ANALYTICAL
BIOCHEMISTRY
Alkylation Sequence Daniel
ti%!-296
(19%)
of Cysteine Analysis’
with Acrylamide
April
and Bochemistv, Tempe, Arizona
and Center for the Study 85287-1604
Press,
of Early
Euents
in Photosynthesis,
13, 1992
Alkylation of cysteine in proteins with acrylamide under mildly alkaline conditions yields a thioether derivative, Cys-S-&propionamide (Cys-S-Pam), which is stable during automated Edman degradation. Its phenylthiohydantoin derivative, PTH-Cys-S-Pam, is easily separated from other PTH-amino acids by HPLC and is thus useful for cysteine identification during protein sequencing. PTH-Cys-S-Pam was first noticed during sequencing polypeptides blotted onto polyvinylidene difluoride membranes from polyacrylamide gels, in which cysteine had reacted with residual unpolymerized acrylamide. Cysteine in proteins is easily alkylated by reaction of proteins in aqueous solution with acrylamide. Methods are also presented for alkylation of cysteine in proteins adsorbed on fiberglass disks in the reaction cartridge of a protein sequencer. Finally, PTH-Cys-S-Pam was synthesized chemically. The synthetic compound is unstable in neutral solution, but can be stabilized by acidification. It has the same HPLC retention time as the product formed from cysteine when sequencing proteins alkylated with acrylamide. @ issz Academic
for Protein
C. Brune
Department of Chemistry Arizona State University,
Received
207,
Inc.
Cysteine is not easy to identify during quencing because its phenylthiohydantoin
protein (PTH)2
sede-
’ The protein sequencer was obtained with funds from NSF Grant BBS%%-04992 (NSF Biological Centers Program). The work described here was partially supported by the Arizona State University Center for the Study of Early Events in Photosynthesis. The Center is funded by U.S. Department of Energy Grant DE-FG-WERl3969 as a part of the USDA/DOE/NSF Plant Science Centers Program. This is Publication 111 from the Arizona State University Center for the Study of Early Events in Photosynthesis. * Abbreviations used BuaP, tributylphosphine; Cys-S-Pam, cysteine-S-fl-propionamide; DTT, dithiothreitoh PAGE, polyacrylamide gel electrophoresis; PITC, phenylisothiocyanate; PTH, phenylthiohydantoin; PVDF, polyvinyhdene difluoride; SDS, sodium dodecyl sulfate. 0003.2697/92 $5.00 Copyright @ 1992 by Academic Press, All rights of reproduction in any form
rivative is unstable, losing H&S to form PTH-dehydroalanine, which in turn degrades further to a variety of products (1). PTH-serine decomposes similarly, but to a lesser extent, by loss of HZO, but in this case some PTHSer remains and can be detected. The problem of PTHCys instability can be overcome by alkylation of the sulfhydryl group to form a more stable thioether. The most widely used alkylating agent is 4-vinyl pyridine, which was introduced by Friedman and co-workers (2) for cysteine determination in analyses of amino acids from hydrolyzed proteins. Several procedures for alkylating cysteine with 4-vinyl pyridine for protein sequencing purposes have recently been described (3-6). When sequencing proteins that had been blotted onto Immobilon P membranes from polyacrylamide gels, I occasionally noticed that a sharp, distinct peak that did not correspond to any of the PTH-amino acid standards occurred on some cycles. In one case (7), homology of the protein being sequenced to known proteins in a data bank indicated that cysteine was expected on those cycles. A brief review of the literature revealed that Friedman and co-workers (8,9) had previously shown that cysteine is readily alkylated by nucleophilic p-addition to the vinyl groups of acrylamide, methyl acrylate, and acrylonitrile under mildly alkaline conditions. A protein of known sequence was therefore denatured, reduced, and allowed to react with acrylamide under appropriate conditions. The expected cysteine derivative was then observed during sequencing. Finally, PTH-Cys-S-ppropionamide (PTH-Cys-S-Pam) was synthesized from cysteine, acrylamide, and phenylisothiocyanate, and shown to have the same HPLC retention time as the product obtained from cysteine in an acrylamide treated protein. Alkylation of cysteine in proteins with acrylamide is useful for protein sequencing because it yields a derivative that is easy to identify. Therefore a procedure for 265
Inc. reserved.
286
DANIEL
C. BRUNE
alkylating proteins dried on fiberglass disks in the reaction cartridge of a protein sequencer was developed and is presented here. This procedure did not decrease either the initial or the repetitive yield when sequencing a standard protein and appears to derivative cysteine quantitatively. MATERIALS
AND
TABLE
Procedure for Washing Protein Samples after Step 1 2 3 4 5 6 7-9 10-12
METHODS
SDS-PAGE was carried out as described by Schagger and von Jagow (lo), and electrophoretically separated polypeptides were blotted onto Immobilon P membranes (Millipore Corp.) as described by Matsudaira (11). The reaction center protein from Heliobacillus mobilus was initially purified by SDS-PAGE, eluted, and cleaved with clostripain, and the fragments were then separated on a second SDS-polyacrylamide gel-see Trost et al. (7) for complete details. As a result of this procedure, cysteines in the reaction center fragments were exposed twice to residual acrylamide in the gels. For protein alkylation in solution, myotoxin a was denatured and reduced by heating for 30 min at 65’C with 2% SDS and 0.1 M DTT in 0.3 M Tris-Cl, pH 8.3. This is identical to the dissociation buffer in which proteins were dissolved for SDS-PAGE, but with the pH increased to facilitate reduction of disulfide bonds. The sample was then diluted lo-fold with distilled water and reconcentrated by centrifugation through a membrane filter (Centrifree, lOOO-MW cutoff) (Amicon Corp.). Acrylamide was then added from a 6 M stock solution to give a final concentration of 2 M and the sample incubated at 37’C for 30 min. The alkylated sample was then washed by two further cycles of concentration and dilution with distilled water in the ultrafiltration unit, and then spotted onto a fiberglass peptide disk (Porton Instruments) for sequencing. For derivatization in the reaction cartridge of the sequencer (Porton Model 209OE), proteins were dissolved in water at a concentration of IO pmol//.Ll (i.e., 10 PM), and IOO-pmol samples were then dried on protein disks. Chymotrypsinogen A was purchased from Sigma, and a-lactalbumin from Porton Instruments. Sequencing was begun and continued until coupling was completed and excess PITC was extracted by two washes with ethyl acetate (into step 54 of Porton procedure 40). Sequencing was then stopped, and the sample dried for 90 s using the cartridge “Drying” function in the manual mode of sequencer operation. Disulfide bonds in tbe protein were then reduced by adding 15 ~1 of 0.1 M tributyl phosphine (Sigma) in 2-propanol. Most of the 2-propanol was evaporated by drying 40 s with nitrogen (using the cartridge “Drying” function). Alkylation was carried out by adding 15 ~1 of 0.5 M acrylamide in 2.75% triethylamine (prepared from used Porton reagent R2; the approximate triethylamine concentration was determined by titration). The reaction cartridge was then
1
Note. acetate.
