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Chemico-Biological Interactions 243 (2016) 54e61
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Chemico-Biological Interactions
journal homepage: www.elsevier .com/locate/chembioint
Comprehensive studies on the nature of interaction between
carboxylated multi-walled carbon nanotubes and bovine serum
albumin
Kai Lou a, b, Zhaohua Zhu b, Hongmei Zhang b, Yanqing Wang b, *, Xiaojiong Wang b,
Jian Cao b, **
a Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing City, Jiangsu Province, 210009, People's Republic of China
b Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province, 224002, People's Republic
of China
a r t i c l e i n f o
Article history:
Received 10 October 2015
Received in revised form
11 November 2015
Accepted 19 November 2015
Available online 30 November 2015
Keywords:
Carboxylated multi-walled carbon
nanotubes
Bovine serum albumin
Binding interaction
Adsorption
* Corresponding author.
** Corresponding author.
E-mail addresses:wyqing76@126.com (Y. Wang), y
http://dx.doi.org/10.1016/j.cbi.2015.11.020
0009-2797/© 2015 Elsevier Ireland Ltd. All rights res
a b s t r a c t
Herein, the interaction between carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) and
bovine serum albumin has been investigated by using circular dichroism, UVevis, and fluorescence
spectroscopic methods and molecular modeling in order to better understand the basic behavior of
carbon nanotubes in biological systems. The spectral results showed that MWCNTs-COOH bound to BSA
and induced the relatively large changes in secondary structure of protein by mainly hydrophobic forces
and p-p stacking interactions. Thermal denaturation of BSA in the presence of MWCNTs-COOH indicated
that carbon nanotubes acted as a structure destabilizer for BSA. In addition, the putative binding site of
MWCNTs-COOH on BSA was near to domain II. With regard to human health, the present study could
provide a better understanding of the biological properties, cytotocicity of surface modified carbon
nanotubes.
© 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Nowadays, carbon nanotubes (CNTs) have been widely used in
many electronics, medicine, space, and military applications due to
their unique and outstanding properties [1e4]. Because of the cell-
penetrating ability, sustained-release and drug targeting, CNTs
were expected to be promising as the basis for potential biomedical
and biotechnological applications, in particular, utilized for a class
of advanced drug and gene delivery [5,6]. Therefore, non-specific
interactions between CNTs and biological macromolecules will
take place when CNTs are introduced into organism [3,7,8].
As one kind of biological macromolecules, proteins are essential
to life and fundamentally components of all living cells. A better
understanding of the interactions of CNTs with different types of
proteins is of central importance for their potential biological ap-
plications and their cytotoxicity. In recent years, many reports
[3,5,9] have emerged about the binding interactions of CNTs with
ctu_caojian@126.com (J. Cao).
erved.
proteins. Liu et al. found that MWCNTs could interact with bovine
serum albumin (BSA) through mainly non-covalent binding force
[10]. Li et al. found that the interactions between BSA and carbox-
ylated single-walled carbon nanotubes were mainly favored by
hydrophobic force [3]. In our previous study, we showed that hy-
droxylated multi-walled carbon nanotubes (MWCNTs-OH) could
interact with BSA and cause secondary and tertiary structure
alteration of the protein [9]. Although interactions of proteins with
CNTs at the molecular level are reported a lot, data for the binding
of carboxylated multi-walled carbon nanotubes (MWCNTs-COOH)
with proteins are extremely deficient.
Here, we have investigated the interactional mechanism of one-
carboxylated multi-walled carbon nanotubes (MWCNTs-COOH)
with BSA by using multi-spectroscopic methods, adsorption ex-
periments and molecular modeling. Then, the results from this
study were expected to provide a better understanding on how
exactly MWCNTs-COOH interacts with biological molecule.
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K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e61 55
2. Material and methods
2.1. Reagents
Bovine serum albumin (A116563, lyophilized powder, � 96%)
was obtained from Aladdin Industrial Corporation. MWCNTs-COOH
(OD, 20e30 nm, Length, 10e30 mm, SSA >110 m2/g, eCOOH, 1.23%,
purity, >95%) were obtained from Chengdu Organic Chemicals Co.
Ltd., Chinese Academy of Sciences. All other chemicals used were of
analytical purity or higher. Water was purified with a Milli-Q pu-
rification system to a specific resistance >18.2 MU/cm.
