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 heme adducts are known to be redox active and are suspected to participate in oxidative tissue injury. In the course of our studies on the toxicological role of these heme adducts, we sought to develop a sensitive assay for their detection and quantitation. We have discovered that protein-bound heme adducts, due to their inherent peroxidase activity, can be detected with the use of enhanced chemiluminescence detection reagents, following SDS-PAGE and electroblotting. The assay is specific for protein-bound heme adducts as we have identified conditions where noncovalently bound hemes are completely dissociated from the protein during electrophoresis. Signal intensity was quantified by laser densitometry and found to be linear over a concentration range of 0.44-22 pmol of protein-bound heme adduct, which represented a 20-fold greater sensitivity than the currently available HPLC method. Moreover, we have identified tris(2-carboxyethyl)phosphine as a thiol reducing agent that does not interfere with the detection of the heme-mediated peroxidase activity. The current method may be utilized to identify heme-binding regions of proteins in addition to the detection of oxidatively modified myoglobin.
Vuletich
J L
JL
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan, 48109-0632, USA.
Osawa
Y
Y
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D008163:Luminescent Measurements; D009211:Myoglobin / Q000032:analysis* / Q000737:chemistry; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014907:Whales
D009211:Myoglobin; D013439:Sulfhydryl Reagents