BSA in different ILs concentration regimes, but it supports the toxicity background of ILs with the different lengths of hydrophobic chain. Acknowledgement We gratefully acknowledge financial support of the Fund from the National Natural Science Foundation of China (Project No. 21571154), the Natural Science Foundation (Grant No. BK20161315, BK20151296), the“333 Project”, “Qinglan Project”, and “Six Talent Peaks Project” of Jiangsu Province. Appendix A. Supplementary Data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2018.02.040. References [1] K. Binnemans, Chem. Rev. 37 (2005) 4148–4204. [2] C.D. Hubbard, P. Illner, R. Eldik van, Chem. Soc. Rev. 40 (2011) 272–290. [3] M.J. Earle, J.M. Esperanc, M.A. Gilea, J.N.C. Lopes, L.P. Rebelo, J.W. Magee, K.R. Seddon, J.A. Widegren, Nature 439 (2006) 831–834.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Impact of the alkyl chain length on binding of imidazolium-based ionic liquids to bovine serum albumin Mengyue Zhang a, Ying Wang a, Hongmei Zhang a, Jian Cao a, Zhenghao Fei a,b, Yanqing Wang a,b,⁎ a
Institute of Environmental Toxicology and Environmental Ecology, Yancheng Teachers University, Yancheng City, Jiangsu Province 224051, People's Republic of China Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers University, Yancheng City, Jiangsu Province 224051, People's Republic of China b
a r t i c l e
i n f o
Article history: Received 5 November 2017 Received in revised form 8 February 2018 Accepted 12 February 2018 Available online 14 February 2018 Keywords: Imidazole-based ionic liquids Bovine serum albumin Binding mechanism Functional properties Thermal denaturation
a b s t r a c t The effects of six imidazolium-based ionic liquids (ILs) with different alkyl chain length ([CnMim]Cl, n = 2, 4, 6, 8, 10, 12) on the structure and functions of bovine serum albumin (BSA) were studied by multi-spectral methods and molecular docking. ILs with the longer alkyl chain length have the stronger binding interaction with BSA and the greater conformational damage to protein. The effects of ILs on the functional properties of BSA were further studied by the determination of non-enzyme esterase activity, β-fibrosis and other properties of BSA. The thermal stability of BSA was reduced, the rate of the formation of beta sheet structures of BSA was lowered, and the esterase-like activity of BSA were decreased with the increase of ILs concentration. Simultaneous molecular modeling technique revealed the favorable binding sites of ILs on protein. The hydrophobic force and polar interactions were the mainly binding forces of them. The calculated results are in a good agreement with the spectroscopic experiments. These studies on the impact of the alkyl chain length on binding of imidazoliumbased ionic liquids to BSA are of great significance for understanding and developing the application of ionic liquid in life and physiological system. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Ionic Liquids, ILs, are a kind of new materials with unique physical and chemical properties [1], such as low melting points, low or negligible vapor pressure, high thermal stability, and good catalytic properties [2,3]. Therefore, they have become increasingly attractive as green solvents for industrial applications [4]. At present, the application of ionic liquids in the field of biochemistry is also very broad, such as biocatalysis, biomass processing, drug delivery [5–7]. However, from an environmental viewpoint, the concomitant toxicological effects to various environments of them in the process of application should not be neglected if they are released into nature [8]. Accordingly, the negative environmental aspects of ILs should be characterized before their safe applications. Numerous toxicity effects of ILs on the environment have been reported. There are some ionic liquids with low to high hazard potential for human being and the environment [9–14]. The toxicity of some ILs on Vibrio fischeri microorganism was found to be more toxic than toluene [15]. The toxicity of some imidazolium-based ionic liquids toward ⁎ Corresponding author at: Institute of Environmental Toxicology and Environmental Ecology, Yancheng Teachers University, Yancheng City, Jiangsu Province 224051, People's Republic of China. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.saa.2018.02.040 1386-1425/© 2018 Elsevier B.V. All rights reserved.
