Kinetic, spectroscopic and in silico characterization of the first step of the reaction between glutathione and selenite

Article abstract of DOI:10.1016/j.ica.2019.119215

Reduction of dietary selenite (SeO₃H^?, SeO₃H₂) is an important process in vivo, which predominantly involves glutathione (GSH). Although the reaction between selenite and thiols has been studied extensively, its mechanism and the identification of products remain controversial. Herein, we present kinetic, spectroscopic and in silico data on the first step of the reaction between GSH and SeO₃^2? in aqueous solutions of varying acidity. We found that the reaction reversibly produces glutathione-S-selenite (GS-SeO₂^?) absorbing at 259 nm in the UV spectrum. Assignment of the absorption maximum at 259 nm to GS-SeO₂^? was performed using TDDFT and mass spectrometry. GS-SeO₂^? undergoes protonation in acidic medium to form the corresponding conjugated acid, GS-SeO₂H (pK_a = 1.9 at 25 °C), which exhibits reduced absorption intensity at 259 nm. According to the kinetic data, the mechanism of GS-SeO₂^?(H⁺) formation includes two pathways: (i) nucleophilic substitution of HO-group in biselenite by the thiolate group of GSH, and (ii) nucleophilic substitution of HO-group in selenous acid by the thiol group of GSH. The complex GS-SeO₂^?(H⁺) is unstable in aqueous medium and undergoes hydrolysis to initial reactants, which is accelerated by an increase in alkalinity.

Full text of DOI:10.1016/j.ica.2019.119215

InorganicaChimicaActa499(2020)119215
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Kinetic, spectroscopic and in silico characterization of the first step of the
reaction between glutathione and selenite
⁎
Ilia A. Dereven'kova, , Luciana Hannibalb, Pavel A. Molodtsova, Adrian M.V. Brânzanicc,
Radu Silaghi-Dumitrescuc, Sergei V. Makarova
a Ivanovo State University of Chemistry and Technology, Sheremetevskiy str. 7, 153000 Ivanovo, Russia
b Laboratory of Clinical Biochemistry and Metabolism, Department of General Pediatrics, Adolescent Medicine and Neonatology, Faculty of Medicine, Medical Center -
University of Freiburg, 79106 Freiburg, Germany
c Department of Chemistry, “Babes-Bolyai” University, Str. Arany Janos Nr. 11, RO-400028 Cluj-Napoca, Romania
A R T I C L E I N F O
A B S T R A C T
Keywords:
Selenium
Glutathione
Selenite
Reduction of dietary selenite (SeO3H−, SeO3H2) is an important process in vivo, which predominantly involves
glutathione (GSH). Although the reaction between selenite and thiols has been studied extensively, its me-
chanism and the identification of products remain controversial. Herein, we present kinetic, spectroscopic and in
silico data on the first step of the reaction between GSH and SeO32− in aqueous solutions of varying acidity. We
found that the reaction reversibly produces glutathione-S-selenite (GS-SeO2−) absorbing at 259 nm in the UV
Mechanisms
Kinetics
−
spectrum. Assignment of the absorption maximum at 259 nm to GS-SeO2 was performed using TDDFT and
−
mass spectrometry. GS-SeO2 undergoes protonation in acidic medium to form the corresponding conjugated
acid, GS-SeO2H (pKa = 1.9 at 25 °C), which exhibits reduced absorption intensity at 259 nm. According to the
kinetic data, the mechanism of GS-SeO2−(H+) formation includes two pathways: (i) nucleophilic substitution of
HO-group in biselenite by the thiolate group of GSH, and (ii) nucleophilic substitution of HO-group in selenous
acid by the thiol group of GSH. The complex GS-SeO2−(H+) is unstable in aqueous medium and undergoes
hydrolysis to initial reactants, which is accelerated by an increase in alkalinity.
1. Introduction
involving glutathione (GSH; Scheme 1) [6,13,14,19] and references
therein]. One of the key intermediates in this process is selenodiglu-
Selenium is an essential ultra-microelement to many animal species.
