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 perox- idases. If an exogenous phenolic substrate is present, Hb promotes its nitration with a fivefold higher efficiency with respect to the perox- idase-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 pathophysiolog-