Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters.

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Catalytic oxidation and reduction reactions of hydrophilic carbon clusters with NADH and cytochrome C: features of an electron transport nanozyme.

Previously, our group reported on the promising efficacy of poly(ethylene glycol)-hydrophilic carbon clusters (PEG-HCCs) to work as broadly active and high capacity antioxidants in brain ischemia and injury models including stroke and traumatic brain injury coupled with hemorrhagic shock. PEG-HCCs are a carbon nanomaterial derived from harsh oxidation of single wall carbon nanotubes and covalently modified with poly(ethylene glycol). They retain no tubular remnants and are composed of a highly oxidized carbon core functionalized with epoxy, peroxyl, quinone, ketone, carboxylate, and hydroxyl groups. HCCs are the redox active carbon core of PEG-HCCs, which have a broad reduction potential range starting at +200 mV and extending to -2 V. Here we describe a new property of these materials: the ability to catalytically transfer electrons between key surrogates and proteins of the mitochondrial electron transport complex in a catalytic fashion consistent with the concept of a nanozyme. The estimated reduction potential of PEG-HCCs is similar to that of ubiquinone and they enabled the catalytic transfer of electrons from low reduction potential species to higher reduction electron transport complex constituents. PEG-HCCs accelerated the reduction of resazurin (a test indicator of mitochondrial viability) and cytochrome c by NADH and ascorbic acid in solution. Kinetic experiments suggested a transient tertiary complex. Electron paramagnetic resonance demonstrated NADH increased the magnitude of PEG-HCCs' intrinsic radical, which then reduced upon subsequent addition of cytochrome c or resazurin. Deconvolution microscopy identified PEG-HCCs in close proximity to mitochondria after brief incubation with cultured SHSY-5Y human neuroblastoma cells. Compared to methylene blue (MB), considered a prototypical small molecule electron transport shuttle, PEG-HCCs were more protective against toxic effects of hydrogen peroxide in vitro and did not demonstrate impaired cell viability as did MB. PEG-HCCs were protective in vitro when cells were exposed to sodium cyanide, a mitochondrial complex IV poison. Because mitochondria are a major source of free radicals in pathology, we suggest that this newly described nanozyme action helps explain their in vivo efficacy in a range of injury models. These findings may also extend their use to mitochondrial disorders.

Critical Comparison of the Superoxide Dismutase-like Activity of Carbon Antioxidant Nanozymes by Direct Superoxide Consumption Kinetic Measurements.

The superoxide dismutase-like activity of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs), anthracite and bituminous graphene quantum dots (PEG-aGQDs and PEG-bGQDs, respectively), and two fullerene carbon nanozymes, tris malonyl-C60 fullerene (C3) and polyhydroxylated-C60 fullerene (C60-OHn), were compared using direct optical stopped-flow kinetic measurements, together with three native superoxide dismutases (SODs), CuZnSOD, MnSOD, and FeSOD, at both pH 12.7 and 8.5. Computer modeling including both SOD catalytic steps and superoxide self-dismutation enabled the best choice of catalyst concentration with minimal contribution to the observed kinetic change from the substrate self-dismutation. Biexponential fitting to the kinetic data ranks the rate constant (M-1 s-1) in the order of PEG-HCCs > CuZnSOD ≈ MnSOD ≈ PEG-aGQDs ≈ PEG-bGQDs > FeSOD ≫ C3 > C60-OHn at pH 12.7 and MnSOD > CuZnSOD ≈ PEG-HCCs > FeSOD > PEG-aGQDs ≈ PEG-bGQDs ≫ C3 ≈ C60-OHn at pH 8.5. Nonlinear regression of the kinetic model above yielded the same ranking as the biexponential fit, but provided better mechanistic insight. The data obtained by freeze-quench EPR direct assay at pH 12.7 also yield the same ranking as stopped-flow data. This is a necessary assessment of a panel of proclaimed carbon nano SOD mimetics using the same two direct methods, revealing a dramatic, 3-4 orders of magnitude difference in SOD activity between PEG-HCCs/PEG-GQDs from soluble fullerenes.

Highly Oxidized Graphene Quantum Dots from Coal as Efficient Antioxidants.

Graphene quantum dots (GQDs) have recently been employed in various fields including medicine as antioxidants, primarily because of favorable biocompatibility in comparison to common inorganic quantum dots, although the structural features that lead to the biological activities of GQDs are poorly understood. Here, we report that coal-derived GQDs and their poly(ethylene glycol)-functionalized derivatives serve as efficient antioxidants, and we evaluate their electrochemical, chemical, and in vitro biological activities.

Perylene Diimide as a Precise Graphene-like Superoxide Dismutase Mimetic.

Here we show that the active portion of a graphitic nanoparticle can be mimicked by a perylene diimide (PDI) to explain the otherwise elusive biological and electrocatalytic activity of the nanoparticle construct. Development of molecular analogues that mimic the antioxidant properties of oxidized graphenes, in this case the poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs), will afford important insights into the highly efficient activity of PEG-HCCs and their graphitic analogues. PEGylated perylene diimides (PEGn-PDI) serve as well-defined molecular analogues of PEG-HCCs and oxidized graphenes in general, and their antioxidant and superoxide dismutase-like (SOD-like) properties were studied. PEGn-PDIs have two reversible reduction peaks, which are more positive than the oxidation peak of superoxide (O2•-). This is similar to the reduction peak of the HCCs. Thus, as with PEG-HCCs, PEGn-PDIs are also strong single-electron oxidants of O2•-. Furthermore, reduced PEGn-PDI, PEGn-PDI•-, in the presence of protons, was shown to reduce O2•- to H2O2 to complete the catalytic cycle in this SOD analogue. The kinetics of the conversion of O2•- to O2 and H2O2 by PEG8-PDI was measured using freeze-trap EPR experiments to provide a turnover number of 133 s-1; the similarity in kinetics further supports that PEG8-PDI is a true SOD mimetic. Finally, PDIs can be used as catalysts in the electrochemical oxygen reduction reaction in water, which proceeds by a two-electron process with the production of H2O2, mimicking graphene oxide nanoparticles that are otherwise difficult to study spectroscopically.

Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin.

Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb-Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 µM(-1) s(-1), irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb-Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb-Hp complexes prepared from unfractionated Hp. All Hb-Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.