- Email: [email protected]
Protective effects of anionic detergents on interferons: Reversible denaturation
Biochimica et Biophysica Acta, 359 (1974) 364-368
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36773 PROTECTIVE EFFECTS OF ANIONIC D E T E R G E N T S ON I N T E R F E R O N S : REVERSIBLE D E N A T U R A T I O N
WILLIAM E. STEWART II, PIERRE DE SOMER and ERIK DE CLERCQ Department of Virology, Rega Institute for Medical Research, University of Leuven, Leuven (Belgium)
(Received February 19th, 1974)
SUMMARY Mouse L-cell interferon preparations were normally quite labile to heat, repeated freezing and thawing and chemical manipulations. In the presence of anionic detergents, decyl-, dodecyl- or tetradecylsulfate, the interferons were partially or completely stabilized against all these denaturants, depending on the intactness or disruption of disulfide bonds, respectively. The requirement for disulfide reduction for stabilization of the majority of the activity of interferons in detergents suggests there are two distinct molecular populations of interferons in the preparations.
INTRODUCTION Interferon preparations differ significantly in their stabilities to physical and chemical manipulation [1, 2]. Indeed, even different interferon preparations from the same species of animal exhibit marked differences in stabilities [3]. It has not yet been resolved whether these variations are reflections of differences in the interferon molecules per se or due to extraneous, non-interferon materials in the preparations. Mogensen and Cantell [4], on the one hand, have presented evidence that purification of interferon from human leucocytes increases its heat stability; Marshall et al. [5], on the other hand, have presented evidence that purification of interferon from human fibroblasts had no effect on its heat lability. Many protein denaturations that occur under conditions not likely to involve breakage of covalent bonds result from intra- and intermolecular reactivities of sulfhydryl groups which lead to inactive conformations or aggregates, respectively [6, 7]. It was, therefore, speculated that if these reactive groups could be reversibly prevented from interacting, proteins should be stable to normally inactivating manipulations. Fish et al. [8] have shown that the sulfhydryl groups of polypeptide chains are protected from the surrounding medium and from each other when the peptide chains are denatured in sodium dodecyl sulfate; this tempted us to speculate that such anionic detergents might afford stability (or renaturability) to interferons. The present communication presents evidence that this speculation was apparently valid.
365 MATERIALS AND METHODS Interferons were prepared in mouse L929-cells inoculated with Newcastle disease virus and contained about 104 N.I.H. reference units of interferon per mg of protein. Partially purified preparations were prepared by acidification to pH 2 and precipitation af extraneous proteins by addition of (NH4)2SO ~ to 20 ~ saturation at 20 °C. The supernatant fluid obtained after centrifugation at 1000 × g for 30 min was adjusted to p H 7.2 by dialysis against 0.01 M Tris-HC1 buffer and contained approximately 106 N.I.H. reference units of interferon per mg of protein. Inlerferons were assayed by plaque reduction against vesicular stomatitis virus on mouse Lgz9-cell. Sodium salts of decyl sulfate, dodecyl sulfate and tetradecyl sulfate were purchased from E. Merck, Darmstadt, Germany. Sodium dodecyl sulfate, electrophoresis purity grade was also purchased from Bio-Rad laboratories, Richmond, Calif. These were dissolved in distilled water to make 10 ~ stock solutions. Urea was purchased from E. Merck, and 2-mercaptoethanol was purchased from Bio-Rad laboratories. RESULTS The crude and partially purified interferons were both equally labile (Table I). Their activities were slightly reduced by either mercaptoethanol or urea, but the combination of these reagents significantly reduced the activities of both preparations. TABLE I INACTIVATION OF INTERFERON PREPARATIONS BY PHYSICAL AND CHEMICAL DENATURANTS Treatment*