It occurs in vivo in the form of selenocysteine (Fig. 1A), selenomethio-
nine (Fig. 1B) and others [1]. Selenocysteine is a proteinogenic amino
acid and a key component of glutathione peroxidase, thioredoxin re-
ductase, iodothyronine deiodinase, methionine sulfoxide reductase and
other enzymes [2–4].
tathione (selenotrisulfide; GS-Se-SG; Scheme 1). Formation of GS-Se-SG
as well as other trisulfides has been unambiguously proved in several
studies [10,12,20,21]. Selenotrisulfides are relatively stable in acidic
conditions and are decomposed in neutral and alkaline medium to Se(0)
and disulfide [21,22]. Nevertheless, data on the structure of the inter-
mediates formed in the course of the first stages of the interaction of
selenite and GSH are speculative. One feature of GSH/selenite systems
is that selenite possesses catalytic properties in redox reactions invol-
ving GSH, e.g. it accelerates formation of reactive oxygen species
[19,23], and increases the rate of resazurin reduction [24]. The cata-
lytic behavior of selenite can be due to the reversible formation of
highly reactive intermediates whose structure and properties require
thorough investigation.
Inorganic salts, i.e. selenites and selenates, are frequently used as a
source of selenium in vitamin-mineral supplements [5]. In the human
body, selenite is reduced to hydrogen selenide (H2Se), which is con-
verted to selenocysteine via the formation of selenophosphate [6]. Ex-
cess selenium is toxic for mammals and it is converted in vivo to sele-
nocyanate [7], selenosugars [8] and various methylated compounds
[9,10]. The reduction of selenite has been highlighted in several works.
For example, selenite can be reduced by super-reduced cobalamin (vi-
tamin B12) [11], thiols [12–14], hydrogen sulfide [15], ascorbic acid
[16], dithionite [17], Fe(0) [18] and others.
Possible mechanisms for the reactions between selenite and thiols
have been highlighted in previous works. The most thorough mechan-
istic study has been performed be Kice et al. [20]. In this work, the
authors suggest a mechanism of reaction of alkylthiols (n- and t-BuSH)
The main in vivo route of the reduction of selenite is the process
⁎ Corresponding author.
E-mail address: derevenkov@gmail.com (I.A. Dereven'kov).
https://doi.org/10.1016/j.ica.2019.119215
Received 21 August 2019; Received in revised form 15 October 2019; Accepted 15 October 2019
Availableonline16October2019
0020-1693/©2019ElsevierB.V.Allrightsreserved.

I.A. Dereven'kov, et al.
InorganicaChimicaActa499(2020)119215
SeO23 + 4 RSH + 2H+
RSSeSR + RSSR + 3H2O.
(7)
Cui and coworkers investigated the reaction between GSH and
SeO32− by mass spectrometry [12]. The authors observed signals in the
mass-spectrum that were assigned as GS-Se-SG and GSSSe−. Formation
of these species in their work was explained by reactions 8–10, which
apparently comprise a complex mechanism including numerous steps.
Fig. 1. Structures of selenocysteine (A) and selenomethionine (B).
4GSH + Na2SeO3
GSSG + GSSeSG + 2 NaOH + H2O,
(8)
2GSH + GSSeSG + 2OH
4GSSG + Se + 2H2O,
(9)
(10)
·
GSSG + Se
GSSSe + G
The present study was motivated by existing controversies on the
mechanisms and the products formed in the reactions between glu-
tathione and selenite. We report kinetic, spectroscopic and in silico
analysis of the first step of the reaction between GSH and selenite, as
well as the characterization of the product of this step, i.e. glutathione-
S-selenite.
2. Experimental
L-glutathione (Sigma; GSH; ≥98%), sodium selenite (Alfa Aesar; ≥
99.5%), 5,5′-dithiobis-(2-nitrobenzoic acid) (Alfa Aesar; ≥ 99.5%) and
hydroxocobalamin hydrochloride (Sigma; ≥ 96%) were used without
additional purification. The stable isotopic variant of GSH with a mass
difference of +3 was purchased from Sigma and used without further
purification (Glutathione-(glycine-13C2, 15N) trifluoroacetate salt, ≥98
atom % 15N, ≥99 atom % 13C, ≥95% (CP), Sigma 683620).
Buffer solutions (acetate, phosphate and borate; 0.1 M) were used to
maintain pH during the measurements. Buffer solutions for mass
spectrometry were prepared using CHROMASOLV™ LC-MS water, pro-
duct Nr. 39253, Honeywell, Germany.
Scheme 1. In vivo pathway of assimilation of inorganic selenium.
with selenite in water/dioxane mixtures, which involves reversible
formation of alkylthioseleninic acid (RSSeO2H) in the course of addi-
tion of selenium dioxide to thiol (reactions (1),2). RSSeO2H is reversibly
+
protonated under acidic conditions to give RSSeO2H2 (reaction (3)).
