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[30] Reduction and reoxidation at disulfide bonds
[30]
REDUCTION AND REOXIDATION AT DISULFIDE BONDS
387
[30] Reduction and Reoxidation at Disulfide Bonds
By FREDERICX H.
WHITE,
JR.
A protein disulfide bond may be cleaved by reagents containing the SH group as follows: Protein
I
S
I +
S
RSH Protein ~" [ I SSR ~ HS SH
Protein
RSH ~
I
HS
I
+ ~SR
(1)
This reaction approaches completion with an excess of the reducing agent, which is specific for disulfide bonds. 1 For cleavage of all such bonds in a given protein, it may be necessary to employ a denaturing agent, such as urea, to render the protein into a conformational state in which the bonds are sufficiently exposed to the reducing agent. Concomitant with seission of the disulfide bonds is loss of the native secondary and tertiary structures as well as biological activity. In general this reaction would be of use under circumstances necessitating the removal of native conformation with no deleterious effect upon the primary structure. Proteolytie cleavage, for example, may not proceed satisfactorily unless the coiling and folding of the native protein are first eliminated. This reaction therefore has found use as a preliminary step for enzymatic degradation associated with investigation of amino acid sequence. Reduction of chicken egg white lysozyme, for example, permitted Canfield 2 to obtain a form of this protein from which he was able to complete the determination of amino acid sequence. Reduction has further been of use in studies on the effects of ionizing radiation. For example, comparative studies of native and reduced proreins have permitted conclusions regarding the effects of conformation on the distribution of carbon free radicals, resulting from irradiation,
1F. H. White, Jr., J. Biol. Chem. 235, 383 (1960). = R. E. Canfield, J. Biol. Chem. 238, 2698 (1963).
388
MODIFICATION REACTIONS
[30]
among the amino acid residues2 Haskill and H u n t 4 found that irradiated ribonuclease (RNase), after full reduction, could be reoxidized to yield an enzyme differing in its K,, value from the native enzyme, thus revealing a "hidden" damage due to irradiation that could not be detected in any other way. Reoxidation of the reduced protein may be readily carried out by exposure in dilute, slightly alkaline solution, to atmospheric oxygen. I t is especially significant that products are obtained in high yield from this reaction which appear to be identical with the native proteins. The reappearance of native conformation in the absence of any biological system demonstrates that the characteristic manner in which a protein chain coils and folds to produce its native conformation during biosynthesis is governed by its amino acid sequence. Beef pancreatic RNase was first observed to exhibit this behavior 5 and then a number of other proteins (see White ~ for summary). More recently the return of antibody specificity on oxidation of reduced antibody fragments 7,8 and of the whole protein 9 demonstrated that this property is controlled by the primary structure of the antibody chain. (For a more detailed discussion see Tanford. 1°) Gutte and Merrifield 11 and Hirschmann et al. 12 simultaneously accomplished for the first time the synthesis of an enzyme (RNase) by employing, in the final step, the principal that the primary structure governs formation of the secondary and tertiary structures. Thus, the fully synthesized protein chain was allowed to oxidize and fold to the native and enzymatically active conformation. Additional
s F. H. White, Jr., P. Riesz, and H. Kon, Radiat. Res. 32, 744 (1967). 4j. S. Haskill and J. W. Hunt, Biochim. Biophys. Acta 105, 333 (1@05). 5F. H. White, Jr., J. Biol. Chem. 236, 1353 (1961). 6F. H. White, Jr., J. Biol. Chem. 239, 1032 (1964). E. Haber, Proc. Nat. Acad. Sci. U.S. 52, 1099 (1964). s p. L. Whitney and C. Tanford, Proc. Nat. Acad. Scl. U.8. 53, 524 (1965). 9M. H. Freedman and M. Sela, J. Biol. Chem. 241, 2383 (1966). ~oC. Tanford, Accounts Chem. Res. 1, 161 (1968). 1, B. Gutte and R. B. Merrifield, J. Amer. Chem. Soc. 91, 501 (1969). R. Hirschmann, R. F. Nutt, D. F. Veber, R. A. Vitali, S. L. Varga, T. A. Jacob, F. W. I.iolly, and R. G. Denkewalter, J. Amer. Chem. Soc. 91, 507 (1969).
