Letters
pubs.acs.org/acschemicalbiology
Persulfide Reactivity in the Detection of Protein S‑Sulfhydration
Jia Pan and Kate S. Carroll*
Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33456, United States
S Supporting Information
*
ABSTRACT: Hydrogen sulfide (H2S) has emerged as a new member of the gaseous transmitter family of signaling molecules
and appears to play a regulatory role in the cardiovascular and nervous systems. Recent studies suggest that protein cysteine Ssulfhydration may function as a mechanism for transforming the H2S signal into a biological response. However, selective
detection of S-sulfhydryl modifications is challenging since the persulfide group (RSSH) exhibits reactivity akin to other sulfur
species, especially thiols. A modification of the biotin switch technique, using S-methyl methanethiosulfonate (MMTS) as an
alkylating reagent, was recently used to identify a large number of proteins that may undergo S-sulfhydration, but the underlying
mechanism of chemical detection was not fully explored. To address this key issue, we have developed a protein persulfide model
and analogue of MMTS, S-4-bromobenzyl methanethiosulfonate (BBMTS). Using these new reagents, we investigated the
chemistry in the modified biotin switch method and examined the reactivity of protein persulfides toward different electrophile/
nucleophile species. Together, our data affirm the nucleophilic properties of the persulfide sulfane sulfur and afford new insights
into protein S-sulfhydryl chemistry, which may be exploited in future detection strategies.
O
results, the structure of reaction products in each step of the
modified BST and the underlying mechanism of thiol vs
persulfide MMTS selectivity was not investigated.
Herein, we report several persulfide models and new MMTS
analogue, S-4-bromobenzyl methanethiosulfonate (BBMTS).
Using these reagents, we investigated the chemistry in the
modified BST and examined the reactivity of protein persulfides
toward different electrophile/nucleophile species. These data
reaffirm the nucleophilic properties of the persulfide sulfane
sulfur and afford new insights into protein S-sulfhydryl
chemistry, which may be exploited in future detection
strategies.
Although selective (albeit slow) methods for H2S detection
have rapidly accumulated,15−18 methods for detecting protein
persulfide modifications have been slower to advance. In this
case, the challenges are 2-fold: (i) the chemical reactivity of the
terminal sulfane sulfur in the protein persulfide is similar to the
thiol and (ii) few examples of robust S-sulfhydryl protein
models have been reported. To this end, we initiated studies to
explore the generation and reactivity of protein persulfides. Our
second objective was to understand the chemical mechanisms
xidative post-translational modification (oxPTM) of
protein cysteines plays an important regulatory role in
many physiological systems.1−3 Of these, protein S-sulfhydration has recently received attention in the context of hydrogen
sulfide (H2S) mediated signaling pathways4−7 involved in
cardiovascular and cerebral vasodilatory reponses.8,9 To date,
the precise mechanisms of H2S action are still the subject of
active investigation. A significant challenge in protein Ssulfhydration research is that the persulfide group (RSSH)
exhibits reactivity akin to other sulfur species, in particular
thiols,10−12 which compounds difficulties in developing
selective detection methods.
In regards to detection, our interest was piqued when a
commonly used alkylating agent, known as S-methyl methanethiosulfonate (MMTS),13 was reported to differentiate
thiols and persulfides using a modified biotin switch technique
(BST),4 originally designed to study protein S-nitrosylation.14
As shown in Figure 1a, in the modified BST thiols are first
alkylated with MMTS, and persulfides were postulated to
remain unreacted and be available for subsequent conjugation
to N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio) propionamide (biotin-HPDP). Using this modified version of the BST, a
large number of proteins were reported as targets of H2S in the
mouse liver, and the basal sulfhydration level of some proteins
was estimated to be as high as 25%.4 Despite these intriguing
© XXXX American Chemical Society
Received: February 13, 2013
Accepted: April 4, 2013
A
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Figure 1. Reported methods for detecting protein persulfide modfiications. (a) Modified biotin switch technique (BST): protein samples are
alkylated by MMTS to block free thiols, while sparing persulfides. In the next step, biotin-HPDP is applied to alkylate persulfides and form protein−
biotin conjugates. (b) Thiols and persulfides are reacted with iodoacetic acid (IAA). The persulfide is then reduced with dithiothreitol (DTT) and
conjugated to biotinylated IAP. (c) Other cysteine oxPTMs that may be identified using the method in b.
Figure 2. Preparation and verification of persulfide formation in GSH and protein models. (a) Formation of glutathione (GSH) persulfide. (b)
HPLC UV trace at 254 nm and corresponding masses from the reaction in a. (c) Formation of protein persulfides. Mass analysis of the reaction
between iodoacetamide (IAM), (d) papain persulfide, and (e) Gpx3 persulfide [*indicates the product derived from alkylation of Gpx3−S−S−SH
(i.e., trisulfide)].
