Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 556–563
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New insight into the binding interaction of hydroxylated carbon nanotubes with bovine serum albumin Yonghui Guan, Hongmei Zhang, Yanqing Wang ⇑ Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Binding interaction of HO-MWCNTs
with BSA was investigated. The binding site of HO-MWCNTs on
BSA was near to domain II and domain I of BSA. HO-MWCNTs acted as a pusher to increase the rate of fibrillation of BSA. The ligand binding and unfolding of BSA were also affected by HOMWCNTs.
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 27 December 2013 Accepted 8 January 2014 Available online 23 January 2014 Keywords: Multi-walled hydroxylated carbon nanotubes Bovine serum albumin Fluorescence spectroscopy Binding mechanism Conformational changes
a b s t r a c t In order to understand the effects of carbon nanotubes on the structural stability of proteins, the ligandbinding ability, fibrillation, and chemical denaturation of bovine serum albumin in the presence of a multi-walled hydroxylated carbon nanotubes (HO-MWCNTs) was characterized by UV–vis, circular dichroism, fluorescence spectroscopy and molecule modeling methods at the molecular level. The experiment results indicated that the fluorescence intensity of BSA was decreased obviously in presence of HOMWCNTs. The binding interaction of HO-MWCNTs with BSA led to the secondary structure changes of BSA. This interaction could not only affect the ligand-binding ability of BSA, but also change the rate of fibrillation and denaturation of BSA. This work gave us some important information about the structures and properties of protein induced by carbon nanotubes. Ó 2014 Elsevier B.V. All rights reserved.
Introduction At the interface of nanomaterials science and biology, the development of carbon nanotubes (CNTs) is one of the most interesting advancements [1]. Owing to their unique and outstanding properties, CNTs have many potential applications in the fields of nanobiotechnology and nanomedicine including molecular imaging, disease diagnosis, advanced drug and gene delivery, and bimolec⇑ Corresponding author. Tel./fax: +86 515 88233188. E-mail address: [email protected] (Y. Wang). 1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2014.01.058
ular assembly [2–4]. Due to the great deal of applications of CNTs, the safe use of CNTs in vivo applications requires a clear understanding of CNTs interface. When CNTs enter the biological fluids, proteins always surround CNTs. The interactions of CNTs with proteins may change the environmental and biological activity of CNTs surface. On the contrary, the surface interactions of proteins with CNTs may be associated with some conformational changes of proteins and affect the biological function of proteins [5]. Therefore, the understanding of the fundamental interactions of CNTs with some blood proteins is of recent interest in the field of biological research [6].
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Serum albumins, the most abundant proteins of blood plasma, constitute the majority of the plasma fluid and play important roles in the transportation and disposition of several endogeneous and exogeneous compounds [7]. As the major transporter-binding proteins, serum albumins are often considered as models for studying the binding interactions of physiological substances with protein in vitro. In recent years, many reports have emerged about the interactions of CNTs with proteins at the molecular level [6,8,9]. Bomboi et al. have studied the interaction of a single walled carbon nanotubes with lysozyme [6]. Valenti et al. have studied the adsorption–desorption process of bovine serum albumin (BSA) on CNTs by reflectometry [8]. Li et al. have used the molecular spectral methods to investigate out the interaction of single-walled carbon nanotubes with human serum albumin [9]. The main aim of above reports is to understand the environmental and biological activity of CNTs. Although interactions between plasma proteins and CNTs at the molecular level are reported a lot, studies on the influences of CNTs on the drug-binding ability, fibrillation, and chemical denaturation of plasma proteins are extremely deficient. The changes of the transporting drugs and other physiological substances ability of serum albumins affect metabolism, membrane penetration, half-life and other pharmacokinetic properties of drugs. Protein fibrillation is involved in many human diseases, such as Alzheimer’s, Creutzfeld-Jacob disease, and dialysis-related amyloidosis [10,11]. In vitro, some protein denaturations have been shown to induce amyloid associated with neurodegenerative diseases. Therefore, the studies about the influence of CNTs on the above properties of serum albumin in depth are helpful to know about the potential biological risks from CNTs and to grow awareness of the nanotoxicology of CNTs [12]. In this paper, we have investigated the interactional mechanisms of one hydroxylated multi-walled carbon nanotubes (HOMWCNTs) with BSA. The effects of HO-MWCNTs on the fibrillation and denaturation of BSA have also been studied. Then, the results of our work were expected to provide a better understanding on how exactly CNTs interact with biological molecule. Materials and methods Materials Bovine serum albumin (A1933, lyophilized powder, P98%), thioflavin T, 1-anilino-8-naphthalenesulfonic acid (ANS), and guanidine hydrochloride (GuHCl) were obtained from Sigma–Aldrich. HO-MWCNTs (OD, 8–15 nm, purity, >95%) were obtained from Chengdu Organic Chemicals Co. Ltd., Chinese Academy of Sciences. The Tris, NaCl, etc. were all of analytical purity. The HCl (36%) were used to adjust pH of buffer solution. The BSA solution was prepared in pH 7.40 Tris–HCl buffer. In this paper, thioflavin T and 1-anilino8-naphthalenesulfonic acid (ANS) were dissolved in methanol to prepare a stock solution (3.0 mM), which were stored at 0–4 °C. Ultrapure water was used throughout. Procedure BSA–HO-MWCNTs interaction studies Fluorescence intensity were measured on LS–50B Spectrofluorimeter (Waltham, Massachusetts, USA) equipped with 1.0 cm quartz cells and a thermostat bath. BSA concentration was kept constant at 2 lM and the HO-MWCNTs concentration have been varied from 0 to 1.750 mg/L. In the fluorescence studies, excitation and emission slit width were set at 3.0 nm, respectively. Fluorescence spectra of all solution upon excitation at 280 nm were recorded from 300 nm to 500 nm. The synchronous fluorescence spectra were recorded at Dk = 15 nm or Dk = 60 nm. The UV–vis
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absorbance spectra were measured on a SPECORD S600 (Jena, Germany). The circular dichroism (CD) spectra were measured by a Chirascan spectrometer (Applied Photophyysics Ltd., Leatherhead, Surrey, UK) using a 1 mm quartz cell, the bandwidth was 1.0 nm. For the CD experiments, a 50 mM phosphate buffer of pH 7.40 was exclusively prepared in ultrapure water. The 2.0 lM BSA solution in presence and absence of HO-MWCNTs were recorded from 190 to 260 nm with three scans averaged and scan speed was set at 20 nm/min for each CD spectrum. The program CDNN was used to analyze CD spectra [13]. Computational modeling study Computational modeling study was carried out to analysis the binding interaction of HO-MWCNTs with BSA. In the present study, the multi-wall hydroxylated carbon nanotubes were generated using Materials Studio 6.0 [14]. The diameter, length, number of walls and wall separation were set at 1.036 nm, 2.254 nm, 3, and 1.1347 Å, respectively. The crystal structure of BSA (PDB ID 3V03) was taken from RCSB Protein Data Bank [15]. HO-MWCNTs were docked to BSA using the Lamarckian Genetic Algorithm provided by Autodock 4.2.3 software obtained from the Scripps Research Institute [16]. During the modeling docking study, we used a grid box of 126–126–126 Å, which included the entire binding site of BSA and provided enough space for HO-MWCNTs translational and rotational walk. The maximum number of energy evaluations and GA population size were set to 2,500,000 and 150, respectively. Other docking parameters were default parameters. The docking data were further analyzed by using the Molegro Molecular Viewer software (Molegro-a CLC bio company, Aarhus, Denmark) [17]. Fibrillation experiment of BSA BSA fibrillation was studied by incubating the sample solutions in absence and presence of HO-MWCNTs. The above sample solutions were prepared in thermostat bath at a constant temperature of 338 K. The complete incubation time was set 24 h. Portions of the incubated BSA solutions in absence and presence of HOMWCNTs were removed periodically using Eppendorf pipette and were placed in 5 mL centrifugal tubes. The samples were monitored by fluorescence measurement. In order to monitor the fibrillation process of BSA, the fluorescence spectra of thioflavin T in BSA-HO-MWCNTs system were measured during the BSA fibrillation. The emission spectra of thioflavin T were detected using an excitation wavelength of kex = 440 nm in the range from 460 nm to 600 nm. Guanidine hydrochloride denaturation of BSA To a fixed volume (3.0 mL) of BSA solution, BSA was previously incubated with different GuHCl concentration for 10 h in absence and presence of HO-MWCNTs at room temperature. BSA concentration was kept constant at 2 lM and the HO-MWCNTs concentrations have been set at 0 mg/L, 0.525 mg/L, and 1.750 mg/L, respectively. In the each BSA-HO-MWCNTs system, the GuHCl concentrations have been set at 0.0 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, 7.0 M and 8.0 M, respectively. After GuHCl denaturation, the fluorescence spectra of all solution upon excitation at 280 nm were recorded from 300 nm to 450 nm. Results and discussion The binding interaction of HO-MWCNTs with BSA BSA has two Trp residues (Trp-134, Trp-213), which have the maximum fluorescence emission peak at 346 nm when the excitation wavelength is fixed at 280 nm. The fluorescence change of Trp
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residue is often used to analyze the binding mechanism of BSA with CNTs and to provide clues about the nature of the binding process. Fig. 1(A) showed the fluorescence spectra of BSA by excitation wavelength at 280 nm in absence and presence of different concentration of HO-MWCNTs. It could be observed that the fluorescence of BSA was obviously quenched after addition of HO-MWCNTs Fig. 1(B). Results from fluorescence quenching data could be used to estimate the binding constant (KA) and binding sites (n) of BSA-HO-MWCNTs system by Eq. (1) [18,19].
