Role Of Mammalian Peroxidases In Oxidative Stress

哺乳动物的过氧化物酶在药物和有毒化学物质的代谢中发挥作用，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在体内发现，过氧化物酶具有非常高的周转数，通常是非特异性的，但很少有工作在细胞或体内发表。缺乏明显的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还原。作为这种还原的结果，可以形成抗坏血酸、谷胱甘肽和NAD（P）H的自由基。后两种物质GS和NAD（P）的自由基代谢物与氧反应形成各种氧衍生物质，称为活性氧物质。我们最近仔细比较了这些生化还原剂的氧化酪氨酰自由基在过氧化物酶系统。此外，我们已经开始了二氯荧光素过氧化物酶氧化的机制的调查。细胞中2-7-二氯荧光素（DCFH）氧化成高荧光产物2-7-二氯荧光素（DCF）已成为氧化应激的流行测定法，近400篇出版物使用该测定法。这种氧化的机制知之甚少，将是调查的主题。根据文献，已知2-7-二氯荧光素的氧化依赖于强氧化剂，例如羟基自由基、过氧亚硝酸根或过氧化物酶的化合物I。我们认为DCFH的过氧化物酶依赖性氧化形成自由基中间体，其与氧反应形成超氧化物。如果这种氧化应激测定在细胞系统中产生活性氧，则其效用有限。使用ESR，耗氧量和荧光，我们将研究细胞和模型过氧化物酶氧化DCFH的机制。

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.