Fundamental investigation for the development of novel types of adsorption gels for immobilized metal affinity Chromatography

新型固定化金属亲和色谱吸附凝胶开发的基础研究

基本信息

批准号：
03650605
负责人：
INOUE Katsutoshi
金额：
$ 1.34万
依托单位：
Saga University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

Immobilized metal affinity chromatography (IMAC) has been in extensive use for separation and purification of proteins, but has been suffering from leakage of loaded metals. In the present work, novel IMAC gel free from this problem was tried to be developed. Two types of IMAC gels containing new ligands to coordinate or immobilize metal ions were developed: 1) the gel containing polyaminocarboxylic acids and 2) that containing porphyrin ring. Trivalent chromium, cobalt and aluminum was immobilized on the polyaminocarboxylic acid gel since the ligand substitution for hydrated water molecules are very slow and, consequently, the leakage of the immobilized metal ions is expected to be negligible. However, contrary to the expectation, nearly same extent of the leakage of copper with from iminodiacetic acid type of gel was observed not only from ethylenediamine triacetic acid gel but also from triethyltetramine pentaacetic acid gel. On the other hand, the leakage of chromium and aluminum i … More mmobilized on these gels were negligibly small, and that of cobalt was also much less than that of copper. The effects of pH and salt concentration on the adsorption of alpha-chymotrypsin, lysozym and alubmin on the ethylenediamine triacetic acid gel immobilized with these metals were examined. Lysozym was most selectively absorbed on every kinds of metals. Maximum adsorption of lysozym was observed between pH = 5 and 8, depending on the kinds of immobilized metal, which suggests the special affinity between the immobilized metal and proteins. Significant salt effect on the absorption of lysozym was observed in its low concentration region, in particular, suggesting that the absorption is greatly controlled by electrostatic interactions.The porphyrin type of gel was prepared by condensating zinc, cobalt or nickel complexes with hematoporphyrin on agarose gel chemically modified with ethylenediamine on its surface. The effects oh pH and salt concentration on the adsorption of alubmin and alpha-chymotrypsin on these gels were examined, and it was found that electrostatic interaction greatly affect on the absorption in addition to the special affinity to the immobilized metal also in these gels. Less

固定的金属亲和力色谱法（IMAC）已广泛用于分离和纯化蛋白质，但一直遭受载荷金属的泄漏。在目前的工作中，新颖的iMac凝胶没有这个问题。开发了两种含有新配体以坐标或固定金属离子的iMac凝胶：1）含有聚氨基羧酸的凝胶和2）含有卟啉环的凝胶。三价铬，钴和铝固定在聚氨基羧酸凝胶上，因为水合水分子的配体取代非常缓慢，因此，预计固定金属离子的泄漏预计可以忽略不计。然而，与预期形成鲜明对比的是，从乙二胺三乙酸三乙酸凝胶中观察到铜的渗漏几乎相同的程度，而且还观察到三乙基四胺五乙酸凝胶。另一方面，铬和铝I的泄漏……在这些凝胶上更易于mmobile，钴的渗漏也忽略不计，而钴的泄漏也远低于铜。研究了pH和盐浓度对α-连晶，溶菌酶和alubmin添加的影响对与这些金属固定固定的乙二胺三乙酸凝胶的影响。溶菌酶最有选择地吸收在各种金属上。根据固定的金属的种类，观察到溶菌酶的最大添加吸收，这表明固定的金属和蛋白质之间的特殊亲和力。特别是在其低浓度区域观察到对溶菌酶的遗憾的显着盐影响，这表明遗憾是通过静电相互作用极大地控制了粘蛋白类型的凝胶类型的凝胶类型，通过将锌，钴或镍复合物与琼脂糖凝胶化学在其表层上化学改性的含量修饰的锌，钴或镍复合物与含量修饰的含量修饰。研究了对这些凝胶的添加Alubmin和α-羟丙烯蛋白的添加作用的作用，并且发现除了对这些凝胶中固定的金属的特殊亲和力外，还发现静电相互作用对抽象产生了很大影响。