[30]
REDUCTIONAND REOXIDATION AT DISULFIDE BONDS
389
significance of this monumental achievement lies in the final elimination of any question that the refolding of a reduced protein might be initiated by a small amount of residual native structure. Little is known of the mechanism by which refolding occurs. Evidence has been found that the process may depend upon attractions between residues TM and upon hydrophobic interactions.1. Friedman et al. 15 have implicated tyrosine at position 115 in the refolding of RNase. The process of refolding, however, must still be regarded as poorly understood. Reduction and Reoxidation Reagents. The reducing agent of choice is mercaptoethanol, which has the advantages of long shelf life, with little oxidation of SH groups, and no reacting groups by which side reactions might occur with the protein. Urea is used as the denaturing agent. Cyanate, which occurs in equilibrium with urea, will react with amino and SH groups in proteins to form the carbamyl derivativesTM and must therefore be eliminated. Crystallization of urea from ethanol and water should be sufficient, but as an additional precaution, cyanate may be decomposed by acidification, 16 or removed by passage through a mixed-bed ion-exchange column.7 A convenient colorimetric test for cyanate is given by WernerY Procedure ]or Reduction. Urea (2.4 g) and mercaptoethanol (0.1 ml) are dissolved in 2.5 ml of water. This solution is adjusted to pH 8.5 with 5% aqueous trimethylamine and used to dissolve 50 mg of RNase. After a final check on the pH, the solution is adjusted to 5 ml with water. Nitrogen is passed through the solution in a test tube for 5 minutes, and the tube is stoppered. Reduction of the four disulfide bonds of RNase is essentially complete after 4 hours at room temperature (22--25°). To separate the reduced protein, the reaction mixture is first brought
is E. Haber and C. B. Anfinsen,J. Biol. Chem. 237, 1839 (1962). 14C. J. Epstein and R. F. Goldberger,J. Biol. Chem. 239, 1087 (1964). 15M. E. Friedman, It. A. Scheraga, and R. F. Goldberger, Biochemistry 5, 3770 (1966). 16G. R. Stark, W. H. Stein, and S. Moore, J. Biol. Chem. 235, 3177 (1960).
1~E. A. Werner, J. Chem. Soc. 123, 2577 (1923).
390
MODIFICATION REACTIONS
[30]
to pH 4 with acetic acid and then applied to a column (2.5 X 25 cm) of Sephadex (Pharmacia, Uppsala, Sweden; G-25, 100-270 mesh) at room temperature, previously equilibrated with 0.1 M acetic acid. For a column of this size, it is advisable not to exceed 5 ml of sample volume. The column is eluted with 0.1 M acetic acid, and 3-ml fractions are collected at a flow rate not greater than 250 ml per hour. The protein peak appears first, followed by the components of lower molecular weight in another peak. Both peaks may be detected at 280 nm, the concentration of reduced RN'ase being determined by an extinction coefficient of 8.55 X 103 M-lcm-12 The volume of the resulting protein solution should be 15-20 ml, at approximately 2 mg/ml. This solution is stable at 5 ° for several days with no detectable loss of SH groups. Alternatively, the reduced protein may be precipitated by a mixture of acetone and HC1, as described earlier. 1 Procedure ]or Reoxidation. An aliquot of the reduced RNase solution is added to 0.1 M tris(hydroxymethyl)aminomethane (Tris)-acetie acid buffer of pH 8, to produce a protein concentration of 20/~g/ml. Oxidation occurs at room temperature on standing in an open beaker. Typically more than half the specific activity of native RNase is regenerated within 1 hour, and the yield of active protein will approach 100% at 3 hours. The assay may be performed with ribonucleic acid or cyclic phosphates as substrates by methods reviewed earlier. 18 To separate the reoxidized protein from the buffer, the volume may first be reduced by lyophilization and the buffer removed by dialysis, as was done previously2 More recently carboxymethyl (CM) Sephadex has been used in this laboratory for concentrating small amounts of reoxidized RNase from large volumes. The reoxidation mixture is diluted to a Tris concentration of 0.05 M and then adjusted to pH 5 by addition of acetic acid. The solution is passed through a column of CM-Sephadex (C-25, 100-270 mesh, 4.4 meq/g), previously equilibrated with 0.05 M ammonium acetate buffer of pH 5. Column dimensions of 2.5 X 10 cm are ample for adsorption of 1 g of RNase. The column is washed with water and then eluted with 0.2 M ammonium bicarbonate to remove the protein. The fractions containing protein, detected at 280 nm, may then be combined and lyophilized. The remaining bicarbonate may be removed by dialysis.
