Labeling of Peroxide-Induced Oxidative Stress Hotspots by Hemin-Catalyzed Tyrosine Click

Tyrosyl radical generation is one of the major factors for hemin/peroxide-induced oxidative stress. A method for trapping tyrosyl radical directly was developed using N-methyl luminol derivative, a tyrosine labeling reagent. N-Methyl luminol derivative selectively forms a covalent bond with a tyrosine residue under the single-electron oxidation condition. This reaction labels oxidative stress hotspots not only at the protein level but also at the level of tyrosine residues undergoing oxidation. Human serum albumin complexed with hemin was labeled at Tyr138, the tyrosine residue closest to the hemin binding site and most strongly subjected to oxidative stress caused by hemin/H₂O₂. Oxidatively damaged proteins were visualized in protein mixtures.