title: The preparation of apo-Cu,Zn superoxide dismutase by ion-exchange chromatography on iminodiacetic acid-sepharose.
abstract: The superoxide dismutases (EC 1.15.1.1) are a family of enzymes that catalyze the dismutation of superoxide radical anion to dioxygen and hydrogen peroxide. The active site contains a critical metal ion such as manganese, iron, or copper. The copper-containing protein also has one zinc ion bound per subunit. The standard method used to remove the metal ions from Cu,Zn superoxide dismutase has been to exhaustively dialyze the protein against chelating agents at low pH. We have developed a new method where the protein is bound to ion-exchange medium based on iminodiacetic acid immobilized on Sepharose. The bound protein is treated with a buffer containing edta at pH 3.5 to remove metal ions; the buffer is then exchanged for acetate buffer to remove edta, after which the protein is eluted by a salt gradient. An advantage of this method is that a single chromatography step is sufficient to produce apo protein. Results are shown for both human and bovine dimeric Cu,Zn superoxide dismutase and the monomeric Escherichia coli Cu,Zn superoxide dismutase. In every case, the metals were removed efficiently.
authors.affiliation: Laboratorium für Anorganische Chemie, Eidgenössische Technische Hochschule, Universitätstrasse 6, Zürich, CH-8092, Switzerland.
authors.forename: B
authors.initials: B
authors.lastname: Sutter
authors.forename: P L
authors.initials: PL
authors.lastname: Bounds
authors.forename: W H
authors.initials: WH
authors.lastname: Koppenol
mesh_terms: D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000302:isolation & purification; D002417:Cattle; D002614:Chelating Agents; D002847:Chromatography, Agarose / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D007098:Imino Acids; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D001051:Apoenzymes; D002614:Chelating Agents; D007098:Imino Acids; D013482:Superoxide Dismutase; C008109:iminodiacetic acid