C. B. Anfinsen and F. H. White, Jr., in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrb~tck, eds.), p. 95. Academic Press, New York, 1961.
[30]
REDUCTION AND REOXIDATION AT DISULFIDE BONDS
391
Discussion Partial Reduction. Reduction of specific disulfide bonds has been reported with other reducing agents. With phosphorothioate, the 4-5 and 3-8 bonds of RNase were selectively cleaved. 19 More recently Sperling et al. 2° reduced the 4-5 bond with either dithiothreitol or dithioerythritol, in the absence of a denaturing agent. Use of the former reagent is treated in detail elsewhere.~1 It may be of interest that, in the experience of this laboratory, mercaptoethanol, in the absence of urea but under conditions otherwise similar to those given herein for full reduction, reduces RNase to approximately the same extent. It remains a subject for further investigation to determine if the same or different disulfide bonds are attacked by these reagents. Determination o] the Extent o] Reduction or Reoxidation. Most commonly these reactions have been followed by measuring the SH content. The spectrophotometric titration with p-chloromercuribenzoate developed by Swenson and Boyer,22 especially as modified by Sela et al., 23 has been satisfactory in this laboratory. Titration with 5,5'-dithiobis (2-nitrobenzoic acid) ~* also has been successful for this purpose. 25 An additional method involves reaction of SH groups with iodoacetate,5 followed by analysis for S-carboxymethylcysteine; it has yielded results that agree well with those obtained by titration. 2~ Other methods of SH determination are reviewed by Cecil and McPhee? ~ Applicability to Other Proteins. It may be necessary to modify the above method for reduction of other proteins. Guanidine hydrochloride (a more effective denaturing agent than urea) may be used in place of urea. Variations in temperature and time of reaction may be necessary, depending upon the ease of solubilization and unfolding of the protein. There are several critical factors to be considered for reoxidation. The concentration of the reduced protein should be as low as possible to minimize intermolecular disulfide bond formation. The yield of active, reoxidized RNase, however, diminishes below 10 ~g/ml, since the reduced
~gH. Neumann, I. Z. Steinberg, J. R. Brown, R. F. Goldberger, and M. Sela, Eur. Y. Biochem. 3, 171 (1967). 2oR. Sperling, Y. Burstein, and I. Z. Steinberg, Biochemistry 8, 3810 (1969). ~1This volume [13]. ~2A. D. Swensonand P. D. Boyer,J. Amer. Chem. Soc. 79, 2174 (1957). M. Sela, F. H. White, Jr., and C. B. Anfinsen, Biochim. Biophys. Acta 31, 417 (1959). HG. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). 2~This volume [37].
