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Investigation on the conformational structure of hemoglobin on graphene oxide
Materials Chemistry and Physics 182 (2016) 272e279
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Investigation on the conformational structure of hemoglobin on graphene oxide Yanqing Wang a, b, *, 1, Zhaohua Zhu b, 1, Hongmei Zhang b, Jian Chen b, Boping Tang a, Jian Cao b, ** a
Jiangsu Key Laboratory for Bioresources of Saline Soils, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People's Republic of China Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People's Republic of China b
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
Multi-noncovalent interactions exist between BHb and GO. GO has high ability of disturbing the secondary of BHb. GO acts as a structure destabilizer during the thermal denaturation process of BHb. GO can obviously prevent the nonenzymatic glycosylation of BHb.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 March 2016 Received in revised form 6 June 2016 Accepted 12 July 2016 Available online 18 July 2016
The binding interaction between bovine hemoglobin (BHb) and a novel two-dimensional carbon nanomaterial, graphene oxide (GO), has been investigated in this work. It is found that GO strongly disturbs the secondary structure of BHb by forming BHb-GO aggregates. The binding affinity between the GO and BHb is shown to be mainly from non-covalent interactions including hydrophobic, hydrogen bonding, van der Waals and electrostatic interactions. In addition, two possible binding modes are proposed, insert binding mode and surface binding mode, as shown by molecular modeling. Our findings also show that the existence of GO can significantly prevent the non-enzymatic glycosylation of BHb and decrease the thermal stability of the protein. This work elucidates the effects of the binding interactions of GO with BHb on some biological properties and functions of heme protein. © 2016 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Surfaces Photoluminescence spectroscopy Thermodynamic properties
1. Introduction Nowadays, carbon nano-materials have been widely used in biological applications due to their unique and outstanding properties [1e3]. A novel two-dimensional (2D) carbon nanomaterial,
* Corresponding author. Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People's Republic of China. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (J. Cao). 1 Yanqing Wang and Zhaohua Zhu contributed equally. http://dx.doi.org/10.1016/j.matchemphys.2016.07.032 0254-0584/© 2016 Elsevier B.V. All rights reserved.
graphene oxide (GO), has shown potential biotechnological applications in drug and gene delivery systems [4e8]. Therefore, the understanding GO's influence on structure and activities of biomolecules are essential for before any biological applications of GO, especially for disease- and drug-related proteins [9,10], cellular bioimaging [11,12], protein adsorption [13,14] etc. Many studies showed that GO strongly interacted with amino acids, peptides, and serum proteins [9]. The adsorption of serum proteins on GO occurred spontaneously and rapidly, leading to significant changes in size, zeta potential, and morphology [15]. However, so far very few studies on the biocompatibility of GO with heme proteins, or on the toxic effects of GO on heme proteins at the molecular level have
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Fig. 1. (A) Circular dichroism (CD) spectra and (B) the percentages of the different structures of free BHb and its GO-BHb complex, c(BHb) ¼ 5 106 mol/L, pH ¼ 7.4, T ¼ 298 K.
been conducted [16,17]. Hemoglobin (Hb) is one of heme proteins that makes up about 92% of the Red blood cells' dry content and plays various roles including in the transport of oxygen, in the dispersion of hydrogen peroxide, and in the affection of electron transfer in human body parts and organs in living beings [18,19]. Hb is oxygen carriers in red blood cells [18]. The amount of Hb in blood is associated with many clinical diseases such as leukemia, anemia, heart diseases, etc [20]. When GO penetrates plasma membrane, the interactions of them with Hb maybe affect human health. In addition, Hb can successfully combine with carboxyl grapheme through condensation reaction to form the bio-nanocomposites that exhibits good ability of oxygen-carrying [17]. For the purpose of investigation about the biocompatibility, bioavailability, and toxicity of GO, the fundamental understandings of the binding behaviors of Hb are of critical importance for the integration of biology with GO. Herein, Hb is used as a good protein model for conformational change studies before and after the assembly on GO surface. The binding affinity, the thermodynamics property, the energetic of the interaction, and the thermal stability of GO-Hb association, the effect of GO on the non-enzymatic glycosylation of BHb was carried out by experimental and calculation studies. According to the study, we expect it not only provides the detailed conformation behavior of Hb molecules on GO but also creates a framework for analyzing the biosafety of GO in terms of the biological behavior of biomacromolecules. In addition, these findings also have shed light respective the design of bio-nanocomposites by comprehensive reconsideration of their interaction with Hb.
Fig. 2. UVevis absorption spectra of BHb, c(BHb) ¼ 5 106 mol/L, pH ¼ 7.4, T ¼ 298 K.
GO,
and
BHb-GO
system,
2. Material and methods 2.1. Reagents Bovine hemoglobin (BHb) was purchased from Sigma (St, Louis, MO, USA) and used without further purification. GO solution (5%) was purchased from Aladdin Industrial Corporation (Shanghai, China). The stock solutions of BHb (5.0 106 mol/L) was dissolved in a 0.05 mol/L potassium phosphate buffer with pH 7.4 by gentle stirring at room temperature and stored at 4 C. All other chemicals were of analytical reagent grade. Ultrapure water was used throughout. 2.2. Methods 2.2.1. UVevis absorption measurements The difference UVevis absorption measurement of BHb (5.0 106 mol/L) in the absence and presence of GO were measured on A SPECORD S600 spectrophotometer equipped with 1 cm quartz cells. A fixed concentration of BHb with different concentration of GO was added to the 1 cm sample cell, Then an equal concentration of GO was added to the reference cell simultaneously [21,22]. The scan speed was set at 200 nm/min and the spectra range was from 200 to 600 nm. 2.2.2. Fluorescence spectra measurements A LSe50B Spectrofluorimeter equipped with 1.0 cm quartz cells and a thermostat bath was used for fluorescence spectra measurements of BHb in the absence and presence of GO. The fluorescence spectra of BHb-GO reaction solutions were recorded in the range of 300e500 nm with the excitation wavelength 280 nm after 1 h equilibrate. Synchronous fluorescence spectra of BHb were recorded at 20 nm or 60 nm. In addition, for three-dimensional fluorescence spectra, the emission wavelengths range was selected from 270 to 500 nm, the initial excitation wavelength was set from 200 to 340 nm with increments of 10 nm. 2.2.3. Circular dichroism spectra measurements The CD spectra of BHb (5.0 106 mol/L) in the absence and presence of GO were acquired on an Applied Photophysics Ltd. Chirascan spectrometer. For each CD spectrum, the program CD spectra deconvolution program (CDNN) was used to obtain the percentage of a-Helix, b-Sheet, b-turn, random coil of BHb in the absence and presence of GO in order to analyze the effect of GO on the secondary structure of protein [http://bioinformatik. biochemtech.uni-halle.de/cdnn] [23]. The temperature of thermal denaturations of BHb in the absence and presence of GO were varied from 20 to 90 C in 5 C steps, with 250 s increments.
