Isolation of two discrete human interferon-γ (immune) subtypes by high-performance liquid chromatography

Abstract

A rapid procedure for isolation of two biologically active human interferon-γ subtypes was developed. Crude interferon-γ produced in a serum-free culture of peripheral blood mononuclear cells by mitogen stimulation was concentrated and partially purified by chromatography on controlled-pore glass. Following desalting and concentration by ultrafiltration, a step of cation-exchange high-performance liquid chromatography was performed. A linear NaCl gradient (0.01–0.4 m) at pH 7 was employed and four peaks of biological activity eluting at 0.17, 0.20, 0.26 (major peak), and 0.3 m were obtained. The major peak of biological activity coincided with two protein peaks. Analysis of one fraction from the major activity peak by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a protein band having an apparent molecular weight of 26.000, while an adjacent fraction of the same activity peak contained a protein band corresponding to a molecular weight of 21.000. The specific activity of both subtypes was 7–10 × 10 6 units/mg.