ANAl
Yl ICAL
BIOCtlEMISIR~
Purification
98, ?14-218
(1979)
of Acid Ethanol-Extracted Human Lymphoid Blue Sepharose Chromatography’ JOHN
S.
ERICKSON
Received
AND
March
KURT
lnterferons
by
PAUCKER
9, 1979
Human leukocyte and lymphoblastoid (Namalva) interferons were purified by a modified acid ethanol extraction procedure and chromatography of the dilute ethanolic interferon solution on Blue Sepharose. Both interferons were purified more than lOOO-fold. with recoveries ranging from 32 to 40%.
Preparations of crude interferon often require extensive purification in order to be suitable for a number of applications. In recent years several purification procedures for human interferons have been described in the literature (1- 13). Of these, the acid ethanol extraction of human leukocyte interferon first reported by Fantes (1) and refined by Cantell and Hirvonen (10,ll) has been useful for the initial purification of large amounts of native interferon. However. in our hands. the acid ethanol procedure gave variable results because extensive losses in activity were frequently sustained during precipitation of the interferon from the ethanolic solution. Therefore, we explored other means to recover the interferon from ethanol. Jankowski, et al. (6) previously noted the affinity of human leukocyte interferon for blue dextran. In the present report. we describe the application of Blue Sepharose chromatography in the purification of ethanolextracted human leukocyte and lymphoblastoid (Namalva) interferons. ’ This work was supported in part by Public Health Service Contract NO1 Al 52520 and Grant ROI AI 13649 from the National Institute of Allergy and Infectious Diseases. 0003-26971791130214.05$02.00/O CopyrIght All rights
0 1979 by Academx Pres\. Inc. or reproductmn I” any term rewrved.
MATERIALS
AND METHODS
Interj~rons. Human leukocyte interferon was induced by Sendai virus (14,15), and received in lyophilized form from the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. The specific activity of the crude interferon ranged from 2-3 x 10” units/mg protein, expressed in terms of the international reference standard G 023-901-527 provided by the Reference Reagents Branch of the National lnstitute for Allergy and Infectious Diseases, Bethesda, Maryland. The interferon was dissolved in PBS at the indicated concentration prior to ethanol extraction. Human lymphoblastoid interferon was induced by Newcastle disease virus and obtained from Dr. K. Zoon, Laboratory of Chemical Biology, National Institute of Health, Bethesda, Maryland. The crude interferon had been concentrated by trichloroacetic acid precipitation and was subsequently passed through Sephadex G-25 equilibrated with PBS to remove the trichloroacetic acid (9). The specific activity of this material 1 The Abbreviations used: PBS, phosphate-buffered saline; 0.15 M NaCl. 0.01 M sodium phosphate. 7.0: buffer A. 0.05 M sodium acetate. pH 5.0.
214
pH
PURIFICATION
OF ACID
ETHANOL-EXTRACTED
was in the range of 30 reference unitslmg protein. Both human interferons were stored at -20°C until used. Etllunol extraction. The acid ethanol extraction procedure of Cantell and Hirvonen ( 10, I 1) was employed, except for the modifications indicated in the text. CIlrornLItO!:r.aph?!. All chromatographies were performed with commercial Blue Sepharose CL-6B (Pharmacia Fine Chemicals, Piscataway, N. J.). The Blue Sepharose was swollen for at least 24 h in bulffer A” and equilibrated with at least 10 column volumes of buffer A prior to use. A.ssa~.s. Interferon was titrated on human foreskin fibroblasts (FS-4 strain) in a microplate system as previously described (16), except for the substitution of encephalomyocarditis virus as challenge agent. Protein concentrations were estimated by absorption at 280 nm, by equating an absorbance of 1.O to 1 .O mgiml of protein. RESULTS
AND DISCUSSION
