The reaction of hydrogen sulfide with disulfides: formation of a stable trisulfide and implications for biological systems

Article abstract of DOI:10.1111/bph.14372

Background and Purpose: The signalling associated with hydrogen sulfide (H₂S) remains to be established, and recent studies have alluded to the possibility that H₂S-derived species play important roles. Of particular interest are hydropersulfides (RSSH) and related polysulfides (RSS_nR, n?>?1). This work elucidates the fundamental chemical relationship between these sulfur species as well as examines their biological effects. Experimental Approach: Using standard analytical techniques (¹H-NMR and MS), the equilibrium reactions between H₂S, disulfides (RSSR), RSSH, dialkyltrisulfides (RSSSR) and thiols (RSH) were examined. Their ability to protect cells from electrophilic and/or oxidative stress was also examined using cell culture. Key Results: H₂S, RSSR, RSSH, RSSSR and RSH are all in a dynamic equilibrium. In a biological system, these species can exist simultaneously, and thus, it is difficult to discern which species is (are) the biological effector(s). Treatment of cells with the dialkyl trisulfide cysteine trisulfide (Cys-SSS-Cys) resulted in high intracellular levels of hydropersulfides and protection from electrophilic stress. Conclusions and Implications: In aqueous systems, the reaction between H₂S and RSSR results in the formation of equilibria whereby H₂S, RSH, RSSR, RSSH and RSSSR are present. In a biological system, any of these species can be responsible for the observed biological activity. These equilibrium species can also be generated via the reaction of RSH with RSSSR. Due to these equilibria, Cys-SSS-Cys can be a method for generating any of the other species. Importantly, HEK293T cells treated with Cys-SSS-Cys results in increased levels of hydropersulfides, allowing examination of the biological effects of RSSH. Linked Articles: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Full text of DOI:10.1111/bph.14372

The Reaction of Hydrogen Sulfide with Disulfides: Formation of a Stable Trisulfide
and Implications to Biological Systems
Christopher L. Bianco1, Takaaki Akaike2, Tomoaki Ida2, Peter Nagy3, Virag Bogdandi3, John
P. Toscano1, Yoshito Kumagai4, Catherine F. Henderson5, Robert N. Goddu5, Joseph Lin5*
and Jon M. Fukuto6*
1Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA,
2Department of Environmental Health Sciences and Molecular Toxicology, Tohoku
University Graduate School of Medicine, Sendai 980-8575, Japan, 3Department of Molecular
Immunology and Toxicology, National Institute of Oncology, 1122 Budapest, Hungary,
4Environmental Biology Section, Faculty of Medicine, University of Tsukuba, Tsukuba,
Ibaraki 305-8575, Japan, 5Department of Biology, Sonoma State University, Rohnert Park
94928, USA and 6Department of Chemistry, Sonoma State University, Rohnert Park, CA
94928, USA
Running title: Dialkyl trisulfide formation and use as a precursor to persulfides
Abbreviations: Cystathionine gamma-lyase (CSE), cystathionine beta-synthase (CBS),
cystine (Cys-SS-Cys), cysteine (Cys-SH), glutathione (GSH), glutathione disulfide (GSSG),
glutathione trisulfide (GSSSG), cysteine trisulfide (Cys-SSS-Cys), 2-mercaptoethanol (2ME),
2-hydroxyethyl disulfide (2HED), 2-hydroxyethyl trisulfide (2HET), 2-ME hydropersulfide
(2MESSH), 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), iodoacetamide (IAM), N-acetyl
penicillamine (NAP), N-ethylmaleimide (NEM), -(4-hydroxyphenyl)ethyl iodoacetamide
(HPE-IAM), S-methoxycarbonyl penicillamine disulfide (MCPD), MCPD hydropersulfide
(MCP-SSH).
*to whom correspondence can be addressed: Jon M. Fukuto (fukuto@sonoma.edu) or
Joseph Lin (linj@sonoma.edu).
Author contributions: Experimental work was carried out by CLB, TI, CFH, RNG and JL.
The manuscript was written by JMF with significant assistance from JL, TA, PN, VB, JPT, YK
and CLB. The project was conceived by CLB, JMF, PN, YK, JL and TA.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.14372
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Abstract
Background and Purpose: The signaling associated with hydrogen sulfide (H2S) remains to
be established and recent studies have alluded to the possibility that H2S-derived species
play important roles. Of particular interest are hydropersulfides (RSSH) and related
polysulfides (RSSnR, n>1). This work elucidates the fundamental chemical relationship
between these sulfur species as well as examine their biological effects.
Experimental Approach: Using standard analytical techniques (1H-NMR and mass
spectrometry), the equilibrium reactions between H2S, disulfides (RSSR), RSSH,
dialkyltrisulfides (RSSSR) and thiols (RSH) were examined. Their ability to protect cells from
electrophilic and/or oxidative stress was also examined using cell culture.
Key Results: H2S, RSSR, RSSH, RSSSR and RSH are all in a dynamic equilibrium. In a
biological system, these species can exist simultaneously and thus it is difficult to discern
which species is(are) the biological effector(s). Treatment of cells with the dialkyl trisulfide
cysteine trisulfide (Cys-SSS-Cys) results in high intracellular levels of hydropersulfides and
protection from electrophilic stress.
Conclusion: In aqueous systems, the reaction between H2S and RSSR results in the
formation of equilbria whereby H2S, RSH, RSSR, RSSH and RSSSR are present. In a
biological system, any of these species can be responsible for the observed biological
activity. These equilibrium species can also be generated via the reaction of RSH with
RSSSR.
Implications: Due to these equilibria, Cys-SSS-Cys can be a method for generating any of
the other species. Importantly, HEK293T cells treated with Cys-SSS-Cys results in
increased levels of hydropersulfides, allowing examination of the biological effects of RSSH.
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Introduction
Hydrogen sulfide (H2S) is an endogenously generated small molecule bioregulator
with a wide array of reported physiological functions [for recent reviews, see Kashfi and
Olson, 2013; Polhemus and Lefer, 2014; Kimura, 2014]. For example, H2S has been
proposed to have a role in long-term potentiation, smooth muscle relaxation, inhibition of
inflammation, activation of KATP channels and protection against ischemia-reperfusion injury,
just to name a few. However, it has also been hypothesized that some of the effects
attributed to H2S may instead be due to the presence of hydropersulfides (RSSH) [for
example, Ida et al., 2014; Ono et al., 2014] since RSSH can be in equilibrium with H2S in the
presence of an oxidized thiol (i.e. a disulfide, RSSR) via Reaction 1 [Cavallini et al., 1970;
Francoleon et al., 2011; Vasas et al., 2015] and, possibly more importantly, the biosynthesis
of H2S can occur via RSSH intermediacy (vide infra). (Note: the term RSSH will be used to
generally describe both the neutral RSSH and the anionic RSS- forms, unless specifically
noted).