392
MODIFICATION REACTIONS
[30]
protein appears to be adsorbed onto the surface of the glass reaction vessel. 2s Temperature must be controlled, since the correct refolding of RNase decreases markedly above room temperature, and at 37 ° the yield of soluble, active protein is only 35% of that at 240. 28 The effects of temperature may be expected to differ with each protein. For chicken egg white lysozyme, for example, the efficiency of refolding is greater at 38 ° than at room temperature. ~ The tendency of some proteins to aggregate in the reduced state may result in a low yield of correctly refolded protein, since intermolecular disulfide bond formation is enhanced by proximity of the reduced chains. The rate of refolding of lysozyme, as well as the yield of active enzyme, has been increased by the addition of mercaptoethanol, thus allowing the incorrectly formed disulfide bonds to break and then recombine correctly.~9 The aggregation of trypsin after reduction has been prevented by complexing the native protein with CM-cellulose2° Freedman and Sela 9 employed an extensive polyalanylation of the e-amino groups of rabbit antibovine serum albumin. The heavy chain, which normally is insoluble after reduction, was thereby rendered soluble, and the 23 disulfide bonds of the native protein could then be reformed with regeneration of immunological activity. A proteolytic enzyme must be safeguarded against autodigestion during reoxidation. The immobilization of trypsin by attachment to CMcellulose has achieved this effect, in addition to prevention of aggregation, as mentioned above. Autolysis might also be prevented by including a specific inhibitor during oxidation, as has been tried by Epstein and Anfinsen31 in the use of lima bean trypsin inhibitor for the oxidation of reduced trypsin.
~A. M. Crestfield, S. Moore, and W. H. Stein, J. Biol. Chem. 238, 622 (1963). ~ It. Cecil and J. It. McPhee, Advan. Protein Chem. 14, 256 (1959). C. J. Epstein, It. F. Goldberger, D. M. Young, and C. B. Anfinsen,Arch. Biochem. Biophys., Suppl. 1, 223 (1962). n C. J. Epstein and R. F. Goldberger,J. Biol. Chem. 238, 1380 (1963). ,oC. J. Epstein and C. B. Anfinsen,J. Biol. Chem. 237, 2175 (1962). C. J. Epstein and C. B. Anfinsen,J. Biol. Chem. 237, 3464 (1962).
REDUCTION AND REOXIDATION AT DISULFIDE BONDS
387
[30] Reduction and Reoxidation at Disulfide Bonds
By FREDERICX H.
WHITE,
JR.
A protein disulfide bond may be cleaved by reagents containing the SH group as follows: Protein
I
S
I +
S
RSH Protein ~" [ I SSR ~ HS SH
Protein
RSH ~
I
HS
I
+ ~SR
(1)
This reaction approaches completion with an excess of the reducing agent, which is specific for disulfide bonds. 1 For cleavage of all such bonds in a given protein, it may be necessary to employ a denaturing agent, such as urea, to render the protein into a conformational state in which the bonds are sufficiently exposed to the reducing agent. Concomitant with seission of the disulfide bonds is loss of the native secondary and tertiary structures as well as biological activity. In general this reaction would be of use under circumstances necessitating the removal of native conformation with no deleterious effect upon the primary structure. Proteolytie cleavage, for example, may not proceed satisfactorily unless the coiling and folding of the native protein are first eliminated. This reaction therefore has found use as a preliminary step for enzymatic degradation associated with investigation of amino acid sequence. Reduction of chicken egg white lysozyme, for example, permitted Canfield 2 to obtain a form of this protein from which he was able to complete the determination of amino acid sequence. Reduction has further been of use in studies on the effects of ionizing radiation. For example, comparative studies of native and reduced proreins have permitted conclusions regarding the effects of conformation on the distribution of carbon free radicals, resulting from irradiation,
1F. H. White, Jr., J. Biol. Chem. 235, 383 (1960). = R. E. Canfield, J. Biol. Chem. 238, 2698 (1963).
