Tuning the Activities and Structures of Enzymes Bound to Graphene Oxide with a Protein Glue

Pattammattel, Ajith; Puglia, Megan K.; Chakraborty, Subhrakanti; Deshapriya, Inoka K.; Dutta, Prabir K.; Kumar, Challa V.

doi:10.1021/la404051c

Cited by 42 publications

(63 citation statements)

References 49 publications

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“…GO quenches BSA-FITC fluorescence and binding has been monitored as a function of increasing concentrations of GO, at fixed BSA-FITC concentration. 10 The quenching data have been analyzed by reported methods (Figure 1) to estimate the binding affinities (K a ).…”

Section: Resultsmentioning

confidence: 99%

“…This might seem trivial as structure retention is essential for activities but it has been noted that GO inhibited the activity of chymotrypsin 39 whereas GO has increased the activities of oxalate oxidase, esterase 5 and cytochrome c . 10 …”

Section: Discussionmentioning

confidence: 99%

“…BSA-FITC emission and FITC emission at 525 nm (485 nm excitation) was used to tabulate the quenching data. The data were fit into modified Stern-Volmer equation, as reported before (equation 1), 10

\frac{F_{0}}{F_{0} - F} = \frac{1}{[(f_{a} K_{a} Q) + \frac{1}{f_{a}}]}

where [Q] = quencher concentration; F 0 = Fluorescence intensity when [Q] = 0; F = Fluorescence intensity at given [Q]; K a = Bimolecular quenching constant, and f a = fraction of the initial fluorescence accessible to the quencher.…”

Section: Methodsmentioning

confidence: 99%

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Biological relevance of oxidative debris present in as-prepared graphene oxide

et al. 2015

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The influence of oxidative debris (OD) present in as-prepared graphene oxide (GO) suspensions on proteins and its toxicity to human embryonic kidney cells (HEK-293T) are reported here. The OD was removed by repeated washing with aqueous ammonia to produce the corresponding base-washed GO (bwGO). The loading (w/w) of bovine serum albumin (BSA) was increased by 85% after base washing, whereas the loading of hemoglobin (Hb) and lysozyme (Lyz), respectively, was decreased by 160% and 100%. The secondary structures of 13 different proteins bound to bwGO were compared with the corresponding proteins bound to GO using the UV circular dichroism spectroscopy. There was a consistent loss of protein secondary structure with bwGO when compared with proteins bound to GO, but no correlation between either the isoelectric point or hydrophobicity of the protein and the extent of structure loss was observed. All enzymes bound to bwGO and GO indicated significant activities, and a strong correlation between the enzymatic activity and the extent of structure retention was noted, regardless of the presence or absence of OD. At low loadings (<100 μg/mL) both GO and bwGO showed excellent cell viability but substantial cytotoxicity (~40% cell death) was observed at high loadings (>100 μg/mL). In control studies, OD by itself did not alter the growth rate even after a 48-h incubation. Thus, the presence of OD in GO played a very important role in controlling the chemical and biological nature of the protein-GO interface and the presence of OD in GO improved its biological compatibility when compared to bwGO.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

\frac{F_{0}}{F_{0} - F} = \frac{1}{[(f_{a} K_{a} Q) + \frac{1}{f_{a}}]}

Section: Methodsmentioning

confidence: 99%

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Biological relevance of oxidative debris present in as-prepared graphene oxide

et al. 2015

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“…510 A simple, novel, and efficient approach was described for improving the structures and activities of enzymes bound to graphene oxide such that bound enzymes are nearly as active as those of the corresponding unbound enzymes. 511 The strategy was to pre-adsorb highly cationized bovine serum albumin (cBSA) to passivate GO, and cBSA/GO (bGO) served as an excellent platform for enzyme binding. The binding of met-hemoglobin, glucose oxidase, horseradish peroxidase, BSA, catalase, lysozyme, and cytochrome c indicated improved binding, structure retention, and activities.…”

