Edwards J J, Reeder D J, Atha D H
Organic Analytical Research Division, National Institute of Standards and Technology, US Department of Commerce, Gaithersburg, Maryland 20899.
Appl Theor Electrophor. 1990;1(4):207-12.
Two-dimensional gel electrophoresis (2DGE) and image processing were used to quantify protein and carbohydrate heterogeneity in human plasma fibronectin (FN) and its enzymatically produced domains. After a 30 minute thermolysin digest of FN, the domains were identified in 2DGE by their known isoelectric points and molecular weights, which were compared to domain standards purified by hydroxyapatite, gel exclusion and heparin-Sepharose chromatography. Three individual species were observed in the cell binding domain which may correspond to the heterogeneity known to result from alternative splicings of the fibronectin gene. In addition, the carbohydrate heterogeneity in the gelatin binding domain was analyzed by 2DGE and isoelectric focusing (IEF) before and after treatment with N-glycanase and neuraminidase to remove selected carbohydrate moieties. Five individual species which differ in carbohydrate structure were observed. The results also indicate a carbohydrate dependent stabilization of the gelatin binding domain with respect to proteolytic digestion.
二维凝胶电泳（2DGE）和图像处理技术被用于量化人血浆纤连蛋白（FN）及其酶促产生的结构域中的蛋白质和碳水化合物异质性。在对FN进行30分钟的嗜热菌蛋白酶消化后，通过已知的等电点和分子量在2DGE中鉴定这些结构域，并将其与通过羟基磷灰石、凝胶排阻和肝素-琼脂糖层析纯化的结构域标准品进行比较。在细胞结合结构域中观察到三种不同的种类，这可能与纤连蛋白基因可变剪接导致的异质性相对应。此外，在用N-聚糖酶和神经氨酸酶处理以去除选定的碳水化合物部分之前和之后，通过2DGE和等电聚焦（IEF）分析明胶结合结构域中的碳水化合物异质性。观察到五种碳水化合物结构不同的种类。结果还表明，明胶结合结构域在蛋白水解消化方面存在碳水化合物依赖性稳定性。
