Effect of phosphate buffer solutions on the reactions of glutathione with hydrogen peroxide and peroxyl radicals

Article abstract of DOI:10.1007/s11172-019-2574-4

Differences in the kinetics and mechanism of the reaction of glutathione (GSH) with hydrogen peroxide (H₂O₂) in deionized water and in phosphate buffer systems with pH ≥ 7 frequently used in biochemical studies were revealed. The formation of GSH dimers and complexes with H₂O₂ in water plays a substantial role in the kinetics of the process, which is manifested as nonlinear dependences of the rate of GSH consumption (W_GSH) and the rate of radical formation (W_i) on the reagent concentrations. In phosphate buffer solutions (PBS), the oxidation of GSH by air oxygen is enhanced and the radical formation rate decreases sharply. An effect of NaCl and KCl in PBS on W_GSH and W_i was observed, unlike a sodium—potassium phosphate buffer mixture (PB). Under other equivalent conditions, W_GSH PBS is several times lower and W_i is higher than those in PB containing no chlorides. It was found that the rate of the thiol-ene reaction of unsaturated phenol resveratrol (RVT) with GSH initiated by the radicals formed in the presence of H₂O₂ in PBS is nearly three times lower than that in water, whereas in PB resveratrol is not consumed under the same conditions. However, in the reactions with peroxyl radicals formed upon the decomposition of 2,2′-azobis(2-methylpropionamidine) dihydrochloride the GSH consumption rate is the same in both phosphate buffer systems.

Full text of DOI:10.1007/s11172-019-2574-4

Russian Chemical Bulletin, International Edition, Vol. 68, No. 7, pp. 1441—1444, July, 2019
1441
Effect of phosphate buffer solutions on the reactions of glutathione
with hydrogen peroxide and peroxyl radicals
K. M. Zinatullina,a O. T. Kasaikina,a V. A. Kuzmin,b N. P. Khrameeva,b and L. M. Pisarenkoa
аN. N. Semenov Institute of Chemical Physics, Russian Academy of Sciences,
4 ul. Kosygina, 119991 Moscow, Russian Federation.
Fax : +7 (495) 651 2191. E-mail: karinazinat11@gmail.com
bN. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences,
4 ul. Kosygina, 119991 Moscow, Russian Federation
Differences in the kinetics and mechanism of the reaction of glutathione (GSH) with
hydrogen peroxide (H2O2) in deionized water and in phosphate buffer systems with pH  7
frequently used in biochemical studies were revealed. The formation of GSH dimers and com-
plexes with H2O2 in water plays a substantial role in the kinetics of the process, which is
manifested as nonlinear dependences of the rate of GSH consumption (WGSH) and the rate of
radical formation (Wi) on the reagent concentrations. In phosphate buffer solutions (PBS), the
oxidation of GSH by air oxygen is enhanced and the radical formation rate decreases sharply.
An effect of NaCl and KCl in PBS on WGSH and Wi was observed, unlike a sodium—potassium
phosphate buffer mixture (PB). Under other equivalent conditions, WGSH in PBS is several
times lower and Wi is higher than those in PB containing no chlorides. It was found that the
