Detection of protein oxidation in rat-1 fibroblasts by fluorescently labeled tyramine.
Oxidative damage to proteins has been postulated as a major cause of various degenerative diseases including the loss of functional capacity during aging. A prominent target for oxidation by reactive oxygen species (ROS) is the tyrosine residue. Here we present a highly sensitive method for the detection of tyrosyl radical formation in cells. The method is based on the fluorescein-labeled tyrosine analogue, tyramine, which upon oxidation may couple to proteins carrying a tyrosyl radical. Coupling of the probe (denoted TyrFluo) to standard proteins could be induced by generating ROS with horseradish peroxidase/hydrogen peroxide, SIN-1 or with peroxides (cumene or hydrogen peroxide) in combination with a transition metal. TyrFluo added to rat-1 fibroblasts remained outside the cell, whereas the acetylated form (acetylTyrFluo) was membrane-permeable and accumulated in the cell. Exposure of the cells to oxidative stress in the presence of either TyrFluo or acetylTyrFluo gave a cellular labeling characteristic for each probe. Western blot analysis confirmed that each probe labeled a specific set of proteins. This new method for the detection of ROS-induced oxidation of proteins may mimic the tendency of oxidized proteins to form dityrosine bonds.
van der Vlies
D
D
Centre for Biomembranes and Lipid Enzymology, Department of Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands. D.vanderVlies@bio.uu.nl
Wirtz
K W
KW
Pap
E H
EH
D000107:Acetylation; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014439:Tyramine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
C115825:6-(fluorescein-5-carboxamido)hexanoic acid succimidyl ester; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005609:Free Radicals; D011506:Proteins; D017382:Reactive Oxygen Species; C408694:tyrosine radical; D014443:Tyrosine; D019793:Fluorescein; D014439:Tyramine