Reaction Cartridge
with
Acrylamide
function
Time
Drying Purging Sl Pressurizing Sl Sl washing Extracting Drying Repeat of 4-6 Second repeat of 4-6
The cartridge temperature Conversion flask function
was 45’C in all steps. inactive in all steps.
(s)
200 20 15 15 20 90 Sl,
ethyl
closed and the sample incubated for 20 min to allow alkylation to proceed to completion. The temperature of the reaction cartridge was maintained at 45’C through all steps of the procedure. The sample was dried and washed tbree times with ethyl acetate (reagent Sl) using the washing program in Table 1. Sequencing was resumed at the start of the second ethyl acetate wash after coupling (step 50 of procedure 40), resulting in a fourth wash before cleavage was initiated, and continued without further modifications. PTH-Cys-S-p-propionamide was synthesized as follows. Cysteine was alkylated as described by Friedman et al. (9) with minor modifications. Free base cysteine was used rather than cysteine HCl and the concentrations of cysteine and acrylamide in the reaction mixture were doubled. The product was then converted to its PTH derivative as described by Edman and Henschen (1). Basically, this procedure is as follows. Cys-S-Pam was reacted with PITC (Sigma) in 50% aqueous pyridine with the pH maintained at 8.6 by adding 2 M NaOH. After extraction of excess reagents with benzene, phenylthiocarbamyl Cys-S-Pam was precipitated by acidification and collected by filtration. This product was then converted to PTH-Cys-S-Pam by boiling in glacial acetic acid, from which it was precipitated by cooling to room temperature. This product was redissolved in boiling acetic acid, crystallized a second time, filtered from solution, and rinsed with distilled water. PTH-amino acids (Pierce) for sequencer calibration (dissolved at a concentration of 1 pmol/pl in 20% acetonitrile containing 1% methanol) were injected manually into the conversion flask of the sequencer. They were then dried, redissolved in 10% acetonitrile (Porton reagent SAC), and injected automatically onto a 0.21 X 20-cm Cl8 reverse-phase column (Hewlett-Packard Hypersil ODS). Two solvents were used for PTH-amino acid separation. Solvent A was 78 mM sodium acetate, pH 4.0, containing 3.5% tetrahydrofuran and 0.01% triethylamine. Solvent B was acetonitrile. The following
CYSTEINE
ALKYLATION
WITH
ACRYLAMIDE
FOR
287
SEQUENCING
I Thr
5
7
8
j 9 10 11 12 13 IL 15 16 17 18 19 20 21 22 23 Time (mid
! 5
6
7
8
1 9 10 11 12 13 I& 15 16 17 18 19 20 21 22 23 Time (mid
FIG. 1. HPLC chromatograms obtained when sequencing a clostripain fragment of the Heliobacilh mobilus reaction center from an Immobilon blot. (A) Cycles 5,6, and 7. (B) Cycles 14,15, and 16. Numbers above each chromatogram (at left) indicate the cycle number, while x’s indicate impurity peaks due to treatment with o-phthaldialdehyde just before cycle 5. The name of the amino acid liberated on each cycle is indicated on the chromatogram above the peak corresponding to its PTH derivative. Numbers in parentheses below the amino acid names indicate the amount of each amino acid in picomoles as determined from the peak area. (The amount of Ser on cycle 16 is overestimated because of a broad background peak that contributes to the PTH-Ser peak area.) The calibration factor for determining the amount of Cys from its peak area was determined using PTH-Cys-S-Pam synthesized as described in the text. Peaks occurring on all cycles at about 19.1 and 16.4 min are due to diphenylthiourea and to a side product of undetermined structure that forms from dithiothreitol in reagent Sl, respectively. Consecutive cycles have been offset along the vertical axis for clarity.
gradient was used: 0 min, 9% B; 1 min, 16% B; 18 min, 40% B; 22 min, 60% B; 25 min, 80% B. The flow rate was 0.2 mUmin and the column temperature 42’C.
