Reaction of hydrogen sulfide with disulfide and Sulfenic acid to form the strongly Nucleophilic Persulfide

Article abstract of DOI:10.1074/jbc.M115.672816

Background: Hydrogen sulfide (H2S) modulates physiological processes in mammals. Results: The reactivity of H2S toward disulfides (RSSR) and albumin sulfenic acid (RSOH) to form persulfides (RSSH) was assessed. Conclusion: H2S is less reactive than thiols. Persulfides have enhanced nucleophilicity. Significance: This kinetic study helps rationalize the contribution of the reactions with oxidized thiol derivatives toH2S biology.

Full text of DOI:10.1074/jbc.M115.672816

JBC Papers in Press. Published on August 12, 2015 as Manuscript M115.672816
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.672816
Reaction of hydrogen sulfide with disulfide and sulfenic acid to form the strongly
nucleophilic persulfide†
Ernesto Cuevasanta‡§, Mike Lange¶, Jenner Bonanata*, E. Laura Coitiño*, Gerardo Ferrer-
Sueta**§, Milos R. Filipovic¶1, Beatriz Alvarez ‡§2
From the ‡Laboratorio de Enzimología, *Laboratorio de Química Teórica y Computacional and
**Laboratorio de Fisicoquímica Biológica, Facultad de Ciencias, and §Center for Free Radical and
Biomedical Research, Universidad de la República, Montevideo, Uruguay; ¶Department of Chemistry
and Pharmacy, Division of Bioinorganic Chemistry, Friedrich-Alexander University of Erlangen–
Nuremberg, Erlangen, Germany
Running title: Reaction of H2S with RSSR and RSOH to form RSSH
1To whom correspondence may be addressed: Milos R. Filipovic, Department of Chemistry and
Pharmacy, Friedrich-Alexander University of Erlangen-Nuremberg, 91058 Erlangen, Germany; Tel:
+49 91318527345; E-mail: milos.filipovic@fau.de.
2To whom correspondence may be addressed: Beatriz Alvarez, Laboratorio de Enzimología, Facultad
de Ciencias, Universidad de la República, 11400 Montevideo, Uruguay; Tel: +598 25250749; E-mail:
beatriz.alvarez@fcien.edu.uy.
Keywords: hydrogen sulfide; disulfide; sulfenic acid; hydrodisulfide; persulfide; thiol; kinetics
Background: Hydrogen sulfide (H2S) modulates HSA was determined. Both reactions of H2S with
physiological processes in mammals.
disulfides and sulfenic acids yield persulfides
Results: The reactivity of H2S toward disulfides (RSSH), recently identified post-translational
(RSSR) and albumin sulfenic acid (RSOH) to modifications. The formation of this derivative in
form persulfides (RSSH) was assessed.
Conclusion: H2S is less reactive than thiols. reactions with a reporter disulfide and with
Persulfides have enhanced nucleophilicity. peroxynitrite revealed that persulfides are better
HSA was determined and the rate constants of its
Significance: This kinetic study helps rationalize nucleophiles than thiols, consistent with the alpha
the contribution of the reactions with oxidized effect. Experiments with cells in culture showed
thiol derivatives to H2S biology.
that treatment with hydrogen peroxide enhanced
the formation of persulfides. Biological
implications are discussed. Our results give light
on the mechanisms of persulfide formation and
provide quantitative evidence for the high
nucleophilicity of these novel derivatives, setting
the stage for understanding the contribution of
the reactions of H2S with oxidized thiol
derivatives to H2S effector processes.
ABSTRACT
Hydrogen sulfide (H2S) is increasingly
recognized to modulate physiological processes
in mammals through mechanisms that are
currently under scrutiny. H2S is not able to react
with reduced thiols (RSH). However, H2S –
precisely, HS-– is able to react with oxidized thiol
derivatives. We performed a systematic study of
the reactivity of HS- toward symmetric low
INTRODUCTION
molecular weight disulfides (RSSR) and mixed Beyond its classical conception as a highly toxic
albumin (HSA) disulfides. Correlations with thiol gas, hydrogen sulfide (H2S)a is now considered a
acidity and computational modeling showed that physiological modulator in mammals. It is
the reaction occurs through
a
concerted produced through the enzymatic activity of
mechanism. Comparison to analogous reactions cystathionine β-synthase and cystathionine γ-
of thiolates indicated that the intrinsic reactivity lyase and probably also of mercaptopyruvate S-
of HS- is one order of magnitude lower than that transferase (1). At neutral pH, the species H2S, a
of thiolates. In addition, H2S is able to react with weak acid (pKa = 7 (2)), is in equilibrium with its
sulfenic acids (RSOH). The rate constant of the conjugate base, the anion hydrosulfide (HS-).
reaction of H2S with the sulfenic acid formed in Simultaneously with the description of its
1
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

biological effects, efforts have been undertaken consumption pathway for H2S, mainly in
to elucidate the underlying molecular extracellular milieu and plasma, where the
mechanisms, yielding slow progress. Hydrogen diversity and high amounts of disulfides present
sulfide cannot react with thiols (RSH). Among a (23) make them a likely target. In plasma, human
range of possible mechanisms that could operate serum albumin (HSA)c is the most abundant
in biological systems, the reaction of H2S -more protein and mixed disulfides of HSA constitute
precisely, the hydrosulfide anion HS- in the predominant disulfides (23). HSA possesses
equilibrium with it- toward disulfides (RSSR) 34 cysteines forming structural disulfides and
and sulfenic acids (RSOH) can be considered a only one reduced cysteine (Cys34, HSA-SH) that
plausible way of unleashing physiological is located in a cleft, hindering intra and
consequences. Furthermore, these reactions intermolecular reactions. These features allow a
produce
hydrodisulfides
constituting possible mechanisms for the so- intermediates generated in thiol oxidation
persulfides
(also
known
as relatively stable sulfenic acid to be formed (HSA-
or
hydropersulfides)b, SOH) (24). Sulfenic acids are transient
called ‘S-sulfhydration’ (3–5).
pathways. They are typically unstable and decay
mainly through reaction with a second thiol to
form disulfides. Since HSA forms a long-lived
and well characterized sulfenic acid (24, 25), it
could open the way to kinetic measurements of
the reactions with H2S. In addition, HSA could
potentially likewise form a stable persulfide
(HSA-SSH).
Persulfides (RSSH) represent novel thiol
modification products of increasing interest in
signaling and catalysis. They are constituent part
of a class of chemical moieties known as
sulfanes, in which sulfur is covalently linked to
another sulfur and/or ionizable protons (6). The
typically
unstable
persulfides
possess
electrophilic character. They also conserve the In this work, we studied the reactivity of H2S
nucleophilic character of the original thiol, or toward symmetrical low molecular weight
actually increase it due to the presence of an (LMW) disulfides and mixed disulfides formed
adjacent sulfur containing unshared pairs of between HSA and LMW thiols, providing the
electrons -alpha effect- (3, 7, 8). Moreover, it has first systematic study that allows comparison of
been proposed that these compounds are the intrinsic reactivity of H2S with respect to
responsible for the biological effects initially thiols. In addition, we report for the first time the
assigned to H2S (9). Persulfides were detected rate constant of the reaction of H2S with a
years ago as naturally occurring products (6) and sulfenic acid, the one formed in HSA. Last, we
proposed to have roles in sulfur transport (10) obtained the HSA persulfide and determined the
and enzymatic catalysis (11, 12). Indeed, animals rate constant of two of its reactions, providing the
have enzymes which produce and transfer first quantitative analysis of the nucleophilicity of
persulfides,
i.e.
cystathionine
γ–lyase, persulfides and its comparison to that of thiols.
cystathionine β–synthase (13), sulfide:quinone Thus, our study contributes to the understanding
oxidoreductase and rhodanese (14), and others of the biochemical reactivity of H2S and helps
which decompose them, like persulfide reveal the impact that its reactions with oxidized
dioxygenase or ETHE1 (15). Recently, thiol derivatives to form hydrosulfides could
persulfides have been produced in a diversity of have in the biology of this recently recognized
systems and detected using analytical methods mediator.
such as cold cyanolisis and dithiothreitol-
EXPERIMENTAL PROCEDURES
dependent release of H2S, together with mass
spectrometry and a variety of selective labeling
methods (16–22).
Preparation of hydrogen sulfide, LMW thiol and
disulfide solutions. Stable Na2S solutions were
prepared by two methods: a) Stock solutions of
Na2S·9H2O (J. T. Baker) were prepared daily in
distilled water in sealed vials and used
immediately. Concentrations were verified by
iodometric titration and remained constant for
several hours. b) Anhydrous Na2S was purchased
from Sigma Aldrich, opened and stored in a
Despite increasing efforts dedicated to
understand the reactivity of H2S, no systematic
kinetic study of the reaction with oxidized thiol
derivatives has been published and few persulfide
models suitable for biochemical characterization
have been proposed (17–19, 22). The reaction
toward disulfides could represent a significant
2

