title: Removing coordinated metal ions from proteins: a fast and mild method in aqueous solution.
abstract: Thermodynamic and kinetic studies of metal binding to proteins require the investigation of metal-free proteins, which are often difficult to obtain. We have developed a very fast and mild method to eliminate metal ions from proteins by column chromatography using a commercially available Ni-NTA-type stationary phase. This material, initially designed for protein purification purposes in biotechnology, acts as a strong cation chelator when Ni2+ ions are removed. We have tested this new method with Ca-ATPase, an integral membrane protein exhibiting a strong affinity for Ca2+. By eluting the protein over the Ni2+-free NTA gel, we could remove 95% of the total Ca2+ and obtain an essentially Ca2+-free protein. This method is efficient with only a small amount of NTA gel, and we suggest that it can be applied in general for removal of metal ions from proteins. Moreover, as this procedure can be carried out under mild conditions, the chosen protein kept its enzymatic activity.
authors.affiliation: Max-Planck-Institut für Biophysik, Max-von-Laue-Str. 3, 60438, Frankfurt am Main, Germany.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Carrer
authors.forename: Michael
authors.initials: M
authors.lastname: Stolz
authors.forename: Erwin
authors.initials: E
authors.lastname: Lewitzki
authors.forename: Claudia
authors.initials: C
authors.lastname: Rittmeyer
authors.forename: Bernd O
authors.initials: BO
authors.lastname: Kolbesen
authors.forename: Ernst
authors.initials: E
authors.lastname: Grell
mesh_terms: D001059:Apoproteins / Q000737:chemistry*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D008670:Metals / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014867:Water / Q000737:chemistry
chemical_list: D001059:Apoproteins; D002614:Chelating Agents; D008670:Metals; D014867:Water; D000252:Calcium-Transporting ATPases