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S-methylation of cysteine residues in peptides and proteins with dimethylsulfate
S-Methylation
of Cysteine Residues in Peptides and Proteins with Dimethylsulfate
JULIUS EYEM,~J~RGEN Department
SJ~DAHL,
AND JOHN !~J~QUIST~
of Medical and Physiological Chemistry, Biomedical Centre, Uppsala, P.O. Box 575, S-751 23 Uppsala, Sweden
University
of
Received November 29, 1975; accepted March 1.5, 1976 A simple, rapid, and quantitative procedure for the protection of reduced S-S bonds in proteins by selective methylation with dimethylsuifate is described. The formed S-methylcysteine is a stable derivative well suited for ion-exchange chromatographic (iec) or gas-liquid chromatographic (glc) analysis. Its methylor phenyl-t~obydantoin is easily analyzed by gas-liquid chromatography after t~methy~silylation or by thin-layer chromatography (tic).
Stabilization of sulfhydryl groups in proteins is usually accomplished by reaction with performic acid, iodoacetic acid, iodoacetamide (l), ethylene imine (2), or sodium tetrathionate (3) giving cysteic acid, ~-carboxymethylcysteine, S-/J-aminoethyl-cysteine, andS-sulfocysteine, respectively. The sulfhydryl groups can also be treated with 2-nitro-5thiocyanobenzoic acid which leads to the formation of S-cyano-cysteine and thereby introduces the possibility of cleavage of the amino peptide bond of the derivatized cysteine residue (4,5). Another method of choice is the alkylation procedure with sodium 2-bromoethane-sulfonate giving S-sulfoethylcysteine (6). The modification of cystine and cysteine residues to a product suitable for both ion-exchange chromatographic (iec) and gas-liquid chromatographi~ (glc) amino acid analysis and for sequential Edman degradation with subsequent glc or thin-layer chromatographic (tic) analysis of the formed amino acid methyl- or phenyl-thiohydantoins can be achieved by a selective methylation of the SH groups. S-methylcysteine is well suited for glc analysis of amino acids as its Nhep~fluorobuty~c (HFB) amino acid n-propyl ester (7). Moreover, it can be released as its methylthiohydantoin derivative (8) by a modified Edman degradation procedure (9) and be analyzed by glc after trimethylsilylation (8). ~-AIkylation of cysteine predominancy by use of alkyl iodides has been described (lo- 16), but application of the methods to reduced proteins was not demonstrated. Rochat et al. (17) used different alkyl iodides for the 1 Present address: LKB-Produkter AB, S-161 25 Bromma, Sweden. 2 To whom correspondence should be addressed. 359 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
360
EYEM, SJdDAHL
AND SJ6QUIST
alkylation of SH groups in proteins, and Heinrikson (18,19) applied methylp-nitrobenzene-sulfonate for the same purpose. This article describes a procedure for the protection of reduced S-S groups by reaction with dimethylsulfate and some applications of the method to peptides and proteins. The formed S-methyl-cysteine residue is well suited for iec or glc analytical methods and for sequence analysis. MATERIALS
AND METHODS
(A) Chemicals. S-Methyl-cysteine, other amino acids, peptides, bovine insulin, and lima bean trypsin inhibitor were from Sigma Chemical Co., St. Louis, MO. The snake venom toxin Naja-4 was kindly provided by Dr. D. Eaker. Methylisothiocyanate (MITC) was obtained from Fluka AG, Switzerland; dioxane and dimethylsulfate from E. Merck, Germany; dithioerythritol (DTE), acetonitrile, n-propanol, heptafluorobutyric acid anhydride (HFBAA), trimethylchlorosilane (TMCS), and bis-trimethylsilyl-trifluoroacetamide (BSTFA) from Pierce Chem. Co., Rockford, 111. The hydrochloric acid used for hydrolysis originated from Riedel-de Ha&n, Germany and Sephadex G-15 from Pharmacia Fine Chemicals AB, Sweden. All other chemicals were of analytical grade. (B) Synthesis of the 3-methyl-2-thiohydantoin
of S-methyl-cysteine
(MT&S-Me-CyS). MTHS-Me-CyS was synthesized according to the method described by Stepanov and Krivtsov (20) with the following modifications. Five millimoles of S-Me-CyS was dissolved in 5 ml of 1 M NaOH, and the pH was adjusted to 9.0 with 1 M HCl. The warm solution (40°C) was treated with 10 mmol of methylisothiocyanate dissolved in 3 ml of peroxidefree dioxane under nitrogen. After stirring the solution at pH 9.0 for 2 hr at room temperature in a pH-stat (Radiometer, Copenhagen), it was adjusted to pH 6.5 with 1 M HCl and lyophilized. The material was dried for 1 hr at 50°C under vacuum (10m2 Torr) and dissolved in 1 ml of acetonitrile and 5 ml of 1 M HCl. It was heated to the azeotropic boiling point (76°C) and maintained at this temperature for 20 min. The cooled sample was lyophilized and the solid material was extracted with acetonitrile. The extract was dried in a stream of nitrogen and the residue was recrystallized three times from warm ethanol. The molar absorptivity (ethanol) l 266nmwas 15,400 and the ratio eZ33n&266nmwas 0.41. The identity was confirmed by mass spectrometry (ms). (C) Derivatization of samplesfor glc and ms. (a) The N-HFB amino acid n-propyl ester derivatives used in quantitative glc amino acid analysis were prepared according to Moss et al. (21). (b) Trimethylsilyl derivatives of lyophilized samples for gas chromatographic and mass spectrometric analyses were prepared as described in (8). (0) Analytical methods. (a) Gas chromatographic analysis of N-HFB amino acid n-propyl esters was performed according to Sjodahl et al. (7).
