Identification of free radicals on hemoglobin from its self-peroxidation using mass spectrometry and immuno-spin trapping - Observation of a histidinyl radical
Identification of free radicals on hemoglobin from its self-peroxidation using mass spectrometry and immuno-spin trapping - Observation of a histidinyl radical
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DOI：
10.1074/jbc.m310704200
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发表时间：
2004-03-19
影响因子：
4.8
通讯作者：
Tomer, KB
中科院分区：
文献类型：
--
作者：
Deterding, LJ;Ramirez, DC;Tomer, KB
为了了解血红蛋白上自由基形成的机制，在自旋陷阱 5,5-二甲基-1-吡咯啉 N-氧化物 (DMPO) 存在下，通过与过氧化氢反应引发血红蛋白上自由基的形成。通过质谱 (MS) 和免疫自旋捕获分析 DMPO 硝酮加合物。然后对自旋捕获的蛋白质加合物进行胰蛋白酶消化和质谱分析。当血红蛋白在 DMPO 存在下与过氧化氢 (H2O2) 反应时，可以通过免疫自旋捕获检测到 DMPO 硝酮加合物。为了验证蛋白质自由基的 DMPO 加合物是否已形成，通过流动注射电喷雾电离质谱 (ESI/MS) 分析反应混合物。血红蛋白/H2O2/DMPO 样品的 ESI 质谱显示血红蛋白的 α 链和 β 链上各有一个加合物，其质量相当于添加一个 DMPO 分子。使用蛋白水解技术探索血红蛋白上形成的自由基的性质，然后进行液相色谱/质谱 (LC/MS) 和串联质谱 (MS/MS) 分析。在血红蛋白上鉴定出以下 DMPO 添加位点：β 链的 Cys-93，以及 α 链的 Tyr-42、Tyr-24 和 His-20。由于 Tyr-24 和 His-20 的 pi-pi 相互作用，未成对电子显然在酪氨酸和组氨酸残基上都离域（pi-pi 堆叠对自由基）。
In an effort to understand the mechanism of radical formation on heme proteins, the formation of radicals on hemoglobin was initiated by reaction with hydrogen peroxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO nitrone adducts were analyzed by mass spectrometry (MS) and immuno-spin trapping. The spin-trapped protein adducts were then subjected to tryptic digestion and MS analyses. When hemoglobin was reacted with hydrogen peroxide (H2O2) in the presence of DMPO, a DMPO nitrone adduct could be detected by immuno-spin trapping. To verify that DMPO adducts of the protein free radicals had been formed, the reaction mixtures were analyzed by flow injection electrospray ionization mass spectrometry (ESI/MS). The ESI mass spectrum of the hemoglobin/H2O2/DMPO sample shows one adduct each on both the alpha chain and the beta chain of hemoglobin which corresponds in mass to the addition of one DMPO molecule. The nature of the radicals formed on hemoglobin was explored using proteolysis techniques followed by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) analyses. The following sites of DMPO addition were identified on hemoglobin: Cys-93 of the beta chain, and Tyr-42, Tyr-24, and His-20 of the alpha chain. Because of the pi-pi interaction of Tyr-24 and His-20, the unpaired electron is apparently delocalized on both the tyrosine and histidine residue (pi-pi stacked pair radical).