388
MODIFICATION REACTIONS
[30]
among the amino acid residues2 Haskill and H u n t 4 found that irradiated ribonuclease (RNase), after full reduction, could be reoxidized to yield an enzyme differing in its K,, value from the native enzyme, thus revealing a "hidden" damage due to irradiation that could not be detected in any other way. Reoxidation of the reduced protein may be readily carried out by exposure in dilute, slightly alkaline solution, to atmospheric oxygen. I t is especially significant that products are obtained in high yield from this reaction which appear to be identical with the native proteins. The reappearance of native conformation in the absence of any biological system demonstrates that the characteristic manner in which a protein chain coils and folds to produce its native conformation during biosynthesis is governed by its amino acid sequence. Beef pancreatic RNase was first observed to exhibit this behavior 5 and then a number of other proteins (see White ~ for summary). More recently the return of antibody specificity on oxidation of reduced antibody fragments 7,8 and of the whole protein 9 demonstrated that this property is controlled by the primary structure of the antibody chain. (For a more detailed discussion see Tanford. 1°) Gutte and Merrifield 11 and Hirschmann et al. 12 simultaneously accomplished for the first time the synthesis of an enzyme (RNase) by employing, in the final step, the principal that the primary structure governs formation of the secondary and tertiary structures. Thus, the fully synthesized protein chain was allowed to oxidize and fold to the native and enzymatically active conformation. Additional
s F. H. White, Jr., P. Riesz, and H. Kon, Radiat. Res. 32, 744 (1967). 4j. S. Haskill and J. W. Hunt, Biochim. Biophys. Acta 105, 333 (1@05). 5F. H. White, Jr., J. Biol. Chem. 236, 1353 (1961). 6F. H. White, Jr., J. Biol. Chem. 239, 1032 (1964). E. Haber, Proc. Nat. Acad. Sci. U.S. 52, 1099 (1964). s p. L. Whitney and C. Tanford, Proc. Nat. Acad. Scl. U.8. 53, 524 (1965). 9M. H. Freedman and M. Sela, J. Biol. Chem. 241, 2383 (1966). ~oC. Tanford, Accounts Chem. Res. 1, 161 (1968). 1, B. Gutte and R. B. Merrifield, J. Amer. Chem. Soc. 91, 501 (1969). R. Hirschmann, R. F. Nutt, D. F. Veber, R. A. Vitali, S. L. Varga, T. A. Jacob, F. W. I.iolly, and R. G. Denkewalter, J. Amer. Chem. Soc. 91, 507 (1969).
[30]
REDUCTIONAND REOXIDATION AT DISULFIDE BONDS
389
significance of this monumental achievement lies in the final elimination of any question that the refolding of a reduced protein might be initiated by a small amount of residual native structure. Little is known of the mechanism by which refolding occurs. Evidence has been found that the process may depend upon attractions between residues TM and upon hydrophobic interactions.1. Friedman et al. 15 have implicated tyrosine at position 115 in the refolding of RNase. The process of refolding, however, must still be regarded as poorly understood. Reduction and Reoxidation Reagents. The reducing agent of choice is mercaptoethanol, which has the advantages of long shelf life, with little oxidation of SH groups, and no reacting groups by which side reactions might occur with the protein. Urea is used as the denaturing agent. Cyanate, which occurs in equilibrium with urea, will react with amino and SH groups in proteins to form the carbamyl derivativesTM and must therefore be eliminated. Crystallization of urea from ethanol and water should be sufficient, but as an additional precaution, cyanate may be decomposed by acidification, 16 or removed by passage through a mixed-bed ion-exchange column.7 A convenient colorimetric test for cyanate is given by WernerY Procedure ]or Reduction. Urea (2.4 g) and mercaptoethanol (0.1 ml) are dissolved in 2.5 ml of water. This solution is adjusted to pH 8.5 with 5% aqueous trimethylamine and used to dissolve 50 mg of RNase. After a final check on the pH, the solution is adjusted to 5 ml with water. Nitrogen is passed through the solution in a test tube for 5 minutes, and the tube is stoppered. Reduction of the four disulfide bonds of RNase is essentially complete after 4 hours at room temperature (22--25°). To separate the reduced protein, the reaction mixture is first brought
is E. Haber and C. B. Anfinsen,J. Biol. Chem. 237, 1839 (1962). 14C. J. Epstein and R. F. Goldberger,J. Biol. Chem. 239, 1087 (1964). 15M. E. Friedman, It. A. Scheraga, and R. F. Goldberger, Biochemistry 5, 3770 (1966). 16G. R. Stark, W. H. Stein, and S. Moore, J. Biol. Chem. 235, 3177 (1960).
