Jan 17, 1992 - From the Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration,. Bethesda, Maryland ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 267, No. 21, Issue of July 25, pp. 15210-15216,1992 Printed in U.S.A.
Purification and Characterizationof Multiple Components of Human Lymphoblastoid Interferon-a* (Received for publication, January 17, 1992)
Kathryn C. ZoonS, Dorothea Miller, Joseph Bekisz, Dorothy zur Nedden,Joan C. Enterline, Nga Y. Nguyen, and Ren-qui Hu From the Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
Twenty-two components of human interferon-a (IFN-a) derived from Sendai virus-induced Namalwa cells werepurified by sequential immunoadsorbent affinity chromatographyusing four monoclonal antibody affinity columns followed by ultrafiltration and reversed-phase high-performance liquid chromatography. The specific activity ranged from 0.2 to 2.6 x lo8 IU/mg protein on Madin-Darby bovine kidney cells, 0.3 to 4.6 X lo* IU/mg protein on human WISH cells, and lo4 to 7 x lo5 units/mg protein on mouse L929 cells. The apparent molecular weights of the components ranged from 17,500 to 23,300 using nonreducing sodium dodecyl polyacrylamide-gel electrophoresis and 17,500 to 27,600 using reducing sodium dodecyl polyacylamide-gel electrophoresis. The amino-terminal amino acid sequences were similar among the components as well as to those reported for the cloned human IFN-a genes (Pestka, S. (1986) Methods Enzymol. 119, 3-14). However, four components, f, i, 1, and m, have amino-terminal amino acid sequences which appear to be unique when compared to those predicted from the cDNA clones. One component, prea, has a potential N-linked glycosylation site on the Asn of residues 2 through 4, Asn-Leu-Ser.
from Sendai or Newcastle disease virus-induced Namalwa (210) and KG-1 (11) cells and peripheral blood lymphocytes from normal individuals (12-16) and those with chronic myelogeneous leukemia (CML) (17) has been achieved. Preliminary evidence suggests that some of the species maybe glycosylated (7, 18-20). It appears that the majority of the natural human IFN-as,like those derived from IFN-a cDNA clones, have 165-166 amino acid residues; however, two species which are truncated at thecarboxyl terminus (155 residues) have been isolated from CML peripheral blood lymphocytes (17) and one species, Interferon Omega 1 (IFN-wl), is approximately 60% similar based on amino acid sequence to the IFN-a family and is larger than members of the IFN-a family, 172 amino acids (21, 22). This report describes the extensive purification and characterization of 22 components of human IFN-a derived from Sendai virus-induced Namalwa cells. The purification scheme is comprised of sequential immunoadsorbent affinity chromatography, using four monoclonal antibody affinitycolumns which recognize different epitopes, followed by ultrafiltration and reversed-phase high performance liquid chromatography (HPLC). Theindividual components have been analyzed for their apparent molecular weights, amino acid compositions, amino-terminal amino acid sequences, and antiviral specific activities.
Interferon-a (IFN-a)’ is one of three major classes of IFN that exhibit antiviral, antiproliferative, and immunomodulatoryactivities (1). The ability to purify and characterize naturally derived IFN-a is still challenging because 1)limited amounts areproduced by cells in response to inducers and 2) IFN-a consists of a large family of structurally similar proteins/glycoproteins. The successful purification of several species of natural IFN-a has been achieved (1-16).However, additional studiesare essential to isolate and characterize the constituents derived from various combinations of cell types and inducers in order to understand the complexity and the biological roles of the IFN-amolecules. Partial characterization of natural human IFN-a derived
MATERIALS ANDMETHODS
Interferon-Preparations of human IFN-a derived from Sendai virus-induced Namalwa cells were obtained from Wellcome Biotechnology Ltd., Beckenham, Kent, England. The antiviral specific activities of the preparations ranged from 2.7 X lo6 (human serum albumin in the preparation) to greater than 1 X 10’ (absence of human serum albumin) IU/mg protein. These IFN preparations were produced and partially purified as previously described (3, 5, 7). Interferon Assays-Antiviral activity was determined as previously described using Madin-Darby bovine kidney (MDBK) cells (ATCC CCL22), WISH cells, a human amniotic cell line (ATCC CCL25), and/or mouse L929cells (23). The assay includes vesicular stomatitis virus as the challenge virus. All IFN units areexpressed with reference to the National Institutes of Healthhuman lymphoblastoid IFN standard Ga23-901-532. SDS-PAGE-SDS-PAGE was performed using a 16% acrylamide, * The costs of publication of this article were defrayed in part by N,N’-methylenebisacrylamideseparating gel