H2S + RSSR
RSSH + RSH
(1)
Although the forward and reverse reactions shown in Reaction 1 can be slow (at least in a
purely chemical system) [Cuevasanta et al., 2015], it appears that H2S and RSSH are
intimately linked and likely to be present simultaneously via Reaction 1 in a biological
system. This makes the assignment of the compound responsible for the biological activity
difficult and/or indicates that multiple effectors can be responsible for the observed effects.
Furthermore, the chemistry represented by Reaction 1 is likely to be greatly oversimplified in
that other reactions can potentially emanate from this initial equilibrium [for example, see
Bogdandi et al., 2018, this issue]. For example, RSSH species are superior nucleophiles
compared to the corresponding RSH species and can react further with other electrophiles in
solution (i.e. RSSR) to give a dialkyltrisulfide (RSSSR, Reaction 2) [for example, Ono et al.,
2014; Bogdandi et al., 2018 this issue].
RSSH + RSSR
RSSSR + RSH
(2)
Clearly, other chemical reactions/equilibria are possible (vide infra), making the simple
reaction of H2S with RSSR potentially more complex than previously recognized.
It is important to note that the generation of RSSSR is not dependent on the
presence of H2S in a biological system. For example, RSSH can be formed via the
conversion of cystine (Cys-SS-Cys) to the corresponding cysteine hydropersulfide (Cys-
SSH) and pyruvate or serine via the actions of cystathionine gamma-lyase (CSE) or
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cystathionine beta-synthase (CBS) (Reaction 3) [for example, Yamanishi and Tuboi, 1981;
Stipanuk, 1986; Ida et al., 2014].
Cys-SS-Cys  Cys-SSH + pyruvate (CSE) or serine (CBS)
(3)
In this system, subsequent reaction of the highly nucleophilic Cys-SSH product with the
electrophilic enzyme substrate Cys-SS-Cys would generate the corresponding cysteine
trisulfide (or thiocystine, Cys-SSS-Cys, Reaction 2) in the absence of H2S. Of course, H2S
can eventually be formed in a biological system where thiols will undoubtedly be present via
the simple reverse of Reaction 1 and the eventual formation of an equilibrium mixture of all
species is likely. More recently, it has been reported that Cys-SSH can also be generated
from cysteine (Cys-SH) via the actions of cysteine t-RNA synthases, leading to translational
incorporation of Cys-SSH into proteins along with release of free Cys-SSH [Akaike et al.,
2017].
Numerous studies have utilized Reaction 1 to generate RSSH in situ and there
seems to be little doubt that RSSH can be generated in this way [for example, Cavallini et
al., 1970; Francoleon et al., 2011; Saund et al., 2015]. However, the potential for Reaction
1 to lead to further chemistry (such as Reaction 2) has not been adequately accounted for in
these previous studies and it is certain that other sulfur species besides those shown in the
equilibrium reaction may be present. It is also possible that biologically generated RSSH
participates in numerous other equilibria as well and a complete description of the chemical
biology of hydropersulfides may require consideration of these other processes. Thus, this
study examines Reaction 1 and evaluates the possibility of further chemistry occurring that
may be relevant to biologically generated RSSH species. Importantly, a companion paper in
this issue [Bogdandi et al., 2018] describes aspects of the equilibrium reactions mentioned
above. The paper herein serves to examine the general scope of these equilibria with other
RSH/RSSR as well as further investigate the generation and potential biological utility of an
important species associated with this chemistry, specifically RSSSR.
Materials and Methods
Chemicals and Solutions: Cystine (Cys-SS-Cys), cysteine (Cys-SH), glutathione (GSH),
glutathione disulfide (GSSG), 2-mercaptoethanol (2ME), 2-hydroxyethyl disulfide (2HED),
4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), iodoacetamide (IAM), N-acetyl
penicillamine (NAP), sulfasalazine and N-ethylmaleimide (NEM) were purchased from
Sigma-Aldrich (St. Louis, MO). Sodium hydrosulfide (NaSH) was purchased from Strem
Chemicals, Inc. (Newburyport, MA). S-Benzyl-L-cysteine was purchased from Wako
Chemicals (Osaka, Japan). -(4-Hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) was
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purchased from Molecular BioSciences (Boulder, CO). All other chemicals were purchased
from commercial sources and were the highest purity available. Glutathione trisulfide
(GSSSG) was a gift from Prof. Herbert Nagasawa and synthesized via previously published
procedures [Savige et al., 1964].
Instrumentation: All NMR analyses were performed using an Agilent 400 MHz NMR
spectrometer running on vnmrj software. Water suppression for all samples was achieved
using a (1H) PreSat method in the vnmrj software. All NMR solvents were 10% D2O in 100
mM phosphate buffer (pH 7.4) containing 1 mM DSS as an internal standard.
Mass spectra of chemical studies were recorded using a Thermo Triple Quadrupole
Electrospray Ionization Mass Spectrometer (ESI-MS) in positive ion mode coupled with
XCalibur 2.1 software. All samples were directly injected via syringe pump at a flow rate of 5
µl/min using N2 as a sheath gas and Ar as a collision gas. Ion optics were optimized using
authentic samples of disulfides. The capillary temperature for all samples was 250°C and
spray voltages were 3000 V for 2HED samples and 2500 V for GSSG samples. Mass
spectral analysis of cell lysates was obtained using a triple quadrupole (Q) mass
spectrometer LCMS-8050 (Shimadzu) coupled to the Nexera UHPLC system (Shimadzu).
Methods:
Synthesis of cysteine trisulfide (Cys-SSS-Cys, thiocystine): Cys-SSS-Cys was synthesized
following the general method of Savige et al. [1964] as described briefly below.
Cystine-S-monoxide was synthesized by dissolving Cys-SS-Cys in 1M H2SO4 (approx. 10
mL of H2SO4 solution/gram of cystine) in an ice bath. Two equivalents of peracetic acid
were then added followed by stirring (on ice) for two hours. After stirring, the pH was
adjusted to 4 using pyridine. Cystine-S-monoxide was then precipitated from solution via the
addition of ethanol (approximately 3 times the volume of the initial H2SO4 solution). The
precipitate was filtered and dried. The cystine-S-monoxide was then dissolved in 1M H2SO4
(approximately 10 mL of H2SO4 solution per gram of cystine-S-monoxide). Two equivalents
of solid NaSH was then added to a separate flask and the cystine-S-monoxide solution
added to the NaSH. After addition the flask was sealed with a septum and stirred at room
temperature for 1 hour. The pH was then adjusted to 3.5 using pyridine. A 50/50 mixture of
ethanol/THF was then added (approximately 2-3 times the volume of the initial H2SO4
solution) to precipitate Cys-SSS-Cys. The precipitate was filtered, washed with 50/50
ethanol/THF and dried. Analysis was carried out using 1H-NMR and ESI/MS. Typical purity
of the resulting product was >90% (by NMR) with cystine as the only detectable impurity.
