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[Heat-induced activation of reducing properties of of sea water]

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It was shown using the Ellman's reagent that chloride and bicarbonate anions are the heat-induced reducing agents of sea-water, and their combined action is more than additive. Sulfate anions do not exhibit these properties. The influence of sea-water anions on the heat-induced production of hydrogen peroxide was studied by enhanced chemiluminescence in a peroxidase-luminol-p-iodophenol system. In NaCl and NaHCO3 solutions, at concentration and pH values equal to those of sea-water, the production of H2O2 upon heating increased, as compared with water, whereas sulfate anions depressed its formation. By using coumarin-3-carboxylic acid as a fluorescent detector of OH radicals, a substantial increase in the production of radicals in the presence of chloride and bicarbonate anions upon heating was shown. The effect is due to the electron donor properties of these anions, which lead to the decomposition of H2O2 with the formation of OH radicals. The results obtained were considered from the viewpoint of the equivalence of heat and electromagnetic radiation of an absolutely black body. It is supposed that the high-energy quanta of its spectrum lead to the dissociation of anions with the formation of a hydrated electron and radicals. Then a recombination of radicals with the formation of various molecular products takes place.
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INTRODUCTION
Physicochemically, natural waters are complex
nonstationary multicomponent redox system [1, 2]. It
is universally accepted that natural waters, including
seawater, are oxidizing media [3]. However, the redox
state of an aqueous medium is determined by a set of
redox reactions which take place in this medium
(some of these reactions yield reducing substances);
therefore, this state is subject to change, depending on
the time of day, season, and biogenic activity, and
varies up to a quasi-reducing state [3]. The oxidizing
properties of natural water are largely determined by
dissolved atmospheric oxygen. The one-electron re
-
duction of oxygen gives rise to reactive oxygen spe
-
cies, which can cause damage to biological mole
-
cules. Therefore, the necessity of protecting from
such a damage emerges [4]. It was previously shown
that heating activates redox processes in water and
aqueous solutions [5–8]. In pure water saturated with
atmospheric gases, an electron donor can be hydroxyl
ions [7]. The electron donor properties of hydroxyl
942
Biophysics, Vol. 48, No. 6, 2003, pp. 942–949. Translated from Biofizika, Vol. 48, No. 6, 2003, pp. 1022–1029.
Original Russian Text Copyright © 2003 by Bruskov, Chernikov, Gudkov, Massalimov.
English Translation Copyright © 2003 by MAIK “Nauka / Interperiodica” (Russia).
MOLECULAR BIOPHYSICS
Thermal Activation of the Reducing Properties
of Seawater Anions
V. I. Bruskov, A. V. Chernikov, S. V. Gudkov, and Zh. K. Massalimov
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences,
Pushchino, Moscow Region, 142290 Russia
E-mail: bruskov_v2000@mail.ru
Received June 11, 2003
A tiny atom in this world of troubles
Don’t be the fierce kT’s subordinate
Don’t stumble over everyday life’s hubbles
Hold on your way in your coordinate
L.A. Blumenfeld
AbstractUsing Ellman’s reagent [5,5-dithiobis(2-nitrobenzoic acid)], it was shown that thermally acti-
vated reducers in seawater are bicarbonate and chloride anions and that their joint effect is superadditive.
Sulfate anions do not exhibit such properties. By studying enhanced chemiluminescence in the
luminol–p-iodophenol–peroxidase system, the formation of hydrogen peroxide and the effect of seawater
anions on the thermally activated hydrogen peroxide production were investigated. In NaCl and NaHCO
3
solutions whose concentrations and pH are close to those for seawater, heating increases the H
2
O
2
produc-
tion more significantly than it increases the H
2
O
2
production in water, with sulfate anions suppressing the
formation of H
2
O
2
. Using coumarin-3-carboxylic acid, a fluorescent probe for hydroxyl radicals, it was
shown that heating considerably increases the OH
.
production in the presence of chloride and bicarbonate
anions. This increase is caused by the electron donor properties of these anions in the decomposition of hy-
drogen peroxide. The results obtained were considered from the standpoint of equivalence between heat and
electromagnetic blackbody radiation. It was assumed that the spectrum of this radiation contains signals of
high-energy quanta causing the dissociation of anions to form a hydrated electron and radicals. These radi
-
cals further recombine to yield various molecular products.
