Detection of specific reaction products is a powerful approach for dissecting out pathways that mediate oxidative damage in vivo.
Eosinophil peroxidase (EPO), an abundant
protein secreted from activated eosinophils, has been implicated in promoting oxidative tissue injury in conditions such as
asthma, allergic inflammatory disorders,
cancer, and helminthic
infections. This unique
heme protein amplifies the oxidizing potential of H2O2 by utilizing plasma levels of Br- as a cosubstrate to form potent brominating agents. Brominated products might thus serve as powerful tools for identifying sites of eosinophil-mediated tissue injury in vivo; however, structural identification and characterization of specific brominated products formed during EPO-catalyzed oxidation have not yet been reported. Here we explore the role of EPO and
myeloperoxidase (MPO), a related leukocyte
protein, in promoting
protein oxidative damage through bromination and demonstrate that
protein tyrosine residues serve as endogenous traps of reactive brominating species forming stable ring-brominated adducts. Exposure of the
amino acid L-tyrosine to EPO, H2O2, and physiological concentrations of halides (100 mM Cl-, </=100 microM Br-) produced two new major products with distinct retention times on reverse phase HPLC. The products were identified as
3-bromotyrosine and 3, 5-dibromotyrosine by electrospray ionization mass spectrometry and multinuclear (1H and 15N) NMR spectroscopy. Formation of the ring-brominated forms of the
amino acid occurred readily at neutral pH with the enzymatic system and a variety of reactive brominating species, including
HOBr/OBr-, N-bromoamines, and N,N-dibromoamines. Addition of primary
amines (e.g., Nalpha-acetyllysine and
taurine) to
L-tyrosine exposed to either
HOBr/OBr- or the EPO-H2O2-Br- system enhanced phenolic ring bromination, suggesting N-bromoamines are preferred brominating intermediates in these reactions. Reduction of N-bromoamines (e.g., Nalpha-acetyl,Nepsilon-bromolysine) by
L-tyrosine was shown to result in the loss of reactive
halogen with a near stoichiometric increase in the level of
tyrosine ring bromination (i.e.,
carbon-
bromine bonds). Although both EPO and MPO could use Br- to halogenate
protein tyrosine residues in vitro, only EPO effectively brominated the
aromatic amino acid at physiological levels of halides and H2O2. Collectively, these results suggest that
3-bromotyrosine and
3,5-dibromotyrosine are attractive candidates for serving as molecular markers for oxidative damage of
proteins by reactive brominating species in vivo. They also suggest that in
biological mixtures where
amine groups are abundant, the trapping of EPO-generated
HOBr/OBr- as N-bromoamines will serve to effectively "funnel" reactive brominating equivalents to stable ring-brominated forms of
tyrosine.