Critical Comparison of the Superoxide Dismutase-Like Activity of Carbon Anti-Oxidant 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 (C-OH), 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. Bi-exponential fitting to the kinetic data ranks the rate constant (M s) in the order of: PEG-HCCs > CuZnSOD ≈ MnSOD ≈ PEG-aGQDs ≈ PEG-bGQDs > FeSOD > C3 > C-OH at pH 12.7, and MnSOD > CuZnSOD ≈ PEG-HCCs > FeSOD > PEG-aGQDs ≈ PEG-bGQDs > C3 ≈ C-OH at pH 8.5. Nonlinear regression of the kinetic model above yielded the same ranking as bi-exponential fit, but provided better mechanistic insight. The data obtained by freeze-quench EPR direct assay at pH 12.7 also yields 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 order of magnitude difference in SOD activity between PEG-HCCs/PEG-GQDs from soluble fullerenes.