Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

May 1991

Abstract

Nitrogen dioxide (NO$\sb2$), 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. To gain information on the mechanism by which NO$\sb2$ damages the lung, in vitro exposures of $\alpha\sb1$-proteinase inhibitor ($\alpha\sb1$-PI), elastin, bovine serum albumin (BSA), human serum albumin (HSA) and synthetic poly-L-lysine were performed. A genetic deficiency of $\alpha\sb1$-PI predisposes humans to emphysema and NO$\sb2$ has been hypothesized to damage $\alpha\sb1$-PI, which would leave proteases such as human neutrophil elastase, (HNE) free to attack lung structural proteins. The ability of $\alpha\sb1$-PI to inhibit HNE declined with exposure to 50% of the control value at molar ratios of NO$\sb2$:$\alpha\sb1$-PI of 400:1 and greater. Exposure of $\alpha\sb1$-PI to NO$\sb2$ resulted in a 50% loss of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO$\sb2$:$\alpha\sb1$-PI of essentially 100:1 and greater. The mechanisms of these effects were investigated via ultraviolet-visible spectroscopy and amino acid analysis. The remaining target molecules were labeled by reductive methylation of amino groups with $\sp3$H-HCHO prior to treatment with NO$\sb2$ in aqueous solutions at physiological pH. Time course exposure of 5 mg $\sp3$H-insoluble bovine ligamentum nuchae elastin suspensions with up to 120 $\mu$moles of NO$\sb2$ resulted in 90% solubilization of the label. Amino acid analysis of the soluble and insoluble fractions from these exposures confirmed that 80% of the $\sp3$H-dimethyllysine residues were in the soluble fraction. Since these results suggested a specific reactivity of NO$\sb2$ with lysine residues, 400 $\mu$g $\sp3$H-poly-L-lysine was exposed to 120 $\mu$moles NO$\sb2$. Gel filtration chromatography revealed that the 50,000 M$\sb{\rm r}$ $\sp3$H-poly-L-lysine had been degraded to small peptides of 1-3,000 M$\sb{\rm r}$. Similar exposures were conducted using $\sp3$H-BSA and $\sp3$H-HSA, followed by gel filtration chromatography and SDS-PAGE with fluorography. The results suggest that NO$\sb2$ preferentially reacts with Lys-Lys or other specific sequences, resulting in peptide bond cleavage. Under the conditions used, 23% of the BSA tyrosine residues were nitrated and aggregates of HSA indicative of bityrosine cross-link formation were observed. These findings are the first indication that NO$\sb2$ can cause protein fragmentation and provide additional data on the potential of NO$\sb2$ to damage lung proteins, such as elastin.

Document Type

Dissertation - unrestricted

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