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Fig. 3. The three-dimensional fluorescence spectral contours of BHb (A), BHb-GO system (B,D), GO (E). c(BHb): A 5.0 106 mol/L, B, 5.0 106 mol/L, C, 5.0 106 mol/L, D, 5.0 106 mol/L, E, 0.0 106 mol/L, c(GO): A, 0.00 mg/mL, B, 0.004 mg/mL, C, 0.010 mg/mL, D, 0.080 mg/mL, E, 0.010 mg/mL.
2.2.4. Molecular modeling The crystal structure of BHb (PDB ID 1G09) was obtained from the Protein Data Bank for molecular modeling [24]. The geometries of GO (20 12 Å2) were optimized at DFT/B3LYP/3-21G (d, p) by Gaussian 09 [25]. The surface of GO was randomly decorated with epoxy and hydroxyl groups. Although the model size of GO was obviously smaller than that used in the experiment, this proposed model will be of service to explain the binding mechanism of GO with BHb. Autodock 4.2.3 Program was used to perform blind docking calculations of BHb with GO, respectively. In the blind docking calculations, a grid box of 126 126 126 Å with spacing of 0.357 Å was used to enclose BHb and GO. The Lamarckian Genetic Algorithm method was used as the searching algorithm. In addition, MMV software was used to analyze the predicated binding mode [26]. 2.2.5. BHb glycosylation and its detection Fructose (1 mol/L) were used to glycosylate BHb. The glycosylation reaction was carried out for 72 h in room temperature, both in the absence and presence of GO. The glycosylation solution of BHbGO system were measured on A SPECORD S600 spectrophotometer equipped with 1 cm quartz cells. 3. Results and discussion 3.1. The influence of GO binding on the conformational structure of BHb The circular dichroism (CD) results can give a useful estimate about the global structural change of protein induced by some molecules [27]. To ascertain the possible of GO binding on the secondary structure of BHb, we have performed CD studies in the
range of 200e300 nm in the absence and presence of GO. Fig. 1 (A) shows the far-UV CD spectra of the free BHb and its GO-BHb complex obtained at pH ¼ 7.4 and T ¼ 298 K. The far-UV spectra of BHb exhibit two negative bands at about 209 nm (pep*) and 222 nm (nep*) indicating the a-Helix structure of protein [28,29]. The binding of GO to BHb causes decrease in band intensity without significant shift of the peaks, which indicates the decrease of the aHelix content in BHb and the peptide strand unfolding even more. In addition, the percentages of the different structures of BHb in the absence and presence of GO is shown in Fig. 1(B), which reveals that the proportion of a-Helix decreased from 53% in native BHb to 39% in the presence of GO (0.08 mg/mL) with increase in random coil from 22% to 29% and in b-Sheet from 9% to 15%. From above results, it is apparent that the effect of GO binding on BHb causes a secondary changes of the protein and induce some perturbations of protein fold by mainly resulted from some hydrogen bonds and hydrophobic interactions [30]. The GO induced conformational change of BHb was also studied with UVevis absorption spectroscopy. The results are shown in Fig. 2. GO displays a maximum absorption at 222 nm due to the pep* transition of aromatic C]C bonds and a shoulder around 300 nm due to the nep* transition of C]O bonds [31]. The absorption spectra of native BHb show several electronic bands at 224 (corresponding to the nep* transition of BHb's characteristic polypeptide backbone structure C]O), 275 nm (corresponding to the pep* transition of the phenyl group of tryptophan (Trp) and tyrosine (Tyr) residues), 407 (belonging to the electronic transition of p/p* of hematoporphyrin), 573, and 630 nm (ligand-to-metal charge transfer, LMCT) [32]. After addition of GO in BHb solution, an alteration in the intensity of these above absorption peaks were observed. The absorbance intensities of bands at about 224 nm decreased from 2.14 to 1.37, indicating the reduction of a-helical
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Fig. 4. Far-UV CD spectra of BHb (A), BHb-GO system (B, C) during thermal denaturation. c(BHb) ¼ 5.0 106 mol/L, c(GO), (A) 0.000 mg/mL; (B) 0.020 mg/mL; (C) 0.080 mg/mL.
Fig. 5. Temperature dependence of the a-helix of BHb as determined by CD measurements in the absence and presence of GO, c(BHb) ¼ 5 106 mol/L, pH ¼ 7.4.
content in BHb and the loosening the BHb skeleton [33]. In addition, the strong Soret band at 407 nm is associated with the porphyrin ring and can provide insight into the environmental influence on the native structural of conformation of BHb [34,35]. The decrease of the intensity of Soret band indicating that GO binding induced the conformational change near heme moiety in BHb and GO are involved in producing a disturbance of the structure and the exposure of the heme group to the aqueous medium. In addition, the absorbance intensities increase from 0.54 to 0.72 and the blue shift (from 275 nm to 268 nm) of the absorption peak at about 275 nm implied that the micro-environmental changes of aromatic acid residues and more aromatic acid residues were extended into the aqueous environment [36]. When GO is added in BHb solution, some water molecules on the surface of protein are replaced by GO that can approach the amino acid residues of BHb [36]. More aromatic acid residues were extended into the aqueous environment
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1.0
pH = 2.0 pH = 7.4 pH = 11.0
0.9
Fcor/F0
0.8 0.7 0.6 0.5 0.4 0.3
0
5
10
15
20
[GO]/(ug/mL) Fig. 8. The quenching of BHb (5.0 106 mol/L) fluorescence intensity by GO in different concentrations. T ¼ 298 K, lex ¼ 280 nm. Fig. 6. UVeVis absorption spectra of the GO-BHb (5.0 106 mol/L) system in glycosylation reaction, pH ¼ 7.4, T ¼ 298 K.
and the tertiary structure of BHb was destroyed [37]. In a word, these observations strongly suggests that binding interactions exist between GO and BHb and GO binding induces the conformational changes in the protein. In addition, three isosbestic points at 236, 329, and 420 nm in the resulting spectra indicate that the binding equilibrium exists between free GO and the bound forms of GO [27]. BHb contains three Trp units (a-14Trp, bTrp-15 and bTrp-37) in each a and b chain. Its three-dimensional Ex/Em fluorescence is presented in Fig. 3. On the basis of the structure and component of BHb, there should be a noncovalent interaction between protein and GO. Indeed, the three-dimensional Ex/Em fluorescence spectral experiments in the present study shows the strong quenching of BHb induced by GO. It could be seen from Fig. 3 (A) that there are two kinds of fluorescence spectral peaks of BHb including endogenous fluorophores peaks (Peak a and Peak b) and the Raleigh scattering peaks (Peak 1 and Peak 2). Peak a is caused by the p/p* transition of aromatic amino acids in BHb, and Peak b is caused by the n/p* transition of BHb's characteristic polypeptide backbone structure C]O [38]. Upon addition of trace amount of GO to BHb solution, some remarkable changes of Peak a and Peak b are observed. the fluorescence intensity of Peak a and Peak b is strongly reduced and the shape of peak b is changed and disappeared, as shown in Fig. 3(BeD). In addition, two new peaks formed from 300 to 500 nm and from 450 to 500 nm. Compared with Fig. 3(E), this
two new fluorescence peak are not come from GO. So there must be some new phenomenon that takes place when BHb binds with GO in solution. Therefore, the newly formed BHb-GO conjunctions by some pep interaction or hydrophobic interaction between protein and plenty of small aromatic areas with sp2 carbons of GO contribute to these two peaks.
