Abstract：
Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals-Fe(II), Cu(II) and Ni(II)-on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.
摘要：
固定化金属离子亲和层析(IMAC)可用于纯化来自多种宿主的组氨酸标记的或富含组氨酸的蛋白质和肽。然而,包括多酚在内的酚类化合物由于对固定在柱状树脂上的过渡金属的高亲和力而干扰IMAC，这阻碍了从基于植物的宿主系统中纯化蛋白质。与对溶液中酚类化合物与过渡金属离子之间相互作用机理的广泛了解相反，了解柱子上的相互作用，过渡金属离子固定在树脂上，仍然难以捉摸。本研究通过改变酚羟基（OH基团）的数量和位置以及使用不同的过渡金属-Fe（II），系统地研究了酚类化合物对过渡金属离子的亲和力，Cu(II)和Ni(II)-在各种IMAC上，其中色谱柱是通过用过渡金属溶液平衡阳离子交换色谱柱来制造的。发现芳香族化合物具有的OH基团越多，对过渡金属离子的亲和力越高；特别是，没食子酸甲酯和邻苯三酚与IMAC柱永久结合，这反映了与过渡金属离子的配位键形成。重要的是,酚类化合物对Ni(II)-IMAC柱无明显亲和力,与Fe(II)-和Cu(II)-IMAC柱相反，而咪唑和组氨酸标记的蛋白质显示出与Ni（II）-IMAC柱的明显结合。因此,Ni(II)-IMAC在从含酚类化合物的体系中分离组氨酸标记的和富含组氨酸的物质方面应该是特别有效的。这些结果表明，色谱柱上酚类化合物与过渡金属离子之间的亲和力与溶液中的结果一致。它们还提供了设计策略以改善来自含有酚类化合物的样品的靶蛋白和肽的IMAC纯化的综合观点。