B
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underlying current chemoproteomic methods of persulfide
detection.
In an effort to identify suitable models, we first investigated
the reported method19 for preparing persulfide derviatives of
the tripeptide glutathione. As illustrated in Figure 2a, the GSH
persulfide was obtained as an equilibrium mixture of
glutathione thiol (GSH, 2) and persulfide (GSSH, 3) after
the reaction of glutathione disulfide (GSSG, 1) with Na2S.
Though GSH persulfide 3 could not be directly observed by
liquid chromatography−mass spectrometry (LC−MS), its
formation was confirmed through conjugation with alkylating
agents such as iodoacetamide (IAM) and N-ethylmaleimide
(NEM), which yielded detectable products in LC−MS analyses
(Figure 2b and Supplementary Figure S1). The inability to
directly observe the persulfide species likely stems from the
instability of the underivatized modification in acidic conditions
and/or poor ionization properties.
In parallel investigations, single protein persulfide modifications were generated via a stepwise procedure in the cysteine
protease, papain, and the C64,82S thiol peroxidase, Gpx3. In
this method, reduced thiol 6 was first reacted with 5,5′dithiobis-2-nitrobenzoic acid (DTNB or Ellman’s reagent) to
form activated disulfide 7, which was then cleaved by Na2S to
generate persulfide 8 (Figure 2c). After conditions such as the
equivalents of DTNB and Na2S were optimized, papain and
Gpx3 persulfides were successfully prepared and confirmed by
intact MS analysis post-trapping with IAM or NEM (Figure
2d). In addition to the desired papain persulfide derivative 9,
we observed signals consistent with papain sulfinic acid 10 (asisolated from the original commercial sample) as well as
persulfide oxidation. Of note, these species were also observed
in the reaction with NEM (Supplementary Figure S2),
indicating these mass signals were not unique to a particular
alkylating reagent. Interestingly, in the case of Gpx3, the
alkylation reaction revealed the presence of both the persulfide
(major derivative) and the trisulfide (minor derviative; Figure
2e and Supplementary Figure S3).
Next, we tested the reaction of the persulfide models with
MMTS. As shown in Figure 3a, the reaction of glutathione
persulfide with MMTS gave a small amount of GSSH derivative
14, with major product 15 derived from GSH, and other
byproducts GSSG (1) and GSSSG (13). The desired papain
persulfide 16 and Gpx3 persulfide 18 products were clearly
observed as the major species, albeit with some overlap
between protein sulfonates and thiosulfates (i.e., papain 17 in
Figure 3b; Gpx3 20 and 21 in Figure 3c). These analyses also
highlight the small mass difference (∼2 Da; Figure 3d) between
byproducts from the oxidation of thiols and persulfides, relative
to the MMTS-modified sulfur, which makes it difficult to
distinguish between these chemically distinct species by intact
protein MS analysis.
To address the above issue and clarify whether the ∼80 Da
mass increase corresponds to −SSMe or −SSO3− forms of
GSH, papain, and Gpx3, we designed and synthesized an
analogue of MMTS, S-4-bromobenzyl methanethiosulfonate
(BBMTS; Figure 4a). Compared to MMTS, the advantage of
this reagent is 2-fold: (i) a larger mass change results from the
thio-BBMTS adduct (+200 Da), and (ii) BBMTS exhibits the
telltale bromine mass pattern (i.e., strong peaks at M − 1 and
M + 1), which can be discerned in low-molecular-weight
peptides, including GSH. Initially, we verified the reactivity of
BBMTS, which proved comparable to MMTS (Supplementary
Tables S1 and S2). We then tested the BBMTS reagent in
Figure 3. Reactivity of GSH and protein persulfide models with
MMTS. (a) HPLC UV trace at 254 nm and corresponding masses
from the reaction of GSH persulfide and MMTS (*the mass in this
peak could not be assigned to a chemical structure). Mass analysis of
the reaction between (b) papain persulfide and MMTS and (c) Gpx3
persulfide and MMTS. (d) Increase in molecular mass that results
from MMTS modification (−SSMe) or oxidation to the thiosulfate
(−SO3−).
reactions with the GSH and protein persulfide models. As
shown in Figure 4b, treatment of glutathione persulfide with
BBMTS, gave a small amount of the desired persulfide
derviative 25 and a large peak of the thiol derviative 26
(both confirmed using the bromine signature afforded by the
new reagent), as well as the thiosulfate byproduct 27 (also
detailed in Supplementary Figures S4−7 and Table S3).
However, the reaction between protein persulfides and
BBMTS clearly showed the formation of the desired persulfide
derivatives, as well as alkyl polysulfides (Figures 4c,d). Taken
together, these data unequivocally establish that the nucleoC
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Figure 4. Reactivity of the GSH and protein persulfide models with BBMTS. (a) Synthesis of S-4-bromobenzyl methanethiosulfonate (BBMTS).