3.2. The influence of GO binding on the thermal stability of BHb The thermal unfolding of BHb in the absence and presence of GO were explored by using CD experiments. BHb consists of two a and two b subunits, each a-chain contains 141 amino acid residues and each b chain contains 146 amino acid residues. At about 20 C the protein retains its native structure in the buffer with two characteristic negative peaks at 209 and 222 nm. The temperaturedependent CD measurements of BHb are shown in Fig. 4. It is found that in buffer the percentages of BHb different structure including a-helix, b-sheet, b-turn, and Random coil change with increasing the temperature. As Fig. 4 A-1,2 shows, the content of ahelix decreased from 53% at 20 C to 16% at 87 C, while b-sheet and random coil obviously increased, indicating that the secondary structure of the protein suffers considerable damage, signifying the one set of an unfolding process [39]. It can also be seen from Fig. 4 B-1,2,C-1,2 that the changes in the secondary structure of BHb in the presence of GO against temperature were different from Hb in the absence of GO. As shown in Fig. 5, the unfolding temperature decreases with increasing GO concentration, revealing that the protein in the presence of GO is more prone towards thermal
Fig. 7. Fluorescence spectra of BHb (5.0 106 mol/L) in different concentrations of GO. c(GO)/(mg/mL): 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20. T ¼ 298 K, lex ¼ 280 nm. (A) pH ¼ 2.0, (B) pH ¼ 7.4, (C) pH ¼ 11.0.
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Fig. 9. The lowest docking energy conformation of the insert binding mode of GO with BHb.
Fig. 10. The lowest docking energy conformation of the surface binding mode of GO with BHb.
Table 1 Docking results of the lowest docking energy conformation of the insert binding mode and the surface binding mode of GO with BHb by using the Autodock program. Rank
Run
DG (kcal/mol)
Einter-mol (kcal/mol)
EVHD (kcal/mol)
Eelec (kcal/mol)
Etotal (kcal/mol)
Etorsional (kcal/mol)
The insert binding mode The surface binding mode
85 18
10.72 10.01
16.98 16.28
14.00 11.36
2.98 4.92
0.07 þ0.03
þ6.26 þ6.26
denaturation.
3.3. The influence of GO binding on the glycosylation of BHb Fig. 6 presents the nonenzymatic glycosylation (NEG) of BHb by fructose in the absence and presence of GO. The spectral profile of BHb in aqueous buffer (Fig. 6 Black line) shows peaks at 407, 573, and 630 nm, which are characteristic of the met state of BHb. The UVevis spectra of GO-BHb system without fructose carried out for 72 h in room temperature are also shown in Fig. 6. The changes of these bands can reflect the microenvironment surrounding heme in BHb-GO bioconjugates by glycosylation. The spectral profile of BHb in the presence of 1 mol/L fructose is shown by the blue-green line in Fig. 6. Compared with the GO-BHb system without fructose, the absorption peaks of BHb-fructose system changes obviously. The UVeVis spectra of GO-BHb system without fructose after 72 h reaction is shown by the blue and red line, respectively, indicating that there is not the glycosylation phenomenon of BHb induced by GO. The Soret band of BHb in the presence of fructose decreased, indicating that the glycosylation remarkable induces the protein structure and destroys the met state of BHb. The decrease of the intensity of Soret band indicating that glycosylation induces the conformational change near heme moiety in BHb. The absorption intensity of peaks at 573 and 630 nm also indicated the glycosylation of BHb. The absorption spectrum of glycosylated BHb in the presence of GO (Fig. 6 yellow, rose lines) to be most identical to that of BHb prior to glycosylation, the Soret band increased with almost complete reversal of the 573 and 630 nm absorption, implying that GO can partly prevent non-enzymatic glycosylation of hemoglobin [40].
3.4. The nature of the binding interactions of BHb with GO Three pH values were used in the experiment of epH 2.0, 7.4, and 11.0. The pH 7.4 can give the native conformation of BHb, and the pH 2.0 and 11.0 can give the extended conformation of BHb binding with GO. Fig. 7(AeC) shows the representative fluorescence emission spectra of BHb in the presence of various concentrations of GO at different pH. Compound emission of BHb is clearly higher at pH 2.0 (BSA in acid extended), indicating pH-induced protein denaturation [41]. In addition, the maximum emission peaks of BHb are at 334, 336, and 340 nm at pH 2.0, 7.4 and 11.0, respectively, indicating the changes in the tertiary structure of BHb in pH 2.0 and 11.0 because the shift in the position of emission maximum reflects the changes in the polarity around the Trp residues [33]. In addition, the effect of GO on fluorescence intensities of BHb were obviously quenched by the addition of GO with not obvious shift of maximum emission wavelength. The decrease of the intensity indicated that GO can bind to BHb. The non-shift of emission wavelength indicates that GO has little influence on the microenvironment of Trp residues of BHb [42]. Fig. 8 depicts the plots of Fcor/F0 versus concentration of GO for various pH values. From the curves, it is clear that the fluorescence quenching extent at pH 7.4 is not obviously different with pH 2. and 11.0 which suggests that the binding affinity of GO to BHb in the physiological condition (pH 7.4), acid (pH 2.0) and alkaline (pH 11.0) pH region are not significantly changed. The changes of pH not only induce the secondary structure changes of BHb, but also cause the ionization state of BHb and GO. The isoelectric points of GO and BHb are about 2.73 and 6.2, respectively [38,42]. At pH ¼ 2.0, both GO and BHb possess more positive. At pH ¼ 7.4 and 11.0, both GO and BHb possess negative charges, so electrostatic interactions are
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not the mainly binding forces between GO and BHb. The fluorescence quenching extent of BHb induced by GO at different pH imply that the hydrophobic interaction between the hydrophobic moiety of aromatic amino residues and carboxylic acid groups at the edge and hydroxyl and epoxy groups on the basal plane of GO play the important role as the mainly possible interaction between GO and amino acid residues during quenching [43]. However, van der Waals interactions and hydrogen bond interactions are not excepted during this binding reaction. In a word, the binding forces of BHb with GO are complex.