1
Cys-SSS-Cys: H-NMR (400 MHz, D2O)  4.015 (dd, J = 8.0, 4.0 Hz, 1H),  3.42 (dd, J =
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15.2, 4.0 Hz, 1H),  3.23 (dd, J = 15.2, 8.0 Hz, 1H). Mass spectral analysis (ESI-MS) m/z [M
+ H]+ calc. for C6H12N2O4S3, 273.37; found 272.98.
Synthesis of S-methoxycarbonyl penicillamine disulfide (MCPD): The hydropersulfide donor
MCPD used previously by our lab [Millikin et al., 2016; Bianco et al. 2016] was synthesized
via the method of Artaud and Galardon [2014].
NMR Analysis of the NaSH/RSSR Equilibrium: Stock solutions of 2HED (200 mM) and
NaSH (50 and 100 mM) were prepared in 100 mM phosphate buffer (pH 7.4). DSS (internal
standard) stock solutions (10 mM) were prepared in D2O. For all experiments, DSS was
diluted in an NMR tube filled with buffer to a final concentration of 1 mM. For reactions
between 2HED and NaSH, the stock solution of 2HED was added to the DSS containing
NMR tube to a final concentration of 10 mM. NaSH was then added to the NMR tube to final
concentrations of 5, 10 or 15 mM. All solutions were equilibrated at room temperature for 30
minutes, followed by 1H-NMR analysis.
For cyanolysis reactions, stock solutions of 2HED and NaSH were diluted in an NMR
tube containing buffer with 1 mM DSS to final concentrations of 20 and 10 mM, respectively.
The solution stood at room temperature for 30 minutes. At this time, a stock solution of
sodium cyanide (100 mM in buffer) was diluted into the NMR tube to a final concentration of
10 mM. The cyanolysis reaction was allowed to proceed at room temperature for 1 hour,
followed by 1H-NMR analysis.
ESI-MS Analysis of the NaSH/RSSR Equilibrium: A stock solution of NaSH (50 mM) was
prepared in 50 mM ammonium bicarbonate buffer (pH 7.4). Disulfide solutions were further
diluted in the same buffer to final concentrations of 1 mM before ESI-MS analysis. For the
reaction of 2HED and NaSH, 2HED was diluted in buffer to a final concentration of 1 mM.
NaSH was then added to a final concentration of 0.5 mM and the mixture allowed to stand at
room temperature for 30 minutes prior to ESI-MS analysis.
NMR Analysis for the Competitive Trapping of Hydropersulfides: Stock solutions of 2HED,
NaSH and DSS were prepared as stated above (NMR Analysis of the NaSH/RSSR
Equilibrium). For reactions of 2HED, 10 mM NaSH was added to an NMR tube of 20 mM
2HED in 100 mM phosphate buffer (pH 7.4) with 1 mM DSS, and the mixture equilibrated for
30 minutes at room temperature. IAM or NEM (1, 2, 3, 4 or 5 mM final concentration) was
then added and the reaction proceeded at room temperature for 1 hour, followed by 1H-NMR
analysis.
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ESI-MS Analysis for the Competitive Trapping of Hydropersulfides: Stock solutions of 2HED
and NaSH were prepared as above (ESI-MS Analysis of the NaSH/RSSR Equilibrium). For
reactions, 0.5 mM NaSH was added to a solution of 1 mM 2HED in 50 mM ammonium
bicarbonate buffer (pH 7.4) and the mixture equilibrated for 30 minutes at room temperature.
IAM (0.5 mM) was then added and the reaction proceeded for 1 hour at room temperature,
followed by ESI-MS analysis.
ESI-MS Analysis of the Trisulfide Equilibrium: Stock solutions of GSSSG (25 mM) and NAP
(25 mM) were prepared in 50 mM ammonium bicarbonate buffer (pH 7.4). For reactions,
250 µM NAP was added to a solution of 250 µM GSSSG in the same buffer and the reaction
proceeded at room temperature for 1 hour, followed by ESI-MS analysis.
ESI-MS Analysis for the reaction of RSSH and RSSR: A stock solution of GSSG (100 mM)
was prepared in 50 mM ammonium bicarbonate buffer (pH 7.4). A stock solution of MCPD
(20 mM) was prepared in deionized (DI) water. For the reaction, 200 µM MCPD was added
to 1 mM GSSG and the reaction proceeded at room temperature for 30 minutes followed by
ESI-MS analysis.
Cell culture and treatments for Cys-SSS-Cys uptake assay: Human embryonic kidney
(HEK) 293T cells (CRL-11268, American Type Culture Collection) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, high-glucose; Sigma-Aldrich) that was
supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, under standard
cell culture conditions (37 °C, humidified, 5% CO2/95% air).
Analysis of intracellular hydropersulfides and other polysulfides with cultured HEK293T cells
using LC-ESI-MS/MS analyses: Intracellular hydropersulfide levels in cultured HEK293T
cells were quantified by LC-MS/MS with beta-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-
IAM) as a trapping agent for hydropersulfides and polysulfides as reported recently
[Numakura et al., 2017; Akaike et al, 2017]. Briefly, the cultured cells were washed with
PBS twice and homogenized in a cold methanol solution containing 1 mM HPE-IAM, after
which the cell lysates were incubated at 37°C for 20 min. After centrifugation, aliquots of the
supernatants of the lysates were diluted 20 times with 0.1% formic acid containing known
amounts of isotope-labeled internal standards, which were then analyzed via LC-ESI-MS/MS
for per/polysulfide determination. A triple quadrupole (Q) mass spectrometer LCMS-8050
(Shimadzu) coupled to the Nexera UHPLC system (Shimadzu) was used to perform LC-ESI-
MS/MS. Per/polysulfide derivatives were separated by means of Nexera UHPLC with a
YMC-Triart C18 column (50 × 2.0 mm inner diameter) under the following elution conditions:
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mobile phases A (0.1% formic acid) with a linear gradient of mobile phases B (0.1% formic
acid in methanol) from 5% to 90% for 15 min at a flow rate of 0.2 ml/min at 40 °C. MS
spectra were obtained with each temperature of the ESI probe, desolvation line, and heat
block at 300 ºC, 250 ºC, and 400 ºC, respectively; and the nebulizer, heating, and drying
nitrogen gas flows were set to 3, 10, and 10 liters/min, respectively. Various per/polysulfide
derivatives were identified and quantified by means of multiple reaction monitoring.
Experiments were carried out 5 separate times (each time in triplicate). For each
experiment, the values from the triplicates were averaged to give a single value.
