Sensitive detection of the redox state of copper proteins using fluorescence.
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.
Schmauder
Ralf
R
Leiden Institute of Physics--Huygens Laboratory, Leiden University, P.O. Box 9504, 2300, Leiden, The Netherlands.
Alagaratnam
Sharmini
S
Chan
Chris
C
Schmidt
Thomas
T
Canters
Gerard W
GW
Aartsma
Thijs J
TJ
D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D002232:Carbocyanines / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry*; D009154:Mutation; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008667:Metalloproteins; C085321:cyanine dye 5; D001400:Azurin; D003300:Copper