None 56 °C, 30 rain 30 Freeze-thaw cycles 100 °C, 2.5 min 2-Mercaptoethanol (1.4.10 -2 M) Urea (5 M) 2-Mercaptoethanol + urea
Residual activity (Loglo units/ml) Crude
Purified
3.8 3.0 2.8 <1.0 3.5 3.3 3.0
4.0 3.0 2.5 <1.0 3.8 3.5 3.0
* Interferon aliquants in glass tubes containing 1 nal. Exposure to heat by immersion in water bath at indicated temperature. Freeze-thaw cycles of solid CO2-alcohol against 37 °C water bath. The addition of dodecyl sulfate itself to the preparations reduced their activities (Table II), but the residual activity was significantly more resistant to further inactivation by heat. The addition of 2-mercaptoethanol and urea, which together significantly inactivated normal interferons, to dodecyl sulfate-containing interferons prevented the loss of activity seen when dodecyl sulfate was added, and the entire interferon activity was resistant to inactivation in this state (Table III). Prior to activity measurements, reagents were removed from the protein solu-
366 TABLE 11 PARTIAL STABILIZATION OF INTERFERON PREPARATIONS BY DODECYL SULFATE Preparation
Treatment*
Residual activity (Log~0 units/ml)
Crude
None Dodecyl sulfate 100 °C, 2.5 rain Dodecyl sulfate ÷ 100 '~C, 2.5 rain
4.0 3.5 -1.0 3.3
Purified
None Dodecyl sulfate 100 °C, 2.5 min Dodecyl sulfate ~ 100 °C, 2.5 rain
4.0 3.3 -: 1.0 3.5
* l-ml aliquants in glass tubes. Dodecyl sulfate added to final concentration of 3.5.10 -3 M.
tions by dialysis at room temperature for 24 h against 0.01 M Tris-HC1 buffer, pH 7.2. This removed urea, mercaptoethanol and excess detergent and eliminated toxicity of the samples for tissue cultures. It likely did not remove i n t e r f e r o n - b o u n d detergent since the samples were still heat-resistant after dialysis. It must be assumed that the i n t e r f e r o n - b o u n d detergent exchanged off onto excess proteins in the serum-containing tissue culture m e d i u m and that liberated interferon then r e n a t u r e d ; evidence for this is that the heat-stable d e t e r g e n t - b o u n d interferon regained its original heat lability after mixing with s e r u m - c o n t a i n i n g medium. TABLE III COMPLETE STABILIZATION OF INTERFERON PREPARATION BYDODECYL SULFATE, 2-MERCAPTOETHANOL AND UREA Residual activity (Log10 units/ml)
Treatment*
None 100 °C, 2.5 min 2-Mercaptoethanol 2-Mercaptoethanol Dodecyl sulfate Dodecyl sulfate ÷ Dodecyl sulfate ÷ Dodecyl sulfate + 100 °C, 2.5 rain
÷ urea ÷ urea ~ 100 °C, 2.5 rain 100 °C, 2.5 rain 2-mercaptoethanol -t- urea 2-mercaptoethanol -t- urea -~
Crude
Purified
4.0 -< 1.0 3.0 < 1.0 3.5 3.3 3.8
4.0 1.0 3.3 < 1.0 3.3 3.5 4.0
4.0
4.0
* Reagents added to final concentrations of: 2-mercaptoethanol, 1.4.10 -2 M; dodecyl sulfate, 3.5.10 -3 M; urea, 5 M. Other a n i o n i c detergents were also f o u n d to afford stability of the interferons, decyl being somewhat less efficient t h a n either dodecyl sulfate or tetradecyl sulfate (Table IV). Presumably the c o n c e n t r a t i o n of detergent required for stabilization would depend on the specific activity of the preparation, as proteins have been shown to bind a b o u t 1.5 times their weight in dodecyl sulfate [9].
