BIOLOGICAL CONSEQUENCES OF PROTEIN OXIDATION

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Reactive oxygen compounds are generated during a wide array of normal and pathologic conditions in vivo. Transient oxidant formation is thought to occur during heart attack and stroke and during acute inflammation while more prolonged oxidant generation may occur in chronic inflammatory conditions. Sources of endogenous oxidants include activated phagocytes (e.g., neutrophils), pro-oxidant enzymes such as xanthine oxidase, and normal aerobic metabolism. The pathology associated with reactive oxygen compounds derives from their ability to modify cellular and extracellular macromolecules through metal-catalyzed reactions. Proteins constitute an important target for oxidative modification. Exposure of a protein to oxidants leads to changes in amino acid sequence and protein structure. The goal of this work is to develop the means to identify when a protein has been oxidatively modified so that the biological consequences of that oxidation can be determined. In previous work, we developed a sensitive and broadly applicable Western blot assay that can identify oxidized proteins within a biological mixture. The approach takes advantage of the fact that protein oxidation results in the formation of carbonyl groups (aldehydes and ketones) on some amino acids and that these can be derivatized with 2,4-dinitrophenylhydrazine. The derivatized proteins are detected with anti-DNP antibodies. Using human plasma as a model, we discovered that when exposed to an oxidant flux, all of the major plasma proteins become modified but to varying degrees. Fibrinogen stood out as being the most highly susceptible to oxidation. Subsequent experiments revealed that this important clotting protein loses function upon oxidative modification; oxidized fibrinogen is unable to undergo thrombin-catalyzed clotting. The results indicate that oxidative processes may play a role in hemostasis. More recently, we have used fibrinogen as a model to determine whether the presence of carbohydrates on a protein affects the detection of protein carbonyls. Because carbohydrate oxidation also leads to the formation of carbonyls (e.g., aldehydes), the possibility exists that some of the moieties detected in a glycoprotein acually reflect sugar oxidation and not protein modification. This would diminish the reliability of carbonyl assays as measures for amino acid oxidation. The data accumulated thusfar suggest however that this is not the case; when glycoproteins are exposed to an oxidant flux, they show the same levels of protein carbonyl groups as their deglycosylated counterparts. Experiments are in progress to confirm this conclusion and to prepare the work for publication.