Nitrative stress causes nitration, oxidation, and subunit cross linking in human hemoglobin

Hemoglobin (Hb) is the most abundant protein in human blood and we showed that under oxidative/nitrative stress conditions it is susceptible to cysteine oxidation, tyrosine nitration, and formation of a dimer of Hb subunits through tyrosine linkage. In the presence of hydrogen peroxide, Hb and its subunits efficiently convert nitrite into reactive nitrogen species, through reactions that are typical of peroxidases. If an exogenous phenolic substrate is present, Hb promotes its nitration with a fivefold higher efficiency with respect to the peroxidase-like phenol coupling reaction. In the absence of an exogenous substrate, the protein itself undergoes covalent modification. Trypsin treatment of Hb modified under conditions mimicking pathophysiological conditions, followed byHPLC-ESI-MS/MS analysis, allowed detection of nitration of Yα24, Yα42, Yβ130 and Yβ145, and conversion of Cα104, Cβ93 and Cβ112 into cysteine sulfinic acids. As additional biomarkers of nitrative stress, we found a covalent dimer of Hb αβ subunits and covalently linked heme-peptide. The dimer is selectively nitrated at Yα42. In a preliminary analysis of samples of human blood we found that low amounts of the dimer of subunits are always present. Therefore, if a correlation between the extent of Hb subunits coupling and pathological states could be established, this dimer could become an easily detectable biomarker of pathological conditions.