Protein Derived Radicals

过氧化氢对组织的氧化损伤已经成为一个反复出现的主题，作为诱导各种医疗条件的机制，包括心肌缺血、癌症、炎症和衰老。蛋白质是这种损伤的目标。大多数含血红素和一些含铜的蛋白质具有过氧化物酶活性，可对多种生物分子造成氧化损伤，包括其自身和膜脂。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/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）的自旋陷阱/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。这种加合物可以用Western blot技术可视化，灵敏度比ESR高10倍。Western blot分析表明，肌红蛋白在大鼠心脏上清液中特异性形成蛋白源性自由基。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，红细胞内检测到血红蛋白衍生的自由基。发现了一种新的组氨酸自由基的质谱小组确定了血红蛋白上自由基形成的位置。

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.