title: Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers.
abstract: A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007371:Interferon-gamma / Q000097:blood / Q000302:isolation & purification*; D007962:Leukocytes / Q000032:analysis; D008970:Molecular Weight
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D007371:Interferon-gamma