rate of the thiol-ene reaction of unsaturated phenol resveratrol (RVT) with GSH initiated by
the radicals formed in the presence of H2O2 in PBS is nearly three times lower than that in
water, whereas in PB resveratrol is not consumed under the same conditions. However, in the
reactions with peroxyl radicals formed upon the decomposition of 2,2´-azobis(2-methylprop-
ionamidine) dihydrochloride the GSH consumption rate is the same in both phosphate buffer
systems.
Key words: glutathione, hydrogen peroxide, peroxyl radicals, phosphate buffer systems,
kinetics, thiol—ene reaction, resveratrol.
Glutathione (GSH) is the most abundant cytosol thiol
classified as an endogenic bioantioxidant synthesized di-
rectly in living organisms. The concentration of GSH in
biological tissues is 0.1—10 mmol L–1, which is signifi-
cantly higher than the concentrations of other potential
bioantioxidants.1—7 The GSH/GSSG redox pair and
H2O2 occupy the central place in determining oxidation—
reduction homeostasis and redox signalization.8—13 It is
known that the reduction of H2O2 by glutathione is ac-
consumption. However, this rate turned out to be sufficient
for initiation of the chain reaction of GSH with unsatu-
rated phenols resveratrol and caffeic acid.23,24 The rates
of radical generation (Wi) were measured by the inhibitor
methods using anionic polymethine dye А (3,3´-di-γ-
sulfopropyl-9-methylthiacarbocyaninebetaine pyridine
salt) as an acceptor. This water-soluble dye is inert toward
thiols and hydrogen peroxide but reacts actively and stoi-
chiometrically with free radicals.25 We used bidistilled and
deionized water as a reaction medium, whereas the most
part of biochemical studies are carried out in phosphate
buffer solutions, since biological fluids are characterized
by pH 7.2—7.4 and represent buffer systems.
companied by the formation of disulfide and water4,14—17
:
2 GSH + H2O2
GSSG + 2 H2O.
(1)
Detailed studies of the mechanism of this reaction
indicate that the complicated process occurred via the
formation of the intermediate GSH—H2O2 complex.18 It
is shown that the rate has the first order with respect to
the concentrations of the reactants4,14,18,19 and depends
on the ratio of GSH and H2O2 concentrations.18,20 We
have recently found21,22 that the reaction of GSH and
H2O2 is accompanied by the formation of radicals with
the rate composing fractions of percent of the rate of GSH
The purpose of this work is to reveal the effect of aque-
ous buffer solutions, isotonic sodium phosphate buffer
PBS (рН 7.4), and sodium—potassium phosphate buffer
PB (рН 7.2, used in electrochemical methods of evaluation
of antioxidants26,27) on the kinetics of GSH consumption
in the reaction with H2O2 and on the yield of radicals in
this reaction. We also have studied the possibility of oc-
currence of the thiol—ene reaction of glutathione with
resveratrol. The latter is an exogenic phenol antioxidant
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1441—1444, July, 2019.
1066-5285/19/6807-1441 © 2019 Springer Science+Business Media, Inc.