3.5. Computational modeling of GO/BHb system In order to confirm the binding nature of GO with BHb, molecular modeling was used in this part. Analyzing from a total of possible conformational clusters obtained from 100 docking runs for the binding site of BHb with GO. These possible conformational clusters are divided two kind of binding modes, including the insert binding mode and the surface binding mode. The lowest docking energy conformation of the insert binding mode and the surface binding mode of GO with BHb are shown in Figs. 9 and 10, respectively. Docking results of the lowest docking energy conformation of the insert binding mode and the surface binding mode of GO with BHb were shown in Table 1. In the insert binding mode shown in Fig. 9, GO enter into the gap formed by b1(Ala-142, Arg104, Asn-139, Asp-94, Glu-90, His-2, 143, 146, Lys-82, 132, 144, Pro-100, Tyr-145, and Val-1), a2(Lys-40, Pro-37, and Thr-38), and b2(Asn-139, Glu-6, 7, His-2, 143, 146, Lys-82, 132, and Tyr-145) of BHb. The insert binding mode indicates the hydrophobic interactions are the mainly binding forces. Among above amino residues, Arg, His, Glu, Lys and Thr are polar amino residues. Therefore, we speculated Van der Waals forces also took part in the binding interaction. In addition, the polar interaction and hydrogen bonds exist between two positively charged amino residues Lys-82 (3.096 Å, 3.164 Å) and Arg-104 (2.707 Å) and the oxygen atom of GO. The pep stacking interactions between His, Tyr residues of BHb and the plenty of small aromatic areas with sp2 carbons of GO also contribute to their binding interaction. Fig. 10 shows the surface binding mode of BHb with GO. There are nineteen amino acid residues of b1 chain (Ala-13, 76, Asn-19, Asp-21, 73, Glu-22, Gly-16, His-77, Leu-75, Lys-8, 17, 65, Ser-9, 72, Thr-12, Trp-15, Val-18, 20) taking part in the binding interaction. Among above amino residues, Val, Ala, and Leu residues are hydrophobic amino residues, indicating that the hydrophobic forces were also involved in the surface binding mode. In addition, the Van der Waals forces may be present because of the presence of Lys, Asp, His, Ser, Thr and Gly (highly polar) [44]. 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), and the torsional free energy (Etorsional) were shown in Table 1. In addition, the desolvo energy is based on new atomic solvation parameters which depend on the absolute partial charge on the atom [45]. Analyzing data in Table 1, we could draw the following conclusions. Firstly, the values of DG indicates that the stable complex between GO and BHb formed. Secondly, EVHD energy including van der Waals energy (Evdw), EHbond and Edesolvo are the main part of binding free energy, implying that Van der Waals, hydrogen bonding and hydrophobic forces are the main forces [46]. Thirdly, electrostatic forces (Eelec) in the Einter-mol are also involved in the binding interactions of BHb with GO, but are not the mainly driving forces. In a word, the complexes form between GO and BHb by hydrogen bonding, hydrophobic, electrostatic, and pep stacking interactions.
4. Conclusion In summary, the effects of GO on the conformational structure, thermal stability, glycosylation, and binding nature of BHb were studied. BHb attach to GO by forming the newly BHb-GO conjunctions at the expose of losing its secondary inducing the exposure of the heme group to the aqueous medium by multinoncovalent interactions including hydrophobic, hydrogen bonds, van der Waals interactions and electrostatic forces. The thermal unfolding experimental results show that BHb is more prone towards thermal denaturation the presence of GO. GO can prevent non-enzymatic glycosylation of BHb. In addition, the binding mode of GO with BHb consists of two kind of binding modes including the insert binding mode and the surface binding mode. We believe that the present study will provide important insight into the biological behaviors of the two-dimensional (2D) carbon nanomaterial. Acknowledgement We gratefully acknowledge financial support of the Fund from the National Natural Science Foundation of China (Project No. 21571154, 21201147, 21307103), the Natural Science Foundation of Jiangsu Province (Grant No.BK20161315, BK20151296, BY201505802), and the Jiangsu Fundament of “Qilan Project” and “333 Project”, and the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents. References [1] D. Depan, R.D.K. Misra, Hybrid nanoparticle architecture for cellular uptake and bioimaging: direct crystallization of polymer immobilized with magnetic nanoparticles on carbon nanotubes, Nanoscale 4 (2012) 6325e6335.. [2] R.D.K. Misra, P. Chaudhari, Osteoblasts response to nylon 6,6 blended with single-walled carbon nanohorn, J. Biomed. Mater. Res. A 101 (2013) 1059e1068. [3] R.D.K. Misra, P. Chaudhari, Cellular interactions ad simulated biological functions mediated by nanostructured carbon for tissue reconstruction and tracheal tubes and sutures, J. Biomed. Mater. Res. A 101 (2013) 528e536. [4] H. Kim, R. Namgung, K. Singha, I. Oh, W.J. Kim, Graphene oxideepolyethylenimine nanoconstruct as a gene delivery vector and bioimaging tool, Bioconjugate Chem. 22 (2011) 2558e2567. [5] A. Paul, A. Hasan, H. Al Kindi, A.K. Gaharwar, V.T.S. Rao, M. Nikkhah, S.R. Shin, D. Krafft, M.R. Dokmeci, D. Shum-Tim, A. Khademhosseini, Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair, ACS Nano 8 (2014) 8050e8062. [6] X. Zhao, L. Liu, X. Li, J. Zeng, X. Jia, P. Liu, Biocompatible graphene oxide nanoparticle-based drug delivery platform for tumor microenvironmentresponsive triggered release of doxorubicin, Langmuir 30 (2014) 10419e10429. [7] G.S. Wang, G.Y. Chen, Z.Y. Wei, X.F. Dong, M. Qi, Multifunctional Fe3O4/graphene oxide nanocomposites for magnetic resonance imaging and drug delivery, Mater. Chem. Phys. 141 (2013) 997e1004. [8] H. Hu, C. Tang, C. Yi, Folate conjugated trimethyl chitosan/grapheme oxide nanocomplexes as potential carriers for drug and gene delivery, Mater. Lett. 125 (2014) 82e85. [9] S. Li, A.N. Aphale, I.G. Macwan, P.K. Patra, W.G. Gonzalez, J. Miksovska, R.M. Leblanc, Graphene oxide as a quencher for fluorescent assay of amino acids, peptides, and proteins, ACS Appl. Mater. Interfaces 4 (2012) 7069e7075. [10] X.L. Wei, Z.Q. Ge, Effect of graphene oxide on conformation and activity of catalase, Carbon 60 (2013) 401e409. [11] P.S. Wate, S.S. Banerjee, A.J. Badwahr, J. Khandare, R.D.K. Misra, Cellular imaging using biocompatible dendrimer-functionalized graphene oxide-based fluorescent probe anchored with magnetic nanoparticles, Nnaotechnology 23 (2012) 415101e445109. [12] B. Girase, J.S. Shah, R.D.K. Misra, Cellular mechanics of modulated osteoblasts functions in graphene oxide reinforced elastomers, Adv. Biommater 14 (2012) 101e111. [13] S.C. Smith, F. Ahmed, K.M. Gutierrez, D.F. Rodrigues, A comparative study of lysozyme adsorption with grapheme, grapheme oxide, and single-walled carbon nanotubes: potential environmental applications, Chem. Eng. J. 240 (2014) 147e154. [14] M. Sopotnik, A. Leonardi, I. Kri zaj, P. Dusak, D. Makovec, T. Mesari c, N.P. Ulrih, I. Junkar, K. Sep ci c, D. Drobne, Comparative study of serum protein binding to three different carbon-based nanomaterials, Carbon 95 (2015) 560e572.
