BIOLOGICAL CONSEQUENCES OF PROTEIN OXIDATION

蛋白质氧化的生物学后果

• 批准号: 3748230
• 负责人: E B SHACTER
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3748230
• 关键词: aldehydes; carbonyl compound; free radical oxygen; human tissue; ketones; metabolism; neutrophil; oxidation; phenylhydrazines; protein metabolism; proteins; western blottings; xanthine oxidase

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.

活性氧化合物是在一系列正常和 体内病理条件。 认为瞬时氧化剂形成 在心脏病发作、中风和急性炎症时发生 虽然在慢性炎症中可能发生更长时间的氧化剂产生， 条件 内源性氧化剂的来源包括活化的吞噬细胞 (e.g.,嗜中性粒细胞），促氧化酶如黄嘌呤氧化酶，和 正常的有氧代谢 与活性氧相关的病理学 化合物来源于它们修饰细胞和细胞外的能力， 大分子通过金属催化反应。 蛋白构成 氧化修饰的重要目标。 蛋白质的暴露 导致氨基酸序列和蛋白质的变化 结构 这项工作的目标是开发识别 当蛋白质已经被氧化修饰使得生物活性物质 可以确定该氧化的结果。 在以前的工作中，我们 开发了一种灵敏且广泛适用的蛋白质印迹法， 鉴定生物混合物中的氧化蛋白质。 的方法 利用了蛋白质氧化导致 在某些氨基酸上形成羰基（醛和酮） 并且它们可以用2，4-二硝基苯肼衍生化。 的 用抗DNP抗体检测衍生的蛋白质。 使用人 等离子体作为模型，我们发现当暴露于氧化剂流时， 所有主要的血浆蛋白都被修饰，但程度不同。 纤维蛋白原是最容易氧化的。 随后的实验表明，这种重要的凝血蛋白 氧化修饰后失去功能;氧化纤维蛋白原无法 进行凝血酶催化的凝血 结果表明 氧化过程可能在止血中起作用。 最近，我们 使用纤维蛋白原作为模型来确定是否存在 蛋白质上的碳水化合物影响蛋白质羰基的检测。 因为碳水化合物的氧化也会导致羰基的形成 (e.g.,醛），存在的可能性是， 在糖蛋白中检测到的一种蛋白质实际上反映了糖的氧化， 蛋白质修饰 这将降低羰基的可靠性 测定作为氨基酸氧化的量度。 积累的数据 然而，这表明情况并非如此;当糖蛋白 暴露在氧化剂流中，它们显示出相同水平的蛋白质羰基， 基团作为其去糖基化的对应物。 实验正在进行 以确认这一结论，并准备出版工作。