BIOLOGICAL CONSEQUENCES OF PROTEIN OXIDATION

活性氧化合物是在一系列正常和 活体内的病理条件。暂时性氧化剂的形成被认为 发生在心脏病发作和中风以及急性炎症期间 而更长时间的氧化剂生成可能发生在慢性炎症性疾病 条件。内源性氧化剂的来源包括激活的吞噬细胞 (例如，中性粒细胞)、黄嘌呤氧化酶等促氧化物酶，以及 正常的有氧代谢。与活性氧相关的病理学 化合物源于它们修饰细胞和细胞外的能力 大分子通过金属催化的反应。蛋白质构成 氧化修饰的一个重要目标。暴露一种蛋白质 氧化剂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 当一种蛋白质被氧化修饰后，生物 这种氧化的后果是可以确定的。在以前的工作中，我们 开发了一种灵敏和广泛适用的蛋白质印迹分析方法，可以 鉴定生物混合物中的氧化蛋白质。该方法 利用了这样一个事实：蛋白质氧化导致 某些氨基酸上的羰基(醛和酮)的形成 这些化合物可以用2，4-二硝基苯肼进行衍生化。这个 用抗DNP抗体检测衍生蛋白。使用人类 以等离子体为模型，我们发现，当暴露在氧化剂熔剂中时， 所有主要的血浆蛋白都发生了不同程度的修饰。 纤维蛋白原是最容易被氧化的。 随后的实验表明，这种重要的凝血蛋白 氧化修饰时失去功能；氧化的纤维蛋白原无法 进行凝血酶催化的凝血。结果表明，该系统具有较高的可靠性。 氧化过程可能在止血中发挥作用。最近，我们 已经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会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 (例如，醛)，存在某些部分存在的可能性 在糖蛋白中检测到的反应是糖的氧化而不是 蛋白质修饰。这会降低羰基化合物的可靠性。 作为氨基酸氧化的检测手段。积累的数据 然而，到目前为止，情况并非如此；当糖蛋白 暴露在氧化剂助熔剂下，它们显示出相同水平的蛋白质羰基 基团作为它们的脱糖基团。实验正在进行中 以确认这一结论，并为出版这部作品做准备。

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.