2.2. Methods
2.2.1. BSA-MWCNTs-COOH interaction studies
Fluorescence intensity were measured on LSe50B Spectrofluo-
rimeter (Waltham, Massachusetts, USA) equipped with 1.0 cm
quartz cells and a thermostat bath to detect the fluorescence
quenching and microenvironment alteration of BSA caused by
MWCNTs-COOH. BSA concentration was kept constant at
5.0 � 10�6 mol L�1 and the MWCNTs-COOH concentrations have
been varied from 0 to 0.04 mg mL�1. Each solution was shaked for
60 min in order to reach binding equilibrium before fluorescence
measurement. In the fluorescence studies, excitation and emission
slit width were set at 3.0 nm with excitation at 280 nm. The syn-
chronous fluorescence spectra were recorded at Dl ¼ 15 nm or
Dl ¼ 60 nm. The circular dichroism (CD) spectra were measured by
a Chirascan spectrometer (Applied Photophyysics Ltd., Leather-
head, Surrey, UK) using a 1 mm quartz cell, the bandwidth was
1.0 nm.
For the CD experiments, a 0.02 mol L�1 phosphate buffer of pH
7.40 was exclusively prepared in ultrapure water. The
2.5 � 10�6 mol L�1 BSA solution in the presence and absence of
MWCNTs-COOH were recorded from 200 to 260 nm with three
scans averaged and scanning speed was set at 30 nm/min for each
CD spectrum. The program CDNN was used to analyze CD spectra.
The temperature of thermal denaturation of BSA in the absence and
presence of MWCNTs-COOH was varied from 20 to 90 �C in 5 �C
steps, with 300 s increments. Themelting temperature (Tm) and the
enthalpy changes at the melting temperature (DHm) of BSA were
obtained by using the Global Analysis Software.
2.2.2. BSA adsorption kinetic on MWCNTs-COOH
BSA adsorption kinetic experiments were performed using a
series of 50 mL flasks containing 30 mg of MWCNTs-COOH and
30 mL of 660 mg L�1 BSA solution. On the regular time intervals (0,
2.5, 5, 7.5, 10, 20, 30, 60, 120, 180 min), suitable aliquots were taken
and filtered using a 0.22 mmglass fiber. The protein concentration in
the solution was measured at 280 nm by using UVevis spectra on a
SPECORD 50 (Jena, Germany) using quartz cells with a 1 cm optical
path. The range of calibration curve of BSA was selected from 0 to
3300 mg L�1 and the calibration curve was y ¼ 0.00171 þ 0.41715x
(R ¼ 0.9999), which indicated that this curve have high linearly
dependent coefficient. The adsorption percentage (ads %) were
calculated based on the following equation:
Ads:% ¼ Ci � Cf
Ci
� 100 (1)
where Ci and Cf are the initial and final concentrations of BSA in
solution phase, respectively.
2.2.3. Molecular docking
The docking study was performed with Autodock 4.2.3 software
obtained from the Scripps Research Institute [11]. The structure of
BSA (PDB ID 3V03) was taken fromRCSB Protein Data Bank [12]. The
chirality (n, m), repeat units, diameter, tubelength, number of
walls, and wall separation were set at 6, 6, 15, 8.14 Å, 3.689 nm, 3,
and 2.0 Å, whichwas optimized by using Gaussian 09 at DFT/B3LYP/
3-21G level [13]. During the modeling docking study, a grid box of
126e126e126 with spacing of 0.700 Å in order to include all
possible binding sites for MWCNTs-COOH on BSA, which included
the entire binding site of BSA and provided enough space for
MWCNTs-COOH translational and rotational walk. All docking pa-
rameters were default parameters. Finally, the best docking results
were further analyzed by using the Molegro Molecular Viewer
software (Molegro-a CLC bio company, Aarhus, Denmark) [14].
3. Results and discussion
3.1. Binding nature of BSA with MWCNTs-COOH
As presented in Fig. 1 (A, B), fluorescence emission wavelength
of BSA had the maximum values at 346 nm when the excitation
wavelength was fixed at 280 nm. BSA had two Trp and eighteen Tyr
residues that have intrinsic fluorescence. It could be observed from
Fig. 1 (C) that the fluorescence of BSAwas obviously quenched after
addition of MWCNTs-COOH, the fluorescence spectra of BSA
experienced a quenching process, indicating that the binding in-
teractions existed betweenMWCNTs-COOH and BSA [15]. Whereas,
there was no obviously changes in the fluorescence emission
wavelength in the presence of MWCNTs-COOH, demonstrating that
microenvironment nearly Trp and Tyr residues is not affected
obviously [16]. Results from fluorescence quenching data could be
used to analysis the binding nature of MWCNTs-COOH with BSA.
