Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709, USA.
Chem Res Toxicol. 2011 Dec 19;24(12):2217-26. doi: 10.1021/tx2003323. Epub 2011 Nov 22.
Nitrone spin traps such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are commonly used for free radical detection. Though proven examples are rare, artifact formation must be considered. For example, the Forrester-Hepburn mechanism yields the same radical adduct as that formed by genuine radical trapping. A hydroxylamine is formed by nucleophilic attack of the substrate on DMPO and subsequently oxidized to the respective nitroxide radical. One potential candidate for this artifact is the sulfur trioxide radical adduct (DMPO/(•)SO(3)(-)), as detected in spin-trapping experiments with horseradish peroxidase and sulfite. It has previously been shown by NMR experiments that the hydroxylamine intermediate does indeed form, but no direct proof for the ESR artifact has been provided. Here, we used isotopically labeled DMPO with horseradish peroxidase and ferricyanide to test for the Forrester-Hepburn artifact directly in a spin-trapping experiment. Besides sulfite, we investigated other nucleophiles such as cyanide, cysteine, and glutathione. Neither sulfite nor biological thiols produced detectable spin-trapping artifacts, but with cyanide the relatively weak signal originated entirely from the nucleophilic reaction. The hydroxylamine intermediate, which is more abundant with cyanide than with sulfite, was identified as cyano-hydroxylamine by means of 2D NMR experiments. Although our study found that spin trapping provided authentic free radical signals with most of the substrates, the occurrence of the Forrester-Hepburn mechanism artifact with cyanide emphasizes the importance of isotope measurements with nucleophile substrates.
氮氧自由基捕获剂如 5,5-二甲基-1-吡咯啉-N-氧化物 (DMPO) 常用于自由基检测。虽然有确凿的实例，但仍必须考虑到假像的形成。例如，福雷斯特-赫本（Forrester-Hepburn）机制产生的自由基加合物与真正的自由基捕获形成的加合物相同。底物对 DMPO 的亲核攻击形成羟胺，随后羟胺被氧化为相应的氮氧自由基。这种假像的一个潜在候选物是三氧化硫自由基加合物（DMPO/(•)SO(3)(-)），它在辣根过氧化物酶和亚硫酸盐的自旋捕获实验中被检测到。以前的 NMR 实验表明，确实形成了羟胺中间体，但没有提供 ESR 假像的直接证据。在这里，我们使用辣根过氧化物酶和铁氰化物与同位素标记的 DMPO 直接在自旋捕获实验中测试福雷斯特-赫本假像。除了亚硫酸盐，我们还研究了其他亲核试剂，如氰化物、半胱氨酸和谷胱甘肽。亚硫酸盐和生物硫醇都没有产生可检测的自旋捕获假像，但氰化物的相对较弱的信号完全来自亲核反应。羟胺中间体与氰化物的丰度比与亚硫酸盐的丰度高，通过二维 NMR 实验确定为氰基羟胺。尽管我们的研究发现，自旋捕获为大多数底物提供了真实的自由基信号，但氰化物的福雷斯特-赫本机制假像的发生强调了亲核试剂底物同位素测量的重要性。