log
F0 F ¼ log K A þ n log½HO-MWCNTs F
ð1Þ
where F and F0 are the fluorescence intensity of BSA in absence and presence of quenching agent (HO-MWCNTs), respectively. KA is the binding constant, [HO-MWCNTs] is the concentration of HOMWCNTs, and n is the number of binding sites of HO-MWCNTs on BSA. Fig. 1(C) showed the plot of log[(F0–F)/F] versus log[HOMWCNTs]. The values of slope and intercept were 1.18 and 0.145, which helped us to determine n = 1.18. This result indicated that a major binding site occurred in the binding site of HO-MWCNTs on BSA. In the BSA-HO-MWCNTs system, the CNTs molecular weight was not definite, so in this work, we used weight concentration multiplied by k as a pseudo molar concentration according to Ref. [19], which value is no more than 1 104. The value of intercept was 0.145, which implied that KA = 1.39 104 M1. The value for the binding constant indicated that there existed moderately strong interaction between HO-MWCNTs and BSA. The absorbance spectrum of BSA was presented in Fig. 2 (line b). Due to the phenyl group of Phe, Trp, and Tyr residues, BSA had an obvious absorption peak at around 278 nm which came from the
Fig. 2. The absorption spectra of BSA-HO-MWCNTs (a), BSA (b) and HO-MWCNTs (c) at room temperature. c(BSA) = 2.0 lM, c(HO-MWCNTs) = 1.750 mg/L.
p–p* electronic transitions [20]. The absorption peak changes are often used to study the structure conformation of BSA. The peak at 278 nm showed a blue shift from 278 to 269 nm, which indicated that the interaction of HO-MWCNTs with BSA induced the spreading of peptide chains and promoted the exposure of Trp and Tyr residues. In order to ascertain the possible effects of HO-MWCNTs binding on the secondary structure of BSA, CD experiments were performed in the absence and presence of HO-MWCNTs (Fig. 3). The analyses for the secondary structural elements were listed in Table 1.
Fig. 1. (A) Fluorescence spectra of BSA quenched by HO-MWCNTs (from top to bottom: c(BSA) = 2 lM, c(HO-MWCNTs) = 0, 0.175, 0.350, 0.525, 0.700, 0.875, 1.051, 1.226, 1.401, 1.576, 1.750 mg/L, pH = 7.40, T = 293 K); (B) the plot of fluorescence quenching for BSA at 346 nm with the concentration of HO-MWCNTs; (C) the logarithmic plot of BSA with the concentration of HO-MWCNTs.
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The binding interaction energy between amino residues and HO-MWCNTs were listed in Fig. 5(C). Hydrophobic interactions between HO-MWCNTs and Val-228, Phe-227, or Phe-325 were involved in the association. In addition, the big value of binding energy of some polar amino acid residues such as Tyr-262, Lys232, and Thr-235 indicated that p–p stacking and electrostatic interactions between BSA and HO-MWCNTs were also justifiable. Effects of HO-MWCNTs on the interaction of BSA with ANS
Fig. 3. The CD spectra of BSA and BSA-HO-MWCNTs system at 293 K. c(BSA) = 2.0 lM, c(HO-MWCNTs) = 1.750 mg/L.
The far-UV CD spectra of BSA (Fig. 3) exhibited two characteristic negative bands at 222 nm and 208 nm, which specified the extent of helicity of protein. As shown in Table 1, BSA had a high degree of a-helical secondary structure (66.5%), little b-turn content (12.1%), b-sheet content (6.2%) and random coil content (14.4%). We observed that the binding interaction of HO-MWCNTs with BSA altered the secondary structure of the protein. There was an obvious decline by 4.7% in a-helical content and the content of random coil partly increased by 2%. These results suggested that the interaction of HO-MWCNTs with BSA led to the loosening and unfolding of the protein skeleton. BSA is made up of three homologous domains (I, II, III) which contain twenty Tyr residues and two Trp residues. In domain I, there are nine Tyr (Tyr-30, 84, 137, 139, 147, 149, 155, 156, and 160) and one Trp residues (Trp-134); In domain II, there are seven Tyr residues (Tyr-262, 318, 331, 333, 352, 340, 352, and 369) and one Trp residues (Trp-213); In domain III, there are only four Tyr residues (Tyr-400, 410, 451, and 496). Of the two Trp residues, Trp-134 locates on the surface of subdomain IB and Trp-213 embeds in the hydrophobic pocket of subdomain IIA. In order to study the effects of HO-MWCNTs on the characteristic information of Trp and Tyr residues respectively, the synchronous fluorescence spectroscopy technique had been used to obtain the characteristic information of Trp or Tyr residues by maintaining a constant wavelength interval between excitation and emission wavelength at 60 nm or 15 nm[21]. It was shown in Fig. 4 that the addition of HO-MWCNTs in BSA solution resulted in strong fluorescence quenching of Trp and Tyr residues with similar decreasing percentages, which implied that the binding site of HO-MWCNT at BSA was near to both Trp and Tyr residues. Therefore, the binding site might locate at subdomain IIA or subdomain IB. In order to further investigate the binding affinity and mode between HO-MWCNTs and BSA, the modeling docking study was carried out using Autodock 4.2.3. The blind docking mode with the lowest binding free energy was shown in Fig. 5. HO-MWCNTs was near to Ala-321, 324, Asn-266, Asp-236, 265, 323, Glu-229, Lys-211, 232, Phe-227, 325, Thr-231, Thr-235, Tyr-262, Val-228. The above amino residues mainly located at domain II of BSA, which implied that the interaction area was near to domain II.
