title: Thermodynamics of imidazole-ligand binding to Ni-nitrilotriacetate in solution and covalently attached to agarose beads: imidazole, his-6 (his-tag) peptide and a new bis-imidazolo-dithiane.
abstract: A new imidazolo- and thiol-containing ligand is prepared and characterized with the intent to serve as surrogate and mimic for the canonical ligands imidazole and hexa-histidinyl peptide (his-6) in immobilized metal affinity chromatography (IMAC) [1,2] systems. The affinity of this ligand: 1,8 bis (N1,N1')imidazolo-octa-(3,6)-dithiane or bImOdS, to Ni-nitrilotriacetic acid (Ni-NTA) complex in solution is measured and compared with that of imidazole and his-6 peptide via isothermal titration calorimetry (ITC). In addition, bImOdS is compared with his-6 binding to the solid-state matrix of Ni-NTA-charged agarose beads, as employed routinely in IMAC. Results reported here include the following: (1) two imidazole moieties bind within a single Ni-NTA complex, while bImOds, being an imidazolo dimer, binds with 1:1, and his-6 peptide binds with 1:3 stoichiometry. (2) Enthalpies of reaction for imidazole and his-6 peptide are reported - these can be utilized to predict changes in affinity in IMAC systems with temperature, should protein unfolding/refolding steps in purification be desired at alternate temperatures. (3) Metal analyses of the Ni-NTA agarose beads suggests that ∼2/3 of the nickel is present in low-affinity sites, which will complicate protein separations at high protein-concentration loading. An improved procedure for subtracting ligand dilution heats from ITC analyses is presented in an Appendix.
authors.affiliation: Mayo Clinic College of Medicine, Rochester, MN 55905, United States. Electronic address: wrkqdeformd@gmail.com.
authors.forename: William R
authors.initials: WR
authors.lastname: Kirk
mesh_terms: D002151:Calorimetry; D002846:Chromatography, Affinity; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry* / Q000378:metabolism; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: C471213:His-His-His-His-His-His; D007093:Imidazoles; D009842:Oligopeptides; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009571:Nitrilotriacetic Acid
keywords: Imidazole ligands; Immobilized metal affinity chromatography; Nickel complexes; Titration calorimetry