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Sensitive detection of the redox state of copper proteins using fluorescence |
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| Authors: | Ralf Schmauder Sharmini Alagaratnam Chris Chan Thomas Schmidt Gerard W Canters Thijs J Aartsma |
| Institution: | (1) Leiden Institute of Physics—Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 Leiden, The Netherlands;(2) Leiden Institute of Chemistry—Gorlaeus Laboratory, Leiden University, P.O. Box 9504, 2300 Leiden, The Netherlands |
| 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 |
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