Key words: water; anions, reducing properties; hyperthermia; hydroxyl radicals; hydrogen production; hy
-
drated electron; thermal electromagnetic radiation; radicals, formation and recombination
Abbreviations: TNA, 5-thio-2-nitrobenzoic acid; 7-OH-3-CCA,
7-hydroxycoumarin-3-carboxylic acid.
BIOPHYSICS Vol. 48 No. 6 2003
ions were experimentally discovered for the first time
by EPR spectroscopy by L.A. Blumenfeld and col
-
leagues forty years ago [9]. However, this achieve
-
ment was not properly appreciated at that time, was
disbelieved, and passed into oblivion. Much later, the
fundamental possibility of the formation of an OH
.
e
aq
electron–radical pair from the hydroxyl ion was theo
-
retically substantiated by Kloss [10] without referring
to Blumenfeld and colleagues [9].
It has recently been shown that heating of sea
-
water gives rise to both hydroxyl radicals and reduc
-
ing substances [8]. It is generally assumed that elec
-
tron donors in biological systems are mainly cations
of transition metals, such as iron and copper. In this
work, we showed that, along with hydroxyl ions
[7–10], the reducing properties are also exhibited by
bicarbonate anions and chloride anions, which are
contained in seawater and can be thermally activated,
acting as electron donors, whereas sulfate anions do
not have such properties. The effect of the reducing
properties of bicarbonate and chloride anions on the
production of hydrogen peroxide and hydroxyl radi-
cals on heating was studied.
EXPERIMENTAL
Artificial seawater was prepared by dissolving
sea salt (Tropical Marine, UK) to a concentration of
34 g/l (pH 8.5) in double distilled water saturated
with air for a day. We also used solutions of the salts
NaCl (special-purity grade, Bio-Chemica, Germany)
and/or NaHCO
3
(USP Grade, Solvey, France),
Na
2
SO
4
(special-purity grade, Reakhim, Russia), and
also p-iodophenol (ICN, USA), peroxidase and super
-
oxide dismutase (Sigma, USA).
As a probe for the formation of electron donors
in a medium, we used Ellman’s reagent (5,5-dithio
-
bis(2-nitrobenzoic acid), Sigma, USA) [11, 12],
whose reduced form (5-thio-2-nitrobenzoic acid) has
acharacteristic absorption spectrum. The optical
density was measured at a wavelength of 412 nm
with a Uvikon 923 B spectrophotometer (Kontron
Instruments, Italy). The concentration of 5-thio-2-
nitrobenzoic acid was calculated at a molar extinc
-
tion coefficient of 12 000 M
–1
cm
–1
at the wave
-
length 412 nm [12].
To study the reducing properties of anions con
-
tained in seawater, a 1 mM 5,5-dithiobis(2-nitro
-
benzoic acid) solution in double distilled water was
prepared. In the experiment, this solution was diluted
with double distilled water to a final concentration of
50 µM (pH 4), and NaCl and/or NaHCO
3
was added
to a concentration of 30.97 g/l (0.53 M) or 0.196 g/l
(2.33 M), respectively, which corresponds to its con
-
centration in seawater [12]. Then, 1 M NaOH was
added until the pH of the solution was 8.5–8.7 and the
resultant solution in polypropylene vials was heated
in a U-10 ultrathermostat (Prüfgeräte-Werk Medin
-
gen, Germany).
Hydroxyl radicals were determined using a reac
-
tion with coumarin-3-carboxylic acid (Aldrich, USA).
