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Related Experiment Videos

New method for removing type 2 copper from Rhus laccase.

A S Klemens1, D R McMillin

  • 1Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.

Journal of Inorganic Biochemistry
|February 1, 1990
PubMed
Summary

Researchers developed a new method to remove type 2 copper from tree laccase, creating a depleted enzyme. This process allows for enzyme reconstitution and offers practical advantages for studying laccase mechanisms.

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Which copper is paramagnetic in the type 2/type 3 cluster of laccase?

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry·1999

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Chemistry

Background:

  • Laccase is a multi-copper oxidase crucial in various biological processes.
  • Understanding the role of individual copper sites, particularly type 2 copper, is vital for elucidating laccase function.
  • Existing methods for copper removal may lack efficiency or practicality.

Purpose of the Study:

  • To describe a novel, practical procedure for preparing type 2 copper-depleted tree laccase.
  • To characterize the resulting enzyme derivative and its properties.
  • To demonstrate the feasibility of reconstituting the enzyme and restoring its activity.

Main Methods:

  • Dialysis of tree laccase against a redox buffer containing ferri- and ferrocyanide ions and EDTA to remove type 2 copper.

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  • Purification of the type 2 copper-depleted fraction using ion-exchange chromatography.
  • Spectroscopic analysis to determine the oxidation state of remaining copper sites (type 1 and type 3).
  • Reconstitution of the enzyme by incubation with Cu(I).

    • Main Results:

    • A procedure successfully removed type 2 copper from tree laccase, yielding a derivative with significantly reduced activity (approx. 5%).
    • The type 1 copper remained oxidized, while the type 3 copper was largely reduced (≥85%), with potential for reoxidation by H2O2.
    • Reconstitution with Cu(I) restored both the activity and spectral properties of the native enzyme.
    • The method removed over 25% of total copper and allowed for easy isolation of the depleted fraction.

    Conclusions:

    • The developed procedure offers a simple and practical method for obtaining type 2 copper-depleted laccase.
    • The study provides insights into the distinct roles of copper sites in laccase activity.
    • The ease of the method facilitates further investigation into the mechanism of copper depletion and laccase function.