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Nonenzymatic formation and isomerization of protein disulfides
[18]
PROTEIN DISULFIDE
FORMATION
301
[18] N o n e n z y m a t i c F o r m a t i o n a n d I s o m e r i z a t i o n o f Protein Disulfides B y DONALD B . WETLAUFER
The formation of protein disulfides can proceed either by enzymatic catalysis r-3 or by uncatalyzed thiol-disulfide exchange. 4,5 It is not yet clear which of these mechanisms predominates in vivo. It has been long known that strong acid can lead to disulfide isomerization. 6 It is also well known that disulfides can be cleaved by sulfitolysis in a mechanism that has formal similarity to thiol-disulfide exchange. 7 We will discuss neither of these further, but will deal with nonenzymatic systems that employ thiol-disulfide exchange reactions between proteins and low molecular weight thiols and/or disulfides. In the covalent reaction (1) it is important to note that the thiolate anion, R'"--S , is the reactive species, not the protonated thiol itself, R'"--SH. The fact that most common alkyl thiols have proton ionization pK values in the range of 8-10 means that the exchange reaction can proceed rapidly under mildly alkaline or neutral conditions; conversely, it is inhibited by acid. [ R ' - - S - - S - - R " ' + R"S R ' - - S - - S - - R " + R"S ~ ~ or l R " - - S - - S - - R " ' + R'S-
(I)
The second point to note is that the reaction is reversible; this ready reversibility proves to be very important. We can, for example, prepare the mixed disulfide between a reduced protein and cysteine (or other low molecular weight disulfide) simply by forcing the equilibrium of Eq. (1) as follows: P ( - - S )~ + 1000x C y - - S - - S - - C y ~ P ( - - S - - S - - C y ) x + xCyS+ (1000x - x ) C y - - S - - S - - C y
(2)
i D. Givol, F. DeLorenzo, R. F. Goldberger, and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 53, 676 (1965). 2 H. M. Katzen and D. Stetten, Jr., Fed. Proc. Fed. Am. Soc. Exp. Biol. 21, 201 (1962); H. M. Katzen and F. Tietze, J. Biol. Chem. 241, 3561 (1966). 3 R. B. Freedman and H. C. Hawkins, Biochem. Soc. Trans. Biochem. Rev. 5, 348 (1977). 4 D. B. Wetlaufer, V. P. Saxena, A. K. Ahmed, S. W. Schaffer, P. W. Pick, K.-J. Oh, and J. D. Peterson, in '"Protein Crosslinking," Part A (M. Friedman, ed.), p. 43. Plenum, New York, 1977. 5 V. P. Saxena and D. B. Wetlaufer, Biochemistry 9, 5015 (1970). 6 A. P. Ryle and F. Sanger, Biochem. J. 60, 535 (1955). 7 W. W. Chan, Biochemistry 7, 4247 (1968).
METHODS IN ENZYMOLOGY, VOL. 107
Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182007-6
302
[18]
OXIDATIONS, HYDROXYLATIONS, AND HALOGENATIONS
Bradshaw e t a l . 8 prepared the mixed disulfide oflysozyme and cysteine in just this fashion. The example in Eq. (2) makes the assumptions that the pK values and the oxidoreduction potentials of the several thiol-disulfide pairs are the same. While this is not strictly true, it is a reasonable approximation under the conditions employed. This mixed disulfide was used as the starting form of the protein in experiments, wherein lysozyme activity was regenerated 8 by the addition of mercaptoethanol, cysteine, or cysteamine to solutions buffered in the pH range 7 to 8.5. Cy--S--S Cy--S--S
S--S--Cy
\/ P
S--S--R
\/ P
+ CyS
\
+ RS ~
Cy
S--S
\
(3)
Ol" S--S--Cy
Cy--S
S
S
\/ P
+ Cy--S
\
S
R
S--S--Cy
Reactions of the type indicated by Eq. (3) can occur with any one of the protein-cysteine mixed disulfides. In addition to the back reaction of Eq. (3), the lower protein product on the right-hand side can undergo intramolecular thiol-disulfide exchange [Eq. (4)]. I Cy--S--S Cy--S--S
S
\/ P
S
\/ P
~
\
S--S--Cy
+ CyS
\
(4)
S
/
or
S~S
\/ P
+ CyS
\ S
S
Cy
It should be obvious that, if one of the two protein products in Eq. (4) represents a native pairing of protein thiols, the other must be nonnative. The back-reaction of Eq. (4) provides a route for removing nonnative disulfides from reaction intermediates in an oxidative regeneration. Often one begins with a completely reduced disulfide protein and wishes to regenerate the native disulfides rapidly and in high yield. The preparation of reduced proteins has been described elsewhere. 9,1° We can see by inspection of the above equations that the rates of some essential s R. A. Bradshaw, L. Kanarek, and R. L. Hill, J. Biol. Chem. 242, 3789 (1967). 9 F. H. White, Jr., this series, Vol. 11, p. 481. 10 S. S. Ristow and D. B. Wetlaufer, Biochem. Biophys. Res. Commun. 50, 544 (1973).