the Channel Catfish Ovary cell was chiefly related to the shape and hydrophobicity parameters of cations [16]. Some proteins have been highly denatured when exposed to ILs [17]. Incidentally, the distribution and metabolism of ILs in the body are correlated with their affinities toward serum albumin. The studies about ILs binding with albumin are of imperative and fundamental importance [18,19,20]. Bovine serum albumin (BSA) is selected as a model protein to study the interaction and its conformational damage induced by ILs because of their low cost, ready availability. BSA consisting of a single chain polypeptide chain of 583 amino acid residues is structurally similar to human serum albumin (HSA) [21]. It is indispensable in the transport and disposition of endogenous and exogenous ligands in vivo, and plays an important role in the life activities of organism [22–24].The interactions between ionic liquid and protein have been discussed in some articles [25–27], but the mechanism of action of globular protein and ionic liquid is still limited. In particular, there is a lack of research on the subsequent changes in protein structure and the ionic liquid tail chain length alters the different effects on protein conformation [28]. In this work, firstly, the intrinsic fluorescence quenching, UV–vis, and circular dichroism(CD) spectra have been used to study the effects of six imidazole-based ionic liquids ([CnMim]Cl, n = 2, 4, 6, 8, 10, 12) with different tail length on the conformational changes of protein. CD signal changes of bilirubin-BSA system, 8-Anilino-1-naphthalenesulfonic acid
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Fig. 1. The fluorescence quenching (A) and the maximum emission wavelength (B) of BSA in the presence of different concentration of ILs, c(BSA) = 5.0 μM, pH = 7.40, T = 298 K, λex = 295 nm.
(ANS) fluorescence probe and molecular modeling techniques were used to analysis the binding site of ILs in BSA. In addition, the non-enzyme
esterase activity and fibrillation of BSA in the presence of ILs were also studied. It is hoped that the information obtained from this paper on
Fig. 2. Effects of ILs on the fluorescence intensity of BSA, Δλ = 15 nm, c(BSA) = 5 μM, pH = 7.40; T = 298 K;The concentration of ILs (A–E) (from 1 to 15): 0.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 mM; (F) (from 1 to 15): 0.0, 0.12, 0.24, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8 mM.
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the binding interactions of serum albumin with ILs will be helpful for the scientific community in choosing and designing ILs in biochemical processes and the toxicity effects of ILs on the environment.
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All other chemicals were of analytical grade. Ultrapure water was used throughout the experiments. 2.2. Fluorescence Quenching Measurements
2. Materials and Methods 2.1. Materials BSA (A1933, lyophilized powder, ≥98%) was purchased from Sigma (St, Louis, MO, USA) was prepared at 10 μM in NaH2PO4 -Na2HPO4 (pH = 7.40), then preserved at 0–4 °C, and diluted as required. 1ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-butyl-3methylimidazolium chloride, ([C4mim]Cl), 1-hexyl-3methylimidazolium chloride ([C6mim]Cl), 1-Oct-3-methylimidazolium chloride([C8mim]Cl), 1-decyl-3-methylimidazolium chloride ([C10mim]Cl), and 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl) with purities of 99% were purchased from MonILs Chem. Eng. Sci. & Tech. (Shanghai) co. The ILs stock solutions were prepared at a concentration of 0.1 M in NaH2PO4-Na2HPO4 (pH = 7.40) buffer.
The samples were placed in a fluorescent curette at room temperature and recorded with fluorescence emission spectra and synchronous fluorescence spectra on a LS-50B fluorescence photometer purchased from Perkin-Elmer(USA), and the light source was a pulsed xenon arc lamp. The scanning range is 300–450 nm, the emission and excitation slit width is 10.0 and 2.5 nm, and the scanning speed is 300 nm/min. Fluorescence spectra were obtained by fluorescence scanning at 295 nm. The fluorescence spectra were obtained by fluorescence scanning with fixed excitation wavelength and emission wavelength spacing of 15 and 60 nm in order to analysis the environment around tyrosine(Tyr) and tryptophan(Trp) residues. In the ANS fluorescence probe experiment, the excitation wavelength was 350 nm with the scan emission wavelength range from 400 to 600 nm. In addition, BSA fibrillation was studied by incubating the sample solutions at 338 K in the absence and presence of ILs. Thioflavin T (ThT) was used as
Fig. 3. Effects of ILs on the fluorescence intensity of BSA, Δλ = 60 nm, c(BSA) = 5 μM, pH = 7.40; T = 298 K; The concentration of ILs (A–E) (from 1 to 15): 0.0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0 mM; (F) (from 1 to 15): 0.0, 0.12, 0.24, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, 4.8 mM.
