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.