In a typical experiment a loo-ml sample of crude human leukocyte interferon (5000 mg of protein, 2000 reference units of interferonimg of protein) was carried through the acid ethanol procedure descrilbed by Cantell and Hirvonen (10,l I), but with some modifications. As pointed out by these authors, the pH of the ethanolic solution must be raised very slowly in order to avoid coprecipitation of interferon. Indeed during the course of these experiments, we observed that the pH of the largely nonaqueous, unbuffered solution was a poor indicator of the amount of protein remaining in solution. Thus, on different trials, ethanolic preparations which had bleen adjusted to pH 5.5 sometimes retained large amounts of protein, whereas at other times very little protein (and quite variable amounts of interferon) remained in solution. Cantell and Hirvonen (10,ll) introduced two rounds of precipitation-centrifugation in order to circumvent co-
HUMAN
LYMPHOID
INTERFERONS
215
precipitation of interferon while raising the pH. For our purposes we found it advisable to modify slightly the pH adjustments and to monitor the amount of protein remaining in solution after each centrifugation. A Chemtrix pH meter, Model 40E equipped with an HO07 electrode, was standardized at 23°C. at pH 7.0 and 4.0. The temperature compensation control was adjusted to 0” for monitoring the pH of the ethanol extract in an ice bath. The pH was slowly raised to 5.4 by dropwise addition of 0.5 1\; NaOH. The resulting precipitate was pelleted by centrifugation (9000 RPM x IO min. GSA Sorvall rotor). Supernatent protein concentration was estimated by measuring the absorption at 280 nm (A,,,,) of a l/10 dilution in PBS. It was determined that an absorbance reading of A,,,, = 0.3 was optimal for removal of extraneous proteins without coprecipitation of interferon. Hence, if the A 2H,,was greater than 0.3, further precipitation was induced by the dropwise addition of 0.1 N NaOH until the pH reached 5.5. The precipitation-centrifugation cycle can be repeated, if necessary, until the A.,,,, of the l/IO dilution reaches 0.30. The following steps were conducted in the cold. The final supernatent was diluted into 12 vol of buffer A. To obtain rapid mixing, the ethanolic solution was introduced into the buffer from a I-mm-i.d. tubing placed directly over a magnetic stirring bar. The resulting solution containing about 7.5% ethanol, was then absorbed overnight to a column of Blue Sepharose CL-6B, 1.5 x 6.0 cm, at a flow rate of 120 ml/h. The breakthrough fraction was collected as a pool which contained less than 2% of the interferon. The column was then developed with successive washes of PBS, 0.5 hi NaCl, 1.0 M NaCl. and 1.0 M NaCl, 50% ethylene glycol (v/v), each being buffered with 0.01 M sodium phosphate. pH 7.0 (Fig. 1A). In a parallel experiment, a human lymphoblastoid interferon preparation (3000 mg of protein, 33 unitsimg of protein) was subjected to the identical steps (Fig. 2A).
216
ERICKSON
20
AND
PAUCKER
I IO
40
20
30
FRACTIONS
FIG. 1. Chromatogram of human leukocyte interferon on Blue Sepharose. (A) Crude interferon was extracted in acid ethanol. diluted to a hnal volume of 1300 ml, and applied to the column (see text for details). (B) Rechromatography of fractions 7-49 from (A). Fraction size was 6.5 ml except in the ethylene glycol region (4.5 ml). The numbered arrows refer to (I) PBS, (2) 0.5 M NaCI, (3) 1 M NaCI, and (4) 1 M NaCl, 50% (v/v) ethylene glycol, each in 0.01 M sodium phosphate, pH 7.0. Interferon, 0: absorbance, -.
The results for the two types of interferon are summarized in Table I. The data show that a 7- to 15fold purification was achieved by the acid ethanol extraction-precipitations alone while the purification for the pooled fractions from the Blue Sepharose columns was 250- and
515fold, respectively, for the leukocyte and lymphoblastoid interferon preparations. The interferon in the pooled fractions lost little or no activity at pH 7.0 and 4°C for at least I month. From other experimentation, we suspetted that the presence of the ethanol in
IO
IO
30
50
IO
20
30
FRACTIONS
FIG. 2. Chromatogram of human lymphoblastoid interferon on Blue Sepharose. (A) Crude interferon was extracted in acid ethanol, diluted to a final volume of 1000 ml. and applied to the column (see text for details). (B) Rechromatography of fractions 33-55 from (A). Fraction size was 6.5 ml except in the ethylene glycol region (4.5 ml). The numbered arrows refer to (I) PBS, (2) 0.5 M NaCI. (3) 1 M NaCI. and (4) 1 M NaCI, 50% (v/v) ethylene glycol, each in 0.01 M sodium phosphate. pH 7.0. Interferon. 0; absorbance -.