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2.5. Molecular Modeling
Fig. 4. ANS Fluorescence spectra analysis of BSA in the absence and presence of Ils. c(BSA) = 5.0 μM, c(ANS) = 5.0 μM, c([C2mim]Cl) = c([C4mim]Cl) = c([C6mim]Cl) = c([C8mim] Cl) = c([C10mim]Cl) = c([C12mim]Cl) = 3.0 mM.
fluorescence probe to monitor the BSA fibrillation. The excitation wavelength of λex = 440 nm in the range from 460 to 600 nm. 2.3. UV–vis Spectroscopy Samples were placed at room temperature in a quartz curette and the UV absorption spectra were recorded on a SPECORD-S600 UV–vis spectrophotometer purchased from Jena Analytical Instruments (Germany). The measurement range is 200–350 nm with reference to the same concentration of ionic liquid solution. 2.4. Circular Dichroism Spectroscopy Samples were placed at room temperature in a curette with a 0.1 cm optical path equipped with temperature controlled quantum purchased from the United Kingdom. The scanning range is 180–260 nm with three scans averaged and scanning speed was set at 30 nm/min for each CD spectrum. After the single scan, select three groups of samples for thermal denaturation experiments. The temperature was changed from 20 to 90 °C in 5 °C step with 240 s increments. Global Analysis Software equipped with the spectrometer was used to obtain the melting temperature (Tm). When measuring, the light source system through the nitrogen to remove air, water vapor. In addition, The scanning range of bilirubin-BSA system was 300–550 nm.
Using the Gaussian09 quantum chemical package, the density functional theory (DFT) B3LYP6-311++G(d,p) method of quantum chemistry was chosen to optimize the structural data of six ILs and obtain the stable configuration [29]. The crystal structure of BSA (PDB ID 3V03) is taken from the protein crystal database [30]. The interactions between ILs with different tail length and BSA were simulated by Autodock 4.2.3 molecular docking software [31]. In the blind docking calculations, a grid box of 126 × 126 × 126 grid points with spacing of 0.457 Å was used. The Lamarckian Genetic Algorithm(GA) method was used as the searching algorithm. The number of GA run and the population size were set at 100 and 150, respectively. In addition, the maximum number of evals, generations, top individuals were set at 150, 2,500,000, 27,000, and 1, respectively. At last, all other parameters were default settings parameters. The calculation structure was further analyzed using the Molegro MolecuLar Viewer software [32]. 3. Results and Discussion 3.1. Fluorescence Spectra of BSA in ILs Solution Fig. S1(A–F) shows the steady-state fluorescence of BSA at 295 nm excitation wavelengths in the presence of ILs. The fluorescence emission peak of BSA is near 346 nm originating mainly from the Trp-134 and Trp-213 tryptophan residues [33]. The fluorescence quenching and the maximum emission wavelength of BSA in the presence of different concentration of ILs were shown in Fig. 1(A,B). The changes of the emission intensity of fluorescence and the shift of the emission wavelength in the presence of different ILs were two main characteristics of the fluorescence spectra of BSA. The results showed that [C2mim]Cl, [C4mim]Cl, and [C6mim]Cl with the increase of ILs concentration, both the fluorescence emission peak intensity and the emission peak shape did not change significantly. In contrast, obviously significant fluorescence quenching as the concentration of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl increases. Concomitantly, the emission peak wavelengths of BSA obviously blue shift, indicating the increase of the hydrophobicity around Trp residues of BSA induced by ILs. Especially [C12mim]Cl, the fluorescence intensity decreased by 90% and the emission peak wavelength blue shift about 19 nm when the concentration of ILs was 4 mM. From the slopes of fluorescence quenching and emission wavelength curves, it can be concluded that ILs with the short tail chain are weak against the protein and did not significantly alter the microenvironment of the Trp residues. And ILs with the long tail chain have a strong effect on proteins and can significantly alter the
Fig. 5. CD spectra of BSA in the absence and presence of Ils. c(BSA) = 5.0 μM, c([C2mim]Cl) = c([C4mim]Cl) = c([C6mim]Cl) = c([C8mim]Cl) = c([C10mim]Cl) = 10.0 mM. c([C12mim]Cl) = 0.8 mM.