and a 3.7% acrylamide, the payment of page charges. This article must therefore be hereby N,N’-methylenebisacrylamidestacking gel by a modification of the marked “advertisement” in accordance with 18 U.S.C. Section 1734 Laemmli method (24, 25). Samples (0.5-5 pg of protein) were heated at 100 “C for 2 min in electrophoresis-solubilizing solution in the solely to indicate this fact. To whom correspondence should be addressed Division of Cy- presence or absence of 3% P-mercaptoethanol. The gels were then tokine Biology, Ctr. for Biologics Evaluation and Research, Bldg. stained and destained as previously described (2). The protein molec29A, Rm. 2D20,8800 Rockville Pike, Bethesda, MD 20892. Tel.: 301- ular weight markers included bovine serum albumin (66,2001, ovalbumin (42,699),carbonic anhydrase (31,000), soybean trypsin inhib496-8245; Fax: 301-402-1659. ‘The abbreviations used are: IFN-a, interferon-a; HU, human; itor (21,500), lysozyme(14,400) or myoglobin(17,000), and cytoPBS, phosphate-buffered saline; HPLC, high-performance liquid chrome c (12, 400). MonoclonalAntibodyAffinity Columns”NK2 monoclonal antichromatography; MDBK, Madin-Darby bovine kidney; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IFN-wl, body-Sepharose 4B was obtained from Celltech, Berkshire, United Kingdom. The bed volume (2-4 ml) varied according to the amount interferon-wl.
15210
Purification and Characterization of Human Interferon a of IFN applied to the column. III/21 (1gGl/~)was made against a synthetic fragment of IFN-a1 as previously described (26, 27). This antibody was purified by protein A-Sepharose and DEAE-Sepharose prior to coupling with cyanogen bromide-activated Sepharose 4B. This antibody recognizes residues 151-165of human IFN-a2 (27). Bed volume of the III/21 column was 5 ml. The M2Q-098 monoclonal antibody was obtained from Hybritech, Inc., San Diego, CA. This antibody was coupled to Affi-Gel 10 and the bed volume was 6 ml. The 4F2 monoclonal antibody made against consensus IFN-a coupled to Sepharose 4B was obtained from Amgen, Inc., Thousand Oaks, CA. Sequential Zmmumdorbent Affinity Chromatography-The NK2-Sepharose 4B column was washed with two bed volumes of citrate buffer, pH 2 (0.067 M citric acid and 0.2 M NaCl) followed by 10 bed volumes of phosphate-buffered saline (PBS; 7.75 g of NaCl, 0.54 g of KHzP04,2.14 g of NazHP04in 1 liter of water). Approximately 1-3 mg of human lymphoblastoid IFN-CYwas applied to the column and the flow-through was collected. The column was then washed sequentially with 10 bed volumes of PBS, eight bed volumes of 0.5 M NaCl, 0.025 M Tris, pH 7.5, containing 0.2% Triton x-100, two bed volumes of 0.15 M NaCl containing 0.1% Triton X-100, four bed volumes of McIlvaine’s citrate phosphate buffer, pH 6 (0.024 M citric acid and 0.051 M NazHP04), and four bed volumes of McIlvaine’s citrate phosphate buffer, pH 5 (0.018 M citric acid and 0.064 M NazHP04). The column was eluted with four bed volumesof citrate buffer, pH 2. The ODm was monitored, and theeluate corresponding to the peak was collected. The pH 2 eluate was neutralized to pH 7 with 1 N NaOH immediately after collection. The column was then washed with 10 bed volumes of PBS followed by the application of the flow-through to thesame NK2 column. The procedures described above for washing and elution was followed for the second NK2 column chromatography as well as for the III/21 affinity column chromatography (twice) and thenonce for the M2Q-098 and the4F2affinity column chromatography, respectively (Fig. 1). HPLC-The neutralized pH 2 eluates from the immunoadsorbent affinity columns were concentrated by ultrafiltration using an Amicon Micro-Volume Stirred Ultrafiltration Cell fitted with a YM-10 (25 mm) membrane and dialyzed against 0.1% trifluoroacetic acid. A Waters HPLC system equipped with two UV detectors monitoring 214 and 280 nm was usedin conjunction with a Vydac reversed-phase C4 column to fractionate the IFN-a components. IFN (5-50 pgof protein) was applied to thecolumn; the column was then eluted with linear gradients made up of 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid for each of the pH 2 eluates as shown in Fig. 2. The flow rate was 1.0 ml/min. The ODzl4peaks were collected manually and thenadjusted to neutral pH with 0.1 M sodium bicarbonate. Protein Determination-Protein concentration was determined by amino acid analysis for the components isolated by HPLC (Applied Biosystems Model 420 A/H derivatizer with automated hydrolysis), by Micro BCA Protein Assay for the starting material and affinity column fractions, and by using bovine serum for the standard curve (Pierce, Rockford, IL). Amino-terminal Amino Acid Sequencing-IFN-a components were HUMAN LYMPHOBLASTOID IFN-ALPHA PREPARATION
PASS THROUGH
PASS THROUGH
UiVafiIQath
ultrafihration
PASS THROUGH
FIG. 1. Purification scheme for the multiple components of human lymphoblastoid IFN-a.