glove box (< 1 ppm O2 and < 1 ppm H2O). Stock prepared by incubating 6 µM HSA-SOH with
solutions (100 mM) were prepared in the glove equimolar Na2S in phosphate buffer (100 mM,
box using argon-bubbled nano-pure water pH 7.4, 0.1 mM dtpa, 25 ºC) during variable
pretreated with Chelex resin-100 and stored in periods of time. Persulfides for kinetic studies
dark glass vials with PTFE septa at +4 ºC. Gas- toward peroxynitrite were prepared by reacting
tight Hamilton syringes were used throughout the HSA-TNB with Na2S. HSA-SSH was quantified
study. 5-Thio-2-nitrobenzoic acid (TNB) based on the amount of TNB released and
solutions were prepared as previously reported purified by gel-filtration on Sephadex G-10
(26). LMW disulfides were purchased from equilibrated with phosphate buffer (30 mM, pH
Sigma or AppliChem. Glutathione disulfide 7.4). Solutions were kept under argon and on ice
(GSSG),
cystinedimethylester
(CysOMe- to minimize decay.
CysOMe) and hydroxyethyldisulfide (HED)
stocks were prepared in distilled water; cystine
(Cys-Cys) and homocystine (Hcy-Hcy) were
diluted in NaOH 0.1 N; 5,5'-dithiobis-(2-
nitrobenzoic acid) (DTNB) stocks were prepared
in ethanol 95 %.
Kinetics of hydrogen sulfide reactions with
disulfides by continuous UV-Vis measurements.
Reactions were initiated by the addition of low
amounts (30-500 µM) of Na2S to disulfides in
pseudo-first order excess in borate (100 mM, pH
9.5, 0.1 mM dtpa) or phosphate (100 mM, pH
Preparation of HSA derivatives. HSA (Sigma) 7.4, 0.1 mM dtpa) buffer using sealed quartz
was delipidated, its thiol was reduced with β– cuvettes with minimal head space. UV-Vis
mercaptoethanol and the concentration of protein spectra were registered in a Varian Cary 50
and thiol were quantified as described previously spectrophotometer at 25 ºC and increases in
(26). Mixed disulfides were prepared by absorbance at 350 nm due to the appearance of
incubating reduced HSA (~1 mM) overnight with sulfane sulfur (28) were fitted to single
the disulfide of interest (up to 20 mM) in acetate exponential functions in order to obtain pseudo-
buffer (100 mM, pH 4.5, 0.1 mM first order rate constants (kobs) for Cys-Cys,
diethylenetriaminepentaacetic acid (dtpa), 4 ºC) CysOMe-CysOMe, GSSG, HED and Hcy-Hcy.
or tris buffer (100 mM, pH 7.4, 0.1 mM dtpa, 4 The reaction with DTNB was followed from
ºC). The pH of the buffer was chosen so as to TNB formation at 412 nm and fitted to double
favor maximum displacement of equilibria exponential functions. The second-order rate
toward mixed disulfide formation: acidic pH constants were determined from the slopes of
when the pKa of the leaving thiol was higher than plots of kobs versus disulfide concentration. The
that of HSA-SH (8.1, (23, 25)) as in the case of pH-independent rate constants were calculated by
Hcy-Hcy, GSSG and HED, and basic pH when correction for HS- taking into account the pH and
the pKa was lower (DTNB, CysOMe-CysOMe) the pKa of H2S.
(27). The mixed disulfides were subjected to gel-
Kinetics of hydrogen sulfide reactions with
disulfides by discrete hydrogen sulfide
measurements. Reaction mixtures containing
excess disulfides in phosphate buffer (100 mM,
pH 7.4, 0.1 mM dtpa) were incubated in closed
vials with minimal head space at 25 ºC. Aliquots
were removed using a gas-tight syringe and
mixed with 4 mM zinc acetate to quench the
reaction. The H2S concentration of the aliquots
was determined using the methylene blue method
(29). In the case of LMW disulfides,
concentration decreases were fitted to single
exponential functions to obtain the corresponding
kobs. In the case of mixed HSA disulfides,
mixtures for methylene blue formation were
centrifuged to remove the precipitated protein
and the rate constants were calculated using the
method of initial rates.
filtration in a PD-10 column (GE) equilibrated
with phosphate buffer (100 mM, pH 7.4, 0.1 mM
dtpa) and the yield of disulfide was quantified by
subtraction of HSA-SH concentration before and
after treatment with LMW disulfides. HSA
preparations enriched in sulfenic acid (HSA-
SOH) were prepared by incubation of reduced
HSA (0.6 mM) with H2O2 (4 mM, 4 min, 37 ºC),
kept on ice and used daily. The concentration of
HSA-SOH was quantified by reaction with TNB
(24). Sulfanes in HSA were produced following
different procedures, depending on the
experiment. For most determinations, mixed
HSA disulfides were incubated with equimolar
Na2S in phosphate buffer (100 mM, pH 7.4, 0.1
mM dtpa, 25 ºC) for a specified period, followed
by gel-filtration. Persulfides for kinetic studies
toward 4,4’-dithiodipyridine (DTDPy) were
3

Kinetics of hydrogen sulfide reactions with 60,000 FWHM. Detection was performed in
disulfides by amperometric hydrogen sulfide negative-ion mode and the source voltage was
measurements. Na2S (10 µM) was injected into 4.0 kV. The flow rates were 180 µL/h. The
phosphate buffer (50 mM, pH 7.4) and its instrument was calibrated by direct infusion of
concentration was followed using a selective H2S the Agilent ESI-TOF low concentration tuning
electrode connected to a Free Radical Analyzer mixture, which provided an m/z range of singly
(World Precision Instruments). The electrode charged peaks up to 2700 Da in both ion modes.
response was continuously recorded. When it
Detection of HSA persulfide by ultra-high
resolution ESI-ToF MS. HSA-TNB was mixed
with equimolar Na2S for 15 min at room
temperature, desalted on Sephadex G10, mixed
with acetonitrile (1/1, v/v) containing 0.5 %
formic acid and continuously sprayed into
reached a maximum, different concentrations of
HSA disulfides were injected. Decay of H2S at 25
ºC followed first order kinetics and the kobs were
plotted versus concentration of the protein
derivative.
Kinetics of hydrogen sulfide reaction with the maXis5G MS. Detection was performed in
sulfenic acid in HSA using a competition positive-ion mode and the source voltage was 4.5
approach. The reaction was evaluated through kV. The flow rates were 180 µL/h. Spectra were
competition with TNB, using a procedure similar post-processed using Data Analysis software
to a previously reported one (24). Sulfenic acid- (Bruker Daltonics).
enriched HSA (~ 0.5 µM HSA-SOH) was mixed
Identification of the persulfidated Cys residue.
HSA-SSH prepared from HSA-TNB was
collected into vials containing 100 mM biotin-
maleimide to ensure the immediate blocking of
both, persulfides and eventually thiols. After 2 h
incubation at 37 ºC, the protein was desalted
again on Sephadex G-10 and mixed with
proteomics grade porcine trypsin (Sigma Aldrich,
1:100 w/w) overnight at 37 ºC. The reaction was
with excess TNB (66 µM) in phosphate buffer
(100 mM, pH 7.4, 0.1 mM dtpa) in the absence or
presence of Na2S (up to 100 µM). The decay of
TNB was recorded at 412 nm and fitted to
exponential plus straight line functions (24). The
second-order rate constant for the reaction of H2S
with HSA-SOH was calculated from the slopes of
plots of kobs versus Na2S concentration.
Sulfane quantification. The protein fraction was stopped by adjusting the pH to 3 and 100 µL of
separated by gel-filtration and sulfane sulfur was the peptide mixture was mixed with 100 µL
quantified by cold cyanolysis. Samples were Pierce™ Streptavidin Agarose beads (Thermo
incubated with cyanide and the thiocyanate Fischer Scientific). The unbound peptides were
formed was determined by the absorbance of the discarded and the beads washed three times with
complex formed with ferric ions at 460 nm (28).
phosphate buffer (50 mM, pH 7.4). The bound
peptides were those modified by biotin-
maleimide, but in the case of peptides initially
containing persulfide, the reaction with biotin-
maleimide creates a disulfide bond. These
peptides were eluted by addition of 100 µL of 10
mM DTT. After centrifugation, the eluted
peptides were analyzed by LC-MS. Reverse
phase high-pressure liquid chromatography
(Dionex, Thermo Fischer Scientific) was
Tag-switch assay. Labeling of persulfides in HSA
preparations (dot-blots) and fixed cells was
performed following an established protocol (19,
30).
2-(Methylsulfonyl)1,3-benzothiazole
(MSBT) was purchased from Santa Cruz
Biotechnology. CN-biotin was synthesized as
previously (19, 30).
Detection of HSA persulfide with tris(2,4-
performed using
a
C18 column and
a
dimethyl-5-sulfophenyl)phosphine
(TDMSP).
combination of water (solvent A) and acetonitrile
(solvent B), both containing 0.5 % formic acid.
The following elution protocol was used: 5 min
of 10 % B and then 45 min of continuous rise to
90 % B. In addition, the peptides were directly
injected into maXis5G using in-source collision-
induced dissociation tandem MS approach
(source voltage was 4.5 kV, source temperature
250 ºC, collision energy 90 eV).
HSA-SSH (20 µM) was prepared from HSA-
TNB by incubation with Na2S at 1:1.5 molar
ratio, purified on Sephadex G-10 and incubated
with a 20-fold excess of TDMSP at room
temperature for 30 min. The reaction mixture was
sprayed into ultra-high resolution ESI-ToF
Bruker Daltonik (Bremen, Germany) maXis5G,
an ESI-ToF MS capable of resolution of at least
4