S-METHYLATION
OF PEF’TIDES AND PROTEINS
361
(b) Gas chromatographic and mass spectrometric analysis of silylated samples were performed according to Eyem and Sjiiquist (8) but using different temperature programs, depending on the type of sample analyzed. The identity of silylated derivative peaks on the chromatograms was confirmed by the appearance of the corresponding molecular ions in the gc-ms analyses (measured with an LKB 2091 instrument). (c) Ionexchange chromatographic analysis of free amino acids was performed on a Biochrom BC-200 analyzer according to Spackman et al. (22). Analyses were performed on samples hydrolyzed in 6 M HCI (1% phenol, w/v) for 20 and 72 hrs. In some cases the samples were oxidized with pet-formic acid (1) prior to hydrolysis. (d) Thin-layer chromatographic identification of phenylthiohydantoin amino acids was done according to Summers et al. (23).
(E) Kinetic studies. (a) Cystine (10 pmol) was dissolved in 1 ml of 0.1 M HCI, and the pH was adjusted with NaOH to 7.0,7.5,8.0,9.0,9.5, or 10.0. Dithioerythritol(30 pmol) in 0.3 ml ofaq. dest. was added and the reaction mixture was stirred for 30 min under nitrogen at 25°C. The dithioerythritol solution was prepared from freshly boiled water cooled under nitrogen. Dimethylsulfate (caution, carcinogenic) in dioxane (30 ~1 of a 1 M solution) was added and the solution was kept at the appropriate pH for 30 min in a pH-stat. The methylation was stopped by adding 0.8 ml of 0.1 M ammonia. A solution of methionine was added as internal standard. The samples were lyophilized, silylated, and analyzed by gas chromatography and mass spectrometry as described above. (b) Cystine samples were treated as above but at pH 7.0for 7.5,15, or 30 min in the methylation reaction. (F) Side reactions on imidazolyl groups. Histidine samples were treated as above (pH 7.0; methylation time, 30 min). The samples were analyzed by iec. (G) Stability test. S-Methyl-cysteine (0.25 pmol) was hydrolyzed for 20 and 72 hr in 6 M HCl at 110°C. Unhydrolyzed and hydrolyzed samples were analyzed with an ion-exchange amino acid analyzer. Three independent samples were prepared for each hydrolysis time. (H) Methylation ofpeptides andproteins. (a) Reduced glutathione (3 mg) and glycyl-histidyl-glycine (3 mg) were dissolved in 1 ml of 0.1 M HCI each. The pH was adjusted to 7.0 with 0.1 M NaOH, and a threefold molar excess of dithioerythritol was added to the stirred solution under nitrogen. After 30 min a threefold molar excess of dimethylsulfate was added and the reaction was allowed to proceed for 30 min at pH 7.0 in the pH-stat. The reaction was terminated by adding 1 ml of 0.1 M ammonia. Internal standard (norleucine) was added, and aliquots were taken for amino acid analysis. (b) Insulin, Naja-4 snake venom toxin, and lima bean trypsin inhibitor (10 pmol of cysteine residues) were dissolved in 1 ml of 6 M guanidine-HCl. The samples were methylated as described above
362
EYEM, SJ(IDAHL
AND SJdQUIST
FIG. 1. Influence of pH on the yield of S-Me-CyS when methylating free reduced cystine samples for 30 min. Experimental details are found in Materials and Methods. 2, iec analysis. 5& gtc analysis.
and desalted by dialysis for 16 hr against running deionized water (insulin) or on a Sephadex G-15 column in 1 M HCOOH. Internal standard was added and aliquots were taken for amino acid analysis. RESULTS
AND DiSCUSStON
Figure 1 shows the results of the pH-dependence followed are:
/ 2
f Q’eS% NH,\ CH--CH,-*-z =L> 1
e\
cod-
studies. The reactions
CH-CH,--SH
_IC
coo’
NH,\+CH-CH,-S-W, coo”
Since the iec analysis can be used only for compounds having free amino groups, all experiments were followed simultaneously by glc. The relative retention times of silylated reagents and reaction products are listed in Table 1, In Fig. 2, a gas chromatogram of the derivatized reaction mixture is shown. Peak numbers correspond to the numbers in Table 1. S-Methylation followed by glc analysis of the derivatized reaction mixture was measured both as an increase of the TMS-S-Me-CyS peak and as a decrease of the TMS-CyS peak in the chromatograms. From these results, pH 7.0 was considered optimal for methylation (Fig. 1). Furthermore, a methylation time of 30 min was judged adequate as the relative yields of S-Me-CyS after 7.5,15, and 30 min were determined to be 78,91, and lOO%,
S-METHYLATION
OF PEPTIDES TABLE
RELATIVE
363
AND PROTEINS
1
RETENTION TIMES (t’R) OF SILYLATED REACTION COMPONENTS (RELATIVE PHENANTHRENE) IN THE GLC-ANALYSIS OF A FREE REDUCED CYSTINE SAMPLE METHYLATED FOR 30 MINUTES AT PH 7.0”
Peak number 2 3 4 5 6 7 8 9 10
Silylated component NJV-dimethylS-methyl-cysteine S-methyl-cysteine N-methyl+methyl-cysteine (Methionine) Cysteine Oxidized DTE Methylated DTE (Phenanthrene) DTE Cystine
TO
I‘R 0.57 0.66 0.72 (0.77) 0.82 0.84 0.93 (1.W 1.05 1.50
D Experimental details are given in Materials and Methods. Temperature program, 80°C for 4 min, then 5Wmin up to 240°C. The mean carrier gas (N,) velocity (k) = 8 cm/set. The components were identified by mass spectrometry.
respectively. The reduction of S-S bonds with DTE (24) is very rapid and quantitative, even with a stoichiometric amount of DTE. The peak corresponding to trimethylsilyl (TMS) cystine disappears completely after reduction for 15 min at room temperature. The aqueous solution of the reagent itself, however, is very sensitive to oxidation by dissolved oxygen, hence the appropriate excess of DTE should be used or the amount of oxidized and reduced DTE in the solution should be tested by glc. The ratio of DTE to its oxidized cyclic disulfide can easily be checked with any gas chromatographic column suitable for the separation of TMS derivatives, since the retention of each substance differs sufficiently (see components 6 and 9 in Table 1). This test is recommended not only for this methylation method but also for other methods utilizing DTE for reduction. The degree ofN-methylation, as determined by gc-ms, is pH dependent and is probably the main reason for the low yield of S-Me-CyS at a pH above 8.5 (Fig. 1). The pH dependence of side reactions giving N-methylated products is obvious, as amino groups can be methylated only in the deprotonated form in which the nitrogen atoms have free electron pairs available for the primary complex formation with dimethylsulfate. By this consideration, the highest probability of N-methylation is at amino or imino groups with low pKa values, such as the N-terminal groups and the imidazolyl groups of histidine residues. However, only 2% N- methylation was observed when histidine was subjected to reaction at pH 7.0 for 30 min. In this case the analyses were performed by iec, as histidine presents certain difficulties in the glc method (7).