BSA plays an important role as a transporter of many endogenous and exogenous ligands [20]. In this work, we used ANS as a ligand to study the effects of HO-MWCNTs on the interaction of BSA with ANS. ANS is one of the most used organic probes in the analysis of protein by fluorescence method. Bound into hydrophobic sites of BSA and surrounded by nonpolar residues, ANS has highly fluorescence. In BSA, the binding sites of ANS locate at subdomain IIA and IIIA, one of them is docked at subdomain IIIA while the two other are docked at subdomain IIA [22]. If the binding site of HO-MWCNTs is near to the ANS binding site or occupies the ANS binding site, HO-MWCNTs maybe effect the fluorescence emission of ANS that binds into the domain of BSA. Fig. 6(A) showed the influence of HO-MWCNTs on the fluorescence emission properties of BSA-ANS system. BSA had a fluorescence emission peak at 346 nm by excitation at 280 nm (Fig. 6A, line a). As the addition of ANS, the fluorescence of BSA decreased obviously together with another fluorescence emission peak at 460 nm, which indicated that ANS entered into the hydrophobic cavity and the fluorescence resonance energy transfer from Trp 213 to ANS happened [22]. After addition of HO-MWCNTs, the fluorescence intensity at both 346 nm and 460 nm decreased in the same proportion. The same phenomenon happened in Fig. 6(B), with addition of HO-MWCNTs in BSA-ANS system, the fluorescence intensity of ANS decreased. These above results implied that HOMWCNTs could displace ANS from its binding site on BSA, or could prohibit the fluorescence resonance energy transfer from BSA to ANS by binding with BSA, which both could result in the ANS fluorescence intensity decrease. The binding site of HO-MWCNTs on BSA was mainly near to domain II or domain III. If we thought of ANS as a model drug, HO-MWCNTs did not share common binding site with ANS. We did also make a conclusion that the binding interaction of HO-MWCNTs with BSA could affect the drug-binding ability of BSA to some extent. Effects of HO-MWCNTs on the fibrillation of BSA In the fields of protein research, the protein fibrillation is often associated with various diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases [23]. Studies on the fibrillation of serum albumin have been received considerable interest [24,25]. Nanoparticles present enormous surface areas and are found to affect the rate of protein fibrillation [12]. Here, we thought of HOMWCNTs as exogenous material to study the role of HO-MWCNTs in the fibrillation process of BSA by fluorescence spectroscopy. As Fig. 7(A) showed, there was a strong increase in thioflavin T fluorescence because thioflavin T selectively bound to amyloid-like fibrils. The maximum thioflavin T fluorescence in BSA solution at
Table 1 Effect of HO-MWCNTs on the percentage of secondary structure elements in BSA. System
BSA BSA + OH-MWCNTs c(HO-MWCNTs) = 1.750 (mg/L)
Secondary structure elements in BSA
a-Helix (%)
b-Sheet (%)
b-Turn (%)
Rndm. coil (%)
66.5 61.8
6.2 7.3
12.1 12.7
14.4 16.4
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Fig. 4. The synchronous fluorescence quenching spectra of BSA by HO-MWCNTs (from top to bottom: c(BSA) = 2 lM, c(HO-MWCNTs) = 0, 0.175, 0.350, 0.525, 0.700, 0.875, 1.051, 1.226, 1.401, 1.576, 1.750 mg/L, pH = 7.40, T = 293 K). (A) Dk = 60 nm, (B) Dk = 15 nm and (C) Dk = 60 nm (j), Dk = 15 nm (d).
different incubation time in absence and presence of HO-MWCNTs at 338 K was reported in Fig. 7(B). In order to obtain statistical analysis of the effect of the HO-MWCNTs on the growth kinetics of fibrillation, Eq. (2) was used by nonlinear square curve fitting [23]. n
F ¼ F 1 þ DF expð½ksp t Þ
ð2Þ
where F, F1, and DF are the observed fluorescence intensity of thioflavin T at time t, the final fluorescence intensity, and the fluorescence amplitude, respectively. ksp is the rate of spontaneous fibril formation. As shown in Table 2, the kinetic parameter (ksp) and n of the fibrillation processes of BSA in absence of HO-MWCNTs was 0.133 h1 and 0.341. When the concentration of HO-MWCNTs were 0.700 mg/L, 1.750 mg/L and 3.502 mg/L, the value of ksp and n were 0.183 h1 and 0.376, 0.219 h1 and 0.456, 0.441 h1 and 0.572, respectively. The ksp value of BSA-HO-MWCNTs system were bigger than that of only BSA, which indicated that the more-efficient aggregation of BSA happened in presence of HO-MWCNTs. HO-MWCNTs acted as a pusher to increase the rate of BSA fibrillation. The interaction of BSA with HO-MWCNTs surfaces could actively promote an appropriate conformational change and induce the change of nature of BSA during its fibril formation process [12]. The interaction of HO-MWCNTs with BSA also maybe changes the concentration of monomers of BSA, which resulted in the rate of BSA fibrillation. The relatively higher n value of BSA in presence of HO-MWCNTs was indicative of a greater cooperative phenomenon in BSA-HO-MWCNTs system [24].
Effects of HO-MWCNTs on the guanidine hydrochloride denaturation of BSA Denaturation of protein has been considerable interest in characterizing the activities and functional properties of protein in different folded/unfolded states. The denaturation of protein often changes its binding activities and abilities to carry therapeutic or diagnostic agents and/or to remove toxic metabolites and xenobiotics [26,27]. In order to study the effects of HO-MWCNTs on the chemical denaturation process of BSA, we measured the fluorescence spectra of BSA and BSA-HO-MWCNTs system in presence of different GuHCl concentrations. Fig. 8(A) shows the fluorescence spectra of BSA in presence of different GuHCl concentrations. As the result showed, the fluorescence emission peak had a red-shift from 346 nm to 353 nm, which could be attributed to the significant alteration of the environment of the Trp residues by GuHCl. The Trp residues were exposed to more solvent. BSA has three domains (domain I, domain II and domain III). GuHCl can unfold these domains. With the increase of GuHCl concentration, domain III is firstly unfolded at about 1.5 M GuHCl [26]. Seen from Fig. 8(B) line a, the completely unfolding of domain III did not obviously effect the fluorescence intensity because the Trp residues of BSA was not located in domain III. When the GuHCl concentration was at 3 M, the fluorescence intensity of BSA began to decrease due to domain I was separated from domain II, domain I was partly unfolded and domain II was not unfolded [26]. After the GuHCl concentration was at 6 M, the fluorescence intensity of BSA did not change obviously, which indicates that the three domains are fully unfolded. However, the fluorescence
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Fig. 5. Binding pose of BSA on the HO-MWCNT according to the Autodock moleculardocking program. (A) The docking structure of BSA-HO-MWCNTs, the protein is colorcoded by subdomain and the HO-MWCNTs is depicted in space-filling representation color-coded by atom-type. (B and C) Detailed illustration of the binding between the HO-MWCNTs and BSA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. (A) Influence of HO-MWCNTs on the fluorescence spectra of BSA-ANS system. Line a: the fluorescence spectrum of BSA in absence of ANS and HO-MWCNTs, c(BSA), 2.0 lM; Line b–e: c(BSA), 2.0 lM; c(ANS), 3.0 lM; c(HO-MWCNTs), 0 mg/L (line b), 0.350 mg/L (line c), 0.875 mg/L (line d), 1.401 mg/L (line e), kex = 280 nm, pH = 7.40, T = 293 K. (B) Fluorescence spectra of BSA-ANS system in the presence of different concentration of HO-MWCNTs, from up to down, c(BSA), 2.0 lM; c(ANS), 3.0 lM; c(HOMWCNTs) (from 1 to 11), 0, 0.175, 0.350, 0.525, 0.700, 0.875, 1.051, 1.226, 1.401, 1.576, 1.750 mg/L, pH = 7.40, T = 293 K, kex = 350 nm.
intensity of BSA does begin to decrease when the GuHCl concentration was at 2 M in presence of HO-MWCNTs. The presence of HOMWCNTs made the separation between domain I and domain II in advance. Comparing to BSA in absence of HO-MWCNTs, the chemical denaturation of BSA became easily in presence of HOMWCNTs. GuHCl causes the conformational changes in the native BSA by destroying hydrogen bonds between the protein peptides [28]. The interaction of HO-MWCNTs with BSA could result in increasing the rate of Trp residues exposing to more solvent. Combining the data of Figs. 1 and 5 with the data of BSA denaturation,
we could draw a conclusion that the binding of BSA with HOMWCNTs mainly near to domain II and domain I.