RESULTS
AND
DISCUSSION
Figure 1 shows chromatograms obtained on cycles 5, 6, 7, 14, 15, and 16 while sequencing a clostripain fragment of the reaction center protein from the gram-positive photosynthetic bacterium H. rnobilis. This polypeptide fragment was purified by SDS-PAGE and electroblotted onto immobilon. [For details of reaction center isolation and proteolysis with clostripain, see Ref. (7)]. Peaks at 11.204, 11.74, and 20,796 min (most prominent on cycle 5) are artifacts due to treatment of the sample with o-phthaldialdehyde as described by Brauer et ul. (12) just prior to cycle 5 to block a minor peptide fragment that copurified with main fragment. Note the sharp peaks at ca. 9.48 min on cycles 6 and 15. These peaks did not correspond to any of the PTH amino acid standards. However, as discussed by Trost et al. (7), comparison of the N-terminal 23 amino acid sequence of this peptide fragment with GenBank sequences revealed over 50% identity with a highly conserved region found in two homologous core proteins of the photosystem I reaction center in plants and algae. Amino acids corresponding to residues 6 and 15 in those sequences are cysteines proposed to bind an atom of
that acts as an electron acceptor within the reaction center. The identity of the peak at 9.48 min as a derivative of cysteine was confirmed by sequencing a lysozyme band that had been run as a molecular weight standard in a similar gel and then blotted onto Immobilon. As expected, a peak eluting at about 9.5 min was observed for amino acid residue 6, which is known to be cysteine in lysozyme (data not shown). The known reactivity of cysteine with acrylamide (8,9) suggested that residual unpolymerized acrylamide in the gels may have reacted with cysteine residues during electrophoresis to form the S-fl-propionamide derivative. The fact that a compound with a retention time of 9.5 min can be formed from cysteine by reaction with acrylamide under conditions like those encountered during SDS-PAGE was demonstrated by experiments on myotoxin a, a 42-amino acid snake venom protein of known primary structure (13). Sequencing gave a sharp peak at 9.45 min on cycle 4 (see Fig. 2), as expected from the known occurrence of cysteine as residue 4 in myotoxin a. A brief account of this work was presented previously (14). Experiments on a myotoxin from the Mojave rattlesnake (Crotalus scutulatw scutulatus) showed that acrylamide-alkylated cysteines were stable when a protein containing them was cleaved at methionine by treatment with CNBr. The results from those experiments will be presented elsewhere. iron
288
DANIEL
6
FIG. 2.
7
8
9
C. BRUNE
10 11 12 13 1L 15 16 17 18 19 20 21 22 23 Time IminI
HPLC chromatograms quencing myotoxin o after reaction are labeled as in Fig. 1.
from with
cycles 3, 4, and 5 when acrylamide. Chromatograms
se-
It is often convenient to alkylate cysteine in proteins directly on the fiberglass disks used in the sequencer. Several methods for doing so using 4-vinyl pyridine as the alkylating reagent have been described (4-6). Analogous methods for alkylation with acrylamide were also developed. Figure 3 shows the first three cycles from sequencing chymotrypsinogen after reduction with tributylphosphine and alkylation with acrylamide. As noted by Friedman et ul. (8), o+ unsaturated compounds can react with amino, as well as sulfhydryl, groups. Although amino group alkylation is typically about 300 times slower, the procedure used here avoids that problem by first derivatizing the N-terminal amino (and lysine e-amino) groups with PITC, as was done by Kruft et al. (6). In one experiment in which reduction and alkylation were done without prior coupling to PITC, the initial yield was almost 40% lower, possibly due to partial alkylation of the N-terminal amino group. 1n &U reduction was also performed with DTT in place of tributylphosphine. In this case, 15 ~1 of 50 mM DTT in 1.7% triethylamine was added to the dried chymotrypsinogen sample which was incubated for 30 min in the closed sequencer reaction cartridge before drying and then adding 0.5 M acrylamide in 2.75% triethylamine. The results were the same as those obtained with tributylphosphine as the reductant. Decreasing the reaction time to 15 min gave equally good results. Increasing the acrylamide concentration to 2 M did not increase the yield of alkylated cysteine, while decreasing it to 0.2 M resulted in a 30% decrease. The only disadvantage of using DTT rather than BURP to reduce disulfide bonds is that reduction must be completed before adding acrylamide, which reacts with sulfhydryl groups of DTT as well as those of cysteine residues in the protein.
To assess the effect of reduction and alkylation on initial and repetitive yields during protein sequencing, parallel experiments were performed on lOO-pmol samples of a-lactalbumin. The sample that was not reduced or alkylated gave an initial yield of 68.3% and a repetitive yield of 93.7%. The sample reduced with BuSP and alkylated with acrylamide after the coupling reaction gave an initial yield of 79.0% and a repetitive yield of 95.1%. Repetitive yields were calculated using Glu residues at positions 1,7, and 11, Leu residues 3,12, and 15, and Lys residues 5 and 16, and averaging the results obtained with each set of residues. Thus reduction and alkylation appear not to have any adverse effect on either initial or repetitive yields. Reduction and alkylation were also performed on a chymotrypsinogen sample dried onto an Immobilon disk that had been wetted with methanol and then water. Because PVDF membranes are less absorbant than fiberglass disks, BuSP was added in two 5-~1 portions, with brief drying until the membrane started to lose its translucent appearance after the first addition. Immediately after the second BURP addition, 10 ~1 of 0.5 M acrylamide was added and sequencing continued after a 20-min incubation as described previously. The results obtained were similar to those shown in Fig. 3. This method also gave good results with peptides attached covalently to sequalon AA membranes (Millipore) (data not shown). Reduction and alkylation of chymotrypsinogen on a fiberglass disk was also tried using 4-vinyl pyridine as the alkylating agent, but without otherwise modifying the procedure used with acrylamide. This gave very unsatisfactory results. A broad off-scale absorption peak
FIG. 3. HPLC when sequencing Fig. 1.
