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。大多数含血红素的蛋白质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的蛋白质衍生的自由基形成的肌红蛋白，血红蛋白和细胞色素c氧化酶，利用我们新的免疫自旋捕获技术。我们的实验室已经产生了多克隆抗体，其特异性地结合到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。用半抗原5，5-二甲基-2-辛酸-1-吡咯啉N-氧化物（OA-DMPO）免疫家兔，制备了抗OA-DMPO的抗体。OA-DMPO是DMPO的结构类似物，具有终止于羧基的额外碳链用于酰胺连接。通过碳二亚胺法将OA-DMPO与鸡蛋白蛋白反应以进行酰胺化来合成抗原。采用ELISA技术测定兔血清的滴度。通过芬顿化学在BSA上产生的蛋白质-DMPO硝酮加合物和由于过氧化氢自身过氧化而在高铁肌红蛋白上产生的加合物，筛选出的多克隆抗体呈阳性。

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 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 cytochrome c oxidase that utilize our new immunological-spin trapping techniques. Our laboratory has produced polyclonal antibodies that bind specifically to the spin trap/protein adduct of 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The antibodies were produced in rabbits immunized against the hapten, 5,5-dimethyl-2-octanoic acid-1-pyrroline N-oxide (OA-DMPO). OA-DMPO is a structural analog of DMPO with an additional carbon chain terminating in a carboxyl-group for amide linking. Antigens were synthesized by reacting OA-DMPO with chicken egg albumin via the carbodiimide method for amidation. Titer of the rabbit serum was determined using ELISA techniques. The polyclonal antibodies screened positive for protein-DMPO nitrone adducts produced on BSA via Fenton chemistry and adducts produced on metmyoglobin as a result of self-peroxidation by hydrogen peroxide.