Advanced IMAC Methodology

F97摘要-分子生物化学Jerker Porath/Roberto Guzman MCB 9726721 1.这项研究旨在探索和改进基于相分布的吸附/解吸方法，因为它们是由固定在凝胶中的蛋白质和诸如交联琼脂糖凝胶中的金属离子之间的相互作用所支配的。与离子交换相比，固定化金属离子亲和力(IMA)原理目前还不发达。目前只有两种简单的螯合配体(亚氨基二乙酸酯(IDA)和三乙酸硝胺(NTA))被广泛使用。他们只能满足适度的要求。本研究中使用的固定化硬、软金属离子具有明显的优势，因为它们可以作为具有碳水化合物专一性、磷酸盐选择性等更高复杂性的高阶亲和吸附剂的核。置换和载体置换层析将在稍后尝试使用一种通过酶降解葡聚糖凝胶衍生物获得的新型亲和置换剂。目前的研究主要集中在用与铁、锰、钙和稀土形成络合物的配体取代的凝胶上的蛋白质层析。为了满足蛋白质在硬固定化金属离子上的层析要求，合成了基于交联琼脂糖基的吸附剂。相互作用的配体应该含有金属配位的氧原子。羧甲基胺、8-羟基喹啉的缩氨基衍生物和水杨醛被选为此类配体的典型例子。Mannich和Michael缩合反应用于在凝胶结合的氨基配体中引入芳香官能团。在存在和不存在金属离子的情况下，含有琼脂糖和聚乙烯酰亚胺的合成杂化凝胶用于交联剂，试图提高金属离子的选择性。通过测定色谱床的选择性、容量和流体力学性质(流速-压力关系)来评估吸附剂的质量。以螯合剂为置换剂，发展了亲和置换层析。不同强度的可溶性螯合剂被用作金属相互作用部位(吸附剂的亲和中心)的竞争对手，而与不同分子大小的载体聚合物结合的螯合剂则作为间隔物置换。这些研究旨在确定分离各种金属结合蛋白和磷蛋白的能力，重点是测试牛脉络丛中存在的铁和锰结合蛋白的分离。牛脉络丛被认为是防止有毒金属离子进入大脑的器官。2.非技术IMAC(固定化金属离子亲和层析)是一种利用样品介质中的溶质与固定在固体颗粒载体上的金属离子之间的强亲和力的层析方法。只有少数金属离子，如锌、铜和镍，对迄今使用的蛋白质中的组氨酸侧链具有特定的亲和力。这项研究的目的是扩大IMAC的研究领域，以包括蛋白质中的其他金属和其他亲和基团。初步研究表明，磷酸基团和羧酸基是固定的“硬”金属离子，如三阳离子铁和钙的亲和靶标。磷蛋白和钙结合蛋白是一类具有重要生物学意义的物质。在本研究中，利用专门合成的亲和洗脱吸附剂，建立了对这类蛋白质进行基团选择性IMAC的方法，从而最大限度地提高了分辨率。不同分子大小和结构的可溶性螯合剂被用作结合的竞争对手。锰是一种可能在钙和铁之间表现出蛋白质亲和行为的金属。使用含有固定铁和/或稀土元素(如Eu)的吸附剂，正在测试从牛脉络丛中分离锰结合蛋白。这些金属结合蛋白可能是保护大脑免受重金属离子损伤的载体，了解它们的性质具有重要的生理学意义。

F97 Abstract - Molecular Biochemistry Jerker Porath/Roberto Guzman MCB 9726721 1. Technical This study is intended to explore and improve adsorption/desorption methods which are based on phase distributions as they are governed by the interactions between proteins and metal ions immobilized in gels such as cross-linked agarose. The Immobilized Metal ion Affinity (IMA) principle is presently underdeveloped in comparison to Ion Exchange. Only two simple chelating ligands (imino diacetate (IDA) and nitrilo triacetate (NTA)) are presently in common use. They meet only modest demands. Immobilized hard and soft metal ions used in this study offer distinct advantages, since they can serve as nuclei for affinity adsorbents of higher order of complexity with carbohydrate specificity, phosphate selectivity and more. Displacement and carrier displacement chromatography will be attempted later with a new kind of affinity displacers obtained by enzymatic degradation of dextran gel derivatives. Present studies are focused on protein chromatography on gels substituted with ligands that form chelates with iron, manganese, calcium and lanthanides. Adsorbents based on cross-linked agarose are synthesized to meet requirements for chromatography of proteins on hard immobilized metal ions of this kind. The interactive ligands should contain metal coordinating oxygen atoms. Carboxymethylated amines, condensed amino derivatives of 8-hydroxyquinoline and salicyl-aldehyde are selected as typical examples of such ligands. Mannich and Michael condensation reactions are used to introduce the aromatic functions into the gel-bound amino ligands. Synthetic hybrid gels containing agarose and polyethylene imide are used in cross-links, in the presence and absence of metal ions, in attempts to increase metal ion selectivity. The quality of the absorbents is assessed by the determination of selectivity, capacity and the hydrodynamic properties of chromatographic beds (flow-rate pressure relationships). Affinity displacement chromatography are developed with chelators as displacers. Soluble chelators of different strength are used as competitors to the substances to be purified for the metal interaction sites (the affinity centers of the absorbent), and chelators bound to carrier polymers of various molecular size as spacer displacers. The studies are aimed at determining the capacity for isolation of various metal binding proteins and phosphoproteins, with priority set to test the separation of iron and manganese binding proteins present in bovine choroid plexus - an organ believed to prevent toxic metal ions from entering the brain. 2. Non-Technical IMAC (Immobilized Metal Ion Affinity Chromatography) is a chromatographic method that utilizes strong affinity between solutes in the sample medium and metal ions fixed to a solid, granular support. Only a few metal ions such as zinc, copper and nickel that have specific affinity for histidine side chains in the proteins that have so far been used. This study is aimed to broaden the field of IMAC to include other metals and other affinity groups in proteins. Preliminary studies have shown that phosphate and carboxylate groups are affinity targets for immobilized "hard" metal ions such as tripositive iron and calcium. Phosphoproteins and calcium binding proteins are substance categories of profound biological importance. Group selective IMAC methods for these kinds of proteins are developed in this study with the aid of specifically synthesized adsorbents for affinity elution, maximizing the resolving power. Soluble chelators of different molecular size and structure are used as competitors for binding. Manganese is a metal likely to show chromatographic protein affinity behavior between calcium and iron. The isolation of manganese bound proteins from bovine choroid plexus is being tested, using adsorbents containing fixed iron and/or lanthanides such as europium. These metal- bindi ng proteins are presumably the vehicles for protecting the brain from heavy metal ion damage, and the understanding of their properties is physiologically important.