Protein Derived Radicals

过氧化氢对组织的氧化损伤已成为一个反复出现的主题，作为诱导各种医学病症（包括心肌缺血、癌症、炎症和衰老）的机制。蛋白质是这种损害的目标。大多数含血红素蛋白和一些含铜蛋白具有过氧化物酶活性，可对多种生物分子（包括自身和膜脂质）造成氧化损伤。我们现在正在研究一系列由肌红蛋白、血红蛋白和超氧化物歧化酶形成的蛋白质衍生自由基，这些自由基利用我们新的免疫学/自旋捕获技术。多克隆抗体特异性结合5，5-二甲基-1-吡咯啉N-氧化物（DMPO）的自旋捕获/蛋白质加合物。肌红蛋白-DMPO自由基的ESR信号在几分钟内衰减，但是可以使用质谱检测到持久的加合物。这种加合物可以使用蛋白质印迹技术以比ESR高10倍的灵敏度可视化。蛋白质印迹分析表明，肌红蛋白特异性地形成一个蛋白质衍生的自由基在大鼠心脏上清液。我们第一次能够证明细胞内自由基的形成。当红细胞暴露于50微摩尔过氧化氢时，在红细胞内检测到血红蛋白衍生的自由基。血红蛋白上自由基形成的位点已经被质谱组确定，他们发现了一种新的组氨酸自由基。

Oxidative damage to tissues by hydrogen peroxide has become a recurring theme as a mechanism for the induction of a variety of medical conditions including myocardial ischemia, cancer, inflammation, and aging. Proteins are a target of such damage. Most heme-containing and some copper-containing proteins have peroxidase activity that can cause oxidative damage to a variety of biological molecules including itself and membrane lipids. We are now in the process of investigating a series of protein-derived radicals formed by myoglobin, hemoglobin and superoxide dismutase that utilize our new immunological/spin-trapping techniques. Polyclonal antibodies bind specifically to the spin trap/protein adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The ESR signal of the myoglobin-DMPO radical decays within minutes, however a persistent adduct can be detected using mass spectroscopy. This adduct can be visualized using Western blot techniques at a sensitivity 10-fold greater than ESR. Western blot analysis demonstrates that myoglobin specifically forms a protein-derived radical in rat heart supernatant. For the first time, we were able to demonstrate the formation of free radicals inside a cell. When red blood cells were exposed to 50-micromolar hydrogen peroxide, hemoglobin-derived radicals were detected inside the red blood cells. The sites of free radical formation on hemoglobin have been determined by the mass spectroscopy group who discovered a novel histidine free radical.