15211
sequenced by Edman Degradation using a Model 475A protein sequencer (AppliedBiosystems) using the 03PRO program. The phenylthiohydantoin amino acid derivatives were analyzed by a Model 120A HPLC (Applied Biosystems) using a 5% tetrahydrofuran/acetonitrile buffer system. Data reduction and analysis were performed using an Applied Biosystems Model 9OOA data reduction system. The amino acid sequences were compared to those found in the Genetics Computer Group Sequence Analysis Software Package (National Institutes of Health, Bethesda, MD). RESULTS
Purification The purification of human lymphoblastoid IFN-a! by sequential immunoadsorbent affinitychromatography is shown in Table I. The antiviral specific activity of the pH 2 eluates of the NK2, 111/21, M2Q-098, and 4F2 monoclonal antibody affinity columns ranged from 2.0 to 2.2 X lo8 IU/mg protein using MDBK cells. Similar specific activities have been reported for other preparations of human lymphoblastoid IFN(Y (2-4, 6-10). Total recovery of applied antiviral activity derived from the four affinity columns ranged from 88 to 120% with the mean being approximately 98%. The nonreducing SDS-PAGE profile of the pH 2 eluates are shown in Fig. 3. A minimum of three bands were observed for the NK2 column eluate between the apparent molecular weights of 17,500 and 22,000, while only one major band was observed for each of the eluates of the 111/21, M2Q-098, and 4F2 columns. They exhibited apparent molecular weights of 17,500, 17,500, and 18,000, respectively. The samples were concentrated and dialyzed by ultrafiltration with no apparent loss in the antiviral activity. The concentrated and dialyzed pH 2 eluates were subsequently fractionated by reversed-phase HPLC (Vydac C4) (using gradients of the solvents, 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid). Fig. 2 shows the HPLC profiles of the pH2 eluates and thegradient conditions employed. The antiviral specific activities of the peak fractions were determined using MDBK cells, and the recovery of the antiviral activity is shown in Table 11. Eight major peaks were identified as pre-a, a, b,b’, c, d, e, f, and two minor peaks were identified b“ and pre-c in the pH 2 eluate of the NK2 column (Fig. 2 A ) . The antiviral specific activities of the major isolated peaks ranged from 1.6 to 2.3 X lo8 IU/ mg protein as shown in Table 11. The two minor components, b” andpre-c, had specific activities of 0.7 x lo8 IU/mg protein and 0.2 X lo8 IU/mg protein, respectively. The average recovery of antiviral activity from the reversed-phase HPLC column was approximately 92%. The pH 2 eluate of the III/21 monoclonal antibody affinitycolumn yielded five major peaks on HPLC analysis, g, h, i, j, andk, and two minor peaks, preg and pre-j (Fig. 2B). The antiviral specific activities of the major peaks on MDBK cells ranged from 1.9 to 2.2 X 10’ IU/ mg protein, and theminor peaks had specific activities of 1.2 x 10’ IU/mg and 0.3 x 10’ IU/mg, respectively. The recovery of these components from the C4column averaged 83% (Table 11). Two peaks were isolated by reversed-phase HPLC from the pH 2 eluate of the M2Q-098 column, 1 and m (Fig. 2C). The antiviral specific activity (MDBK cells) of those two peaks were 1.3 and 1.5 X lo8 IU/mg protein. The recovery of the antiviralactivity from the M2Q-098 monoclonal antibody column was the lowest, 67% (Table 11). It was critical to perform HPLC analysison this pH 2 eluate immediately following concentration and dialysis by ultrafiltration to minimize the formation of a precipitate which can lead to poor yields. The pH 2 eluate from the 4F2 monoclonal antibody affinity column yielded three peaks, n, 0,and p, when analyzed by HPLC (Fig. 20). The antiviral specific activities of the
onitrile
Purification and Characterizationof Human InterferonCY
15212
-50-
Panel C 0.6-
--!
r”
0.5-
*
”-”
0.4-
“ 0 5 ) -40
”””
m
3
I
-30
R
-10
f
-m B2.
