DIFFERENTIAL SUSCEPTIBILITY OF PLASMA-PROTEINS TO OXIDATIVE MODIFICATION - EXAMINATION BY WESTERN-BLOT IMMUNOASSAY
DIFFERENTIAL SUSCEPTIBILITY OF PLASMA-PROTEINS TO OXIDATIVE MODIFICATION - EXAMINATION BY WESTERN-BLOT IMMUNOASSAY
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DOI：
10.1016/0891-5849(94)90169-4
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发表时间：
1994-11-01
影响因子：
7.4
通讯作者：
LEVINE, RL
中科院分区：
文献类型：
--
作者：
SHACTER, E;WILLIAMS, JA;LEVINE, RL
血浆蛋白在体内的各种情况下暴露于氧化剂，例如在组织损伤和炎症期间。在本报告中，通过将整个血浆暴露于金属催化的自由基生成系统并使用新型蛋白质印迹免疫测定法检测氧化（蛋白质羰基），评估了每种主要血浆蛋白对氧化修饰的相对敏感性。用二硝基苯肼衍生蛋白质，通过SDS-凝胶电泳分离，并用抗二硝基苯基团的抗体筛选。可以检测到低至1 pmol的蛋白质相关羰基（100 ng的50 kD蛋白质含有0.5 mol羰基/mol蛋白质）。通过与标准品的共迁移、与特异性抗体的交叉反应性以及血浆与血清的比较来鉴别个体血浆蛋白。使用这种方法，我们发现，血浆纤维蛋白原是更容易氧化修饰相比，其他主要的血浆蛋白，白蛋白，免疫球蛋白和转铁蛋白。结果强调了这种方法用于研究细胞提取物和组织中蛋白质氧化的实用性，并表明氧化对纤维蛋白原功能的影响的实验是值得的。
Plasma proteins are exposed to oxidants in a variety of circumstances in vivo, such as during tissue injury and inflammation. In this report, the relative susceptibility of each of the major plasma proteins to oxidative modification was assessed by exposing whole plasma to a metal-catalyzed radical generating system and detecting oxidation (protein carbonyl groups) using a novel Western blot immunoassay. Proteins were derivitized with dinitrophenylhydrazine, separated by SDS-gel electrophoresis, and screened with antibodies against dinitrophenyl groups. As little as I pmol of protein-associated carbonyls could be detected (100 ng of a 50 kD protein containing 0.5 mol carbonyl/mol protein). Individual plasma proteins were identified by their comigration with standards, crossreactivity with specific antibodies, and by comparison of plasma to serum. Using this approach, we found that plasma fibrinogen was much more susceptible to oxidative modification compared to the other major plasma proteins, albumin, immunoglobulins, and transferrin. The results emphasize the utility of this method for studying oxidation of proteins in cell extracts and tissues and indicate that experiments on the effects of oxidation on fibrinogen function are merited.