367 TABLE IV STABILIZATIONS OF INTERFERON PREPARATIONS BY ANIONIC DETERGENTS DECYL-AND TETRADECYL SULFATE Treatment*
None 100 °C, 2.5 rain Decyl sulfate Decyl sulfate + 2-mercaptoethanol ÷ urea Decyl sulfate ÷ 100 °C, 2.5 min Decyl sulfate + 2-mercaptoethanol + urea ÷ 100 °C, 2.5 min Tetradecyl sulfate Tetradecyl sulfate + 2-mercaptoethanol ÷ urea Tetradecyl sulfate ÷ 100 °C, 2.5 min Tetradecyl sulfate ÷ 2-mercaptoethanol ÷ urea ÷ 100 °C, 2.5 rain
Residual activity (Log10 units/ml) Crude
Puriiied
4.0
4.(I <1.0 3.5 3.5 3.0 3.5 3.(I 4.(I 3.3 4.0
* Reagents added to final concentrations of: 2-mercaptoethanol, 1.4.10 -z M; urea, 5 M; decyl sulfate, 3.10 -3 M ; tetradecyl sulfate, 3.10 -3 M.
DISCUSSION The most likely explanation for the finding that it is necessary to unfold and to reduce the interferons for them to be fully stabilized by anionic detergents is that the interferons are more able to revert to their native conformations if their amino acid sequences are in a linear r a n d o m coil. This seems to be the situation required for most proteins to renature [6, 7]. Therefore, detergent-stabilized interferons are more likely to renature u p o n removal of the detergent if they are in the reduced forms, lacking disulfide cross-links. The finding that detergent alone stabilized a part o f the activity of interferons that are not reduced can be interpreted in two ways: (1) interferon can renature to some extent from cross-linked peptide chains; or, (2) there are two distinct molecular populations o f interferons ih the preparations, the minor molecular species renaturable from detergent without reduction (perhaps lacking cross-links) and the major molecular species containing cross-links and renaturable only from the reduced state. The finding that interferons can be recovered from the protein-dissociating solution of' boiling dodecyl sulfate, mercaptoethanol and urea, has enabled us (by use of dodecyl sulfate-polyacrylamide gel electrophoresis) to resolve~these alternatives in favor of the latter interpretation [12]. Recently, h u m a n leukocyte interferon has been reported to be stabilized against heat inactivation by dodecyl sulfate alone [10]. However, the activity of this interferon was destroyed more than 95 ~o by treatment with dodecyl sulfate, mercapteethanol and urea. Interestingly, Edy et al. [3] have found that h u m a n fibroblast interferons contain only a minor c o m p o n e n t of activity exhibiting the stability characteristics of human leukocyte interferon. Also, we have found [11] that h u m a n fibroblast interferon in reduced and unreduced forms exhibits characteristics with regard to dodecyl sulfate almost identical to those described here for mouse L-cell interferons.
368 REFERENCES 1 Ng, N. H. and Vilcek, J. (1972) Adv. Protein Chem. 26, 173-124 2 Weil, R. and Dorner, F. (1973) in Selective lnhibitors of Viral Functions (Carter, W., ed.), pp. 107-121, CRC Press, Cleveland 3 Edy, V., Billiau, A., Joniau, M. and De Somer, P. (1974) Proc. Soc. Exp. Biol. Med., in the press, 4 Mogensen, K. E. and Cantell, K. (1973) Acta Pathol. Microbiol. Scand. 81, 382-383 5 Marshall, L. W., Pitha, P. and Carter, W. D. (1973) Virology 48, 607-611 6 Tanford, C. (1968) Adv. Protein Chem. 23, 121-282 7 Tanford, C. (1970) Adv. Protein Chem. 24, 1-85 8 Fish, W. W., Reynolds, J. A. and Tanford, C. (1970) J. Biol. Chem. 245, 5166-5168 9 Maizel, J. V., Jr. (1970) in Methods in Virology (Mararnorosch, K. and Koprowski, H., eds), pp. 179-246, Academic Press, New York 10 Mogensen, K. E. and Cantell, K. (1974) J. Gen. Virol. 22, 95-103 11 Stewart I1, W. E., De Clercq, E. and De Somer, P. (1974) Nature 249, 460-461 12 Stewart I1, W. E. (1974) Virology, in the press
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36773 PROTECTIVE EFFECTS OF ANIONIC D E T E R G E N T S ON I N T E R F E R O N S : REVERSIBLE D E N A T U R A T I O N
WILLIAM E. STEWART II, PIERRE DE SOMER and ERIK DE CLERCQ Department of Virology, Rega Institute for Medical Research, University of Leuven, Leuven (Belgium)
(Received February 19th, 1974)
SUMMARY Mouse L-cell interferon preparations were normally quite labile to heat, repeated freezing and thawing and chemical manipulations. In the presence of anionic detergents, decyl-, dodecyl- or tetradecylsulfate, the interferons were partially or completely stabilized against all these denaturants, depending on the intactness or disruption of disulfide bonds, respectively. The requirement for disulfide reduction for stabilization of the majority of the activity of interferons in detergents suggests there are two distinct molecular populations of interferons in the preparations.
