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Sensitive detection of the redox state of copper proteins using fluorescence

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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.

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Abbreviations

FRET:

Fluorescence resonant energy transfer

DMSO:

Dimethylsulfoxide

DTT:

Dithiothreitol

PBS solution:

Phosphate buffered saline solution

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Acknowledgements

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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Authors and Affiliations

  1. Leiden Institute of Physics—Huygens Laboratory, Leiden University, P.O. Box 9504, 2300, Leiden, The Netherlands

    Ralf Schmauder, Thomas Schmidt & Thijs J. Aartsma

  2. Leiden Institute of Chemistry—Gorlaeus Laboratory, Leiden University, P.O. Box 9504, 2300, Leiden, The Netherlands

    Sharmini Alagaratnam, Chris Chan & Gerard W. Canters

Authors
  1. Ralf Schmauder
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  2. Sharmini Alagaratnam
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  3. Chris Chan
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  4. Thomas Schmidt
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  5. Gerard W. Canters
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  6. Thijs J. Aartsma
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Correspondence to Thijs J. Aartsma.

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Schmauder, R., Alagaratnam, S., Chan, C. et al. Sensitive detection of the redox state of copper proteins using fluorescence. J Biol Inorg Chem 10, 683–687 (2005). https://doi.org/10.1007/s00775-005-0020-6

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  • DOI: https://doi.org/10.1007/s00775-005-0020-6

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