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Fig. 6. CD spectra of bilirubin-BSA system (A) and the radio of millidegrees θ/θ0(426 nm) value of bilirubin-BSA system (B) in the absence and presence of ILs, c(BSA) = c (bilirubin) = 5.0 μM, pH = 7.40, T = 298 K.
microenvironment of Trp residues [34]. In a word, the binding ability of ILs with BSA became stronger with increasing the length of alkyl-chain of ILs. In addition, ILs with longer alkyl-chain could pose more of a health threat than those with shorter alkyl-chain [35]. To make the trends more clear of the effects of ILs on the aromatic amino acids of BSA, synchronous fluorescence spectral method was used in this work [36]. The change of the fluorescence emission peak position of the protein and the change of the peak reflect the change of the microenvironment of the amino acid residue, and the change of the protein conformation can be judged by changing the emission wavelength [37]. The fluorescence spectra of Tyr and Trp residues were separated by synchronous fluorescence with wavelength difference of 15 and 60 nm, respectively [38,39]. The synchronous fluorescence spectra of Tyr and Trp residues of BSA in the presence of ILs were shown in Fig. 2(A–F) and Fig. 3(A–F), respectively. As can be seen from Fig. 2 and Fig. 3 that the fluorescence emission peak intensity did not change significantly and the peak shape of the emission peak did not change obviously in the presence of [C2mim]Cl, [C4mim]Cl, and [C6mim]Cl, indicating that the effect of the short-tailed ionic liquid on the microenvironment of Tyr and Trp residues in BSA is weak. In contrast, with the increase of the concentration, the fluorescence intensity changed significantly and the maximum emission wavelength blue shifted, implying that the hydrophobicity of the microenvironment of Tyr and Trp residues are enhanced. These results prove that the presence of long tailed ionic liquids changes the tertiary structure of BSA. In addition, Fig. 2(E,F) showed that the intensity of Tyr residues increased first and then decreased with the increase of concentration in the synchronous fluorescence of Δλ = 15 nm in the presence of [C10Mim]Cl, [C12Mim]Cl, confirming that different binding ability of ILs with the different length of alkyl-chain with protein lead to different effects on the tertiary structure of protein [40]. The results from the synchronous fluorescence spectra showed good agreement with the steady-state fluorescence spectra data. The longer alkyl-tail ILs have, the stronger binding ability with BSA ILs have.
reduced the fluorescence intensity of ANS by 38.9% and 78.5%, which implied that [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl changed the structure and the original properties of the proteins. This shows that the hydrophobic patches of BSA for ANS binding was gradual unfolding by the presence of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl. BSA becomes loose in the presence of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl. At the same concentration of ILs, other ILs caused less fluorescence intensity decrease than that of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl, implying that the existence of some intermediate state which is not completely unfolded of BSA in the presence of [C2mim]Cl, [C4mim]Cl, and [C6mim] Cl. The results are in agreement with the results of the previous fluorescence spectral results. 3.3. CD Spectra of BSA in ILs Solution The polypeptide backbone structure of BSA in the presence of ILs was investaged by using CD spectroscopy, which is a powerful technique to analysis the secondary structural changes of proteins. Fig. 5 (A) showed CD spectra of BSA in the absence and presence of ILs. BSA shows two characteristic negative peaks in the UV region at 208 and 222 nm, which are characteristic of α-helical structure [44]. As shown in Fig. 5(A), the presence of ILs significantly changes the CD spectra of BSA. The quantitatively measured secondary structure variation of BSA is recorded in Fig. 5(B). BSA in native form exhibits 67.4% α-helical
3.2. ANS Fluorescence Spectra of BSA in ILs Solution ANS fluorescence probe is often used to explore the characterization of intermediate states and aggregates, unfolding state, tertiary structure changes of protein [41]. When ANS bound to the hydrophobicity cavity of protein and other substances, its the fluorescence intensity will significantly increase, which can be used to characterize the protein surface hydrophobicity [42,43]. Fig. 4 showed the fluorescence spectra of ANS of BSA in the absence and presence of ILs. The presence of different ILs induced varying degree reduction of the ANS fluorescence intensity. Especially, the presence of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl
Fig. 7. Variation in Tm values of BSA in buffer (green) and in various ILs which is obtained from CD analysis with 10 mM (red), and 20 mM (blue). The values of [C12mim]Cl are 0.8 mM (red), and 2.4 mM (blue).