Eqs (2)e(5) were used to estimate the binding constant, binding
sites, binding thermal parameters (DHB, DSB, and DGB).
log
�
F0 � F
F
�
¼ log KA þ nlog½Q � (2)
ln
ðKAÞ2
ðKAÞ1
¼ DH
Ο
R
�
1
T1
� 1
T2
�
(3)
DGΟ ¼ �RT ln KA (4)
DSΟ ¼ DH
Ο � DGΟ
T
(5)
where F and F0 are the fluorescence intensity of BSA in the absence
and presence of quenching agent (MWCNTs-COOH), respectively.
KA, n and [Q] are the binding constant, the number of binding sites
of MWCNTs-COOH on BSA and the concentration of MWCNTs-
COOH. Fig. 1(D) showed the plots of log [(F0eF)/F] versus log[Q].
The calculated results were shown in Table 1. In addition, the mo-
lecular weight of MWCNTs-COOH was not definite in the BSA-
MWCNTs-COOH system, a pseudo molar concentration (l) was
used according to Ref [17]. The value of l is no more than 1 � 10�8
according to the molecular size of MWCNTs-COOH in this paper.
Table 1 demonstrated that the value of binding constant and the
number of binding sites: KA ¼ 173.07/l, n ¼ 1.14 at 298 K, and
KA ¼ 213.80/l, n ¼ 1.23 at 310 K, which indicated that a major
binding site occurred in the binding site of MWCNTs-COOH on BSA.
The larger correlation coefficients are approximately equal to 1
demonstrating the occurrence of independent binding sites [3]. In
addition, the order of magnitude of KA of BSAwith MWCNTs-COOH
is about 107 if l is no more than 1 � 10�8, which indicated that
strong interactions existed between MWCNTs-COOH and BSA,
which is obviously stronger than the binding interaction of BSA
Fig. 1. (A, B) Effects of MWCNTs-COOH on the steady-state fluorescence of BSA, c (BSA) ¼ 5.0 � 10�6 mol L�1, c (MWCNTs-COOH) (from a to k) ¼ 0, 0.004, 0.008, 0.012, 0.016, 0.020,
0.024, 0.028, 0.032, 0.036, 0.040 mg mL�1, pH ¼ 7.40, A, T ¼ 298 K, B, T ¼ 310 K; (C) the plot of fluorescence quenching for BSA at 346 nmwith the concentration of MWCNTs-COOH;
(D) the logarithmic plot of BSA with the concentration of MWCNTs-COOH.
Table 1
Thermodynamic parameters for the binding interaction of BSA with MWCNTs-COOH.
T(K) KA L/mol n R DHB (kJ mol�1) DGB (kJ mol�1) DSB (J mol�1 K�1)
298 (173.07 ± 1.142)/l 1.14(±0.052) 0.9916 13.53 12.77 þ 2.48lnl 2.55e112.60 lnl
310 (213.80 ± 1.145)/l 1.23(±0.033) 0.9971 13.29 þ 2.58lnl
K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e6156
with carboxylated single-walled canbon nanotubes (SWCNTs-
COOH) (9.48 � 104 L mol�1) [3]. The surface area, the number of
eCOOH groups, and the particle size may affect the binding ability
of carbon nanotube with protein. Compared to SWCNTs, the
MWCNTs have higher mechanical strength and are able to absorb
more photons per nanotube due to the higher mass density of the
multiwalls [18]. The structure of MWCNTs takes the form of a stack
of concentrically rolled grapheme sheets [18]. In addition, BSA may
interact with SWNTs-COOH through amines with eCOOH which is
in negative state at pH7.4 [3]. These possibilities may result in the
differences on binding of the single and multi-wall carbon nano-
tubes to BSA. The binding constant increased with increasing the
temperature, meaning that at higher temperature, the interaction
between MWCNTs-COOH and BSA was stronger. In this report,
the binding constants at different temperature were used in Eqs
(3)e(5) to obtain the enthalpy change (DH�), entropy change (DS�)
and free energy change (DG�) in order to further study the bind-
ing nature of them. In Table 1, DH� > 0 and DS� > 0 were observed,
indicating that the binding interaction of BSA with MWCNTs-
COOH is endothermic and favored by hydrophobic interaction [19].