0.3-
=.
FIG.2. High-performanceliquid chromatography of the pH2 eluates derived from the monoclonal antibody affinity columns. A, NK2 pH 2 eluate; III/21 pH 2 C, M2Qeluate; R, 098 pH 2 eluate;D, 4F2 pH 2 eluate. The Vydac C4 columnis developed witha 0.1% trifluoroacetic acid and 0.1% trifluoroacetic gradient as shown lines).(dashed The absorbance is monitored at 214 nm (solid lines).
Minutes
Minutes
Panel B
Panel D
0.7-
”/_/”
0.6-
0.5-
2
I
I
0.3-
/
/
mI
1
40
60
0
I
TABLE I Purification of HuIFN-cu from Namalwa cells by monoclonal antibody-affinity chromatography
IU X
Specific activitpb IU/mgprotein
mg
10’
24.8
8.1 X
1
2
3
4
TABLEI1 High-performance liquid chromatography of the p H 2 eluates of the monoclonal antibodv affinitv columns IFN-n component
NK2 pH 2 eluate pre-a a b b’ b” pre-c
60 25 7 6
C
d e f Total
5
III/21 pH 2 eluate U pre-g g h
68-
1
43-
pre-j 1
k Total
Each peak is designated as component pre-a, a,b, etc.
X
2.3 1.8 2.1 2.3 1.7 0.9 0.2 2.2 1.6 2.2 1.7 1.8 1.2 2.0 1.9 2.1 0.3 2.0 2.2
M2Q-098 pH 2 eluate 1 m Total
2.0 1.5 1.3
4F2 pH 2 eluate n
1.9 2.0 2.6 1.6
IO-’
Recoveryb %
8 28 22 4 2 0.3 11 6 5 6 92.3 6 18 15 12 3 16 13 83 22 45 67
9 54 7 P 70 Total a Activity was determined on MDBK cells; specific activity based on the average of seven to nine experiments forNK2 and III/21 derived components, four experiments for M2Q-098 derived components, and three experimentsfor 4F2-derived components. Recovery was based on the antiviral activity and represents the average of six experiments for NK2 and III/21 derived components, three experiments for M2Q-098 derived components, and two experiments for 4F2-derived components. 0
collected peaks ranged from 1.6 to 2.6 X lo8 IU/mg protein (MDBK cells) asshowninTable 11; the recovery of the antiviral activity from the C4 column was 70%.’ Although
Specific activity”
IU/mg
%
lo6
FIG. 3. SDS-PAGE o f p H 2 eluates from monoclonal antibody affinity columns.Lane I , molecular weight standards; lane 2, NK2 pH 2 eluate; lane 3, III/21 pH 2 eluate; lane 4, M2Q-098 pH2 eluate; lane 5, 4F2 pH2 eluate. The higher molecular weight bands observed in lane 2 are an artifactof the SDS-PAGE.
30
Minutes
Recovery Per step‘
pH 2 eluates NK2 1.2 X 10’ 0.60 2.0 X 10’ III/21 5.1 X lo7 0.26 2.0 X lo8 M2Q-098 1.3 X lo7 0.06 2.2 X 10’ 1.1 X 107 0.05 2.2 X lo8 4F2 Activity was determined on MDBKcells. * Values represent an average of five experiments. e Recovery based on antiviral activity.
mI
10
Minutes
2.0
9
3
/
0
Starting material
0
0
1
0 0.2-
Antiviral activity”.’
G o-
4 s
-40
I-”
/ec
0.4-
Step
-
-0
‘
Purification and Characterization of Human Interferon qualitatively each major peak was present in every batch, variations in the quantity of each peak were observed for each of the lots of pH 2 eluates. However, components a and b were consistently the most abundant components in all preparations examined. Component o exhibited the greatest antiviral specific activity on MDBK cells.
4F2
M, x10-3
p; I
!