Conclusion In this paper, the interaction of the hydroxylated multi-walled carbon nanotubes (HO-MWCNTs) with BSA was investigated by using fluorescence, UV–vis, CD spectroscopy and molecule modeling methods. The result indicated that the binding site of
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Fig. 7. (A) Aggregation of BSA leads to a marked increase in thioflavin T fluorescence. pH = 7.40, T = 338 K, kex = 440 nm, the final analysis sample concentration: c(BSA), 2.0 lM, c(thioflavin T), 40.0 lM. (B) Thioflavin T fluorescence over time measured at 482 nm for BSA incubated at 338 K in absence and presence of HO-MWCNTs. Line (a): 0 mg/L, line (b): 0.700 mg/L, line (c): 1.750 mg/L and line (d): 3.502 mg/L.
Table 2 Kinetic parameters of the fibrillation processes of BSA solution. Sample
ksp (h1)
n
R
BSA BSA + HO-MWCNTs c(HO-MWCNTs) = 0.700 (mg/L) BSA + MWCNTs c(HO-MWCNTs) = 1.750 (mg/L) BSA + MWCNTs c(HO-MWCNTs) = 3.502 (mg/L)
0.133 0.183 0.219 0.441
0.341 0.376 0.456 0.572
0.9956 0.9943 0.9955 0.9919
Fig. 8. (A) The fluorescence spectra of BSA in presence of different concentrations of GuHCl, pH = 7.40, kex = 280 nm, the final analysis sample concentration: c(BSA), 2.0 lM. (B) The fluorescence spectra of BSA measured at the maximum fluorescence emission peak for BSA.
HO-MWCNT at BSA was near to both Trp and Tyr residues and induced quenching the fluorescence intensity of BSA. There existed moderately strong interaction between HO-MWCNTs and BSA. The HO-MWCNTs affected the hydrophobic site of ANS on BSA and the rate of BSA fibrillation. HO-MWCNTS weaken the conformational stability of BSA. In conclusion, HO-MWCNTS had influences on the structure and properties of BSA by the surface interaction of them. This work provides the insights of the potential applications and biocompatible properties of carbon nanotubes, which may help to evaluate the undesirable risks of carbon nanotubes, and to modulate the function of this protein in the presence of other nano-materials.
Acknowledgements We gratefully acknowledge financial support of the Fund for the National Natural Science Foundation of China (Project No. 21201147), the Natural Science Foundation of Jiangsu Province (Grant Nos. BK2011422, BK2012671), and the Natural Science
Foundation of Education Department of Jiangsu Province (Grant No. 11KJB150019), the Jiangsu Fundament of ‘‘Qilan Project’’ and ‘‘333 Project’’, and the Scientific Foundation of Yancheng Teachers University.
References [1] D. Richards, A. Ivanisevic, Chem. Soc. Rev. 41 (2012) 2052–2060. [2] L. Cao, X. Wang, M.J. Meziani, F. Lu, H. Wang, P.G. Luo, Y. Lin, B.A. Harruff, L.M. Veca, D. Murray, S.Y. Xie, Y.P. Sun, J. Am. Chem. Soc. 129 (2007) 11318–11319. [3] T.W. Kim, P.W. Chung, I.I. Slowing, M. Tsunoda, E.S. Yeung, V.S. Lin, Nano Lett. 8 (2008) 3724–3727. [4] C. Thauvin, S. Rickling, P. Schultz, H. Celia, S. Meunier, Nat. Nanotechnol. 3 (2008) 743–748. [5] P. Roach, D. Farrar, C.C. Perry, J. Am. Chem. Soc. 127 (2005) 8168–8173. [6] F. Bomboi, F. Tardani, D. Gazzoli, A. Bonincontro, C.L. Mesa, Colloids Surf. B 108 (2013) 16–22. [7] U. Kragh-Hansen, Pharm. Rev. 33 (1981) 17–53. [8] L.E. Valenti, P.A. Fiorito, C.D. García, C.E. Giacomelli, J. Colloids Interface Sci. 307 (2007) 349–356. [9] L.L. Li, R. Lin, H. He, L. Jiang, M.M. Gao, Spectrochim. Acta. Part A. 105 (2013) 45–51.
Y. Guan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 556–563 [10] M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K.A. Dawson, Proc. Natl. Acad. Sci. USA 105 (2008) 14265–14270. [11] E. Herczenik, M.F.B.G. Gebbink, FASEB J. 22 (2008) 2115–2133. [12] S. Linse, C. Cabaleiro-Lago, W.F. Xue, I. Lynch, S. Lindman, E. Thulin, S.E. Radford, K.A. Dawson, Proc. Natl. Acad. Sci. USA 104 (2007) 8691–8696. [13] G. Böhm, R. Muhr, R. Jaenicke, Protein Eng. 5 (1992) 191–195. [14] Accelrys Inc. InsightII 2000 and Materials Studio, San Diego, CA. [15] . [16] . [17] . [18] F. Cañaveras, R. Madueño, J.M. Sevilla, M. Blázquez, T. Pineda, J. Phys. Chem. C 116 (2012) 10430–10437. [19] X. Zhao, R. Liu, Z. Chi, Y. Teng, P. Qin, J. Phys. Chem. B 111 (2010) 5625–5631. [20] F. Ding, J.X. Diao, Y. Sun, Y. Sun, J. Agric. Food. Chem. 60 (2012) 7218–7228.
563
[21] Z. Chi, R. Liu, H. Zhang, Biomacromolecules 11 (2010) 2454–2459. [22] D.I. Cattoni, S.B. Kaufman, F.L.G. Flecha, Biochim. Biophys. Acta 1794 (2009) 1700–1708. [23] J. Juárez, S.G. López, A. Cambón, P. Taboada, V. Mosquera, J. Phys. Chem. B 113 (2009) 10521–10529. [24] N.K. Pandey, S. Ghosh, S. Dasgupta, J. Phys. Chem. B 114 (2010) 10228–10233. [25] N.K. Holm, S.K. Jespersen, L.V. Thomassen, T.Y. Wolff, P. Sehgal, L.A. Thomsen, G. Christiansen, C.B. Andersen, A.D. Knudsen, D.E. Otzen, Biochim. Biophys. Acta 1774 (2007) 1128–1138. [26] B. Ahmad, M.Z. Ahmed, S.K. Haq, R.H. Khan, Biochim. Biophys. Acta 1750 (2005) 93–102. [27] H. Gao, X. Geng, B. Wang, Y. Zhou, J. Colloids Interface Sci. 344 (2010) 468–474. [28] D. Shortle, J. Biol. Chem. 264 (1989) 5315–5318.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
New insight into the binding interaction of hydroxylated carbon nanotubes with bovine serum albumin Yonghui Guan, Hongmei Zhang, Yanqing Wang ⇑ Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People’s Republic of China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Binding interaction of HO-MWCNTs
with BSA was investigated. The binding site of HO-MWCNTs on
BSA was near to domain II and domain I of BSA. HO-MWCNTs acted as a pusher to increase the rate of fibrillation of BSA. The ligand binding and unfolding of BSA were also affected by HOMWCNTs.