Reactivity
of
HSA
persulfide
toward Corrector integrator algorithm (38), 50 points
peroxynitrite. Peroxynitrite was synthesized each side of the transition state with a step size of
asdescribed (31). Kinetic data were obtained by 0.05 Bohr. Thermochemistry at 298 K and 1 atm
recording time-resolved UV–vis spectra using a was assessed at the same level of theory,
modified μSFM-20 Bio-Logic stopped-flow applying usual approximations of statistical
module equipped with a J&M TIDAS high-speed thermodynamics and unscaled zero point
diode array spectrometer with combined vibrational energies, as implemented in the
deuterium and tungsten lamps (200–1015 nm Gaussian09 suite (35).
wavelength range). The syringes were controlled
Computational comparison of the global
reactivity and nucleophilicity of HS– and Cys
persulfide with respect to LMW thiolates. The
structures of five LMW thiolates of known
by separate drives, allowing the variation of the
ratio of mixing volumes used in the kinetic runs.
Data were analyzed in the Specfit/32™ program.
At least five kinetic runs were recorded for all
conditions. Final concentration of peroxynitrite
was 9 µM and the concentrations of HSA-SSH
and HSA-SH were in the range of 2-60 µM. The
yield of nitrotyrosine was determined by
measuring absorbance at 430 nm (ε = 4400 M-1
cm-1) after alkalinization to pH 10, 5 min after
mixing 183 µM protein with 500 µM
peroxynitrite in phosphate buffer (50 mM, pH
7.4).
basicity
(cysteinemethylester,
CysOMe;
cysteinylglycine, Cys-Gly; cysteine, Cys; -
mercaptoethanol, -ME; homocysteine, Hcy) as
well as cysteine persulfide anion (CysSS–) were
determined at the same level of theory in aqueous
solution as described above. The energy of the
highest occupied Kohn-Sham (KS) orbital,
EHOMO, was taken as an indicator of the
nucleophilicity
of
each
species.
The
corresponding global reactivity was quantified by
the chemical hardness (η), calculated under the
finite differences approximation with the
following equation (39):
Reactivity of HSA persulfide toward DTDPy. An
Applied Photophysics RX2000 stopped-flow
accessory
coupled
to
a
Varian
50
spectrophotometer was used. Preparations
enriched in HSA-SOH were incubated with
equimolar Na2S in one syringe and mixed at
increasing time points with excess DTDPy
contained in the other syringe, in phosphate
buffer (100 mM, pH 7.4, 0.1 mM dtpa) at 25 ºC.
The time course of thiopyridone appearance was
followed at 324 nm (ε = 21400 M-1 cm-1). The
traces were fitted to double exponential functions
to obtain the corresponding kobs.
η = (ELUMO – EHOMO)/2
(Eq. 1)
where ELUMO represents the energy of the lowest
unoccupied KS orbital.
Computational prediction of the pKa of cysteine
persulfide. Using the same level of theory
described above, a prediction of the pKa shift
(ΔpKa) corresponding to CysSSH with respect to
CysSH was obtained by applying the
proton-exchange method proposed by Yu et al.
Computational modeling of the reaction
mechanism of HS– with
a
representative
(40) with the calculated free-energy, ΔGexch
,
corresponding to the reaction:
symmetrical LMW disulfide (zwitterionic Cys-
Cys) in aqueous solution. The structures of the
reactant and product complexes and the
corresponding transition state including two
explicit water molecules were fully optimized
and verified by inspection of the eigenvalues of
the Hessian matrix at the IEFPCM/ωB97X-D/6-
31+G(d) (32–34) level of theory, as implemented
in Gaussian09 rev. D.01 (35). Water was also
described as a continuum solvent (ε = 78.5),
placing solutes in molecular-shaped cavities
constructed using Bondi’s radii (36). The
reaction path was followed by the intrinsic
reaction coordinate (37) calculated at the same
level of theory with the Hessian-based Predictor-
CysSSH + CysS- → CysSS- + CysSH (Eq. 2)
from which ΔpKa is obtained as follows:
ΔpKa = ΔGexch/(2.303 RT)
(Eq. 3)
Cell experiments. Human umbilical vein
endothelial cells (HUVEC) (PromoCell) were
grown in endothelial cell growth medium 2
(ready-to-use, PromoCell) on ibidi dishes (ibidi,
Martinsried, Germany) at 5 % CO2 and 20 %
oxygen until they reached > 50 % confluency.
Human neuroblastoma cells (SH-SY5Y, ECACC,
Sigma Aldrich) were grown in Ham’s
5

F12:EMEM (EBSS) (1:1) medium supplemented at alkaline pHs the predominant species is
with 2 mM glutamine, 1 % non-essential amino cystine2- and not the zwitterion, as in our
acids + 15 % fetal bovine serum FBS / FCS, on conditions. In addition, HS- may ionize to S2-. A
ibidi dishes. Inhibition of endogenous production change in the charge in the reacting species could
of H2S was performed by using oxamic acid (2 affect the reaction rate constant by two orders of
mM) in SH-SY5Y cultures or propargylglycine magnitude (43). Recently, Vasas et al (44)
(2 mM) in HUVEC cultures. Fluorescence kinetically characterized the reaction of H2S with
microscopy was carried out using Carl Zeiss DTNB. The second-order rate constant reported
Axiovert 40 CLF inverted microscope, equipped at pH 7.4 is 881 M-1 s-1, consistent with our
with green fluorescent filters and AxioCam determination (861 M-1 s-1, Table 1). They also
ICm1. Images were post processed in ImageJ report that the kinetics of reaction with cysteine
software.
and glutathione are significantly slower.
Data processing. Data were processed using Kinetics of reaction of hydrogen sulfide toward
OriginPro 8 (Microcal Software). Numerical asymmetric disulfides of HSA formed with LMW
fitting of data to reaction sequences was thiols. Second-order rate constant were obtained
performed with Gepasi (41).
for the reactions of H2S with several mixed
disulfides (Table 2) by measuring initial rates of
H2S consumption using the methylene blue
method (Fig. 2A). The values were in the order of
10-1-101 M-1 s-1 for the disulfides formed between
HSA and alkyl thiols. Controls were performed
using HSA-SH to rule out the reduction of
structural disulfides as well as other decay
pathways of H2S such as autooxidation. Rate
constants were also determined by following the
disappearance of H2S amperometrically, with
similar results (Fig. 2B).
RESULTS AND DISCUSSION
Kinetics of reaction of hydrogen sulfide with
symmetric LMW disulfides. In order to evaluate
the reaction of H2S toward LMW disulfides,
kinetic measurements were performed. Parallel
approaches were used in most cases. Under
pseudo-first order conditions with disulfides in
excess, exponential decays in H2S concentration
were observed (Fig. 1A). The reactions are likely
to reach equilibrium situations, but under our
conditions the consumption of H2S was close to
total, probably because of the excess disulfide
reagent and because of further reactions of the
products. The observed rate constants showed a
linear dependence on disulfide concentration
(Fig. 1B). The second-order rate constants for six
disulfides are summarized in Table 1. The values
were in the order of 10-2 to 100 M-1 s-1 for alkyl
disulfides. Since HS- is the species responsible
for reactivity, the rate constants determined at pH
7.4 were corrected to obtain the pH-independent
values, which represent the rate constants that
Mechanism of the reaction of hydrogen sulfide
toward disulfides. The data obtained for H2S can
be rationalized in the context of reported data for
thiol-disulfide interchange reactions. These
interchange reactions occur by single concerted
SN2 mechanisms where the reactive species
involved is the thiolate anion (45). When the pH-
independent rate constants are plotted against the
pKas of a variety of attacking thiols and target
disulfides, Brønsted correlations are obtained
according to the following equation:
would be measured if all the H2S was ionized to log k = βnuc pKanuc + βc pKac + βlg pKalg + C (Eq. 4)
HS-. Results obtained by initial rate processing or
where βnuc, βc, βlg are the Brønsted coefficients
for the nucleophilic sulfur, central sulfur and
leaving group sulfur, respectively. Analogously,
for the reaction studied herein of HS- with
symmetrical disulfides producing a thiolate
(leaving group) and a persulfide, the logarithm of
the pH-independent rate constants obtained show
a negative linear correlation with the pKa of the
leaving thiol (Fig. 3A), supporting a simple
concerted SN2 mechanism and indicating that
acidic thiols -with lower pKas- are better leaving
by UV-Vis absorbance measurements were
consistent with H2S consumption measurements.
As precedent, rate constants of (6.2  0.7) x 10-2
and 1.3 x 10-2 M-1 s-1 have been reported for the
reaction of cystine with excess H2S in 50 mM
borate buffer at pH 10.0 or NaOH 0.1 M,
respectively (42). These values are lower, by
factors of 15-70, than the pH-independent rate
constant determined in this work, (9  2) x 10-1
M-1 s-1. The difference can be explained because
6