Mass analyses of the reaction between (b) GSH persulfide and BBMTS, (c) papain persulfide and BBMTS [+papain−SO3− (sulfonic acid);
*papain−S−SO3− (thiosulfate)], and (d) Gpx3 persulfide and BBMTS.
Figure 5. Sequential labeling reactions of protein persulfides with MMTS or BBMTS and biotin-HPDP analogue, N-acetylcysteine pyridyldisulfide
(NACP). Mass analyses of the reaction between (a) papain persulfide, MMTS and then NACP, b() papain persulfide, BBMTS, and then NACP
[+papain−SO3− (sulfonic acid); *papain−S−SO3− (thiosulfate)], (c) Gpx3 persulfide, MMTS, and then NACP [*Gpx3−S−S-S-SMe (tetrasulfide)],
and (d) Gpx3 persulfide, BBMTS, and then NACP as described in the Methods section [*Gpx3−S−S-S−SCH2C6H4Br (tetrasulfide)].
philic sulfane sulfur of protein persulfides reacts with
electrophilic MMTS and BBMTS reagents.
Since the MMTS and BBMTS electrophiles react with
protein persulfides, an important question arises: what does the
biotin signal in the modified BST stem from? To address this
issue, we treated papain and Gpx3 products of the MMTS/
BBMTS alkyation step with the biotin-HPDP analogue, Nacetylcysteine pyridyldisulfide (NACP). Ensuing intact MS
analysis of these reactions indicated the following: (i) no
product corresponding to a NACP-labeled persulfide was
detected in papain or Gpx3, (ii) a small amount of NACPlabeled thiol was observed in papain (32; Figures 5a,b), and
(iii) no NACP-labeled thiol was observed in Gpx3 (Figures
5c,d). These studies suggest that the protein-NACP labeling
product is derived from thiol alkylation.
Finally, since little has been reported about the chemical
reactivity of protein persulfides, we investigated the behavior of
the Gpx3-SSH sulfane sulfur with various nucleophilic and
D
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Table 1. Reactivity of Gpx3 Persulfide Towards Electrophilic and Nucleophilic Reagents
carbon nucleophiles (Supplementary Figures S9 and S13F−K
and Table S4).
We have investigated GSH and protein persulfide models for
chemical reactivity studies. Among these prototypes, GSH and
papain persulfides have been previously described; 19 however,
the protein persulfides were not evaluated or confirmed by
mass analysis. Using this early study as inspiration, in this work,
we have examined the utility of a new persulfide protein model,
based on a single-cysteine mutant of thiol peroxidase, Gpx3.
This model proved quite facile for the generation of protein
persulfides, and intact MS analysis was not complicated by the
presence of a large fraction sulfinic acid-modified protein, in
contrast to that observed for commercial sources of papain.
With respect to the GSH persulfide model, we observed only a
small amount of persulfide IAM and NEM alkylation products.
The most likely explanation for these data is that the reaction of
sterically unhindered persulfide derviatives 14 and 25 with low
molecular weight thiols (i.e., GSH and GSSH) is rapid, relative
to chemical alkylation. Thus, compared to protein persulfide
electrophilic reagents (Table 1 and Supplementary Figure S8).
The nucleophilicity of the sulfane sulfur was reaffirmed in
reactions with electrophilic alkylating reagents such as IAM,
NEM, and MMTS (Entry 1−3), as well as disulfide-containing
compounds, like DTNB and NACP (Entry 4−5). As an
electrophile, the sulfane sulfur could be reduced to the free
thiol form by DTT (Entry 6), GSH (note free thiol and mixed
disulfide forms; Entry 7), and NAC (Entry 8). By contrast,
weaker sulfur-based nucleophiles like sulfite or methyl sulfinate
did not reduce the Gpx3 persulfide (Entry 9−10). We also
tested the reaction of Gpx3 persulfide with carbon nucleophiles
known to react with sulfenic acid (RSOH), but not disulfides or
nitrosothiols (RSNO). These studies show that dimedone,
malononitrile, methyl acetoacetate, and barbituric acid carbon
nucleophiles exhibited no reactivity toward the Gpx3 persulfide
(Entries 11−14). As before, thiosulfate formation was also
noted in the persulfide samples, most likely resulting from
oxidation by trace metal ions in the presence of oxygen.20 The
thiosulfate species was stable and also did not react with the
E
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Figure 6. Proposed labeling mechanism of protein persulfide, methanthiosulfonate (MMTS or BBMTS), and pyridydisulfide NACP. Two possible
models are possible. (a) Free thiols may be incompletely blocked in the first MMTS alkylation step and subsequently react with the pyridyldisulfide
biotin reagent. (b) Alternatively, or in addition, labeling may be achieved via stepwise thiol-disulfide exchange in a reaction catalyzed by trace free
thiols (RSH).