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 27 December 2013 Accepted 8 January 2014 Available online 23 January 2014 Keywords: Multi-walled hydroxylated carbon nanotubes Bovine serum albumin Fluorescence spectroscopy Binding mechanism Conformational changes
a b s t r a c t In order to understand the effects of carbon nanotubes on the structural stability of proteins, the ligandbinding ability, fibrillation, and chemical denaturation of bovine serum albumin in the presence of a multi-walled hydroxylated carbon nanotubes (HO-MWCNTs) was characterized by UV–vis, circular dichroism, fluorescence spectroscopy and molecule modeling methods at the molecular level. The experiment results indicated that the fluorescence intensity of BSA was decreased obviously in presence of HOMWCNTs. The binding interaction of HO-MWCNTs with BSA led to the secondary structure changes of BSA. This interaction could not only affect the ligand-binding ability of BSA, but also change the rate of fibrillation and denaturation of BSA. This work gave us some important information about the structures and properties of protein induced by carbon nanotubes. Ó 2014 Elsevier B.V. All rights reserved.
Introduction At the interface of nanomaterials science and biology, the development of carbon nanotubes (CNTs) is one of the most interesting advancements [1]. Owing to their unique and outstanding properties, CNTs have many potential applications in the fields of nanobiotechnology and nanomedicine including molecular imaging, disease diagnosis, advanced drug and gene delivery, and bimolec⇑ Corresponding author. Tel./fax: +86 515 88233188. E-mail address: [email protected] (Y. Wang). 1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2014.01.058
ular assembly [2–4]. Due to the great deal of applications of CNTs, the safe use of CNTs in vivo applications requires a clear understanding of CNTs interface. When CNTs enter the biological fluids, proteins always surround CNTs. The interactions of CNTs with proteins may change the environmental and biological activity of CNTs surface. On the contrary, the surface interactions of proteins with CNTs may be associated with some conformational changes of proteins and affect the biological function of proteins [5]. Therefore, the understanding of the fundamental interactions of CNTs with some blood proteins is of recent interest in the field of biological research [6].
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Serum albumins, the most abundant proteins of blood plasma, constitute the majority of the plasma fluid and play important roles in the transportation and disposition of several endogeneous and exogeneous compounds [7]. As the major transporter-binding proteins, serum albumins are often considered as models for studying the binding interactions of physiological substances with protein in vitro. In recent years, many reports have emerged about the interactions of CNTs with proteins at the molecular level [6,8,9]. Bomboi et al. have studied the interaction of a single walled carbon nanotubes with lysozyme [6]. Valenti et al. have studied the adsorption–desorption process of bovine serum albumin (BSA) on CNTs by reflectometry [8]. Li et al. have used the molecular spectral methods to investigate out the interaction of single-walled carbon nanotubes with human serum albumin [9]. The main aim of above reports is to understand the environmental and biological activity of CNTs. Although interactions between plasma proteins and CNTs at the molecular level are reported a lot, studies on the influences of CNTs on the drug-binding ability, fibrillation, and chemical denaturation of plasma proteins are extremely deficient. The changes of the transporting drugs and other physiological substances ability of serum albumins affect metabolism, membrane penetration, half-life and other pharmacokinetic properties of drugs. Protein fibrillation is involved in many human diseases, such as Alzheimer’s, Creutzfeld-Jacob disease, and dialysis-related amyloidosis [10,11]. In vitro, some protein denaturations have been shown to induce amyloid associated with neurodegenerative diseases. Therefore, the studies about the influence of CNTs on the above properties of serum albumin in depth are helpful to know about the potential biological risks from CNTs and to grow awareness of the nanotoxicology of CNTs [12]. In this paper, we have investigated the interactional mechanisms of one hydroxylated multi-walled carbon nanotubes (HOMWCNTs) with BSA. The effects of HO-MWCNTs on the fibrillation and denaturation of BSA have also been studied. Then, the results of our work were expected to provide a better understanding on how exactly CNTs interact with biological molecule. Materials and methods Materials Bovine serum albumin (A1933, lyophilized powder, P98%), thioflavin T, 1-anilino-8-naphthalenesulfonic acid (ANS), and guanidine hydrochloride (GuHCl) were obtained from Sigma–Aldrich. HO-MWCNTs (OD, 8–15 nm, purity, >95%) were obtained from Chengdu Organic Chemicals Co. Ltd., Chinese Academy of Sciences. The Tris, NaCl, etc. were all of analytical purity. The HCl (36%) were used to adjust pH of buffer solution. The BSA solution was prepared in pH 7.40 Tris–HCl buffer. In this paper, thioflavin T and 1-anilino8-naphthalenesulfonic acid (ANS) were dissolved in methanol to prepare a stock solution (3.0 mM), which were stored at 0–4 °C. Ultrapure water was used throughout. Procedure BSA–HO-MWCNTs interaction studies Fluorescence intensity were measured on LS–50B Spectrofluorimeter (Waltham, Massachusetts, USA) equipped with 1.0 cm quartz cells and a thermostat bath. BSA concentration was kept constant at 2 lM and the HO-MWCNTs concentration have been varied from 0 to 1.750 mg/L. In the fluorescence studies, excitation and emission slit width were set at 3.0 nm, respectively. Fluorescence spectra of all solution upon excitation at 280 nm were recorded from 300 nm to 500 nm. The synchronous fluorescence spectra were recorded at Dk = 15 nm or Dk = 60 nm. The UV–vis
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absorbance spectra were measured on a SPECORD S600 (Jena, Germany). The circular dichroism (CD) spectra were measured by a Chirascan spectrometer (Applied Photophyysics Ltd., Leatherhead, Surrey, UK) using a 1 mm quartz cell, the bandwidth was 1.0 nm. For the CD experiments, a 50 mM phosphate buffer of pH 7.40 was exclusively prepared in ultrapure water. The 2.0 lM BSA solution in presence and absence of HO-MWCNTs were recorded from 190 to 260 nm with three scans averaged and scan speed was set at 20 nm/min for each CD spectrum. The program CDNN was used to analyze CD spectra [13]. Computational modeling study Computational modeling study was carried out to analysis the binding interaction of HO-MWCNTs with BSA. In the present study, the multi-wall hydroxylated carbon nanotubes were generated using Materials Studio 6.0 [14]. The diameter, length, number of walls and wall separation were set at 1.036 nm, 2.254 nm, 3, and 1.1347 Å, respectively. The crystal structure of BSA (PDB ID 3V03) was taken from RCSB Protein Data Bank [15]. HO-MWCNTs were docked to BSA using the Lamarckian Genetic Algorithm provided by Autodock 4.2.3 software obtained from the Scripps Research Institute [16]. During the modeling docking study, we used a grid box of 126–126–126 Å, which included the entire binding site of BSA and provided enough space for HO-MWCNTs translational and rotational walk. The maximum number of energy evaluations and GA population size were set to 2,500,000 and 150, respectively. Other docking parameters were default parameters. The docking data were further analyzed by using the Molegro Molecular Viewer software (Molegro-a CLC bio company, Aarhus, Denmark) [17]. Fibrillation experiment of BSA BSA fibrillation was studied by incubating the sample solutions in absence and presence of HO-MWCNTs. The above sample solutions were prepared in thermostat bath at a constant temperature of 338 K. The complete incubation time was set 24 h. Portions of the incubated BSA solutions in absence and presence of HOMWCNTs were removed periodically using Eppendorf pipette and were placed in 5 mL centrifugal tubes. The samples were monitored by fluorescence measurement. In order to monitor the fibrillation process of BSA, the fluorescence spectra of thioflavin T in BSA-HO-MWCNTs system were measured during the BSA fibrillation. The emission spectra of thioflavin T were detected using an excitation wavelength of kex = 440 nm in the range from 460 nm to 600 nm. Guanidine hydrochloride denaturation of BSA To a fixed volume (3.0 mL) of BSA solution, BSA was previously incubated with different GuHCl concentration for 10 h in absence and presence of HO-MWCNTs at room temperature. BSA concentration was kept constant at 2 lM and the HO-MWCNTs concentrations have been set at 0 mg/L, 0.525 mg/L, and 1.750 mg/L, respectively. In the each BSA-HO-MWCNTs system, the GuHCl concentrations have been set at 0.0 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, 7.0 M and 8.0 M, respectively. After GuHCl denaturation, the fluorescence spectra of all solution upon excitation at 280 nm were recorded from 300 nm to 450 nm. Results and discussion The binding interaction of HO-MWCNTs with BSA BSA has two Trp residues (Trp-134, Trp-213), which have the maximum fluorescence emission peak at 346 nm when the excitation wavelength is fixed at 280 nm. The fluorescence change of Trp
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residue is often used to analyze the binding mechanism of BSA with CNTs and to provide clues about the nature of the binding process. Fig. 1(A) showed the fluorescence spectra of BSA by excitation wavelength at 280 nm in absence and presence of different concentration of HO-MWCNTs. It could be observed that the fluorescence of BSA was obviously quenched after addition of HO-MWCNTs Fig. 1(B). Results from fluorescence quenching data could be used to estimate the binding constant (KA) and binding sites (n) of BSA-HO-MWCNTs system by Eq. (1) [18,19].