groups. The slope of this curve represents the The effect of the pKa of the nucleophile on the
addition of the Brønsted coefficients of central reaction with a disulfide is evident from Fig. 5,
and leaving sulfurs (βc + βlg) and has a value of - where the pH-independent rate constants of thiol
0.75, consistent with the values reported for the disulfide interchange reactions for different
reaction with thiolates (45). In the case of mixed thiolates with DTNB or GSSG, taken from the
disulfides of HSA and LMW thiols, the slope of literature (45, 47), are compared with the value
the Brønsted plot was -0.6, reflecting βc or βlg obtained for HS-. Again, the intrinsic reactivity of
(Fig. 3B). The leaving group can be either HSA- HS- is lower than that of thiolates, by factors of
S- or RS-. In the absence of steric effects, the 20-25.
most acidic groups will be the preferred leaving
groups.
Although the intrinsic reactivity of HS- is lower
than that of thiolates, at pH 7.4 its availability is
Computational modeling. The structures of the higher. Indeed, the ratio HS-/H2S is 2.51 at pH
reactant complex, transition state and product 7.4 due to the low pKa of H2S, while the ratio
complex for the reaction mechanism of HS– with thiolate/thiol for cysteine and glutathione are 0.13
cystine -as a representative LMW disulfide- are and 0.03, respectively. The Brønsted correlation
shown in Fig. 4. The reaction exhibits a can be used to obtain a theoretical optimum pKa
concerted linear transition state (with an for maximizing the observed rate for reactions at
associated imaginary frequency of 233i cm–1) a certain pH (43).
consistent with a typical SN2 mechanism. The
(1-βnuc)/βnuc = 10(pH-pKa)
(Eq. 5)
explicit water molecules present in the model are
not directly involved in the reaction coordinate
but play an ancillary role stabilizing the
participant moieties through hydrogen bonding.
The Gibbs free-energy of reaction is predicted to
be of 13.4 kcal mol–1. Although formation of the
Considering a βnuc value of 0.38 (Fig. 5A), the
optimal pKa for the reaction at pH 7.4 is 7.2. This
means that at physiological pH the nucleophilic
capacity of H2S is maximally exploited. In other
words, although H2S is a relatively weak
nucleophile, its reactivity is maximized at
physiological pHs.
products
would
thus
appear
to
be
thermodynamically disfavored, fast acid base
equilibration of the products is expected to drive
the outcome of the process. Precisely, since
CysSH has a pKa of 8.29 and the predicted pKa
for CysSSH as obtained from Eq. 3 is 4.34 (ΔpKa
of -3.96, Table 3), the products at physiological
pH become CysSS- and CysSH, preventing the
reverse reaction. The activation Gibbs free-
energy is calculated to be of 19.9 kcal mol–1. This
value is comparable to the experimental one of
17.5 kcal mol–1 that can be derived from the rate
constant by applying the Eyring-Polanyi
equation, confirming the consistency between
theory and experiment in the description of the
reaction.
As shown by inspection of the calculated
descriptors
of
global
reactivity
and
nucleophilicity collected in Table 3, HS– is
harder and a weaker nucleophile than the
thiolates. The nucleophilicities (estimated
through the KS-HOMO energies, EHOMO, as a
measure of the facility of each species to donate
electrons) of the five thiolates linearly correlate
with the corresponding experimental pKa (Fig. 6)
mirroring the trend emerging from the
experimental data and showing that more basic
thiolates are better nucleophiles (Fig. 5). In
contrast, the nucleophilicity of HS– falls below
that of a thiolate of comparable basicity.
Comparison of nucleophilicities of hydrosulfide
and thiolates. The pH-independent rate constants
obtained for HS- reactions with the six symmetric
disulfides studied are about one order of
magnitude lower than those reported for alkyl
thiolates of comparable pKa (Fig. 3A). This
decreased nucleophilic character of HS- could be
due to the lack of the inductive effect of the
adjacent methylene of the sulfur, changes in the
polarizability of the sulfur or solvation effects (8,
46).
Detection of HSA persulfide. The formation of a
persulfide in HSA could not only represent a
potential physiological product, but could also be
a useful model to study its features, as in the
example of sulfenic acids (26). Using the method
of cold cyanolisis, the formation of sulfane
sulfur, a group of compounds that includes
persulfide, was evaluated. Sulfane sulfur was
detected in reaction mixtures of asymmetric HSA
7

disulfides with Na2S and coeluted with protein in Nevertheless, the mass differences are in
gel-filtration separations (Fig. 7A). agreement with the changes expected.
In addition, a selective method for persulfide Finally, we also proved that the actual site of
detection has been recently developed, the tag- HSA persulfidation is Cys34 (Figs. 9D, 9E and
switch assay, which labels protein persulfides 9F). To do so we designed an approach, adapted
with biotin (19, 30). HSA-SSH formed through from (5), in which HSA-SSH, generated in the
the reaction of several mixed disulfides with reaction between HSA-TNB and H2S, was mixed
Na2S were analyzed for the level of biotin with biotin-maleimide. This alkylated free thiols
labeling by dot-blot assay to confirm that indeed, and also reacted with persulfides forming a
in all tested cases, HSA-SSH is formed (Fig. 7B). mixed disulfide. Protein was then trypsinized and
Control experiments performed with the mixed streptavidin beads added into the peptide mixture.
disulfides in the absence of Na2S gave negative Only labeled peptides are expected to bind to the
results, with the only exception of HSA-TNB that streptavidin beads. After careful washout, the
showed a weak signal (data not shown).
peptide which originally contained persulfide was
eluted with DTT, which reduces the mixed
disulfide formed in the reaction with biotin-
maleimide (Fig. 9D). The peptide was then
analyzed by HPLC-MS. The positive-ion mode
analysis revealed the presence of the Cys34
peptide ALVLIAFAQYLQQCPFEDHVK as the
doubly charged species (Fig 9E). Subsequent
collision-induced dissociation analysis further
confirmed it (Fig. 9F).
Sulfane sulfur reacts with triphenylphosphine and
its derivatives to give the corresponding sulfide.
This reaction has been recently used as a basis for
a new method for sulfane sulfur quantification
(48). We used a water soluble phosphine,
TDMSP, to prove HSA-SSH formation. HSA-
SSH prepared from HSA-TNB incubation with
Na2S was treated with TDMSP and analyzed by
negative-ion mode ultra-high resolution ESI-TOF
MS. The spectrum of the reaction mixture (Figs. Kinetics of reaction of hydrogen sulfide with the
8A and B, for two different m/z ranges) suggested sulfenic acid of HSA. The reaction of H2S toward
formation of TDMSP sulfide (TDMSP=S). sulfenic acid provides an alternative way to form
Namely, although the strongest signals were persulfide. To kinetically evaluate this reaction in
assigned to unreacted TDMSP, flying as HSA, an experimental design involving competi-
[TDMSP + Na]2- (Fig. 8C) and [TDMSP + 2Na]- tion between H2S and TNB for HSA-SOH was
(Fig. 8D), the presence of TDMSP=S with one used. The kobs of TNB consumption increased and
sodium (Fig. 8E) or two sodium cations (Fig. 8F) the amplitude decreased with Na2S concentration
was also observed. The peak intensity was ~4.5 (Figs. 10A and 10B). From the slope obtained
%
matching
nicely
the
assumed (Eq. 6), we could calculate a rate constant of (2.7
± 0.8) x 102 M-1 s-1 for the reaction of H2S with
HSA-SOH at pH 7.4 and a pH-independent rate
TDMSP/persulfide ratio.
constant of (4 ± 1) x 102 M-1 s-1 (assuming that all
HSA-SOH was protonated).
HSA-SSH was also analyzed by MS before and
after the equimolar addition of Na2S to HSA-
TNB. An obvious shift toward lower m/z could
be observed in the sample of HSA-TNB treated
with H2S (Figs. 9A and 9B) suggesting the loss
of the TNB moiety. Spectral deconvolution
confirmed this as shown in Fig 9C. The
appearance of a new peak corresponded well to
the loss of the TNB moiety and the addition of
sulfur, i.e. formation of HSA-SSH. In addition, a
mass change consistent with the formation of
kobs = kTNB [TNB] + kH2S [H2S]
(Eq. 6)
This is to our knowledge the first kinetic report
regarding the reactivity of H2S toward a sulfenic
acid. In contrast to the trend observed for LMW
disulfides (Figs 3 and 5), the pH-independent rate
constant of HS- reaction with HSA-SOH is higher
than those of Cys, GSH and TNB (1.1, 1.0 and
1.9 x 102 M-1 s-1, respectively, (24) and similar to
Hcy (4.8 x 102 M-1 s-1), which pKa is two units
higher. This difference can be explained by steric
hindrance, since Cys34 is partially buried in the
protein surface and HS- could have easier access
than thiolates.
-
HSA-SSO3 was observed, in accordance with
previous observations on bovine serum albumin
and glutathione peroxidase (18, 19). The mass of
the protein was higher than expected, probably
because of the presence of sodium ions.
8

Reactivity of hydrogen sulfide toward disulfides (Fig. 12). This value is likely an underestimation
and sulfenic acids in cellular context. Oxidized due to HSA-SSH instability and to the difficulty
thiols that could constitute molecular targets for in exactly knowing its concentration. For
H2S (i.e. disulfides and sulfenic acids) are scarce comparison, HSA-SH reacted more slowly with
in the cytoplasmic environment. Accordingly, the peroxynitrite, (2.7 ± 0.3) x 103 M-1 s-1, under the
majority of protein persulfides has been found to same conditions, confirming the higher reactivity
be located in the endoplasmic reticulum, an of the persulfide with respect to the thiol. It is
organelle rich in protein oxidized thiols (19). The worth mentioning that along with the increased
influence of oxidative conditions could increase kinetics for the reaction with peroxynitrite, we
the probability of inducing the formation of observed decreased nitrotyrosine levels (0.041 vs
persulfides in cells. We addressed this possibility 0.071 moles nitrotyrosine/mol protein for HSA-
by treating cells in culture with H2O2, with or SSH vs HSA-SH, respectively).
without the prior inhibition of the intracellular
In order to improve the quantitative assessment
of the nucleophilicity of HSA-SSH, we then
evaluated its reactivity toward DTDPy. The use
of the disulfide DTDPy as target presents
advantages: it has high intrinsic reactivity, the
reaction can be followed through thiopyridone
absorbance, and it can be used in pseudo first-
order excess, eliminating the need for accurately
knowing the concentration of the persulfide.
When equimolar amounts of HSA-SOH and
Na2S were incubated for a certain time and
aliquots mixed with excess DTDPy in a stopped-
flow device, an initial fast increment in
thiopyridone absorbance was detected (Fig. 13A).
Controls using HSA-SH and HSA-SOH
preparations in the absence of H2S also showed
an absorbance increase but lacked the first fast
phase. For the latter, the absorbance increase
production of H2S. We used the tag-switch assay
to detect persulfide levels in HUVEC and SH-
SY5Y cells (Fig. 11). In both cell lines the
treatment with H2O2 led to a significant increase
in the total persulfide levels as evidenced by
fluorescence microscopy. More importantly, we
assessed the effect of the inhibition of the
endogenous production of H2S. For HUVEC,
propargylglycine was used, a relatively specific
inhibitor of cystathionine γ-lyase. For SH-SY5Y
cells, oxamic acid was used, which inhibits both
cystathionine β-synthase and cystathionine γ-
lyase, as shown recently (49). Inhibition of
endogenous H2S production abolished H2O2-
induced effects suggesting that the reaction of
H2S with oxidized thiols could be an important
source of endogenous persulfides (Fig. 11).
Kinetics of reaction of the persulfide of HSA likely represented remnant thiols. The observed
toward electrophiles. Both, reactions of H2S with rate constants were linearly dependent on DTDPy
disulfides and with sulfenic acids yield concentration, and a second-order rate constant of
persulfides. These products are likely to have (1.7 ± 0.1) x 104 M-1 s-1 for HSA-SSH was
enhanced nucleophilic reactivity with respect to determined (pH 7.4, 25 ºC), higher than those
thiolates. Several publications suggest that observed
for
HSA-SH
or
HSA-SOH
persulfides are better nucleophiles than thiols, but preparations: (7.6 ± 0.4) x 102 M-1 s-1 and (6.4 ±
so far there are no quantitative kinetic analysis 0.1) x 102 M-1 s-1, respectively (Fig. 13B). In
reported (13, 17).
addition, an increase in the amplitude of the
initial fast phase with time was observed,
indicative of the time course of HSA-SSH
formation (Figs. 13C-D). The rate constant of the
reaction between HSA-SOH and H2S was
estimated from the fit of the amplitude versus
We first studied the reactivity of HSA-SSH
toward peroxynitrite (ONOO-/ONOOH), the
product of the reaction of nitric oxide and
superoxide radicals. For these experiments, the
persulfide concentration was judged based on the
yield of TNB released form HSA-TNB after it
reacted with Na2S and prior to desalting. An
initial rate approach was used to analyze the
kinetic traces obtained (50), since a pseudo-first
order excess of HSA-SSH could not be reached.
The initial rate increased linearly with the
assumed concentration of HSA-SSH, leading to a
rate constant of (1.2 ± 0.4) x 104 M-1 s-1 at 20 ºC
incubation time plot using
a
Levenberg-
Marquardt algorithm included in Gepasi,
considering also the spontaneous decay of HSA-
SOH (5.6 x 10-4 s-1 at 25 ºC (23)) (Fig. 13D). A
value of (4.7 ± 0.2) x 102 M-1 s-1 was obtained,
consistent with that determined by competition
with TNB.
9

