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Tryptophan-to-dye fluorescence energy transfer applied to oxygen sensing by using type-3 copper proteins.

Chemistry (Weinheim an der Bergstrasse, Germany) (2007-06-20)
Gerhild Zauner, Emanuela Lonardi, Luigi Bubacco, Thijs J Aartsma, Gerard W Canters, Armand W J W Tepper
ABSTRACT

A fluorescence-based system to sense oxygen in solution is described. The method exploits the sensitivity of the endogenous fluorescence of type-3 copper proteins towards the presence of oxygen by translating the near-UV emission of the protein to label fluorescence in the visible range through a FRET mechanism. The main protein in this study, a recombinant tyrosinase from the soil bacterium Streptomyces antibioticus, has been covalently labeled with a variety of fluorescent dye molecules with emission maxima spanning the whole visible wavelength range. In all cases, the emission of the label varied considerably between O2-bound and O2-free protein with a contrast exceeding that of the Trp emission for some labels. It is shown that different constructs may be simultaneously observed using a single excitation wavelength. Next to the described application in oxygen sensing, the method may be applicable to any protein showing variations in tryptophan fluorescence, for example as a function of ligand binding or catalysis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 655, BioReagent, suitable for fluorescence, ≥85% (HPLC)
Sigma-Aldrich
Atto 655 NHS ester, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 655-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 655 amine, suitable for fluorescence
Sigma-Aldrich
Atto 390 azide, suitable for fluorescence
Supelco
Atto 390 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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