INTRODUCTION Interferon preparations differ significantly in their stabilities to physical and chemical manipulation [1, 2]. Indeed, even different interferon preparations from the same species of animal exhibit marked differences in stabilities [3]. It has not yet been resolved whether these variations are reflections of differences in the interferon molecules per se or due to extraneous, non-interferon materials in the preparations. Mogensen and Cantell [4], on the one hand, have presented evidence that purification of interferon from human leucocytes increases its heat stability; Marshall et al. [5], on the other hand, have presented evidence that purification of interferon from human fibroblasts had no effect on its heat lability. Many protein denaturations that occur under conditions not likely to involve breakage of covalent bonds result from intra- and intermolecular reactivities of sulfhydryl groups which lead to inactive conformations or aggregates, respectively [6, 7]. It was, therefore, speculated that if these reactive groups could be reversibly prevented from interacting, proteins should be stable to normally inactivating manipulations. Fish et al. [8] have shown that the sulfhydryl groups of polypeptide chains are protected from the surrounding medium and from each other when the peptide chains are denatured in sodium dodecyl sulfate; this tempted us to speculate that such anionic detergents might afford stability (or renaturability) to interferons. The present communication presents evidence that this speculation was apparently valid.
365 MATERIALS AND METHODS Interferons were prepared in mouse L929-cells inoculated with Newcastle disease virus and contained about 104 N.I.H. reference units of interferon per mg of protein. Partially purified preparations were prepared by acidification to pH 2 and precipitation af extraneous proteins by addition of (NH4)2SO ~ to 20 ~ saturation at 20 °C. The supernatant fluid obtained after centrifugation at 1000 × g for 30 min was adjusted to p H 7.2 by dialysis against 0.01 M Tris-HC1 buffer and contained approximately 106 N.I.H. reference units of interferon per mg of protein. Inlerferons were assayed by plaque reduction against vesicular stomatitis virus on mouse Lgz9-cell. Sodium salts of decyl sulfate, dodecyl sulfate and tetradecyl sulfate were purchased from E. Merck, Darmstadt, Germany. Sodium dodecyl sulfate, electrophoresis purity grade was also purchased from Bio-Rad laboratories, Richmond, Calif. These were dissolved in distilled water to make 10 ~ stock solutions. Urea was purchased from E. Merck, and 2-mercaptoethanol was purchased from Bio-Rad laboratories. RESULTS The crude and partially purified interferons were both equally labile (Table I). Their activities were slightly reduced by either mercaptoethanol or urea, but the combination of these reagents significantly reduced the activities of both preparations. TABLE I INACTIVATION OF INTERFERON PREPARATIONS BY PHYSICAL AND CHEMICAL DENATURANTS Treatment*
None 56 °C, 30 rain 30 Freeze-thaw cycles 100 °C, 2.5 min 2-Mercaptoethanol (1.4.10 -2 M) Urea (5 M) 2-Mercaptoethanol + urea
