Immobilized metal-ion affinity chromatography: imidazole proton pump and chromatographic sequelae. I. Proton pump.
Complexation of imidazole (Im) with an iminodiacetate (IDA) metal chelate [IDA-M(II)] ligand of chelating gel results in an acidification of the mobile phase. The scope of the action of this IDA-M(II)Im 'proton pump' in IMAC is determined by: (a) IDA-M(II) density of the gel; (b) concentration of applied Im; and (c) the buffering capacity of the mobile phase. Application of Im onto a metal chelate column in a gradient rather than in a stepwise manner, mitigates the proton pump's action, as it does an increase of buffer concentration in the mobile phase. However, only an antecedent conversion of the metal chelate gel, IDA-M(II), to its Im derivative, IDA-M(II) Im, can effectively circumscribe the action of the proton pump. The same holds true, as anticipated, when another chelating ligand (nitrilotriacetate) is used.
Sulkowski
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Roswell Park Cancer Institute, Department of Molecular and Cellular Biology, Buffalo, NY 14263, USA.
D002021:Buffers; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003035:Cobalt; D003300:Copper; D007093:Imidazoles / Q000737:chemistry*; D007098:Imino Acids / Q000737:chemistry*; D008670:Metals; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D017494:Proton Pumps; D012685:Sepharose; D015032:Zinc
D002021:Buffers; D002614:Chelating Agents; D007093:Imidazoles; D007098:Imino Acids; D008670:Metals; D017494:Proton Pumps; D003035:Cobalt; D003300:Copper; C029899:imidazole; D009532:Nickel; D012685:Sepharose; D015032:Zinc; D009571:Nitrilotriacetic Acid; C008109:iminodiacetic acid