Interferon-α2Produced by Normal Human Leukocytes Is Predominantly Interferon-α2b

Abstract

Peripheral blood leukocytes, isolated from the buffy coats of greater than 10,700 normal healthy donors, were induced with Sendai virus to produce biologically active interferon a (IFN-α). The IFN-α was purified to near homogeneity by immunoaffinity chromatography, followed by size-exclusion chromatography. The resultant product, IFN-α_n3, is reproducibly ≥98% pure (to be reported elsewhere). The different IFN-α proteins in IFN-αn3 were separated by reverse-phase high performance liquid chromatography (RP-HPLC) and the identity of the IFN-α₂ isolated by HPLC was determined by amino-terminal sequencing. IFN-α₂ was found to migrate as two closely eluting peaks on RP-HPLC, and they have been designated as peaks 1.1 and 1.2. Distinction among the three possible variants of IFN-α₂, i.e., IFN-α_2a, IFN-α_2b,, and IFN-α_2c, was determined by amino-terminal sequencing of the first 35 amino acids in peaks 1.1 and 1.2. Protein sequence data showed that the discriminating amino acids found at positions 23 and 34 are Arg and His, respectively. The presence of Arg and not Lys at amino acid position 23 and His at amino acid position 34 argues that IFN-α_2b, is the major component in the Sendai virus-induced leukocyte IFN-α₂ and that IFN-α_2a is not present. These findings were verified by subjecting RP-HPLC peaks 1.1 and 1.2 to CNBr cleavage, followed by separation of the fragments by RP-HPLC and sequencing. The sequence of CNBr fragment 3 from both peaks, consisting of a peptide from amino acid positions 22–59, revealed Arg at amino acid position 23 and predominantly His at position 34. Tryptic digestion of isolated CNBr fragment 3, followed by HPLC separation and sequencing, demonstrated the presence of a tryptic peptide with sequence DRHDF..., corresponding to IFN-α_2b. Another peptide with sequence DFGF..., which corresponds to IFN-α_2c., was also found to be present in a very low concentration. From these data we conclude that the majority of IFN-α₂ found in IFN-α_n3 is IFN-α_2b,, with a minor contribution by IFN-α_2c, and that IFN-α_2a is not present.