log
F0 F ¼ log K A þ n log½HO-MWCNTs F
ð1Þ
where F and F0 are the fluorescence intensity of BSA in absence and presence of quenching agent (HO-MWCNTs), respectively. KA is the binding constant, [HO-MWCNTs] is the concentration of HOMWCNTs, and n is the number of binding sites of HO-MWCNTs on BSA. Fig. 1(C) showed the plot of log[(F0–F)/F] versus log[HOMWCNTs]. The values of slope and intercept were 1.18 and 0.145, which helped us to determine n = 1.18. This result indicated that a major binding site occurred in the binding site of HO-MWCNTs on BSA. In the BSA-HO-MWCNTs system, the CNTs molecular weight was not definite, so in this work, we used weight concentration multiplied by k as a pseudo molar concentration according to Ref. [19], which value is no more than 1 104. The value of intercept was 0.145, which implied that KA = 1.39 104 M1. The value for the binding constant indicated that there existed moderately strong interaction between HO-MWCNTs and BSA. The absorbance spectrum of BSA was presented in Fig. 2 (line b). Due to the phenyl group of Phe, Trp, and Tyr residues, BSA had an obvious absorption peak at around 278 nm which came from the
Fig. 2. The absorption spectra of BSA-HO-MWCNTs (a), BSA (b) and HO-MWCNTs (c) at room temperature. c(BSA) = 2.0 lM, c(HO-MWCNTs) = 1.750 mg/L.
p–p* electronic transitions [20]. The absorption peak changes are often used to study the structure conformation of BSA. The peak at 278 nm showed a blue shift from 278 to 269 nm, which indicated that the interaction of HO-MWCNTs with BSA induced the spreading of peptide chains and promoted the exposure of Trp and Tyr residues. In order to ascertain the possible effects of HO-MWCNTs binding on the secondary structure of BSA, CD experiments were performed in the absence and presence of HO-MWCNTs (Fig. 3). The analyses for the secondary structural elements were listed in Table 1.
Fig. 1. (A) Fluorescence spectra of BSA quenched by HO-MWCNTs (from top to bottom: c(BSA) = 2 lM, c(HO-MWCNTs) = 0, 0.175, 0.350, 0.525, 0.700, 0.875, 1.051, 1.226, 1.401, 1.576, 1.750 mg/L, pH = 7.40, T = 293 K); (B) the plot of fluorescence quenching for BSA at 346 nm with the concentration of HO-MWCNTs; (C) the logarithmic plot of BSA with the concentration of HO-MWCNTs.
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The binding interaction energy between amino residues and HO-MWCNTs were listed in Fig. 5(C). Hydrophobic interactions between HO-MWCNTs and Val-228, Phe-227, or Phe-325 were involved in the association. In addition, the big value of binding energy of some polar amino acid residues such as Tyr-262, Lys232, and Thr-235 indicated that p–p stacking and electrostatic interactions between BSA and HO-MWCNTs were also justifiable. Effects of HO-MWCNTs on the interaction of BSA with ANS
Fig. 3. The CD spectra of BSA and BSA-HO-MWCNTs system at 293 K. c(BSA) = 2.0 lM, c(HO-MWCNTs) = 1.750 mg/L.
The far-UV CD spectra of BSA (Fig. 3) exhibited two characteristic negative bands at 222 nm and 208 nm, which specified the extent of helicity of protein. As shown in Table 1, BSA had a high degree of a-helical secondary structure (66.5%), little b-turn content (12.1%), b-sheet content (6.2%) and random coil content (14.4%). We observed that the binding interaction of HO-MWCNTs with BSA altered the secondary structure of the protein. There was an obvious decline by 4.7% in a-helical content and the content of random coil partly increased by 2%. These results suggested that the interaction of HO-MWCNTs with BSA led to the loosening and unfolding of the protein skeleton. BSA is made up of three homologous domains (I, II, III) which contain twenty Tyr residues and two Trp residues. In domain I, there are nine Tyr (Tyr-30, 84, 137, 139, 147, 149, 155, 156, and 160) and one Trp residues (Trp-134); In domain II, there are seven Tyr residues (Tyr-262, 318, 331, 333, 352, 340, 352, and 369) and one Trp residues (Trp-213); In domain III, there are only four Tyr residues (Tyr-400, 410, 451, and 496). Of the two Trp residues, Trp-134 locates on the surface of subdomain IB and Trp-213 embeds in the hydrophobic pocket of subdomain IIA. In order to study the effects of HO-MWCNTs on the characteristic information of Trp and Tyr residues respectively, the synchronous fluorescence spectroscopy technique had been used to obtain the characteristic information of Trp or Tyr residues by maintaining a constant wavelength interval between excitation and emission wavelength at 60 nm or 15 nm[21]. It was shown in Fig. 4 that the addition of HO-MWCNTs in BSA solution resulted in strong fluorescence quenching of Trp and Tyr residues with similar decreasing percentages, which implied that the binding site of HO-MWCNT at BSA was near to both Trp and Tyr residues. Therefore, the binding site might locate at subdomain IIA or subdomain IB. In order to further investigate the binding affinity and mode between HO-MWCNTs and BSA, the modeling docking study was carried out using Autodock 4.2.3. The blind docking mode with the lowest binding free energy was shown in Fig. 5. HO-MWCNTs was near to Ala-321, 324, Asn-266, Asp-236, 265, 323, Glu-229, Lys-211, 232, Phe-227, 325, Thr-231, Thr-235, Tyr-262, Val-228. The above amino residues mainly located at domain II of BSA, which implied that the interaction area was near to domain II.
