The long-range goal of this research is to define the role of the lipid bilayer in mediating oxidative damage to membrane-associated proteins. The data resulting from the preliminary experiments proposed here will be the basis of a regular proposal to NSF. That proposal will be to use the model system consisting of cytochrome b5 and cytochrome b5 reductase incorporated separately and together into the bilayer of liposomes and to characterize, in terms of protein structure and function, the effects of free radical generation within the bilayer and from the acqueous media. Changes in protein structure will be determined by peptide mapping on HPLC, amino acid analysis and, ultimately, by protein sequencing. Oxidative damage to a cysteine or lysine residue essential for the binding of NADH by cytochrome b5 reductase would be expected to result in impaired enzymic activity. In addition,, the oxidation of other amino acid residues including histidine, arginine and proline as well as cysteine or lysine residues of either protein, not involved at the active site, could potentially impair function because of alterations in conformation. Finally, cytochrome b5 contains heme iron which may serve as a nucleation site for oxidative damage of amino acid residues in which vicinity of the heme or on a neighboring cytochrome b5 reductase molecule. Once the amino acid residues susceptible to oxidative damage are known, careful studies can be done on the effect of changing the lipid composition of the liposomes and contrasting the effects of free radicals generated within the bilayer to those generated in the surrounding aqueous compartment. %%% The studies proposed here will establish conditions for examining oxidative modifications to membrane-associated enzymes in a model system. Oxidant species may originate exogenously from components of tobacco smoke and air pollutants as well as exposure to radiation. They may arise endogenously from normal metabolic reactions and through the metabolism of certain solvents, drugs and pesticides. Highly reactive oxidant molecules can cause tissue damage by reacting with polyunsaturated fatty acids in cell membranes, nucleotides in DNA and cytosolic proteins. However, little is known about the extent of oxidation of proteins associated with lipid bilayers. The potential for impaired function is great as membrane-associated proteins are responsible for a host of reactions necessary to maintain viability. //