thiols. Likewise, the γ-sulfur of persulfide is a weak electrophile
that shares some reactivity with disulfides (also alluded to by
sulfur transfer mechanisms in Fe−S biosynthetic pathways12)
but is chemically distinct from the sulfur atom in sulfenic acid,
which is significantly more electrophilic in nature. Collectively,
these data reaffirm the nucleophilic properties of the persulfide
sulfane sulfur and afford new insights into protein S-sulfhydryl
chemistry, which can serve as a resource in developing future
detection strategies.
models, the GSH persulfide may have less utility in reactivity
studies.
In addition, we have examined the chemistry underlying the
derivitization steps in the modified BST using the GSH and
protein persulfide model systems. In contrast to earlier
supposition,4 we demonstrate that the terminal persulfide
sulfur undergoes facile reaction with both MMTS and BBMTS
and that these alkylated forms do not react with the NACP
pyridyldisulfide. These findings thus reaffirm the nucleophilic
reactivity of the sulfane sulfur atom in protein persulfides and
are consistent with physical organic studies10,11 and the IAM/
NEM labeling observed in this work. In fact, the only NACPlabeled species that we observed in this study corresponded to
the reaction product derived from thiol, as opposed to the
persulfide. As shown in Figure 6, two possible models could
account for this finding. In one scenario, free thiols may be
incompletely blocked in the first MMTS alkylation step and
subsequently react with the pyridyldisulfide biotin reagent
(Figure 6a). Alternatively, or in addition, biotin labeling may be
achieved via stepwise thiol-disulfide exchange in a reaction
catalyzed by trace free thiols (Figure 6b). A common element
in both models is that not all free thiols are blocked during the
MMTS labeling step.
Recently, it was also reported that the persulfide-modified
active site cysteine of protein tyrosine phosphatase 1B
(PTP1B) can be alkylated by iodoacetic acid (IAA).
Subsequent reduction of the persulfide adducts and labeling
with iodoacetamide-linked biotin (IAP) has also been proposed
as an indirect approach for detecting protein S-sulfhydration
(Figure 1b).6 However, it is not apparent how this method
distinguishes persulfide modifications from other DTTreducible modifications, such as disulfide bonds, sulfenic
acids, and nitrosothiols (Figure 1c). Neverthless, IAA
derivitization of the PTP1B persulfide provides further evidence
that the persulfide is nucleophilic with similar reactivity to
■
METHODS
Sodium sulfide nonahydrate (Na2S·9H2O), extra pure, was obtained
from ACROS. DTNB was obtained from Sigma, and the purity was
≥98%. Papain 2× crystallized was purchased from Sigma. All other
chemicals were purchased from Sigma-Aldrich or Fisher at the highest
available purity.
Preparation of Persulfides. (1). Glutathione Persulfide.19 To a
solution of freshly prepared glutathione (oxidized form, 1 mM final
concentration) in 1 mL of Tris-HCl buffer (100 mM, pH 7.4) was
added freshly prepared sodium sulfide (1 mM final concentration),
and the solution was stirred at 37 °C for 15 min. The resulting
glutathione persulfide (1 mM final concentration) was directly used
without further purification.
(2). Papain Persulfide.19 A solution of papain (10 mg) in 1 mL of
Tris-HCl (pH 7.4, 100 mM, degassed with N2 for 10 min) was
incubated with cysteine (1.7 mg) at rt for 10 min, and the protein was
purified through a PD10 column (GE Healthcare) containing G-25
Sephadex. Protein-containing fractions were pooled from this column
(500 μL, 270 μM) and incubated with DTNB (50 μL, 4 mM) at rt for
20 min, and purified by passing through a PD-10 column. A fraction of
the pooled protein (200 μL, 120 μM) was then incubated in a solution
of Na2S (4 μL, 30 mM) at rt for 10 min and purified by PD-10 column
to give the papain persulfide, which was immediately used in further
reactions.
(3). Gpx3 Persulfide. C64,82S Gpx3 was prepared as previously
described.21,22 A solution of C64,82S Gpx3 (100 μM, 100 μL) was
buffer exchanged through a P-30 spin filtration column (BioRad) that
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sulfhydration of NF-κB mediates its antiapoptotic actions. Mol. Cell 45,
13−24.
(8) Zhao, W., Zhang, J., Lu, Y., and Wang, R. (2001) The
vasorelaxant effect of H2S as novel endogenous gaseous gaseous KATP
channel opener. EMBO J. 20, 6008−6016.
