Dalle-Donne Isabella, Rossi Ranieri, Giustarini Daniela, Milzani Aldo, Colombo Roberto
Department of Biology, University of Milan, Italy.
Clin Chim Acta. 2003 Mar;329(1-2):23-38. doi: 10.1016/s0009-8981(03)00003-2.
Oxidative stress, an imbalance toward the pro-oxidant side of the pro-oxidant/antioxidant homeostasis, occurs in several human diseases. Among these diseases are those in which high levels of protein carbonyl (CO) groups have been observed, including Alzheimer's disease (AD), rheumatoid arthritis, diabetes, sepsis, chronic renal failure, and respiratory distress syndrome. What relationships might be among high level of protein CO groups, oxidative stress, and diseases remain uncertain.The usage of protein CO groups as biomarkers of oxidative stress has some advantages in comparison with the measurement of other oxidation products because of the relative early formation and the relative stability of carbonylated proteins. Most of the assays for detection of protein CO groups involve derivatisation of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl (DNP) hydrazone product. This then can be detected by various means, such as spectrophotometric assay, enzyme-linked immunosorbent assay (ELISA), and one-dimensional or two-dimensional electrophoresis followed by Western blot immunoassay. At present, the measurement of protein CO groups after their derivatisation with DNPH is the most widely utilized measure of protein oxidation.
氧化应激是指促氧化剂/抗氧化剂稳态向促氧化剂一侧失衡，在多种人类疾病中都会出现。在这些疾病中，包括阿尔茨海默病（AD）、类风湿性关节炎、糖尿病、败血症、慢性肾衰竭和呼吸窘迫综合征等，都观察到了高水平的蛋白质羰基（CO）基团。蛋白质CO基团水平升高、氧化应激和疾病之间可能存在何种关系仍不确定。与其他氧化产物的检测相比，将蛋白质CO基团用作氧化应激的生物标志物具有一些优势，这是因为羰基化蛋白质形成相对较早且相对稳定。大多数检测蛋白质CO基团的方法都涉及用2,4-二硝基苯肼（DNPH）对羰基进行衍生化，这会导致形成稳定的二硝基苯基（DNP）腙产物。然后可以通过各种方法检测该产物，如分光光度法、酶联免疫吸附测定（ELISA）以及一维或二维电泳后进行蛋白质免疫印迹分析。目前，用DNPH对蛋白质进行衍生化后检测蛋白质CO基团是最广泛使用的蛋白质氧化检测方法。