[18]
PROTEIN DISULFIDE FORMATION
303
reactions will depend on the concentration of R - - S - - S - - R and others will depend on the concentration of R - - S - . However, all the reactions are expressed by Eq. (1); that is, they are all thiol-disulfide exchange reactions? Empirically we have found5,1El2 that a concentration of R - - S of approximately 1-3 mM in the presence of one-fifth to one-tenth of that concentration of R - - S - - S - - R provides nearly optimal conditions for the oxidative re-formation of native protein disulfides. Typical conditions for such a reaction follow: Tris acetate buffer, 0.02 M, pH 8.0, 25°, 1 /~M reduced protein, 3.0 mM reduced glutathione, 0.3 mM oxidized glutathione, 1 mM EDTA. If the regeneration requires more than a few hours, it may prove advantageous to exclude atmospheric oxygen. Re-formation of protein disulfides may be monitored by a number of analytical methods. 8'1° Half-times for formation of protein disulfides have been found between a few minutes and 2 hr. It may be more relevant to monitor the appearance of a specific biological activity. The latter requires a rapid specific assay under conditions inhibitory to further thioldisulfide exchange--for example, a few minutes at pH 5. 5,j1,~2 Typically, the biological activity appears more slowly than the protein disulfide. 5,8,~1,~2 The above regeneration system has proved to be relatively insensitive to temperature over the range 25-40 °. Regenerations of hen egg white lysozyme with a glutathione system as described above showed substantially less than one protein-glutathione mixed disulfide throughout the whole course of the reaction. ~3 Means and Feeney 14 have noted, and we have also observed, that reduced proteins have a strong tendency to aggregate. This is why we recommend that oxidative renaturation be carried out at low protein concentrations. In the case of RNase A, much higher concentrations can be used, but the regeneration is very sensitive to the presence of specific ions in the buffer (sulfate and phosphate accelerate; bromide and thiocyanate inhibit).15 In addition to glutathione, several other low molecular weight thiol-disulfide pairs can promote oxidative renaturation. These include cysteine and cystine, 8 cysteamine and cystamine,8,12 reduced and oxidized 2-mercaptoethanol, 8,9,16 and reduced and oxidized dithiothreitol, j7 u W. L. Anderson and D. B. Wetlaufer, J. Biol. Chem. 251, 3147 (1976). ~2 K. Oh-Johanson, D. B. Wetlaufer, R. Reed, and T. Peters, Jr., J. Biol. Chem. 256, 445 (1981). ~3p. W. Pick, Ph.D. Thesis, University of Minnesota, 1974. Manuscript in preparation. t4 G. E. Means and R. E. Feeney, "Chemical Modification of Proteins." Holden-Day, San Francisco, California, 1971. t5 S. W. Schaffer, A. K. Ahmed, and D. B. Wetlaufer, J. Biol. Chem. 250, 8483 (1975). t6 p. Branca, M.S. Thesis, University of Delaware, 1982. Manuscript in preparation. t7 T. E. Creighton, J. Mol. Biol. 87, 563 (1974).
