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The type 2 copper of ascorbate oxidase
B Reinhammar1, R Aasa, T Vänngård
1Department of Biochemistry and Biophysics, Göteborg University, Sweden. bengt.reinhammar@bcbp.gu.se
Biochimica Et Biophysica Acta
|February 8, 1997
Summary
Ascorbate oxidase activity is higher in phosphate buffer but decreases significantly after cold storage. Hepes buffer better preserves enzyme activity and EPR spectra, suggesting phosphate buffer alters the copper sites.
Area of Science:
- Biochemistry
- Biophysics
- Enzymology
Background:
- Ascorbate oxidase is a copper-containing enzyme crucial for plant defense and cell wall metabolism.
- Enzyme stability and copper site integrity are vital for its catalytic function.
- Understanding buffer effects on enzyme stability is important for experimental design and storage.
Purpose of the Study:
- To investigate the effect of different buffer systems (HEPES and sodium phosphate) on ascorbate oxidase stability.
- To analyze changes in enzyme activity and copper site integrity using EPR spectroscopy after cold storage.
- To identify potential causes for observed changes in enzyme performance and spectral characteristics.
Main Methods:
- Ascorbate oxidase activity assays.
- Electron Paramagnetic Resonance (EPR) spectroscopy.
- Copper content analysis.
- Storage of enzyme solutions at -30°C and 77 K.
Main Results:
- Specific activity was higher in phosphate buffer than HEPES buffer.
- Storage at -30°C significantly reduced activity in phosphate buffer, but less so in HEPES buffer.
- EPR spectra showed alterations in the type 2 copper site in phosphate buffer after storage, which were reversible upon thawing and refreezing.
- Copper analysis indicated approximately 50% of total copper was EPR-detectable, with type 2 Cu2+ contributing about 25% of total EPR intensity.
- Proposed existence of trinuclear copper centers contributing to the EPR signal.
Conclusions:
- HEPES buffer provides better protection for ascorbate oxidase activity and copper site integrity during cold storage compared to sodium phosphate buffer.
- Sodium phosphate buffer may induce alterations in the type 2 copper site, affecting enzyme stability.
- The EPR data suggests the presence of additional copper species, possibly trinuclear centers, beyond the well-characterized type 1 and type 2 copper sites.