INTERFERON-ALPHA(2), PRODUCED BY NORMAL HUMAN-LEUKOCYTES IS PREDOMINANTLY INTERFERON-ALPHA(2B)

Peripheral blood leukocytes, isolated from the buffy coats of greater than 10,700 normal healthy donors, were induced with Sendai virus to p roduce biologically active interferon alpha (IFN-alpha). The IFN-alpha was purified to near homogeneity by immunoaffinity chromatography, fo llowed by size-exclusion chromatography. The resultant product, IFN-al pha(n3), is reproducibly greater than or equal to 98% pure (to be repo rted elsewhere). The different IFN-alpha proteins in IFN-alpha(n3), we re separated by reverse-phase high performance liquid chromatography ( RP-HPLC) and the identity of the IFN-alpha(2) isolated by HPLC was det ermined by amino-terminal sequencing, IFN-alpha(2) was found to migrat e as two closely eluting peaks on RP-HPLC, and they have been designat ed as peaks 1.1 and 1.2, Distinction among the three possible variants of IFN-alpha(2), i.e., IFN-alpha(2a), IFN-alpha(2b), and IFN-alpha(2) , was determined by amino-terminal sequencing of the first 35 amino ac ids in peaks 1.1 and 1.2. Protein sequence data showed that the discri minating amino acids found at positions 23 and 34 are Arg and His, res pectively. The presence of Arg and not Lys at amino acid position 23 a nd His at amino acid position 34 argues that IFN-alpha(2b), is the maj or component in the Sendai virus-induced leukocyte IFN-alpha(2) and th at IFN-alpha(2b), is not present. These findings were verified by subj ecting RPHPLC peaks 1.1 and 1.2 to CNBr cleavage, followed by separati on of the fragments by RP-HPLC and sequencing. The sequence of CNBr fr agment 3 from both peaks, consisting of a peptide from amino acid posi tions 22-59, revealed Arg at amino acid position 23 and predominantly His at position 34. Tryptic digestion of isolated CNBr fragment 3, fol lowed by HPLC separation and sequencing, demonstrated the presence of a tryptic peptide with sequence DRHDF..., corresponding to IFN-alpha(2 b),. Another peptide with sequence DFGF..., which corresponds to IFN-a lpha(2c), was also found to be present in a very low concentration. Fr om these data we conclude that the majority of IFN-alpha(2) found in I FN-alpha(n3), is IFN-alpha(2b), with a minor contribution by IFN-alpha (2c), and that IFN-alpha(2a), is not present.