The evaluation of nucleophilicity by means of and that methemoglobin in red blood cells can
DTDPy demonstrates twenty-fold increased participate in its clearance. Recently,
reactivity of the persulfide at pH 7.4 in consumption rates for H2S by methemoglobin
comparison to thiol. The pKa of HSA-SSH is consistent with a half-life of 4 min in blood were
likely to be several units lower than the original reported (55). This value is within one order of
HSA-SH (Table 3) resulting in an estimated pH- magnitude of that ascribed to disulfides. Thus,
independent rate constant close to the value variations in the levels of methemoglobin or
determined at pH 7.4, 2 x 104 M-1 s-1. On the
disulfides could produce changes in the relative
contributions of disulfides and red blood cells to
H2S decay rate.
other hand, the pH-independent rate constant for
HSA-SH can be calculated considering the pKa of
8.1, as 7 x 103 M-1 s-1. This value is lower than
that determined for HSA-SSH by a factor of ~3.
The enhanced reactivity of a nucleophilic atom
when it is adjacent to an atom containing one or
more unshared pairs of electrons is a classic
observation, referred to as the alpha effect (7, 8).
For another reaction, that of different
nucleophiles with the ester p-nitrophenyl acetate,
the alpha effect is associated with increases in
reactivity by factors of 10-100 (7, 51). By
comparison, the extent of the effect in our case is
more modest. The higher reactivity of HSA-SSH
with respect to HSA-SH is consistent with the
computational calculations performed for
cysteine persulfide, that show a higher intrinsic
nucleophilicity than the corresponding thiol
(increased KS-HOMO energy) as well as
decreased chemical hardness (Table 3 and Fig.
6). The latter could confer preferential reactivity
toward soft electrophiles such as heavy metal
cations (52), alkyl halides and Michael
electrophiles (18). Our study is, to our
knowledge, the first evaluation of the magnitude
of the alpha effect for a persulfide.
Targets in the extravascular milieu can also be
involved in H2S clearance. The high permeability
of membranes allows mitochondria to become a
sink for H2S, limiting its half-life in the cell.
Formation of persulfides by direct reaction of
H2S with disulfides and sulfenic acids is probably
under kinetic competition with mitochondrial
consumption. In this regard, an interesting
scenario could be set in the nervous system,
where H2S scavenging by mitochondria is
inefficient due to the lack of sulfide:quinone
oxidoreductase activity (56), the enzyme
responsible for the first step of the mitochondrial
catabolism of H2S, and effects of endogenous
H2S on non-mitochondrial persulfide generation
are probably maximized.
In the cytoplasmic environment, the reaction of
H2S with LMW and typical protein disulfides or
sulfenic acids is kinetically challenged due to the
low concentration of oxidized thiols and the
relatively low rate constants, in the context of
high concentrations of reduced thiols that might
compete with H2S. Thus, situations that lead to
increased steady state levels of oxidized thiols
increase persulfide formation. Based in our
experiments with cells in culture treated with
H2O2, the reaction of H2S with oxidized thiols is
a plausible pathway for persulfide production in
cytoplasmic and reticulum environments in
response to oxidative signals. In addition to the
direct reaction of H2S with oxidized thiols,
alternative pathways for persulfide formation in
vivo can involve the consumption of cystine by
cystathionine β-synthase or cystathionine γ-lyase
(13). Furthermore, proteins that participate in
Biological implications. Consistent with the
limited intrinsic nucleophilicity of H2S, the rate
constants of the reactions with LMW and HSA
disulfides are relatively low. In order to
extrapolate how these reactions could contribute
to H2S decay, factors of rate constant times
concentration together with competing pathways
and compartmentalization aspects need to be
taken into account.
In the intravascular space, a compartment where
disulfides are abundant, factors of rate constant
times concentration can be evaluated considering
the total concentration levels of LMW and HSA
disulfides (~ 250 µM (23)) and the rate constants
now known (Tables 1 and 2). Thus, a global half-
life of 26 min can be estimated for this process in
plasma. However, from previous work (53, 54) it
is known that H2S can traverse membranes easily
H2S
oxidation
pathways
can
generate
intermediate persulfides that can subsequently be
transferred to other targets (57).
It is important to bear in mind that the rate
constants of H2S reactions can be accelerated in
the case of particular protein contexts. Proof of
10

concept for the potential acceleration of the - The intrinsic reactivity observed for HS- toward
reaction of H2S with a disulfide by the protein disulfides is lower than that reported for thiolates,
environment is provided by sulfide:quinone probably due to the absence of the inductive
oxidoreductase. This flavoprotein catalyzes the effect of the methylene adjacent to the sulfur
oxidation of H2S to sulfane sulfur at the expense atom, increased chemical hardness or solvation.
of ubiquinone. The process starts with the - Hydrogen sulfide is able to react with the
reaction of H2S with a critical protein disulfide, sulfenic acid in HSA, proving another pathway
and values of kcat/KM,sulfide of 107 M-1 s-1 (58, 59) for persulfide formation.
demonstrate an extraordinary acceleration with - Experiments with cells in culture indicate that
respect to free cystine (0.6 M-1 s-1). Likewise, persulfide formation increases upon exposure to
wide variations in rate constants are expected for H2O2, consistent with the proposed processes.
the reactions of H2S with different protein - Kinetic determinations evidence that the
sulfenic acids. To achieve specificity, one aspect persulfide has increased nucleophilic reactivity
that H2S could invoke, as suggested by the case than the parent thiol, as expected from the alpha
of HSA-SOH, is a steric advantage for reaction effect.
with constrained disulfides and sulfenic acids due
Overall, our study sets the stage for
understanding the reactivity of H2S toward
oxidized thiol derivatives and the possible
contribution of persulfides to the biological
effects of this novel endogenous modulator.
to its small size in comparison to other cellular
nucleophiles. In fact, from the point of view of
biological function, the low rate constants of the
uncatalyzed processes constitute actually an
asset, opening the way to kinetic control.
ACKNOWLEDGMENTS
Persulfides are promising candidates to constitute
intermediate species in the modulation of cellular
functions by H2S. The levels of persulfides are so
far hard to evaluate in biological samples due to
the instability of these compounds and to the lack
of rapid and selective analytical methods, which
are still under development. Persulfides have
been proposed to protect the original thiol from
irreversible oxidative modifications (60). They
have been reported to participate in the synthesis
of sulfur-containing molecules (11) and to be
involved in enzymatic catalysis and regulation
(6). The twenty-fold increased reactivity of the
HSA persulfide relative to the HSA thiol at pH
7.4 underscores the improved nucleophilicity of
these derivatives with respect to the parent thiol
and to H2S. As functions for persulfides in
biological systems continue to be unraveled, this
increased nucleophilicity is likely to play a key
role in explaining the underlying molecular
mechanisms.
We thank Matías N. Möller, Laura Antmann,
Lucía Turell, Martina Steglich (Universidad de la
República, Uruguay) and Jan Miljkovic
(University of Erlangen–Nuremberg, Germany)
for helpful discussions and technical assistance.
The authors are grateful to professor Ivana
Ivanovic-Burmazovic (FAU) for the use of the
equipment.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of
interest with the contents of this article.
AUTHOR CONTRIBUTIONS
E.C. designed, performed and analyzed
experiments and wrote the manuscript. J.B. and
E.L.C. designed, performed and analized the
computational experiments, and wrote the
corresponding parts. G.F.S. helped design and
analyze experiments and edited the manuscript.
M.R.F. designed, performed and analyzed mass
spectrometry, tag-switch, amperometric kinetics,
peroxynitrite and cell experiments with the
assistance of M.L., and co-wrote the manuscript.
B.A. conceived, designed and analyzed
experiments and wrote the manuscript. All
authors approved the final version.
CONCLUSIONS
Our study reveals several aspects relative to the
reactivity of H2S, summarized as follows:
- Hydrogen sulfide reacts with LMW and mixed
protein disulfides to produce persulfides.
- The reaction occurs through a concerted
mechanism, similar to the thiol-disulfide
interchange reaction.
11

