The primary structures of three major species of human leukocyte. interferon differ from the structure predicted from the DNA sequence of recombinants containing leukocyte interferon-coding regions. Compared to the recombinant interferon produced in bacteria, three of the purified natural proteins isolated from leukocytes lack the 10 COOH-terminal amino acids suggested by the DNA sequence.Although interferon was discovered 23 years ago (1), the structure of the genes and proteins are only now being elucidated with the aid of recombinant DNA technology, DNA sequence analysis, and advances in protein purification and sequence determination. These results indicate that human leukocyte interferon consists of a family of proteins with similar primary structures.Sensitive methods for protein sequence analysis at the nanomole level (2-5) have revealed NH2-terminal amino acid sequences for lymphoblastoid (6) and leukocyte (7) interferon that differ in 2 out of 20 positions. Powerful protein purification techniques involving high-performance liquid chromatography (HPLC) have been used to resolve at least 10 different species of human leukocyte interferon, and tryptic maps of this family of proteins exhibit remarkable homology (8). Amino acid sequence analysis of tryptic and chymotryptic peptides from human lymphoblastoid interferon suggests the existence ofat least five species (9).The successful cloning of human leukocyte interferon has provided additional evidence in support of this diversity. Recombinant bacterial plasmids containing interferon cDNAs have been analyzed and reveal different restriction maps and DNA sequences (10-14). Extensive nucleic acid sequence determination of interferon cDNAs from a virus-induced myeloblast cell line indicates that at least eight distinct species of leukocyte interferon are transcribed during the induction process (14), and this result is corroborated by restriction endonuclease mapping ofinterferon sequences in a human gene bank (15)(16)(17). All of these reports suggest that every active species of human leukocyte interferon is 165 or 166 amino acids in length, even though many individual amino acid assignments differ within the family of proteins. We report here the partial amino acid sequence ofmajor species ofhuman leukocyte interferon. These proteins, which represent a significant fraction of the active interferon produced by these cells, lack the 10 COOH-terminal amino acids suggested previously (11-14) from the DNA sequences. Each of the three species is active although lacking the 10 COOH-terminal amino acids.EXPERIMENTAL PROCEDURES Human leukocyte interferon species a,, a2, and 81 were isolated and purified from chronic myelogenous leukemia (CML) cells as has been described (8,(18)(19)(20). Samples of interferon (7-12 nmol) were digested with tosylphenylalanine chloromethyl ketone-treated trypsin (TPCK-trypsin, 0.2 nmol, Worthington) for 19 hr at 370C in 50 ul of 0.2 M NaHCO3. Then, 2-mercaptoethanol (2 pb1) was added and the sample was incubated for 1 hr at 37...