3.2. Effect of MWCNTs-COOH on BSA conformation
Synchronous fluorescence spectroscopy has beenwidely used to
investigate the tertiary changes of protein, for this spectroscopy
effectively provides information on the vicinity of the fluorescence
groups like Tyr and Trp residues [5]. When the distinction between
excitation and emission wavelengths is set at 15 nm, the fluores-
cence spectra are characteristics of Tyr residues whereas
Dl ¼ 60 nm indicates that of Trp residues. The effects of MWCNTs-
COOH on the synchronous fluorescence spectra of BSAwere shown
in Fig. 2 (A, B). As the data indicated, the maximum emission
wavelengths of Tyr and Trp residues in BSA did not show obvious
shift upon MWCNTs-COOH addition, for the changes in maximum
emissionwavelengths are indicators of the polarity of surroundings
of Trp and Tyr residues. This result was coincided with the stead-
state fluorescence spectra data that the binding of MWCNTs-
COOH to BSA did not perturb the environment of Try and Trp res-
idues in BSA [20]. It has been also shown in Fig. 2 that the reduced
degree of the fluorescence intensity of Tyr and Trp residues were
90.4% and 88.9%, respectively, indicating that partly Trp and Tyr
residues were near to MWCNTs-COOH.
In addition, circular dichroism (CD) spectra of BSA in the
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Fig. 2. Effects of MWCNTs-COOH on the synchronous fluorescence spectra of BSA, c (BSA) ¼ 5.0 � 10�6 mol L�1, c (MWCNTs-COOH) (from up to down) ¼ 0, 0.004, 0.008, 0.012,
0.016, 0.020, 0.024, 0.028, 0.032, 0.036, 0.040 mg mL�1, pH ¼ 7.40, T ¼ 298 K (A, Dl ¼ 15 nm; B, Dl ¼ 60 nm).
K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e61 57
absence and presence of MWCNTs-COOH were also recorded. The
results were shown in Fig. 3. As shown in line 1 of Fig. 3 A, two
negative bands at 208 and 222 nm of BSA indicates itself a-helical
structure. With the increase of MWCNTs-COOH concentrations, the
CD signal of BSA changed obviously, which indicated that the
binding interaction of MWCNTs-COOH with BSA induced the sec-
ondary structure of protein. The changes of the percentages of a-
helix, b-sheet, b-turn, and random coil in BSA with the increase of
MWCNTs-COOH concentrations are plotted in Fig. 3(B). It can be
seen that the content of a-helix decreased to reach 52.3% from
69.8%, the content of b-sheet, b-turn, and random coil all increased,
indicating that the protein a-helix conformation partly changed
into b-sheet, b-turn, and Random coil and MWCNTs-COOH partly
induced the denaturation and unfolding of BSA. Therefore, the
thermal denaturation behavior of BSA in the presence of MWCNTs-
COOH is helpful to understand the binding mechanism of them.
In this work, CD spectroscopy was used to characterize the
impact of above thermal denaturation on the secondary structure
and unfolding transition of BSA. First, Fig. 4(A, B) showedthe Far-
UV CD spectra of BSA in the absence and presence of MWCNTs-
COOH during thermal denaturation. The results from Fig. 4(A-2,
B-2) showed that the content of a-Helix decreased obviously and
the contents of b-sheet, b-turn, and random coil increased with the
increasing temperature. Compared with the thermal denaturation
process of BSA in the absence of MWCNTs-COOH, the change trend
in the thermal denaturation process of BSA-MWCNTs-COOH system
Fig. 3. (A) Effect of MWCNTs-COOH on the CD spectrum of BSA (2.5 � 10�6 mol L�1). MWCN
0.050, and 0.080 mg mL�1. (B) Plots of the percentages of the different structures of BSA in
did not change obviously, implying that the presence of MWCNTs-
COOH had not an enormous capacity for changing the thermal
denaturation behavior of BSA. Secondly, the melting temperature
(Tm) and the enthalpy changes at the melting temperature (DHm) of
BSAwere obtained by using Global Analysis Software. The values of
Tm and DHm of BSA in the absence of MWCNTs-COOH were
68.6 ± 0.2 �C and 138.2 ± 3.7 kJ M�1, and those of BSA in the
presence of MWCNTs-COOH were 64.8 ± 0.2 �C and
149.3 ± 2.6 kJ M�1, respectively. The values of Tm were reduced by
3.8 �C, which indicated that the thermal stability of BSA decreased
and MWCNTs-COOH acted as a structure destabilizer. The main
reason for the decrease of Tm might be the loss of ordered water
coating the peptide or the backbone changes of eNH or eC]O
groups in protein induced by the presence of MWCNTs-COOH near
the peptide [21].