Residual activity (Loglo units/ml) Crude
Purified
3.8 3.0 2.8 <1.0 3.5 3.3 3.0
4.0 3.0 2.5 <1.0 3.8 3.5 3.0
* Interferon aliquants in glass tubes containing 1 nal. Exposure to heat by immersion in water bath at indicated temperature. Freeze-thaw cycles of solid CO2-alcohol against 37 °C water bath. The addition of dodecyl sulfate itself to the preparations reduced their activities (Table II), but the residual activity was significantly more resistant to further inactivation by heat. The addition of 2-mercaptoethanol and urea, which together significantly inactivated normal interferons, to dodecyl sulfate-containing interferons prevented the loss of activity seen when dodecyl sulfate was added, and the entire interferon activity was resistant to inactivation in this state (Table III). Prior to activity measurements, reagents were removed from the protein solu-
366 TABLE 11 PARTIAL STABILIZATION OF INTERFERON PREPARATIONS BY DODECYL SULFATE Preparation
Treatment*
Residual activity (Log~0 units/ml)
Crude
None Dodecyl sulfate 100 °C, 2.5 rain Dodecyl sulfate ÷ 100 '~C, 2.5 rain
4.0 3.5 -1.0 3.3
Purified
None Dodecyl sulfate 100 °C, 2.5 min Dodecyl sulfate ~ 100 °C, 2.5 rain
4.0 3.3 -: 1.0 3.5
* l-ml aliquants in glass tubes. Dodecyl sulfate added to final concentration of 3.5.10 -3 M.
tions by dialysis at room temperature for 24 h against 0.01 M Tris-HC1 buffer, pH 7.2. This removed urea, mercaptoethanol and excess detergent and eliminated toxicity of the samples for tissue cultures. It likely did not remove i n t e r f e r o n - b o u n d detergent since the samples were still heat-resistant after dialysis. It must be assumed that the i n t e r f e r o n - b o u n d detergent exchanged off onto excess proteins in the serum-containing tissue culture m e d i u m and that liberated interferon then r e n a t u r e d ; evidence for this is that the heat-stable d e t e r g e n t - b o u n d interferon regained its original heat lability after mixing with s e r u m - c o n t a i n i n g medium. TABLE III COMPLETE STABILIZATION OF INTERFERON PREPARATION BYDODECYL SULFATE, 2-MERCAPTOETHANOL AND UREA Residual activity (Log10 units/ml)
Treatment*
None 100 °C, 2.5 min 2-Mercaptoethanol 2-Mercaptoethanol Dodecyl sulfate Dodecyl sulfate ÷ Dodecyl sulfate ÷ Dodecyl sulfate + 100 °C, 2.5 rain
÷ urea ÷ urea ~ 100 °C, 2.5 rain 100 °C, 2.5 rain 2-mercaptoethanol -t- urea 2-mercaptoethanol -t- urea -~
Crude
Purified
4.0 -< 1.0 3.0 < 1.0 3.5 3.3 3.8
4.0 1.0 3.3 < 1.0 3.3 3.5 4.0
4.0
4.0
* Reagents added to final concentrations of: 2-mercaptoethanol, 1.4.10 -2 M; dodecyl sulfate, 3.5.10 -3 M; urea, 5 M. Other a n i o n i c detergents were also f o u n d to afford stability of the interferons, decyl being somewhat less efficient t h a n either dodecyl sulfate or tetradecyl sulfate (Table IV). Presumably the c o n c e n t r a t i o n of detergent required for stabilization would depend on the specific activity of the preparation, as proteins have been shown to bind a b o u t 1.5 times their weight in dodecyl sulfate [9].