Experimental results are presented as the averages of the five individual experiments for an
n=5. Experimental protocols did not require sample blinding or randomization since sample
analyses were not subjective and not susceptible to experimental bias.
Examination of Cys-SSS-Cys pretreatment on cell viability: HEK293T cells were plated at 3
x 104 cells/well in a 96-well plate. The following day, media was removed and replaced with
DMEM w/o Cys/Met (Gibco) alone or with 1 mM Cys-SSS-Cys. After 1.5 hrs, media was
replaced and cells treated with H2O2 (Fisher Sci) or NEM (Sigma) for another 6 hrs in
complete media. For WST8 assays (Cayman Chemical Co.), the reagent was added for the
final 2 hrs and then the absorbance read at 450 nm. For Sytox Green assays (Life
Technologies), the reagent was added after the 6 hrs and then the fluorescence (485/528
nm) measured. All treatments were performed in triplicate and samples read on a Synergy
HT plate reader (BioTek Instruments). Experiments were carried out 5 separate times. For
each experiment, the values from the triplicates were averaged to give a single value.
Experimental results are presented as the averages of the five individual experiments for an
n=5. Experimental protocols did not require sample blinding or randomization since sample
analyses were not subjective and not susceptible to experimental bias.
Data and statistical analysis: Statistical analyses were performed with GraphPad Prism
software ver. 6.0 (GraphPad Software, San Diego, CA). Results are presented as means
from 5 experiments run separately. For statistical comparisons, a two-way ANOVA was
utilized followed by Bonferroni post-tests, with significance set at P < 0.05. Post-hoc tests
were run only if F achieved P<0.05 and there was no significant variance inhomogeneity. In
general, experimental design and statistical analyses of the biological experiments follow the
recommendations prescribed by Curtis et al. [2015].
Nomenclature of targets and ligands: Key protein targets and ligands in this article are
hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common
portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY [Harding et al., 2018],
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and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18
[Alexander et al., 2017].
Results
Analysis of the NaSH/RSSR Equilibrium: Bogdandi and coworkers [2018, this issue]
have described the dynamic equilibrium that establishes between numerous sulfur and
polysulfur-containing species. For example, the reaction of GSSG with H2S results in the
formation of GSSH, GSH and GSSSG as equilibrium partners with the initial reactants
(Reactions 1 and 2). To determine whether these reactions are specific to GSSG or are
generally applicable to other disulfides, a series of studies analogous to those reported by
Bogdandi et al. [2018, this issue] using a chemically simple disulfide have been carried out.
Thus, the reaction of NaSH with 2-hydroxyethyl disulfide (HOCH2CH2SSCH2CH2OH, 2HED),
the disulfide of the thiol 2-mercaptoethanol (HOCH2CH2SH, 2ME) was examined. Increasing
additions of NaSH (5-15 mM) to 2HED (10 mM) caused subsequent concentration-
dependent increases in the formation of two new species after equilibration (Figure 1a).
Comparison of the newly formed species with standard samples (data not shown) indicates
these species are 2ME (δ = 2.67-2.70 and 3.68-3.72 ppm) and 2-hydroxyethyl trisulfide
(HOCH2CH2SSSCH2CH2OH, 2HET) (δ = 3.08-3.11 and 3.91-3.94 ppm). Of additional note
is that the ratio of formed 2ME:2HET is 2:1, which would be expected for formation of 2HET
via Reactions 1 and 2, as described below). Interestingly, addition of 10 mM NaCN to an
equilibrated sample of 20 mM 2HED + 10 mM NaSH (Figure 1b, bottom spectrum) causes
the disappearance of 2HET (Figure 1b, top spectrum), supporting the reaction of CN- with
RSSSR.
Figure 1: (a) Selected region for the stacked 1H-NMR spectra of equilibrated
samples (post 30 min) of 10 mM 2HED with increasing additions of NaSH (0,
5, 10 and 15 mM). The newly formed species correspond to 2HET (δ = 3.08-
3.11 and 3.91-3.94 ppm) and 2ME (δ = 2.67-2.70 and 3.68-3.72 ppm) and
increase in concentration with increasing additions of NaSH. (b) Stacked 1H-
NMR spectra of an equilibrated sample (post 30 min) of 20 mM 2HED + 10
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mM NaSH (bottom spectrum) and addition of 10 mM NaCN (top spectrum) to
this equilibrated sample. Addition of NaCN eliminates 2HET (δ = 3.08-3.11
and 3.91-3.94 ppm), making it absent in the top spectrum. All spectra were
measured in 100 mM phosphate buffer (pH 7.4) with 10% D2O and 1 mM
DSS as an internal reference.
Data collected from NMR analysis (Figure 1) indicate the addition of NaSH to 2HED results
in the formation of RSH and RSSSR species after equilibration (consistent with the work of
Bogdandi and coworkers [2018, this issue] with GSSG), indicating the general nature of this
chemistry. ESI-MS analysis of the 2HED/NaSH reaction also confirms the generation of the
corresponding 2HET, which is consistent with the results of NMR analysis here and of
Bogdandi and coworkers for GSSG/NaSH [2018, this issue] (data not shown).
Analysis for the Competitive Trapping of Hydropersulfides: The results above and
those of Bogdandi et al., [2018, this issue] indicate that addition of NaSH to RSSR results in
the formation of the corresponding RSH and RSSSR species as a general reaction. It has
been widely accepted that the initial reaction of H2S with RSSR results in an equilibrated
mixture with the corresponding RSH and RSSH species (Reaction 1). However, lack of
detection of RSSH in the equilibrated systems herein (described above) implies that under
the conditions of these experiments the RSSH species are subject to further reaction. This is
also consistent with the detection of RSSSR (vide supra). It is therefore reasonable to
consider that RSSH is fleeting and participates in a second equilibrium that ultimately results
in RSSSR formation (Reaction 2). If indeed a highly nucleophilic RSSH is fleeting due to
subsequent reaction with electrophiles present in the reaction mixture (e.g. RSSR, Reaction
2), it is expected that RSSSR formation could be avoided or lessened by the presence of an
alternate electrophile capable of trapping the intermediate RSSH (Reaction 4). In other
words, trapping of RSSH by an exogenous electrophile (Reaction 4) would be competitive
with trapping of RSSH by RSSR (Reaction 2) (as reported by Bogdandi for GSSG [2018,
this issue]), and thus by increasing the concentration of this exogenous electrophile, a
leftward shift in the equilibrium shown in Reaction 2 would be expected and RSSSR levels
would decrease.
RSSH + E+  RSS-E + H+ (E+ = electrophile)
(4)
Indeed, increased addition of the electrophile iodoacetamide (IAM) (0.5-2.5 mM) to
an equilibrated sample of 20 mM 2HED + 10 mM NaSH caused subsequent decreases in
the concentration 2HET formed (δ = 3.08-3.11 and 3.91-3.94 ppm) (Figure 2a).