chromatograms chymotrypsinogen
obtained on the first A. Chromatograms
three cycles labeled asin
CYSTEINE
ALKYLATION
WITH
centered at about 12-13 min appeared on the first cycle and persisted to a gradually decreasing extent through several subsequent cycles. This is consistent with the requirement for extensive washes with 1-chlorobutane and heptane as well as ethyl acetate in the procedure of Kruft et ul. (6). Even so, they reported a broad background peak coeluting with PTH-Ala that could interfere with identification of PTH-Ala or PTH-His on the first cycle when sequencing low picomole amounts of protein. This problem appears to be somewhat alleviated in the vapor-phase alkylation procedure of Amons (5), although fairly extensive solvent washes prior to the onset of sequencing are also recommended in that procedure. As is apparent from Fig. 3, extraneous peaks due to the alkylating reagents are clearly not a problem when acrylamide is used. that PTH-Cys-S-4-ethylpyridine Amons noted (formed from cysteine residues alkylated with 4-vinyl pyridine) gave a small peak relative to that of other amino acids in proteins being sequenced (5). This result is also seen in the data presented by Kruft et al. (6), and is consistent with my own observations when sequencing proteins alkylated with 4-vinyl pyridine. Amons (5) attributed this to incomplete extraction of PTH-Cys-S4-ethyl pyridine from the fiberglass disk on which the protein was absorbed. A final advantage of alkylating cysteine in proteins with acrylamide is that this problem is avoided; PTH-Cys-S-Pam is efficiently extracted and yields a sharp HPLC peak similar in size to that of PTH-Glu or PTH-Gin, for example.
0.
rn
240
280 Wovelength
320 tnrn)
‘.-~
,
360
400
FIG. 4. Absorption spectrum of synthetic PTH-Cys-S-Pam. Solid line, PTH-Cys-S-Pam was dissolved in ethanol containing 0.1% trifluoroacetic acid. Dashed line, Approximately the same concentration of PTH-Cys-S-Pam dissolved in ethanol. In the absence of acid, PTH-Cys-S-Pam decomposed, resulting in a gradual increase in 320nm absorbance and a decrease in 269-nm absorbance.
ACRYLAMIDE
FOR
,
289
SEQUENCING
,
,
!
2
c
2
I
I
11
5 6 7 8 9 10 11 12 13 lb 15 16 17 18 19 20 21 22 3 Time (min) FIG. 5. HPLC chromatogram acid standards supplemented just before the PTH-Cys-S-Pam standards.
with
showing separation of PTH amino PTH-Cys-S-Pam. The shoulders peak are due to impurities in the
PTH-Cys-S-Pam was synthesized in order to better characterize it and as a final confirmation of the product formed when sequencing proteins alkylated with acrylamide. Figure 4 (solid line) shows an absorption spectrum of the synthetic compound in ethanol acidified with 0.1% trifluoroacetic acid. This spectrum is typical for PTH-amino acids. The molar extinction coefficient was measured to be 16,000 at 269 nm, and the 245 nml269 nm absorbance ratio was found to be 0.38 [cf. Table 8-l in Ref. (l)]. Unlike most other PTH-amino acids, PTH-Cys-S-Pam was found to be unstable in ethanol that was not acidified. In neutral ethanol (or acetonitrile), PTH-Cys-S-Pam gradually decomposed to give a product absorbing maximally at 320 nm (Fig. 4, dashed line). This product may be PTH-dehydroalanine, which is known to absorb maximally at 320 nm (1) and could form from PTH-Cys-S-Pam by loss of p-mercaptopropionamide. Formation of this product does not occur to any noticeable extent during protein sequencing. Figure 5 shows separation of PTH-amino acid standards supplemented with PTH-Cys-S-Pam. Although some breakdown of the standards has occurred due to prolonged storage, it is clear that PTH-Cys-S-Pam is well resolved from the other standards. In some chromatograms of PTH-Cys-S-Pam, a minor peak at 11.0 min due to PTH-Cys-S-propionic acid was observed. This compound is also well separated from other PTHamino acids, the closest of which is PTH-Ala at 11.3 min, Although PTH-Cys-S-Pam was well separated from other PTH amino acids and sequencing byproducts in
290
DANIEL
the experiments described here, it was recently brought to my attention that it may not always be separated from N,N-dimethylphenylthiourea, a side product formed with other sequencing chemistries. Replacing acrylamide with N,N-dimethylacrylamide, a more hydrophobic derivative that would yield a later eluting alkylated form of PTH-Cys, might be useful in this case. In conclusion, alkylating cysteine in proteins with acrylamide yields PTH-Cys-S+propionamide during sequencing, a product that is well resolved by HPLC and easily identified. This procedure has two advantages over alkylation with 4-vinyl pyridine. One is that the PTH derivative gives a larger peak on HPLC chromatograms, and the other is that strongly absorbing residues that give interfering peaks in the initial chromatograms are avoided. Acrylamide is readily available in most biochemistry laboratories, and if disulfide bonds are reduced with DTT, no special reagents are required for the alkylation reaction. ACKNOWLEDGMENTS Shahrzad Zarezadeh, a student from McClintock High School participating in Project SEED (a summer research program sponsored by the American Chemical Society), helped with sequencing work on myotoxin o and synthesis of PTH-Cys-S-Pam. Reaction center fragments from H. m0bili.s were prepared by Jeff Trost, and myotoxin cz was provided by Allan Bieber.
C. BRUNE REFERENCES 1. Edman, nation York.
P., and Henschen, A. (1975) in Protein Sequence (NeedIernan, S., Ed.), pp. 232-279, Springer-Verlag,
2. Friedman, M., Krull, 245,386%38X.
L. H., and Cavins,
J. F. (1970)
3. Wilson, K. J., and Yuan, P. M. (1989) in Protein Practical Approach (Findlay, J. B. C. and Geisow, l-41, IRL Press, Oxford. 4. Andrews, 528.
P. C., and Dixon,
5. Amons,
R. (1987)
FEBS
J. E. (1987)
Anal.
DetermiNew
J. &ol.
C&m.
Sequencing, M. J., Eds.),
A pp.
Biochem.
161,524-
B. (1991)
Anal.
212,68-72.
Lett.
6. Kruft, them.
V., Kapp,
U., and Wittmann-Liebold,
7. Trost, synth.
J. T., Brune, D. C., and Res. 32, 11-22.
Bio-
193,306-309.