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Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Investigation on the conformational structure of hemoglobin on graphene oxide Yanqing Wang a, b, *, 1, Zhaohua Zhu b, 1, Hongmei Zhang b, Jian Chen b, Boping Tang a, Jian Cao b, ** a
Jiangsu Key Laboratory for Bioresources of Saline Soils, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People's Republic of China Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People's Republic of China b
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
Multi-noncovalent interactions exist between BHb and GO. GO has high ability of disturbing the secondary of BHb. GO acts as a structure destabilizer during the thermal denaturation process of BHb. GO can obviously prevent the nonenzymatic glycosylation of BHb.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 March 2016 Received in revised form 6 June 2016 Accepted 12 July 2016 Available online 18 July 2016
The binding interaction between bovine hemoglobin (BHb) and a novel two-dimensional carbon nanomaterial, graphene oxide (GO), has been investigated in this work. It is found that GO strongly disturbs the secondary structure of BHb by forming BHb-GO aggregates. The binding affinity between the GO and BHb is shown to be mainly from non-covalent interactions including hydrophobic, hydrogen bonding, van der Waals and electrostatic interactions. In addition, two possible binding modes are proposed, insert binding mode and surface binding mode, as shown by molecular modeling. Our findings also show that the existence of GO can significantly prevent the non-enzymatic glycosylation of BHb and decrease the thermal stability of the protein. This work elucidates the effects of the binding interactions of GO with BHb on some biological properties and functions of heme protein. © 2016 Elsevier B.V. All rights reserved.
Keywords: Biomaterials Surfaces Photoluminescence spectroscopy Thermodynamic properties
1. Introduction Nowadays, carbon nano-materials have been widely used in biological applications due to their unique and outstanding properties [1e3]. A novel two-dimensional (2D) carbon nanomaterial,
* Corresponding author. Institute of Applied Chemistry and Environmental Engineering, Yancheng Teachers University, Yancheng City, Jiangsu Province 224002, People's Republic of China. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (J. Cao). 1 Yanqing Wang and Zhaohua Zhu contributed equally. http://dx.doi.org/10.1016/j.matchemphys.2016.07.032 0254-0584/© 2016 Elsevier B.V. All rights reserved.
graphene oxide (GO), has shown potential biotechnological applications in drug and gene delivery systems [4e8]. Therefore, the understanding GO's influence on structure and activities of biomolecules are essential for before any biological applications of GO, especially for disease- and drug-related proteins [9,10], cellular bioimaging [11,12], protein adsorption [13,14] etc. Many studies showed that GO strongly interacted with amino acids, peptides, and serum proteins [9]. The adsorption of serum proteins on GO occurred spontaneously and rapidly, leading to significant changes in size, zeta potential, and morphology [15]. However, so far very few studies on the biocompatibility of GO with heme proteins, or on the toxic effects of GO on heme proteins at the molecular level have
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Fig. 1. (A) Circular dichroism (CD) spectra and (B) the percentages of the different structures of free BHb and its GO-BHb complex, c(BHb) ¼ 5 106 mol/L, pH ¼ 7.4, T ¼ 298 K.
been conducted [16,17]. Hemoglobin (Hb) is one of heme proteins that makes up about 92% of the Red blood cells' dry content and plays various roles including in the transport of oxygen, in the dispersion of hydrogen peroxide, and in the affection of electron transfer in human body parts and organs in living beings [18,19]. Hb is oxygen carriers in red blood cells [18]. The amount of Hb in blood is associated with many clinical diseases such as leukemia, anemia, heart diseases, etc [20]. When GO penetrates plasma membrane, the interactions of them with Hb maybe affect human health. In addition, Hb can successfully combine with carboxyl grapheme through condensation reaction to form the bio-nanocomposites that exhibits good ability of oxygen-carrying [17]. For the purpose of investigation about the biocompatibility, bioavailability, and toxicity of GO, the fundamental understandings of the binding behaviors of Hb are of critical importance for the integration of biology with GO. Herein, Hb is used as a good protein model for conformational change studies before and after the assembly on GO surface. The binding affinity, the thermodynamics property, the energetic of the interaction, and the thermal stability of GO-Hb association, the effect of GO on the non-enzymatic glycosylation of BHb was carried out by experimental and calculation studies. According to the study, we expect it not only provides the detailed conformation behavior of Hb molecules on GO but also creates a framework for analyzing the biosafety of GO in terms of the biological behavior of biomacromolecules. In addition, these findings also have shed light respective the design of bio-nanocomposites by comprehensive reconsideration of their interaction with Hb.
Fig. 2. UVevis absorption spectra of BHb, c(BHb) ¼ 5 106 mol/L, pH ¼ 7.4, T ¼ 298 K.
GO,
and
BHb-GO
system,
2. Material and methods 2.1. Reagents Bovine hemoglobin (BHb) was purchased from Sigma (St, Louis, MO, USA) and used without further purification. GO solution (5%) was purchased from Aladdin Industrial Corporation (Shanghai, China). The stock solutions of BHb (5.0 106 mol/L) was dissolved in a 0.05 mol/L potassium phosphate buffer with pH 7.4 by gentle stirring at room temperature and stored at 4 C. All other chemicals were of analytical reagent grade. Ultrapure water was used throughout. 2.2. Methods 2.2.1. UVevis absorption measurements The difference UVevis absorption measurement of BHb (5.0 106 mol/L) in the absence and presence of GO were measured on A SPECORD S600 spectrophotometer equipped with 1 cm quartz cells. A fixed concentration of BHb with different concentration of GO was added to the 1 cm sample cell, Then an equal concentration of GO was added to the reference cell simultaneously [21,22]. The scan speed was set at 200 nm/min and the spectra range was from 200 to 600 nm. 2.2.2. Fluorescence spectra measurements A LSe50B Spectrofluorimeter equipped with 1.0 cm quartz cells and a thermostat bath was used for fluorescence spectra measurements of BHb in the absence and presence of GO. The fluorescence spectra of BHb-GO reaction solutions were recorded in the range of 300e500 nm with the excitation wavelength 280 nm after 1 h equilibrate. Synchronous fluorescence spectra of BHb were recorded at 20 nm or 60 nm. In addition, for three-dimensional fluorescence spectra, the emission wavelengths range was selected from 270 to 500 nm, the initial excitation wavelength was set from 200 to 340 nm with increments of 10 nm. 2.2.3. Circular dichroism spectra measurements The CD spectra of BHb (5.0 106 mol/L) in the absence and presence of GO were acquired on an Applied Photophysics Ltd. Chirascan spectrometer. For each CD spectrum, the program CD spectra deconvolution program (CDNN) was used to obtain the percentage of a-Helix, b-Sheet, b-turn, random coil of BHb in the absence and presence of GO in order to analyze the effect of GO on the secondary structure of protein [http://bioinformatik. biochemtech.uni-halle.de/cdnn] [23]. The temperature of thermal denaturations of BHb in the absence and presence of GO were varied from 20 to 90 C in 5 C steps, with 250 s increments.
