Singh V, Kar S K
Institute of Self-Organising Systems and Biophysics, North-Eastern Hill University, Shillong, Meghalaya, India.
Indian J Biochem Biophys. 1992 Feb;29(1):31-41.
Ribosome-inactivating protein, gelonin, isolated from an Indian plant Gelonium multiflorum of Euphorbiaceae family has been used to design and synthesize immunotoxins and hormonotoxins for selective targeting purposes. Since gelonin isolated by aqueous extraction, cation-exchange chromatography and gel-filtration chromatography (Method I), contains non-proteinous material absorbing at 280 nm, the ammonium sulphate precipitation method (Method II) and Cibacron blue affinity chromatography method. (Method III) have been used to purify gelonin from the dry seeds. Three batches of gelonin purified by each method were prepared and subjected to extensive physico-chemical and immunochemical characterization. The molecular weight was determined by gel-filtration chromatography on a pre-calibrated Sephadex G-100, TSK-G4000 TW on HPLC or Superose-12 on fast protein liquid chromatography. In all cases, the molecular weight was approximately 30,000Da. The SDS-PAGE also revealed a homogeneous protein of 30kDa molecular weight. In Method II, the non-proteinous material which binds to CMC-gel in association of gelonin was substantially removed during ammonium sulphate fractionation. A careful analysis clearly revealed that Method II, although yielded low protein, gave gelonin devoid of the non-proteinous material. The SPDP modification of epsilon-NH2 groups of gelonin obtained from Methods I, II, and III was also carried out and its effect on immunoreactivity was studied.
核糖体失活蛋白——去甲基金缕梅毒素，是从大戟科印度植物多花金缕梅中分离得到的，已被用于设计和合成免疫毒素及激素毒素，以实现选择性靶向作用。由于通过水提取、阳离子交换色谱法和凝胶过滤色谱法（方法一）分离得到的去甲基金缕梅毒素含有在280nm处有吸收的非蛋白质物质，因此采用硫酸铵沉淀法（方法二）和汽巴克隆蓝亲和色谱法（方法三）从干燥种子中纯化去甲基金缕梅毒素。通过每种方法制备了三批纯化的去甲基金缕梅毒素，并对其进行了广泛的物理化学和免疫化学表征。分子量通过在预先校准的葡聚糖凝胶G - 100、高效液相色谱上的TSK - G4000 TW或快速蛋白质液相色谱上的Superose - 12上进行凝胶过滤色谱法测定。在所有情况下，分子量约为30,000Da。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳也显示出一种分子量为30kDa的均一蛋白质。在方法二中，与去甲基金缕梅毒素结合的与羧甲基纤维素凝胶结合的非蛋白质物质在硫酸铵分级分离过程中被大量去除。仔细分析清楚地表明，方法二虽然蛋白质产量低，但得到的去甲基金缕梅毒素不含非蛋白质物质。还对从方法一、二和三中获得的去甲基金缕梅毒素的ε - NH2基团进行了SPDP修饰，并研究了其对免疫反应性的影响。
