We have detected seemingly uninduced interferons (IFNs) Chromatographic Products. Sephadex G-25 medium in pre-packed PD 10 columns, concanavalin A (Con A)-Sepharose and Blue Sepharose CL-6B (group-specific adsorbents), cyanogen bromide-activated Sepharose 4B, and activated CH-Sepharose 4B (coupling gels for antiserum immobilization), and acrylamide were purchased from Pharmacia. The standard proteins used for calibration of the slab gels were also from Pharmacia.Biological Preparations. Human placentas were collected shortly after caesarian section between the 37th week (calculated on the basis of amenorrhea and sonar exploration) and delivery. Most of the women were in the 39th week, corresponding to a fetal age of 37 weeks. In two additional cases, products of therapeutic abortion by vacuum aspiration in the 10th and 17th weeks of development were studied.Fetal membranes were separated from the placenta, washed carefully one to five times in 500 ml of saline (0.15 M NaCl, pH 7), then incubated with Eagle's minimal essential medium (MEM), supplemented with 10%o heat-inactivated newborn calf serum, at 370C for 18 hr.Blood from the umbilical cord was collected. The whole placenta was then compressed for 3 hr under 0.15 kg/cm2 (15 kPa) to squeeze out the blood, which was analyzed.Cells and Viruses. Human diploid fibroblasts (F7000, Flow Laboratories) were grown in Eagle's basal medium (BME) supplemented with nonessential amino acids, 2 mM glutamine, and 10% heat-inactivated fetal calf serum. Human amnion (WISH), bovine kidney (MDBK), and mouse L929 cells, in continuous cultivation, and rat fibroblasts (REF) in secondary subcultures, were grown in Eagle's MEM supplemented with 10% heat-inactivated newborn calf serum. Normal rat kidney (NRK) cells were grown in RPMI 1629 medium, supplemented with 10% fetal calf serum and 4 mM glutamine.Vesicular stomatitis virus (VSV), Indiana strain, and encephalomyocarditis (EMC) virus were grown on L929 cells and titrated by plaque-forming unit assay. EMC virus titer was also evaluated by human erythrocyte (O+ group) agglutination.Affinity Chromatography on Con A-Sepharose. IFN preparations were applied to a Sephadex G-25 M PD 10 column (1.5 x 5 cm), previously equilibrated with 0.02 M sodium phosphate, pH 7.2/1 M NaCl (buffer EO) and eluted with the same buffer. 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 5010The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Human leukocyte interferon can be separated into two classes of subspecies by polynucleotide-agarose affinity chromatography; 30-40% of the molecular species have the polynucleotide-binding property and 60-70% lack affinity for the polynucleotide ligand. When analyzed on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the former class of interferon has a slower mobility corresponding to the migration of a polypeptide of 21,000 daltons, while the latter class has a faster mobility corresponding to a polypeptide of 13,500-15,000 daltons. By analogy to the behavior of other interferons and a class of nucleotidyl transferases on the polynucleotide-agarose chromatography, we suggest that the human leukocyte interferon having the polynucleotide-binding site is in a possibly "native" conformation and the loss of affinity for polynucleotide results from a degradative alteration of the native molecules. Moreover, the alteration of interferon is accompanied by an increase in heterospecific activity on bovine cells. It is suggested that the polypeptide domain responsible for species specificity may be closely related to the polynucleotide binding area. The modified interferon molecule, however, still conserves its antiviral activity. The simplicity and the high capacity of polynucleotide-agarose chromatography make this a powerful technique for the purification of interferon. The easy separation of these two classes of human leukocyte interferon makes the purification procedures more rational and will facilitate the preparation of both subspecies to a high degree of molecular homogeneity. Human leukocyte interferon (Hu Le IF) contains two subpopulations of active molecules, which are differentiated by their molecular sizes, charge properties, and even biological activities (1-6). Such heterogeneity, resulting from the presence of molecular species having different physicochemical properties, complicates the purification of leukocyte interferon.In particular, the strategy for purification has been mostly orientated by the recovery of as much initial activity as possible, necessitating copurification of the two subpopulations.We have recently described an affinity chromatography with blue dextran or polynucleotide as ligand for proteins interacting with nucleic acids (7). Such chromatography not only allows the separation of proteins having a polynucleotide-binding site from other classes of proteins but also can be used to separate one native enzyme from a degraded enzyme that has lost the polynucleotide attachment site (7). The methodology was also applied to the characterization and purification of both viral and immune interferons (8-11). We postulate that the polynucleotide-binding property, observed with mouse types I and II and human interferons, could be a basic characteristic of this antiviral protein. This property also could reveal a structural homology between the mouse types I and II interferons (10). In this paper, we show that Hu Le IF can be separated into two classes of molecules according...