RSSeO2H (or RSSeO2H2+) undergoes reaction with a second thiol mo-
lecule to give RSSe(O)SR (reaction (4)), which is further transformed to
RSSeSR (reactions (5),6).
The pH values of solutions were determined using Multitest IPL-103
pH-meter (SEMICO) equipped with ESK-10601/7 electrode
(Izmeritelnaya tekhnika) filled by 3.0 M KCl solution. The electrode was
preliminarily calibrated using standard buffer solutions (pH
1.65–12.45).
H2SeO3
SeO2 + H2O
(1)
SeO2 + RSH
RSSeO2H,
(2)
RSSeO2 H+ H+
RSSeO2H+2 ,
Ultraviolet–visible (UV–vis) spectra were recorded on a cryother-
mostated ( 0.1 °C) Cary 50 UV–Vis spectrophotometer in quartz cells
under aerobic conditions. Preliminary experiments showed that the
presence of oxygen does not affect the reaction. Kinetics of the reaction
between GSH and selenite was studied on a thermostated ( 0.1 °C)
RX2000 (Applied Photophysics, UK) rapid mixing stopped-flow acces-
sory connected to Cary 50 spectrophotometer. Experimental data were
analyzed using Origin 7.5 software.
(3)
RSSeO2H(or RSSeO2H+2 ) + RSH
RS (Se = O) SR + H2O,
RSSeOH + RSSR,
(4)
(5)
(6)
RS (Se = O) SR + RSH
RSSeOH + RSH
RSSeSR + H2O.
A Similar mechanism for the reaction between selenite and cysteine
has been suggested by Forastiere et al. [25], i.e. RSSeO2H is formed in
neutral and basic media and is further decomposed via two parallel
routes, which may or may not involve a second thiol molecule.
The following disadvantages can be drawn out for these mechan-
isms. (i) Selenium dioxide formation [20] is unlikely from selenite in
weakly acidic, neutral and alkaline media, however, the reaction pro-
ceeds at considerable rate (even more rapidly than in strongly acidic
conditions) under these conditions. (ii) The work presented in [20,25]
did not provide direct, unequivocal proof for the formation of RSSeO2H
species. (iii) The existence of the product of the first step in protonated
form (RSSeO2H) in neutral or alkaline media is unlikely: acid-base
properties of RSSeO2H are expected to be comparable with that of se-
leninic acids, which are protonated in weakly acidic medium (e.g., pKa
of selenohypotaurine is 5.4 [26]).
Equilibrium constants were calculated using Eq. (11) [27].
A0 + A K [SeO32
]
A =
,
1 + K [SeO23
]
(11)
[SeO32−] is the selenite concentration in solution, M; A, A0, A∞ are
absorbances at the monitoring wavelength for the complex of selenite
with GSH at a particular selenite concentration, for the starting GSH,
and for the final complex, respectively; K is equilibrium constant, M−1
Calculation of pKa value was performed using Eq. (12).
.
10pH
A = A1 + (A2 A1)
,
10pH + 10pK
a
(12)
A, A1, A2 are absorbances at the monitoring wavelength for the
compound at a particular pH, for the protonated species, and for the
deprotonated species, respectively.
Work by Gennari et al focused on the reversibility of the first step of
the reaction between cysteine and selenite [14]. However, authors as-
signed the formed product as selenotrisulfide. Formation of seleno-
trisulfide requires reaction of selenite with four thiol molecules (reac-
tion (7)) that cannot be a simple and reversible reaction. Likewise, no
direct evidence was provided for the identification of the reaction
product.
For DFT calculations, the B3LYP functional and the def2-SV(P) basis
set were employed, in geometry optimizations and subsequent time-
dependent (TDDFT) (N = 10 states) procedures within the Gaussian
software package [28].
ESI mass spectrometry measurements were performed on a Sciex
6500 + triple quadrupole mass spectrometer (Sciex). The instrument
2

I.A. Dereven'kov, et al.