3.3. BSA adsorption onto MWCNTs-COOH
As discussed in fluorescence spectral experiment, the fluores-
cence emission of BSA was quenched by MWCNTs-COOH. In order
to observe the binding process, the adsorption kinetic was analyzed
in this report. The variation of the adsorption amount with
adsorption time at 298 K was described in Fig. 5. As the result
showed, the BSA adsorption on MWCNTs-COOH could reach equi-
librium after 60 min, indicating that this adsorption process was a
fast adsorption. In addition, the pseudo-first-order and pseudo-
Ts-COOH concentrations from line 1 to line 8 were 0, 0.005, 0.010, 0.020, 0.030, 0.040,
the absence and presence of MWCNTs-COOH.
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Fig. 4. Far-UV CD spectra of the BSA (A) and BSA-MWCNTs-COOH system (B) during thermal denaturation. c(BSA) ¼ 2.5 � 10�6 mol L�1, c(MWCNTs-COOH) ¼ 0.040 mg mL�1.
Fig. 5. Adsorption capacity of BSA on MWCNTs-COOH versus time. MWCNTs-COOH,
1000 mg L�1; BSA, 660 mg L�1; pH ¼ 7.40, T ¼ 298 K.
K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e6158
second-order models were used to investigate the adsorption
process of BSA on MWCNTs-COOH. The pseudo-first-order and
pseudo-second-order models were presented as Eqs (6) and (7),
respectively.
logðqe � qtÞ ¼ log qe � k1t2:303 (6)
t
qt
¼ 1
k2q2e
þ t
qe
(7)
where qe, and qt are the amount of BSA adsorbed on the sorbent
(mg g�1) at equilibrium and tome t, respectively. k1 and k2 are the
rate constants of the first- and second-order adsorptions, which
were obtained from the plots of log(qe-qt) against t and t/qt versus t,
respectively. The plots of the first- and second-order models were
shown in Fig. 6 (A, B). The results of the kinetic parameters and the
calculated initial sorption rate values were listed in Table 2. As the
data showed, the linear regression coefficients of the first- and
second-order models were 0.9577 and 0.9998, respectively, indi-
cating that the pseudo-second-order model could describe the
adsorption of BSA. In addition, the values of k2 was 0.0008 min�1,
which implying that the adsorption of BSA on MWCNTs-COOH was
a fast process and BSA had the high density of activity site for
MWCNTs-COOH adsorption [22].
In addition, the WebereMorris intraparticle diffusion model
was used to deeply analysis the BSA adsorption onMWCNTs-COOH,
which is represented as follows [23]:
qt ¼ Kdt1=2 þ I (8)
where qt is the amount of BSA adsorbed at time t (min) and Kd is the
rate constant for intraparticle diffusion. In addition, values of I give
an idea about the thickness of the boundary layer [24]. The plots of
qt versus t1/2 for sorption of BSA were shown in Fig. 7 and the
related parameters were listed in Table 3. As the result showed, qt
versus t1/2 plots had two distinct regions. The sorption period of the
first and second linear portions were 0.0e6.0 and 8.0e14.0 min,
respectively. The high values of Kd1 represented external mass
transfer and binding of BSA by those active sites distributed onto
the outer surface of MWCNTs-COOHwith high affinity and this step
could be treated as a rate-limiting step. The second linear portions
denoted establishment of the equilibriumwith the low affinity sites
of BSA on MWCNTs-COOH. In addition, the value of I2 is larger than
that of I1 indicating that the boundary layer effect of second step is
greater [22].
Fig. 6. The plots of log (qe-qt) against t (A) and t/qt versus t (B).
Table 2
Kinetic parameters for BSA adsorption on MWCNTs-COOH at 298 K.
Equation Parameters
pseudo-first order model k1(min�1) 0.0297
qe(mg g�1) 77.09
R 0.9577
Pseudo-second order model k2(min�1) 0.0008
qe(mg g�1) 140.65
R 0.9998
Fig. 7. WebereMorris plots of BSA adsorption on MWCNTs-COOH.
Table 3
Kinetic parameters calculated from the WebereMorris kinetic model.