PURIFICATION
OF ACID
ETHANOLEXTRACTED
HUMAN
TABLE
LYMPHOID
INTERFERONS
217
I
PVRIFICAIION OF HUMAN LFUKOCYTF. AND L~MPHOBI.ASTOID~NIERFERONS BY ACID ETHANOI--BLUE SEPHAROSECHROMA~OGRAPHI
Interferon preparation Leukocyte, crude Ethanol, pH 5.5 Blue Sepharose 1. total #14-49 Blue Sepharose 2, total #I6620 Lymphoblastoid, crude Ethanol. pH 5.5 Blue Sepharose I, total #33-54 Blue Sepharose 2. total #I9925
Total volume (ml) 100 100 234 32.5
Units/ ml” 1 x lo” 1 x lo” 2.5 Y IO’ 1 x IO’
100 100
I x lo:’ 6 % IO’
143
5.7 x IO’
45.5
8.6 x lo’
Units total
Protein
(mgiml)
I x 1x10’ 7.4 x 5.8 x 4.2 x 3.3 Y
IO’
1 6 I 8.2 5.7 3.9
IO:’ IO 10’ IO’ IO’ IO’
” Units are expressed in terms of reference standard ” Expressed as percentage of initial concentration.
the applied sample was affecting the elution position of the interferon. Therefore, the pooled fractions of each type of interferon were concentrated by means of a membrane concentrator (PM-10 membrane) to 56 ml (leukocyte) and 50 ml (lymphoblastoid), dialyzed twice against 1 liter of buffer A, for 2 days. and rechtomatographed on a Blue Sepharose column, (1 x 9.5 cm). The corresponding elution profiles are shown in Figures IB and 2B and the data are summarized in Table 1. The elution pattern of both interferons was more sharply defined and that of lymphoblastoid interferon displaced with respect to leukocyte interferon. The bulk of leukocyte interferon (837~~) eluted in 0.5 M NaCl. whereas most of the lymphoblastoid interferon (68%) was desorbed in 1.0 M NaCl. Overall purification in both cases exceeded lOOO-fold. The specific activity of the leukocyte interferon pool was 2.6 x 10” units/ mg protein and thus comparable to values reported by Cantell and Hirvonen ( 10,l 1), except that our starting material was of
x x x x x x
IO” IO” IO” lo”
50 3
Specific activity (unitsimg protein)
Purification
2 % lo:’ 3 x IOJ
15X
0.048
5 * lo”
250 x
0.039
2.6 x IO”
30 2.5
33 240
1.300‘*
7x
0.034
1.7 x IO’
515x
0.025
3.4 x IO’
1.030x
Recovery” (5 1
100 74 58 40 35
60 100 x2 57 39
G023-901-527.
lesser purity. In the case of peak fraction 17 (Fig. lB), purification was 3450-fold and the specific activity 6.9 x 10” unitsimg protein. The present report describes a relatively simple procedure to overcome the variable losses we have encountered with the traditional acid ethanol extraction of human leukocyte interferon. In the modification described, the amount of protein in solution was constantly monitored and precipitation terminated when an experimentally determined “endpoint” was attained (A,,,, ZY 0.3 of a l/l0 dilution). When the A,,,, exceeded 0.48, certain protein contaminants that were retained could not be separated from interferon by Blue Sepharose chromatography and the resulting purification was approximately 20-fold less. Blue Sepharose chromatography can complement the ethanol extraction-precipitation technique because we have found that serum albumin, the major impurity in ethanol-extracted leukocyte interferon (11). is readily eluted by PBS in advance of the bulk of the interferon,
218
ERICKSON
and a second major class of impurities requires ethylene glycol for elution (Fig. 1A). The method lends itself also to the purification of human lymphoblastoid interferon except that the Namalva interferon exhibited greater affinity for Blue Sepharose than leukocyte interferon and required a higher salt concentration or ethylene glycof for efficient elution. It is, therefore, suspected that these two lymphoid interferons may not be structurally analogous. They have been shown previously to contain different proportions of the Le and F antigenic species (17-20). A more thorough analysis of these and other human interferons fractionated by Blue Sepharose chromatography will be published elsewhere (manuscript in preparation). REFERENCES 1. Fantes, K. (1970) Ann. N. Y. Acud. Sci. 173, 118121. 2. Anfinsen, C.. Bose, S.. Corley, L., and GuarariRotman. D. (1974) Proc. Nut. Aud. SC;. USA 71, 3139-3142. 3. Cantell. K.. Hirvonen, S.. Mogensen. K. E., and Pyhala. L. (1974) in In Vitro Monograph: (Waymouth, C., ed.), Vol. 3, pp. 35-38, Tissue Culture Association, Rockville, Md. 4. Matsuo, A.. Hayashi, S., and Kishida, T. (1974) Jupun. J. Mic,robiol. 18, 21-7-7. 5. Berg, K.. Ogburn. C., Paucker. K., Mogensen, K. E., and Cantell, K. (1975) J. Immunul. 114, 640-644.