(9) Olson, K. R., Dombkowski, R. A., Russell, M. J., Doellman, M. M.,
Head, S. K., Whitfield, N. L., and Madden, J. A. (2006) Hydrogen
sulfide as an oxygen sensor/transducer in vertebrate hypoxic
vasoconstriction and hypoxic vasodilation. J. Exp. Biol. 209, 4011−
4023.
(10) Flavin, M. (1962) Microbial transsulfuration: the mechanism of
an enzymatic disulfide elimination reaction. J. Biol. Chem. 237, 768−
777.
(11) Heimer, N. E. (1981) Biologically oriented organic sulfur
chemistry. 21. Hydrogendisulfide of a penicillamine derivative and
related compounds. J. Org. Chem. 46, 1374−1377.
(12) Mueller, E. G. (2006) Trafficking in persulfides: delivering sulfur
in biosynthetic pathways. Nat. Chem. Biol. 2, 185−194.
(13) Smith, D. J., Maggio, E. T., and Kenyon, G. L. (1975) Simple
alkanethiol groups for temporary blocking of sulfhydryl groups of
enzymes. Biochemistry 14, 766−771.
(14) Jaffrey, S. R., and Snyder, S. H. (2001) The biotin switch
method for the detection of S-nitrosylated proteins. Sci. STKE 86, l1.
(15) Lippert, A. R., New, E. J., and Chang, C. J. (2011) Reactionbased fluorescent probes for selective imaging of hydrogen sulfide in
living cells. J. Am. Chem. Soc. 133, 10078−10080.
(16) Liu, C., Pan, J., Li, S., Zhao, Y., Wu, L. Y., Berkman, C. E.,
Whorton, A. R., and Xian, M. (2011) Capture and visualization of
hydrogen sulfide by a fluorescent probe. Angew. Chem., Int. Ed. 50,
10327−10329.
(17) Qian, Y., Karpus, J., Kabil, O., Zhang, S., Zhu, H., Banerjee, R.,
Zhao, J., and He, C. (2011) Selective fluorescent probes for live-cell
monitoring of sulphide. Nat. Commun. 2, 495.
(18) Lin, V. S., and Chang, C. J. (2012) Fluorescent probes for
sensing and imaging biological hydrogen sulfide. Curr. Opin. Chem.
Biol. 16, 595−601.
(19) Francoleon, N. E., Carrington, S. J., and Fukuto, J. M. (2011)
The reactivity of H2S with oxidized thiols: Generation of persulfides
and implications to H2S biology. Arch. Biochem. Biophys. 516, 146−
153.
(20) Everett, S. A., and Wardman, P. (1995) Perthiols as
antioxidants: radical-scavenging and prooxidative mechanisms. Methods Enzymol. 251, 55−69.
(21) Mason, J. T., Kim, S. K., Knaff, D. B., and Wood, M. J. (2006)
Thermodynamic basis for redox regulation of the Yap1 signal
transduction pathway. Biochemistry 45, 13409−13417.
(22) Paulsen, C. E., and Carroll, K. S. (2009) Chemical dissection of
an essential redox switch in yeast. Chem. Biol. 16, 217−225.
had been equilibrated in Tris-HCl buffer (pH 7.4, 100 mM, degassed
with N2 for 10 min). After determination of protein concentration (80
μM), the solution was incubated with DTNB (4 μL, 4 mM) at rt for
20 min and purified again by spin filtration. The resulting protein (100
μL, 62 μM) was then incubated with a Na2S solution (1 μL, 30 mM)
at rt for 10 min and purified by spin column to give the Gpx3
persulfide, which was immediately used in downstream reactions.
Verification of Persulfide Formation and Analysis of
Chemical Reactivity. Freshly prepared GSH or protein persulfide
(final concentration 1 mM for glutathione; 20 μM for protein) was
incubated with IAM or other nucleophilic or electrophilic reagents
(final concentration 4 mM for glutathione; 2 mM for protein) at rt for
1 h. After purification, aliquots of the samples were analyzed by
electrospray LC−MS on a Poroshell 120 (Agilent) or ZIC-pHILIC
HPLC column (the Nest group).
Reaction of GSSH with MMTS or BBMTS. To a solution of
freshly prepared glutathione persulfide (1 mM final concentration) in
1 mL of Tris-HCl buffer (100 mM, pH 7.4) was added MMTS or
BBMTS (4 mM final concentration), and the solution was stirred at rt
for 30 min. Sample aliquots were generated at each step and analyzed
by LC−MS.
Reaction of Protein Persulfides with MMTS or BBMTS
Followed by NACP. A freshly prepared protein persulfide solution
(20 μM final concentration) was incubated with the methanethiosulfonate (2 mM final concentration) at rt for 30 min and then purified
through a P-30 spin column. The sample solution was then incubated
with NACP (2 mM final concentration) at rt for 3 h and purified as
before. Sample aliquots were generated at each step and analyzed by
LC−MS.
