JBIC Journal of Biological Inorganic Chemistry

, Volume 10, Issue 6, pp 683–687 | Cite as

Sensitive detection of the redox state of copper proteins using fluorescence

  • Ralf Schmauder
  • Sharmini Alagaratnam
  • Chris Chan
  • Thomas Schmidt
  • Gerard W. Canters
  • Thijs J. Aartsma
Original Article


The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.


Energy transfer FRET Enzymes Cu protein Azurin Fluorescence quenching Fluorescent probes Redox chemistry Single molecule 



Fluorescence resonant energy transfer





PBS solution

Phosphate buffered saline solution



We thank Ellen de Waal, M.Sc. and Thyra de Jongh, M.Sc. for expert advice on protein purification and sample handling, and Dr. G. Blab for helpful discussions. This work is part of the research program “Biomolecular Physics” of the Stichting voor Fundamenteel Onderzoek der Materie (FOM) and of the joint program “Physical Biology” of FOM and the Stichting Aard en Levenswetenschappen (ALW), which are financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). The work was performed in part under the auspices of the BIOMAC Research School of Leiden University


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Copyright information

© SBIC 2005

Authors and Affiliations

  • Ralf Schmauder
    • 1
  • Sharmini Alagaratnam
    • 2
  • Chris Chan
    • 2
  • Thomas Schmidt
    • 1
  • Gerard W. Canters
    • 2
  • Thijs J. Aartsma
    • 1
  1. 1.Leiden Institute of Physics—Huygens LaboratoryLeiden UniversityLeidenThe Netherlands
  2. 2.Leiden Institute of Chemistry—Gorlaeus LaboratoryLeiden UniversityLeidenThe Netherlands

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