Linear portion Parameters
Initial linear portion Kd1(mg g�1 min�1/2) 18.12
I1 13.55
R 0.9799
second linear portion Kd2(mg g�1 min�1/2) 1.10
I2 119.56
R 0.9945
K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e61 59
3.4. Computational modeling of the MWCNTseCOOHeBSA complex
The binding interaction of MWCNTs-COOH with BSA was also
studied by using molecular docking technique. From the docking
results, seven possible conformations with minimum binding en-
ergy were obtained from 50 docking runs for the binding sites of
MWCNTs-COOH on BSA. The binding energy including the final
intermolecular energy (vdw þ Hbond þ desolvo
energyþ electrostatic energy) (1), the final total internal energy (2),
the torsional free energy (3) and the unbound system's energy (4)
were obtained from the Autodock calculation result of them were
showed in Table 4. The estimated free energy of
binding¼ (1) þ (2) þ (3)e(4). The estimated free energy of binding
(DG), the final intermolecular energy (Einter-mol),
vdw þ Hbond þ desolvo energy (EVHD), the electrostatic energy
(Eelec), the final total internal energy (Etotal), the torsional free en-
ergy (Etorsional), the unbound system's energy (Eunbound), and the
binding constant calculated from DG were also shown in Table 4.
Analyzing data in Table 4, we found that BSA had more than one
possible binding site to bind with MWCNTs-COOH. The large values
of Kb indicated that the strong interactions existed between BSA
and MWCNTs-COOH by hydrophobic, vdw, hydrogen-bonding
forces, for EVHD energy including van der Waals energy (Evdw),
EHbond and Edesolvo were the main part of binding free energy [25].
In addition, electrostatic forces (Eelec) were not the mainly driving
forces. The predicated binding model with the lowest docking en-
ergy and the largest value of binding constant of BSA with
MWCNTs-COOH was shown in Fig. 8(A, B). There were 29 amino
acid residues of domain II of BSA taking part in the binding in-
teractions of BSA with MWCNTs-COOH. These amino acid residues
were Glu-293, Lys-294, Asp-295, Ala-296, Ile-297, Pro-298, Glu-
299, Asn-300, Leu-301, Pro-302, Pro-303, Leu-304, Thr-305, Glu-
310, Arg-336, His-337, Pro-338, Glu-339, Tyr-340, Ser-370, Thr-371,
Phe-373, Asp-374, Lys-377, Val-380, Asp-381, Pro-440, Ser-442, and
Glu-443. Among above amino acid residues, Ala, Leu, Pro, Phe, and
Val residues are hydrophobic amino acid residues, especially five
Pro residues. Tyr and Phe residues were aromatic acid residues. So
the binding forces of BSAwith BSA should include hydrophobic and
p-p stacking interactions. These results were in accordance with
the fluorescence experimental results.
4. Conclusion
In the present manuscript, we have studied the bindingin-
teractions of MWCNTs-COOH with BSA by using CD, UVevis, fluo-
rescence spectroscopy and molecular modeling. The features are as
follows. MWCNTs-COOH acted as a structure destabilizer for BSA
and induced the decrease of the stable helical structures of protein.
The complex between MWCNTs-COOH and BSA easily formed with
high values of binding constants by mainly hydrophobic force and
p-p stacking interactions. In addition, the pseudo-second-order
model could describe the adsorption of BSA on MWCNTs-COOH.
This work not only provides useful information for characterizing
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Table 4
Docking results of BSA with MWCNTs-COOH by using Autodock program generated different ligand conformations.
Rank DG (kcal/mol) E inter-mol (kcal/mol) EVHD (kcal/mol) E elec (kcal/mol) E total (kcal/mol) E torsional (kcal/mol) E unbound (kcal/mol) Kb (L/mol)
1 �12.50 �17.57 �20.60 þ3.03 þ5.10 þ5.07 þ5.10 1.48 � 109
2 �12.21 �17.29 �20.28 þ2.99 þ5.05 þ5.07 þ5.05 9.06 � 108
3 �11.15 �16.22 �18.90 þ2.68 þ5.10 þ5.07 þ5.10 1.51 � 108
4 �11.00 �16.07 �19.96 þ3.89 þ5.12 þ5.07 þ5.12 1.17 � 108
5 �10.91 �15.98 �20.46 þ4.48 þ5.64 þ5.07 þ5.64 1.01 � 108
6 �9.25 �14.32 �17.73 þ3.40 þ5.64 þ5.07 þ5.64 6.10 � 106
7 �8.89 �13.96 �17.90 þ3.94 þ5.14 þ5.07 þ5.14 3.32 � 106
Fig. 8. Predicted orientation of the binding conformation of MWCNTs-COOHwith BSA, (A) the cartoon representation of the lowest docking energy conformation of MWCNTs-COOH
with BSA; (B) the representation of the amino acid residues of MWCNTs-COOH with BSA.