InorganicaChimicaActa499(2020)119215
was set up on the low-mass detection mode to monitor m/z from 100 to
1000 Da. Detection of glutathione and selenite species was performed in
the negative mode. The temperature was set at 100 °C, the declustering
potential at −60 Volts and the entrance potential at −10 Volts. Each
measurement was an average of 10 scans. Direct injection of the re-
actants upon mixing was performed with minimal delay (30–45 s) using
a gas-tight Hamilton syringe hosted at and driven by an automated
infusion accessory. All the experiments were performed at room tem-
perature (23–25 °C). Data analysis was performed with built-in software
Analyst (Sciex). Experiments were performed either in the presence of
0.1% (w/v) ammonium formate pH 6.8 (neutral conditions) or 0.1% (v/
v) formic acid pH 2.1 (acidic conditions) as the solvents. Stock solutions
of 100 mM sodium selenite (Sigma S5261-10G) were prepared freshly
by dissolving solid sodium selenite in water (CHROMASOLV™ LC-MS
water, 39253 Honeywell, Germany). Stock solutions of 50 mM glu-
tathione or its isotope 15N-13C2-GSH were made freshly by dissolving
the appropriate solid reduced glutathione in water (CHROMASOLV™
LC-MS water, 39253 Honeywell, Germany). Both stock solutions of GSH
and selenite were kept on ice and brought to room temperature right
before their mixing. Reaction mixtures contained equimolar quantities
of GSH and selenite (0.5 mM). MS spectra were collected at intervals of
1 min after mixing for the first 5–10 min, and then at 45 min or 90 min
(aging of the reaction mix at room temperature).
Fig. 3. Plot of maximum absorbance at 259 nm vs. [SeO32−].
[GSH]0 = 1.2·10−4 mM; pH 7.2; 25.0 °C.
spectrum [30]. We found that UV–vis spectral changes in the course of
the reaction of the GSH/selenite complex with Ellman’s reagent (Fig.
S1) or aquacobalamin (Fig. S2) are identical to those for the processes
involving free GSH, although the latter reactions proceed more rapidly.
Using eq. (11), we determined equilibrium constants for complex
formation between GSH and selenite in solutions of varying acidity. The
maximum equilibrium constant (ca. 4000 M−1 at 25 °C) is observed at
pH 1…3 and 6.5…7.2 (Fig. 4).
3. Results and discussion
The reaction of GSH with selenite at pH 7.2 generates a first-step
product with absorbance maximum at 259 nm in UV spectrum (Fig. 2).
This observation has been reported in earlier works [12–14,20,25]. The
same peak is observed in acidic and alkaline media with the exception
of strongly acidic and strongly alkaline conditions, where formation of
this product is negligible. However, we found that the maximum in-
tensity of the peak at 259 nm depends strongly on selenite concentra-
tion and reaches maximum intensity with excess selenite over GSH
(Fig. 3). This fact is typical to equilibrium processes, i.e. GSH and se-
lenite are capable of reversibly binding within the first step of the
process.
Next, we studied the kinetics of the reaction between GSH and an
excess of selenite. Kinetics of a reversible pseudo-first-order reaction
can be confused with two schemes, i.e. with (i) a consecutive pathway
(A → B; B + A → P) and (ii) parallel reactions (C → P1; C + D → P2)
including the decomposition of reactant taken in excess (C → P1)
[31,32]. Using Ellman’s reagent and aquacobalamin, we clearly showed
that the product of the studied reaction regenerates GSH that allows to
analyze the interaction between GSH and excess selenite as the re-
versible pseudo-first-order reaction.
To support regeneration of GSH from its complex with selenite, we
mixed GSH with a significant excess of selenite in order to transform
GSH to its bound state and then added Ellman’s reagent (5,5′-dithiobis-
(2-nitrobenzoic acid)) or aquacobalamin. Reaction of thiols with
Ellman’s reagent results in formation of the product with absorption
peak at 412 nm, whereas aquacobalamin tightly binds GSH to give
glutathionylcobalamin [29], a complex with characteristic UV–vis
Kinetic curves are best described by an exponential equation (Fig. 5)
that indicates first order with respect to GSH. The plot of observed rate
constants versus [SeO32−] is provided in Fig. 6. The linear dependence
indicates first order with respect to selenite. The value of intercept is
indistinguishable from zero at near neutral media and becomes more
Fig. 2. Maximum of absorption in the UV–vis spectra of GSH/SeO32− mixtures
collected at pH 7.2; 25.0 °C. [GSH]0 = 1.2·10−4 M; [SeO32−]0 = 5.0·10−5
(lower spectrum) … 8.0·10−3 M (upper spectrum).
M
Fig. 4. Dependence of equilibrium constant (K) for the reaction between GSH
and selenite on pH at 25.0 °C.
3

I.A. Dereven'kov, et al.
InorganicaChimicaActa499(2020)119215
2−
Fig. 5. Typical kinetic curve of the reaction between GSH (0.1 mM) and SeO3
(1.0 mM) at pH 7.5; 25.0 °C.
