A new purification procedure for fumarase based of affinity chromatography. Isolation and characterization of pig-liver fumarase.
A new purification procedure for fumarase based of affinity chromatography. Isolation and characterization of pig-liver fumarase.
复制标题
基于亲和层析的延胡索酸酶的新纯化方法。
DOI：
10.1111/j.1432-1033.1977.tb11756.x
复制
发表时间：
1977
期刊：
影响因子：
--
通讯作者：
L. Kanarek
中科院分区：
文献类型：
--
作者：
S. Beeckmans;L. Kanarek
已开发出一种分离延胡索酸酶的新程序 (EC 4.2.1.2)。它被描述用于纯化猪心和肝酶。均苯四酸已与 Sepharose-4B 共价偶联，并以二氨基丙醇作为间隔基。当将透析后的 0.55 饱和硫酸铵沉淀施加到柱上时，在 Tris-乙酸盐缓冲液（pH 7.3）中，延胡索酸酶保持定量结合。它与一些其他蛋白质一起被天然产物 L-苹果酸竞争洗脱。通过透析从洗出液中除去苹果酸。经过这个高效的纯化步骤后，酶很容易结晶。猪心和猪肝制剂的最终收率均为 67%。发现从两种组织中纯化的延胡索酸酶的比活性是相同的。在十二烷基硫酸中的聚丙烯酰胺凝胶电泳显示出一条对应于48500的亚基分子量的单条带。通过在酸性尿素中的电泳也获得了一条单条带。这种基于生物特异性亲和层析的新程序可以快速、轻松地制备克量的延胡索酸酶。
A new procedure has been developed for the isolation of fumarase (EC 4.2.1.2). It is described for the purification of pigheart and liver enzyme. Pyromellitic acid has been covalently coupled to Sepharose-4B with diaminopropanol as spacer arr. When a dialysed 0.55 saturated ammonium sulphate precipitate is applied to the column, in Tris-acetate buffer, pH 7.3, fumarase remains quantitatively bound. It is eluted by competition, together with a few other proteins, by the natural product L-malate. Malate is removed from the eluate by dialysis. After this highly efficient purification step the enzyme is very easily crystallized. The final yield by 67% for both pig heart and liver preparations. The specific activity of fumarase purified from both tissues is found to be the same. Polyacrylamide gel electrophoresis in dodecylsulphate shows one single band corresponding with a subunit molecular weightof 48500. A single band is also obtained by electrophoresis in acid urea. This new procedure based on biospecific affinity chromatography allows a fast and easy preparation of gram quantities of fumarase.