364
EYEM, SJODAHL
AND SJt)QUIST 6
I
I
5
10
1
15 20 MINUTES
1
I
25
30
FIG. 2. Gas-liquid chromatography of a silylated reaction mixture after reduction and methylation of cystine. Methylation was performed at pH 7 for 30 min. Experimental details are given in Materials and Methods. Numbering of peaks corresponds to that in Table 1. Temperature program, 80°C for 4 min, then S”C/min up to 240°C.
The stability of S-Me-CyS towards the normally employed acid hydrolysis conditions for amino acid analysis was tested. It has been claimed (19) that 10% decomposition occurs during the first 22 hr of hydrolysis. In our experiments yields of 97 and 91% were observed after 20- and 72-hr hydrolysis, respectively. A good approximation would be the use of a decomposition correction factor of O.l4%/hr of hydrolysis. This factor should be dependent on the hydrolysis technique, the presence of interfering substances, etc. The iec analysis of,!?-Me-CyS does not present any difficulties; the amino acid emerges next to proline in the chromatogram. The results from the methylation studies on different peptides and proteins dissolved in 6 M guanidine-HCl are illustrated in Table 2. Although no conclusions could be drawn as to whether acid-labile modifications had occurred, such modifications were not observed in the methylation studies on free cystine and histidine described above. The complete disappearance of cysteine was observed in all analyses of methylated samples
S-METHYLATION
OF PEPTIDES TABLE
AND
365
PROTEINS
2
AMINO AC~DCOMP~SITIONSOFMETHYLATEDANDNONMETHYLATEDPE~IDESAND PROTEIN@ Lima bean Reduced Amino acid
Gly-His-Gly (Meth iec)
LYS His A*g CyS03H ASP Thr Se* Glu Pro S-Me-CyS GUY Ala Vd Met Ile
1.01 -
LW TY* Phe
2.00 -
Theory
iec
---(1) 0.93 ---1 0.99 (1) 1 1.01 ----------------
glutathione
Insulin
Meth iec
Meth glc
-
_
1.00 1.00 1.03 -
1.00 1.05 O.% -
-
-
-
-
-
-
-
-
Theory
Naja-4 toxin
iec
Meth glc
Theory”
iec
Metb iec
3 1 3 7 1 (6) 4 3 5
1.1 1.9 1.0 5.6 3.0 1.2 2.8 7.2 1.2 4.0 2.9 4.6
1.1 1.9 I.1 31 1.2 3.0 7.4 1.2 5.9 3.9 2.9 4.5
4 1 6 (10) 9 9 4 1 6 (10) 5 2 4
4.0 1.0 5.8 8.9 9.4 8.7 4.1 1.6 6.2 5.1 2.0 3.7
1 6 4 3
0.9 6.0 4.0 2.8
0.9 6.0 4.0 2.7
4 1 1 3
3.7 1.3 1.1 2.7
1 2 1
(6)
trypsin
inhibitor iec
Meth kc
Meth &
3.9 1.0 5.6 9.3 8.6 4.2 1.7 6.2 9.5 5.2 2.0 3.6
4.2 6.3 2.4 13.4 15.8 5.1 16.5 8.7 8.3 1.5 3.9 1.5
4.5 6.2 2.7 15.6 5.3 15.6 8.7 7.7 14.4 1.6 4.1 1.4
5.1 6.0 2.3 15.9 5.4 16.8 9.5 7.7 14.8 1.4 4.1 1.4
3.7 1.3 1.1 2.7
5.0 3.0 1.5 2.4
5.0 3.0 1.5 2.4
4.7 3.0 1.6 2.8
“Ibe vahx.s given for threonine and wine are extrapolated to zero time, the values for valine and isoleucine are the 72shr values. The cyst&e-plus-cystine contents in the nonmethylated samples were determined after perfonnic acid oxidation and a subsequent 20.hr hydrolysis and are expressed as cysteic acid. S-Me-CyS-values were corrected to zero time (see text). For all other amino acids the mean values are used. Tryptophan was not determined and is therefore omitted. The analyses of glycyl-histidyl-glycine, reduced glutathione, insulin, Naja-4 toxin, and lima bean trypsin inhibitor were namrdized to hvo glycine, one glutamic acid, six leucine, two r&nine, and three leucine residues, respectively. Meth, methylated sample; iec. analyzed by iec; glc, analyzed by glc; theory, theoretic values. ’ The theoretical values for Nr&x-4 toxin were personally communicated by Dr. D. Eaker.
originally containing this amino acid, and the appearance of equivalent amounts of S-methyl-cysteine was observed. In all cases these values exceeded the values for cysteic acid in the oxidized samples, but the differences cannot be considered significant. Generally, the analyses of methylated samples were in better agreement with the theoretical values for the cysteine content than the analyses of oxidized samples. It is also clear from the results presented in Table 2 that the quantitative glc amino acid analysis, when employed, gave results entirely compatible with the iec analysis. Furthermore, no extra peaks were obtained in the gas chromatograms. This fact, together with the excellent agreement of the analyses of methylated and nonmethylated material, strongly supports the statement of a methylation reaction specific for cysteine residues. It goes without saying that solubility problems encountered, for example, in the desalting step after methylation are shared by all the different methods generating the same product. Larger peptides and proteins are easily handled by dialysis if such problems arise, but smaller
366
EYEM, SJGDAHL
L
.
