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 native and two chemically modified hemoglobins that are being developed as oxygen-carrying reperfusion agents for use in a variety of clinical conditions. Reactions o...

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Bibliographic Details
Published in:	Archives of biochemistry and biophysics Vol. 349; no. 1; pp. 65 - 73
Main Authors:	Alayash, Abdu I., Ryan, Beth A.Brockner, Cashon, Robert E.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-01-1998
Subjects:	Heme - chemistry Heme - metabolism hemoglobin-based blood substitutes Hemoglobins - chemistry Hemoglobins - drug effects Hemoglobins - metabolism Humans Nitrates - pharmacology Oxidants - pharmacology Oxidation-Reduction peroxynitrite hemoglobin-based blood substitutes peroxynitrite
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Summary:	Peroxynitrite (ONOO−) has been shown to play a critical role in tissue reperfusion injury. We have studied the reactions of ONOO−with native and two chemically modified hemoglobins that are being developed as oxygen-carrying reperfusion agents for use in a variety of clinical conditions. Reactions of native and chemically modified oxyhemoglobins (oxyHb) at 7.4 with ONOO−lead to a rapid oxidation of the heme iron to ferric (HbFe3+) form. Addition of excess molar ratios of ONOO−to the ferryl (HbFe4+) heme protein induced a spectral change indicative of the reduction of HbFe4+to the HbFe3+oxidation state. No major spectral changes were noted when ONOO−was added to methemoglobin (HbFe3+) or cyanomethemoglobin (Hb3+CN−), whereas the carbonmonoxy derivative of ferrous hemoglobin (HbCO) underwent an immediate spectral change suggesting the displacement of the CO ligand and oxidation of 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 native, cross-linked and polymerized, cross-linked hemoglobins as a function of ONOO−concentration were linear, yielding a single second-order rate for all hemoglobins of between 2 to 3 × 104M−1s−1, independent of the oxygen affinities and molecular sizes of the proteins. Oxidative modifications of the protein by ONOO−, occuring primarily at the β subunits, were observed in reaction mixtures of oxyHb and ONOO−using reverse-phase HPLC. The immunodetection method confirms that nitration of tyrosine residues by ONOO−occurs on the hemoglobin molecule and contributes to the modifications observed. We postulate that the presence of hemoglobin in close proximity to ONOO−production sites in the vasculature can contribute to possiblein vivotoxicity by a two-step mechanism involving (i) direct oxidation of the heme iron and (ii) nitration of the tyrosine residues on the molecule, leading to subsequent instability and heme loss from the protein.
ISSN:	0003-9861 1096-0384
DOI:	10.1006/abbi.1997.0449