BSA plays an important role as a transporter of many endogenous and exogenous ligands [20]. In this work, we used ANS as a ligand to study the effects of HO-MWCNTs on the interaction of BSA with ANS. ANS is one of the most used organic probes in the analysis of protein by fluorescence method. Bound into hydrophobic sites of BSA and surrounded by nonpolar residues, ANS has highly fluorescence. In BSA, the binding sites of ANS locate at subdomain IIA and IIIA, one of them is docked at subdomain IIIA while the two other are docked at subdomain IIA [22]. If the binding site of HO-MWCNTs is near to the ANS binding site or occupies the ANS binding site, HO-MWCNTs maybe effect the fluorescence emission of ANS that binds into the domain of BSA. Fig. 6(A) showed the influence of HO-MWCNTs on the fluorescence emission properties of BSA-ANS system. BSA had a fluorescence emission peak at 346 nm by excitation at 280 nm (Fig. 6A, line a). As the addition of ANS, the fluorescence of BSA decreased obviously together with another fluorescence emission peak at 460 nm, which indicated that ANS entered into the hydrophobic cavity and the fluorescence resonance energy transfer from Trp 213 to ANS happened [22]. After addition of HO-MWCNTs, the fluorescence intensity at both 346 nm and 460 nm decreased in the same proportion. The same phenomenon happened in Fig. 6(B), with addition of HO-MWCNTs in BSA-ANS system, the fluorescence intensity of ANS decreased. These above results implied that HOMWCNTs could displace ANS from its binding site on BSA, or could prohibit the fluorescence resonance energy transfer from BSA to ANS by binding with BSA, which both could result in the ANS fluorescence intensity decrease. The binding site of HO-MWCNTs on BSA was mainly near to domain II or domain III. If we thought of ANS as a model drug, HO-MWCNTs did not share common binding site with ANS. We did also make a conclusion that the binding interaction of HO-MWCNTs with BSA could affect the drug-binding ability of BSA to some extent. Effects of HO-MWCNTs on the fibrillation of BSA In the fields of protein research, the protein fibrillation is often associated with various diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases [23]. Studies on the fibrillation of serum albumin have been received considerable interest [24,25]. Nanoparticles present enormous surface areas and are found to affect the rate of protein fibrillation [12]. Here, we thought of HOMWCNTs as exogenous material to study the role of HO-MWCNTs in the fibrillation process of BSA by fluorescence spectroscopy. As Fig. 7(A) showed, there was a strong increase in thioflavin T fluorescence because thioflavin T selectively bound to amyloid-like fibrils. The maximum thioflavin T fluorescence in BSA solution at
Table 1 Effect of HO-MWCNTs on the percentage of secondary structure elements in BSA. System
BSA BSA + OH-MWCNTs c(HO-MWCNTs) = 1.750 (mg/L)
Secondary structure elements in BSA
a-Helix (%)
b-Sheet (%)
b-Turn (%)
Rndm. coil (%)
66.5 61.8
6.2 7.3
12.1 12.7
14.4 16.4
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Fig. 4. The synchronous fluorescence quenching spectra of BSA by HO-MWCNTs (from top to bottom: c(BSA) = 2 lM, c(HO-MWCNTs) = 0, 0.175, 0.350, 0.525, 0.700, 0.875, 1.051, 1.226, 1.401, 1.576, 1.750 mg/L, pH = 7.40, T = 293 K). (A) Dk = 60 nm, (B) Dk = 15 nm and (C) Dk = 60 nm (j), Dk = 15 nm (d).
different incubation time in absence and presence of HO-MWCNTs at 338 K was reported in Fig. 7(B). In order to obtain statistical analysis of the effect of the HO-MWCNTs on the growth kinetics of fibrillation, Eq. (2) was used by nonlinear square curve fitting [23]. n
F ¼ F 1 þ DF expð½ksp t Þ
ð2Þ
where F, F1, and DF are the observed fluorescence intensity of thioflavin T at time t, the final fluorescence intensity, and the fluorescence amplitude, respectively. ksp is the rate of spontaneous fibril formation. As shown in Table 2, the kinetic parameter (ksp) and n of the fibrillation processes of BSA in absence of HO-MWCNTs was 0.133 h1 and 0.341. When the concentration of HO-MWCNTs were 0.700 mg/L, 1.750 mg/L and 3.502 mg/L, the value of ksp and n were 0.183 h1 and 0.376, 0.219 h1 and 0.456, 0.441 h1 and 0.572, respectively. The ksp value of BSA-HO-MWCNTs system were bigger than that of only BSA, which indicated that the more-efficient aggregation of BSA happened in presence of HO-MWCNTs. HO-MWCNTs acted as a pusher to increase the rate of BSA fibrillation. The interaction of BSA with HO-MWCNTs surfaces could actively promote an appropriate conformational change and induce the change of nature of BSA during its fibril formation process [12]. The interaction of HO-MWCNTs with BSA also maybe changes the concentration of monomers of BSA, which resulted in the rate of BSA fibrillation. The relatively higher n value of BSA in presence of HO-MWCNTs was indicative of a greater cooperative phenomenon in BSA-HO-MWCNTs system [24].
Effects of HO-MWCNTs on the guanidine hydrochloride denaturation of BSA Denaturation of protein has been considerable interest in characterizing the activities and functional properties of protein in different folded/unfolded states. The denaturation of protein often changes its binding activities and abilities to carry therapeutic or diagnostic agents and/or to remove toxic metabolites and xenobiotics [26,27]. In order to study the effects of HO-MWCNTs on the chemical denaturation process of BSA, we measured the fluorescence spectra of BSA and BSA-HO-MWCNTs system in presence of different GuHCl concentrations. Fig. 8(A) shows the fluorescence spectra of BSA in presence of different GuHCl concentrations. As the result showed, the fluorescence emission peak had a red-shift from 346 nm to 353 nm, which could be attributed to the significant alteration of the environment of the Trp residues by GuHCl. The Trp residues were exposed to more solvent. BSA has three domains (domain I, domain II and domain III). GuHCl can unfold these domains. With the increase of GuHCl concentration, domain III is firstly unfolded at about 1.5 M GuHCl [26]. Seen from Fig. 8(B) line a, the completely unfolding of domain III did not obviously effect the fluorescence intensity because the Trp residues of BSA was not located in domain III. When the GuHCl concentration was at 3 M, the fluorescence intensity of BSA began to decrease due to domain I was separated from domain II, domain I was partly unfolded and domain II was not unfolded [26]. After the GuHCl concentration was at 6 M, the fluorescence intensity of BSA did not change obviously, which indicates that the three domains are fully unfolded. However, the fluorescence
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Fig. 5. Binding pose of BSA on the HO-MWCNT according to the Autodock moleculardocking program. (A) The docking structure of BSA-HO-MWCNTs, the protein is colorcoded by subdomain and the HO-MWCNTs is depicted in space-filling representation color-coded by atom-type. (B and C) Detailed illustration of the binding between the HO-MWCNTs and BSA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. (A) Influence of HO-MWCNTs on the fluorescence spectra of BSA-ANS system. Line a: the fluorescence spectrum of BSA in absence of ANS and HO-MWCNTs, c(BSA), 2.0 lM; Line b–e: c(BSA), 2.0 lM; c(ANS), 3.0 lM; c(HO-MWCNTs), 0 mg/L (line b), 0.350 mg/L (line c), 0.875 mg/L (line d), 1.401 mg/L (line e), kex = 280 nm, pH = 7.40, T = 293 K. (B) Fluorescence spectra of BSA-ANS system in the presence of different concentration of HO-MWCNTs, from up to down, c(BSA), 2.0 lM; c(ANS), 3.0 lM; c(HOMWCNTs) (from 1 to 11), 0, 0.175, 0.350, 0.525, 0.700, 0.875, 1.051, 1.226, 1.401, 1.576, 1.750 mg/L, pH = 7.40, T = 293 K, kex = 350 nm.
intensity of BSA does begin to decrease when the GuHCl concentration was at 2 M in presence of HO-MWCNTs. The presence of HOMWCNTs made the separation between domain I and domain II in advance. Comparing to BSA in absence of HO-MWCNTs, the chemical denaturation of BSA became easily in presence of HOMWCNTs. GuHCl causes the conformational changes in the native BSA by destroying hydrogen bonds between the protein peptides [28]. The interaction of HO-MWCNTs with BSA could result in increasing the rate of Trp residues exposing to more solvent. Combining the data of Figs. 1 and 5 with the data of BSA denaturation,
we could draw a conclusion that the binding of BSA with HOMWCNTs mainly near to domain II and domain I.
