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-C 60 fullerene (C3) and polyhydroxylated-C 60 fullerene (C 60 -OH n ), 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 > C 60 -OH n at pH 12.7 and MnSOD > CuZnSOD ≈ PEG-HCCs > FeSOD > PEG-aGQDs ≈ PEG-bGQDs ≫ C3 ≈ C 60 -OH n 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.