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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

固定化金属亲和色谱法（IMAC）在蛋白质分离纯化中得到了广泛的应用，但一直存在负载金属泄漏的问题。本文试图研制一种新型的不存在这一问题的IMAC凝胶。开发了两种新型配体的IMAC凝胶：含聚氨基羧酸的凝胶和含卟啉环的凝胶。将三价铬、钴和铝固定在聚氨基羧酸凝胶上，由于配体对水合水分子的取代非常缓慢，因此固定金属离子的泄漏可以忽略不计。然而，与预期相反的是，无论是乙二胺三乙酸凝胶还是三乙基四胺五乙酸凝胶，铜的泄漏程度都与亚氨基二乙酸凝胶几乎相同。另一方面，铬和铝在这些凝胶上的固定化程度可以忽略不计，钴的泄漏也比铜少得多。考察了pH和盐浓度对三乙酸乙二胺凝胶吸附α -凝乳胰蛋白酶、溶菌酶和白蛋白的影响。溶菌酶对各种金属的选择性吸收效果最好。根据固定化金属的种类不同，溶菌酶在pH = 5 ~ 8之间的吸附量最大，说明固定化金属与蛋白质之间具有特殊的亲和力。在溶菌酶的低浓度区，盐对溶菌酶的吸收有明显的影响，说明溶菌酶的吸收在很大程度上受静电作用的控制。在表面用乙二胺进行化学修饰的琼脂糖凝胶上，将血卟啉与锌、钴或镍配合物缩合成卟啉型凝胶。考察了pH和盐浓度对白蛋白和α -凝乳胰蛋白酶在凝胶上吸附的影响，发现除了对固定金属具有特殊的亲和力外，静电相互作用对吸附也有很大影响。少