Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization

Abstract

Human leukocyte interferon (HL-IF) produced by mixed leukocytes infected with Newcastle disease virus was resolved into 3 distinct fractions when chromatographed on concanavalin [Con] A-agarose. The major portion (70-75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25-30%) was displayed from the column as 2 peaks: the 1st was eluted with 0.1 M methyl .alpha.-D-mannoside, yielding 15-20% of the interferon activity (.alpha.-MM fraction), and the 2nd by including ethylene glycol (70%) in the eluant, yielding the remaining 5-15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on Con A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The 3 fractions of interferon (BT, .alpha.-MM and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral [vesicular stomatitis virus] activity and neutralization by specific antisera. The BT fraction contains exclusively the 16,000 MW component of human leukocyte interferon. The majority of the .alpha.-MM fraction (90%) is the 21,000 MW component. The EG fraction contains the 16,000 and 21,000-23,000 MW components in essentially equal proportions. Based on cross-species antiviral activity and neutralization by specific antisera, the BT and .alpha.-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.