REFERENCES
1.
Kabil, O., and Banerjee, R. (2010) Redox biochemistry of hydrogen sulfide. J. Biol. Chem. 285,
21903–7
2.
3.
Cotton, F., and Wilkinson, G. (1988) Advanced Inorganic Chemistry, New York
Paulsen, C. E., and Carroll, K. S. (2013) Cysteine-mediated redox signaling: chemistry, biology,
and tools for discovery. Chem. Rev. 113, 4633–4679
4.
5.
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
Sen, N., Paul, B. D., Gadalla, M. M., Mustafa, A. K., Sen, T., Xu, R., Kim, S., and Snyder, S. H.
(2012) Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions. Mol.
Cell. 45, 13–24
6.
7.
8.
9.
Toohey, J. I. (1989) Sulphane sulphur in biological systems: a possible regulatory role. Biochem.
J. 264, 625–632
Jencks, W. P., and Carriuolo, J. (1960) Reactivity of nucleophilic reagents toward esters. J. Am.
Chem. Soc. 82, 1778–1786
Edwards, J. O., and Pearson, R. G. (1962) The factors determining nucleophilic reactivities. J.
Am. Chem. Soc. 84, 16–24
Toohey, J. I. (2011) Sulfur signaling: is the agent sulfide or sulfane? Anal. Biochem. 413, 1–7
10. Olson, K. R. (2009) Is hydrogen sulfide a circulating “gasotransmitter” in vertebrate blood?
Biochim. Biophys. Acta. 1787, 856–863
11. Wright, C. M., Christman, G. D., Snellinger, A. M., Johnston, M. V., and Mueller, E. G. (2006)
Direct evidence for enzyme persulfide and disulfide intermediates during 4-thiouridine
biosynthesis. Chem. Commun. (Camb.). (29), 3104–310610
12. Mueller, E. G. (2006) Trafficking in persulfides: delivering sulfur in biosynthetic pathways. Nat.
Chem. Biol. 2, 185–194
13. Ida, T., Sawa, T., Ihara, H., Tsuchiya, Y., Watanabe, Y., Kumagai, Y., Suematsu, M.,
Motohashi, H., Fujii, S., Matsunaga, T., Yamamoto, M., Ono, K., Devarie-Baez, N. O., Xian,
M., Fukuto, J. M., and Akaike, T. (2014) Reactive cysteine persulfides and S-polythiolation
regulate oxidative stress and redox signaling. Proc. Natl. Acad. Sci. U.S.A. 111, 7606–7611
14. Hildebrandt, T. M., and Grieshaber, M. K. (2008) Three enzymatic activities catalyze the
oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria. Febs J. 275,
3352–61
15. Kabil, O., and Banerjee, R. (2012) Characterization of patient mutations in human persulfide
dioxygenase (ETHE1) involved in H2S catabolism. J. Biol. Chem. 287, 44561–44567
16. Ubuka, T. (2002) Assay methods and biological roles of labile sulfur in animal tissues. J
Chromatogr. B Analyt. Technol. Biomed. Life Sci. 781, 227–49
17. Francoleon, N. E., Carrington, S. J., and Fukuto, J. M. (2011) The reaction of H(2)S with
oxidized thiols: generation of persulfides and implications to H(2)S biology. Arch. Biochem.
Biophys. 516, 146–153
18. Pan, J., and Carroll, K. S. (2013) Persulfide reactivity in the detection of protein S-sulfhydration.
ACS Chem. Biol. 8, 1110–1106
19. Zhang, D., Macinkovic, I., Devarie-Baez, N. O., Pan, J., Park, C.-M., Carroll, K. S., Filipovic,
M. R., and Xian, M. (2014) Detection of protein S-sulfhydration by a tag-switch technique.
Angew. Chem. Int. Ed. Engl. 53, 575–581
20. Nagahara, N., Nirasawa, T., Yoshii, T., and Niimura, Y. (2012) Is novel signal transducer sulfur
oxide involved in the redox cycle of persulfide at the catalytic site cysteine in a stable reaction
intermediate of mercaptopyruvate sulfurtransferase? Antioxid. Redox Signal. 16, 747–753
21. Chatterji, T., Keerthi, K., and Gates, K. S. (2005) Generation of reactive oxygen species by a
persulfide (BnSSH). Bioorg. Med. Chem. Lett. 15, 3921–3924
22. Bailey, T. S., Zakharov, L. N., and Pluth, M. D. (2014) Understanding hydrogen sulfide storage:
probing conditions for sulfide release from hydrodisulfides. J. Am. Chem. Soc. 136, 10573–
10576
12

23. Turell, L., Radi, R., and Alvarez, B. (2013) The thiol pool in human plasma: the central
contribution of albumin to redox processes. Free Radic. Biol. Med. 65, 244–253
24. Turell, L., Botti, H., Carballal, S., Ferrer-Sueta, G., Souza, J. M., Durán, R., Freeman, B. A.,
Radi, R., and Alvarez, B. (2008) Reactivity of sulfenic acid in human serum albumin.
Biochemistry. 47, 358–367
25. Torres, M. J., Turell, L., Botti, H., Antmann, L., Carballal, S., Ferrer-Sueta, G., Radi, R., and
Alvarez, B. (2012) Modulation of the reactivity of the thiol of human serum albumin and its
sulfenic derivative by fatty acids. Arch. Biochem. Biophys. 521, 102–110
26. Alvarez, B., Carballal, S., Turell, L., and Radi, R. (2010) Formation and reactions of sulfenic
acid in human serum albumin. Meth. Enzymol. 473, 117–136
27. Iversen, R., Andersen, P. A., Jensen, K. S., Winther, J. R., and Sigurskjold, B. W. (2010) Thiol-
disulfide exchange between glutaredoxin and glutathione. Biochemistry. 49, 810–820
28. Wood, J. L. (1987) Sulfane sulfur. Meth. Enzymol. 143, 25–29
29. Carballal, S., Trujillo, M., Cuevasanta, E., Bartesaghi, S., Möller, M. N., Folkes, L. K., García-
Bereguiaín, M. A., Gutiérrez-Merino, C., Wardman, P., Denicola, A., Radi, R., and Alvarez, B.
(2011) Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological
interest. Free Radic. Biol. Med. 50, 196–205
30. Park, C.-M., Macinkovic, I., Filipovic, M. R., and Xian, M. (2015) Use of the “tag-switch”
method for the detection of protein S-sulfhydration. Meth. Enzymol. 555, 39–56
31. Saha, A., Goldstein, S., Cabelli, D., and Czapski, G. (1998) Determination of optimal conditions
for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite. Free Radic.
Biol. Med. 24, 653–9
32. Chai, J.-D., and Head-Gordon, M. (2008) Long-range corrected hybrid density functionals with
damped atom-atom dispersion corrections. Phys. Chem. Chem. Phys. 10, 6615–6620
33. Tomasi, J., Mennucci, B., and Cancès, E. (1999) The IEF version of the PCM solvation method:
an overview of a new method addressed to study molecular solutes at the QM ab initio level. J.
Mol. Struct.: Theochem. 464, 211–226
34. Ditchfield, R., Hehre, W. J., and Pople, J. A. (1971) Self‐consistent molecular‐orbital methods.
IX. An extended Gaussian‐type basis for molecular‐orbital studies of organic molecules. J.
Chem. Phys. 54, 724–728
35. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G., Robb, M. A., Cheeseman, J. R.,
Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li, X.,
Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G., Sonnenberg, J. ., Hada, M., Ehara, M.,
Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H.,
Vreven, T., Montgomery, J. A., Peralta, J. E., Ogliaro, F., Bearpark, M., Heyd, J. J., Brothers, E.,
Kudin, K. N., Staroverov, V. N., Keith, T., Kobayashi, R., Normand, J., Raghavachari, K.,
Rendell, A., Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene,
M., Knox, J. E., Cross, J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.
E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L.,
Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S.,
Daniels, A. D., Farkas, O., Foresman, J. B., Ortiz, J. V., Cioslowski, J., and Fox, D. J. (2013)
Gaussian 09, Revision D.01 ed., Gaussian Inc.; Wallingford, CT
36. Bondi, A. (1964) van der Waals volumes and radii. J. Phys. Chem. 68, 441–451
37. Fukui, K. (1981) The path of chemical reactions - the IRC approach. Acc. Chem. Res. 14, 363–
368
38. Hratchian, H. P., and Schlegel, H. B. (2005) Using Hessian updating to increase the efficiency of
a Hessian based predictor-corrector reaction path following method. J. Chem. Theory Comput. 1,
61–69
39. Geerlings, P., Fias, S., Boisdenghien, Z., and De Proft, F. (2014) Conceptual DFT: chemistry
from the linear response function. Chem. Soc. Rev. 43, 4989–5008
40. Yu, H.-Z., Yang, Y.-M., Zhang, L., Dang, Z.-M., and Hu, G.-H. (2014) Quantum-chemical
predictions of pKa’s of thiols in DMSO. J. Phys. Chem. A. 118, 606–622
41. Mendes, P. (1993) GEPASI: a software package for modelling the dynamics, steady states and
control of biochemical and other systems. Comput. Appl. Biosci. 9, 563–71.
13