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which is comparable with reported literature values [45]. It has been observed that BSA undergoes a consecutive unfolding process in the presence of ILs. There are decreases of 54.0%, 53.7%, 56.5%, 60.8%, and 70.3% α-helical content in the presence of 10 mM [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, and [C10mim]Cl, respectively. Especially [C12mim]Cl, there is a decreases of 89.6% in the presence of 0.8 mM. Since most of the α-helices of BSA are present in subdomains, the decrease of α-helical indicating that BSA undergoes a consecutive unfolding process and the presence of ILs opens up the structure of BSA and exposes the helices sequentially. In addition, [C12mim]Cl shows the strong destruction of the secondary structure of BSA, which implied that the hydrophobic chain of ILs played an important role in the binding of ILs with BSA. This behavior corroborates to the blueshift in fluorescence wavelength of BSA with added ILs with the longer alkyl-tail [46]. 3.4. CD Spectra of Bilirubin-BSA System in ILs Solution The unfolding of BSA may indicate the decrease of the binding capacity of BSA with small molecules. Bilirubin is bound to BSA causing it to
Fig. 9. The esterase-like activity of BSA in the absence and presence of ILs. c(BSA) = 5 μM, pH = 7.40, T = 310 K.
Fig. 8. (A–G)ThT fluorescence spectra of BSA in the absence and presence of ILs(A, BSA; B, BSA + [C2mim]Cl; C, BSA + [C4mim]Cl; D, BSA + [C6mim]Cl; E, BSA + [C8mim]Cl; F, BSA + [C10mim]Cl; G, BSA + [C12mim]Cl) after different incubation time at 338 K, (H) Change in ThT fluorescence intensity at 485 nm of BSA at 338 K with time in the absence and presence of ILs(10 mM), pH = 7.40, λex = 440 nm.
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become optically active. CD spectroscopy is a method inherently sensitive to the structure of bilirubin binding with BSA [47]. The CD spectra of bilirubin-BSA system in the absence and presence of ILs were shown in Fig. 6(A). There was a negative CD couplet with maxima at 475(−)/426 (+)nm, which is characteristic for M-helical conformer of bilirubin binding with BSA [47]. The presence of ILs in bilirubin-BSA system change the CD signal, especially ILs with the hydrophobic chain, [C10mim]Cl and [C12mim]Cl. We suggested that the presence of ILs led to variation in the angle between two dipyrrinone chromophores of bilirubin, or destroy the binding domain of BSA with bilirubin. Fig. 6 (B) presents the change of the CD signal at 426 nm of bilirubin-BSA system with increase of ILs concentration. As evident from Fig. 6(B), there are appreciable decrease in the radio of millidegrees θ/θ0(426 nm) value of bilirubin-BSA in the presence of ILs. In the same concentration range of ILs, the value of θ/θ0(426 nm) decrease sharply for [C10mim] Cl and [C12mim]Cl. When the concentration is b0.4 mM, the value of θ/ θ0(426 nm) change little in the presence of [C2mim]Cl, [C4mim]Cl, and [C6mim]Cl. The decrease in the θ/θ0(426 nm) values with increase
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cation chain length indicated the order of unfolding BSA to be [C12mim]Cl N [C10mim]Cl N [C8mim]Cl N [C6mim]Cl N [C4mim]Cl N [C2mim]Cl. 3.5. Thermal Stability of BSA in ILs Solution Fig. 7 shows the transition temperature (Tm) of BSA in ILs obtained by using Global Analysis Software. The value of Tm of BSA in tuffer was found to be 64.9 ± 0.2 °C. However, the value of Tm in the presence of ILs was different from that of BSA in buffer. [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, and [C8mim]Cl shows a similar pattern. The desorption temperature of BSA increased first in the presence of 10 mM [Cnmim]Cl (n = 2, 4, 6, 8) and then decreased in the presence of 20 mM ILs. It was found that [Cnmim]Cl (n = 2, 4, 6, 8) stabilized BSA at low concentration and destroyed at high concentration. As for [C10mim]Cl and [C12mim]Cl, the value of Tm in the presence of ILs decrease sharply. [Cnmim]Cl (n = 10, 12) acted as the destabilizer of BSA. The results show that the decrease in Tm was more pronounced by ILs with the longer hydrophobic