■
ASSOCIATED CONTENT
S Supporting Information
*
Synthesis/preparation of the materials; LC−MS analysis; NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*(K.S.C.) E-mail: kcarroll@scripps.edu. Phone: (561) 2282460. Fax: (561) 228-2919.
Notes
The authors declare no competing financial interest.
■
■
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health
(Grant No. GM102187).
REFERENCES
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Protein post transilational modifications: the chemistry of proteome
diversifications. Angew. Chem., Int. Ed. 44, 7342−7372.
(2) Reddie, K. G., and Carroll, K. S. (2008) Expanding the functional
diversity of proteins through cysteine oxidation. Curr. Opin. Chem. Biol.
12, 746−754.
(3) Mieyal, J. J., and Chock, P. B. (2012) Posttranslational
modification of cysteine in redox signaling and oxidative stress:
focus on S-glutathionylation. Antioxid. Redox Signaling 16, 471−475.
(4) Mustafa, A. K., Gadalla, M. M., Sen, N., Kim, S., Mu, W., Gazi, S.
K., Barrow, R. K., Yang, G., Wang, R., and Snyder, S. H. (2009) H2S
signals through protein S-sulfhydration. Sci. Signal. 2, ra72.
(5) Kabil, O., and Banerjee, R. (2010) Redox biochemistry of
hydrogen sulfide. J. Biol. Chem. 285, 21903−21907.
(6) Krishnan, N., Fu, C., Pappin, D. J., and Tonks, N. K. (2011) H2Sinduced sulfhydration of the phosphatase PTP1B and its role in the
endoplasmic reticulum stress response. Sci. Signal. 4 4, ra86.
(7) Sen, N., Paul, B. D., Gadalla, M. M., Mustafa, A. K., Sen, T., Xu,
R., Kim, S., and Synder, S. H. (2012) Hydrogen sulfide-linked
G
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Persulfide Reactivity in the Detection of Protein S-sulfhydration
Jia Pan and Kate S. Carroll*
Supporting information
Synthesis/preparation of the materials ………………..…………………………………………………S2-S3
LC-MS analysis …………………………………………………………………………………………S4-S13
References………………………………………………………………………………..........................S14
NMR Spectrum …………………………………………….……………………………………………S15-S18
S1 Materials and Methods: Unless otherwise noted, all reactions were performed under an argon atmosphere in
oven-dried glassware. All purchased materials were used without further purification. Thin layer
chromatography (TLC) was carried out using Analtech Uniplate silica gel plates. Yields refer to
chromatographically and spectroscopically pure compounds, unless otherwise stated. NMR spectra were
obtained on a Varian Inova 400 (400 MHz for 1H; 100 MHz for
13
C). 1H and
13
C NMR chemical shifts are
reported in parts per million (ppm) relative to chloroform (δ 7.26) for 1H NMR and chloroform (δ 77.0) for 13C
NMR. Low-resolution electrospray ionization (ESI) mass spectra of small-molecules were analyzed by an
Agilent 6120 Quadruple LC/MS spectrometer on Poroshell 120 (Agilent) or ZIC®-pHILIC HPLC column (the
Nest group). Protein intact mass analysis was performed on a Thermo LTQ XL coupled with Agilent 1100 mass
spectrometer.
Synthesis of S-4-Bromobenzyl methanethiosulfonate (BBMTS)
A solution of sodium methanethiosulfonate (134 mg, 1 mmol), 4-bromobenzylmercaptan (250 mg, 1 mmol) and
sodium iodide (7.5 mg, 0.05 mmol) in methanol (7 mL) was stirred and refluxed for 12 hours. After the reaction
was done, the solution was partitioned between ethylacetate and saturated ammonium chloride solution. The
organic layer was washed with water and brine, separated and dried over anhydrous magnesium sulfate. After
filtration and evaporation, the residue was purified through flash chromatography to give product as a white
solid (101 mg, 35%). m.p. 76-77 ℃; 1H NMR (400 MHz, CDCl3): δ 7.42 (2H, d, J = 8.4 Hz), 7.21 (2H, d, J =
8.8 Hz), 4.25 (2H, s), 2.91 (3H, s);
13
C NMR (75 MHz, CDCl3): δ 134.2, 132.1, 130.7, 122.3, 51.2, 40.0; IR
(thin film) cm-1 2924, 1590, 1487, 1404, 1315, 1129, 1070, 1011, 954, 819, 804, 743.
Synthesis of N-acetylcysteine pyridyldisulfide (NACP)
S2 To a stirred solution of pyridyldisulfide (280 mg, 1 mmol) in THF (3 mL) was added dropwise a solution of
N-acetylcysteine (163 mg, 1 mmol) in THF (2 mL), and the solution was stirred at room temperature for 1 hour.
