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Cite this: Chem. Commun., 2013, 49, 8611 Received 20th June 2013, Accepted 30th July 2013

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Contrasting modulation of enzyme activity exhibited by graphene oxide and reduced graphene† Xinjian Yang,ab Chuanqi Zhao,a Enguo Ju,ab Jinsong Ren*a and Xiaogang Qu*a

DOI: 10.1039/c3cc44632h www.rsc.org/chemcomm

Here we demonstrate that GO and RGO exhibit contrasting effects on modulation of the peroxidase activity of cyt c. GO could dramatically improve the enzyme activity of cyt c, while RGO showed a strong inhibition effect on cyt c.

As a novel one-atom-thick planar sheet of sp2-bonded carbon atoms, graphene has received much attention in recent years in materials science and biotechnology due to its extraordinary electronic, optical and thermal properties.1 Especially, in the area of biomedicine, graphene and its derivatives have shown great promise in the development of novel biosensors, tissue engineering scaffolds, nanocarriers for drug and gene delivery, and photo therapies of cancer.2 Recently, Dravid et al. investigated the efficacy of graphene oxide (GO) as an artificial protein inhibitor in modulating enzymatic activity of a-chymotrypsin.3 Subsequently, Peng and Liu et al. found that functionalized GO nanosheets could act as efficient enzyme positive modulators with great selectivity.4 Although little work has been done to study graphene–protein interactions,3–5 understanding the interaction of enzymes with graphene and its derivatives will be fundamentally important to the design and creation of new hierarchical material assemblies and functional devices and to elucidate potential health effects, either positive or negative, of nanoscale materials at the molecular level.6 Cytochrome c (cyt c) is a globular protein with a unique covalently bound heme active centre that can be probed spectroscopically via peroxidase activity.7 GO contains functional groups such as epoxide, carboxyl, and hydroxyl groups, which can undergo covalent, electrostatic, or hydrogen bonding with proteins.5a,8 RGO, which is the reduced counterpart of GO, holding only carboxyl mainly at the periphery, could adsorb proteins by hydrophobic interaction.9 Herein, we used cyt c as a model protein to assess local perturbations resulting from protein-support (GO–RGO) interactions that depend on surface conditions. Interestingly, we found that GO and RGO a

State Key laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. E-mail: [email protected], [email protected] b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cc44632h

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Scheme 1

Illustration of the peroxidase activity changes by GO and RGO.

could contrastingly change the peroxidase activity of the adsorbed cyt c. As shown in Scheme 1, GO was able to enhance the peroxidase activity of cyt c significantly, in contrast, RGO exhibited a strong inhibition effect. Further study showed that these different effects were attributed to the changes in the heme microenvironment induced by different interaction between cyt c and GO–RGO. GO was synthesized from natural graphite powder using a modified Hummers method.10 RGO was prepared by reduction of GO as previously described.11 As shown in Fig. S1 (ESI†), the color of GO changed from yellowish brown to black after reduction. The UV/Vis absorption peak at 230 nm for GO also red-shifted to 265 nm for RGO, and the absorption intensity of the RGO, especially in the region above 300 nm, increased dramatically, suggesting the reduction of GO. Zeta-potential measurements were performed to monitor the evolution of the nanosheet surface charges. GO showed a zeta potential of 60.9 mV owing to oxygen-containing groups. In contrast, the zeta-potential increased to 21.2 mV after reduction. FTIR results further confirmed the reduction of the GO. Fig. S2 (ESI†) showed that the FTIR peaks of epoxide and carbonyl groups disappeared, which revealed the removal of surface groups. Chem. Commun., 2013, 49, 8611--8613

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Fig. 2 The peroxidase activity of cyt c in the presence of different concentrations of GO (0–1 mg mL 1) by monitoring the absorbance change of ABTS at 418 nm. The concentrations of cyt c, ABTS and H2O2 are 0.2 mM, 0.5 mM and 6 mM, respectively.

Fig. 1 AFM images of cyt c on GO and RGO (a and b) and the height of cyt c on GO and RGO (c and d). The scale bar is 200 nm.

The interaction between proteins and GO materials is a critical factor for enzyme loading, enzyme activity and stability. The immobilization of cyt c was carried out by incubating GO–RGO with cyt c in Tris–HCl buffer at pH 7.0. Compared with many other immobilization supports, the GO surface is enriched with oxygencontaining functional groups, such as hydroxyl, carbonyl, and epoxy groups. The isoelectric point (PI) of cyt c is 10, and the protein is positively charged under the experimental conditions. Thus the interaction between GO and cyt c is mainly electrostatic interaction and the protein immobilization can be performed directly. The image of the immobilized cyt c on GO was obtained by atomic force microscopy (AFM) (Fig. 1a and c). The height of GO increased from 1 nm to 6–7 nm (Fig. S3, ESI†). From the image one can directly observe the loaded protein, which is not possible for most solid substrates. Since the interaction between GO and protein is electrostatic interaction, the enzyme loading can be tuned by controlling the pH value of the buffer. As for RGO, most of its surface groups disappeared owing to reduction, leaving a greatly hydrophobic surface. So the adsorption of cyt c on RGO was realized through hydrophobic interaction. The AFM image showed the immobilization of cyt c on RGO (Fig. 1b and d). To investigate the influence of supports on the enzyme activity of immobilized proteins, we examined the peroxidase activity of cyt c for the free form and the adsorbed on the GO and RGO. Previous studies demonstrated that ‘‘native’’ hexa-coordinated low spin cytochrome c does not show any peroxidase activity. Only the protein unfolded or with a ‘‘perturbed’’ environment of the metal centre shows a significant increase in the peroxidase activity toward the catalytic reduction of hydrogen peroxide.12 H2O2 acting as the catalytic substrate could activate cyt c, resulting in weak catalytic activity. Fig. 2 showed the time-dependent absorption changes against concentrations of GO in 1 mM Tris–HCl buffer at pH 7.0. With the increasing of GO, the absorption increased. When the concentration of added GO reached 0.6 mg ml 1, the absorption leveled off. In our previous work, carboxyl-modified GO exhibited intrinsic peroxidase-like activity that could catalyze the peroxidase substrate ABTS in the presence of H2O2 to produce a bluegreen color reaction. To eliminate the impact of GO (noncarboxyl-modified) on the peroxidase activity, control experiment was carried out, 8612