0
5
AND SJGQUIST
I
10
1s
20
25 MINUTES
30
35
10
15
50
FIG. 3. Gas-liquid chromatography of silylated MTH derivatives of S-Me-CyS and other amino acids. Experimental details are given in Materials and Methods. Temperature program, 120°C for 8 min, then Z.S”C/min up to 250°C.
peptides may require desalting by gel chromatography in a suitable solvent. When necessary, an organic solvent could be added to the reaction mixture. The methylation of cysteine residues has been proposed by Gross (25) to be useful in order to get a derivative suitable for bromine cyanide cleavage at cysteine, analogous to the cleavage at methionine residues
H 5 > 5 v)
ILE,LEU 0 PRO PHE OOVAL ME P f30AL~ S-Me-C% 0 (31-y GLN LYSO GLUO 8 d;N 0 SER TYRO OASP d HIS, ARG
X
Solvent
II
FIG. 4. Thin-layer chromatography of FM-IS-Me-CyS and other PTH-amino acids on a 5 x S-cm polyamide thin-layer sheet. Solvent I, toluene:n-pentane:glacial acetic acid, 60:30:35 (v/v). Solvent II, 35% aqueous acetic acid.
S-METHYLATION
0
5
OF PEPTIDES
10
15 20 MINUTES
AND PROTEINS
25
30
367
35
FIG. 5. Separation of the N-HFB amino acid n-propyl ester derivatives of S-Me-CyS and other amino acids. Experimental details are given in Materials and Methods. Temperature program, 70°C for 4 min, then S”C/min up to 250°C. The peak behind S-Me-CyS represents cysteine (impurity from cystine).
with this reagent. This type of fragmentation has been utilized to cleave an S-methylated peptide of bovine pancreatic ribonuclease A with the formation of the expected fragments in 88% yield (26). In sequence studies, the possibility of using S-Me-CyS for glc analysis is an advantage. In Fig. 3, a gas chromatogram from an analysis of TMS derivatives of different methylthiohydantoin amino acids, including MTH-S-Me-CyS, is illustrated. Preliminary experiments with phenylthiohydantoin (PTH)-S-Me-CyS showed that even this derivative is well suited for glc analysis. Furthermore, this derivative is easily analyzed by common thin-layer chromatographic techniques. In Fig. 4, the separation of PTH-S-Me-CyS from other PTH-amino acids on a 5 x 5-cm polyamide thin-layer sheet (23) is shown. Figure 5 shows the elution profile obtained when analyzing S-Me-CyS and other amino acids gas chromatographically as the N-HFB amino acid n-propyl esters (7). To summarize, the methylation of cysteine residues with dimethylsulfate is simple, rapid, quantitative, and selective and should be a good alternative to other methylating procedures employed. A derivative is formed, which is well suited for both iec and glc amino acid
368
EYEM,
SJODAHL
AND SJOQUIST
analysis as well as glc or tic analysis of the MTH or PTH derivatives. Furthermore, it has been shown (26) that S-methylation introduces a peptide bond sensitive to the action of bromine cyanide. Therefore, we believe that this modification procedure should be applicable in sequence work. ACKNOWLEDGMENTS This investigation was supported by grants from the Swedish Medical Research Council (Project No. 13X-2518) and the Swedish Natural Science Research Council (Project No. K2%1-001). We thank Mrs. M. Gustafson for skillful secretarial work.
REFERENCES 1. Hirs, C. H. W. (1%7) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 197-203, Academic Press, New York. 2. Cole, R. D. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.) Vol. 11, pp. 315-317, Academic Press, New York. 3. Inglis, A. S., and Liu, T. Y. (1970) J. Bid. Chem. 245, 112-116. 4. Jacobson, G. R., Schaffer, M. H., Stark, G. R., and Vanaman, T. C. (1973) J. Biol. Chem. 248, 6583-6591. 5. Degani, Y., and Patchomic, A. (1974) Biochemisfry 13, l-11. 6. Niketik, V., Thomsen, J., and Kristiansen, K. (1974) Eur. J. Biochem. 46, 547-551. 7. Jiinsson, J., Eyem, J., and Sjoquist, J. (1973) Anal. Biochem. 51, 204-219. (The first author’s name is now Sjiidahl.) 8. Eyem, J., and Sjiiquist, J. (1973) Anal. Biochem. 52, 255-271. 9. Richards, F. F., Barnes, W. T., Lovins, R. E., Salomone, R., and Waterfleld, M.D. (1969) Nature (London) 221, 1241-1244. 10. Pirie, N. W. (1932) Biochem. J. 26, 2041-2045. 11. de Vigneaud, V., Loring, H. S., and Craft, H. A. (1934) J. Biol. Chem. 105,481-488. 12. Wood, J. L., and du Vigneaud, V. (1934) J. Biol. Chem. 130, 109-l 14. 13. Zbarsky, S. H., and Young, L. (1943)J. Biol. Chem. 151,211-215. 14. Stoll, A., and Seebeck, E. (1949) Helv. Chim. Acta 32, 866-876. 15. Armstrong, M. D., and Lewis, J. D. (1951) J. Org. Chem. 16, 749-753. 16. Banks, T. E., and Shafir, J. A. (1970) Biochemistry 9,3343-3348. 17. Rochat, C., Rochat, H., and Edman, P. (1970) Anal. Biochem. 37, 259-267. 18. Heinrikson, R. L. (1970) Biochem. Biophys. Res. Commun. 41,967-972. 19. Heimikson, R. L. (1971) J. Biol. Chem. 246, 4090-4096. 20. Stepanov, M. V., and Krivtsov, V. F. (1965) Zh. Obsch. Khim. 35,982-986. 21. Moss, C. W., Lambert, M. A., and Diaz, F. J. (197l)J. Chromatogr. 60, 134-136. 22. Spa&man, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. 23. Summers, M. R., Smythers, G. W., and Oroszlan, S. (1973) Anal. Biochem. 53, 624-628. 24. Cleland, W. W. (1964) Biochemistry 3,480-482. 25. Gross, E., Morel], J. L., and Lee, P. Q. (1%7) Seventh Znt. Congr. Biochem. (Tokyo) 11, 535-536. 26. Gross, E., and Morel& J. L. (1974) Biochem. Biophys. Res. Commun. 59, 1145-1150.
of Cysteine Residues in Peptides and Proteins with Dimethylsulfate
JULIUS EYEM,~J~RGEN Department
SJ~DAHL,
AND JOHN !~J~QUIST~
of Medical and Physiological Chemistry, Biomedical Centre, Uppsala, P.O. Box 575, S-751 23 Uppsala, Sweden
University
of
Received November 29, 1975; accepted March 1.5, 1976 A simple, rapid, and quantitative procedure for the protection of reduced S-S bonds in proteins by selective methylation with dimethylsuifate is described. The formed S-methylcysteine is a stable derivative well suited for ion-exchange chromatographic (iec) or gas-liquid chromatographic (glc) analysis. Its methylor phenyl-t~obydantoin is easily analyzed by gas-liquid chromatography after t~methy~silylation or by thin-layer chromatography (tic).