304
OXIDATIONS, HYDROXYLATIONS,AND HALOGENATIONS
[18]
N o n e of these systems has shown substantial advantages in terms of regeneration rate and yield. White 9 has described the air oxidation methods early used in Anfinsen's laboratory. Air oxidation has been studied also in our laboratory. ~°,16,~8,19 We believe that the use of thiol-disulfide combinations is preferable because of the greater speed of the overall reactions, the often higher yields of active protein, and the greater reproducibility of the results. L e t us reexamine the regeneration of p r o t e i n - c y s t e i n e and mixed disulfides with mercaptoethanol, as carried out by Bradshaw e t a l . 8 It seems likely that that system is rapidly transformed by thiol-disulfide exchanges, into a regeneration system very similar to what we have detailed above (where at zero time we have completely reduced protein and a mixture o f low molecular weight thiols and disulfides). That is, from different beginnings, the two reaction systems converge to produce similar reaction intermediates and products. Likewise, oxidation of a reduced protein with d e h y d r o a s c o r b a t e in the presence of excess added thiol 2° will rapidly produce a mixture of low molecular weight thiol, its conjugate disulfide, and mixed disulfides. Again, this would appear to produce a convergent reaction pathway or network. It should be apparent that the reaction formulated in Eq. (4) does not require that the role of C y S - be played by a low molecular weight thiol; it can also be played by a protein thiol. This provides a mechanism for shuffling - - S - - S - - bonds even in the absence of added low molecular weight thiol. The much lower reaction rates observed under these conditions probably result from the limited concentration of protein thiol and an unfavorable stereochemistry for shuffling reactions. One final note: commercial preparations of 2-mercaptoethanol contain variable amounts of disulfide as received from the supplier and are susceptible to air oxidation once the container has been opened. Moreover, in the regeneration solution itself, even in the presence of E D T A , air oxidation is rapid enough to increase substantially the ratio of [ - - S - - S - - ] to [ - - S H ] o v e r the course of a few hours.J6,2J
~8j._p. Perraudin, T. Torchia, and D. B. Wetlaufer, J. Biol. Chem. (in press). x9A. K. Ahmed, S. W. Schaffer, and D. B. Wetlaufer, J. Biol. Chem. 250, 8477 (1975). 2oD. Givol, R. F. Goldberger, and C. B. Anfinsen, J. Biol. Chem. 239, 3114 (1964). 2i p. Branca, E. Chen, and D. B. Wetlaufer, Fed. Proc. Fed. A m . Soc. Exp. Biol. 42, Abstr. 1415 (1983).
PROTEIN DISULFIDE
FORMATION
301
[18] N o n e n z y m a t i c F o r m a t i o n a n d I s o m e r i z a t i o n o f Protein Disulfides B y DONALD B . WETLAUFER
The formation of protein disulfides can proceed either by enzymatic catalysis r-3 or by uncatalyzed thiol-disulfide exchange. 4,5 It is not yet clear which of these mechanisms predominates in vivo. It has been long known that strong acid can lead to disulfide isomerization. 6 It is also well known that disulfides can be cleaved by sulfitolysis in a mechanism that has formal similarity to thiol-disulfide exchange. 7 We will discuss neither of these further, but will deal with nonenzymatic systems that employ thiol-disulfide exchange reactions between proteins and low molecular weight thiols and/or disulfides. In the covalent reaction (1) it is important to note that the thiolate anion, R'"--S , is the reactive species, not the protonated thiol itself, R'"--SH. The fact that most common alkyl thiols have proton ionization pK values in the range of 8-10 means that the exchange reaction can proceed rapidly under mildly alkaline or neutral conditions; conversely, it is inhibited by acid. [ R ' - - S - - S - - R " ' + R"S R ' - - S - - S - - R " + R"S ~ ~ or l R " - - S - - S - - R " ' + R'S-
(I)
The second point to note is that the reaction is reversible; this ready reversibility proves to be very important. We can, for example, prepare the mixed disulfide between a reduced protein and cysteine (or other low molecular weight disulfide) simply by forcing the equilibrium of Eq. (1) as follows: P ( - - S )~ + 1000x C y - - S - - S - - C y ~ P ( - - S - - S - - C y ) x + xCyS+ (1000x - x ) C y - - S - - S - - C y
(2)
i D. Givol, F. DeLorenzo, R. F. Goldberger, and C. B. Anfinsen, Proc. Natl. Acad. Sci. U.S.A. 53, 676 (1965). 2 H. M. Katzen and D. Stetten, Jr., Fed. Proc. Fed. Am. Soc. Exp. Biol. 21, 201 (1962); H. M. Katzen and F. Tietze, J. Biol. Chem. 241, 3561 (1966). 3 R. B. Freedman and H. C. Hawkins, Biochem. Soc. Trans. Biochem. Rev. 5, 348 (1977). 4 D. B. Wetlaufer, V. P. Saxena, A. K. Ahmed, S. W. Schaffer, P. W. Pick, K.-J. Oh, and J. D. Peterson, in '"Protein Crosslinking," Part A (M. Friedman, ed.), p. 43. Plenum, New York, 1977. 5 V. P. Saxena and D. B. Wetlaufer, Biochemistry 9, 5015 (1970). 6 A. P. Ryle and F. Sanger, Biochem. J. 60, 535 (1955). 7 W. W. Chan, Biochemistry 7, 4247 (1968).
