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

Abstract

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-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 > C₆₀-OH_n at pH 12.7 and MnSOD > CuZnSOD ≈ PEG-HCCs > FeSOD > PEG-aGQDs ≈ PEG-bGQDs ≫ C3 ≈ C₆₀-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.

Keywords: comparative study; freeze−quench EPR; nanozymes; stopped-flow; superoxide dismutase activity.

Figure 1.

Time courses of SO self-dismutation at different pHs. The time courses of A₂₄₄ were followed after mixing 5 mM of KO₂ in DMSO with buffers at different pHs ([KO₂]_final = 192 μM): 12.7 (black), 8.5 (red), 8.0 (blue), and 7.5 (green). Data at times shorter than 5 ms are not shown due to contamination by mixing artifacts. Each time course is labelled with the 2^nd-order rate constants k in M⁻¹s⁻¹, obtained by fitting to eq 6 (see Experimental Methods section). Inset, log k vs pH and linear fit.

Figure 2.

SO quenching activities of SODs and nanozymes at pH 12.7. Black lines in each panel: time course of self dismutation of 192 μM KO₂. (A) Time courses of SO quenching by 20 nM SOD: CuZnSOD, MnSOD (blue), and FeSOD (green). (B) Time courses of SO quenching by nanozymes: 20 nM PEG-HCCs (red), 20 nM of PEG-aGQDs (green), 20 nM of PEG-bGQDs (blue), 2 μM C3 (yellow), and 30 μM C₆₀-OH_n (purple). The time courses with PEG-HCCs, PEG-aGQDs, and PEG-bGQDs were vertically adjusted to remove the background absorbance of these nanozymes. Due to the high concentrations of C3 and C₆₀-OH_n required in the reactions, their time courses showed large vertical backgrounds and were vertically shifted arbitrarily to bring the traces into y-axis scale range. Data at time shorter than 5 ms are not shown due to contamination by mixing artifacts.

Figure 3.

Computer simulation of SO dismutation with different [SO]. The time courses of SO optical signal were simulated using SCoP based on a kinetic model including simultaneously progressing irreversible self-dismutation (eq 1) and catalyzed dismutation (eq 2 and 3). The SO self-dismutation rate constant was set at measured value at pH 8.5, k_s = 3.3 × 10⁴ M⁻¹s⁻¹ (Figure 1); the two rate constants of a catalyzed SO dismutation were set identical k_r = k_o; [catalyst] was set at 20 nM; [SO] varied from 10 nM to 100 μM. The time course of self-dismutation is represented by black circles and those of catalyzed reactions by solid lines. Ranges of rate constants k_r/k_o varied from: 1 × 10⁵ M⁻¹s⁻¹, black; 1 × 10⁶ M⁻¹s⁻¹, red; 1 × 10⁷ M⁻¹s⁻¹, green;1 × 10⁸ M⁻¹s⁻¹, yellow; 1 × 10⁹ M⁻¹s⁻¹, blue. [O₂⁻•] is marked in each panel: 100 μM (A); 10 μM (B); 1 μM (C); 100 nM (D); 10 nM (E).

Figure 3.

Computer simulation of SO dismutation with different [SO]. The time courses of SO optical signal were simulated using SCoP based on a kinetic model including simultaneously progressing irreversible self-dismutation (eq 1) and catalyzed dismutation (eq 2 and 3). The SO self-dismutation rate constant was set at measured value at pH 8.5, k_s = 3.3 × 10⁴ M⁻¹s⁻¹ (Figure 1); the two rate constants of a catalyzed SO dismutation were set identical k_r = k_o; [catalyst] was set at 20 nM; [SO] varied from 10 nM to 100 μM. The time course of self-dismutation is represented by black circles and those of catalyzed reactions by solid lines. Ranges of rate constants k_r/k_o varied from: 1 × 10⁵ M⁻¹s⁻¹, black; 1 × 10⁶ M⁻¹s⁻¹, red; 1 × 10⁷ M⁻¹s⁻¹, green;1 × 10⁸ M⁻¹s⁻¹, yellow; 1 × 10⁹ M⁻¹s⁻¹, blue. [O₂⁻•] is marked in each panel: 100 μM (A); 10 μM (B); 1 μM (C); 100 nM (D); 10 nM (E).

Figure 4.

Computer simulation of SO dismutation with different [catalyst]. The time courses of SO optical signal were simulated using SCoP based on a kinetic model including parallel irreversible self-dismutation (eq 1) and catalyzed dismutation (eq 2 and 3). The SO self-dismutation rate constant was set at measured value at pH 8.5, k_s = 3.3 × 10⁴ M⁻¹s⁻¹ (Figure 1); the rate constants of catalyzed SO dismutation were set at identical k_r = k_o; [SO] was set at 100 μM and [catalyst] varied from 20 nM to 20 μM. The time course of self-dismutation is represented by black circles and those of catalyzed reactions by solid lines. The [catalyst] used in simulations are marked in each panel: 20 nM (A) & (F); 100 nM (B); 500 nM (C); 2 μM (D); 20 μM (E).The varying rate constants used in each panel: 1 × 10⁵ M⁻¹s⁻¹, black; 1 × 10⁶ M⁻¹s⁻¹, red; 1 × 10⁷ M⁻¹s⁻¹, green;1 × 10⁸ M⁻¹s⁻¹, yellow; 1 × 10⁹ M⁻¹s⁻¹, blue. Panel F: biphasic exponential fits to the simulated time courses (same as in (A) represented with open circles) using the same color scheme as above. The fit rate constants are divided by [catalyst] = 20 nM to obtain the 2^nd-order rate constants: 3.7 × 10⁸/9.0 × 10⁷ M⁻¹s⁻¹ (black); 3.7 × 10⁸/9.0 × 10⁷ M⁻¹s⁻¹ (red); 3.8 × 10⁸/9.2 × 10⁷ M⁻¹s⁻¹ (green); 4.8 × 10⁸/1.4 × 10⁸ M⁻¹s⁻¹ (yellow); 2.2 × 10⁹/9.8 × 10⁸ M⁻¹s⁻¹ (blue).

Figure 5.

SO quenching activities of SODs and nanozymes at pH 8.5. Black circles in each panel: self dismutation of 192 μM KO₂. (A) SO consumption activities of SODs (circles): 40 nM CuZnSOD (red), 40 nM MnSOD (blue), and 200 nM FeSOD (green). (B) SO consumption activities of nanozymes (circles): 100 nM PEG-HCCs (red), 500 nM of PEG-aGQDs (green), 500 nM of PEG-bGQDs (blue), 13.5 μM C3 (yellow), and 30 μM C₆₀-OH_n (purple). The time courses with PEG-HCCs, PEG-aGQDs, and PEG-bGQDs were vertically adjusted to remove the background absorbance of these nanozymes. Due to the high concentrations of C3 and C₆₀-OH_n required in the reactions, their time courses showed large vertical backgrounds and were vertically shifted arbitrarily to bring the traces into y-axis scale range. Data at time shorter than 5 ms are not shown due to mixing artifacts. Black lines in both (A) and (B), fit by 2^nd-order function (eq 6) for SO self-dismutation; other lines are SCoP fits to the experimental data: (A) CuZnSOD (red), MnSOD (blue), and FeSOD (green), (B) PEG-HCCs (red), PEG-aGQDs (green), and PEG-bGQDs (blue).