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Fig. 3. The three-dimensional fluorescence spectral contours of BHb (A), BHb-GO system (B,D), GO (E). c(BHb): A 5.0 106 mol/L, B, 5.0 106 mol/L, C, 5.0 106 mol/L, D, 5.0 106 mol/L, E, 0.0 106 mol/L, c(GO): A, 0.00 mg/mL, B, 0.004 mg/mL, C, 0.010 mg/mL, D, 0.080 mg/mL, E, 0.010 mg/mL.
2.2.4. Molecular modeling The crystal structure of BHb (PDB ID 1G09) was obtained from the Protein Data Bank for molecular modeling [24]. The geometries of GO (20 12 Å2) were optimized at DFT/B3LYP/3-21G (d, p) by Gaussian 09 [25]. The surface of GO was randomly decorated with epoxy and hydroxyl groups. Although the model size of GO was obviously smaller than that used in the experiment, this proposed model will be of service to explain the binding mechanism of GO with BHb. Autodock 4.2.3 Program was used to perform blind docking calculations of BHb with GO, respectively. In the blind docking calculations, a grid box of 126 126 126 Å with spacing of 0.357 Å was used to enclose BHb and GO. The Lamarckian Genetic Algorithm method was used as the searching algorithm. In addition, MMV software was used to analyze the predicated binding mode [26]. 2.2.5. BHb glycosylation and its detection Fructose (1 mol/L) were used to glycosylate BHb. The glycosylation reaction was carried out for 72 h in room temperature, both in the absence and presence of GO. The glycosylation solution of BHbGO system were measured on A SPECORD S600 spectrophotometer equipped with 1 cm quartz cells. 3. Results and discussion 3.1. The influence of GO binding on the conformational structure of BHb The circular dichroism (CD) results can give a useful estimate about the global structural change of protein induced by some molecules [27]. To ascertain the possible of GO binding on the secondary structure of BHb, we have performed CD studies in the
range of 200e300 nm in the absence and presence of GO. Fig. 1 (A) shows the far-UV CD spectra of the free BHb and its GO-BHb complex obtained at pH ¼ 7.4 and T ¼ 298 K. The far-UV spectra of BHb exhibit two negative bands at about 209 nm (pep*) and 222 nm (nep*) indicating the a-Helix structure of protein [28,29]. The binding of GO to BHb causes decrease in band intensity without significant shift of the peaks, which indicates the decrease of the aHelix content in BHb and the peptide strand unfolding even more. In addition, the percentages of the different structures of BHb in the absence and presence of GO is shown in Fig. 1(B), which reveals that the proportion of a-Helix decreased from 53% in native BHb to 39% in the presence of GO (0.08 mg/mL) with increase in random coil from 22% to 29% and in b-Sheet from 9% to 15%. From above results, it is apparent that the effect of GO binding on BHb causes a secondary changes of the protein and induce some perturbations of protein fold by mainly resulted from some hydrogen bonds and hydrophobic interactions [30]. The GO induced conformational change of BHb was also studied with UVevis absorption spectroscopy. The results are shown in Fig. 2. GO displays a maximum absorption at 222 nm due to the pep* transition of aromatic C]C bonds and a shoulder around 300 nm due to the nep* transition of C]O bonds [31]. The absorption spectra of native BHb show several electronic bands at 224 (corresponding to the nep* transition of BHb's characteristic polypeptide backbone structure C]O), 275 nm (corresponding to the pep* transition of the phenyl group of tryptophan (Trp) and tyrosine (Tyr) residues), 407 (belonging to the electronic transition of p/p* of hematoporphyrin), 573, and 630 nm (ligand-to-metal charge transfer, LMCT) [32]. After addition of GO in BHb solution, an alteration in the intensity of these above absorption peaks were observed. The absorbance intensities of bands at about 224 nm decreased from 2.14 to 1.37, indicating the reduction of a-helical
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Fig. 4. Far-UV CD spectra of BHb (A), BHb-GO system (B, C) during thermal denaturation. c(BHb) ¼ 5.0 106 mol/L, c(GO), (A) 0.000 mg/mL; (B) 0.020 mg/mL; (C) 0.080 mg/mL.
Fig. 5. Temperature dependence of the a-helix of BHb as determined by CD measurements in the absence and presence of GO, c(BHb) ¼ 5 106 mol/L, pH ¼ 7.4.
content in BHb and the loosening the BHb skeleton [33]. In addition, the strong Soret band at 407 nm is associated with the porphyrin ring and can provide insight into the environmental influence on the native structural of conformation of BHb [34,35]. The decrease of the intensity of Soret band indicating that GO binding induced the conformational change near heme moiety in BHb and GO are involved in producing a disturbance of the structure and the exposure of the heme group to the aqueous medium. In addition, the absorbance intensities increase from 0.54 to 0.72 and the blue shift (from 275 nm to 268 nm) of the absorption peak at about 275 nm implied that the micro-environmental changes of aromatic acid residues and more aromatic acid residues were extended into the aqueous environment [36]. When GO is added in BHb solution, some water molecules on the surface of protein are replaced by GO that can approach the amino acid residues of BHb [36]. More aromatic acid residues were extended into the aqueous environment
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1.0
pH = 2.0 pH = 7.4 pH = 11.0
0.9
Fcor/F0
0.8 0.7 0.6 0.5 0.4 0.3
0
5
10
15
20
[GO]/(ug/mL) Fig. 8. The quenching of BHb (5.0 106 mol/L) fluorescence intensity by GO in different concentrations. T ¼ 298 K, lex ¼ 280 nm. Fig. 6. UVeVis absorption spectra of the GO-BHb (5.0 106 mol/L) system in glycosylation reaction, pH ¼ 7.4, T ¼ 298 K.
and the tertiary structure of BHb was destroyed [37]. In a word, these observations strongly suggests that binding interactions exist between GO and BHb and GO binding induces the conformational changes in the protein. In addition, three isosbestic points at 236, 329, and 420 nm in the resulting spectra indicate that the binding equilibrium exists between free GO and the bound forms of GO [27]. BHb contains three Trp units (a-14Trp, bTrp-15 and bTrp-37) in each a and b chain. Its three-dimensional Ex/Em fluorescence is presented in Fig. 3. On the basis of the structure and component of BHb, there should be a noncovalent interaction between protein and GO. Indeed, the three-dimensional Ex/Em fluorescence spectral experiments in the present study shows the strong quenching of BHb induced by GO. It could be seen from Fig. 3 (A) that there are two kinds of fluorescence spectral peaks of BHb including endogenous fluorophores peaks (Peak a and Peak b) and the Raleigh scattering peaks (Peak 1 and Peak 2). Peak a is caused by the p/p* transition of aromatic amino acids in BHb, and Peak b is caused by the n/p* transition of BHb's characteristic polypeptide backbone structure C]O [38]. Upon addition of trace amount of GO to BHb solution, some remarkable changes of Peak a and Peak b are observed. the fluorescence intensity of Peak a and Peak b is strongly reduced and the shape of peak b is changed and disappeared, as shown in Fig. 3(BeD). In addition, two new peaks formed from 300 to 500 nm and from 450 to 500 nm. Compared with Fig. 3(E), this
two new fluorescence peak are not come from GO. So there must be some new phenomenon that takes place when BHb binds with GO in solution. Therefore, the newly formed BHb-GO conjunctions by some pep interaction or hydrophobic interaction between protein and plenty of small aromatic areas with sp2 carbons of GO contribute to these two peaks.
