Inactivation of .alpha.1-proteinase inhibitor by peroxynitrite

Abstract

We here report the reactions of peroxynitrite (O=NOO-) with alpha 1-proteinase inhibitor (alpha 1PI) and with a synthetic decapeptide (MER10) containing the sequence of amino acids found in the active site of alpha 1PI (Pro-Met-Ser-Ile-Pro-Pro-Glu-Val-Lys-Phe). Peroxynitrite inactivates alpha 1PI at both pH 7.4 and 12.0. Thiourea and methionine protect alpha 1PI against inactivation by peroxynitrite, while mannitol and benzoate fail to afford effective protection. The major product isolated from the reaction between peroxynitrite and MER10 was analyzed by NMR, mass spectrometry, and amino acid analysis. These analyses indicate that peroxynitrite primarily oxidizes the methionine residue in the peptide. We detect neither smaller molecular weight peptides, which would indicate cleavage of MER10, nor hydroxylation or nitration of the phenylalanine residue. Our results suggest that peroxynitrite is capable of oxidizing methionine residues in proteins without the involvement of the hydroxyl radical or nitrogen dioxide. The implications of these observations on lung diseases attributed to cigarette smoke are discussed.