8. Friedman, M., Cavins, Sot. 87,3672-3682.
Blankenship,
J. F., and
9. Cavins, J. F., and Friedman, 3360. 10. Schagger, H., and van Jagow,
Wall,
M. (1968) G. (1987)
R. E. (1992)
J. S. (1965) J. Biol. Ad.
J. Am.
Chem. Biochem.
PhotoChem.
243,3357-
166,368-
379. 11. Matsudaira, 12. Brauer, Biochem.
P. (1987) A. W.,
Cfzem.
C. L., and
262,
10035-10038.
Margulies,
M.
M. (1984)
And.
137,134-142.
13. Fox, J. W., 678-684. 14. Brune,
J. Biol.
Oman,
Elzinga,
M.,
D. C. (1991)
ABRF
and
Tu,
News
A. T. (1979) 2(3),
6.
Biochemistry
18,
BIOCHEMISTRY
Alkylation Sequence Daniel
ti%!-296
(19%)
of Cysteine Analysis’
with Acrylamide
April
and Bochemistv, Tempe, Arizona
and Center for the Study 85287-1604
Press,
of Early
Euents
in Photosynthesis,
13, 1992
Alkylation of cysteine in proteins with acrylamide under mildly alkaline conditions yields a thioether derivative, Cys-S-&propionamide (Cys-S-Pam), which is stable during automated Edman degradation. Its phenylthiohydantoin derivative, PTH-Cys-S-Pam, is easily separated from other PTH-amino acids by HPLC and is thus useful for cysteine identification during protein sequencing. PTH-Cys-S-Pam was first noticed during sequencing polypeptides blotted onto polyvinylidene difluoride membranes from polyacrylamide gels, in which cysteine had reacted with residual unpolymerized acrylamide. Cysteine in proteins is easily alkylated by reaction of proteins in aqueous solution with acrylamide. Methods are also presented for alkylation of cysteine in proteins adsorbed on fiberglass disks in the reaction cartridge of a protein sequencer. Finally, PTH-Cys-S-Pam was synthesized chemically. The synthetic compound is unstable in neutral solution, but can be stabilized by acidification. It has the same HPLC retention time as the product formed from cysteine when sequencing proteins alkylated with acrylamide. @ issz Academic
for Protein
C. Brune
Department of Chemistry Arizona State University,
Received
207,
Inc.
Cysteine is not easy to identify during quencing because its phenylthiohydantoin
protein (PTH)2
sede-
’ The protein sequencer was obtained with funds from NSF Grant BBS%%-04992 (NSF Biological Centers Program). The work described here was partially supported by the Arizona State University Center for the Study of Early Events in Photosynthesis. The Center is funded by U.S. Department of Energy Grant DE-FG-WERl3969 as a part of the USDA/DOE/NSF Plant Science Centers Program. This is Publication 111 from the Arizona State University Center for the Study of Early Events in Photosynthesis. * Abbreviations used BuaP, tributylphosphine; Cys-S-Pam, cysteine-S-fl-propionamide; DTT, dithiothreitoh PAGE, polyacrylamide gel electrophoresis; PITC, phenylisothiocyanate; PTH, phenylthiohydantoin; PVDF, polyvinyhdene difluoride; SDS, sodium dodecyl sulfate. 0003.2697/92 $5.00 Copyright @ 1992 by Academic Press, All rights of reproduction in any form
rivative is unstable, losing H&S to form PTH-dehydroalanine, which in turn degrades further to a variety of products (1). PTH-serine decomposes similarly, but to a lesser extent, by loss of HZO, but in this case some PTHSer remains and can be detected. The problem of PTHCys instability can be overcome by alkylation of the sulfhydryl group to form a more stable thioether. The most widely used alkylating agent is 4-vinyl pyridine, which was introduced by Friedman and co-workers (2) for cysteine determination in analyses of amino acids from hydrolyzed proteins. Several procedures for alkylating cysteine with 4-vinyl pyridine for protein sequencing purposes have recently been described (3-6). When sequencing proteins that had been blotted onto Immobilon P membranes from polyacrylamide gels, I occasionally noticed that a sharp, distinct peak that did not correspond to any of the PTH-amino acid standards occurred on some cycles. In one case (7), homology of the protein being sequenced to known proteins in a data bank indicated that cysteine was expected on those cycles. A brief review of the literature revealed that Friedman and co-workers (8,9) had previously shown that cysteine is readily alkylated by nucleophilic p-addition to the vinyl groups of acrylamide, methyl acrylate, and acrylonitrile under mildly alkaline conditions. A protein of known sequence was therefore denatured, reduced, and allowed to react with acrylamide under appropriate conditions. The expected cysteine derivative was then observed during sequencing. Finally, PTH-Cys-S-ppropionamide (PTH-Cys-S-Pam) was synthesized from cysteine, acrylamide, and phenylisothiocyanate, and shown to have the same HPLC retention time as the product obtained from cysteine in an acrylamide treated protein. Alkylation of cysteine in proteins with acrylamide is useful for protein sequencing because it yields a derivative that is easy to identify. Therefore a procedure for 265
Inc. reserved.