3.2. The influence of GO binding on the thermal stability of BHb The thermal unfolding of BHb in the absence and presence of GO were explored by using CD experiments. BHb consists of two a and two b subunits, each a-chain contains 141 amino acid residues and each b chain contains 146 amino acid residues. At about 20 C the protein retains its native structure in the buffer with two characteristic negative peaks at 209 and 222 nm. The temperaturedependent CD measurements of BHb are shown in Fig. 4. It is found that in buffer the percentages of BHb different structure including a-helix, b-sheet, b-turn, and Random coil change with increasing the temperature. As Fig. 4 A-1,2 shows, the content of ahelix decreased from 53% at 20 C to 16% at 87 C, while b-sheet and random coil obviously increased, indicating that the secondary structure of the protein suffers considerable damage, signifying the one set of an unfolding process [39]. It can also be seen from Fig. 4 B-1,2,C-1,2 that the changes in the secondary structure of BHb in the presence of GO against temperature were different from Hb in the absence of GO. As shown in Fig. 5, the unfolding temperature decreases with increasing GO concentration, revealing that the protein in the presence of GO is more prone towards thermal
Fig. 7. Fluorescence spectra of BHb (5.0 106 mol/L) in different concentrations of GO. c(GO)/(mg/mL): 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20. T ¼ 298 K, lex ¼ 280 nm. (A) pH ¼ 2.0, (B) pH ¼ 7.4, (C) pH ¼ 11.0.
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277
Fig. 9. The lowest docking energy conformation of the insert binding mode of GO with BHb.
Fig. 10. The lowest docking energy conformation of the surface binding mode of GO with BHb.
Table 1 Docking results of the lowest docking energy conformation of the insert binding mode and the surface binding mode of GO with BHb by using the Autodock program. Rank
Run
DG (kcal/mol)
Einter-mol (kcal/mol)
EVHD (kcal/mol)
Eelec (kcal/mol)
Etotal (kcal/mol)
Etorsional (kcal/mol)
The insert binding mode The surface binding mode
85 18
10.72 10.01
16.98 16.28
14.00 11.36
2.98 4.92
0.07 þ0.03
þ6.26 þ6.26
denaturation.
3.3. The influence of GO binding on the glycosylation of BHb Fig. 6 presents the nonenzymatic glycosylation (NEG) of BHb by fructose in the absence and presence of GO. The spectral profile of BHb in aqueous buffer (Fig. 6 Black line) shows peaks at 407, 573, and 630 nm, which are characteristic of the met state of BHb. The UVevis spectra of GO-BHb system without fructose carried out for 72 h in room temperature are also shown in Fig. 6. The changes of these bands can reflect the microenvironment surrounding heme in BHb-GO bioconjugates by glycosylation. The spectral profile of BHb in the presence of 1 mol/L fructose is shown by the blue-green line in Fig. 6. Compared with the GO-BHb system without fructose, the absorption peaks of BHb-fructose system changes obviously. The UVeVis spectra of GO-BHb system without fructose after 72 h reaction is shown by the blue and red line, respectively, indicating that there is not the glycosylation phenomenon of BHb induced by GO. The Soret band of BHb in the presence of fructose decreased, indicating that the glycosylation remarkable induces the protein structure and destroys the met state of BHb. The decrease of the intensity of Soret band indicating that glycosylation induces the conformational change near heme moiety in BHb. The absorption intensity of peaks at 573 and 630 nm also indicated the glycosylation of BHb. The absorption spectrum of glycosylated BHb in the presence of GO (Fig. 6 yellow, rose lines) to be most identical to that of BHb prior to glycosylation, the Soret band increased with almost complete reversal of the 573 and 630 nm absorption, implying that GO can partly prevent non-enzymatic glycosylation of hemoglobin [40].
3.4. The nature of the binding interactions of BHb with GO Three pH values were used in the experiment of epH 2.0, 7.4, and 11.0. The pH 7.4 can give the native conformation of BHb, and the pH 2.0 and 11.0 can give the extended conformation of BHb binding with GO. Fig. 7(AeC) shows the representative fluorescence emission spectra of BHb in the presence of various concentrations of GO at different pH. Compound emission of BHb is clearly higher at pH 2.0 (BSA in acid extended), indicating pH-induced protein denaturation [41]. In addition, the maximum emission peaks of BHb are at 334, 336, and 340 nm at pH 2.0, 7.4 and 11.0, respectively, indicating the changes in the tertiary structure of BHb in pH 2.0 and 11.0 because the shift in the position of emission maximum reflects the changes in the polarity around the Trp residues [33]. In addition, the effect of GO on fluorescence intensities of BHb were obviously quenched by the addition of GO with not obvious shift of maximum emission wavelength. The decrease of the intensity indicated that GO can bind to BHb. The non-shift of emission wavelength indicates that GO has little influence on the microenvironment of Trp residues of BHb [42]. Fig. 8 depicts the plots of Fcor/F0 versus concentration of GO for various pH values. From the curves, it is clear that the fluorescence quenching extent at pH 7.4 is not obviously different with pH 2. and 11.0 which suggests that the binding affinity of GO to BHb in the physiological condition (pH 7.4), acid (pH 2.0) and alkaline (pH 11.0) pH region are not significantly changed. The changes of pH not only induce the secondary structure changes of BHb, but also cause the ionization state of BHb and GO. The isoelectric points of GO and BHb are about 2.73 and 6.2, respectively [38,42]. At pH ¼ 2.0, both GO and BHb possess more positive. At pH ¼ 7.4 and 11.0, both GO and BHb possess negative charges, so electrostatic interactions are
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not the mainly binding forces between GO and BHb. The fluorescence quenching extent of BHb induced by GO at different pH imply that the hydrophobic interaction between the hydrophobic moiety of aromatic amino residues and carboxylic acid groups at the edge and hydroxyl and epoxy groups on the basal plane of GO play the important role as the mainly possible interaction between GO and amino acid residues during quenching [43]. However, van der Waals interactions and hydrogen bond interactions are not excepted during this binding reaction. In a word, the binding forces of BHb with GO are complex.
