NSF Award
9505806
9505806 Hood This research is based on the hypothesis that the chronic effect of NO2 on protein structure and function is due, in part, to the reaction of this oxidant pollutant with specific basic amino acid side chains and that such reactions are potentiated by formation of oxidant radicals resulting from the peroxidation of polyunsaturated fatty acids (PUFAs). Preliminary data suggest that NO2 reacts with the E-amino group of lysine residues and with the amide nitrogen of surface exposed lysine and arginine residues in the polypeptide backbone which can result in polypeptide bond cleavage. In vitro NO2 exposures of a series of decapeptides followed by exposures of recombinant 1-PI (a 53kDa protein which protects the critical structural protein, elastin, from proteolysis by elastase) will be performed to determine the susceptibility of these polypeptides to pollutant oxidants. NO2 exposures of selected decapeptides and of proteins, followed by exposures of inhibitors and unsaturated hydrocarbons as well as membrane entrapped inhibitors analyzed by Electrospray Ionization Mass Spectrometry, 15N-NMR, 13C15N-NMR, and 1H-NMR and Gas Chromatography will identify specific NO2 derived adducts. These studies will also determine whether the autoxidation of unsaturated fatty acids contributes to polypeptide inactivation. The in vitro exposure studies will be conducted using well defined systems, providing accurate quantitation and biochemical characterization. The target molecules chosen are relevant to the mechanistic study of conformational transitions in proteins as a result of oxidant exposure. %%% Nitrogen dioxide (NO2), and its associated NO (x) (nitroxide) species are examples of atmospheric pollutants derived from fossil fuel combustion and cigarette smoke. Combustion processes initially produce nitric oxide, which is then oxidized in the atmosphere to the much more reactive NO2 species. This photochemical process is responsible for the daily variations i n nitroxide concentrations in large metropolitian areas. Very little effort has been directed at the possibility that oxidants, such as NO2, may directly alter the structure of proteins. Based on the oxidation-reduction potential of NO2 in neutral aqueous solutions, it has been postulated that NO2 exhibits a high degree of selectivity among the molecular constituents of living cells. This research will examine the effects of NO2 exposure on selected peptides and proteins. Structural changes will be determined and the susceptibility of these protein structural changes to different conditions of oxidant exposure will be evaluated. ***