1
4 3 - 1
Apparent molecular weight IFN-a Nonreducing Reducing component SDS-PAGE SDS-PAGE
NK-2 pre-a a
b b’ b” pre-c C
d e f 111/21 pre-g g
h
1
?re-j J
k
M2Q-098 1
m 4F2 n 0
P
27,600 21,300/18,000 21,600/18,000 22,700 24,000 19,000 20,000 18,000 19,000 27,000
23,300 20,200 19,300 19,700 19,700 17,500 17,500 17,500 17,500 20,500
22,500 18,300 18,300 18,300 21,500 17,500 18,300
18,500 17,500 17,500 17,500 17,500 17,500 18,000
21,400 21,400
18,000 18,400
18,300 19,000 19,000
19,300 18,500 17,800
o
p
pq
e
3
4
, I
I
.; . I,
Chemical Characterization
TABLE I11 Apparent molecular weightsof human lymphoblastoid interferon-as
n
68--,
,
Apparent Molecular Weights-The individual components isolated by reversed-phase HPLC were analyzed for purity, and their apparent molecular weights were determined by SDS-PAGEunder reducing and nonreducing conditions (Table 111). Analysis using nonreducing conditions resulted in a single band for each component. However, doublets were observed by SDS-PAGE using reducing conditions for components a (21,300 and 18,000) and b (21,600 and 18,000); all other components migrated as single bands. As shown in Table 111, the apparentmolecular weights under nonreducing conditions range from 17,500 (pre-c, c, d, e, g, h, i, pre-j, and j) to 23,300 (pre-a) with only pre-a, a, and f having apparent molecular weights greater than 20,000. The mobility differences between some of the similarly sized proteins are illustrated in Fig. 4. Under reducing conditions, the apparent molecular weights ranged from 17,500 (j) to 27,600 (pre-a). A majority (17/22) of the components had apparent molecular weights in the range of 17,500 to 21,600. The exceptions were pre-a, b’, b”, f, and pre-g which hadapparent molecular weights of 27,600, 22,700, 24,000, 27,000, and 22,500, respectively. Amino-terminal Amino AcidSequences-The amino-terminal amino acid sequences for the 22 components of human lymphoblastoid IFN-a are shown in Fig. 5. All but one component (8) analyzed yielded a single major sequence. The repetitive yield for the amino-terminal amino acid sequence analyses ranged from 95 to 99% . The X values at positions 1 and 29 probably represent cysteines as deduced from the
15213
a
1712.4-
,
,
1
2
FIG. 4. Nonreducing SDS-PAGE of IFN-a components from the 4F2 monoclonal antibody affinity column. Lane 1,4F2 pH2 eluate; lane 2, IFN-a (n);lane 3, IFN-a ( 0 ) ; lane 4, IFN-a (p).
cDNA sequences (28-31). The sequences obtained for these components were similar to those reported for other recombinant and natural human IFN-as as shown in Table IV. However, four components, f, i,l, and m, have amino-terminal amino acid sequences that, while similar in their sequences to otherhuman IFN-as, appear to be unique in that they are not identical to those found in the Genetics Computer Group Sequence Analysis Software Package obtained from the National Institutes of Health (Table IV). Components a and b which are themost abundant forms of human IFN-a are thus far indistinguishable from IFN-a2b. Of interest, pre-a, which is thus far identical to the IFN-aH/a14 gene, has thesequence Asn-Leu-Ser (residues 2 through 4) in which the Asn is a potential site for N-linked glycosylation. This is also the component which has an apparent molecular weight of 27,600 (reducing SDS-PAGE), thus suggesting that this component may be glycosylated. Biological Characterization Species Specificity of the Human IFN-a Components-As shown in Table V, the antiviral specific activity varies with the cell line used in the assay system. The specific activities of the major components on MDBK cells are similar (within 2-fold) with a range of 1.3 X IO8 IU/mg protein (m) to 2.6 x lo8 IU/mg protein (0).The minor components b”, pre-c, preg, and pre-j had lower specific activities on MDBK ranging from 0.2 to 1.0 X 10’ IU/mg protein. In contrast to the results on MDBK cells, the major components exhibited a broader range (15-fold) of specific activities, 0.1 X 10* IU/mg protein (pre-j) to 4.6 X 10* IU/mg protein (o), on the human amnion cell line WISH. The ratios of the specific activities, MDBK to WISH, vary from 0.1 (pre-c) to 10 (pre-j). The human IFN-a components aremuch less active (approximately 1000fold) on the murine cell line, L929, thus supporting the concept of species specificity. It is interesting that the range of specific activities on the L929 cells was very broad (70fold) from less than IO4units/mg protein (m) to 7X IO5units/ mg protein (pre-a).Several components isolated from the III/ 21 monoclonal antibody column exhibited relatively high specific activities on the L929 cells (pre-g, g, j, and k). Likewise, the range, 257 (pre-a) togreater than 23,000 (b), of the ratios of the MDBK to L929 specific activities are also very broad.