367 TABLE IV STABILIZATIONS OF INTERFERON PREPARATIONS BY ANIONIC DETERGENTS DECYL-AND TETRADECYL SULFATE Treatment*
None 100 °C, 2.5 rain Decyl sulfate Decyl sulfate + 2-mercaptoethanol ÷ urea Decyl sulfate ÷ 100 °C, 2.5 min Decyl sulfate + 2-mercaptoethanol + urea ÷ 100 °C, 2.5 min Tetradecyl sulfate Tetradecyl sulfate + 2-mercaptoethanol ÷ urea Tetradecyl sulfate ÷ 100 °C, 2.5 min Tetradecyl sulfate ÷ 2-mercaptoethanol ÷ urea ÷ 100 °C, 2.5 rain
Residual activity (Log10 units/ml) Crude
Puriiied
4.0
4.(I <1.0 3.5 3.5 3.0 3.5 3.(I 4.(I 3.3 4.0
* Reagents added to final concentrations of: 2-mercaptoethanol, 1.4.10 -z M; urea, 5 M; decyl sulfate, 3.10 -3 M ; tetradecyl sulfate, 3.10 -3 M.
DISCUSSION The most likely explanation for the finding that it is necessary to unfold and to reduce the interferons for them to be fully stabilized by anionic detergents is that the interferons are more able to revert to their native conformations if their amino acid sequences are in a linear r a n d o m coil. This seems to be the situation required for most proteins to renature [6, 7]. Therefore, detergent-stabilized interferons are more likely to renature u p o n removal of the detergent if they are in the reduced forms, lacking disulfide cross-links. The finding that detergent alone stabilized a part o f the activity of interferons that are not reduced can be interpreted in two ways: (1) interferon can renature to some extent from cross-linked peptide chains; or, (2) there are two distinct molecular populations o f interferons ih the preparations, the minor molecular species renaturable from detergent without reduction (perhaps lacking cross-links) and the major molecular species containing cross-links and renaturable only from the reduced state. The finding that interferons can be recovered from the protein-dissociating solution of' boiling dodecyl sulfate, mercaptoethanol and urea, has enabled us (by use of dodecyl sulfate-polyacrylamide gel electrophoresis) to resolve~these alternatives in favor of the latter interpretation [12]. Recently, h u m a n leukocyte interferon has been reported to be stabilized against heat inactivation by dodecyl sulfate alone [10]. However, the activity of this interferon was destroyed more than 95 ~o by treatment with dodecyl sulfate, mercapteethanol and urea. Interestingly, Edy et al. [3] have found that h u m a n fibroblast interferons contain only a minor c o m p o n e n t of activity exhibiting the stability characteristics of human leukocyte interferon. Also, we have found [11] that h u m a n fibroblast interferon in reduced and unreduced forms exhibits characteristics with regard to dodecyl sulfate almost identical to those described here for mouse L-cell interferons.
368 REFERENCES 1 Ng, N. H. and Vilcek, J. (1972) Adv. Protein Chem. 26, 173-124 2 Weil, R. and Dorner, F. (1973) in Selective lnhibitors of Viral Functions (Carter, W., ed.), pp. 107-121, CRC Press, Cleveland 3 Edy, V., Billiau, A., Joniau, M. and De Somer, P. (1974) Proc. Soc. Exp. Biol. Med., in the press, 4 Mogensen, K. E. and Cantell, K. (1973) Acta Pathol. Microbiol. Scand. 81, 382-383 5 Marshall, L. W., Pitha, P. and Carter, W. D. (1973) Virology 48, 607-611 6 Tanford, C. (1968) Adv. Protein Chem. 23, 121-282 7 Tanford, C. (1970) Adv. Protein Chem. 24, 1-85 8 Fish, W. W., Reynolds, J. A. and Tanford, C. (1970) J. Biol. Chem. 245, 5166-5168 9 Maizel, J. V., Jr. (1970) in Methods in Virology (Mararnorosch, K. and Koprowski, H., eds), pp. 179-246, Academic Press, New York 10 Mogensen, K. E. and Cantell, K. (1974) J. Gen. Virol. 22, 95-103 11 Stewart I1, W. E., De Clercq, E. and De Somer, P. (1974) Nature 249, 460-461 12 Stewart I1, W. E. (1974) Virology, in the press