The isolation and amino acid/sugar composition of human fibroblastoid interferon.

Human fibroblastoid interferon produced from an established human cell line was purified by controlled-pore glass and concanavalin A-Sepharose column chromatography followed by preparative two-dimensional gel electrophoresis. The purification procedure provided a 10% recovery of pure interferon with good reproducibility. The purified protein was homogeneous with respect to its molecular weight of 20,000 and net electrical charge at pH 2.5. Interferon of high specific activity of 5 x 10(8) units/mg of protein was directly demonstrated in the polyacrylamide gel before staining with Coomassie brilliant blue. Parallel purification of a sham-induced interferon preparation did not yield an equivalent product indicating the purified interferon is not derived from uninduced cells or from the fetal calf serum of the tissue culture growth medium. Pure interferon was radioiodinated by Bolton-Hunter reagent. Amino acid analysis of the pure preparation shows interferon to be a leucine-rich glycoprotein containing a high percentage of glutamic/glutamine residues and that disulfide bridges(s) are important for its biological activity.

Human fibroblastoid interferon produced from an established human cell line was purified by controlledpore glass and concanavalin A-Sepharose column chromatography followed by preparative two-dimensional gel electrophoresis.
The purification procedure provided a 10% recovery of pure interferon with good reproducibility.
The purified protein was homogeneous with respect to its molecular weight of 20,000 and net electrical charge at pH 2.5. Interferon of high specific activity of 5 x 10' unit.s/mg of protein was directly demonstrated in the polyacrylamide gel before staining with Coomassie brilliant blue. Parallel purification of a sham-induced interferon preparation did not yield an equivalent product indicating the purified interferon is not derived from uninduced cells or from the fetal calf serum of the tissue culture growth medium. Pure interferon was radioiodinated by Bolton-Hunter reagent. Amino acid analysis of the pure preparation shows interferon to be a leucine-rich glycoprotein containing a high percentage of glutamic/glutamine residues and that disulfide bridge(s) are important for its biological activity.
The large scale purification of interferon to homogeneity has eluded investigators for almost two decades. Initially, the difficulty in the purification of interferon was the scarcity of this protein. In recent years, concerted efforts have been made to produce adequate amounts of interferon for the development of procedures for its purification (l-lo). One criticism of these procedures concerns the use of a single parameter to show the purified product is homogeneous, i.e. the demonstration of interferon activity in one or more protein fractions after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The problem with introducing another parameter such as the homogeneity of net electrical charge is that interferon usually does not migrate as a singular discrete fraction in electrophoresis (10). Furthermore, interferon is polydisperse during electrofocusing (8,9). This polydispersity was attributed to differences in the degree of glycosylation, possible microheterogeneity in the peptide composition, and even differential removal of sodium dodecyl sulfate from interferon previously exposed to sodium dodecyl sulfate during electrophoresis (10). We now report the purification of interferon to homogeneity with respect to its molecular weight and with respect to its net electrical charge at pH 2. 5  Preliminary Purification to 10% Purity-Our approach was to find a method that would provide a preliminary purification of interferon to about 10% purity with nearly quantitative recovery and then to purify it to homogeneity by two-dimensional gel electrophoresis.
The preliminary purification procedure consists of chromatography on controlled-pore glass column followed by chromatography on concanavalin A-Sepharose column. Interferon bound to the CPG* column at neutral pH is eluted by lowering the pH of the eluting buffer to 3.0. The CPG-purified interferon is stable at pH 3.0, and if the pH is adjusted to neutrality most of the interferon is irreversibly lost in co-precipitation with other proteins. For this reason, it was necessary to maintain the pH of CPG-purified interferon at an acid pH not exceeding 4.5 to avoid the irreversible loss of interferon through precipitation.
The CPG-purified interferon is applied to a Con A column to which it binds strongly at pH 4.5 but most of the non-interferon proteins do not. Interferon is not eluted from the column by buffers of high ionic concentrations and only a small percentage (5%) is eluted by 100 mM a-methylmannoside.
However, most (80%) of the interferon activity applied to the Con A column is recovered in 50% ethylene glycol. The binding of interferon to CPG and to Con A is most probably due to a combination of hydrophobic and ionic interactions.
This explanation is consistent with the analysis given in Table  II, that interferon is rich in hydrophobic amino acid residues and contains charged amino acids as well as amino sugars. The impurities of the interferon preparation derived from this preliminary purification are shown in Fig. 1. The specific activity of the partially purified interferon is nearly 1 x 10' reference units," with a recovery of  (Table I). However, this preliminary procedure introduces concanavalin A and its peptides into the preparation. We had previously reported that phenyl-Sepharose column chromatography could remove these extraneous concanavalin A contaminants (10) but on closer examination a small amount of the M, = 20,000 concanavalin A fragment may sometimes be present. Consequently, other procedures were investigated, and a method which removes the concanavalin A contaminants as well as separating interferon from the other proteins is the two-dimensional gel electrophoretic step described below.