1~E. A. Werner, J. Chem. Soc. 123, 2577 (1923).
390
MODIFICATION REACTIONS
[30]
to pH 4 with acetic acid and then applied to a column (2.5 X 25 cm) of Sephadex (Pharmacia, Uppsala, Sweden; G-25, 100-270 mesh) at room temperature, previously equilibrated with 0.1 M acetic acid. For a column of this size, it is advisable not to exceed 5 ml of sample volume. The column is eluted with 0.1 M acetic acid, and 3-ml fractions are collected at a flow rate not greater than 250 ml per hour. The protein peak appears first, followed by the components of lower molecular weight in another peak. Both peaks may be detected at 280 nm, the concentration of reduced RN'ase being determined by an extinction coefficient of 8.55 X 103 M-lcm-12 The volume of the resulting protein solution should be 15-20 ml, at approximately 2 mg/ml. This solution is stable at 5 ° for several days with no detectable loss of SH groups. Alternatively, the reduced protein may be precipitated by a mixture of acetone and HC1, as described earlier. 1 Procedure ]or Reoxidation. An aliquot of the reduced RNase solution is added to 0.1 M tris(hydroxymethyl)aminomethane (Tris)-acetie acid buffer of pH 8, to produce a protein concentration of 20/~g/ml. Oxidation occurs at room temperature on standing in an open beaker. Typically more than half the specific activity of native RNase is regenerated within 1 hour, and the yield of active protein will approach 100% at 3 hours. The assay may be performed with ribonucleic acid or cyclic phosphates as substrates by methods reviewed earlier. 18 To separate the reoxidized protein from the buffer, the volume may first be reduced by lyophilization and the buffer removed by dialysis, as was done previously2 More recently carboxymethyl (CM) Sephadex has been used in this laboratory for concentrating small amounts of reoxidized RNase from large volumes. The reoxidation mixture is diluted to a Tris concentration of 0.05 M and then adjusted to pH 5 by addition of acetic acid. The solution is passed through a column of CM-Sephadex (C-25, 100-270 mesh, 4.4 meq/g), previously equilibrated with 0.05 M ammonium acetate buffer of pH 5. Column dimensions of 2.5 X 10 cm are ample for adsorption of 1 g of RNase. The column is washed with water and then eluted with 0.2 M ammonium bicarbonate to remove the protein. The fractions containing protein, detected at 280 nm, may then be combined and lyophilized. The remaining bicarbonate may be removed by dialysis.
C. B. Anfinsen and F. H. White, Jr., in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrb~tck, eds.), p. 95. Academic Press, New York, 1961.