Fig. 10. Cluster analyses of the AutoDock docking runs of ILs in the binding sites of BSA with ILs (a-1, [C2mim]Cl; b-1, [C4mim]Cl; c-1, [C6mim]Cl; d-1, [C8mim]Cl; e-1, [C10mim]Cl; f-1, [C12mim]Cl); the binding sites for the five BSA–ILs most stable complexes(a-2, [C2mim]Cl; b-2, [C4mim]Cl; c-2, [C6mim]Cl; d-2, [C8mim]Cl; e-2, [C10mim]Cl; f-2, [C12mim]Cl).
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Fig. 10 (continued).
chain. The drop of the value of Tm implied that the presence of high concentration of ILs could reduce the thermal stability of BSA by accelerating the rate of thermal denaturation process [48]. 3.6. The Fibrillation Process of BSA in ILs As discussed earlier, the presence of surfactants may play a significant role in the protein fibrillation pathway [49]. The formation of fibrils by BSA in the absence and presence of ILs was verified using ThT fluorescence probe [50]. As indicated in Fig. 8(A), the fluorescence intensity of ThT upon binding to BSA was increased significantly with increase of incubation time, indicating the fibrillar aggregates of BSA. The increase shift at about 485 nm peak indicated the increase in beta sheet assemblies. As indicated in Fig. 8(B–G), the presence of [Cnmim]Cl with the different length hydrophobic chain did cause varying degrees of influence on the fibrillation process of BSA. Fig. 8(H) showed the kinetic of amyloid aggregation for BSA-ILs solutions by curving the fluorescence emission of ThT at 485 nm versus incubation time. [Cnmim]Cl (n = 2,
4, 6) (10 mM) had little effect on the slope and intensity of ThT fluorescence emission kinetics. However, [Cnmim]Cl (n = 8, 10, 12) (10 mM) decreased significantly the slope and intensity of ThT fluorescence kinetic, indicating that the rate of the formation of beta sheet structures of BSA was lover in the presence of ILs with the longer hydrophobic chain, especially [C10mim]Cl and [C12mim]Cl. These results implied that the nature of functionalization of alkyl chain of ILs has a significant effect on their interactions with BSA and a variety of structurally distinct complexes between ILSs and BSA formed [51]. Ils with the longer hydrophobic chain have led to the formation of unordered large selfassembled structures of BSA. 3.7. The Esterase Activity of BSA in ILs Solution The retention of BSA activity after ligand binding is of particular importance for any biological application [52]. The esterase-like activity is an interesting enzymatic property of BSA, which is evaluated by monitoring the hydrolysis of p-nitrophenyl acetate to p-nitrophenol by the
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action of BSA [46]. In order to study the possibility of the ILs binding affecting on the enzymatic activity of BSA, the evaluation of esterase-like activity of BSA in the absence and presence of ILs are depicted in Fig. 9. The results showed that the esterase-like activity of BSA were decreased with the increase of ILs concentration. Especially, the binding interactions with [Cnmim]Cl (n = 8, 10, 12) is associated with a discernible reduction in the esterase-like activity of BSA. Such loss of BSA activity in the presence of [Cnmim]Cl (n = 8, 10, 12) is consistent with the breakdown of the native protein structure. The presence of ILs opens up the structure of BSA and exposes the helices sequentially, which affected the activity pocket of protein. In addition, ILs maybe bind into the activity pocket of protein. The influence of ILs on the activity of BSA can prove useful for explaining the previously observed physiological phenomena such as the bioaccumulation potential and toxicity of ILs. 3.8. Molecular Modeling Study Molecular docking was often used to analyze the putative binding sites and the intermolecular interactions of some toxicity molecule with serum albumin [53–56]. To determine the binding sites of BSA with ILs, the