1442 Russ. Chem. Bull., Int. Ed., Vol. 68, No. 7, July, 2019
Zinatullina et al.
D
that comes to the organism with feeding products and
biologically active additives,28—31 and the reaction with
resveratrol proceeds via the chain mechanism in the pres-
ence of H2O2 in an aqueous medium.23,24
0.5
0.4
0.3
0.2
0.1
Experimental
Glutathione, Ellman reagent (5,5´-dithio-bis(2-nitrobenzoic
acid), DTNB (both from Sigma-Aldrich), hydrogen peroxide
Н2О2 (Usol´khimprom, Russia), azoinitiator 2,2´-azobis(2-
methylpropionamide) dihydrochloride (AAPH, Fluka), and
trans-resveratrol (RVT, ABCR GmbH) were used as received.
Bidistilled deionized water and two phosphate buffer solu-
tions, PBS and PB, were used as reaction media. Buffer PBS
(рН 7.4) is an isotonic phosphate—salt buffer solution containing
sodium and potassium chlorides, sodium hydrophosphate, and
potassium dihydrophosphate in which the concentration of ions
in the solution corresponds to the concentration in the human
blood; PB is a buffer mixture of KH2PO4 and Na2HPO4•12H2O
with рН 7.2. The reactions of GSH with Н2О2 were carried out
at a physiological temperature of 37 С in a temperature-main-
tained vessel with magnetic stirring, from which samples (9 L)
were taken during the reaction. The taken samples were diluted
in 3 mL of phosphate buffer (PBS, рН 7.4) containing the
Ellman reagent (0.1 mmol). The concentration of formed
2-nitro-5-thiobenzoic acid equal to GSH concentration was
determined spectrophotometrically:  = 0.14•105 L mol–1 cm–1
at max = 412 nm.32—34
The rate of radical formation in the reaction GSH + Н2О2
(Wi) was determined by the inhibitor method. Polymethine dye
А was used as an acceptor of free radicals, and its concentration
was detected spectrophotometrically:  = 0.77•105 L mol–1 cm–1
at max = 543 nm. The reaction was carried out directly in
the temperature-maintained cell of a Ultraspec 1100 Pro spec-
trophotometer (l = 1 cm). The thiol—ene reaction of GSH
with unsaturated phenol RVT was conducted in the spectro-
photometer cell in which the RVT consumption was detected
( = 0.3•105 L mol–1 cm–1 at max = 304—308 nm).
1
3
5
2
4
6
2
4
6
8
10 12 14 16 18 t/min
Fig. 1. Kinetic curves of consumption of acceptor А
(0.0065 mmol L–1) in the reaction with the radicals formed upon
the decomposition of ААРН (18 mmol L–1) in water (1, 2),
PBS (рН 7.4) (3, 4), and phosphate buffer solution РВ (рН 7.2)
(5, 6) in the absence of glutathione (1, 3, 5) and with the addition
of glutathione: 0.015 (2), 0.012 (4), and 0.011mmol L–1 (6).
to the rate of consumption of A in water and to the rate of
radical initiation (Wi). The latter remained almost un-
changed in the range of pH 5—8.35,36 This means that in
PBS and РВ for А f = Wi/(–d[A]/dt) = 2. The addition of
GSH (see Fig. 1, curve 2) decreases the consumption rate
of А in water, indicating the competition between А
and GSH. The rate constant of the reaction of GSH
with peroxyl radicals formed upon the decomposition
of ААРН in an aqueous medium at 37 С (kROO
=
= 0.84•105 L mol–1 s–1) was determined22 by the method
of competing reactions. The additives of GSH to PBS and
РВ lead to induction periods ( = 12 min), after the end
of which А is consumed nearly with the rate of the non-
inhibited reaction (see Fig. 1, cf. curves 4 and 3, 6 and 5).
The pronounced induction periods in the presence of small
concentrations of GSH ([GSH] < [A]) indicate that in
PBS and РВ glutathione reacts with radicals more actively
than with А, and both buffer solutions affect the antiradi-
cal activities of А and GSH in the same way. Since the
equation [GSH]0/  Wi is fulfilled, the stoichiometric
coefficient for GSH in PBS and РВ is equal to 1.
Bidistilled water was used to prepare basis solutions of accep-
tor А, and basis solutions of RVT were prepared in ethanol
(Medkhimprom, Russia). The basis solutions were added to the
reaction mixture in amounts of 1—10 L. The concentration of
H2O2 (in the absence of glutathione) was monitored by the iodo-
metric method. The error in rate measurements did not exceed 10%.
Results and Discussion
Reaction of glutathione with Н2О2. The effect of buffer
solutions PBS and РВ on the consumption of GSH in the
reaction with H2O2 is presented in Fig. 2. The influence
of buffer solutions PBS and PB on the yield of radicals in
this reaction, which was measured by the inhibitor method
from the consumption of acceptor А is shown in Fig. 3.
It is seen that in phosphate buffer solutions the rate of
GSH consumption (WGSH) increases and the rate of
radical formation decreases sharply. The oxidation of GSH
by air oxygen is a reason for increasing WGSH in PBS and
especially in РВ. In buffer solutions GSH was consumed
in a larger amount (5 mmol L–1) than it is required ac-
Reaction of glutathione with peroxyl radicals. The ki-
netic curves of consumption of anionic polymethine dye
А in the reaction with peroxyl radicals derived from AAPH
decomposition in water and in phosphate buffers PBS and
РВ are presented in Fig. 1. The kinetic characteristics of
the reaction of А with the formed peroxyl radicals have
been determined previously25: stoichiometric coefficient
f = 1 and rate constant of the reaction with peroxyl radicals
kROO = 5.4•105 L mol–1 s–1
.
The data in Fig. 1 show that in buffer solutions А is
consumed with the same rate, which is halved compared

