Immobilized metal-chelate affinity

Serafica, G. C., Pimbley, J., and Belfort, G. (1994). Protein fractionation using fast flow immobilized metal chelate affinity membranes. Biotechnol. Bioeng. 43, 21-36. 

Burba, P., Jakubowski, B., Kuckuk, R., Kiillmer, K., and Heumann, K. G., Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron (lll)-loaded ion exchangers, Fresenius J. Anal Chem., 368, 689-696, 2000. 

Hynek, R. Kozak, A. Drab, V. Sajdok, J. Kas, J. Charecterization of Casein phosphopeptides in cheese extracts using a combination of immobilized metal chelate affinity and leversed-phase high-performance liquid chromatography. Adv. Food Sci. (CMTL) 1999, 21 (5/6), 192. 

Epoxy resins with alternative spacer group chemistries have been used for the preparation of resins for immobilized metal-chelate affinity chromatography (IMAC). The following materials have been synthesized [56] ... 

Metal-chelate affinity chromatography is a powerful purification technique whereby proteins or other molecules can be separated based upon their ability to form coordination complexes with immobilized metal ions (Porath et al., 1975 Lonnerdal and Keen, 1982 Porath and Belew, 1983 Porath and Olin, 1983 Sulkowski, 1985 Kagedal, 1989). The metal ions are stabilized on a matrix through the use of chelating compounds which usually have multivalent points of interaction with the metal atoms. To form useful affinity supports, these metal ion complexes must have some free or weakly associated and exchangeable coordination sites. These exchangeable sites then can form complexes with coordination sites on proteins or other molecules. Substances that are able to interact with the immobilized metals will bind and be retained on... 

Figure 6.17 Schematic representation of the basic principles of metal chelate affinity chromatography. Certain proteins are retained on the column via the formation of coordinate bonds with the immobilized metal ion (a). The actual structure of the most commonly used metal chelator, iminodiacetic acid, is presented in (b)...

Metal-chelate affinity chromatography (Immobilized-metal (Ion) affinity chromatography)... 

In 1970s, first application of metal-chelate affinity chromatography which is later named as "immobilized-metal (ion) affinity chromatography (IMAC) was perfomed. Metal-chelate chromatography technique exploits selective interactions and affinity between transition metal immobilized on a solid support (resin) via a metal chelator and amino acid residues which act as electron donors in the protein of interest [25-26]. As well as aromatic and heterocyclic compounds, proteins such as histidine, tyrosine, tyriptophane and phenylalanine posses affinity to transition metals which form complexes with compounds rich in electrons [25,27]. 

The protein-chemical characterisation revealed a homotetrameric enzyme with a molecular mass of 362 kDa, an isoelectric point of pi 6.16 and a blocked N-terminus in Edman degradation studies [353], In biochemical studies we investigated the effect of free and chelated first-row transition metal ions (Cu2+, Ni2+, Zn2+ and Co2+) on SuSy activity [355]. Further experiments on the binding behaviour of SuSy in immobilized metal ion affinity chromatography (I MAC) gave an insight into the topography of sucrose synthase from rice... 

Smith, M. C., Furman, T. C., Ingolia, T. D., and Pidgeon, C. (1988) Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins. J. Biol. Chem. 263(15), 7211-7215. 

Metal chelate affinity chromatography is influenced by several parameters such as the nature of immobilized chelating group, the immobilized metal ion, the pH environment as well as the ionic strength. 

There are several types of affinity chromatography. Two typical types, immobilized dye chromatography and metal chelate affinity chromatography, are described. 

Metal chelate affinity chromatography is a kind of separation method which has, as a ligand, a metal ion. Some proteins and peptides are purified on the basis of affinity for metal ions immobilized by chelation on the adsorbents. Histidine and cysteine form complexes with the chelated metals around neutral pH. Biological proteins include many histidines as well as recombinant proteins as polyhistidine fusions for instance, His-tag proteins have a specific metal chelate affinity. The adsorbent is prepared by coupling a metal chelate ligand with an iminodiacetic acid group, which forms a chelate with divalent metal ions such as Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, Fe2+, etc. 

Immobilized metal ion affinity chromatography (IMAC) rehes on the ability of certain amino acid side chains to form coordinate bonds with immobilized metal ion complexes [6,181-186]. The adsorption of proteins mainly takes place through interactions with the imidazole ring of histidines. For other amino acids with electron donor atoms in their side chains, binding tends to be weak. Cysteines in natural proteins, are rarely available in an appropriate reduced form for binding to chelated metal ions. 

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