possible conformations of BSA-ILs system were modeled by using Autodock method. Firstly, the cluster analyses were performed for the possible binding sites of ILs on BSA. The statistical results selected from the minimum energy conformers from 100 runs were shown in Fig. 10(a-1, b-1, c-1, d-1, e-1, and f-1). The total of 16, 24, 40, 46, 51, and 52 multimember conformational clusters for BSA binding with [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl, respectively, indicating that BSA has multi possible binding sites for ILs. In addition, the number of the possible binding sites were increased from 16 to 52 implied that ILs with the longer hydrophobic chain are prone to binding with BSA (Fig. 11), which was in good agreement with the binding energy shown in Fig. 11. The lowest binding energy of [C2mim]Cl, [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl with BSA were −3.92, −4.34, −4.47, −4.72, −5.07, and −5.12 kcal/mol, respectively, implying that the binding ability of ILs with BSA increased sequentially with the increase of the number of the carbon atom of the hydrophobic chain. The experimental results are supported by the calculation results. In order to deep insight into the binding amino acids of BSA with ILs, the binding sites for the five BSA-Ils most stable complexes were analyzed and the results were shown in Fig. 10(a-2, b-2, c-2, d-2, e-2, and f-2). As shown in Fig. 10, it was found that site A located the joint gap between subdomain IA and subdomain IIA, site B was close to subdomain IIIA, which is known as “indole-benzodiazepine binding site” [57]. Site C was located in subdomain IIIA and B. In addition, both site D and site E was near subdomain IB. Fig. 12(A–E) presented a sketch of the five binding sites A–E of [C2mim]Cl with BSA. In site A, [C2mim]Cl entered into the binding cavity of BSA formed by Leu-22, Val-23, Ala-26, Leu-46, Leu-66, His-67, Phe70, Gly-247, Asp-248, Leu-249, Leu-250 amino acid residues. Among them, Leu, Val and Phe residues were hydrophobic amino residues, especially, five Leu residues were involved in the binding interaction of BSA with [C2mim]Cl, implying that the hydrophobic force was the main binding force between [C2mim]Cl and BSA, and the hydrophobic core cavity is able to host the hydrophobic carbon chain of ILs. In site B, [C2mim]Cl entered into subdomain IIIA, thirteen amino acid residues including Leu-197, Ser-343, Arg-347, Glu-449, Leu-452, Ser-453, Leu456, Asn-456, Asn-457, Leu-480, Val-481, Arg-483, Arg-484, and Pro485 took part in the binding interaction of BSA with [C2mim]Cl. Besides of Leu, Val, Pro, and other hydrophobic amino residues taking part in the binding interaction, the polar head of imidazole ring with positive charge interacted with Ser-453, Asn-457, Arg-483, and Arg-484. In site C, Thr-411, Arg-412, Pro-415, Pro-492, Asp-493, Glu-494, Tyr-496, Leu-532, Lys-533, Pro-536, and Lys-537 took part in the BSA-[C2mim] Cl complex formation. In sits D, Asp-108, His-145, Pro-146, Tyr-147, Phe-148, Ser-192, Ala-193, Glu-195, Arg-196, and Arg-458 interacted
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with [C2mim]Cl. In site E, Tyr-137, Ile-141, Tyr-160, Ile-181, Met-184, Arg-185, Val-188 were involved the binding interaction of BSA with [C2mim]Cl. In summary, the hydrophobic amino residues are main Leu, Pro, Phe, and Val residues, and the polar amino residues are main Arg, Ser, and Lys residues. The hydrophobic forces and polar interaction are the mainly binding forces of BSA with [C2mim]Cl. The binding sites for the five most stable complexes of the other four Ils with BSA were shown in Fig. 10(b-2, c-2, d-2, e-2, and f-2), and their sketch of the five binding sites A-E of [C4mim]Cl, [C6mim]Cl, [C8mim]Cl, [C10mim]Cl, [C12mim]Cl with BSA were presented in Fig. S2–6. It was found that there were similar binding sites of ILs in BSA. The subdomain IIIA and subdomain IB are the common sites of BSA binding with ILs. With the increase of the hydrophobic carbon