Purification of Natural Human Interferon-Gamma by Antibody Affinity Chromatography: Analysis of Constituent Protein Species in the Dimers

A simple procedure for purifying human interferon-γ 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)×107 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 MW 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.