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Related Experiment Videos

Relation between ESR-detectable Cu(II) and superoxide dismutase activity.

J Kajihara1, M Enomoto, K Katoh

  • 1Research Laboratories, Nippon Kayaku Co., Ltd., Tokyo.

Journal of Biochemistry
|November 1, 1988
PubMed
Summary

Electron spin resonance (ESR) accurately measures copper(II) in superoxide dismutase (SOD). This ESR-detectable copper(II) directly correlates with SOD enzyme activity, unlike total copper content.

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Area of Science:

  • Biochemistry
  • Biophysics
  • Enzymology

Background:

  • Copper, zinc-superoxide dismutase (Cu,Zn-SOD) is a critical antioxidant enzyme.
  • Accurate quantification of active enzyme sites is essential for understanding enzyme function.
  • Previous methods for assessing SOD activity may not precisely reflect the active copper content.

Purpose of the Study:

  • To investigate the relationship between ESR-detectable Cu(II) and the activity of Cu,Zn-SOD.
  • To establish ESR as a reliable method for quantifying active copper in SOD.
  • To compare ESR measurements with traditional methods for SOD activity assessment.

Main Methods:

  • Electron spin resonance (ESR) spectroscopy was used to quantify Cu(II) in various SOD preparations.
  • Cu,Zn-SOD activity was measured and compared with ESR-determined Cu(II) levels.

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  • ESR measurements were performed across a pH range of 4.0-10.0 at sub-zero temperatures.
  • Results were correlated with the xanthine-xanthine oxidase assay method.

    • Main Results:

    • A consistent amount of ESR-detectable Cu(II) (6.26 x 10^12 spins/unit SOD) was found in recombinant and native Cu,Zn-SOD preparations.
    • ESR measurements showed good correlation (r=0.995) with the established xanthine-xanthine oxidase method for SOD activity.
    • Specific SOD activity was directly proportional to the ESR-measured Cu(II) content.
    • Total copper content, measured by atomic absorption, did not correlate with specific SOD activity.

    Conclusions:

    • ESR spectroscopy is a valid and sensitive method for quantifying the active Cu(II) sites in Cu,Zn-SOD.
    • Measuring ESR-detectable Cu(II) provides a more accurate assessment of Cu,Zn-SOD activity than measuring total copper.
    • This finding highlights the importance of quantifying specific metal ion states for enzyme activity determination.