PEROXYNITRITE-MEDIATED HEME OXIDATION AND PROTEIN MODIFICATION OF NATIVE AND CHEMICALLY-MODIFIED HEMOGLOBINS

Peroxynitrite (ONOO-) has been shown to play a critical role in tissue reperfusion injury. We have studied the reactions of ONOO- with nativ e and two chemically modified hemoglobins that are being developed as oxygen-carrying reperfusion agents for use in a variety of clinical co nditions. Reactions of native and chemically modified oxyhemoglobins ( oxyHb) at 7.4 with ONOO- lead to a rapid oxidation of the heme iron to ferric (HbFe(3+)) form. Addition of excess molar ratios of ONOO- to t he ferryl (HbFe(4+)) heme protein induced a spectral change indicative of the reduction of HbFe(4+) to the HbFe(3+) oxidation state. No majo r spectral changes were noted when ONOO- was added to methemoglobin (H bFe(3+)) or cyanomethemoglobin (Hb(3+)CN(-)), whereas the carbonmonoxy derivative of ferrous hemoglobin (HbCO) underwent an immediate spectr al change suggesting the displacement of the CO ligand and oxidation o f the heme iron. Rapid mixing of ONOO- with oxyHb in the stopped-flow spectrophotometer yielded biphasic kinetic plots for the oxidation of the ferrous iron (Fe2+). Replots of the apparent rate constants for na tive, cross-linked and polymerized, cross-linked hemoglobins as a func tion of ONOO concentration were linear, yielding a single second-order rate for all hemoglobins of between 2 to 3 x 10(4) M-1 s(-1), indepen dent of the oxygen affinities and molecular sizes of the proteins. Oxi dative modifications of the protein by ONOO-, occuring primarily at th e beta subunits, were observed in reaction mixtures of oxyHb and ONOO- using reverse-phase HPLC, The immunodetection method confirms that ni tration of tyrosine residues by ONOO- occurs on the hemoglobin molecul e and contributes to the modifications observed. We postulate that the presence of hemoglobin in close proximity to ONOO- production sites i n the vasculature can contribute to possible in vivo toxicity by a two -step mechanism involving (i) direct oxidation of the heme iron and (i i) nitration of the tyrosine residues on the molecule, leading to subs equent instability and heme loss from the protein. (C) 1998 Academic P ress.