Section: Cnts For Enzyme Immobilizationmentioning

confidence: 99%

Heterogeneous Catalysis on Nanostructured Carbon Material Supported Catalysts

2015

Nanostructured Carbon Materials for Catalysis

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For almost twenty years there has been an increasing interest in the use of nanostructured carbon materials as catalyst supports. 1-16 Following the nanotechnology wave, a wide variety of carbon nanomaterials has been investigated as catalyst supports, including fullerenes, CNTs, CNFs, carbon onions, nanodiamonds, FLG or GO. CNTs and CNFs, which constitute a new family of support offering a good compromise between the advantages of AC and high surface area graphite have been particularly studied. The main advantages offered by CNT or CNF supports are: 17 (i) the high purity of the material can avoid self-poisoning; (ii) the mesoporous nature of these supports can be of interest for liquid-phase reactions, thus limiting the mass transfer; (iii) the high thermal conductivity that limits hot-spots that can damage the catalyst; (iv) their well-defined and tunable structure; (v) their rich surface chemistry that offers numerous perspectives for adsorption and dispersion of the active phase; and (vi) the specific metal support interactions, due to high electrical conductivity of the support and to the tunable orientation of the graphene layers, that exist and that can directly affect catalytic activity and selectivity. The possibility to perform catalysis in a confined space is also of great interest. 6,18 From a theoretical viewpoint, graphene provides the ultimate two-dimensional model of a catalytic support. The reason is related to its high theoretical surface area (ca. 2630 m 2 g −1 ), high conductivity, unique graphitized basal plane structure and chemical tolerance. FLG,

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“…1 PEGylated GO has been used to engage serine proteases for enzyme engineering, 12 while bovine serum albumin has been used as a "protein glue" to bind enzymes and metal nanoparticle catalysts. 13,14 Covalent functionalization of GO occurs at its oxidized regions. These modifications risk disrupting GO's aqueous dispersibility and leave the graphitic areas solvent exposed, 15,16 such that they may denature biomolecules or quench fluorescent labels.…”

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confidence: 99%

Retaining the Activity of Enzymes and Fluorophores Attached to Graphene Oxide

et al. 2015

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Graphene oxide (GO) has drawn interest for impermeable coatings, water purification, drug delivery vectors, and other applications that involve functionalizing its surface with molecular, polymeric, or biomolecular species. Existing GO functionalization strategies rely on covalent attachment to polar functionalities at its oxidized regions or weak nonspecific absorption to the graphitic regions. These modifications risk disrupting GO's aqueous dispersibility and leave its hydrophobic patches available to disrupt protein structure and quench the emission of fluorophores. Here, we demonstrate a general strategy to functionalize GO noncovalently using tripodal binding motifs, which present three pyrene moieties that bind to the hydrophobic regions. Tripods immobilize the serine protease enzyme chymotrypsin (ChT) onto GO and preserve its native structure and activity. In contrast, unmodified GO is one of the strongest known ChT inhibitors, which we show to arise from interactions with both GO's hydrophilic regions and hydrophobic regions. Furthermore, GO quenches the photoemission of many fluorescent probes, and its weak inherent photoemission is inconvenient for imaging via fluorescence microscopy. When presented on the GO surface through a tripod, the fluorescent dye Alexa Fluor 488 retains its fluorescence and allows the GO sheets to be imaged using a standard fluorescence microscope. As such, tripod-binding groups represent a useful strategy to functionalize GO with biomolecules and study its interactions with cells and living organisms. G raphene oxide (GO) is one of the most intensely studied nanostructured materials of the past decade because of its two-dimensional structure, low cost, water dispersibility, facile processability, and ability to be reduced to graphene with acceptable conductivity for many applications. 1,2 Improved methods to interface active functionalities with its surface, such as biomolecules or fluorophores, can increase its utility for nanomedicine and clean technologies. 3−7 GO is composed of nanometer-sized islands of graphitic carbon surrounded by hydrophilic oxidized areas and edges, 8 such that it may engage fluorophores, cells, and biomolecules through a full complement of noncovalent interactions. 9−11 A few strategies have emerged to functionalize GO or limit nonspecific protein interactions. 1 PEGylated GO has been used to engage serine proteases for enzyme engineering, 12 while bovine serum albumin has been used as a "protein glue" to bind enzymes and metal nanoparticle catalysts. 13,14 Covalent functionalization of GO occurs at its oxidized regions. These modifications risk disrupting GO's aqueous dispersibility and leave the graphitic areas solvent exposed, 15,16 such that they may denature biomolecules or quench fluorescent labels. 17 Modular binding motifs capable of anchoring active functionalities promise improved generality and operational simplicity. Here, we functionalize GO with a model enzyme and fluorophore onto molecular tripods 18,19 adsorbed to the hydrophobic reg...

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Tuning the Activities and Structures of Enzymes Bound to Graphene Oxide with a Protein Glue

Cited by 42 publications

References 49 publications

Biological relevance of oxidative debris present in as-prepared graphene oxide

Biological relevance of oxidative debris present in as-prepared graphene oxide

Heterogeneous Catalysis on Nanostructured Carbon Material Supported Catalysts

Retaining the Activity of Enzymes and Fluorophores Attached to Graphene Oxide

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