AND
PAUCKER
6. Jankowski. W.. Von Muenchhausen. W., Sulkowski. E.. and Carter, W. (1976) Bictc~hetni.rtr:\ 15. 5182-5187. 7. Sulkowski. E.. Davey, M.. and Carter. W. (1976) J. Bid. c/lc~tn. 251, 5381-5385. 8. Torma, E., and Paucker. K. (1976) J. Biol. C’llem. 251, 4310-4316. 9. Bridgen. P.. Antinsen, C.. Corley. L.. Bose, S., Zoon, K., and Ruegg, U. (1977) J. Biol. C/~orrr. 252. 6585-6587. 10. Cantell, K.. and Hirvonen. S. (1977) 7e.ro.s Rep. Bid. Med. 35. 1388144. 11. Cantell. K.. and Hirvonen, S. (1978)5. Gelr. viral. 39, 541-543. 12. Lin. L., Wiranowska-Stewart. M.. and Stewart 11. W. (1978) Ahst. Annu. Mrct. Amer. Sot. Microbial. 246. 13. Rubinstein. M.. Rubinstein. S.. Familletti. P.. Gross. M.. Miller. R., Waldman. A., and Pestka. S. (1978) Scirncr 202, 1289- 1290. 14. Strander. H.. and Cantell, K. (1966) Ann. Med. Exp. Biol. t
Yl ICAL
BIOCtlEMISIR~
Purification
98, ?14-218
(1979)
of Acid Ethanol-Extracted Human Lymphoid Blue Sepharose Chromatography’ JOHN
S.
ERICKSON
Received
AND
March
KURT
lnterferons
by
PAUCKER
9, 1979
Human leukocyte and lymphoblastoid (Namalva) interferons were purified by a modified acid ethanol extraction procedure and chromatography of the dilute ethanolic interferon solution on Blue Sepharose. Both interferons were purified more than lOOO-fold. with recoveries ranging from 32 to 40%.
Preparations of crude interferon often require extensive purification in order to be suitable for a number of applications. In recent years several purification procedures for human interferons have been described in the literature (1- 13). Of these, the acid ethanol extraction of human leukocyte interferon first reported by Fantes (1) and refined by Cantell and Hirvonen (10,ll) has been useful for the initial purification of large amounts of native interferon. However. in our hands. the acid ethanol procedure gave variable results because extensive losses in activity were frequently sustained during precipitation of the interferon from the ethanolic solution. Therefore, we explored other means to recover the interferon from ethanol. Jankowski, et al. (6) previously noted the affinity of human leukocyte interferon for blue dextran. In the present report. we describe the application of Blue Sepharose chromatography in the purification of ethanolextracted human leukocyte and lymphoblastoid (Namalva) interferons. ’ This work was supported in part by Public Health Service Contract NO1 Al 52520 and Grant ROI AI 13649 from the National Institute of Allergy and Infectious Diseases. 0003-26971791130214.05$02.00/O CopyrIght All rights
0 1979 by Academx Pres\. Inc. or reproductmn I” any term rewrved.
