International Journal of Biological Macromolecules

[Pages:26]International Journal of Biological Macromolecules 84 (2016) 227?235

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International Journal of Biological Macromolecules

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Graphene oxide derivatives with variable alkyl chain length and terminal functional groups as supports for stabilization of cytochrome c

Michaela Patila a, Ioannis V. Pavlidis b, Antonios Kouloumpis c, Konstantinos Dimos c, Konstantinos Spyrou c, Petros Katapodis a, Dimitrios Gournis c, Haralambos Stamatis a,

a Biotechnology Laboratory, Department of Biological Applications and Technologies, University of Ioannina, 45110 Ioannina, Greece b Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, 17487 Greifswald, Germany c Department of Material Science and Engineering, University of Ioannina, 45110 Ioannina, Greece

article info

Article history: Received 29 April 2015 Received in revised form 8 December 2015 Accepted 10 December 2015 Available online 17 December 2015

Keywords: Graphene-oxide Nanomaterials Cytochrome c Immobilization Nanobiocatalysis

a b s t r a c t

In this study we report the ability of reduced and non-reduced graphene oxide-based nanomaterials (GONs), modified with variable alkyl chain length and terminal functional groups, to act as effective scaffolds for the immobilization of cytochrome c (cyt c) using different immobilization procedures. The GONs/cyt c conjugates are characterized by a combination of techniques, namely atomic force microscopy, X-ray photoelectron and FT-IR spectroscopies as well as thermo-gravimetric and differential thermal analysis. The effect of the structure of functional groups and the surface chemistry of GONs on the immobilization efficiency, the peroxidase activity and the stability of the cyt c was investigated and correlated with conformational changes on the protein molecule upon immobilization. The enhanced thermal stability (up to 2-fold) and increased tolerance (up to 25-fold) against denaturing agents observed for immobilized cyt c, indicates that these functionalized GONs are suitable as nanoscaffolds for the development of robust nanobiocatalysts.

? 2015 Elsevier B.V. All rights reserved.

1. Introduction

Graphene, a two-dimensional carbon sheet with single-atom thickness, has attracted the scientific interest since its discovery in 2004 [1]. Due to its unique structure and geometry, graphene possesses remarkable physical?chemical properties, including high surface area, strong mechanical stability and flexibility, excellent electrical and thermal conductivity, fast mobility of charge carriers and biocompatibility [2,3].

Graphene oxide (GO) is the oxidized form of graphene and consists of a large surface area with random distributed hydroxyl, epoxy, ketone, carboxyl groups at edges and basal planes. GO can be further functionalized with different kind of chemical groups, which can lead to the formation of new graphene oxide-based nanomaterials (GONs) with novel properties targeted specific applications such as the development of biosensors and biofuel cells [4,5], the delivery of drugs [6], tissue engineering [7], and the development of effective nanobiocatalysts [8]. GO is an excel-

Corresponding author. E-mail address: hstamati@uoi.gr (H. Stamatis).

0141-8130/? 2015 Elsevier B.V. All rights reserved.

lent support for protein or enzyme immobilization which can be attributed to their surface and physicochemical properties and the high water solubility [9]. Moreover, the use of GO and its functionalized derivatives as nanoscaffolds for enzyme immobilization offers the possibility of manipulation of the microenvironment of the enzymes, enhancing their catalytic behavior and operational stability which result in the development of robust nanobiocalysts [10,11]. Various GONs have been employed for the immobilization of enzymes for the development of nanobiocatalytic systems with application in wastewater treatment [12], phenol compound removal [13], in situ protein digestion [14], as well as various biocatalytic transformations with industrial interest [15].

Both non-specific physical adsorption and covalent binding have been used to immobilize enzymes on GONs [10,13,16]. By nonspecific binding via physical adsorption, the enzymes interact with the surface of GONs mostly through van der Walls and electrostatic forces, hydrophobic or ? stacking interactions [17]. The catalytic activity of the immobilized enzyme is usually retained but the nonspecific adsorption could lead to gradual leakage of the enzyme from the surface of the nanomaterial. On the other hand, the covalent binding of enzymes significantly reduces the leakage from the carrier and lead to higher stability owing to the increased robust-

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M. Patila et al. / International Journal of Biological Macromolecules 84 (2016) 227?235

ness [8]. However, the formation of the covalent bond between the enzyme and GONs may result in loss in activity and changes in the structure of the biomolecule [18]. The immobilization procedure followed leads in most cases to conformational changes of the protein molecule which may result in lower enzyme activity [19], although an improvement in catalytic performance of enzymes has also been reported [10]. The interactions between GONs and enzymes which affect the catalytic and structural characteristics of immobilized enzymes mainly depend on the surface chemistry of the nanomaterials used and on the immobilization approach followed [8,20]. Therefore it is crucial to deepen our understanding on the effect of these factors on the biocatalytic function and structure of the attached biomolecules.

