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Human interferon binds to a omega-carboxpentyl-agarose column at low ionic strength (0.15 M NaCl) and is still retained when the ionic strength is raised (to 1.0 M NaCl). The binding can be reversed, however, by ethylene glycol, indicating a hydrophobic interaction. The binding of human interferon to omega-aminohexyl-agarose is weak, even at a low ionic strength, and is probably exclusively electrostatic. This disparate binding behavior may be caused by the presence of a positive charge, adjacent to the hydrophobic binding site, on human interferon. The interaction of human interferon with omega-carboxypentyl-agarose is quite selective, inasmuch as the majority of proteins present in interferon preparations pass through the column unretained. Hydrophobic chromatography of human interferon may thus be useful in its purification.

Interferons of human, mouse, and rabbit origin bind to straight chain hydrocarbons immobilized on agarose. The hydrophobic nature of binding is established by the following observations: (a) a positive correlation between the length of hydrocarbon ligand and the strength of interaction; (b) a stronger interaction with hydrocarbon ligands terminated with apolar rather than polar head groups; (c) a lack of dependence of binding on ionic strength and pH of the solvent; (d) a reversal of binding by ethylene glycol, a hydrophobic solute; (e) an increasing eluting efficacy of tetraalkylammonium ions with the length of their alkyl substituents. The hydrophobic interactions of human interferon underlie the efficiency of two-step chromatographic procedures. For example, human embryo kidney interferon can be purified about 3,600-fold by sequential chromatography on (a) concanavalin A-agarose, (b) octyl-agarose. Another two-step procedure: (a) concanavalin A-agarose, (b) L-tryptophan-agarose, gives about 10,000-fold purification. The overall recovery of interferon in both cases in close to 90%.

Structural differences between human leukocyte virus-induced interferon and human fibroblast polyinosinic-polycytidylic acid (rIn-rCn)-induced interferon have been noted in previous studies. This study reports the behavior of human leukocyte and fibroblast interferon, induced by virus and by rIn-rCn, in several lectin and hydrophobic chromatographic systems. Differences in both glycosylation and in hydrophobicity of human leukocyte and fibroblast interferons are documented. Human fibroblast interferon is a glycoprotein, whereas our evidence suggests that human leukocyte interferon probably is not. Also, fibroblast interferon is more hydrophobic than leukocyte interferon, as probed on several hydrophobic adsorbents. The possible relationships of these differences to each other and to antigenic variations are discussed. Generally, the differences appear to be attributable to the cell type in which the interferon was induced. However, our results suggest that at least subtle differences in the processing of the induction signal (virus or rIn-rCn) within the same cell type may occur, slightly altering some structural features.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.

Several novel selective sorbents for mouse interferon are described that exploit the hydrophobic property and glycoprotein nature of this molecule. Low-molecular-weight ligands (hydrocarbons) and high-molecular-weight ligands (bovine serum albumin) immobilized on agarose bind selectively mouse L-cell interferon. The high selectivity of binding is due primarily to a hydrophobic effect, although electrostatic forces are also apparently involved. Mouse L-cell interferon binds to immobilized serum albumin and can be completely recovered by raising the ionic strength of the eluant. The specific activity of interferon preparations can be increased 2,000-fold to a value of 3 x 10(8) reference units per mg of protein in a single step with full recovery of the antiviral activity. A selective adsorption, although to a lesser degree, can be also obtained on hydrocarbon-coated agarose (Affi-Gel 202), resulting in 300-fold purification on desorption. The existence of two major components of mouse interferon was revealed upon its chromatography on the following sorbents: (i) bovine serum albumin-agarose, (ii) omega-carboxypentyl-agarose; and (iii) Bandeiraea simplicifolia lectin-agarose. This report thus provides for the first time a means for efficient and clear-cut separation of interferon components, thus enabling their further characterization.

Human fibroblast interferon binds to L-tryptophan, D-tryptophan, L-phenylalanine, and L-tyrosine, all immobilized directly to cyanogen bromide-activated agarose, as well as to L-tryptophan and D-tryptophan methyl ester, both immobilized via molecular arms. The retention of fibroblast interferon is selective and results in a 2300-fold purification. Human leukocyte interferon binds neither to L-tryptophan attached directly to an agarose matrix nor to L-tryptophan immobilized via a molecular arm; it binds, however, to immobilized L-tryptophyl-L-tryptophan and L-tryptophyl-L-tryrosine. When retained, both interferons cannot be displaced unless ethylene glycol is included in the eluant, indicating a hydrophobic interaction. The interaction takes place under physiologic solvent conditions, thus revealing the high intrinsic hydrophobicity of both interferons.