After evaporation of the solvent, the residue was purified through flash chromatography to give product as a
light yellow oil (156 mg, 57%). 1H NMR (400 MHz, MeOD-d4): δ 8.26 (1H, d, J = 4.0 Hz), 7.65 (2H, d, J = 2.4
Hz), 7.08 (1H, d, J = 3.2 Hz), 4.55 (1H, q, J = 4.0 Hz), 3.20 (1H, dd, J1 = 14.0 Hz, J2 = 4.0 Hz), 3.02 (1H, dd, J1
= 14.0 Hz, J2 = 8.4 Hz), 1.84 (3H, s); 13C NMR (75 MHz, MeOD-d4): δ 173.4, 160.8, 150.6, 139.3, 122.7, 121.6,
53.4, 41.6, 22.6; IR (thin film) cm-1 3258, 2061, 2926, 1723, 1643, 1574, 1561, 1447, 1417, 1375, 1292, 1223,
1118, 1043, 762, 717; MS m/z 271.0 [M-H+].
S3 Figure S1. Mass spectrum of the reaction between glutathione persulfide and and N-ethylmaleimide (* the mass
in this peak could not be assigned to a chemical structure).
Figure S2. Mass spectrum of the reaction between papain persulfide and N-ethylmaleimide.
Figure S3. Mass spectrum of the reaction between Gpx3 persulfide and N-ethylmaleimide (*the mass in this
peak could not be assigned to a chemical structure).
S4 Due to the difficulty to measure the fast kinetics of the reactions between thiols and MMTS/BBMTS, a
competitive reactivity study was carried out. Biologically relevant thiols - glutathione (GSH) and
N-acetylcysteine (NAC) reacted with MMTS and BBMTS to give product ratios close to 1:1, which proved the
similar reactivity of BBMTS to MMTS.
Table S1. Competitive reaction of MMTS and BBMTS with glutathione (GSH). To a mixed solution of
MMTS and BBMTS (500 µM final concentration each) was added GSH (20, 40, and 100 µM final
concentration, respectively) or N-acetylcysteine (NAC) (50, 100, and 200 µM final concentration, respectively),
and the solutions were stirred at room temperature for 30 minutes before taken into LC-MS analysis. The
concentration of the products was calculated based on the standard curve of the UV absorption of the products
versus the sulfhydryl concentration.
Entry 1 2 3 GSH / uM 20 40 100 Compound 15 / uM 7.75 13.8 42.9 Compound 33 / uM 6.25 24.2 69.1 Compound 15 Compound 33 1 2 3 S5 Table S2. Competitive reaction of MMTS and BBMTS with N-acetylcysteine. To a mixed solution of
MMTS and BBMTS (500 µM final concentration each) was added N-acetylcysteine (NAC) (50, 100, and 200
µM final concentration, respectively), and the solutions were stirred at room temperature for 30 minutes before
taken into LC-MS analysis. The concentration of the products was calculated based on the standard curve of the
UV absorption of the products versus the sulfhydryl concentration.
Entry 1 2 3 GSH / uM 50 100 200 Compound 34 / uM * * 77 Compound 35 / uM * 34.4 85.3 Compound 34 Compound 35 1 2 3 * undetectable concentration due to the weak UV absorption or ionization capability of the N-acetylcysteine
derivatives
S6 Formation of thiosulfate 27.
Thiosulfate 27 was proposed to be the oxidation product of 4-bromobenzyl persulfide 36, a byproduct that could
be generated via different pathways in the reaction of GSSH with BBMTS.
Figure S4. Proposed mechanism of thiosulfate 27 formation in the reaction between glutathione persulfide and
BBMTS.
Further treatment of BBMTS with Na2S also yielded thiosulfate 27, which suggests that the sulfane sulfur of the
persulfide species (such as 36) is highly susceptible to aerial oxidation to form thiosulfate (oxidation of
persulfide sulfane sulfur to sulfate in aerobic conditions has been previously reported) [2].
When Na2S was the limiting reagent, the sulfate concentration (from the sulfane sulfur in both 27 and 39)
correlates linearly with the concentration of Na2S (figure S6). However, when excess Na2S was used, the sulfate
concentration decreased with the increasing concentration of Na2S (figure S7).
S7 140000
y = 347.52x -­‐ 12880
R² = 0.9971
120000
100000
80000
60000
40000
20000
0
0
Figure S5.
100
200
300
400
500
Linear correlation between the sulfate formation (27+39) and the concentration of Na2S
(limiting reagent). 500 µL each of BBMTS (1 mM final concentration) in Tris (100 mM, pH 7.4) was mixed
with Na2S (40 µM, 100 µM , 200 µM, 400 µM and 1 mM final concentration, respectively) at room temperature
for 30 minutes.
