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

Abstract

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.

Keywords

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

Abbreviations

FRET

Fluorescence resonant energy transfer

DMSO

Dimethylsulfoxide

DTT

Dithiothreitol

PBS solution

Phosphate buffered saline solution

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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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