Electron Paramagnetic Resonance Detection of Free Tyrosyl Radical Generated by Myeloperoxidase, Lactoperoxidase, and

Phagocytes secrete the heme protein myeloperoxi-dase, which is present and active in human atheroscle-rotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end prod-ucts. When this system acts on L-tyrosine in vitro, it forms o,o*-dityrosine, which is enriched in atheroscle-rotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking their L-tyro-sine residues. In these studies, we used electron para-magnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rap-idly mix and examine solutions containing horseradish peroxidase, H2O2, and L-tyrosine, we detected free ty-rosyl radical (a2,6 H 5 6.3 G, a3,5 H 5 1.6 G, and ab H 5 15.0 G). We then used spin trapping techniques with 2-meth-yl-2-nitrosopropane (MNP) to further identify this inter-mediate. The resulting three-line spectrum (aN 5 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeled L-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN 5 15.6 G,