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Fig. 3 The peroxidase activity of cyt c in the presence of different concentrations of RGO (0–2 mg mL 1) by monitoring the absorbance change of ABTS at 418 nm. The concentrations of cyt c, ABTS and H2O2 used are 0.2 mM, 0.5 mM and 6 mM, respectively.

and results showed that GO exhibited little catalytic properties (Fig. S4, ESI†). These results indicated that the catalytic reaction could be enhanced by addition of GO. Interestingly, dramatic decrease in the catalytic velocity was observed in the presence of RGO (Fig. 3). It was worth noting that more RGO was needed to reach the optimal effect. This was because that cyt c loading efficiency of RGO was lower than that of GO.9b The reaction was completely inhibited at 1.2 mg ml 1 RGO. Previous reports have demonstrated that the peroxidase activity of cyt c was strongly dependent on the heme environment.6e a-Helix content of the protein regulates the catalytic activity of the enzyme.13 Thus, we measured the CD spectra of cyt c to evaluate the structural change of cyt c induced by GO or RGO. The electrostatic interaction of cyt c with GO may be responsible for the structural unfolding of cyt c. As shown in Fig. 3a, the heme microenvironment underwent dramatic structural perturbation when cyt c was adsorbed onto the GO. With the increasing of GO, the negative peak at 222 nm decreased obviously, which indicated the loss of a-helix content of adsorbed cyt c.6e It should be noted that cyt c does not have a sufficiently solvent accessible heme due to the requirements for the heme group to be enclosed in a crevice structure to form the necessary covalent and coordinate-covalent bonds with the polypeptide chain. This restricted the interaction of the heme with the peroxidase substrates. Therefore, cyt c possesses weak peroxidatic activity. The denaturing of cyt c on GO increased the access of solvent to heme, resulting in the increasing of the catalytic rate. To further confirm electrostatic interaction was the main force that induced the denaturing of cyt c, control experiments were carried out. As shown in Fig. S5 (ESI†), negatively charged G-PSS (poly(sodium 4-styrenesulfonate) modified GO) could enhance the catalytic This journal is

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Communication cyt c on GO. Whereas the hydrophobic interaction was the main force for the adsorption of cyt c on RGO. Each of the interaction changed the accessibility of substrates to heme accordingly, resulting in the increase or decrease in enzyme activity of cyt c. We anticipated that this fundamental study could open the door for the designing and creating of new hierarchical material assemblies and functional devices in the field of enzyme engineering, enzyme-based biosensing and bioassay. Financial support was provided by the National Basic Research Program of China (Grant 2012CB720602, 2011CB936004) and the National Natural Science Foundation of China (Grants 21210002, 91613006, 21072182).

Notes and references

Fig. 4 CD spectra of cyt c in the presence of different concentrations of GO (a) and RGO (b). The concentrations of cyt c, GO and RGO are 2 mM, 0–8 mg mL 1 and 0–16 mg mL 1.

rate, while positively charged G-PAH (poly(allylamine hydrochloride) modified GO) had not any impact on cyt c peroxidase activity. Under the experimental conditions, cyt c was free for the electrostatic repulsion from G-PAH, while it could be adsorbed onto G-PSS and unfolded (Fig. S6, ESI†). But the decrease in a-helix content was not obvious comparing with that on GO, resulting in relatively little change in peroxidase activity. All these results demonstrated that GO could greatly enhance the peroxidase activity through unfolding of cyt c by electrostatic interaction. Different conformational changes were induced in the case of the a-helix fraction by RGO (Fig. 4b). Comparing the native conformation of cyt c, a more compact conformation could be induced due to the increase of a-helix when cyt c was adsorbed on RGO by hydrophobic interaction. This resulted in that the reactive centre became more difficult to contact with peroxidase substrates. Meanwhile, the adsorption of cyt c on RGO may block the reactive centre, and prevent the access of solvent to heme, resulting in the inhibition of catalytic reaction. Although further work is required to investigate the detailed mechanism, our work highlights that the peroxidase activity of cyt c could be regulated by nanomaterials. In summary, for the first time, we demonstrated that GO and RGO showed different interaction with protein and exhibited contrasting effects on modulation of the enzymatic activity. By using cyt c as a model system, we found that GO could dramatically improve the enzyme activity, contrastingly, RGO showed a strong inhibition effect on cyt c. A detailed study showed that these different effects were mainly attributed to the changes in the heme microenvironment induced by different interactions between cyt c and GO–RGO. The electrostatic interaction was the main force causing the adsorption of

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Chem. Commun., 2013, 49, 8611--8613

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Contrasting modulation of enzyme activity exhibited by graphene oxide and reduced graphene.

Here we demonstrate that GO and RGO exhibit contrasting effects on modulation of the peroxidase activity of cyt c. GO could dramatically improve the e...
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