Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan.
Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Koto, Tokyo 135-0064, Japan.
J Chromatogr A. 2022 Aug 2;1676:463277. doi: 10.1016/j.chroma.2022.463277. Epub 2022 Jun 24.
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.
固定化金属离子亲和层析（IMAC）在从各种宿主中纯化组氨酸标记或富含组氨酸的蛋白质和肽方面非常有用。然而，包括多酚在内的酚类化合物由于与固定在柱树脂上的过渡金属具有高亲和力，会干扰 IMAC，从而阻碍了从植物宿主系统中纯化蛋白质。与溶液中酚类化合物与过渡金属离子相互作用的机制的广泛知识相比，对于固定在树脂上的过渡金属离子的柱上相互作用的理解仍然难以捉摸。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)、Cu(II)和 Ni(II)）在各种 IMAC 上，系统地研究了酚类化合物对过渡金属离子的亲和力，其中柱是通过将阳离子交换柱与过渡金属溶液平衡来制备的。结果发现，芳香族化合物中 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焦没食子酸与 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，与 Fe(II)-和 Cu(II)-IMAC 柱相比，酚类化合物对 Ni(II)-IMAC 柱没有明显的亲和力，而咪唑和组氨酸标记的蛋白质对 Ni(II)-IMAC 柱有明显的结合。因此，Ni(II)-IMAC 应该特别有效地从含有酚类化合物的系统中分离组氨酸标记和富含组氨酸的物质。这些结果表明，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。它们还为设计从含有酚类化合物的样品中纯化目标蛋白质和肽的 IMAC 策略提供了全面的视角。