Additionally, increasing additions of the alternate electrophile N-ethylmaleimide (NEM) (0-5
mM) also caused subsequent decreases in the amount of 2HET formed (Figure 2b, black
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diamonds), albeit to a lesser extent compared to IAM (Figure 2b, black squares). Again, as
was the case for GSSH [Bogdandi et al., this issue], it appears that the hydropersulfide of
2ME (2MESSH) preferentially reacts with 2HED in the presence of NEM, and this preference
occurs to a lesser extent in the presence of IAM.
Figure 2: Selected region for stacked 1H-NMR spectra of 20 mM 2HED + 10
mM NaSH with increasing additions of IAM (1-5 mM, bottom to top spectrum,
respectively). Increasing additions of IAM causes subsequent decreases in
the concentration of 2ME (δ = 2.67-2.70 and 3.68-3.72 ppm) and 2HET (δ =
3.08-3.11 and 3.91-3.94 ppm) because IAM competes with 2HED for the
trapping of the intermediate 2ME-SSH. (b) Plot for [2HET] as a function of
increasing concentrations of exogenous electrophiles IAM (black squares) or
NEM (black diamonds) (0-5 mM). Increasing addition of both electrophiles
causes a subsequent decrease in the concentration of 2HET formed. All 1H-
NMR experiments were performed in 100 mM phosphate buffer (pH 7.4) with
10% D2O and 1 mM DSS as an internal reference.
ESI-MS Analysis for the Competitive Trapping of Hydropersulfides: The above data
indicate that exogenous addition of an electrophile competes with RSSR for the trapping of
RSSH. To verify this further, the products formed after addition of 0.5 mM IAM to an
equilibrated sample of 1 mM 2HED + 0.5 mM NaSH were analyzed via ESI-MS. The
spectrum shown in Figure 3 indicates that exogenously added IAM to an equilibrated
solution of 2HED + NaSH traps 2MESSH, resulting in the 2MESSH-acetamide trapped
adduct ([2MESS+AM+H]+, m/z = 168.05). It should also be noted that 2HET is no longer
present in the ESI-MS spectrum of 2HED + NaSH treated with IAM, indicating that trapping
of 2MESSH by IAM causes a leftward shift in Reaction 2 with subsequent loss of 2HET,
consistent with the data presented in Figure 2.
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A
B
HO
NH2
H2N
NH2
HO
NH2
SS
S
S
O
O
O
O
2MESS-AM (m/z = 168 for 2MESS-AM·H+
)
S(AM)2 (m/z = 149 for S(AM)2·H+
)
2ME-AM (m/z = 137 for 2ME-AM·H+
)
NH2
NH2
I
NH2
I
O
I
O
O
IAM
IAM
IAM
OH
HO
HO
OH
+
H2S
+
HS
SSH
S
S
+
2HED (m/z = 172 for 2HED·NH4
)
HO
+
OH
OH
HO
OH
HO
HS
SSS
+
S S
SSH
+
2HED (m/z = 172 for 2HED·NH4
)
NH2
NH2
I
I
O
O
IAM
IAM
HO
NH2
HO
NH2
S
SS
O
O
2ME-AM (m/z = 137 for 2ME-AM·H+
)
2MESS-AM (m/z = 168 for 2MESS-AM·H+
)
Figure 3: A) ESI-MS spectrum resulting from the addition of 0.5 mM IAM to
an equilibrated solution (post 30 min) of 1 mM 2HED + 0.5 mM NaSH.
Addition of IAM reacts with, and traps HS- ([S(AM)2+H]+), 2ME
([2ME+AM+H]+) and 2MESSH, ([2MESS+AM+H]+). B) Scheme of equilibrium
and trapped species.
ESI-MS Analysis of the Trisulfide Equilibrium: Summation of Reaction 1 and 2 leads
to the overall equilibrium of Reaction 5.
2RSSR + H2S
RSSSR + 2RSH
(5)
Consistent with the relevance of Reaction 5, Figure 1a shows that treatment of 2HED with
NaSH forms both RSSSR and RSH in an approximate 1:2 ratio, respectively. Additionally,
the equilibrium of Reaction 5 implies that reaction of RSH with RSSSR should lead to
formation of the corresponding RSSR and H2S (the reverse of Reaction 5). To test this, 250
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µM GSSSG was reacted with 250 µM of the thiol N-acetyl penicillamine (NAP) and analyzed
by ESI-MS. Figure 4a shows that treatment of GSSSG with NAP (post 3 hours) results in
the mixed NAP-SS-G disulfide ([NAP+GS+H]+, m/z = 497.18) with no other alkyl polysulfur
species formed, consistent with the reverse of Reaction 5.
O
O
SSH
+
G S S G
SH
GSSSG
O
NH
+
NH
O
O
MCP-SSH
N-Acetyl penicillamine (NAP,
m/z = 192 for NAP·H+)
O
O
O
S S G
NH
+ GSH
SSSG
GSSH +
O
NH
O
NAP-GS (m/z = 497 for NAP-GS·H+)
MCPSS-GS (m/z = 545 for
MCPSS-GS·H+)
Figure 4: ESI-MS spectrum for the reaction of (a) 250 µM NAP + 250 µM
GSSSG (post 3 hours). The mixed disulfide ([NAP+GS+H]+, m/z = 497.18) is
the only resulting species from this reaction. (b) Reaction of 200 µM MCP-
SSH + 1 mM GSSG. The mixed trisulfide ([MCPSS+GS+H]+, m/z = 545.29)
and GSH ([GSH+H]+, m/z = 308.15) are the only resulting species from the
reaction. All spectra were recorded in 50 mM ammonium bicarbonate buffer
(pH 7.4). Relevant chemistry and mass assignments are shown directly
below the spectra.
ESI-MS Analysis for the reaction of RSSH and RSSR: To this point, RSSH
intermediates resulting from reaction of NaSH with RSSR have not been directly detected.
