Ramirez Dario C, Chen Yeong-Renn, Mason Ronald P
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27713, USA.
Free Radic Biol Med. 2003 Apr 1;34(7):830-9. doi: 10.1016/s0891-5849(02)01437-5.
To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.
为了研究血红蛋白自由基在人氧合血红蛋白（oxyHb）或高铁血红蛋白/H₂O₂系统中的作用，我们采用了一种名为“免疫自旋捕获”的新方法，该方法结合了自旋捕获和抗原：抗体相互作用的特异性和敏感性。此前，我们实验室开发并验证了一种新型兔多克隆抗DMPO硝酮加合物抗血清，它能特异性识别蛋白质自由基衍生的硝酮加合物。在本研究中，通过酶联免疫吸附测定（ELISA）和蛋白质印迹法确定，血红蛋白上硝酮加合物的形成取决于铁血红素的氧化状态、H₂O₂和DMPO的浓度以及时间。还原型谷胱甘肽或L-抗坏血酸的存在以剂量依赖的方式显著降低了高铁血红蛋白上硝酮加合物的水平。为了证实ELISA结果，蛋白质印迹分析表明只有完整系统（oxy-或metHb/DMPO/H₂O₂）产生抗血清识别的表位。碘化对高铁血红蛋白上酪氨酸残基的特异性修饰几乎消除了抗体结合，而半胱氨酸残基的硫醇化导致硝酮加合物量有小幅但可重复的减少。这些发现强烈表明酪氨酸残基是免疫化学可检测的血红蛋白自由基衍生硝酮加合物的形成位点。此外，我们能够证明在暴露于H₂O₂和DMPO的红细胞内存在血红蛋白自由基衍生的硝酮加合物。总之，我们的新方法通过证明在人oxyHb/metHb中以比电子顺磁共振（EPR）更低的浓度形成酪氨酸自由基衍生的硝酮加合物，并检测暴露于H₂O₂的红细胞内的自由基，显示出相对于用抗DMPO硝酮加合物抗血清进行EPR自旋捕获的几个优势。