We reprt the amino-terminal sequence of the first 22 amino acids of human leukocyte interferon. These and other results indicate that human leukocyte interferon consists of many individual species. We, therefore, postulate that diversity in this protein is routinely present and that the human leukocyte interferons represent a multigene family. Although 23 years have passed since the discovery of interferon (1), purification of several interferons has been attained only in the last few years. We previously reported the purification and amino acid composition of human leukocyte interferon (2). Within the past few months, the amino-terminal amino acid sequences of several interferons were reported (3-5). The major obstacle in determining the sequence of interferon has been the availability of sufficient amounts of pure material. Recent advances in manual (6, 7) and automatic (8, 9) sequence analysis have made possible the determination of the primary structure of microgram amounts of protein. We report here the aminoterminal sequence of human leukocyte interferon as determined by automatic and manual sequencing of the native protein and tryptic fragments. In addition, we extend the recently reported amino-terminal sequence and describe two significant differences between our data and those of Zoon et al. (4). EXPERIMENTAL PROCEDURESThe purification of human leukocyte interferon from chronic myelogenous leukemia (CML) cells has been described (2, 10, 11). The sequences of human leukocyte interferon species al and 132 were determined with a modified Beckman spinningcup sequenator. Peptide fragments of a1, a2, and #,-species were produced by digestion with trypsin and purified by high-performance liquid chromatography (HPLC). The sequences of tryptic peptides were determined manually by the Edman reaction (12), and the phenylthiohydantoin derivatives of the amino acids were identified by HPLC (13-15).Automatic Edman degradations were performed in a modified Beckman 890C sequenator on 1.7 nmol of species al of human leukocyte interferon. The modifications, which are similar to those described by and Hun-kapiller and Hood (16), include an improved vacuum system, improved reagent and solvent delivery system, extensive solvent and reagent purification, and a device (17) that automatically converts anilinothiazolinone to phenylthiohydantoin derivatives of amino acids. Proteins are retained in the spinning cup with 6 mg of Polybrene, which, together with 100 nmol of glycylglycine, has been subjected to seven precycles of Edman degradation. Phenylthiohydantoin amino acids were analyzed by HPLC on Du Pont Zorbax octadecylsilica or cyanopropylsilica columns on a Waters Associates chromatograph, by monitoring absorbance at 254 nm and 313 nm. Peak assignments, except for serine, were made by chromatography on a Zorbax octadecylsilica column. Phenylthiohydantoin serine was identified as the "dehydro" derivative on a cyanopropylsilica column. Peaks were integrated and gradient elution was controlled by a Spectra Physic...

The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met- Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-PheGln-X-Gln-Lys.Other laboratories have reported on the purification and partial structural analysis of human fibroblast interferon (1, 2). We present a novel purification and some additional sequences of human fibroblast interferon. Interferon used in our studies was prepared in serum-free medium and was purified by a procedure based on the combination of affinity chromatography and high-performance liquid chromatography (HPLC).EXPERIMENTAL PROCEDURES Interferon Production and Assay. Crude fibroblast interferon was produced as described by Havell and Vilcek (3), except that serum was omitted from the overnight induction medium. Interferon titers were determined by a cytopathic effect inhibition assay that was modified so that the entire assay could be performed within 16 hr (4). All interferon titers were expressed in terms of reference units/ml, calibrated against the reference standard for human leukocyte interferon (G-023-901-527) provided by the Antiviral Substances Program of the National Institute of Allergy and Infectious Diseases, Bethesda, MD.Purification of Human Fibroblast Interferon on Blue Sepharose. Sodium chloride was added to medium containing interferon to a final concentration of 1 M, and the solution was then pumped onto a 25-ml Blue Sepharose CL-6B (Pharmacia) column (1, 5) at room temperature at a rate of 2.5 ml/min. The unfractionated interferon was maintained on ice during the loading process. The column was washed with 250 ml of sodium phosphate buffer (50 mM Na2HPO4 adjusted to pH 7.2 with HCI) containing 1 M NaCl and 30% (vol/vol) ethylene glycol. The interferon was eluted with the same solution containing 50% (vol/vol) ethylene glycol. Peak fractions of activity were pooled and stored at 40C until used (Fig. 1). Activity appeared to be stable for at least 3 mo at 4VC or in liquid nitrogen.In preparations having a low initial titer, a second passage through Blue Sepharose was required. In these instances, when a total of 25 liters of culture medium containing interferon had been chromatographed, the peak fractions from five columns were pooled and adjusted to 10% (vol/vol) ethylene glycol, 2 M NaCl, and 50 mM Na2HPO4 (pH 7.2). This material was then applied to another Blue Sepharose column. The column was then washed with 250 ml of the sodium phosphate buffer containing 2 M NaCl and 30% ethylene glycol. Some interferon was eluted at this step. The remaining interferon was eluted with the same solution containing 50% ethylene glycol. Interferon that eluted from the second Blue Sepharose column with both 30% and 50% ethylene glycol was satisfactory for use in the next step of the purification. The specific activity of the interferon that was deemed sati...

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