In the present work, we investigate the ability of functionalized reduced (rGONs) and non-reduced GO-derivatives, to act as effective nanoscaffolds for immobilization of a model protein such as cytochrome c (cyt c) using different immobilization procedures. Cyt c is a small heme protein with peroxidase activity which is among the best characterized redox proteins with biotechnological interest, including the development of effective biosensors [21] and the degradation of pollutants [22]. The novel nanobiocatalysts formed were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and thermo-gravimetric (TG) and differential thermal analysis (DTA). The effect of the functionalization of graphene-based nanoscaffolds on the immobilization efficiency, the peroxidase activity and the operational stability of the cyt c was investigated and correlated with conformational changes on the protein molecule upon immobilization.

2. Experimental

2.1. Materials

Cytochrome c from equine heart was purchased from Sigma?Aldrich (>98%, St. Louis, MO) and used without further purification. Guaiacol (2-methoxyphenol) was purchased from Sigma?Aldrich (St. Louis, MO). Hydrogen peroxide (30% w/v, H2O2) was obtained from Fluka. N-Hydroxysuccinimide (NHS), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and HEPES were obtained from Sigma. Graphite was from Fluka. Aminopropanoic acid, aminovaleric acid, aminoundecanoic, ethylene diamine, diaminobutane and hexamethylenediamine used for the functionalization of GO were purchased from Sigma?Aldrich (St. Louis, MO). All other solvents and reagents were of HPLC or analytical grade.

2.2. Functionalization and determination of GOs

Aqueous dispersions of GO were produced using a modified Staudenmaier's method from graphite powder [23]. GO was functionalized with aminopropanoic acid, aminovaleric acid, aminoundecanoic acid, ethylene diamine, diaminobutane and hexamethylenediamine for the addition of carboxyl and amino groups, as described in previous works [11,20,24]. Reduced GO derivatives were prepared by chemical reduction of GO as described elsewhere [25].

2.3. Non covalent immobilization of cyt c

In a typical procedure, 3 mg of functionalized GONs in 5.7 mL of phosphate buffer (50 mM, pH 7.0) were sonicated for 30 min. Then 1 mL of cyt c solution (3 mg cyt) was added and the mixture was incubated under stirring for 1 h at 30 C and then overnight at 4 C. The nanomaterial-cyt c conjugates were separated by centrifugation at 6000 rpm and then were washed three times with buffer

solution to remove loosely bound protein. The immobilized cyt c was dried over silica gel and was stored at 4 C until used.

2.4. Covalent immobilization of cyt c using glutaraldehyde as cross-linking agent

3 mg of amine-functionalized GONs in 9.13 mL of phosphate buffer (50 mM, pH 7.0) were sonicated for 30 min in the presence of 110 L Tween-20. After the dispersion of nanomaterials, 1.76 mL of glutaraldehyde was added and the mixture was incubated under stirring for 1 h at 30 C. The modified nanomaterials were separated by centrifugation at 6000 rpm and washed three times with buffer solution to remove the excess of glutaraldehyde. Then 11 mL of buffer solution containing cyt c (3 mg) was added and the mixture was treated in a similar manner as described for the non covalent procedure.

2.5. Covalent immobilization of cyt c via diimide-activated amidation

2 mg of carboxyl-functionalized GONs in 5 mL HPLC water were sonicated for 30 min. Then 1 mL of HEPES buffer (50 mM, pH 7.0) and 1.2 mL of a 10 mg/mL aqueous solution EDC were added to the above suspension. Under fast stirring, 2.3 mL of a 50 mg/mL NHS aqueous solution were added quickly and the mixture was incubated for 30 min at 30 C. The activated nanomaterials were separated by centrifugation at 6000 rpm and washed three times with HEPES buffer to remove the excess of EDC. The activated nanomaterials were re-dispersed in 9 mL HEPES buffer solution. Then, 1 mL of protein solution in HEPES buffer (containing 2 mg cyt c) was added and the mixture was treated as described for non covalent procedure.

2.6. Atomic force microscopy studies

AFM images were obtained in tapping mode with a 3D Multimode Nanoscope, using Tap-300G silicon cantilevels with a tip radius ................
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