Metal-Ion Affinity Chromatography

Immobilized metal-affinity chromatography (IMAC) is also known as metal-chelate affinity chromatography (MCAC). This method was first proposed by Porath et al. in 1975 [63] and is based on the specific interactions between immobilized metal ions and amino acid residues, such as histidine, fiyptophan, and cysteine in proteins or peptides [63]. IMAC has become an important tool for the detection and purification of metalloproteins, histidine-tagged proteins, and phosphorylated proteins. Areas in which this method is now used include proteomics [64—66], work with recombinant proteins [67—69], and disease diagnosis [70,71]. 

The stationary phase in IMAC consists of an immobilized chelating ligand that is complexed with a metal ion. The chelating ligand is attached covalently to a support and used to entrap metal ions via coordinate binding. Agarose was the first support utilized in IMAC, but supports based on other materials, such as silica, are also now common [72,73]. Recent reports have further described the development of supports for IMAC based on cryogels, silica monoliths, and polymethacrylate monoliths [74-76]. 

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Li, S. and Dass, C., Iron(III)-Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry for the Purification and Characterization of Synthetic Phosphopeptides, Anal. Biochem., 270, 9, 1999. 

Amini, A., Chakraborty, A., Regnier, F.E. (2002). Simplification of complex tryptic digests for capillary electrophoresis by affinity selection of histidine-containing peptides with immobilized metal ion affinity chromatography. J. Chromatogr. B 772, 35-44. 

Porath, J. (1992) Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263-281. 

Porath, J., and Olin, B. (1983) Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22, 1621-1630. 

Cao P. and Stults J.T. (1999), Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry, J. Chromatogr. 853(1-2), 225-235. 

Todorova, D. and Vijayalakshmi, M. A., Immobilized metal-ion affinity chromatography, in Handbook of Affinity Chromatography, 2nd edn., Hage, D.S., Ed., CRC Press, Boca Raton, FL, 2005, Chap. 10. 

Chaga, G.S., Twenty-five years of immobilized metal ion affinity chromatography Past, present and future, J. Biochem. Biophys. Methods, 49, 313-334, 2001. 

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]. 

R. Woker, B. Champluvier, M. R. Kula, Purification of S-oxynitrilase from Sorghum hicolor by immobilized metal ion affinity chromatography on different carrier materials./. Chromatogr. 1992, 584, 85-92. 

The technique immobilized metal ion affinity chromatography (IMAC) is widely used to purify proteins (Experiment 4). Find two proteins that have recently been purified by this technique and briefly describe the methods for isolation. 

R. Boyer,/. Chem. Educ. 68, 430-432 (1991). Purification of Milk Whey a-Lactal-bumin by Immobilized Metal-Ion Affinity Chromatography. ... 

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. 

A very interesting application of affinity chromatography to the purification of halophilic enzymes was reported by Sundquist and Fahey (1988). These authors have purified the enzymes bis-y-glu-tamylcysteine reductase and dihydrolipoamide dehydrogenase from H. halohium using immobilized metal ion affinity chromatography in high-salt buffers. 

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