42. Liu, D. K., and Chang, S. G. (1987) Kinetic study of the reaction between cystine and sulfide in
alkaline solutions. Can. J. Chem. 65, 770–774
43. Singh, R., and Whitesides, G. M. (1993) Thiol—disulfide interchange. in Sulphur-Containing
Functional Groups (1993), pp. 633–658, John Wiley & Sons, Inc.
44. Vasas, A., Dóka, É., Fábián, I., and Nagy, P. (2015) Kinetic and thermodynamic studies on the
disulfide-bond reducing potential of hydrogen sulfide. Nitric Oxide. 46, 93–101
45. Szajewski, R. P., and Whitesides, G. M. (1980) Rate constants and equilibrium constants for
thiol-disulfide interchange reactions involving oxidized glutathione. J. Am. Chem. Soc. 102,
2011–2026
46. Bunnett, J. F. (1963) Nucleophilic Reactivity. Annu. Rev. Phys. Chem. 14, 271–290
47. Wilson, J. M., Bayer, R. J., and Hupe, D. J. (1977) Structure-reactivity correlations for the thiol-
disulfide interchange reaction. J. Am. Chem. Soc. 99, 7922–7926
48. Liu, C., Zhang, F., Munske, G., Zhang, H., and Xian, M. (2014) Isotope dilution mass
spectrometry for the quantification of sulfane sulfurs. Free Radic. Biol. Med. 76, 200–207
49. Asimakopoulou, A., Panopoulos, P., Chasapis, C. T., Coletta, C., Zhou, Z., Cirino, G., Giannis,
A., Szabo, C., Spyroulias, G. A., and Papapetropoulos, A. (2013) Selectivity of commonly used
pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE).
Br. J. Pharmacol. 169, 922–932
50. Alvarez, B., Ferrer-Sueta, G., Freeman, B. A., and Radi, R. (1999) Kinetics of peroxynitrite
reaction with amino acids and human serum albumin. J. Biol. Chem. 274, 842–8
51. Frey, P. A., and Hegeman, A. D. (2007) Enzymatic reaction mechanisms, Oxford University
Press, USA
52. Abiko, Y., Yoshida, E., Ishii, I., Fukuto, J. M., Akaike, T., and Kumagai, Y. (2015) Involvement
of reactive persulfides in biological bismethylmercury sulfide formation. Chem. Res. Toxicol. 28,
1301–1306
53. Cuevasanta, E., Denicola, A., Alvarez, B., and Möller, M. N. (2012) Solubility and permeation
of hydrogen sulfide in lipid membranes. PLoS ONE. 7, e34562
54. Mathai, J. C., Missner, A., Kugler, P., Saparov, S. M., Zeidel, M. L., Lee, J. K., and Pohl, P.
(2009) No facilitator required for membrane transport of hydrogen sulfide. Proc. Natl. Acad. Sci.
U S A. 106, 16633–8
55. Vitvitsky, V., Yadav, P. K., Kurthen, A., and Banerjee, R. (2015) Sulfide oxidation by a
noncanonical pathway in red blood cells generates thiosulfate and polysulfides. J. Biol. Chem.
290, 8310–8320
56. Lagoutte, E., Mimoun, S., Andriamihaja, M., Chaumontet, C., Blachier, F., and Bouillaud, F.
(2010) Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse
electron transfer in colonocytes. Biochim. Biophys. Acta. 1797, 1500–1511
57. Mishanina, T. V., Libiad, M., and Banerjee, R. (2015) Biogenesis of reactive sulfur species for
signaling by hydrogen sulfide oxidation pathways. Nat. Chem. Biol. 11, 457–464
58. Jackson, M. R., Melideo, S. L., and Jorns, M. S. (2012) Human sulfide:quinone oxidoreductase
catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite.
Biochemistry. 51, 6804–6815
59. Libiad, M., Yadav, P. K., Vitvitsky, V., Martinov, M., and Banerjee, R. (2014) Organization of
the human mitochondrial hydrogen sulfide oxidation pathway. J. Biol. Chem. 289, 30901–30910
60. Ono, K., Akaike, T., Sawa, T., Kumagai, Y., Wink, D. A., Tantillo, D. J., Hobbs, A. J., Nagy, P.,
Xian, M., Lin, J., and Fukuto, J. M. (2014) Redox chemistry and chemical biology of H2S,
hydropersulfides, and derived species: implications of their possible biological activity and
utility. Free Radic. Biol. Med. 77, 82–94
61. Creighton, T. E. (1975) Interactions between cysteine residues as probes of protein
conformation: the disulphide bond between Cys-14 and Cys-38 of the pancreatic trypsin
inhibitor. J. Mol. Biol. 96, 767–776
62. Whitesides, G. M., Lilburn, J. E., and Szajewski, R. P. (1977) Rates of thiol-disulfide
interchange reactions between mono- and dithiols and Ellman’s reagent. J. Org. Chem. 42, 332–
338
14

63. Keire, D. A., Strauss, E., Guo, W., Noszal, B., and Rabenstein, D. L. (1992) Kinetics and
equilibria of thiol/disulfide interchange reactions of selected biological thiols and related
molecules with oxidized glutathione. J. Org. Chem. 57, 123–127
64. Portillo-Ledesma, S., Sardi, F., Manta, B., Tourn, M. V., Clippe, A., Knoops, B., Alvarez, B.,
Coitino, E. L., and Ferrer-Sueta, G. (2014) Deconstructing the catalytic efficiency of
peroxiredoxin-5 peroxidatic cysteine. Biochemistry 53, 6113-6125.
15

FOOTNOTES
† This work was financed by grants from CSIC (Universidad de la República), and L'Oréal–UNESCO,
Uruguay (to B.A.). E.C. and J.B. were supported by fellowships from the Agencia Nacional de
Investigación e Innovación and by PEDECIBA, Uruguay. M.L. and M.R.F. acknowledge the support
from the Emerging Field Initiative intramural grant form FAU Erlangen-Nuremberg (MRIC).
a
The mixture of H2S and HS– present in aqueous solution according to the working pH is referred as
‘H2S’, unless otherwise specified. The IUPAC-recommended names are sulfane and hydrogen sulfide
for H2S, and sulfanide and hydrogen(sulfide)(1-) for hydrosulfide anion, HS-.
b
The term ‘persulfides’ is used in this text for the mixture of RSSH (hydrodisulfide derivative) and
RSS- (disulfanidyl derivative) in solution according to the working pH, unless otherwise specified. The
IUPAC-recommended names for RSSH are disulfanyl and dithiohydroperoxide.
c The abbreviations used are: HSA, human serum albumin; LMW, low molecular weight; DTNB, 5,5'-
dithiobis-(2-nitrobenzoic acid); dtpa, diethylenetriaminepentaacetic acid; TNB, 5-thio-2-nitrobenzoic
acid; GSSG, glutathione disulfide; GSH, glutathione; Cys-Cys, cysteine disulfide; Cys, cysteine;
CysOMe-CysOMe, cystinedimethylester; CysOMe, cysteinemethylester; HED, hydroxyethyldisulfide;
β-ME, β-mercaptoethanol; Hcy-Hcy, homocystine; Hcy, homocysteine; DTDPy, 4,4’-dithiodipyridine;
DTT, dithiothreitol; TDMSP, tris(2,4-dimethyl-5-sulfophenyl)phosphine.
16

FIGURE LEGENDS
Figure 1. Kinetics of hydrogen sulfide consumption in the presence of glutathione disulfide. A)
Na2S (250 µM) was mixed with 2.0, 5.25 and 10.5 mM GSSG in phosphate buffer (100 mM, pH 7.4,
0.1 mM dtpa, 25 ºC), in closed vials. Aliquots were analyzed for H2S content with the methylene blue
method. B) Observed rate constants for the reaction of HS- with GSSG obtained from the fitting of
time courses to single exponential equations.
Figure 2. Kinetics of reaction of hydrogen sulfide with HSA mixed disulfides. A) Na2S (476 µM)
was incubated with HSA-Cys (470 µM, black circles). Controls were prepared using Na2S alone (476
µM, red squares) or Na2S (118 µM) with HSA-SH (439 µM, blue diamonds) in tris buffer (0.2 M, pH
7.4, 0.1 mM dtpa, 25 ºC). Aliquots were analyzed for H2S content with the methylene blue method. B)
Kinetics of H2S decay in the presence of HSA-TNB, measured by selective H2S electrode. Inset:
original traces of H2S decay upon addition of 34.5 µM (black line), 51.7 µM (red line), 172.4 µM
(green line) and 344.7 µM (blue line) HSA-TNB into 10 µM Na2S in phosphate buffer (50 mM, pH
7.4, 25 ºC).
Figure 3. Comparison of the reactivities of HS- and thiolates toward disulfides depending on the
pKa of the leaving thiol. A) pH-independent rate constants for the reactions of LMW disulfides with
HS- (this work) or with LMW thiolates (data reported in (45, 61, 47, 62)). B) pH-independent rate
constants of the reactions of HS- with mixed HSA disulfides.
Figure 4. Computational modeling of the reaction of hydrosulfide toward cystine. DFT optimized
structures in water of the reactant complex, transition state and product complex for the reaction
between cystine and HS-. Values in parentheses correspond to relative Gibbs free-energies at 298 K
calculated respect to RC.
Figure 5. Comparison of the reactivities of HS- and thiolates toward disulfides depending on the
pKa of the attacking thiol. A) DTNB as electrophile. pH-independent rate constants for the reactions
of HS- (this work) or thiolates (45, 47, 62) with DTNB. Data are grouped for thiocarboxyls (green
squares), aryl thiols (red circles), alkyl thiols (blue triangles) and H2S (black circle). Slopes (βnuc) are
0.30, 0.38 and 0.46 for attacking thiocarboxyles, thioaryles and thioalkyls, respectively. B) GSSG as
electrophile. pH-independent rate constants for alkyl thiols (blue triangles, (45, 61, 63)) and H2S
(black circle, this work).
Figure 6. KS-HOMO energies for thiolates, HS– and CysSS– calculated at the IEFPCM/ωB97X-
D/6-31+G(d) level of theory as a function of pKa of conjugated acid.
Figure 7. Product characterization. A) Detection of sulfane sulfur. HSA-Hcy (350 µM) was
incubated with Na2S (350 µM) during 3 hours. The reaction mixture was gel-filtrated using a PD-10
column and sulfanes were quantified in elution fractions by cold cyanolisis. B) Dot-blot detection of
HSA-SSH using the tag-switch assay. Different mixed disulfides of HSA were treated with Na2S to
produce persulfides which were then labeled by tag-switch method. Duplicates of aliquots (5 µL) from
the samples (30 µM) were added to a nitrocellulose membrane and the presence of biotinylation was
visualized on radiographic films using chemiluminescent reagents.
Figure 8. Detection of HSA persulfide with tris(2,4-dimethyl-5-sulfophenyl)phosphine (TDMSP).
A-B) Mass spectrum of the reaction mixture obtained by mixing HSA-SSH (20 µM) with TDMSP
(400 µM) during 30 min at 25 ºC. Two different m/z ranges are shown from the same spectrum, m/z
200-500 (A) and m/z 500-800 (B). The peak assignment (C-F) reveals that the most intense peaks
correspond to unreacted TDMSP flying in cluster with one or two sodium cations (C-D). However,
formation of TDMSP sulfide (TDMSP=S) could also be detected (E-F). Observed peaks (in blue)
match perfectly the predicted isotopic distribution (in red). For TDMSP, the observed and predicted
masses are 303.0111 and 303.0109, respectively, for the monosodium species, while the
corresponding values for the disodium species are 629.0111 and 629.0116. For TDMSP=S, the
17