Purification and Characterization of Human Interferon a
15214
FIG. 5 . Amino-terminal amino acid sequence of the human lymphoblastoid interferon a. -, identity; X,unknown. IFN-a88 is the amino acid sequence deduced from the cDNA of human lymphoblastoid IFN-a from Namalwa cells (35).
TABLE IV Correlation of N-terminal aminoacid sequences of Namalwa humaninterferon components to those predicted from nucleic acid sequences Search run using Genetics Computer Group/Sequence Analysis Software Package, Convex system, DCRT, NIH, Bethesda, MD. IFN-a components
pre-a a b b’ b“ pre-c C
d e f g
sequences sequences Components acid nucleic Known acid nucleic Known
H, a 14 a-2 a-2 as 8,4b,F,16,1,b,88,1fand WA gene as F,16,1,4b,f,1’,88and WA gene as C,F,8,1,16,4b,1’,88and WA gene as F,1,1’,88 as F,1,88,1’ as l,F,1’,88 None Not evaluable a
h 1
pre-j J k
1 m n 0
P
a D and a-1 None 01 D, a-1 01
D, a-1 D, a-1
None None as 8,4b,16,1,F,b,1’,88and WA gene as F,1,1’,88 as 8,B
analysis, since this type of doublet formation was observed with purified recombinant human IFN-a2 upon this type of The purification of a mixture of human IFN-as derived .~ further examination, two-dimensional analfrom Sendai virus-induced Namalwa cells have resulted in the a n a l y ~ i s Upon yses of components a and b (Protein Databases, Inc., Hunisolation of 22 IFN-a components. Immunoadsorbent affinity tington Station, NY) yielded multiple spots at the indicated chromatography using four monoclonal antibodies with apapparent molecularweights (18,000-21,600) between pH 5 parently differentrecognition sites for human IFN-a, incom- and 7. Again, although the nature of the heterogeneity is bination with reversed-phase HPLC, hasproven to be a very currentlyunknown,such microheterogeneitycould result powerful procedure to accomplish this separation. This apfromincomplete fractionation of theIFN-aconstituents, proach may have a much broader application for the identi- minor modifications to the IFNs during isolation, e.g. deamification and isolation/characterization of other families of dation and/orglycosylation and/or an artifact of the analysis. structurally related proteins. Most of the isolated human IFNThe aminoacid composition of each componentwas detera components yield single bands by reducingand nonreducing mined(datanotshown).The compositions were similar SDS-PAGE with the exception of components a and b which, amongthemselvesas well as tothosereported for other under reducing conditions, appear to migrate as doublets. natural and recombinant DNA derived human IFN-a prepaThese doublets may be the result of incomplete separation of rations; all the components exhibiteda large number of gluthe IFN-a gene products or posttranslational modifications. tamic acid/glutamine andleucine residues (20, 28). Examination of the amino-terminal amino acid sequences Our preliminary analysesof the carbohydrate contentof the IFN-a components suggests that pre-a, a, andb are glycosy- of the 22 components of IFN-a are similar to those reported lated; thus, thismay contribute to the apparent heterogeneity previously for other human natural and recombinant IFN-a of a and b (33). Another plausible explanation for the heterof the SDS-PAGE S. Pestka, personal communication. ogeneity of a and b is that it is an artifact DISCUSSION
Purification and Characterization of Human Interferon a
15215
TABLEV SDecies soecificitv of human lvmohoblnstoid interferon-a Specific antiviral activity
IFN-a components WISHb
MDBK"
L92gb MDBK/L929 WISH/L929 MDBKIWISH IUfmg X IO-5
IU/mg X IO-'
pre-a a b b' 1.5 b" pre-c C
d e f pre-g g h 1
?re-j
0.1
0.2 0.3 5.0
1.8 2.1 2.3 1.7 0.7 0.2 2.2 1.6 2.2 1.7 0.9 2.0 1.9 2.1
2.4 1.2 1.9 1.0 0.5 0.1
1.5 1.5 1.4 1.8 1.3 2.3 1.2 0.4 0.3 0.1 0.4 0.3 1.1 1.7 0.9 4.6 0.7
1.0 2.0 2.2 1.5 m 1.3 n 2.0 0 2.6 P 1.6 Average of three to nine experiments. * Average of two to four experiments. e ND, not detectable at
