PURIFICATION AND CHARACTERIZATION OF RAT FIBROBLAST DERIVED INTERFERON

PAUL KAPLAN, Fordham University

Abstract

Rat fibroblast interferon (RfIFN) induced by Newcastle disease virus was purified 2000-fold to 88% purity by a simple method that involved two chromatographic steps: controlled pore glass and phenyl agarose chromatography. The average specific activity was 3.7 x 10('8) units/mg with over 70% of the initial activity recovered. Gel electrophoresis (SDS- and LiDS-PAGE) and silver staining of the purified interferon revealed two major bands of RfIFN activity corresponding to Mr of 26,000 and 22,500 daltons, respectively. A very small amount of RfIFN was also recovered from a region of the gel corresponding to a Mr of 18,500 daltons. Several components of RfIFN activity were resolved by isoelectric focusing, with major peaks of RfIFN activity at pI 5.1 and 6.0. A reduction in size and charge heterogeneity was observed when RfIFN was produced in the presence of tunicamycin, an inhibitor of glycosylation. Gel electrophoresis and isoelectric focusing of RfIFN produced in the presence of tunicamycin revealed only one peak of interferon activity corresponding to a Mr of 16,800 daltons with a pI of 7.2, respectively. A change in the chromatographic behavior of RfIFN due to the absence of sugar residues was found in the loss of affinity for the lectin concanavalin A-agarose. However, the affinity of nonglycosylated RfIFN for various other ligands and its apparent hydrophobicity were unchanged. Removal of the carbohydrate moiety by treatment of RfIFN with glycosidases or periodate also resulted in a reduction in size and charge heterogeneity. RfIFN exhibited cross-species activity, however, the various subtypes differed in their capacity to protect heterologous cells. Removal of the carbohydrate moiety by treatment with glycosidases or periodate, or by inhibition of glycosylation during its biosynthesis resulted in the same cross-reactivity as native RfIFN. ^

Subject Area

Biology|Immunology

Recommended Citation

KAPLAN, PAUL, "PURIFICATION AND CHARACTERIZATION OF RAT FIBROBLAST DERIVED INTERFERON" (1986). ETD Collection for Fordham University. AAI8615733.
http://fordham.bepress.com/dissertations/AAI8615733

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