Stabilization of sulfhydryl groups in proteins is usually accomplished by reaction with performic acid, iodoacetic acid, iodoacetamide (l), ethylene imine (2), or sodium tetrathionate (3) giving cysteic acid, ~-carboxymethylcysteine, S-/J-aminoethyl-cysteine, andS-sulfocysteine, respectively. The sulfhydryl groups can also be treated with 2-nitro-5thiocyanobenzoic acid which leads to the formation of S-cyano-cysteine and thereby introduces the possibility of cleavage of the amino peptide bond of the derivatized cysteine residue (4,5). Another method of choice is the alkylation procedure with sodium 2-bromoethane-sulfonate giving S-sulfoethylcysteine (6). The modification of cystine and cysteine residues to a product suitable for both ion-exchange chromatographic (iec) and gas-liquid chromatographi~ (glc) amino acid analysis and for sequential Edman degradation with subsequent glc or thin-layer chromatographic (tic) analysis of the formed amino acid methyl- or phenyl-thiohydantoins can be achieved by a selective methylation of the SH groups. S-methylcysteine is well suited for glc analysis of amino acids as its Nhep~fluorobuty~c (HFB) amino acid n-propyl ester (7). Moreover, it can be released as its methylthiohydantoin derivative (8) by a modified Edman degradation procedure (9) and be analyzed by glc after trimethylsilylation (8). ~-AIkylation of cysteine predominancy by use of alkyl iodides has been described (lo- 16), but application of the methods to reduced proteins was not demonstrated. Rochat et al. (17) used different alkyl iodides for the 1 Present address: LKB-Produkter AB, S-161 25 Bromma, Sweden. 2 To whom correspondence should be addressed. 359 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
360
EYEM, SJdDAHL
AND SJ6QUIST
alkylation of SH groups in proteins, and Heinrikson (18,19) applied methylp-nitrobenzene-sulfonate for the same purpose. This article describes a procedure for the protection of reduced S-S groups by reaction with dimethylsulfate and some applications of the method to peptides and proteins. The formed S-methyl-cysteine residue is well suited for iec or glc analytical methods and for sequence analysis. MATERIALS
AND METHODS
(A) Chemicals. S-Methyl-cysteine, other amino acids, peptides, bovine insulin, and lima bean trypsin inhibitor were from Sigma Chemical Co., St. Louis, MO. The snake venom toxin Naja-4 was kindly provided by Dr. D. Eaker. Methylisothiocyanate (MITC) was obtained from Fluka AG, Switzerland; dioxane and dimethylsulfate from E. Merck, Germany; dithioerythritol (DTE), acetonitrile, n-propanol, heptafluorobutyric acid anhydride (HFBAA), trimethylchlorosilane (TMCS), and bis-trimethylsilyl-trifluoroacetamide (BSTFA) from Pierce Chem. Co., Rockford, 111. The hydrochloric acid used for hydrolysis originated from Riedel-de Ha&n, Germany and Sephadex G-15 from Pharmacia Fine Chemicals AB, Sweden. All other chemicals were of analytical grade. (B) Synthesis of the 3-methyl-2-thiohydantoin
of S-methyl-cysteine
(MT&S-Me-CyS). MTHS-Me-CyS was synthesized according to the method described by Stepanov and Krivtsov (20) with the following modifications. Five millimoles of S-Me-CyS was dissolved in 5 ml of 1 M NaOH, and the pH was adjusted to 9.0 with 1 M HCl. The warm solution (40°C) was treated with 10 mmol of methylisothiocyanate dissolved in 3 ml of peroxidefree dioxane under nitrogen. After stirring the solution at pH 9.0 for 2 hr at room temperature in a pH-stat (Radiometer, Copenhagen), it was adjusted to pH 6.5 with 1 M HCl and lyophilized. The material was dried for 1 hr at 50°C under vacuum (10m2 Torr) and dissolved in 1 ml of acetonitrile and 5 ml of 1 M HCl. It was heated to the azeotropic boiling point (76°C) and maintained at this temperature for 20 min. The cooled sample was lyophilized and the solid material was extracted with acetonitrile. The extract was dried in a stream of nitrogen and the residue was recrystallized three times from warm ethanol. The molar absorptivity (ethanol) l 266nmwas 15,400 and the ratio eZ33n&266nmwas 0.41. The identity was confirmed by mass spectrometry (ms). (C) Derivatization of samplesfor glc and ms. (a) The N-HFB amino acid n-propyl ester derivatives used in quantitative glc amino acid analysis were prepared according to Moss et al. (21). (b) Trimethylsilyl derivatives of lyophilized samples for gas chromatographic and mass spectrometric analyses were prepared as described in (8). (0) Analytical methods. (a) Gas chromatographic analysis of N-HFB amino acid n-propyl esters was performed according to Sjodahl et al. (7).
S-METHYLATION
OF PEF’TIDES AND PROTEINS
361
(b) Gas chromatographic and mass spectrometric analysis of silylated samples were performed according to Eyem and Sjiiquist (8) but using different temperature programs, depending on the type of sample analyzed. The identity of silylated derivative peaks on the chromatograms was confirmed by the appearance of the corresponding molecular ions in the gc-ms analyses (measured with an LKB 2091 instrument). (c) Ionexchange chromatographic analysis of free amino acids was performed on a Biochrom BC-200 analyzer according to Spackman et al. (22). Analyses were performed on samples hydrolyzed in 6 M HCI (1% phenol, w/v) for 20 and 72 hrs. In some cases the samples were oxidized with pet-formic acid (1) prior to hydrolysis. (d) Thin-layer chromatographic identification of phenylthiohydantoin amino acids was done according to Summers et al. (23).