Fig. 7. Dependence of the rate constants of the forward reaction of GSH with
selenite (k’; slopes of concentration dependencies) on pH at 25.0 °C.
(kint.; Fig. S5) reactions on pH explains profile of plot of equilibrium
constant (K = k’/kint.) versus pH (Fig. 4). Plot of K versus pH exhibits
minimum at pH 3.0…6.5 that coincides with minimum of plot of k’
versus pH in this range. Decrease of K at pH > 7.5 is predominantly
provided by an increase of kint. in this medium.
(13)
SeO3H
SeO3H2
SeO23 + H+
(14)
(15)
SeO3H + H+
Taking into account these equilibria, Eq. (16) was expressed.
pK (SeO H )+pK (GSH)
a
3
H
2
a
10
k
= k1 (10 + 10
+
pH
pK (SeO
)
pH
pK (GSH)
a
)
a
3
2
)(10 + 10
pH+pK (SeO
H )
a
)
3
10
+ k
2
pH
pK (SeO
H
pH
pK (GSH)
a
)
a
3
(10 + 10
)(10 + 10
(16)
Fig. 6. Plot of observed rate constants (kobs.) vs. [SeO32−]. [GSH]0 = 0.1 mM;
where pKa(SeO3H−), pKa(SeO3H2) and pKa(GSH) are acid dissociation
constants for SeO3H−, SeO3H2 and the thiol group in GSH, respectively;
k1 is rate constant for the reaction between GSH with protonated thiol
group and SeO3H2; k2 is rate constant for the reaction between
GS−(thiolate form of GSH) and SeO3H−. Using pKa(GSH) = 8.9 and
pKa(SeO3H2) = 2.7 (pKa(SeO3H−) = 7.5 was determined during fit-
pH 7.5; 25.0 °C.
pronounced in acidic (Fig. S3) and alkaline (Fig. S4) conditions. The
intercept can be explained by the presence of reverse reaction, i.e. de-
composition of the complex between GSH and selenite. The value of the
intercept increased upon alkalinization (Fig. S5), which is explained by
hydrolysis of the complex.
ting), k1 = (1.8
0.1)·102 and k2 = (6.7
0.2)·104 M−1 s−1 were
obtained.
The dependence of reaction rate on pH in weakly acidic, neutral and
weakly alkaline conditions is bell-shaped (Fig. 7), suggesting an influ-
ence of acid-base properties of both reactants on kinetics. Apparently,
one of acid-base equilibria affecting the kinetics is deprotonation of
thiol group of GSH (13; pKa = 8.9 at 25.0 °C [33]). The second process
involves protonation of selenite to biselenite (14; pKa ranges from 6.6 to
8.5 depending on conditions at 25.0 °C [34]). Thus, the reactive species
in weakly acidic, neutral and weakly alkaline media are thiolate form of
GSH and monoprotonated form of selenite. The plot of rate constant
versus pH indicates slight increase upon acidification at pH 1…3, which
can be due to the protonation of biselenite to selenous acid (15;
pKa = 2.7 at 25.0 °C [34]) and the reaction between selenous acid and
GSH with protonated thiol group.Fig. 8.
Finally, we assessed the rate of the subsequent step of the reaction
between GSH and selenite accompanied by a reduction of absorbance
intensity at 259 nm. As can be seen from Fig. S6, this step proceeds
much slower than the first step and the primary product of the reaction
between GSH and selenite is more stable in the presence of higher se-
lenite concentrations.
To identify the product, we performed MS measurements under
acidic (pH 2.1) and neutral conditions (pH 6.8). Formation of a species
with m/z at 418 was observed upon mixing GSH with an equimolar
amount of selenite both under acidic and neutral conditions (Figs. 8 and
S7). Use of isotopically labeled glutathione with a mass difference of
3+ (15N-13C2-GSH) permitted the identification of a species at m/z 421.
−
These m/z can be attributed to GSSeO2 adduct formed upon con-
Dependencies of rate constant of forward (k’; Fig. 7) and reverse
jugation of selenite with GSH. Evidence for the formation of the product
4

I.A. Dereven'kov, et al.
InorganicaChimicaActa499(2020)119215
Fig. 8. Mass-spectra of GSH (upper spectrum, panel A), GSH/selenite mixture (bottom spectrum, panel A, and upper spectrum, panel B) and 15N-13C2-GSH/selenite
mixture (bottom spectrum, panel B) recorded after mixing of reactants at pH 6.8. Signals of GSH and GSSG ions or their Na-derived versions and NH3-adducts are
indicated by • and ♦, respectively.