METHODS IN ENZYMOLOGY, VOL. 107
Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182007-6
302
[18]
OXIDATIONS, HYDROXYLATIONS, AND HALOGENATIONS
Bradshaw e t a l . 8 prepared the mixed disulfide oflysozyme and cysteine in just this fashion. The example in Eq. (2) makes the assumptions that the pK values and the oxidoreduction potentials of the several thiol-disulfide pairs are the same. While this is not strictly true, it is a reasonable approximation under the conditions employed. This mixed disulfide was used as the starting form of the protein in experiments, wherein lysozyme activity was regenerated 8 by the addition of mercaptoethanol, cysteine, or cysteamine to solutions buffered in the pH range 7 to 8.5. Cy--S--S Cy--S--S
S--S--Cy
\/ P
S--S--R
\/ P
+ CyS
\
+ RS ~
Cy
S--S
\
(3)
Ol" S--S--Cy
Cy--S
S
S
\/ P
+ Cy--S
\
S
R
S--S--Cy
Reactions of the type indicated by Eq. (3) can occur with any one of the protein-cysteine mixed disulfides. In addition to the back reaction of Eq. (3), the lower protein product on the right-hand side can undergo intramolecular thiol-disulfide exchange [Eq. (4)]. I Cy--S--S Cy--S--S
S
\/ P
S
\/ P
~
\
S--S--Cy
+ CyS
\
(4)
S
/
or
S~S
\/ P
+ CyS
\ S
S
Cy
It should be obvious that, if one of the two protein products in Eq. (4) represents a native pairing of protein thiols, the other must be nonnative. The back-reaction of Eq. (4) provides a route for removing nonnative disulfides from reaction intermediates in an oxidative regeneration. Often one begins with a completely reduced disulfide protein and wishes to regenerate the native disulfides rapidly and in high yield. The preparation of reduced proteins has been described elsewhere. 9,1° We can see by inspection of the above equations that the rates of some essential s R. A. Bradshaw, L. Kanarek, and R. L. Hill, J. Biol. Chem. 242, 3789 (1967). 9 F. H. White, Jr., this series, Vol. 11, p. 481. 10 S. S. Ristow and D. B. Wetlaufer, Biochem. Biophys. Res. Commun. 50, 544 (1973).
[18]
PROTEIN DISULFIDE FORMATION
303
reactions will depend on the concentration of R - - S - - S - - R and others will depend on the concentration of R - - S - . However, all the reactions are expressed by Eq. (1); that is, they are all thiol-disulfide exchange reactions? Empirically we have found5,1El2 that a concentration of R - - S of approximately 1-3 mM in the presence of one-fifth to one-tenth of that concentration of R - - S - - S - - R provides nearly optimal conditions for the oxidative re-formation of native protein disulfides. Typical conditions for such a reaction follow: Tris acetate buffer, 0.02 M, pH 8.0, 25°, 1 /~M reduced protein, 3.0 mM reduced glutathione, 0.3 mM oxidized glutathione, 1 mM EDTA. If the regeneration requires more than a few hours, it may prove advantageous to exclude atmospheric oxygen. Re-formation of protein disulfides may be monitored by a number of analytical methods. 8'1° Half-times for formation of protein disulfides have been found between a few minutes and 2 hr. It may be more relevant to monitor the appearance of a specific biological activity. The latter requires a rapid specific assay under conditions inhibitory to further thioldisulfide exchange--for example, a few minutes at pH 5. 5,j1,~2 Typically, the biological activity appears more slowly than the protein disulfide. 