NITROGEN-DIOXIDE REACTIVITY WITH PROTEINS - EFFECTS ON ACTIVITY AND IMMUNOREACTIVITY WITH ALPHA-1-PROTEINASE INHIBITOR AND IMPLICATIONS FOR NO2-MEDIATED PEPTIDE DEGRADATION

Nitrogen dioxide (NO2), an air pollutant produced by burning fossil fuels and a component of cigarette smoke, is thought to contribute to the pathogenesis of pulmonary diseases, such as emphysema. In order to gain information on the mechanism by which NO2 damages the lung and proteins vital to its function, as well as its reaction with proteins in general, in vitro exposures of α-1-proteinase inhibitor (α1PI), elastin, poly-L-lysine, and poly-L-arginine were performed. The ability of α1PI to inhibit its natural physiological target, human neutrophil elastase (HNE), declined with exposure to 54% of the control value at molar ratios of NO:α12PI of 400:1 and greater. Exposure of α1PI to NO2 resulted in a 50% loss of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO:α12PI of 100:1 and greater. The results of parallel O-phthalaldehyde and bicinchoninic acid protein assays as well as amino acid analysis on control and NO2-exposed α1PI suggested a reactivity of NO2 with lysine residues. Elastin and poly-L-lysine were labeled by reductive methylation of amino groups with [3H]HCHO prior to treatment with NO2 in aqueous solutions at physiological pH. NO2 exposure of elastin resulted in the solubilization of 84% of the associated radioactivity of which 79% was identified as [3H]methyllysine by amino acid analysis. After NO2 exposure of poly-L-[3H]lysine, gel filtration chromatography revealed that the 50,000 Mr poly-L-[3H]lysine had been degraded to small peptides of 1-3000 Mr. Similarly, after NO2 exposure of unlabeled poly-L-arginine, gel filtration chromatography, and total peptide analysis revealed that the 47,500 Mr peptide was also partially degraded to peptides. These results suggest that NO2 reacts with the ε{lunate}-amino groups of Lys residues (primary amines) and with the amide nitrogen (secondary amines) of surface-exposed Lys and Arg residues in the peptide backbone to result in peptide bond cleavage. These findings are the first indication of NO2-mediated peptide degradation and provide additional data on the potential of NO2 to damage proteins vital to the function of the lung in an in vitro exposure system. © 1993 Academic Press, Inc.