chain, [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl entered into the hydrophobic cavity of subdomain IIA. Trp 213 residue was involved in the binding interaction of BSA with [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl, which favorable supported for the view that there were obviously significant fluorescence quenching as the concentration of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl increases. For BSA-[C12mim]Cl system, Trp-134 was involved in their binding interaction each other (Fig. S6 E), therefore, the fluorescence intensity decreased by 90% and the emission peak wavelength blue shift about 19 nm when the concentration of ILs was 4 mM in [C12mim]Cl-BSA system. In addition, with the increase of the hydrophobic carbon chain of ILs, their chain flexibility are enhanced. It can seen from Fig. S4–6 that there are different degree of blending of the hydrophobic carbon chain of [C8mim]Cl, [C10mim]Cl, and [C12mim]Cl in order to move into the active site. This conformational modifications of carbon chain indicated the importance of the hydrophobic chain of ILs during the binding interactions of them with BSA [58]. 4. Conclusion In this work, the impact of the alkyl chain length on binding of imidazolium-based ionic liquids to BSA was studied through three spectroscopic techniques including fluorescence, UV–vis, and CD and molecular modeling. On the basis of the results, it was found from fluorescence spectral data that ILs with the longer alkyl chain length have the stronger binding interaction with BSA and the greater conformational damage to protein. The different binding ability of ILs with the different length of alkyl-chain with protein leads to different effects on the tertiary structure of protein. The CD spectral results indicated that the order of unfolding BSA was to be [C12mim]Cl N [C10mim]Cl N [C8mim]Cl N [C6mim]Cl N [C4mim]Cl N [C2mim]Cl. [C12mim]Cl shows the strong destruction of the secondary structure of BSA. The drop of the value of Tm implied that the presence of high concentration of ILs
Fig. 11. The plots of the lowest binding energy (red line) and the conformational clusters (blue line) as a function of the number of carbon atom of the hydrophobic chain of ILs.
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Fig. 12. Details of the binding sites for the five BSA–[C2mim]Cl most stable complexes.
could reduce the thermal stability of BSA by accelerating the rate of thermal denaturation process. ThT fluorescence probe data showed that rate of the formation of beta sheet structures of BSA was lower in the presence of ILs with the longer hydrophobic chain, especially [C10mim]Cl and [C12mim]Cl. The nature of functionalization of alkyl chain of ILs has a significant effect on their interactions with protein. The esterase-like activity of BSA were decreased with the increase of ILs concentration. In addition, molecular modeling information indicated that ILs with the longer hydrophobic chain are prone to binding with BSA by the hydrophobic forces and polar interaction. This study not only provides important insights into the mechanism of interaction between ILs and BSA in different ILs concentration regimes, but it supports the toxicity background of ILs with the different lengths of hydrophobic chain. Acknowledgement We gratefully acknowledge financial support of the Fund from the National Natural Science Foundation of China (Project No. 21571154), the Natural Science Foundation (Grant No. BK20161315, BK20151296), the“333 Project”, “Qinglan Project”, and “Six Talent Peaks Project” of Jiangsu Province. Appendix A. Supplementary Data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2018.02.040. References [1] K. Binnemans, Chem. Rev. 37 (2005) 4148–4204. [2] C.D. Hubbard, P. Illner, R. Eldik van, Chem. Soc. Rev. 40 (2011) 272–290. [3] M.J. Earle, J.M. Esperanc, M.A. Gilea, J.N.C. Lopes, L.P. Rebelo, J.W. Magee, K.R. Seddon, J.A. Widegren, Nature 439 (2006) 831–834.
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