Two-Dimensional Gel Electrophoresis-The first step consists of electrophoresis in a 15% polyacrylamide gel containing 2.5 M urea (acid-urea). The second step is polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at pH 8.8. Proteins are separated in the first dimension according to their charge and in the second dimension according to their molecular weight. In the acid-urea gel, interferon migrates well ahead of concanavalin A and its peptides and most of the extraneous proteins remaining in the Con A-purified preparation ( Fig. 2A). Interferon obtained from the acid-urea gel is already highly purified and the pure protein is isolated in the final step by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purity of the final product in Figs. 2 and 3 was examined by Coomassie blue staining as well as by radioiodination with the Greenwood-Hunter or the Bolton-Hunter reagent (11,12). In the case of radioiodination by the Bolton-Hunter reagent, it was possible to correlate the biological activity of interferon with its radioactive label.
Homogeneity, Polydispersity, and Monodispersity-The definition of pure interferon requires some discussion. A pure or homogeneous preparation usually is one which consists of identical molecules, but this is an uncommon situation even with highly pure native proteins. For example, several pure proteins possess more than one isoelectric point. Evidently, this is the rule rather than the exception for most glycoproteins (14). Consequently, a protein preparation can be pure and yet polydisperse, meaning the molecules in the prepara- tion have the same overall chemical structure but vary in detail with respect to shape, size, or charge. Thus, in current glycoprotein purification literature, the term homogeneity refers to the monodispersity of a protein with respect to a specific parameter such as the molecular weight and a net charge under certain conditions. The purity of most interferon preparations until now has relied on the demonstration of interferon activity with a monodisperse population of molecules of the same molecular weight. In this study, we extend the criterion of homogeneity to its electrical charge in an acidurea gel. In this system, the antiviral activity of interferon can be either eluted from a polyacrylamide gel column (Uniphor LKB, Sweden) with an RF of 0.44 or excised from a slab gel (I in Fig. 2A). In either case, electrophoresis in the second dimension shows the fraction containing interferon activity to be a protein of 20,000 molecular weight (Figs. 2 and 3). A similar co-migration of interferon activity with protein can be demonstrated by gel electrophoresis at higher pH values (10) but the protein fraction is diffuse indicating a degree of polydispersity in its net charge at alkaline pH. In these studies, we purified sham-induced interferon preparations to determine whether a protein of the same molecular weight as interferon is obtainable. We regard this to be an important control to eliminate the possibility that another protein may have co-purified with interferon. The control preparation was obtained by treating cells with 2.5 pg/ml of actinomycin D for 45 min in the absence of poly(r1). poly(rC) and cycloheximide and harvesting the culture medium 24 h later. After this treatment, many endogenous cell proteins are released into the medium as also occurs during actual interferon production, but the amount of interferon produced is only 0.01% that of the induced cells. Purification of the shaminduced preparation did not yield an equivalent M, = 20,000 component with the charge characteristics of the protein isolated from the medium of induced cultures (Fig. 4), indicating that the putative interferon is derived only from induced cells and is unlikely to be a serum or endogenous cell protein or a contaminant introduced during the purification The molecular weight of both biological activity and labeled protein is 21,000, slightly higher than the unlabeled and radioiodinated (Greenwood-Hunter) M, = 20,000 concanavalin A fragment used as a marker (not shown). An unassayed amount of ""I-labeled interferon was applied on the gel. The specific radioactivity of the interferon extracted from the gel was 93.3 dpm/unit of antiviral activity. procedure.
In contrast, purification of an interferon preparation yielded two ll& = 20,000 protein fractions containing human interferon activity in the second dimension (Fig. 4). The two distinct protein fractions are identified in the gel as a2 and 1'. Fraction a2 is derived from the solubilization of aggregated protein designated a at the top of the acid-urea gel and fraction 1' is derived from the protein fraction 1 of the acid-urea gel. These data strongly indicate that the purified product of M, = 20,000 having antiviral activity is interferon.
Recouery-There is a loss of 60 to 90% of interferon activity during electrophoresis in acid/urea gel. This loss is recoverable by simply adding sodium dodecyl sulfate to the inactivated interferon such that the final concentration of sodium dodecyl sulfate is 1%. The rescue of interferon activity by sodium dodecyl sulfate is an empirical observation for which no suitable explanation is currently available from this laboratory. Bearing this inactivation and reactivation of interferon in mind, the overall yield of pure interferon by this procedure is 10% or better (Table I) Amino Acid and Amino Sugar Composition of Interferon-Two different methods were used to prepare the pure interferon for acid hydrolysis (see miniprint).