Reaction of glutathione with H2O2 in PBS
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 7, July, 2019
1443
[GSH]/mmol L–1
10
alkyl radical, the product of thiyl radical addition to the
double bond of phenol.
Scheme 1
9
8
7
1
6
3
2
5
50
100
150
200 t/min
Fig. 2. Kinetic curves of consumption of glutathione (10 mmol L–1
)
in the reaction with H2O2 (2 mmol L–1) in water (1), PBS (2),
and phosphate buffer РВ (3).
D
In buffer solutions PBS and РВ the radical generation
rates are substantially lower than those in water and, hence,
a decrease in the rate of the thiol—ene reaction of GSH and
RVT can be expected. The consumption rates of GSH,
acceptor А, and RVT at the same initial concentrations of
the reactants are compared in Table 1. The consumption
rate of acceptor А in water and its doubled rate in buffer
solutions PBS and РВ determine the rate of radical ini-
tiation (Wi), and the WRVT/Wi ratio characterizes the
chain length () of the thiol—ene reaction. The data in
Table 1 show that the rate of RVT consumption decreased
less than by three times in spite of the strong decrease in
Wi in PBS. However, RVT is not consumed at all in phos-
phate buffer PB.
3
2
0.7
0.6
0.5
0.4
0.3
0.2
0.1
1
5
10
15
20
25
30
35
t/min
To conclude, these results explain in part why in nu-
merous publications on glutathione almost nobody men-
tions the formation of radicals in the reaction of GSH with
Н2О2 and the thiol—ene reaction with unsaturated phe-
nols. The unexpected substantial differences in effects of
PBS and РВ, which are close in ionic strength, on the
Fig. 3. Kinetic curves of consumption of acceptor А
(0.009 mmol L–1) in the reaction of GSH (10 mmol L–1
)
with H2O2 (2 mmol L–1) in water (1), PBS (2), and phosphate
buffer РВ (3).
cording to the stoichiometry of Eq. (1), whereas less than
4 mmol L–1 were consumed in water. A substantial effect
of NaCl and KCl in PBS (unlike sodium—potassium
phosphate buffer mixture PB) on the rate of GSH con-
sumption and Wi is remarkable. Under other equivalent
conditions, WGSH in PBS is seven times lower than that in
PB containing no chlorides, while Wi is three times higher.
Thiol—ene reaction of glutathione with resveratrol in
the presence of Н2О2. Thiyl radicals, which are formed
with relatively low rates in the reactions of GSH with
H2O2, can initiate chain processes of cis—trans-isomeri-
zation and thiol—ene reaction of GSH with unsaturated
compounds. We have previously shown23,24 that the reac-
tion of GSH with unsaturated phenol RVT in water is
satisfactorily described by Scheme 1, where RVT* is the
Table 1. Consumption rates of GSH, acceptor А, and RVT
upon the interaction of glutathione (10 mmol L–1) and Н2О2
(2 mmol L–1) in various aqueous media
Medium
Consumption rate
/mol L–1 s–1
*
GSH
А
RVT
Bidistilled water
18•10–7 5.3•10–9 7.8•10–8 14.7
Phosphate buffer—NaCl, 11•10–7 4.5•10–10 2.9•10–8 32
рН 7.4 (PBS)
Phosphate buffer
рН 7.2 (PB)
70•10–7 1.6•10–10 ~0
~0
* Chain length of the thiol—ene reaction.

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with RVT indicate a high sensitivity of GSH and GSH—
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This work was financially supported by the Russian
Foundation for Basic Research (Project No. 18-33-00742)
and in the framework of the state task of the Federal
Agency for Scientific Organizations of Russia (Project
No. 0082-2018-0006, registration No. AAAA-A18-
118020890097-1).
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Received February 9, 2019;
in revised form March 21, 2019;
accepted May 23, 2019