title: Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography.
abstract: Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363-4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.
authors.affiliation: Bulgarian Academy of Sciences, Central Laboratory of Biophysics, Sofia.
authors.forename: L
authors.initials: L
authors.lastname: Smilenov
authors.forename: E
authors.initials: E
authors.lastname: Forsberg
authors.forename: I
authors.initials: I
authors.lastname: Zeligman
authors.forename: M
authors.initials: M
authors.lastname: Sparrman
authors.forename: S
authors.initials: S
authors.lastname: Johansson
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D003035:Cobalt; D003300:Copper; D005290:Ferric Compounds; D005353:Fibronectins / Q000302:isolation & purification*; D018093:Gelatinases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009532:Nickel; D009994:Osmolar Concentration; D010434:Pepsin A / Q000097:blood*; D015032:Zinc
chemical_list: D005290:Ferric Compounds; D005353:Fibronectins; D003035:Cobalt; D003300:Copper; D009532:Nickel; D010434:Pepsin A; D018093:Gelatinases; D015032:Zinc