As an indication of their presence, RSSH intermediates have been trapped via addition of
the electrophiles IAM and NEM (Figures 2 and 3). To further confirm reaction of RSSH with
RSSR, ultimately leading to formation of the corresponding RSSSR (Reaction 2), a model
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H2S-independent RSSH donor MCPD was used, as previously described by us [Millikin et
al., 2016]. The ESI-MS spectrum corresponding to the decomposition of MCPD (releasing
the MCP-SSH hydropersulfide) in the presence of GSSG is shown in Figure 4b. As
displayed in the spectrum, reaction of MCP-SSH with GSSG leads to the mixed MCP-SSS-G
trisulfide (Figure 4b, [MCPSS+GS+H]+, m/z = 545.29). No other MCP-SSH derived
polysulfur species are detected, which is consistent with the primary fate of RSSH in the
presence of RSSR being RSSSR formation (Reaction 2). Interestingly, the persulfide donor
MCPD is also a disulfide and yet no adduct associated with the persulfide reacting with the
precursor donor disulfide species is detected. Thus, GSSG appears to outcompete this
reaction since the only trisulfide that was readily detectable was the mixed trisulfide with
GSSG (MCPSS+GS). It can also be envisioned that the persulfide formed in this experiment
could have also reacted with a protonated hydropersulfide resulting in the corresponding
trisulfide, RSSSR and HS- (RSS- + RSSH  RSSSR + HS-). However, the pKa of RSSH
has been reported to be 6.2 – 4.3 [Everett and Wardman, 1995; Cuevasanta et al., 2015].
Thus, under the conditions of these experiments (pH 7.4), this reaction is unlikely to be
competitive since the predominant species will be the nucleophilic anionic RSS- species with
lesser amounts of the electrophilic protonated RSSH species present.
Dialkyltrisulfides as biological sources of hydropersulfides: Thus far, the results
presented are consistent with the idea that RSSSR is in a dynamic equilibrium with RSSR,
H2S, RSH and RSSH via Reactions 1 and 2. Moreover, RSSH levels are kept low in this
system and higher order polysulfides are not readily generated (a rationale for this is given
below). These results indicate that the equilibria set up via Reactions 1 and 2 (and
summarized by Reaction 5) can be approached starting from either RSSR/H2S or
RSSSR/RSH (Reaction 5). That is, RSSH can be generated starting with either equilibrium
pair. In order to examine this in a biological system, cells were treated with cysteine
trisulfide (Cys-SSS-Cys), with the hope that uptake would occur, thus establishing an
intracellular RSSH equilibrium. Indeed, it was found that HEK293T cells treated with 0.2, 0.5
and 1 mM Cys-SSS-Cys for 1.5 hours results in a concentration-dependent and significant
increase in intracellular levels of Cys-SSH and also GSSH (Figure 5). Importantly,
treatment with Cys-SS-Cys alone did not result in increased RSSH levels, indicating that the
observed increases in intracellular RSSH were not due to CSE/CBS-mediated conversion of
Cys-SS-Cys to Cys-SSH.
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Figure 5: RSH and RSSH levels in HEK293T cells pretreated with either cystine
(Cys-SS-Cys) or cysteine trisulfide (Cys-SSS-Cys). (a) Cysteine levels resulting from
pretreatment with Cys-SS-Cys or Cys-SSS-Cys; (b) Cysteine hydropersulfide (Cys-
SSH) levels in cells pretreated with Cys-SS-Cys or Cys-SSS-Cys; (c) Glutathione
levels in cells pretreated with Cys-SS-Cys or Cys-SSS-Cys and (d) Glutathione
hydropersulfide (GSSH) levels in cells pretreated with Cys-SS-Cys or Cys-SSS-Cys.
Each experiment was carried out 5 separate times (each time in triplicate). For each
experiment, the values from the triplicates were averaged to give a single value.
Data represents average ± SD (n = 5), *P< 0.05. Statistical analysis was performed
as described in the Methods section.
Dialkyltrisulfide-mediated cellular protection against oxidative and electrophilic stress:
The results shown in Figure 5 indicate that HEK293T cells pre-treated with Cys-SSS-Cys
results in significant increases in intracellular Cys-SSH and GSSH. It has been postulated
that due to their unique chemistry [for example, Ono et al., 2014; Millikin et al., 2016] a
possible function of intracellular RSSH species is to protect cells from oxidative and/or
electrophilic stress. Thus, Cys-SSS-Cys represents a novel method for examining the ability
of increased intracellular RSSH levels in HEK293T cells to protect against electrophilic
and/or oxidative stress. To test this, HEK293T cells were pre-treated with 1 mM Cys-SSS-
Cys for 1.5 hours followed by exposure to either 0-1 mM H2O2 (oxidative/electrophilic stress)
or 0-1 mM NEM (electrophilic stress). After 6 hours of treatment with the stressor, cell
viability was assessed using a WST8 assay [Ishiyama et al., 1997]. The data indicate that
pretreatment with Cys-SSS-Cys results in significant protection against NEM-mediated
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toxicity (Figure 6b) but only marginal effects on H2O2 toxicity were observed (Figure 6a).
Since viability measured by WST8 is actually an indicator of cellular reductive capacity
(NADH/NADPH levels), another method for cell viability was used that interrogates another
cell function in order to help confirm these results. Thus, the membrane impermeable nucleic
acid dye, Sytox Green was also used as a measure of cytotoxicity (a measure of cell
membrane integrity [Jones and Singer, 2001]). Cells were again treated as before, and
Sytox Green added after 6 hours (Figure 6c). As before, pretreatment with Cys-SSS-Cys
protected cells against NEM-mediated toxicity confirming the previous results using WST8.
Figure 6: HEK293T cells were pre-incubated with 1mM cysteine trisulfide (Cys-SSS-
Cys) for 1.5 hrs. Media was then replaced and cells treated with the indicated
amount of
for (a) and
(b), graphs
H2O2 (a) or NEM (b,c) for 6 hrs. The cell viability indicator, WST-8, was used
(b), while the cell death indicator Sytox Green was used in (c). For (a) and
show data normalized to 100% viability for either untreated control or Cys-
SSS-Cys only background, whereas insets show data normalized to 100% viability only in
untreated
controls. Each experiment was carried out 5 separate times (each time in
each experiment, the values from the triplicates were averaged to give
Experimental results are presented as the averages ± SEM of the five
experiments, *P < 0.05. Statistical analysis was performed as described in the
triplicate). For
a single value.
individual
Methods section.
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Discussion
Recent studies have alluded to the possible importance of RSSH species in
biological signaling and in cellular protection against oxidative and/or electrophilic stress [for
example, Ono et al., 2014; Ida et al., 2014; Millikin et al., 2016; Saund et al., 2015]. It is
becoming increasingly apparent that H2S, RSSH and other polysulfides are all biologically
prevalent and their chemistries intimately intertwined [for example, Ida et al., 2014; Doka et
al., 2016]. As mentioned previously, numerous studies have utilized the reaction of H2S with
RSSR (Reaction 1) to generate RSSH for study in both chemical and biological systems.
However, the work presented herein indicates this may be an oversimplified view of this
reaction. It is clear that the RSSH product of Reaction 1 is very nucleophilic (most likely as
the RSS- anion) and reacts further with the electrophilic RSSR to form RSSSR (Reaction 2).
