Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
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DOI：
10.3791/2810
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发表时间：
2012-08-01
影响因子：
1.2
通讯作者：
Villamena, Frederick A.
中科院分区：
文献类型：
--
作者：
Gopalakrishnan, Bhavani;Nash, Kevin M.;Villamena, Frederick A.
低浓度的活性氮/氧物质（ROS/RNS）在调节细胞功能、信号传导和免疫应答中起重要作用，但在未调节的浓度下对细胞活力有害（1，2）。虽然生命系统已经进化出内源性和膳食抗氧化防御机制来调节ROS的产生，但ROS作为正常氧代谢的天然副产物持续产生，并可对生物分子造成氧化损伤，导致蛋白质功能丧失、DNA裂解或脂质过氧化（3），并最终导致氧化应激，导致细胞损伤或死亡（4）。（O-2中心点-）是一些已知存在于生物系统中的最高氧化性物质的主要前体，如过氧亚硝酸根和羟基自由基。O-2中心点的产生是氧化爆发的第一个信号，因此，它在生物系统中的检测和/或隔离是重要的。在该演示中，O-2中心点-是由多形核中性粒细胞（PMNs）产生的。通过佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）的趋化刺激，PMN通过激活烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶（5）产生O-2中心点。一氧化氮（NO）合酶有三种亚型，分别为诱导型、神经元型和内皮型NOS，或iNOS、nNOS或eNOS，催化L-精氨酸转化为L-瓜氨酸，使用NADPH产生NO 6。在这里，我们从内皮细胞产生NO。在氧化应激条件下，eNOS例如可以在称为解偶联的过程中从产生NO转换为O-2中心点，这被认为是由血红素（7）或辅因子四氢生物蝶呤（BH 4）（8）的氧化引起的。自旋捕集通常用于鉴定自由基，涉及自由基与自旋捕集器的加成反应，形成可通过电子顺磁共振（EPR）光谱检测的持久自旋加合物。各种自由基加合物显示出独特的光谱，其可用于鉴定所产生的自由基，并且可提供关于自由基产生的性质和动力学的丰富信息（9）。环状硝酮，5，5-二甲基-吡咯啉-N-氧化物，DMPO 10，磷酰基取代的DEPMPO 11，以及酯取代的EMPO 12和BMPO 13，已被广泛用作自旋陷阱--后者的自旋陷阱对于O-2中心点加合物显示出更长的半衰期。铁（II）-N-甲基-D-葡糖胺二硫代氨基甲酸盐，Fe（MGD）2通常用于捕获NO，这是由于加合物形成的高速率和自旋加合物的高稳定性（14）。
Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability(1, 2). While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation(3), and ultimately to oxidative stress leading to cell injury or death(4).Superoxide radical anion (O-2 center dot-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O-2 center dot- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O-2 center dot- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O-2 center dot- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase(5).Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal-and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L-arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O-2 center dot- in a process called uncoupling, which is believed to be caused by oxidation of heme(7) or the co-factor, tetrahydrobiopterin (BH4)(8).There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production(9).The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O-2 center dot- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD) 2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct(14).