Conclusion In this paper, the interaction of the hydroxylated multi-walled carbon nanotubes (HO-MWCNTs) with BSA was investigated by using fluorescence, UV–vis, CD spectroscopy and molecule modeling methods. The result indicated that the binding site of
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Fig. 7. (A) Aggregation of BSA leads to a marked increase in thioflavin T fluorescence. pH = 7.40, T = 338 K, kex = 440 nm, the final analysis sample concentration: c(BSA), 2.0 lM, c(thioflavin T), 40.0 lM. (B) Thioflavin T fluorescence over time measured at 482 nm for BSA incubated at 338 K in absence and presence of HO-MWCNTs. Line (a): 0 mg/L, line (b): 0.700 mg/L, line (c): 1.750 mg/L and line (d): 3.502 mg/L.
Table 2 Kinetic parameters of the fibrillation processes of BSA solution. Sample
ksp (h1)
n
R
BSA BSA + HO-MWCNTs c(HO-MWCNTs) = 0.700 (mg/L) BSA + MWCNTs c(HO-MWCNTs) = 1.750 (mg/L) BSA + MWCNTs c(HO-MWCNTs) = 3.502 (mg/L)
0.133 0.183 0.219 0.441
0.341 0.376 0.456 0.572
0.9956 0.9943 0.9955 0.9919
Fig. 8. (A) The fluorescence spectra of BSA in presence of different concentrations of GuHCl, pH = 7.40, kex = 280 nm, the final analysis sample concentration: c(BSA), 2.0 lM. (B) The fluorescence spectra of BSA measured at the maximum fluorescence emission peak for BSA.
HO-MWCNT at BSA was near to both Trp and Tyr residues and induced quenching the fluorescence intensity of BSA. There existed moderately strong interaction between HO-MWCNTs and BSA. The HO-MWCNTs affected the hydrophobic site of ANS on BSA and the rate of BSA fibrillation. HO-MWCNTS weaken the conformational stability of BSA. In conclusion, HO-MWCNTS had influences on the structure and properties of BSA by the surface interaction of them. This work provides the insights of the potential applications and biocompatible properties of carbon nanotubes, which may help to evaluate the undesirable risks of carbon nanotubes, and to modulate the function of this protein in the presence of other nano-materials.
Acknowledgements We gratefully acknowledge financial support of the Fund for the National Natural Science Foundation of China (Project No. 21201147), the Natural Science Foundation of Jiangsu Province (Grant Nos. BK2011422, BK2012671), and the Natural Science
Foundation of Education Department of Jiangsu Province (Grant No. 11KJB150019), the Jiangsu Fundament of ‘‘Qilan Project’’ and ‘‘333 Project’’, and the Scientific Foundation of Yancheng Teachers University.
References [1] D. Richards, A. Ivanisevic, Chem. Soc. Rev. 41 (2012) 2052–2060. [2] L. Cao, X. Wang, M.J. Meziani, F. Lu, H. Wang, P.G. Luo, Y. Lin, B.A. Harruff, L.M. Veca, D. Murray, S.Y. Xie, Y.P. Sun, J. Am. Chem. Soc. 129 (2007) 11318–11319. [3] T.W. Kim, P.W. Chung, I.I. Slowing, M. Tsunoda, E.S. Yeung, V.S. Lin, Nano Lett. 8 (2008) 3724–3727. [4] C. Thauvin, S. Rickling, P. Schultz, H. Celia, S. Meunier, Nat. Nanotechnol. 3 (2008) 743–748. [5] P. Roach, D. Farrar, C.C. Perry, J. Am. Chem. Soc. 127 (2005) 8168–8173. [6] F. Bomboi, F. Tardani, D. Gazzoli, A. Bonincontro, C.L. Mesa, Colloids Surf. B 108 (2013) 16–22. [7] U. Kragh-Hansen, Pharm. Rev. 33 (1981) 17–53. [8] L.E. Valenti, P.A. Fiorito, C.D. García, C.E. Giacomelli, J. Colloids Interface Sci. 307 (2007) 349–356. [9] L.L. Li, R. Lin, H. He, L. Jiang, M.M. Gao, Spectrochim. Acta. Part A. 105 (2013) 45–51.
Y. Guan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 556–563 [10] M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K.A. Dawson, Proc. Natl. Acad. Sci. USA 105 (2008) 14265–14270. [11] E. Herczenik, M.F.B.G. Gebbink, FASEB J. 22 (2008) 2115–2133. [12] S. Linse, C. Cabaleiro-Lago, W.F. Xue, I. Lynch, S. Lindman, E. Thulin, S.E. Radford, K.A. Dawson, Proc. Natl. Acad. Sci. USA 104 (2007) 8691–8696. [13] G. Böhm, R. Muhr, R. Jaenicke, Protein Eng. 5 (1992) 191–195. [14] Accelrys Inc. InsightII 2000 and Materials Studio, San Diego, CA. [15] . [16] . [17] . [18] F. Cañaveras, R. Madueño, J.M. Sevilla, M. Blázquez, T. Pineda, J. Phys. Chem. C 116 (2012) 10430–10437. [19] X. Zhao, R. Liu, Z. Chi, Y. Teng, P. Qin, J. Phys. Chem. B 111 (2010) 5625–5631. [20] F. Ding, J.X. Diao, Y. Sun, Y. Sun, J. Agric. Food. Chem. 60 (2012) 7218–7228.
563
[21] Z. Chi, R. Liu, H. Zhang, Biomacromolecules 11 (2010) 2454–2459. [22] D.I. Cattoni, S.B. Kaufman, F.L.G. Flecha, Biochim. Biophys. Acta 1794 (2009) 1700–1708. [23] J. Juárez, S.G. López, A. Cambón, P. Taboada, V. Mosquera, J. Phys. Chem. B 113 (2009) 10521–10529. [24] N.K. Pandey, S. Ghosh, S. Dasgupta, J. Phys. Chem. B 114 (2010) 10228–10233. [25] N.K. Holm, S.K. Jespersen, L.V. Thomassen, T.Y. Wolff, P. Sehgal, L.A. Thomsen, G. Christiansen, C.B. Andersen, A.D. Knudsen, D.E. Otzen, Biochim. Biophys. Acta 1774 (2007) 1128–1138. [26] B. Ahmad, M.Z. Ahmed, S.K. Haq, R.H. Khan, Biochim. Biophys. Acta 1750 (2005) 93–102. [27] H. Gao, X. Geng, B. Wang, Y. Zhou, J. Colloids Interface Sci. 344 (2010) 468–474. [28] D. Shortle, J. Biol. Chem. 264 (1989) 5315–5318.