5,8,~1,~2 The above regeneration system has proved to be relatively insensitive to temperature over the range 25-40 °. Regenerations of hen egg white lysozyme with a glutathione system as described above showed substantially less than one protein-glutathione mixed disulfide throughout the whole course of the reaction. ~3 Means and Feeney 14 have noted, and we have also observed, that reduced proteins have a strong tendency to aggregate. This is why we recommend that oxidative renaturation be carried out at low protein concentrations. In the case of RNase A, much higher concentrations can be used, but the regeneration is very sensitive to the presence of specific ions in the buffer (sulfate and phosphate accelerate; bromide and thiocyanate inhibit).15 In addition to glutathione, several other low molecular weight thiol-disulfide pairs can promote oxidative renaturation. These include cysteine and cystine, 8 cysteamine and cystamine,8,12 reduced and oxidized 2-mercaptoethanol, 8,9,16 and reduced and oxidized dithiothreitol, j7 u W. L. Anderson and D. B. Wetlaufer, J. Biol. Chem. 251, 3147 (1976). ~2 K. Oh-Johanson, D. B. Wetlaufer, R. Reed, and T. Peters, Jr., J. Biol. Chem. 256, 445 (1981). ~3p. W. Pick, Ph.D. Thesis, University of Minnesota, 1974. Manuscript in preparation. t4 G. E. Means and R. E. Feeney, "Chemical Modification of Proteins." Holden-Day, San Francisco, California, 1971. t5 S. W. Schaffer, A. K. Ahmed, and D. B. Wetlaufer, J. Biol. Chem. 250, 8483 (1975). t6 p. Branca, M.S. Thesis, University of Delaware, 1982. Manuscript in preparation. t7 T. E. Creighton, J. Mol. Biol. 87, 563 (1974).
304
OXIDATIONS, HYDROXYLATIONS,AND HALOGENATIONS
[18]
N o n e of these systems has shown substantial advantages in terms of regeneration rate and yield. White 9 has described the air oxidation methods early used in Anfinsen's laboratory. Air oxidation has been studied also in our laboratory. ~°,16,~8,19 We believe that the use of thiol-disulfide combinations is preferable because of the greater speed of the overall reactions, the often higher yields of active protein, and the greater reproducibility of the results. L e t us reexamine the regeneration of p r o t e i n - c y s t e i n e and mixed disulfides with mercaptoethanol, as carried out by Bradshaw e t a l . 8 It seems likely that that system is rapidly transformed by thiol-disulfide exchanges, into a regeneration system very similar to what we have detailed above (where at zero time we have completely reduced protein and a mixture o f low molecular weight thiols and disulfides). That is, from different beginnings, the two reaction systems converge to produce similar reaction intermediates and products. Likewise, oxidation of a reduced protein with d e h y d r o a s c o r b a t e in the presence of excess added thiol 2° will rapidly produce a mixture of low molecular weight thiol, its conjugate disulfide, and mixed disulfides. Again, this would appear to produce a convergent reaction pathway or network. It should be apparent that the reaction formulated in Eq. (4) does not require that the role of C y S - be played by a low molecular weight thiol; it can also be played by a protein thiol. This provides a mechanism for shuffling - - S - - S - - bonds even in the absence of added low molecular weight thiol. The much lower reaction rates observed under these conditions probably result from the limited concentration of protein thiol and an unfavorable stereochemistry for shuffling reactions. One final note: commercial preparations of 2-mercaptoethanol contain variable amounts of disulfide as received from the supplier and are susceptible to air oxidation once the container has been opened. Moreover, in the regeneration solution itself, even in the presence of E D T A , air oxidation is rapid enough to increase substantially the ratio of [ - - S - - S - - ] to [ - - S H ] o v e r the course of a few hours.J6,2J
~8j._p. Perraudin, T. Torchia, and D. B. Wetlaufer, J. Biol. Chem. (in press). x9A. K. Ahmed, S. W. Schaffer, and D. B. Wetlaufer, J. Biol. Chem. 250, 8477 (1975). 2oD. Givol, R. F. Goldberger, and C. B. Anfinsen, J. Biol. Chem. 239, 3114 (1964). 2i p. Branca, E. Chen, and D. B. Wetlaufer, Fed. Proc. Fed. A m . Soc. Exp. Biol. 42, Abstr. 1415 (1983).