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A coelenterazine-based luminescence assay to quantify high-molecular-weight superoxide anion scavenger activities

Abstract

In all living cells, levels of reactive oxygen species are kept in check by antioxidative activities. Superoxide radicals are dismutated by superoxide dismutases, by other enzymes and by nonenzymatic compounds. This protocol describes the quantification of superoxide scavenging activities (SOSA). It is based on the inhibition of chemiluminescence emitted by coelenterazine when oxidized by superoxide. SOSA is a summary parameter comprising all high-molecular-weight superoxide scavengers in a biological sample. Enzymes and nonenzymatic scavengers can also be distinguished. The SOSA assay is quick, reproducible and applicable to fields as diverse as medical diagnostics, food sciences, or agriculture. The protocol presented here requires about 2 working days to complete.

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Figure 1: XOD reaction and CTZ luminescence.
Figure 2: SOSA assay procedure and data evaluation.
Figure 3: SOSA calibration curve.
Figure 4: Superoxide scavenging activities in biological samples.
Figure 5: Changes in SOSA in 5-d-old Lepidium after abiotic stress treatment.
Figure 6: SOSA increases in culture medium of HepG2 cells.

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Acknowledgements

We thank L. Shaw (Kiel) for critically reading the paper. We also thank S. Vollbehr, G. Weppner and S. Anderson for technical assistance; J. Scheller (Institute for Biochemistry, University of Kiel) for providing HepG2 cells; B. Bryan of Prolume for the generous gift of coelenterazine; and A. Scheidig (Structural Biology Group, Kiel) and U.-P. Hansen (Biophysics Group, Kiel) for their generous support. We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (Grant no. PL253/5), as well as access to the core facilities of the Zentrum für Biochemie und Molekularbiologie, Christian-Albrechts-Universität, Kiel.

Author information

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Authors

Contributions

L.S. carried out the experiments reported in the main paper, performed data processing and participated in amending the draft. C.P. conceived of the protocol, carried out the experiments shown in the supplementary information and wrote the paper. Both authors approved the final version.

Corresponding author

Correspondence to Christoph Plieth.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Coelenterazine analogues, their generic names, synonyms, and commercial sources. (PDF 10 kb)

Supplementary Note 1

Acetaldehyde is a less favourable substrate for xanthine oxidase in the SOSA assay. Anaerobic organisms may contain superoxide reductase instead of SOD. (PDF 12 kb)

Supplementary Note 2

The signal to background ratio of coelenterazine luminescence produced by different CTZ analogues. (PDF 19 kb)

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Saleh, L., Plieth, C. A coelenterazine-based luminescence assay to quantify high-molecular-weight superoxide anion scavenger activities. Nat Protoc 5, 1635–1641 (2010). https://doi.org/10.1038/nprot.2010.121

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