(E) Kinetic studies. (a) Cystine (10 pmol) was dissolved in 1 ml of 0.1 M HCI, and the pH was adjusted with NaOH to 7.0,7.5,8.0,9.0,9.5, or 10.0. Dithioerythritol(30 pmol) in 0.3 ml ofaq. dest. was added and the reaction mixture was stirred for 30 min under nitrogen at 25°C. The dithioerythritol solution was prepared from freshly boiled water cooled under nitrogen. Dimethylsulfate (caution, carcinogenic) in dioxane (30 ~1 of a 1 M solution) was added and the solution was kept at the appropriate pH for 30 min in a pH-stat. The methylation was stopped by adding 0.8 ml of 0.1 M ammonia. A solution of methionine was added as internal standard. The samples were lyophilized, silylated, and analyzed by gas chromatography and mass spectrometry as described above. (b) Cystine samples were treated as above but at pH 7.0for 7.5,15, or 30 min in the methylation reaction. (F) Side reactions on imidazolyl groups. Histidine samples were treated as above (pH 7.0; methylation time, 30 min). The samples were analyzed by iec. (G) Stability test. S-Methyl-cysteine (0.25 pmol) was hydrolyzed for 20 and 72 hr in 6 M HCl at 110°C. Unhydrolyzed and hydrolyzed samples were analyzed with an ion-exchange amino acid analyzer. Three independent samples were prepared for each hydrolysis time. (H) Methylation ofpeptides andproteins. (a) Reduced glutathione (3 mg) and glycyl-histidyl-glycine (3 mg) were dissolved in 1 ml of 0.1 M HCI each. The pH was adjusted to 7.0 with 0.1 M NaOH, and a threefold molar excess of dithioerythritol was added to the stirred solution under nitrogen. After 30 min a threefold molar excess of dimethylsulfate was added and the reaction was allowed to proceed for 30 min at pH 7.0 in the pH-stat. The reaction was terminated by adding 1 ml of 0.1 M ammonia. Internal standard (norleucine) was added, and aliquots were taken for amino acid analysis. (b) Insulin, Naja-4 snake venom toxin, and lima bean trypsin inhibitor (10 pmol of cysteine residues) were dissolved in 1 ml of 6 M guanidine-HCl. The samples were methylated as described above
362
EYEM, SJ(IDAHL
AND SJdQUIST
FIG. 1. Influence of pH on the yield of S-Me-CyS when methylating free reduced cystine samples for 30 min. Experimental details are found in Materials and Methods. 2, iec analysis. 5& gtc analysis.
and desalted by dialysis for 16 hr against running deionized water (insulin) or on a Sephadex G-15 column in 1 M HCOOH. Internal standard was added and aliquots were taken for amino acid analysis. RESULTS
AND DiSCUSStON
Figure 1 shows the results of the pH-dependence followed are:
/ 2
f Q’eS% NH,\ CH--CH,-*-z =L> 1
e\
cod-
studies. The reactions
CH-CH,--SH
_IC
coo’
NH,\+CH-CH,-S-W, coo”
Since the iec analysis can be used only for compounds having free amino groups, all experiments were followed simultaneously by glc. The relative retention times of silylated reagents and reaction products are listed in Table 1, In Fig. 2, a gas chromatogram of the derivatized reaction mixture is shown. Peak numbers correspond to the numbers in Table 1. S-Methylation followed by glc analysis of the derivatized reaction mixture was measured both as an increase of the TMS-S-Me-CyS peak and as a decrease of the TMS-CyS peak in the chromatograms. From these results, pH 7.0 was considered optimal for methylation (Fig. 1). Furthermore, a methylation time of 30 min was judged adequate as the relative yields of S-Me-CyS after 7.5,15, and 30 min were determined to be 78,91, and lOO%,
S-METHYLATION
OF PEPTIDES TABLE
RELATIVE
363
AND PROTEINS
1
RETENTION TIMES (t’R) OF SILYLATED REACTION COMPONENTS (RELATIVE PHENANTHRENE) IN THE GLC-ANALYSIS OF A FREE REDUCED CYSTINE SAMPLE METHYLATED FOR 30 MINUTES AT PH 7.0”
Peak number 2 3 4 5 6 7 8 9 10
Silylated component NJV-dimethylS-methyl-cysteine S-methyl-cysteine N-methyl+methyl-cysteine (Methionine) Cysteine Oxidized DTE Methylated DTE (Phenanthrene) DTE Cystine
TO
I‘R 0.57 0.66 0.72 (0.77) 0.82 0.84 0.93 (1.W 1.05 1.50
D Experimental details are given in Materials and Methods. Temperature program, 80°C for 4 min, then 5Wmin up to 240°C. The mean carrier gas (N,) velocity (k) = 8 cm/set. The components were identified by mass spectrometry.
respectively. The reduction of S-S bonds with DTE (24) is very rapid and quantitative, even with a stoichiometric amount of DTE. The peak corresponding to trimethylsilyl (TMS) cystine disappears completely after reduction for 15 min at room temperature. The aqueous solution of the reagent itself, however, is very sensitive to oxidation by dissolved oxygen, hence the appropriate excess of DTE should be used or the amount of oxidized and reduced DTE in the solution should be tested by glc. The ratio of DTE to its oxidized cyclic disulfide can easily be checked with any gas chromatographic column suitable for the separation of TMS derivatives, since the retention of each substance differs sufficiently (see components 6 and 9 in Table 1). This test is recommended not only for this methylation method but also for other methods utilizing DTE for reduction. The degree ofN-methylation, as determined by gc-ms, is pH dependent and is probably the main reason for the low yield of S-Me-CyS at a pH above 8.5 (Fig. 1). The pH dependence of side reactions giving N-methylated products is obvious, as amino groups can be methylated only in the deprotonated form in which the nitrogen atoms have free electron pairs available for the primary complex formation with dimethylsulfate. By this consideration, the highest probability of N-methylation is at amino or imino groups with low pKa values, such as the N-terminal groups and the imidazolyl groups of histidine residues. However, only 2% N- methylation was observed when histidine was subjected to reaction at pH 7.0 for 30 min. In this case the analyses were performed by iec, as histidine presents certain difficulties in the glc method (7).
