I.A. Dereven'kov, et al.
InorganicaChimicaActa499(2020)119215
(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).
GSSG +
GSSeSG +
2 NaOH +
H2O,
(8)
(9)
(10)
·
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
(1)
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
(4)
(5)
(6)
A Similar mechanism for the reaction between selenite and cysteine
has been suggested by Forastiere et al. [25], i.e. RSSeO2H is formed in
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
]
,
]
(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
(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-
trisulfide requires reaction of selenite with four thiol molecules (reac-
tion (7)) that cannot be a simple and reversible reaction. Likewise, no
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
ESI mass spectrometry measurements were performed on a Sciex
6500 + triple quadrupole mass spectrometer (Sciex). The instrument
2