Fig. 9. UV spectra of GS-SeO2−(H+) collected at pH 0.5 to 7.2 (A) and the plot of absorbance at 259 nm versus pH (B). GS-SeO2−(H+) was prepared by mixing GSH
(1·10−4 M) with selenite (2·10−2 M) at 25.0 °C.
−
Fig. 10. Main orbitals that contribute to the DFT-computed UV–vis spectrum of GS-SeO2
.
with m/z = 418 was presented in an earlier ESI-MS study [12], but this
peak was attributed to GSSSe−. However, since species with m/z = 418
is formed very rapidly, after the mixing of glutathione and selenite, we
SeO2H) = (1.9
0.1) (25.0 °C) was determined by fitting plot of ab-
sorbance versus pH (Fig. 9B) to eq. (12).
The assignment of the experimentally-observed UV peak to GS-
−
assume that it cannot be GSSSe−
.
SeO2 was further verified using time-dependent DFT (TDDFT) calcu-
Further examination of the equilibrium between protonated and
deprotonated species of GS-SeO2−(H+) was conducted by recording UV
spectra at pH 0.5 to 8.0. The intensity of the peak at 259 nm is sub-
stantially decreased at pH < 3 (Fig. 9A; extinction coefficients for
deprotonated and protonated species are 5700 and 1900 M−1 cm−1 at
lations. The strongest contribution to the DFT-derived UV–vis spectrum
−
of GS-SeO2
is at 296 nm (computed extinction coefficient:
3235 M−1cm−1), arising primarily from a HOMO-4 → LUMO excita-
tion. The second strongest contribution, closely overlapping at 278 nm
(computed extinction coefficient: 1325 M−1cm−1) arises primarily
from a HOMO-6 → LUMO excitation. Other contributions are sig-
nificantly lower in intensity. These computed wavelengths are in rea-
sonable agreement with experiment, considering the accuracy of the
−
259 nm), which can be due to the protonation of SeO2 group of GS-
SeO2−. It is unlikely that protonation of groups distant from the S-Se
motif (i.e., carboxylates) can be reflected in the UV spectrum. pKa(GS-
5

I.A. Dereven'kov, et al.
InorganicaChimicaActa499(2020)119215
Scheme 2. Mechanism of the first step of the reaction between selenite and glutathione in weakly acidic, neutral, weakly alkaline (A) and strongly acidic (B) media.
DFT method [35]. As seen in Fig. 10, the calculated electronic transi-
tions amount to a σ → σ* and n → σ* character of the main band ex-
pected in the UV–vis spectrum of GS-SeO2−. On the other hand, the
DFT-derived UV–vis spectrum of the protonated version (GS-SeO2H) is
found to be one order of magnitude weaker and redshifted by
100–150 nm and is therefore not further discussed.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
The mechanism of the reaction is shown in Scheme 2. Taking into
account the type of reactive species elucidated upon fitting of the plot of
rate constant versus pH (Fig. 7), two routes depending on pH can be
suggested. In weakly acidic, neutral and weakly alkaline conditions, the
reaction proceeds between the thiolate form of GSH and the mono-
protonated form of selenite. Deprotonation of the thiol group enhances
its nucleophilicity and enables substitution of HO-group in biselenite.
Deprotonated selenite species (SeO32−) is unlikely to be involved in
reaction with GSH or its deprotonated version due to their large ne-
gative charge hampering the contact with nucleophiles. In more acidic
conditions the reacting species are selenous acid and the thiol form of
GSH. Second protonation of selenite increases its electrophilicity and
facilitates its interaction with the protonated thiol group. The products
of the interaction are glutathione-S-selenite (GS-SeO2−) and its con-
jugated acid (GS-SeO2H). S-Se bond in GS-SeO2−(H+) is capable of
reacting with water or hydroxide that result in formation of initial re-
actants.
Acknowledgements
This work was supported by grant of Council on grants of the
President of the Russian Federation for state support of young Russian
researchers (project MК-1083.2019.3).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ica.2019.119215.
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This study provides direct proof that the reaction of selenite with
glutathione reversibly produces glutathione-S-selenite (GSSeO2−) or its
conjugated acidic form (GSSeO2H). The reaction involves nucleophilic
substitution of the HO-group of biselenite by the thiolate glutathione
species or substitution of the HO-group in selenous acid by the thiol
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solutions and undergoes decomposition to selenite and GSH and can be
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