MATERIALS
AND METHODS
Interj~rons. Human leukocyte interferon was induced by Sendai virus (14,15), and received in lyophilized form from the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. The specific activity of the crude interferon ranged from 2-3 x 10” units/mg protein, expressed in terms of the international reference standard G 023-901-527 provided by the Reference Reagents Branch of the National lnstitute for Allergy and Infectious Diseases, Bethesda, Maryland. The interferon was dissolved in PBS at the indicated concentration prior to ethanol extraction. Human lymphoblastoid interferon was induced by Newcastle disease virus and obtained from Dr. K. Zoon, Laboratory of Chemical Biology, National Institute of Health, Bethesda, Maryland. The crude interferon had been concentrated by trichloroacetic acid precipitation and was subsequently passed through Sephadex G-25 equilibrated with PBS to remove the trichloroacetic acid (9). The specific activity of this material 1 The Abbreviations used: PBS, phosphate-buffered saline; 0.15 M NaCl. 0.01 M sodium phosphate. 7.0: buffer A. 0.05 M sodium acetate. pH 5.0.
214
pH
PURIFICATION
OF ACID
ETHANOL-EXTRACTED
was in the range of 30 reference unitslmg protein. Both human interferons were stored at -20°C until used. Etllunol extraction. The acid ethanol extraction procedure of Cantell and Hirvonen ( 10, I 1) was employed, except for the modifications indicated in the text. CIlrornLItO!:r.aph?!. All chromatographies were performed with commercial Blue Sepharose CL-6B (Pharmacia Fine Chemicals, Piscataway, N. J.). The Blue Sepharose was swollen for at least 24 h in bulffer A” and equilibrated with at least 10 column volumes of buffer A prior to use. A.ssa~.s. Interferon was titrated on human foreskin fibroblasts (FS-4 strain) in a microplate system as previously described (16), except for the substitution of encephalomyocarditis virus as challenge agent. Protein concentrations were estimated by absorption at 280 nm, by equating an absorbance of 1.O to 1 .O mgiml of protein. RESULTS
AND DISCUSSION
In a typical experiment a loo-ml sample of crude human leukocyte interferon (5000 mg of protein, 2000 reference units of interferonimg of protein) was carried through the acid ethanol procedure descrilbed by Cantell and Hirvonen (10,l I), but with some modifications. As pointed out by these authors, the pH of the ethanolic solution must be raised very slowly in order to avoid coprecipitation of interferon. Indeed during the course of these experiments, we observed that the pH of the largely nonaqueous, unbuffered solution was a poor indicator of the amount of protein remaining in solution. Thus, on different trials, ethanolic preparations which had bleen adjusted to pH 5.5 sometimes retained large amounts of protein, whereas at other times very little protein (and quite variable amounts of interferon) remained in solution. Cantell and Hirvonen (10,ll) introduced two rounds of precipitation-centrifugation in order to circumvent co-
HUMAN
LYMPHOID
INTERFERONS
215
precipitation of interferon while raising the pH. For our purposes we found it advisable to modify slightly the pH adjustments and to monitor the amount of protein remaining in solution after each centrifugation. A Chemtrix pH meter, Model 40E equipped with an HO07 electrode, was standardized at 23°C. at pH 7.0 and 4.0. The temperature compensation control was adjusted to 0” for monitoring the pH of the ethanol extract in an ice bath. The pH was slowly raised to 5.4 by dropwise addition of 0.5 1\; NaOH. The resulting precipitate was pelleted by centrifugation (9000 RPM x IO min. GSA Sorvall rotor). Supernatent protein concentration was estimated by measuring the absorption at 280 nm (A,,,,) of a l/10 dilution in PBS. It was determined that an absorbance reading of A,,,, = 0.3 was optimal for removal of extraneous proteins without coprecipitation of interferon. Hence, if the A 2H,,was greater than 0.3, further precipitation was induced by the dropwise addition of 0.1 N NaOH until the pH reached 5.5. The precipitation-centrifugation cycle can be repeated, if necessary, until the A.,,,, of the l/IO dilution reaches 0.30. The following steps were conducted in the cold. The final supernatent was diluted into 12 vol of buffer A. To obtain rapid mixing, the ethanolic solution was introduced into the buffer from a I-mm-i.d. tubing placed directly over a magnetic stirring bar. The resulting solution containing about 7.5% ethanol, was then absorbed overnight to a column of Blue Sepharose CL-6B, 1.5 x 6.0 cm, at a flow rate of 120 ml/h. The breakthrough fraction was collected as a pool which contained less than 2% of the interferon. The column was then developed with successive washes of PBS, 0.5 hi NaCl, 1.0 M NaCl. and 1.0 M NaCl, 50% ethylene glycol (v/v), each being buffered with 0.01 M sodium phosphate. pH 7.0 (Fig. 1A). In a parallel experiment, a human lymphoblastoid interferon preparation (3000 mg of protein, 33 unitsimg of protein) was subjected to the identical steps (Fig. 2A).
