Binding of human interferons to immobilized Cibacron Blue F3GA: the nature of molecular interaction

Abstract

Blue Dextran (Cibacron Blue F3GA-dextran) was immobilized on cyanogen bromide activated agarose and used as a ligand for human fibroblast and leukocyte interferons in a solvent of phosphate-buffered (pH 7.4), physiological saline (0.15 M NaCl). Fibroblast interferon binds completely and is not displaced from the column by an increase in ionic strength of the solvent (1.0 M NaCl); it can be, however, recovered with ethylene glycol, indicating the hydrophobic nature of interaction. Leukocyte interferon also binds to Blue Dextran-agarose but it can be recovered simply by an increase in the ionic strength of the solvent, indicating primarily the electrostatic nature of binding. Attempts to displace both interferons selectively with nucleosides and aromatic amino acids were unsuccessful. When Cibacron Blue F3GA is immobilized directly to agarose matrix or via molecular arm, the strength of binding of fibroblast interfern is significantly decreased, although ethylene glycol is still required for its displacement from the column. Leukocyte interferon, by contrast, does not bind at all under the same solvent conditions; it does bind when the pH value of the solvent is in the range 3-5 i.e., below its isoelectric point. Human fibroblast interferon binds completely to: aminobenzene, aminonaphthalene, and aminoanthracene, all immobilized on agarose, and it can be recovered with ethylene glycol. In contrast, human leukocyte interferon does not bind to benzene-agarose; it is retarded on naphthalene-agarose and completely retained on an anthracene-agarose column. All data point to a higher intrinsic hydrophobicity of human fibroblast interferon vis-á-vis human leukocyte interferon. Selective binding of human fibroblast interferon of Cibacron Blue F3GA-agarose results in a significant purification, about 800-fold.

Binding of mouse interferon to polynucleotides.

“…The strong affinity of interferon molecules for the dye Cibacron blue F3GA has been recently reported, independently, from three laboratories (1)(2)(3). A practical application of this observation is the purification of interferon by affinity chromatography on blue dextran-Sepharose (1, 2).…”

Unusual apparently constitutive interferons and antagonists in human placental blood.

“…The breakthrough active fractions (5 ml) were applied on 5 ml of Con A-Sepharose in a column (IBF 11, 1.14 x 10 cm) equilibrated with buffer EO at room temperature at a flow rate of 4.5 ml/hr, according to a previously described procedure (10,11 F3G-A (Blue Sepharose CL-6B). After buffer exchange with Sephadex G-25 M, as described above, samples (3-4 ml) were applied to 3.5-ml Blue Sepharose CL-6B gel (12) in an IBF-11 column (1.14 x 10 cm), equilibrated with starting buffer (EO), and recycled continuously overnight at a flow rate of 4.5 ml/hr. After washing, the bound substances were eluted at a flow rate of 20 ml/hr by increasing the ionic strength of the buffer to 2 M NaCl (buffer El').…”

Binding of 125I-labeled recombinant beta interferon (IFN-beta Ser17) to human cells.

“…A consistent observation in our laboratory has been an almost quantitative inactivation of IFN-1 Ser17 by Bolton-Hunter reagent. The stoichiometry of the inactivation suggests a site-specific modification, perhaps a result of interaction between the hydrophobic reagent and a hydrophobic site on the protein (10,14,23,24). Although Bolton-Hunter-labeled a and interferons have been reported to bind to receptors (11,27 Our competition results with labeled P and unlabeled a interferon support the concept of a common binding site for a and 3 interferon, a concept previously suggested by competition experiments between labeled a and unlabeled 1 interferon (7,15).…”