286
DANIEL
C. BRUNE
alkylating proteins dried on fiberglass disks in the reaction cartridge of a protein sequencer was developed and is presented here. This procedure did not decrease either the initial or the repetitive yield when sequencing a standard protein and appears to derivative cysteine quantitatively. MATERIALS
AND
TABLE
Procedure for Washing Protein Samples after Step 1 2 3 4 5 6 7-9 10-12
METHODS
SDS-PAGE was carried out as described by Schagger and von Jagow (lo), and electrophoretically separated polypeptides were blotted onto Immobilon P membranes (Millipore Corp.) as described by Matsudaira (11). The reaction center protein from Heliobacillus mobilus was initially purified by SDS-PAGE, eluted, and cleaved with clostripain, and the fragments were then separated on a second SDS-polyacrylamide gel-see Trost et al. (7) for complete details. As a result of this procedure, cysteines in the reaction center fragments were exposed twice to residual acrylamide in the gels. For protein alkylation in solution, myotoxin a was denatured and reduced by heating for 30 min at 65’C with 2% SDS and 0.1 M DTT in 0.3 M Tris-Cl, pH 8.3. This is identical to the dissociation buffer in which proteins were dissolved for SDS-PAGE, but with the pH increased to facilitate reduction of disulfide bonds. The sample was then diluted lo-fold with distilled water and reconcentrated by centrifugation through a membrane filter (Centrifree, lOOO-MW cutoff) (Amicon Corp.). Acrylamide was then added from a 6 M stock solution to give a final concentration of 2 M and the sample incubated at 37’C for 30 min. The alkylated sample was then washed by two further cycles of concentration and dilution with distilled water in the ultrafiltration unit, and then spotted onto a fiberglass peptide disk (Porton Instruments) for sequencing. For derivatization in the reaction cartridge of the sequencer (Porton Model 209OE), proteins were dissolved in water at a concentration of IO pmol//.Ll (i.e., 10 PM), and IOO-pmol samples were then dried on protein disks. Chymotrypsinogen A was purchased from Sigma, and a-lactalbumin from Porton Instruments. Sequencing was begun and continued until coupling was completed and excess PITC was extracted by two washes with ethyl acetate (into step 54 of Porton procedure 40). Sequencing was then stopped, and the sample dried for 90 s using the cartridge “Drying” function in the manual mode of sequencer operation. Disulfide bonds in tbe protein were then reduced by adding 15 ~1 of 0.1 M tributyl phosphine (Sigma) in 2-propanol. Most of the 2-propanol was evaporated by drying 40 s with nitrogen (using the cartridge “Drying” function). Alkylation was carried out by adding 15 ~1 of 0.5 M acrylamide in 2.75% triethylamine (prepared from used Porton reagent R2; the approximate triethylamine concentration was determined by titration). The reaction cartridge was then
1
Note. acetate.
Reaction Cartridge
with
Acrylamide
function
Time
Drying Purging Sl Pressurizing Sl Sl washing Extracting Drying Repeat of 4-6 Second repeat of 4-6
The cartridge temperature Conversion flask function
was 45’C in all steps. inactive in all steps.
(s)
200 20 15 15 20 90 Sl,
ethyl
closed and the sample incubated for 20 min to allow alkylation to proceed to completion. The temperature of the reaction cartridge was maintained at 45’C through all steps of the procedure. The sample was dried and washed tbree times with ethyl acetate (reagent Sl) using the washing program in Table 1. Sequencing was resumed at the start of the second ethyl acetate wash after coupling (step 50 of procedure 40), resulting in a fourth wash before cleavage was initiated, and continued without further modifications. PTH-Cys-S-p-propionamide was synthesized as follows. Cysteine was alkylated as described by Friedman et al. (9) with minor modifications. Free base cysteine was used rather than cysteine HCl and the concentrations of cysteine and acrylamide in the reaction mixture were doubled. The product was then converted to its PTH derivative as described by Edman and Henschen (1). Basically, this procedure is as follows. Cys-S-Pam was reacted with PITC (Sigma) in 50% aqueous pyridine with the pH maintained at 8.6 by adding 2 M NaOH. After extraction of excess reagents with benzene, phenylthiocarbamyl Cys-S-Pam was precipitated by acidification and collected by filtration. This product was then converted to PTH-Cys-S-Pam by boiling in glacial acetic acid, from which it was precipitated by cooling to room temperature. This product was redissolved in boiling acetic acid, crystallized a second time, filtered from solution, and rinsed with distilled water. PTH-amino acids (Pierce) for sequencer calibration (dissolved at a concentration of 1 pmol/pl in 20% acetonitrile containing 1% methanol) were injected manually into the conversion flask of the sequencer. They were then dried, redissolved in 10% acetonitrile (Porton reagent SAC), and injected automatically onto a 0.21 X 20-cm Cl8 reverse-phase column (Hewlett-Packard Hypersil ODS). Two solvents were used for PTH-amino acid separation. Solvent A was 78 mM sodium acetate, pH 4.0, containing 3.5% tetrahydrofuran and 0.01% triethylamine. Solvent B was acetonitrile. The following
CYSTEINE
ALKYLATION
WITH
ACRYLAMIDE
FOR
287
SEQUENCING
I Thr
5
7
8
j 9 10 11 12 13 IL 15 16 17 18 19 20 21 22 23 Time (mid
! 5
6
7
8
1 9 10 11 12 13 I& 15 16 17 18 19 20 21 22 23 Time (mid
FIG. 1. HPLC chromatograms obtained when sequencing a clostripain fragment of the Heliobacilh mobilus reaction center from an Immobilon blot. (A) Cycles 5,6, and 7. (B) Cycles 14,15, and 16. Numbers above each chromatogram (at left) indicate the cycle number, while x’s indicate impurity peaks due to treatment with o-phthaldialdehyde just before cycle 5. The name of the amino acid liberated on each cycle is indicated on the chromatogram above the peak corresponding to its PTH derivative. Numbers in parentheses below the amino acid names indicate the amount of each amino acid in picomoles as determined from the peak area. (The amount of Ser on cycle 16 is overestimated because of a broad background peak that contributes to the PTH-Ser peak area.) The calibration factor for determining the amount of Cys from its peak area was determined using PTH-Cys-S-Pam synthesized as described in the text. Peaks occurring on all cycles at about 19.1 and 16.4 min are due to diphenylthiourea and to a side product of undetermined structure that forms from dithiothreitol in reagent Sl, respectively. Consecutive cycles have been offset along the vertical axis for clarity.
gradient was used: 0 min, 9% B; 1 min, 16% B; 18 min, 40% B; 22 min, 60% B; 25 min, 80% B. The flow rate was 0.2 mUmin and the column temperature 42’C.