The results of these analyses are given in Table II. The amino acid compositions derived by either method of sample preparation are compa-Berthold et al. (10). The labeled protein was subjected to electrophoresis in a 12% polyacrylamide gel as in D. Kodak X-omatic film was exposed to the dried gel for 12 h and the resulting autoradiograph is presented.
The molecular weight of the radioiodinated interferon was found to correspond to that of the 20,000 concanavalin A fragment (not shown) used as a molecular weight marker. rable (see Table II and miniprint). The analyses agree with previous observations on the physicochemical properties of fibroblast interferon. For example, hydrophobic amino acids (valine, phenylalanine, leucine, and isoleucine) are relatively abundant (30%) which is consistent with the well known hydrophobicity of the molecule. Fibroblast interferon also contains a moderate amount (15%) of the basic amino acids histidine, lysine, and arginine. These residues would provide a relatively strong net positive charge at low pH. This is what we observe since interferon migrates faster than other proteins in the acid-urea gel system ( Fig. 2A).
Although the two-dimensional gel system should separate concanavalin A and its fragments from interferon, it is possible that a small amount of the M, = 20,000 fragment is still present. To test this possibility, we isolated this protein from a sodium dodecyl sulfate-polyacrylamide gel and subjected it to amino acid analysis. The results idicate that its primary structure is very different from that of interferon (Table II). For example, the M, = 20,000 concanavalin A fragment is rich in serine and threonine, contains tryptophan, and lacks cysteine. In contrast, our interferon preparation contains cysteine and lacks tryptophan.
This strongly suggests that our preparations are free of concanavalin A contaminants.
When human interferon was analyzed on the Beckman amino acid analyzer, a ninhydrin-positive fraction which did not correspond to an amino acid residue was eluted from the column shortly after tyrosine and phenylalanine.
However, this peak did coincide with the galactosamine and the mannosamine standards which elute so close to each other that they overlap. This observation suggests that interferon contains galactosamine and/or mannosamine. These amino sugars are derived from either hexosamine and/or N-acetylhexosamine linked to interferon protein. These results are qualitative since the acid treatment carried out to hydrolyze the protein results in some degradation of amino sugars. Such degradation is known for other glycopeptides and glycoproteins hydrolyzed under conditions used for amino acid analysis of proteins (14). In order to quantitate the amino sugar content of interferon, it will be necessary to select conditions of acid hydrolysis conducive to the optimal release of amino sugars with minimum degradation.
In addition, other analytical procedures must be tested to independently determine interferon's N-acetylhexosamine and sialic acid content (18).  ' Cysteine residues were converted to cysteic acid by performic acid oxidation before acid hydrolysis. ' The glycine residues are not integrated due to the high glycine ' These ninhydrin-positive fractions correspond to either galactosbackground which originates from the incomplete removal of Tris/ amine or mannosamine and the given value was obtained when about glycine by extensive dialysis. 7 pg of interferonh was analyzed. I Methionine value includes that for methionine sulfone.
tecting protein contaminants that might not be shown by staining with Coomassie brilliant blue. Iodination of interferon by the Greenwood-Hunter reaction resulted in a loss of 80 to 90% of activity during the oxidative step and a total loss of activity during the subsequent iodination of the tyrosyl residue. This observation led us to describe the importance of the tyrosine residue for interferon activity (13). Presently, the possibility that the oxidation of other amino acid residues such as cysteine, methionine, or tryptophan residues contributes to the loss of activity during the Greenwood-Hunter labeling of interferon is considered. The lack of tryptophan in interferon (Table II) and resistance to cyanogen bromide (Fig.  5) eliminate the possibility that tryptophan and methionine are involved. Treatment with iodoacetamide or N-ethylmaleimide did not result in any loss of interferon activity indicating that free sulfhydryl groups are not involved either. Exposure to reducing agents like mercaptoethanol and dithiothreitol did not always inactivate interferon, but reduction with mercaptoethanol or dithiothreitol followed by blocking the resulting free sulfhydryl group(s) with iodoacetamide did produce a loss of interferon activity (Table III). A further loss of activity occurred when urea-treated interferon was exposed to dithiothreitol and then blocking reagent (Table III). These results suggest that S-S bonds in addition to tyrosyl residue(s) (13) play an important role in maintaining an active conformation for interferon, a hypothesis consistent with the presence of cysteine residues in interferon (Table II).
