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

点击翻译按钮获取中文摘要

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