30000
25000
20000
15000
10000
5000
0
0
Figure S6.
1000
2000
3000
4000
5000
Relationship between the sulfate formation (39) and the concentration of Na2S (in excess):
500 µL each of BBMTS (200 µM final concentration) in Tris (100 mM, pH 7.4) was mixed with Na2S (200 µM,
400 µM , 1 mM
2 mM and 5 mM final concentration, respectively) at room temperature for 30 minutes.
To explain why thiosulfate 27 did not exist in the excess Na2S and the concentration of thiosulfate 39 decreased
with increasing concentration of Na2S, we proposed that excess Na2S reduced both thiosulfate 27 and 39 back to
S8 persulfide intermediate 40, which undergo oxidation to form only thiosulfate 39. Although thiosulfate 39 and
persulfide 40 should be maintained to similar level under the equilibrium, the nucleophilic substitution from 39
to 40 must be more kinetically and thermodynamically significant than the aerial oxidation from 40 to 39,
therefore led to a decreasing trend of thiosulfate 39 as well.
Figure S7. Proposed mechanism of the reaction between BBMTS and Na2S. Freshly prepared Na2S solution (1
mM final concentration) and BBMTS (1 mM final concentration) were mixed for 10 minutes at room
temperature, and the products were analyzed in LC-MS.
S9 Persulfide has been reported to have anti-oxidant property in biological systems [3-4] .To clarify the oxidation
pathways, various anti-oxidative conditions have been tested, and found to inhibit the thiosulfate formation,
similar to a recent report by Gate, K. S. et al[5]. As shown in table S3, compared to the reaction condition
without any additive, the thiosulfate formation was suppressed in the presence of radical scavenger MeOH and
DMSO, metal chelator EDTA, and H2O2 decomposer catalase. Those data suggest that the sulfane sulfur of the
persulfide intermediate may be susceptible to different oxidation pathways. In addition, persulfide was known
to react with oxygen molecule to generate sulfate and thiosulfate anions.[6] All those evidence may also explain
the thiosulfate formation in the protein persulfide experiments.
Table S3. Thiosulfate formation in the presence of antioxidants. To a solution of BBMTS (1 mM final
concentration) in 500 µL Tris (100 mM, pH 7.4) with conditions: a. MeOH (1 M); b. DMSO (1 M); c. EDTA
(20 mM); d. catalase (100 U/mL); and e. no additive, was added Na2S (500 µM final concentration). After 30
minutes, the product formation was measured in LC-MS.
No addi9ve MeOH (1 M) DMSO (1 M) EDTA (20 mM) catalase (100 U/mL) S10 Figure S8. Mass spectrum of the Gpx3 persulfide reaction with a NACP; b DTNB; c DTT; d GSH; e NAC; f
sulfite; g methane sulfinate; h dimedone; i malononitrile; j methyl acetoacetate; k N,N’-dimethyl barbituric
acid.
S11 Reactivity of thiosulfate towards nucleophiles:
Table S4. The reactivity of Fmoc-cysteine thiosulfate towards nucleophiles.
S12 Figure S9. The reaction of Fmoc-cysteine thiosulfate with A. DTT; B. GSH; C. NAC; D. methyl sulfinate; E.
thiourea; F. dimedone and G. malononitrile (* represent peaks with unidentified mass).
S13 References
[1] Francoleon, N. E.; Carrington, S. J.; Fukuto, J. M. (2011) The reaction of H2S with oxidized thiols:
Generation of persulfides and implications to H2S biology. Arch. Biochem. Biophys. 516, 146.
[2] Everett, S. A.; Schoneich, C.; Stewart, J. H.; Asmus, K. (1992) Perthiyl radicals, trisulfide radical ions, and
sulfate formation. A combined photolysis and radiolysis study on redox processes with organic di- and
trisulfides. J. Phys. Chem. 96, 306.
[3] Everett, S. A.; Folkers, L. K.; Wardman, P. (1994) Free-radical repair by a novel perthiol: reversible
hydrogen transfer and perthiyl radical formation. Free Rad. Res. 20, 387.
[4] Everett, S. A.; Wardman, P. (1995) Perthiols as antioxidants: radical-scavenging and prooxidative
mechanisms. Methods in Enzymology 251, 55-69.
[5] Chatterji, T.; Keerthi, K.; Gates, K. S. (2005) Generation of reactive oxygen species by a persulfide
(BnSSH). Bioorg. Med. Chem. Lett. 15, 3921-3924.
[6] Kabil, O.; Banerjee, R. (2012) Characterization of patient mutations in human persulfide dioxygenase
(ETHE1) involved in H2S catabolism. J. Biol. Chem. 287, 44561.
S14 S15 S16 S17 S18