Human y (immune) interferon (IFN-y) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-yis a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 ± 3000. A purification process for IFN-y was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 104 (crude culture fluid) to an estimated 107 units per mg of protein with a cumulative recovery of about 40% of the IFN activity.

Interferon (IFN)-y was produced in cultures of human leukocytes by combined stimulation with 12-0-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA).IFN-ywas purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAESephacel at pH 8.0. Treatment of such partially purified IFN-y preparations with the anionic detergent NaDodSO4 (0.1% at 20-250C) decreased biological activity to approximately 5-20%.When analyzed by NaDodSO4polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-y. Native IFN-y was found to have a lower affinity for alkyl agarose columns than human IFN-a or IFN-fi did, suggesting that IFN-y is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-y to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.Until recently virtually no information was available about physicochemical properties ofthe interferon (IFN) species produced in human lymphocytes upon stimulation with specific antigens (1) or with various other mitogens (2, 3). The identification of mitogen-induced IFN as IFN-y and its differentiation from the other two major species, termed IFN-a and IFN-/3 (4), is based mainly on antigenic differences among the three IFN species and on the known relative instability of IFN-y at pH 2 (3).The scarcity of information about IFN-y was mainly due to the lack of suitable methods for its production and purification. Recently, we reported a method of human IFN-y production based on combined stimulation of human lymphocytes with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA) (5, 6). This method enables the production of IFN-y preparations with relatively high initial specific activity. The use of lymphocyte-rich plateletpheresis residues as the cell source for production of IFN-y makes it possible to prepare batches yielding several liters of starting material for purification. We showed earlier that a relatively simple three-step purification procedure led to approximately a 1,000-fold purification of IFN from this starting material (7).Another factor that contributed to the successful purification of IFN-y described in this paper was the recent demonstration that the biological activity of human IFN-y was not completely destroyed by treatment with NaDodSO4 (8). Residual biological activity after NaDodSO4 treatment could thus be used as a marker for the identification of IFN-y proteins under conditions precluding noncovalent interactions am...

The molecular weight (as determined by molecular sieve chromatography) of human gamma interferon, formerly referred to as immune or type II interferon, is between 40,000 and 70,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gamma interferon activity was recovered mainly from two regions of the gels corresponding to molecular weights of 20,000 and 25,000. The results suggest that in native form human gamma interferon may be aggregated.

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