[30]
REDUCTION AND REOXIDATION AT DISULFIDE BONDS
391
Discussion Partial Reduction. Reduction of specific disulfide bonds has been reported with other reducing agents. With phosphorothioate, the 4-5 and 3-8 bonds of RNase were selectively cleaved. 19 More recently Sperling et al. 2° reduced the 4-5 bond with either dithiothreitol or dithioerythritol, in the absence of a denaturing agent. Use of the former reagent is treated in detail elsewhere.~1 It may be of interest that, in the experience of this laboratory, mercaptoethanol, in the absence of urea but under conditions otherwise similar to those given herein for full reduction, reduces RNase to approximately the same extent. It remains a subject for further investigation to determine if the same or different disulfide bonds are attacked by these reagents. Determination o] the Extent o] Reduction or Reoxidation. Most commonly these reactions have been followed by measuring the SH content. The spectrophotometric titration with p-chloromercuribenzoate developed by Swenson and Boyer,22 especially as modified by Sela et al., 23 has been satisfactory in this laboratory. Titration with 5,5'-dithiobis (2-nitrobenzoic acid) ~* also has been successful for this purpose. 25 An additional method involves reaction of SH groups with iodoacetate,5 followed by analysis for S-carboxymethylcysteine; it has yielded results that agree well with those obtained by titration. 2~ Other methods of SH determination are reviewed by Cecil and McPhee? ~ Applicability to Other Proteins. It may be necessary to modify the above method for reduction of other proteins. Guanidine hydrochloride (a more effective denaturing agent than urea) may be used in place of urea. Variations in temperature and time of reaction may be necessary, depending upon the ease of solubilization and unfolding of the protein. There are several critical factors to be considered for reoxidation. The concentration of the reduced protein should be as low as possible to minimize intermolecular disulfide bond formation. The yield of active, reoxidized RNase, however, diminishes below 10 ~g/ml, since the reduced
~gH. Neumann, I. Z. Steinberg, J. R. Brown, R. F. Goldberger, and M. Sela, Eur. Y. Biochem. 3, 171 (1967). 2oR. Sperling, Y. Burstein, and I. Z. Steinberg, Biochemistry 8, 3810 (1969). ~1This volume [13]. ~2A. D. Swensonand P. D. Boyer,J. Amer. Chem. Soc. 79, 2174 (1957). M. Sela, F. H. White, Jr., and C. B. Anfinsen, Biochim. Biophys. Acta 31, 417 (1959). HG. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). 2~This volume [37].
392
MODIFICATION REACTIONS
[30]
protein appears to be adsorbed onto the surface of the glass reaction vessel. 2s Temperature must be controlled, since the correct refolding of RNase decreases markedly above room temperature, and at 37 ° the yield of soluble, active protein is only 35% of that at 240. 28 The effects of temperature may be expected to differ with each protein. For chicken egg white lysozyme, for example, the efficiency of refolding is greater at 38 ° than at room temperature. ~ The tendency of some proteins to aggregate in the reduced state may result in a low yield of correctly refolded protein, since intermolecular disulfide bond formation is enhanced by proximity of the reduced chains. The rate of refolding of lysozyme, as well as the yield of active enzyme, has been increased by the addition of mercaptoethanol, thus allowing the incorrectly formed disulfide bonds to break and then recombine correctly.~9 The aggregation of trypsin after reduction has been prevented by complexing the native protein with CM-cellulose2° Freedman and Sela 9 employed an extensive polyalanylation of the e-amino groups of rabbit antibovine serum albumin. The heavy chain, which normally is insoluble after reduction, was thereby rendered soluble, and the 23 disulfide bonds of the native protein could then be reformed with regeneration of immunological activity. A proteolytic enzyme must be safeguarded against autodigestion during reoxidation. The immobilization of trypsin by attachment to CMcellulose has achieved this effect, in addition to prevention of aggregation, as mentioned above. Autolysis might also be prevented by including a specific inhibitor during oxidation, as has been tried by Epstein and Anfinsen31 in the use of lima bean trypsin inhibitor for the oxidation of reduced trypsin.
~A. M. Crestfield, S. Moore, and W. H. Stein, J. Biol. Chem. 238, 622 (1963). ~ It. Cecil and J. It. McPhee, Advan. Protein Chem. 14, 256 (1959). C. J. Epstein, It. F. Goldberger, D. M. Young, and C. B. Anfinsen,Arch. Biochem. Biophys., Suppl. 1, 223 (1962). n C. J. Epstein and R. F. Goldberger,J. Biol. Chem. 238, 1380 (1963). ,oC. J. Epstein and C. B. Anfinsen,J. Biol. Chem. 237, 2175 (1962). C. J. Epstein and C. B. Anfinsen,J. Biol. Chem. 237, 3464 (1962).