Role Of Mammalian Peroxidases In Oxidative Stress

哺乳动物过氧化物酶在氧化应激中的作用

• 批准号: 6535046
• 负责人: RONALD P MASON
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/6535046
• 关键词: NAD(H) phosphate; cells; electron spin resonance spectroscopy; enzyme activity; fluoresceins; fluorescence; free radical oxygen; glutathione; hydrogen peroxide; oxidative stress; oxygen consumption; peroxidases

Mammalian peroxidases have a role in the metabolism of drugs and toxic chemicals, but to date there has been very little interest shown in this one-electron, oxidative metabolism. Although both hydrogen peroxide and hydroperoxides are found in vivo and peroxidases have a very high turnoover number and are generally nonspecific, little work has been published in cells or in vivo. The primary reason for the lack of apparent peroxidase metabolism is that the free radical metabolites are reduced by biochemical reducing agents regenerating the parent substrate. As a consequence of this reduction, free radicals of ascorbate, glutathione, and NAD(P)H may be formed. The radical metabolites of the latter two species GS and NAD(P) react with oxygen to form a variety of oxygen-derived species known as reactive oxygen species. We have recently carefully compared the oxidation of these biochemical reducing agents by the tyrosyl radical in a peroxidase system. In addition, we have begun an investigation of the mechanisms of dichlorofluorescein peroxidase oxidation. The oxidation of 2-7-dichlorofluorescin (DCFH) in cells to the highly fluorescent product 2-7-dichlorofluorescein (DCF) has become a popular assay for oxidative stress with nearly 400 publications using this assay. The mechaniam of this oxidation is poorly understood and will be the subject of investigation. From the literature, the oxidation of 2-7-dichlorofluorescin is known to be dependent on a strong oxidant such as hydroxyl radical, peroxynitrite, or compund I of a peroxidase. We believe the peroxidase-dependent oxidation of DCFH forms a free radical intermediate, which reacts with oxygen to form superoxide. If this assay for oxidative stress creates reactive oxygen species in cellular systems, it will be of limited utility. Using ESR, oxygen consumption, and fluorescence, we will investigate the mechanisms of DCFH oxidation by cells and a model peroxidase.

哺乳动物过氧化物酶在药物和有毒化学物质的代谢中起作用，但迄今为止，对这种单电子氧化代谢的兴趣很少。虽然过氧化氢和氢过氧化物都存在于体内，而且过氧化物酶的周转率很高，通常是非特异性的，但在细胞或体内的研究很少发表。缺乏明显过氧化物酶代谢的主要原因是自由基代谢物被再生母体底物的生化还原剂所减少。由于这种减少，抗坏血酸、谷胱甘肽和NAD(P)H的自由基可能形成。后两种自由基代谢产物GS和NAD(P)与氧反应形成多种氧源物质，称为活性氧。我们最近仔细比较了过氧化物酶系统中酪氨酸自由基对这些生化还原剂的氧化作用。此外，我们已经开始了二氯荧光素过氧化物酶氧化机制的研究。细胞中2-7-二氯荧光素（DCFH）氧化为高荧光产物2-7-二氯荧光素（DCF）已成为一种流行的氧化应激检测方法，近400篇论文使用了这种检测方法。这种氧化的机制尚不清楚，将是研究的主题。从文献来看，已知2-7-二氯荧光素的氧化依赖于强氧化剂，如羟基自由基、过氧亚硝酸盐或过氧化物酶的化合物I。我们认为DCFH的过氧化物酶依赖氧化形成自由基中间体，它与氧反应形成超氧化物。如果这种氧化应激试验在细胞系统中产生活性氧，它将是有限的效用。利用ESR，耗氧量和荧光，我们将研究DCFH被细胞和模型过氧化物酶氧化的机制。