K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e6160
and understanding the binding interactions of MWCNTs-COOH
with serum albumin, but also provides important insight into the
biological properties, cytotocicity of other modified surface carbon
nanotubes.
Acknowledgment
We gratefully acknowledge financial support of the Fund for the
National Natural Science Foundation of China (Project No.
21571154, 21201147), the Natural Science Foundation of Jiangsu
Province (Grant No. BK2012671, BK20151296), and the Jiangsu
Fundament of “Qilan Project” and “333 Project”, and the sponsor-
ship of Jiangsu Overseas Research & Training Program for University
Prominent Young & Middle-aged Teachers and Presidents.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2015.11.020.
References
[1] O.G. Apul, Y. Zhou, T. Karanfil, Mechanisms and modeling of halogenated
aliphatic contaminant adsorption by carbon nanotubes, J. Hazard. Mater 295
(2015) 138e144.
[2] M.S. Mauter, M. Elimelech, Environmental applications of carbon-based
nanomaterials, Environ. Sci. Technol. 42 (16) (2008) 5843e5859.
[3] L. Li, R. Lin, H. He, L. Jiang, M. Gao, Interaction of carboxylated single-walled
carbon nanotubes with bovine serum albumin, Spectrochim. Acta A 105
(2013) 45e51.
[4] Q.X. Mu, D.L. Broughton, B. Yan, Endosomal leakage and nuclear translocation
of multiwalled carbon nanotubes: developing a model for cell uptake, Nano
Lett. 9 (2009) 4370e4375.
[5] X. Zhao, D. Lu, F. Hao, R. Liu, Exploring the diameter and surface dependent
conformational changes in carbon nanotube-protein corona and the related
cytotoxicity, J. Hazard. Mater 292 (2015) 98e107.
[6] D. Nepal, K.E. Geckeler, Proteins and carbon nanotubes: close encounter in
water, Small 3 (2007) 1259e1265.
[7] H. Ding, Y. Ma, Computer simulation of the role of protein corona in cellular
delivery of nanoparticles, Biomaterials 35 (2014) 8703e8710.
[8] H.J. Eom, C.P. Roca, J. Roh, N. Chatterjee, J. Jeong, I. Shim, H. Kim, P. Kim,
K. Choi, F. Giralt, J. Choi, A systems toxicology approach on the mechanism of
uptake and toxicity of MWCNT in Caenorhabditis elegans, Chem-Biol. Interact.
239 (2015) 153e163.
[9] Y. Guan, H. Zhang, Y. Wang, New insight into the binding interaction of of
hydroxylated carbon nanotubes with bovine serum albumin, Spectrochim.
Acta A 124 (2014) 556e563.
[10] X.C. Zhao, R.T. Liu, Z.X. Chi, Y. Teng, P.F. Qin, New insights into the behavior of
bovine serum albumin adsorbed onto carbon nanotubes: comprehensive
spectroscopic studies, J. Phys. Chem. B 114 (2010) 5625e5631.
[11] <http://www.scripps.edu/mb/olson/doc/autodock>.
[12] K.A. Majorek, P.J. Porebski, M. Chruszcz, S.C. Almo, W. Minor, Structural and
immunologic characterization of bovine, horse, and rabbit serum albumins,
Mol. Immunol. 52 (2012) 174e182.
[13] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson,
H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino,
G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven,
J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers,
K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand,
K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi,
N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi,
C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski,
G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas,
J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision D.01,
Gaussian Inc., Wallingford, CT, 2013.
[14] http://www.clcbio.com/products/molegro/.
[15] X. Li, Z. Yang, Interaction of oridonin with human serum albumin by
isothermal titration calorimetry and spectroscopic techniques, Chem-Biol
Interact. 232 (2015) 77e84.
[16] N. Gull, P. Sen, R.H. Khan, K. Din, Interaction of bovine (BSA), rabbit (RSA), and
porcine (PSA) serum albumins with cationic single-chain/gemini surfactants:
A comparative study, Langmuir 25 (2009) 11686e11691.
