Advanced IMAC Methodology

先进的 IMAC 方法论

• 批准号: 9726721
• 负责人: Jerker Porath
• 金额: $ 31万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9726721&HistoricalAwards=false
• 关键词: Advanced; IMAC; Methodology

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.

F97摘要-分子生物化学Jerker Porath/Roberto Guzman MCB 9726721 1.技术 本研究的目的是探索和改进吸附/解吸方法，这是基于相分布，因为它们是由蛋白质和金属离子之间的相互作用固定在凝胶，如交联琼脂糖。与离子交换相比，固定化金属离子亲和（IMA）原理目前尚不发达。目前只有两种简单的螯合配体（亚氨基二乙酸酯（IDA）和次氮基三乙酸酯（NTA））是常用的。他们只满足适度的要求。 本研究中使用的固定化硬和软金属离子提供了明显的优势，因为它们可以作为具有碳水化合物特异性、磷酸盐选择性等更高复杂性的亲和吸附剂的核。 随后将尝试置换和载体置换色谱法，用酶降解葡聚糖凝胶衍生物获得一种新的亲和置换剂。 目前的研究主要集中在蛋白质色谱凝胶取代配体形成螯合物与铁，锰，钙和镧系元素。合成基于交联琼脂糖的吸附剂以满足在这种硬固定化金属离子上进行蛋白质层析的要求。相互作用的配体应含有金属配位氧原子。羧甲基化胺、8-羟基喹啉和水杨醛的缩合氨基衍生物被选为此类配体的典型实例。Mannich和Michael缩合反应用于将芳族官能团引入到凝胶结合的氨基配体中。含有琼脂糖和聚乙烯酰亚胺的合成混合凝胶用于交联，在存在和不存在金属离子的情况下，试图增加金属离子的选择性。通过测定色谱床的选择性、容量和流体动力学特性（流速-压力关系）来评估吸收剂的质量。亲和置换色谱是以螯合剂作为置换剂而发展起来的。不同强度的可溶性螯合剂用作待纯化物质的金属相互作用位点（吸收剂的亲和中心）的竞争者，并且螯合剂结合到各种分子大小的载体聚合物上作为间隔置换剂。 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，优先考虑测试牛脉络丛中存在的铁和锰结合蛋白的分离-一种被认为可以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色谱法）是一种色谱方法，利用样品介质中的溶质与固定在固体颗粒支持物上的金属离子之间的强亲和力。 迄今为止，只有少数金属离子如锌、铜和镍对蛋白质中的组氨酸侧链具有特异性亲和力。 本研究的目的是拓宽IMAC的领域，包括其他金属和蛋白质中的其他亲和基团。初步研究表明，磷酸盐和羧酸盐基团是固定的“硬”金属离子如三价铁和钙的亲和靶。磷蛋白和钙结合蛋白是具有深刻生物学意义的物质类别。在这项研究中，开发了这些种类的蛋白质的组选择性IMAC方法，特别是合成的亲和洗脱吸附剂的帮助下，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者。 锰是一种可能显示钙和铁之间的色谱蛋白亲和行为的金属。使用含有固定铁和/或镧系元素如铕的吸附剂，从牛脉络丛中分离锰结合蛋白正在进行测试。 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意义。