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

固定化金属亲和色谱（Immobilized metal affinity chromatography，IMAC）已广泛应用于蛋白质的分离和纯化，但存在负载金属的泄漏问题。在目前的工作中，新的IMAC凝胶从这个问题的自由试图被开发。研制了两种含新型配体的IMAC凝胶：1）含聚氨基羧酸的IMAC凝胶和2）含卟啉环的IMAC凝胶。三价铬、钴和铝被固定在聚氨基羧酸凝胶上，因为配体取代水合水分子非常慢，因此，预计固定的金属离子的泄漏可以忽略不计。然而，与预期相反，不仅从乙二胺三乙酸凝胶而且从三乙基四胺五乙酸凝胶观察到几乎相同程度的铜从亚氨基二乙酸型凝胶的泄漏。另一方面，铬和铝的泄漏， ...更多信息 固定在这些凝胶上的钴的量小得可以忽略，并且钴的量也远小于铜的量。研究了pH和盐浓度对固定化金属离子的乙二胺三乙酸凝胶吸附α-糜蛋白酶、溶菌酶和白蛋白的影响。溶菌酶对各种金属离子的吸附选择性最强。溶菌酶的最大吸附在pH = 5和8之间，这取决于固定化金属的种类，这表明固定化金属与蛋白质之间的特殊亲和力。溶菌酶在低浓度区的吸附受盐的影响较大，说明溶菌酶的吸附主要受静电作用的控制。将锌、钴或镍与血卟啉的配合物缩合在表面用乙二胺修饰的琼脂糖凝胶上，制备了卟啉型凝胶。研究了pH和盐浓度对Alubmin和α-糜蛋白酶在这些凝胶上吸附的影响，发现静电相互作用对吸附有很大影响，而且这些凝胶对固定化金属也有特殊的亲和力。少

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