364
EYEM, SJODAHL
AND SJt)QUIST 6
I
I
5
10
1
15 20 MINUTES
1
I
25
30
FIG. 2. Gas-liquid chromatography of a silylated reaction mixture after reduction and methylation of cystine. Methylation was performed at pH 7 for 30 min. Experimental details are given in Materials and Methods. Numbering of peaks corresponds to that in Table 1. Temperature program, 80°C for 4 min, then S”C/min up to 240°C.
The stability of S-Me-CyS towards the normally employed acid hydrolysis conditions for amino acid analysis was tested. It has been claimed (19) that 10% decomposition occurs during the first 22 hr of hydrolysis. In our experiments yields of 97 and 91% were observed after 20- and 72-hr hydrolysis, respectively. A good approximation would be the use of a decomposition correction factor of O.l4%/hr of hydrolysis. This factor should be dependent on the hydrolysis technique, the presence of interfering substances, etc. The iec analysis of,!?-Me-CyS does not present any difficulties; the amino acid emerges next to proline in the chromatogram. The results from the methylation studies on different peptides and proteins dissolved in 6 M guanidine-HCl are illustrated in Table 2. Although no conclusions could be drawn as to whether acid-labile modifications had occurred, such modifications were not observed in the methylation studies on free cystine and histidine described above. The complete disappearance of cysteine was observed in all analyses of methylated samples
S-METHYLATION
OF PEPTIDES TABLE
AND
365
PROTEINS
2
AMINO AC~DCOMP~SITIONSOFMETHYLATEDANDNONMETHYLATEDPE~IDESAND PROTEIN@ Lima bean Reduced Amino acid
Gly-His-Gly (Meth iec)
LYS His A*g CyS03H ASP Thr Se* Glu Pro S-Me-CyS GUY Ala Vd Met Ile
1.01 -
LW TY* Phe
2.00 -
Theory
iec
---(1) 0.93 ---1 0.99 (1) 1 1.01 ----------------
glutathione
Insulin
Meth iec
Meth glc
-
_
1.00 1.00 1.03 -
1.00 1.05 O.% -
-
-
-
-
-
-
-
-
Theory
Naja-4 toxin
iec
Meth glc
Theory”
iec
Metb iec
3 1 3 7 1 (6) 4 3 5
1.1 1.9 1.0 5.6 3.0 1.2 2.8 7.2 1.2 4.0 2.9 4.6
1.1 1.9 I.1 31 1.2 3.0 7.4 1.2 5.9 3.9 2.9 4.5
4 1 6 (10) 9 9 4 1 6 (10) 5 2 4
4.0 1.0 5.8 8.9 9.4 8.7 4.1 1.6 6.2 5.1 2.0 3.7
1 6 4 3
0.9 6.0 4.0 2.8
0.9 6.0 4.0 2.7
4 1 1 3
3.7 1.3 1.1 2.7
1 2 1
(6)
trypsin
inhibitor iec
Meth kc
Meth &
3.9 1.0 5.6 9.3 8.6 4.2 1.7 6.2 9.5 5.2 2.0 3.6
4.2 6.3 2.4 13.4 15.8 5.1 16.5 8.7 8.3 1.5 3.9 1.5
4.5 6.2 2.7 15.6 5.3 15.6 8.7 7.7 14.4 1.6 4.1 1.4
5.1 6.0 2.3 15.9 5.4 16.8 9.5 7.7 14.8 1.4 4.1 1.4
3.7 1.3 1.1 2.7
5.0 3.0 1.5 2.4
5.0 3.0 1.5 2.4
4.7 3.0 1.6 2.8
“Ibe vahx.s given for threonine and wine are extrapolated to zero time, the values for valine and isoleucine are the 72shr values. The cyst&e-plus-cystine contents in the nonmethylated samples were determined after perfonnic acid oxidation and a subsequent 20.hr hydrolysis and are expressed as cysteic acid. S-Me-CyS-values were corrected to zero time (see text). For all other amino acids the mean values are used. Tryptophan was not determined and is therefore omitted. The analyses of glycyl-histidyl-glycine, reduced glutathione, insulin, Naja-4 toxin, and lima bean trypsin inhibitor were namrdized to hvo glycine, one glutamic acid, six leucine, two r&nine, and three leucine residues, respectively. Meth, methylated sample; iec. analyzed by iec; glc, analyzed by glc; theory, theoretic values. ’ The theoretical values for Nr&x-4 toxin were personally communicated by Dr. D. Eaker.
originally containing this amino acid, and the appearance of equivalent amounts of S-methyl-cysteine was observed. In all cases these values exceeded the values for cysteic acid in the oxidized samples, but the differences cannot be considered significant. Generally, the analyses of methylated samples were in better agreement with the theoretical values for the cysteine content than the analyses of oxidized samples. It is also clear from the results presented in Table 2 that the quantitative glc amino acid analysis, when employed, gave results entirely compatible with the iec analysis. Furthermore, no extra peaks were obtained in the gas chromatograms. This fact, together with the excellent agreement of the analyses of methylated and nonmethylated material, strongly supports the statement of a methylation reaction specific for cysteine residues. It goes without saying that solubility problems encountered, for example, in the desalting step after methylation are shared by all the different methods generating the same product. Larger peptides and proteins are easily handled by dialysis if such problems arise, but smaller
366
EYEM, SJGDAHL
L
.