Not all radioiodination results in the destruction of its biological activity. Interferon can be radioiodinated with minimal loss of biological activity by acylating terminal amino groups with Bolton-Hunter reagent. This reaction conjugates the N-hydroxysuccinimide ester ofp-hydroxyphenylpropionic acid to the lysyl residue(s) of the interferon protein. For each lysyl residue acylated, one would expect the molecular weight of interferon to increase by 380. The iodinated interferon was found to have a slightly higher molecular weight of 21,000 instead of a molecular weight of 20,000 characteristic for both unlabeled interferon and ""I-tyrosyl interferon labeled by the Greenwood-Hunter reagent (Figs. 2 and 3). This increase in the molecular weight of '*"I-lysyl interferon may be the result of the relative abundance of lysyl residues (Table II) as well as the size of the acylating group in the labeled interferon molecule. The ['2"I]lysyl-labeled interferon is 61 times more effective in inducing an antiviral state in human skin fibroblast trisomic for human chromosome 21 than in fibroblasts which are monosomic for this chromosome. The chromosome 21 dosage effect on the antiviral action of interferon was reported with respect to the mapping of the human interferon activity gene to this chromosome (15,16). The retention of such a dosage effect in the ""I-lysyl interferon is indicative that the lysyl residues are not critical for its activity. No doubt, there is always some loss of activity when pure interferon is labeled with the Bolton-Hunter reagent. However, the same amount of activity is lost when a similar quantity of pure interferon is processed in a sham label control indicating the observed loss is due largely to a physical one when a small amount (4 pg) of pure interferon is handled. Furthermore, the fact that biological activity was found to co-migrate sharply with the ""I-1ysyl interferon during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have a slightly higher molecular weight of 21,000 than the unlabeled interferon (20,000) suggests that radioiodination by Bolton-Hunter reagent does not abolish biological activity (Figs. 2 and 3).
Since interferon is a scarce commodity, the synthesis of a This molecular weight value is 20,000 based on the migration of concanavalin A and its fragments which served as standards.    (Table  II) should be useful in determining the composition of a radioactive amino acid mixture to use in the labeling of interferon in uiuo as well as in uitro.
The preparation of a radioactive interferon with high radio specific activity will be most useful in the microsequencing of interferon.
i 0. lntederon wa;;luted by Sblution e and before applying ,t t; a concanavahn .&Sepharose (Con A, column (1". 211 the preparatl"" was ad,usted to 20 mu sodium acetate, pH 4.35. The WC-purified interferon was appbed to a small Con A column (3 ml, at a "ow rate weight reagents and radioactive iodme. The dmlyzed radioiodmated interferon was lyophdizrd to dryness. Hadioactwity measurements of the iodinated proteins were performed by auloradmgraphy usmg Kodak RP-Somatic paper. Radioiodination was also performed by the method of Greenwood and Hunter as previously described by Berthold et al. (10).
Amrno Actd Analyses-Batches of purified mterferon contammng about 10 pg of protein were obtained from each batch of crude harvest. Two different methods were used to prepare samples for ammu acid analyses. AU reagents used were of analytwd grade and steps were taken to prevent the powble contamination of the solutions by extrsneou~ amino acids and/or proteins One method was to extract interferon from the gel K3.75 $1 by homogenizmg the gel in a Ilounce homogenizer wth 1 ml of 1% sodurn dodecyl sulfate (&o-Had, Ca., electrophoresis grade). The extract was dialyzed agam~t a 1000.fold volume of 0.001% sodium dodecyl sulfate for 9 days with a change of distilled water dady. The dialysis tubing was prepared by boiling m I% sodum bicarbonate and 1% EDT& washing m 1% sodium dodecyl sulfate, and thoroughly rinsing m glass-distdled water to remove as much of the trace amino acids that may be present m thu matenal In spite of these preeaut~~s, analysis of control gel eruaets showed that small amounts of amino aads and a significant amount of glycme persist (Table II). The high glycine background IS most likely due to Tris/glycme buffer in the gel. The other amino acid residues are probably derived from a combination of sources such a the polyacrylanude gel, extraction buffer. and dialysis tubing Alternatively. interferon was fued in the gel by metbanol:glaeial acetic acidwater. 5:~. and stained with Coomassie brilliant blue. The gel (0 75 $1) eontaming the staned protein was thoroughly washed in 7s acetic acld and then in glass-distilled water. The gel was then hydrolyzed in 6 N HCI. Thu procedure WBS found to yield an extremely clean background in control gel not containmg protem, though a small amount of glycmr persisted.
The amino acid composition of a protein of known sequence was determined by the above method usmg an equwalent amount of hma bean trypsin mhtbltor. The resulting valur~ are in close agreement with the prewously reported composition of this protem (22) indicating that the method of analysts used here should be r&able for interferon as well.