216
ERICKSON
20
AND
PAUCKER
I IO
40
20
30
FRACTIONS
FIG. 1. Chromatogram of human leukocyte interferon on Blue Sepharose. (A) Crude interferon was extracted in acid ethanol. diluted to a hnal volume of 1300 ml, and applied to the column (see text for details). (B) Rechromatography of fractions 7-49 from (A). Fraction size was 6.5 ml except in the ethylene glycol region (4.5 ml). The numbered arrows refer to (I) PBS, (2) 0.5 M NaCI, (3) 1 M NaCI, and (4) 1 M NaCl, 50% (v/v) ethylene glycol, each in 0.01 M sodium phosphate, pH 7.0. Interferon, 0: absorbance, -.
The results for the two types of interferon are summarized in Table I. The data show that a 7- to 15fold purification was achieved by the acid ethanol extraction-precipitations alone while the purification for the pooled fractions from the Blue Sepharose columns was 250- and
515fold, respectively, for the leukocyte and lymphoblastoid interferon preparations. The interferon in the pooled fractions lost little or no activity at pH 7.0 and 4°C for at least I month. From other experimentation, we suspetted that the presence of the ethanol in
IO
IO
30
50
IO
20
30
FRACTIONS
FIG. 2. Chromatogram of human lymphoblastoid interferon on Blue Sepharose. (A) Crude interferon was extracted in acid ethanol, diluted to a final volume of 1000 ml. and applied to the column (see text for details). (B) Rechromatography of fractions 33-55 from (A). Fraction size was 6.5 ml except in the ethylene glycol region (4.5 ml). The numbered arrows refer to (I) PBS, (2) 0.5 M NaCI. (3) 1 M NaCI. and (4) 1 M NaCI, 50% (v/v) ethylene glycol, each in 0.01 M sodium phosphate. pH 7.0. Interferon. 0; absorbance -.
PURIFICATION
OF ACID
ETHANOLEXTRACTED
HUMAN
TABLE
LYMPHOID
INTERFERONS
217
I
PVRIFICAIION OF HUMAN LFUKOCYTF. AND L~MPHOBI.ASTOID~NIERFERONS BY ACID ETHANOI--BLUE SEPHAROSECHROMA~OGRAPHI
Interferon preparation Leukocyte, crude Ethanol, pH 5.5 Blue Sepharose 1. total #14-49 Blue Sepharose 2, total #I6620 Lymphoblastoid, crude Ethanol. pH 5.5 Blue Sepharose I, total #33-54 Blue Sepharose 2. total #I9925
Total volume (ml) 100 100 234 32.5
Units/ ml” 1 x lo” 1 x lo” 2.5 Y IO’ 1 x IO’
100 100
I x lo:’ 6 % IO’
143
5.7 x IO’
45.5
8.6 x lo’
Units total
Protein
(mgiml)
I x 1x10’ 7.4 x 5.8 x 4.2 x 3.3 Y
IO’
1 6 I 8.2 5.7 3.9
IO:’ IO 10’ IO’ IO’ IO’
” Units are expressed in terms of reference standard ” Expressed as percentage of initial concentration.
the applied sample was affecting the elution position of the interferon. Therefore, the pooled fractions of each type of interferon were concentrated by means of a membrane concentrator (PM-10 membrane) to 56 ml (leukocyte) and 50 ml (lymphoblastoid), dialyzed twice against 1 liter of buffer A, for 2 days. and rechtomatographed on a Blue Sepharose column, (1 x 9.5 cm). The corresponding elution profiles are shown in Figures IB and 2B and the data are summarized in Table 1. The elution pattern of both interferons was more sharply defined and that of lymphoblastoid interferon displaced with respect to leukocyte interferon. The bulk of leukocyte interferon (837~~) eluted in 0.5 M NaCl. whereas most of the lymphoblastoid interferon (68%) was desorbed in 1.0 M NaCl. Overall purification in both cases exceeded lOOO-fold. The specific activity of the leukocyte interferon pool was 2.6 x 10” units/ mg protein and thus comparable to values reported by Cantell and Hirvonen ( 10,l 1), except that our starting material was of
x x x x x x
IO” IO” IO” lo”
50 3
Specific activity (unitsimg protein)
Purification
2 % lo:’ 3 x IOJ
15X
0.048
5 * lo”
250 x
0.039
2.6 x IO”
30 2.5
33 240
1.300‘*
7x
0.034
1.7 x IO’
515x
0.025
3.4 x IO’
1.030x
Recovery” (5 1
100 74 58 40 35
60 100 x2 57 39
G023-901-527.