RESULTS
AND
DISCUSSION
Figure 1 shows chromatograms obtained on cycles 5, 6, 7, 14, 15, and 16 while sequencing a clostripain fragment of the reaction center protein from the gram-positive photosynthetic bacterium H. rnobilis. This polypeptide fragment was purified by SDS-PAGE and electroblotted onto immobilon. [For details of reaction center isolation and proteolysis with clostripain, see Ref. (7)]. Peaks at 11.204, 11.74, and 20,796 min (most prominent on cycle 5) are artifacts due to treatment of the sample with o-phthaldialdehyde as described by Brauer et ul. (12) just prior to cycle 5 to block a minor peptide fragment that copurified with main fragment. Note the sharp peaks at ca. 9.48 min on cycles 6 and 15. These peaks did not correspond to any of the PTH amino acid standards. However, as discussed by Trost et al. (7), comparison of the N-terminal 23 amino acid sequence of this peptide fragment with GenBank sequences revealed over 50% identity with a highly conserved region found in two homologous core proteins of the photosystem I reaction center in plants and algae. Amino acids corresponding to residues 6 and 15 in those sequences are cysteines proposed to bind an atom of
that acts as an electron acceptor within the reaction center. The identity of the peak at 9.48 min as a derivative of cysteine was confirmed by sequencing a lysozyme band that had been run as a molecular weight standard in a similar gel and then blotted onto Immobilon. As expected, a peak eluting at about 9.5 min was observed for amino acid residue 6, which is known to be cysteine in lysozyme (data not shown). The known reactivity of cysteine with acrylamide (8,9) suggested that residual unpolymerized acrylamide in the gels may have reacted with cysteine residues during electrophoresis to form the S-fl-propionamide derivative. The fact that a compound with a retention time of 9.5 min can be formed from cysteine by reaction with acrylamide under conditions like those encountered during SDS-PAGE was demonstrated by experiments on myotoxin a, a 42-amino acid snake venom protein of known primary structure (13). Sequencing gave a sharp peak at 9.45 min on cycle 4 (see Fig. 2), as expected from the known occurrence of cysteine as residue 4 in myotoxin a. A brief account of this work was presented previously (14). Experiments on a myotoxin from the Mojave rattlesnake (Crotalus scutulatw scutulatus) showed that acrylamide-alkylated cysteines were stable when a protein containing them was cleaved at methionine by treatment with CNBr. The results from those experiments will be presented elsewhere. iron
288
DANIEL
6
FIG. 2.
7
8
9
C. BRUNE
10 11 12 13 1L 15 16 17 18 19 20 21 22 23 Time IminI
HPLC chromatograms quencing myotoxin o after reaction are labeled as in Fig. 1.
from with
cycles 3, 4, and 5 when acrylamide. Chromatograms
se-
It is often convenient to alkylate cysteine in proteins directly on the fiberglass disks used in the sequencer. Several methods for doing so using 4-vinyl pyridine as the alkylating reagent have been described (4-6). Analogous methods for alkylation with acrylamide were also developed. Figure 3 shows the first three cycles from sequencing chymotrypsinogen after reduction with tributylphosphine and alkylation with acrylamide. As noted by Friedman et ul. (8), o+ unsaturated compounds can react with amino, as well as sulfhydryl, groups. Although amino group alkylation is typically about 300 times slower, the procedure used here avoids that problem by first derivatizing the N-terminal amino (and lysine e-amino) groups with PITC, as was done by Kruft et al. (6). In one experiment in which reduction and alkylation were done without prior coupling to PITC, the initial yield was almost 40% lower, possibly due to partial alkylation of the N-terminal amino group. 1n &U reduction was also performed with DTT in place of tributylphosphine. In this case, 15 ~1 of 50 mM DTT in 1.7% triethylamine was added to the dried chymotrypsinogen sample which was incubated for 30 min in the closed sequencer reaction cartridge before drying and then adding 0.5 M acrylamide in 2.75% triethylamine. The results were the same as those obtained with tributylphosphine as the reductant. Decreasing the reaction time to 15 min gave equally good results. Increasing the acrylamide concentration to 2 M did not increase the yield of alkylated cysteine, while decreasing it to 0.2 M resulted in a 30% decrease. The only disadvantage of using DTT rather than BURP to reduce disulfide bonds is that reduction must be completed before adding acrylamide, which reacts with sulfhydryl groups of DTT as well as those of cysteine residues in the protein.
To assess the effect of reduction and alkylation on initial and repetitive yields during protein sequencing, parallel experiments were performed on lOO-pmol samples of a-lactalbumin. The sample that was not reduced or alkylated gave an initial yield of 68.3% and a repetitive yield of 93.7%. The sample reduced with BuSP and alkylated with acrylamide after the coupling reaction gave an initial yield of 79.0% and a repetitive yield of 95.1%. Repetitive yields were calculated using Glu residues at positions 1,7, and 11, Leu residues 3,12, and 15, and Lys residues 5 and 16, and averaging the results obtained with each set of residues. Thus reduction and alkylation appear not to have any adverse effect on either initial or repetitive yields. Reduction and alkylation were also performed on a chymotrypsinogen sample dried onto an Immobilon disk that had been wetted with methanol and then water. Because PVDF membranes are less absorbant than fiberglass disks, BuSP was added in two 5-~1 portions, with brief drying until the membrane started to lose its translucent appearance after the first addition. Immediately after the second BURP addition, 10 ~1 of 0.5 M acrylamide was added and sequencing continued after a 20-min incubation as described previously. The results obtained were similar to those shown in Fig. 3. This method also gave good results with peptides attached covalently to sequalon AA membranes (Millipore) (data not shown). Reduction and alkylation of chymotrypsinogen on a fiberglass disk was also tried using 4-vinyl pyridine as the alkylating agent, but without otherwise modifying the procedure used with acrylamide. This gave very unsatisfactory results. A broad off-scale absorption peak
FIG. 3. HPLC when sequencing Fig. 1.