observed and predicted masses are 318.9972 and 318.9970, respectively, for the monosodium, or
660.9831 and 660.9837 for the disodium species.
Figure 9. Mass spectrometry of HSA persulfide. A) Mass spectrum of HSA-TNB (black) and HSA-
TNB mixed with equimolar amounts of Na2S (red). B) Zoomed overlay of the MS spectra showing
difference. C) Deconvoluted mass spectra of HSA-TNB (black) and HSA-TNB + Na2S (red). The
spectrum shows the mass difference of 166 ± 1 suggesting the formation of protein persulfide. In
addition, a peak assigned to HSA-SSO3H could be also observed. D) Methodological approach to label
and elute the peptide containing persulfidated cysteine. E) Observed (black) and simulated isotopic
pattern of a peptide eluted following the protocol depicted in D). The mass is consistent with the
doubly charged ALVLIAFAQYLQQ34CPFEDHVK peptide. F) MS/MS spectrum confirms the
sequence.
Figure 10. Competition kinetics for the reaction of HSA sulfenic acid with hydrogen sulfide.
HSA-SOH (0.5 µM) was incubated with TNB (66 µM) and increasing concentrations of Na2S in
phosphate buffer (0.1 M, pH 7.4, 0.1 mM dtpa, 25 ºC). A) Time course of TNB consumption at 412
nm. B) Correlation of kobs versus concentration of Na2S.
Figure 11. Effect of H2O2 on protein persulfidation in SH-SY5Y and HUVEC cells. A) SH-SY5Y
cells were preincubated or not with 2 mM oxamic acid (OXA, inhibitor of cystathionine β-synthase
and cystathionine γ-lyase) and treated with 300 µM H2O2 for 30 min. Cells were fixed with methanol
and labeled for persulfide detection using the tag-switch assay. The biotinylation of the proteins was
visualized by fluorescence microscopy using Dylight 488 streptavidin. Nuclei were stained with
DAPI. B) The change in the fluorescence was semi-quantified using ImageJ, n = 100 from the
experiment done in triplicates. Columns represent the mean ± S.E.M. * p < 0.01 versus control by
analysis of variance (simple paired student’s t-test). C) HUVEC were preincubated or not with 2 mM
propargylglycine (PG, inhibitor of cystathionine γ-lyase) for 1.5 h and treated with 500 µM H2O2 for
30 min. Cells were fixed, labeled and visualized as in A). D) The change in the fluorescence was semi-
quantified using ImageJ, n = 20 from the experiment done in triplicates. Columns represent the mean ±
S.E.M. * p < 0.05 versus control or ** p = 0.053 versus H2O2 by analysis of variance (simple paired
student’s t-test).
Figure 12. Kinetics of the reaction of HSA persulfide with peroxynitrite. A) Kinetic traces of
peroxynitrite decay (9 µM) in the presence of 7 (red), 20 (blue) and 40 µM (black) HSA-SSH. B)
Analysis of kinetic traces using an initial rate approach for HSA-SSH (red diamonds) and HSA-SH
(black squares). Error bars represent the standard deviation (n = 5).
Figure 13. Kinetics of the reaction of HSA persulfide with DTDPy. A) Kinetic traces of
thiopyridone formation at 324 nm during the incubation of ~ 3 µM HSA-SSH (red) with 32 µM
DTDPy are compared with those produced by 18 µM HSA-SH (black) and 3 µM HSA-SOH (blue)
preparations. B) Dependence of kobs on the concentration of DTDPy for HSA-SSH (red diamonds),
HSA-SH (black squares) and HSA-SOH (blue triangles) preparations. C) Kinetic traces when HSA-
SOH (6 µM, initial) was incubated with Na2S (6 µM, initial) during 50 seconds and mixed with 32 µM
DTDPy every 3 minutes. D) Red points: amplitudes obtained from fits of the first phase produced by
incubation of DTDPy (32–207 µM) with the mixture of HSA-SOH and Na2S (final concentration: 3
µM each) at different incubation times. Black line: numerical determination of the second-order rate
constant between HSA-SOH and H2S. The spontaneous decay of HSA-SOH was also considered. Rate
constant estimated: (4.7 ± 0.2) x 102 M-1 s-1.
18

TABLES
Table 1. Second-order rate constants for the reaction of hydrogen sulfide with symmetric LMW
disulfides in 100 mM phosphate buffer at 25 ºC.
Reaction: HS- + RSSR -> RSS- + RSH
pKa
of the thiol
k pH 7.4
k pH-indep
Disulfide
(M-1 s-1)
(M-1 s-1)
DTNB
4.4a
6.45b
8.29c
8.94c
9.1c
(8.61  0.04) x 102
3.3  0.7
(1.20  0.01) x 103
5  1
CysOMe-CysOMe
Cys-Cys
GSSG
(6  1) x 10-1
(9  2) x 10-1
(2.2  0.1) x 10-1
(4.5  0.6) x 10-1
(1.6  0.1) x 10-1
(3.2  0.4) x 10-1
Hcy-Hcy
HED
9.6 c
(3.92  0.03) x 10-2 (5.48  0.04) x 10-2
aData from (25). bThe pKa of CysOMe was determined by UV titration as in ref (64). cData from (64).
19

Table 2. Second-order rate constants for the reaction of hydrogen sulfide toward mixed
disulfides formed between Cys34 of HSA and LMW thiols in 100 mM phosphate buffer at 25 ºC.
Reaction: HS- + HSA-SSR -> HSA-SS- + RSH or HSA-SH + RSS-
pKa of
the thiol
k pH 7.4
k pH-indep
Disulfide
(M-1 s-1)
(M-1 s-1)
HSA-TNB
HSA-CysOMe
HSA-Cys
4.4a
6.45b
8.29c
8.94c
9.1c
(3.5  0.8) x 102
(5.5  1.3) x 101
3.0  1.1
(5  1) x 102
(8  2) x 101
4  2
2  1
(3  1) x 10-1
HSA-GSH
HSA-Hcy
1.7  1.1
(2.3  0.8) x 10-1
HSA-βME
9.6c
(5.1  0.4) x 10-1 (7.2  0.6) x 10-1
aData from (25). bThe pKa of CysOMe was determined by UV titration as in ref (64). cData from (64).
20

Table 3. Comparison of basicity, nucleophilicity (KS-HOMO energies) and chemical hardness
(η) values for thiolates, HS– and CysSS– calculated at the DFT level in aqueous solution.
Species
CysOMe-
HS-
CysGly-
Cys-
Hcy-
pKa
6.45a
7.00b
7.95c
8.29c
9.1c
EHOMO (eV)
–7.63
η (eV)
4.53
4.94
4.67
4.72
4.58
4.75
4.16
–7.84
–7.57
–7.50
–7.33
9.60c
–7.39
β-ME-
CysSS-
(4.34)d
–6.97
aThis work. bData from (2). cData from (64). dEstimated here from the predicted shift respect to Cys of
-3.96 (Eq. 3).
21

Figure 1
A
250
200
150
100
50
[GSSG]
10.5 mM
5.25 mM
2.0 mM
0
0
2000
4000
6000
Time (s)
B
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
0
2
4
6
8
10
12
[GSSG] (mM)
22

Figure 2
A
100
80
60
40
20
0
H2S
H2S + HSA-SH
H2S + HSA-Cys
0
1000 2000 3000 4000 5000 6000
Time (s)
B
0.20
0.15
0.10
0.05
0.00
0
50 100 150 200 250 300 350
[HSA-TNB] (M)
23

Figure 3
A
Reducing species
dithiotreitol
mercaptoethanol
glycol-dimercaptoacetate
hydrosulfide
6
4
DTNB
2
CysOMe-CysOMe
0
Hcy-Hcy
HED
Cys-Cys
GSSG
-2
3
4
5
6
7
8
9
10
11
pKa of leaving thiol
B
3
2
HSA-TNB
HSA-CysOMe
1
HSA-GSH
HSA-Hcy
HSA-Cys
0
HSA-ME
-1
3
4
5
6
7
8
9
10
pKa of LMW thiol
24

Figure 4
25

Figure 5
A
6
5
4
3
2
thiocarboxyl
thioaryl
thioalkyl
hydrosulfide
2
3
4
5
6
7
8
9
10 11 12
pKa of nucleophile
B
3
2
1
0
thioalkyl
hydrosulfide
-1
6
7
8
9
10
11
12
pKa of nucleophile
26

Figure 6
-6.8
-7.0
-7.2
-7.4
-7.6
-7.8
-8.0
CysSS–
Hcy–
ME–
Cys–
CysOMe–
CysGly–
HS–
7
4
5
6
8
9
10
11
pKa
27

Figure 7
A
600
400
200
0
Cyanolizable sulfur
HSA
0
5
10
15
20
Elution fraction
B
28

Figure 8
29

Figure 9
30