3.5. Computational modeling of GO/BHb system In order to confirm the binding nature of GO with BHb, molecular modeling was used in this part. Analyzing from a total of possible conformational clusters obtained from 100 docking runs for the binding site of BHb with GO. These possible conformational clusters are divided two kind of binding modes, including the insert binding mode and the surface binding mode. The lowest docking energy conformation of the insert binding mode and the surface binding mode of GO with BHb are shown in Figs. 9 and 10, respectively. Docking results of the lowest docking energy conformation of the insert binding mode and the surface binding mode of GO with BHb were shown in Table 1. In the insert binding mode shown in Fig. 9, GO enter into the gap formed by b1(Ala-142, Arg104, Asn-139, Asp-94, Glu-90, His-2, 143, 146, Lys-82, 132, 144, Pro-100, Tyr-145, and Val-1), a2(Lys-40, Pro-37, and Thr-38), and b2(Asn-139, Glu-6, 7, His-2, 143, 146, Lys-82, 132, and Tyr-145) of BHb. The insert binding mode indicates the hydrophobic interactions are the mainly binding forces. Among above amino residues, Arg, His, Glu, Lys and Thr are polar amino residues. Therefore, we speculated Van der Waals forces also took part in the binding interaction. In addition, the polar interaction and hydrogen bonds exist between two positively charged amino residues Lys-82 (3.096 Å, 3.164 Å) and Arg-104 (2.707 Å) and the oxygen atom of GO. The pep stacking interactions between His, Tyr residues of BHb and the plenty of small aromatic areas with sp2 carbons of GO also contribute to their binding interaction. Fig. 10 shows the surface binding mode of BHb with GO. There are nineteen amino acid residues of b1 chain (Ala-13, 76, Asn-19, Asp-21, 73, Glu-22, Gly-16, His-77, Leu-75, Lys-8, 17, 65, Ser-9, 72, Thr-12, Trp-15, Val-18, 20) taking part in the binding interaction. Among above amino residues, Val, Ala, and Leu residues are hydrophobic amino residues, indicating that the hydrophobic forces were also involved in the surface binding mode. In addition, the Van der Waals forces may be present because of the presence of Lys, Asp, His, Ser, Thr and Gly (highly polar) [44]. 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), and the torsional free energy (Etorsional) were shown in Table 1. In addition, the desolvo energy is based on new atomic solvation parameters which depend on the absolute partial charge on the atom [45]. Analyzing data in Table 1, we could draw the following conclusions. Firstly, the values of DG indicates that the stable complex between GO and BHb formed. Secondly, EVHD energy including van der Waals energy (Evdw), EHbond and Edesolvo are the main part of binding free energy, implying that Van der Waals, hydrogen bonding and hydrophobic forces are the main forces [46]. Thirdly, electrostatic forces (Eelec) in the Einter-mol are also involved in the binding interactions of BHb with GO, but are not the mainly driving forces. In a word, the complexes form between GO and BHb by hydrogen bonding, hydrophobic, electrostatic, and pep stacking interactions.
4. Conclusion In summary, the effects of GO on the conformational structure, thermal stability, glycosylation, and binding nature of BHb were studied. BHb attach to GO by forming the newly BHb-GO conjunctions at the expose of losing its secondary inducing the exposure of the heme group to the aqueous medium by multinoncovalent interactions including hydrophobic, hydrogen bonds, van der Waals interactions and electrostatic forces. The thermal unfolding experimental results show that BHb is more prone towards thermal denaturation the presence of GO. GO can prevent non-enzymatic glycosylation of BHb. In addition, the binding mode of GO with BHb consists of two kind of binding modes including the insert binding mode and the surface binding mode. We believe that the present study will provide important insight into the biological behaviors of the two-dimensional (2D) carbon nanomaterial. Acknowledgement We gratefully acknowledge financial support of the Fund from the National Natural Science Foundation of China (Project No. 21571154, 21201147, 21307103), the Natural Science Foundation of Jiangsu Province (Grant No.BK20161315, BK20151296, BY201505802), and the Jiangsu Fundament of “Qilan Project” and “333 Project”, and the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents. References [1] D. Depan, R.D.K. Misra, Hybrid nanoparticle architecture for cellular uptake and bioimaging: direct crystallization of polymer immobilized with magnetic nanoparticles on carbon nanotubes, Nanoscale 4 (2012) 6325e6335.. [2] R.D.K. Misra, P. Chaudhari, Osteoblasts response to nylon 6,6 blended with single-walled carbon nanohorn, J. Biomed. Mater. Res. A 101 (2013) 1059e1068. [3] R.D.K. Misra, P. Chaudhari, Cellular interactions ad simulated biological functions mediated by nanostructured carbon for tissue reconstruction and tracheal tubes and sutures, J. Biomed. Mater. Res. A 101 (2013) 528e536. [4] H. Kim, R. Namgung, K. Singha, I. Oh, W.J. Kim, Graphene oxideepolyethylenimine nanoconstruct as a gene delivery vector and bioimaging tool, Bioconjugate Chem. 22 (2011) 2558e2567. [5] A. Paul, A. Hasan, H. Al Kindi, A.K. Gaharwar, V.T.S. Rao, M. Nikkhah, S.R. Shin, D. Krafft, M.R. Dokmeci, D. Shum-Tim, A. Khademhosseini, Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair, ACS Nano 8 (2014) 8050e8062. [6] X. Zhao, L. Liu, X. Li, J. Zeng, X. Jia, P. Liu, Biocompatible graphene oxide nanoparticle-based drug delivery platform for tumor microenvironmentresponsive triggered release of doxorubicin, Langmuir 30 (2014) 10419e10429. [7] G.S. Wang, G.Y. Chen, Z.Y. Wei, X.F. Dong, M. Qi, Multifunctional Fe3O4/graphene oxide nanocomposites for magnetic resonance imaging and drug delivery, Mater. Chem. Phys. 141 (2013) 997e1004. [8] H. Hu, C. Tang, C. Yi, Folate conjugated trimethyl chitosan/grapheme oxide nanocomplexes as potential carriers for drug and gene delivery, Mater. Lett. 125 (2014) 82e85. [9] S. Li, A.N. Aphale, I.G. Macwan, P.K. Patra, W.G. Gonzalez, J. Miksovska, R.M. Leblanc, Graphene oxide as a quencher for fluorescent assay of amino acids, peptides, and proteins, ACS Appl. Mater. Interfaces 4 (2012) 7069e7075. [10] X.L. Wei, Z.Q. Ge, Effect of graphene oxide on conformation and activity of catalase, Carbon 60 (2013) 401e409. [11] P.S. Wate, S.S. Banerjee, A.J. Badwahr, J. Khandare, R.D.K. Misra, Cellular imaging using biocompatible dendrimer-functionalized graphene oxide-based fluorescent probe anchored with magnetic nanoparticles, Nnaotechnology 23 (2012) 415101e445109. [12] B. Girase, J.S. Shah, R.D.K. Misra, Cellular mechanics of modulated osteoblasts functions in graphene oxide reinforced elastomers, Adv. Biommater 14 (2012) 101e111. [13] S.C. Smith, F. Ahmed, K.M. Gutierrez, D.F. Rodrigues, A comparative study of lysozyme adsorption with grapheme, grapheme oxide, and single-walled carbon nanotubes: potential environmental applications, Chem. Eng. J. 240 (2014) 147e154. [14] M. Sopotnik, A. Leonardi, I. Kri zaj, P. Dusak, D. Makovec, T. Mesari c, N.P. Ulrih, I. Junkar, K. Sep ci c, D. Drobne, Comparative study of serum protein binding to three different carbon-based nanomaterials, Carbon 95 (2015) 560e572.
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