[17] X. Zhao, R. Liu, Z. Chi, Y. Teng, P. Qin, New insights into the behavior of bovine
serum albumin adsorbed onto carbon nanotubes: comprehensive spectro-
scopic studies, J. Phys. Chem. B 111 (2010) 5625e5631.
[18] M.H.M. Ahmed, N.M. Ali, Z.S. Salleh, A.A. Rahman, S.W. Harun, M. Manaf,
H. Arof, Q-switched erbium doped fiber laser based on single and multiple
walled carbon nanotubes embedded in polyethylene oxide film as saturable
absorber, Opt. Laser Technol. 65 (2015) 25e28.
http://dx.doi.org/10.1016/j.cbi.2015.11.020
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref1
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref1
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref1
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref1
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref2
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref2
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref2
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref3
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref3
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref3
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref3
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref4
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref4
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref4
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref4
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref5
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref5
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref5
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref5
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref6
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref6
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref6
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref7
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref7
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref7
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref8
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref8
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref8
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref8
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref8http://refhub.elsevier.com/S0009-2797(15)30122-8/sref9
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref9
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref9
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref9
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref10
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref10
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref10
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref10
http://www.scripps.edu/mb/olson/doc/autodock
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref12
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref12
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref12
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref12
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref13
http://www.clcbio.com/products/molegro/
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref15
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref15
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref15
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref15
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref16
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref16
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref16
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref16
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref17
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref17
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref17
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref17
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref18
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref18
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref18
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref18
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref18
K. Lou et al. / Chemico-Biological Interactions 243 (2016) 54e61 61
[19] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions:
forces contributing to stability, Biochemistry 20 (1981) 3096e3102.
[20] Y.Q. Wang, H.M. Zhang, Q.H. Zhou, H.L. Xu, A study of the binding of colloidal
Fe3O4 with bovine hemoglobin using optical spectroscopy, Colloid. Surf. A
Physicochem. Eng. Asp. 337 (2009) 102e108.
[21] E. Blanco, J.M. Ruso, J. Sabín, G. Prieto, F. Sarmiento, Thermodynamic study of
the thermal denaturation of a globular protein in the presence of different
ligand, J. Therm. Anal. Calorim. 87 (2007) 143e147.
[22] L. Zhang, P. Fang, L. Yang, J. Zhang, X. Wang, Rapid method for the separation
and recovery of endocrine-disrupting compound bisphenol AP from
wastewater, Langmuir 29 (2013) 3968e3975.
[23] V.S. Mane, I.D. Mall, V.C. Srivastava, Kinetic and equilibrium isotherm studies
for the adsorptive removal of Brilliant Green dye from aqueous solution by
rice husk ash, J. Environ. Manage 84 (2007) 390e400.
[24] A. Khenifi, B. Zohra, B. Kahina, H. Houari, D. Zoubir, Removal of 2,4-DCP from
wastewater by CTAB/bentonite using one-step and two-step methods: A
comparative, Chem. Eng. J. 146 (2009) 345e354.
[25] K.A. Connors, The stability of cyclodextrin complexes in solution, Chem. Rev.
97 (1997) 1325e1358.
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref19
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref19
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref19
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref20
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref20
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref20
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref20
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref20
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref20
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref21
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref21
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref21
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref21
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref22
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref22
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref22
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref22
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref23
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref23
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref23
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref23
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref24
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref24
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref24
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref24
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref25
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref25
http://refhub.elsevier.com/S0009-2797(15)30122-8/sref25
	Comprehensive studies on the nature of interaction between carboxylated multi-walled carbon nanotubes and bovine serum albumin
	1. Introduction
	2. Material and methods
	2.1. Reagents
	2.2. Methods
	2.2.1. BSA-MWCNTs-COOH interaction studies
	2.2.2. BSA adsorption kinetic on MWCNTs-COOH
	2.2.3. Molecular docking
	3. Results and discussion
	3.1. Binding nature of BSA with MWCNTs-COOH
	3.2. Effect of MWCNTs-COOH on BSA conformation
	3.3. BSA adsorption onto MWCNTs-COOH
	3.4. Computational modeling of the MWCNTs–COOH–BSA complex
	4. Conclusion
	Acknowledgment
	Transparency document
	References