It is important to note that RSSH is more acidic than the corresponding RSH by
approximately 1-2 pKa units (and possibly more [Cuevasanta et al., 2015]), indicating that a
higher percentage of the nucleophilic anionic RSS- species will exist compared to the thiolate
(RS-). It is also important to note that the higher levels of RSS- (compared to the protonated
and electrophilic RSSH) will greatly decrease the rate of the reverse of Reaction 1 since an
electrophilic RSSH is required in this reaction [Cuevasanta et al., 2015].
RSSSR generated from Reaction 2 is likely to be even more electrophilic than the
corresponding RSSR (vide infra) indicating it may react further under these reaction
conditions. In the case of a disulfide, RSSR, nucleophilic attack at either sulfur atom
releases RSH/RS- as the leaving group (Reaction 6). In the case of RSSSR, there are two
types of sulfur atoms, a sulfane sulfur atom and non-sulfane sulfur atoms (the bolded atoms
of RSSSR). Nucleophilic attack at the sulfane sulfur atom results in an RS- leaving group
(Reaction 7) and attack at a non-sulfane sulfur atoms results in expulsion of an RSS-
leaving group (Reaction 8).
RSSR + Nuc-  RS-Nuc + RS-
RSSSR + Nuc-  RSS-Nuc + RS-
RSSSR + Nuc-  RS-Nuc + RSS-
(6)
(7)
(8)
Previous studies examining the nucleophilic cleavage of mixed disulfides (Reaction 6 where
the R groups are different), found that the better leaving group (the weaker RS- base)
governed the regiochemistry [Hiskey and Carroll, 1961]. That is, the sulfur atom attacked
was the one that had the better leaving group attached. Extrapolating this idea to the
nucleophilic attack on RSSSR, it would be expected that attack at the non-sulfane sulfur
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sites would predominate since RSS- is a significantly weaker base than RS- and thus a
better leaving group [for example, Nagy, 2013; Cuevasanta et al., 2015]. Thus, in the
reactions examined herein, the attack of a highly nucleophilic RSS- species on RSSSR is
non-productive since it gives back the same reactants (Reaction 9).
R’SSSR’ + RSSH
R’SSSR + R’SSH
(9)
(note: prime notation given to follow reactants)
Of course other nucleophiles are present in solution that can also react with the electrophilic
RSSSR species, namely H2S and RSH. The reaction of HS- with RSSSR should also occur
on the non-sulfane sulfur atoms, leading to the generation of RSSH (Reaction 10) and the
reaction of RS- with RSSSR will lead to the formation of the corresponding RSSR and RSSH
(Reaction 11).
RSSSR + H2S
2 RSSH
(10)
(11)
RSSSR + R’SH
RSSR’ + RSSH
(note: prime notation given to follow reactants)
In the case of the reaction of H2S with RSSR (Reaction 1) and subsequent reactions
(Reactions 2, 9, 10 and 11) formation of the equilibrium partners RSSR, H2S, RSH, RSSSR
and RSSH are expected without the generation of higher order polysulfides (i.e. RSSSnR or
RSSnH (n > 1). Although chemical reactions can be envisioned that could lead to higher
order polysulfides (Reactions 12 and 13), based on the results and arguments presented
here, these reactions appear unfavorable since the sulfane sulfur of RSSSR would have to
be attacked.
RSSSR + RSSH
RSSSR + H2S
RSSSSR + RSH
RSSSH + RSH
(12)
(13)
Figure 7 schematically depicts the rationale explaining the lack of formation of higher order
polysulfides (e.g. tetrasulfides, RSSSSR) from the reaction of RSS- with RSSSR. In all
cases the favored reaction involves nucleophilic attack at the non-sulfane sulfur atoms
(Figure 7, pathway b).
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R
S
S
S
R
R
S
S
S
R
R'
S
S
R'
S
S
a
b
R-S-S-S-S-R' + RS
R-S-S-S-R' + RSS
Figure 7: Reaction of a nucleophilic hydropersulfide with a trisulfide. (a)
nucleophilic attack at the sulfane sulfur atom, (b) nucleophilic attack at a non-
sulfane sulfur atom.
Another factor that may favor nucleophilic attack on the non-sulfane sulfur atoms of
RSSSR may involve electron pair repulsion between the attacking nucleophile and the lone
pair electrons on the sulfur atoms of RSSSR. Nucleophilic attack on the central sulfane
sulfur atom of RSSSR by RSS- (Figure 7, pathway a) would be inhibited by repulsion
between the nucleophile and the lone pairs of electrons on both of the two adjacent sulfur
atoms whereas nucleophilic attack on the non-sulfane sulfur atoms (Figure 7, pathway b)
involves repulsion from only one adjacent sulfur atom. Regardless of which effect
predominates, both favor nucleophilic attack on the non-sulfane sulfur sites of an RSSSR
species in this purely chemical system. Scheme 1 illustrates the likely integrated chemistry
that describes the results herein.
R-S-S-E
R-S-S-S-S-R + R-S-H (unfavorable products)
4
E+ (electrophile)
CSE
not favorable*
3
1
R-S-S-R + H2S
R-S-S-H + R-S-H
2
R-S-S-S-R + R-S-S-H (non-productive)**
* non-favorable pathway due to poor leaving group.
** non-productive reaction since reactants and products are the same.
R-S-H + R-S-S-S-R
Scheme 1: Integrated reaction pathways.
Support for the chemistry depicted in Scheme 1 is as follows:
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1) Equilibrium 1 is a previously established reaction, which has been used by several labs to
generate RSSH species in situ [Rao and Gorin, 1959; Cavalini et al., 1970; Francoleon et al.,
2011].
2) Equilibrium 2 is evidenced by results presented in this study and the study of Bogdandi et
al., [2018, this issue]. Both NMR and mass spectral analysis indicate that the reaction of a
disulfide (e.g. 2HED) with H2S results in detectable levels of thiol (2ME) and the
corresponding trisulfide (2HET). Interestingly, RSSH species are not detected using either
method of analysis, indicating that they may be at extremely low levels under the conditions
of these experiments.
3) Reaction 3 (tetrasulfide, RSSSSR formation, Reaction 12, Figure 7a) appears
unfavorable (vide supra) and, expectedly, RSSSSR species are not observed.
4) Reaction 4 the trapping of RSSH by an exogenous electrophile, occurs readily and
serves as evidence for the proposed equilibria (although RSSH species are not directly
detected).
