Chemiluminescence assay for oxidatively modified myoglobin

Treatment of myoglobin with H2O2 results in covalent alteration of the heme prosthetic group, in part, to protein-bound adducts. These protein-bound h eme adducts are known to be redox active and are suspected to participate i n oxidative tissue injury. In the course of our studies on the toxicologica l role of these heme adducts, we sought to develop a sensitive assay for th eir detection and quantitation. We have discovered that protein-bound heme adducts, due to their inherent peroxidase activity, can be detected with th e use of enhanced chemiluminescence detection reagents, following SDS-PAGE and electroblotting, The assay is specific for protein-bound heme adducts a s we have identified conditions where noncovalently bound hemes are complet ely dissociated from the protein during electrophoresis. Signal intensity w as quantified by laser densitometry and found to be linear over a concentra tion range of 0.44-22 pmol of protein-bound heme adduct, which represented a 20-fold greater sensitivity than the currently available HPLC method. Mor eover, we have identified tris(2-carboxyethyl)phosphine as a thiol reducing agent that does not interfere with the detection of the heme-mediated pero xidase activity. The current method may be utilized to identify heme-bindin g regions of proteins in addition to the detection of oxidatively modified myoglobin. (C) 1998 Academic Press.