lesser purity. In the case of peak fraction 17 (Fig. lB), purification was 3450-fold and the specific activity 6.9 x 10” unitsimg protein. The present report describes a relatively simple procedure to overcome the variable losses we have encountered with the traditional acid ethanol extraction of human leukocyte interferon. In the modification described, the amount of protein in solution was constantly monitored and precipitation terminated when an experimentally determined “endpoint” was attained (A,,,, ZY 0.3 of a l/l0 dilution). When the A,,,, exceeded 0.48, certain protein contaminants that were retained could not be separated from interferon by Blue Sepharose chromatography and the resulting purification was approximately 20-fold less. Blue Sepharose chromatography can complement the ethanol extraction-precipitation technique because we have found that serum albumin, the major impurity in ethanol-extracted leukocyte interferon (11). is readily eluted by PBS in advance of the bulk of the interferon,
218
ERICKSON
and a second major class of impurities requires ethylene glycol for elution (Fig. 1A). The method lends itself also to the purification of human lymphoblastoid interferon except that the Namalva interferon exhibited greater affinity for Blue Sepharose than leukocyte interferon and required a higher salt concentration or ethylene glycof for efficient elution. It is, therefore, suspected that these two lymphoid interferons may not be structurally analogous. They have been shown previously to contain different proportions of the Le and F antigenic species (17-20). A more thorough analysis of these and other human interferons fractionated by Blue Sepharose chromatography will be published elsewhere (manuscript in preparation). REFERENCES 1. Fantes, K. (1970) Ann. N. Y. Acud. Sci. 173, 118121. 2. Anfinsen, C.. Bose, S.. Corley, L., and GuarariRotman. D. (1974) Proc. Nut. Aud. SC;. USA 71, 3139-3142. 3. Cantell. K.. Hirvonen, S.. Mogensen. K. E., and Pyhala. L. (1974) in In Vitro Monograph: (Waymouth, C., ed.), Vol. 3, pp. 35-38, Tissue Culture Association, Rockville, Md. 4. Matsuo, A.. Hayashi, S., and Kishida, T. (1974) Jupun. J. Mic,robiol. 18, 21-7-7. 5. Berg, K.. Ogburn. C., Paucker. K., Mogensen, K. E., and Cantell, K. (1975) J. Immunul. 114, 640-644.
AND
PAUCKER
6. Jankowski. W.. Von Muenchhausen. W., Sulkowski. E.. and Carter, W. (1976) Bictc~hetni.rtr:\ 15. 5182-5187. 7. Sulkowski. E.. Davey, M.. and Carter. W. (1976) J. Bid. c/lc~tn. 251, 5381-5385. 8. Torma, E., and Paucker. K. (1976) J. Biol. C’llem. 251, 4310-4316. 9. Bridgen. P.. Antinsen, C.. Corley. L.. Bose, S., Zoon, K., and Ruegg, U. (1977) J. Biol. C/~orrr. 252. 6585-6587. 10. Cantell, K.. and Hirvonen. S. (1977) 7e.ro.s Rep. Bid. Med. 35. 1388144. 11. Cantell. K.. and Hirvonen, S. (1978)5. Gelr. viral. 39, 541-543. 12. Lin. L., Wiranowska-Stewart. M.. and Stewart 11. W. (1978) Ahst. Annu. Mrct. Amer. Sot. Microbial. 246. 13. Rubinstein. M.. Rubinstein. S.. Familletti. P.. Gross. M.. Miller. R., Waldman. A., and Pestka. S. (1978) Scirncr 202, 1289- 1290. 14. Strander. H.. and Cantell, K. (1966) Ann. Med. Exp. Biol. t