0
5
AND SJGQUIST
I
10
1s
20
25 MINUTES
30
35
10
15
50
FIG. 3. Gas-liquid chromatography of silylated MTH derivatives of S-Me-CyS and other amino acids. Experimental details are given in Materials and Methods. Temperature program, 120°C for 8 min, then Z.S”C/min up to 250°C.
peptides may require desalting by gel chromatography in a suitable solvent. When necessary, an organic solvent could be added to the reaction mixture. The methylation of cysteine residues has been proposed by Gross (25) to be useful in order to get a derivative suitable for bromine cyanide cleavage at cysteine, analogous to the cleavage at methionine residues
H 5 > 5 v)
ILE,LEU 0 PRO PHE OOVAL ME P f30AL~ S-Me-C% 0 (31-y GLN LYSO GLUO 8 d;N 0 SER TYRO OASP d HIS, ARG
X
Solvent
II
FIG. 4. Thin-layer chromatography of FM-IS-Me-CyS and other PTH-amino acids on a 5 x S-cm polyamide thin-layer sheet. Solvent I, toluene:n-pentane:glacial acetic acid, 60:30:35 (v/v). Solvent II, 35% aqueous acetic acid.
S-METHYLATION
0
5
OF PEPTIDES
10
15 20 MINUTES
AND PROTEINS
25
30
367
35
FIG. 5. Separation of the N-HFB amino acid n-propyl ester derivatives of S-Me-CyS and other amino acids. Experimental details are given in Materials and Methods. Temperature program, 70°C for 4 min, then S”C/min up to 250°C. The peak behind S-Me-CyS represents cysteine (impurity from cystine).
with this reagent. This type of fragmentation has been utilized to cleave an S-methylated peptide of bovine pancreatic ribonuclease A with the formation of the expected fragments in 88% yield (26). In sequence studies, the possibility of using S-Me-CyS for glc analysis is an advantage. In Fig. 3, a gas chromatogram from an analysis of TMS derivatives of different methylthiohydantoin amino acids, including MTH-S-Me-CyS, is illustrated. Preliminary experiments with phenylthiohydantoin (PTH)-S-Me-CyS showed that even this derivative is well suited for glc analysis. Furthermore, this derivative is easily analyzed by common thin-layer chromatographic techniques. In Fig. 4, the separation of PTH-S-Me-CyS from other PTH-amino acids on a 5 x 5-cm polyamide thin-layer sheet (23) is shown. Figure 5 shows the elution profile obtained when analyzing S-Me-CyS and other amino acids gas chromatographically as the N-HFB amino acid n-propyl esters (7). To summarize, the methylation of cysteine residues with dimethylsulfate is simple, rapid, quantitative, and selective and should be a good alternative to other methylating procedures employed. A derivative is formed, which is well suited for both iec and glc amino acid
368
EYEM,
SJODAHL
AND SJOQUIST
analysis as well as glc or tic analysis of the MTH or PTH derivatives. Furthermore, it has been shown (26) that S-methylation introduces a peptide bond sensitive to the action of bromine cyanide. Therefore, we believe that this modification procedure should be applicable in sequence work. ACKNOWLEDGMENTS This investigation was supported by grants from the Swedish Medical Research Council (Project No. 13X-2518) and the Swedish Natural Science Research Council (Project No. K2%1-001). We thank Mrs. M. Gustafson for skillful secretarial work.
REFERENCES 1. Hirs, C. H. W. (1%7) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 197-203, Academic Press, New York. 2. Cole, R. D. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.) Vol. 11, pp. 315-317, Academic Press, New York. 3. Inglis, A. S., and Liu, T. Y. (1970) J. Bid. Chem. 245, 112-116. 4. Jacobson, G. R., Schaffer, M. H., Stark, G. R., and Vanaman, T. C. (1973) J. Biol. Chem. 248, 6583-6591. 5. Degani, Y., and Patchomic, A. (1974) Biochemisfry 13, l-11. 6. Niketik, V., Thomsen, J., and Kristiansen, K. (1974) Eur. J. Biochem. 46, 547-551. 7. Jiinsson, J., Eyem, J., and Sjoquist, J. (1973) Anal. Biochem. 51, 204-219. (The first author’s name is now Sjiidahl.) 8. Eyem, J., and Sjiiquist, J. (1973) Anal. Biochem. 52, 255-271. 9. Richards, F. F., Barnes, W. T., Lovins, R. E., Salomone, R., and Waterfleld, M.D. (1969) Nature (London) 221, 1241-1244. 10. Pirie, N. W. (1932) Biochem. J. 26, 2041-2045. 11. de Vigneaud, V., Loring, H. S., and Craft, H. A. (1934) J. Biol. Chem. 105,481-488. 12. Wood, J. L., and du Vigneaud, V. (1934) J. Biol. Chem. 130, 109-l 14. 13. Zbarsky, S. H., and Young, L. (1943)J. Biol. Chem. 151,211-215. 14. Stoll, A., and Seebeck, E. (1949) Helv. Chim. Acta 32, 866-876. 15. Armstrong, M. D., and Lewis, J. D. (1951) J. Org. Chem. 16, 749-753. 16. Banks, T. E., and Shafir, J. A. (1970) Biochemistry 9,3343-3348. 17. Rochat, C., Rochat, H., and Edman, P. (1970) Anal. Biochem. 37, 259-267. 18. Heinrikson, R. L. (1970) Biochem. Biophys. Res. Commun. 41,967-972. 19. Heimikson, R. L. (1971) J. Biol. Chem. 246, 4090-4096. 20. Stepanov, M. V., and Krivtsov, V. F. (1965) Zh. Obsch. Khim. 35,982-986. 21. Moss, C. W., Lambert, M. A., and Diaz, F. J. (197l)J. Chromatogr. 60, 134-136. 22. Spa&man, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190-1206. 23. Summers, M. R., Smythers, G. W., and Oroszlan, S. (1973) Anal. Biochem. 53, 624-628. 24. Cleland, W. W. (1964) Biochemistry 3,480-482. 25. Gross, E., Morel], J. L., and Lee, P. Q. (1%7) Seventh Znt. Congr. Biochem. (Tokyo) 11, 535-536. 26. Gross, E., and Morel& J. L. (1974) Biochem. Biophys. Res. Commun. 59, 1145-1150.