chromatograms chymotrypsinogen
obtained on the first A. Chromatograms
three cycles labeled asin
CYSTEINE
ALKYLATION
WITH
centered at about 12-13 min appeared on the first cycle and persisted to a gradually decreasing extent through several subsequent cycles. This is consistent with the requirement for extensive washes with 1-chlorobutane and heptane as well as ethyl acetate in the procedure of Kruft et ul. (6). Even so, they reported a broad background peak coeluting with PTH-Ala that could interfere with identification of PTH-Ala or PTH-His on the first cycle when sequencing low picomole amounts of protein. This problem appears to be somewhat alleviated in the vapor-phase alkylation procedure of Amons (5), although fairly extensive solvent washes prior to the onset of sequencing are also recommended in that procedure. As is apparent from Fig. 3, extraneous peaks due to the alkylating reagents are clearly not a problem when acrylamide is used. that PTH-Cys-S-4-ethylpyridine Amons noted (formed from cysteine residues alkylated with 4-vinyl pyridine) gave a small peak relative to that of other amino acids in proteins being sequenced (5). This result is also seen in the data presented by Kruft et al. (6), and is consistent with my own observations when sequencing proteins alkylated with 4-vinyl pyridine. Amons (5) attributed this to incomplete extraction of PTH-Cys-S4-ethyl pyridine from the fiberglass disk on which the protein was absorbed. A final advantage of alkylating cysteine in proteins with acrylamide is that this problem is avoided; PTH-Cys-S-Pam is efficiently extracted and yields a sharp HPLC peak similar in size to that of PTH-Glu or PTH-Gin, for example.
0.
rn
240
280 Wovelength
320 tnrn)
‘.-~
,
360
400
FIG. 4. Absorption spectrum of synthetic PTH-Cys-S-Pam. Solid line, PTH-Cys-S-Pam was dissolved in ethanol containing 0.1% trifluoroacetic acid. Dashed line, Approximately the same concentration of PTH-Cys-S-Pam dissolved in ethanol. In the absence of acid, PTH-Cys-S-Pam decomposed, resulting in a gradual increase in 320nm absorbance and a decrease in 269-nm absorbance.
ACRYLAMIDE
FOR
,
289
SEQUENCING
,
,
!
2
c
2
I
I
11
5 6 7 8 9 10 11 12 13 lb 15 16 17 18 19 20 21 22 3 Time (min) FIG. 5. HPLC chromatogram acid standards supplemented just before the PTH-Cys-S-Pam standards.
with
showing separation of PTH amino PTH-Cys-S-Pam. The shoulders peak are due to impurities in the
PTH-Cys-S-Pam was synthesized in order to better characterize it and as a final confirmation of the product formed when sequencing proteins alkylated with acrylamide. Figure 4 (solid line) shows an absorption spectrum of the synthetic compound in ethanol acidified with 0.1% trifluoroacetic acid. This spectrum is typical for PTH-amino acids. The molar extinction coefficient was measured to be 16,000 at 269 nm, and the 245 nml269 nm absorbance ratio was found to be 0.38 [cf. Table 8-l in Ref. (l)]. Unlike most other PTH-amino acids, PTH-Cys-S-Pam was found to be unstable in ethanol that was not acidified. In neutral ethanol (or acetonitrile), PTH-Cys-S-Pam gradually decomposed to give a product absorbing maximally at 320 nm (Fig. 4, dashed line). This product may be PTH-dehydroalanine, which is known to absorb maximally at 320 nm (1) and could form from PTH-Cys-S-Pam by loss of p-mercaptopropionamide. Formation of this product does not occur to any noticeable extent during protein sequencing. Figure 5 shows separation of PTH-amino acid standards supplemented with PTH-Cys-S-Pam. Although some breakdown of the standards has occurred due to prolonged storage, it is clear that PTH-Cys-S-Pam is well resolved from the other standards. In some chromatograms of PTH-Cys-S-Pam, a minor peak at 11.0 min due to PTH-Cys-S-propionic acid was observed. This compound is also well separated from other PTHamino acids, the closest of which is PTH-Ala at 11.3 min, Although PTH-Cys-S-Pam was well separated from other PTH amino acids and sequencing byproducts in
290
DANIEL
the experiments described here, it was recently brought to my attention that it may not always be separated from N,N-dimethylphenylthiourea, a side product formed with other sequencing chemistries. Replacing acrylamide with N,N-dimethylacrylamide, a more hydrophobic derivative that would yield a later eluting alkylated form of PTH-Cys, might be useful in this case. In conclusion, alkylating cysteine in proteins with acrylamide yields PTH-Cys-S+propionamide during sequencing, a product that is well resolved by HPLC and easily identified. This procedure has two advantages over alkylation with 4-vinyl pyridine. One is that the PTH derivative gives a larger peak on HPLC chromatograms, and the other is that strongly absorbing residues that give interfering peaks in the initial chromatograms are avoided. Acrylamide is readily available in most biochemistry laboratories, and if disulfide bonds are reduced with DTT, no special reagents are required for the alkylation reaction. ACKNOWLEDGMENTS Shahrzad Zarezadeh, a student from McClintock High School participating in Project SEED (a summer research program sponsored by the American Chemical Society), helped with sequencing work on myotoxin o and synthesis of PTH-Cys-S-Pam. Reaction center fragments from H. m0bili.s were prepared by Jeff Trost, and myotoxin cz was provided by Allan Bieber.
C. BRUNE REFERENCES 1. Edman, nation York.
P., and Henschen, A. (1975) in Protein Sequence (NeedIernan, S., Ed.), pp. 232-279, Springer-Verlag,
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L. H., and Cavins,
J. F. (1970)
3. Wilson, K. J., and Yuan, P. M. (1989) in Protein Practical Approach (Findlay, J. B. C. and Geisow, l-41, IRL Press, Oxford. 4. Andrews, 528.
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