It is important to note that care must be taken when extrapolating the chemical
results described herein to a biological system. The kinetics of all the reactions examined
(e.g. the forward and reverse reactions of Reactions 1 and 2) are not firmly established and
it is possible (if not likely) that competing reactions of the equilibrium species with other
available biological molecules can occur. For example, RSSH species are highly redox
active [Bianco et al., 2017; Francoleon et al., 2011] and can be intercepted by mild biological
oxidants before it can participate in the equilibrium reactions described herein. Also,
enzymatic conversion of, for example, RSSSR species via reduction processes to other
species can occur, thus removing trisulfides from the equilibrium reactions. It is also worth
noting that previous studies have examined the kinetics associated with the forward process
of Reaction 1 (the reaction of H2S with a disulfide) and the rate constants are reported to be
highly variable and dependent on the nature of the reactants [Vasas et al., 2015;
Cuevasanta et al., 2015]. For example, second order rate constants ranging from
approximately 103 M-1s-1 to 10-2 M-1s-1 for the reaction of H2S with 5,5-dithiobis-(2-
nitrobenzoic acid) (DTNB) and 2HED, respectively, are reported. Since RSSH species are
considered to be superior nucleophiles compared to thiols (and H2S), it is expected that they
can react faster with electrophilic species compared to the corresponding thiol or H2S.
Indeed, Cuevasanta and coworkers [2015] estimate that RSSH species can be as much as
20-fold more reactive than the corresponding thiol at physiological pH.
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Treatment of HEK293T cells with Cys-SSS-Cys led to a significant increase in the
intracellular hydropersulfides Cys-SSH and GSSH (Figure 5). It is established that the
uptake of Cys-SS-Cys in mammalian cells occurs via the activity of the antiporter xc-, which
exports glutamate while it takes up Cys-SS-Cys from extracellular solution [Conrad and
Sato, 2012; Lewerenz et al., 2013]. However, it is not known whether Cys-SSS-Cys is also
transported via xc-. Regardless, Cys-SSS-Cys may represent a possible
pharmacological/research tool for raising intracellular hydropersulfides (although more cell
types need to be examined to determine if this is a general phenomenon). Importantly,
trisulfides (e.g. Cys-SSS-Cys) are stable and can be stored and used similarly to disulfides
(e.g. Cys-SS-Cys). In our hands, Cys-SSS-Cys is stable to acidic conditions, can be stored
at room temperature, is stable to decomposition for months and is more soluble
(approximately 10x) than Cys-SS-Cys in aqueous solution. On the other hand,
hydropersulfides are inherently unstable to disproportionation and cannot be easily stored
[Rao and Gorin, 1959].
Consistent with the idea that hydropersulfides can protect against specific stressors,
HEK293T cells pretreated with Cys-SSS-Cys (which leads to increased intracellular levels of
hydropersulfides, Figure 5) were significantly protected from the toxicity of the electrophile
NEM (Figure 6b). However, a marginal effect (if any, depending on how samples are
normalized) was observed with H2O2 treatment (Figure 6a). In the case of NEM, the
increased levels of the highly nucleophilic hydropersulfides may be capable of
intercepting/scavenging an otherwise toxic electrophile before it is able to modify critical
cellular sites. Alternatively, hydropersulfide formation on critical protein thiols could be
protected from irreversible electrophilic modification since modification will occur on the
sacrificial sulfane sulfur atom, which can be reductively removed [Ono et al., 2014; Millikin et
al., 2016]. Importantly, persulfide-mediated protection against thiophilic metal toxins has
also been reported [Abiko et al., 2015]. Although much more work remains to determine
whether generation of per- and/or poly-sulfides represents a protective mechanism against
oxidative and/or electrophilic stress, the utility of Cys-SSS-Cys as a means of
pharmacologically altering intracellular hydropersulfide levels may be an important tool in
this regard.
Differences in the observed protection by Cys-SSS-Cys between H2O2 and NEM is
intriguing. It is well established that NEM toxicity is primarily the result of thiol protein
alkylation [for example, Vimard et al., 2011]. Thus, considering the high nucleophilicity of
RSSH it is not surprising that increased intracellular RSSH results in protection against
NEM. Although H2O2-mediated physiology (i.e. signaling) can either directly or indirectly
involve oxidative thiol modification [for example, Winterbourn, 2013; Jones, 2008] the toxicity
of H2O2 is thought to primarily involve metal-mediated generation of potent and
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indiscriminate oxidants (i.e. via the Fenton reaction) [Winterbourn, 1995]. Importantly, the
oxidants generated in this way are thought to be too reactive to be trapped or scavenged
since they are immediately reactive and do not diffuse away from their site of generation.
Thus, RSSH may have little effect on Fenton-type oxidants.
It is important to note that intracellular increases in RSSH species may also lead to
higher intracellular levels of RSSR, RSH and H2S via the equilibrium processes described
above. Thus, it is naïve and even ill-advised to conclude categorically that the observed
protection is the sole result of increased intracellular RSSH levels. Since treatment of cells
with Cys-SSS-Cys does not dramatically increase levels of RSH species (Figure 5) the
observed protection is clearly not due to RSH. However, it remains possible that the
protection results, at least partially, from increased intracellular H2S levels. Indeed, based
on the results herein and those of Bogdandi and coworkers [2018, this issue], it is currently
difficult to distinguish between the actions of RSSH from H2S in a biological system since
they are equilibrium partners and should be present simultaneously. To be sure, there are
chemical rationale for predicting protective functions associated with RSSH species [for
example, Ono et al., 2014; Millikin et al., 2016], but this remains to be unequivocally
established.
Finally, it is interesting to note that treatment of cells with Cys-SSS-Cys alone leads
to an increase in apparent “viability” (as measured by the WST8 assay) (Figure 6a and 6b)
in control samples. That is, Cys-SSS-Cys treatment on its own appears to result in an
increase in cellular reducing equivalents/reduction capacity (what WST8 ultimately measures
[Ishiyama et al., 1997]), which is construed as an increase in viability. It is noteworthy that
hydropersulfides are considered to be good reductants (superior to, for example, thiols and
akin to ascorbate and/or tocopherols [for example, Bianco et al., 2016; Chauvin et al.,
2017]). Thus, in spite of the fact that RSSH species are formally oxidized with respect of the
corresponding RSH, they are significantly more reducing and may possibly be responsible
for the greater reducing capacity observed. Interestingly, Akaike and coworkers [2017]
reported that hydropersulfide generation in mitochondria is an important aspect of
mitochondrial regulation and activity and thus it is intriguing to speculate that since NADH
(one of the primary species assayed by WST8) is used by mitochondria as an electron
source, the apparent increases in NADH levels in Cys-SSS-Cys pre-treated cells may be the
result of other reductants, possibly RSSH species, serving as alternative mitochondrial
electron sources (proposed previously [Akaike et al., 2017]) allowing NADH levels to rise.
Acknowledgements: The authors would like to acknowledge Dr. Michael Bartberger for
helpful discussions regarding the chemical arguments presented. J.P.T. would like to
acknowledge the NSF (CHE-1566065) for support of this research. P. N. would like to
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acknowledge National Research, Development and Innovation Office (grants No.: KH17-
126766 and K 109843) for support.
Conflict of Interest: The authors declare no conflicts of interest.
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