Bhushan R, Mahesh V K, Mallikharjun P V
Department of Chemistry, University of Roorkee, India.
Int J Pept Protein Res. 1990 Nov;36(5):445-9. doi: 10.1111/j.1399-3011.1990.tb01304.x.
A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.
从油菜籽高分子量蛋白质中分离并纯化出一个亚基（分子量15,600）；确定了其纯度和均一性。用溴化氰、胰蛋白酶、糜蛋白酶和金黄色葡萄球菌V8蛋白酶对该亚基进行切割。通过聚丙烯酰胺凝胶电泳、凝胶过滤、Dowex 1 x 2柱色谱和纸电泳对片段进行分离和纯化。测定了完整亚基以及酶切和化学切割得到的不同片段的氨基酸组成。通过手动埃德曼法对亚基及其片段进行测序。通过薄层色谱和紫外光谱对每一步后得到的苯硫代乙内酰脲氨基酸进行鉴定。通过重叠法确定了由125个氨基酸残基组成的亚基的完整氨基酸序列。