7-Hydroxycoumarin-3-carboxylic acid, a product of
hydroxylation of coumarin-3-carboxylic acid, is a
convenient fluorescent probe for the formation of
these radicals [8]. The experimental procedure was
described in detail previously [8]. The formation of
hydrogen peroxide was determined by studying en
-
hanced chemiluminescence in the luminol–p-iodophe
-
nol–peroxidase system as described earlier [8]. Fresh
double distilled water had a conductivity of 1.7 µS. In
the experiments, water saturated with atmospheric
gases for a day was used. After saturation, the con-
ductivity was 4 µSand pH was 5.6. To eliminate the
effect of possible trace contaminants of transition
metal compounds, water and the salt solutions were
additionally treated with the chelator Chelex-100
(Bio-Rad, USA). Such treatment did not influence the
experimental results.
RESULTS
Heating of seawater samples containing 5,5-di
-
thiobis(2-nitrobenzoic acid) in the temperature range
of 40 to 60°Ccauses an increase in the absorption
A
412
, which is caused by the reduction of 5,5-dithio
-
bis(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid
[8]. The kinetics of this process is described by an
equation of a (pseudo)first-order reaction, which is in
-
dicated by the linearization of the kinetic curves in
semilogarithmic coordinates. The activation energy of
formation of a reducing substance on heating of sea
-
water was 20 kcal/mol. Introduction of 5,5-dithio
-
bis(2-nitrobenzoic acid) into seawater in various time
intervals after heating allowed us to determine the
half-life of the reducing substance, which was about
4 min. Transition metal ions in seawater are mainly in
their higher oxidation states [14]; therefore, their par
-
ticipation in the observed reaction as electron donors
is unlikely [8]. Seawater contains a significant
THERMAL ACTIVATION OF REDUCING PROPERTIES OF SEAWATER ANIONS 943
BIOPHYSICS Vol. 48 No. 6 2003
amount of carbon dioxide, whose solubility is many
times higher than the solubilities of nitrogen and oxy-
gen. Carbon dioxide dissolved in seawater at pH val-
ues characteristic of seawater is primarily in the form
of the bicarbonate anion
HCO
3
[14]. It is supposed
that the
HCO
3
ion is capable of one-electron oxida
-
tion and exhibits the properties of a reducer to yield
bicarbonate anion radical [15].
To check the ability of the bicarbonate anion
and also other inorganic anions contained in seawa
-
ter to act as reducers, the reducing properties of its
individual components were studied. The concentra
-
tions of salts in samples (0.53 M NaCl and 2.33 mM
NaHCO
3
)corresponded to their concentrations in
seawater [13], and the pH of the samples (8.5–8.7)
was taken to be equal to the pH of artificial seawater.
Figure 1 shows that the presence of both the chloride
anion (Fig. 1b) and the bicarbonate anion (Fig. 1c)at
the given concentrations in the heated samples of the
aqueous solution of 5,5-dithiobis(2-nitrobenzoic
acid) leads to the reduction of 5,5-dithiobis(2-nitro
-
benzoic acid) and to an increase in the 5-thio-2-
nitrobenzoic acid concentration as compared with
reference samples containing no inorganic anions
(Fig. 1a). Under the joint action of sodium chloride
and bicarbonate (Fig. 1d), the concentration of the
forming 5-thio-2-nitrobenzoic acid exceeds its con
-
centration reached in seawater on heating at the same
temperature for the same time (Fig. 1e). Note that
the joint effect of Cl
and
HCO
3
is superadditive.
Similar experiments with sulfate anions at a concen
-
tration of 28.87 mM, which is characteristic of sea
-
water [13], gave negative results.
We previously showed that heating of water and
aqueous solutions gives rise to hydrogen peroxide
[5–7]. By studying enhanced chemiluminescence in
the luminol–p-iodophenol–peroxidase system [5–7],
the formation of hydrogen peroxide in seawater on
heating and the effect of seawater anions on the hy
-
drogen peroxide production were investigated. The
kinetics of the reaction of formation of H
2
O
2
in sea
-
water has a complex quasi-oscillating character, as in
water [7] and phosphate buffer [5, 6]. In the initial lin
-
ear portion of the kinetic curve, the kinetics is de-
scribed by an equation of a pseudofirst-order reaction.
In this portion at temperatures of 40 to 65°C, the rate
constant varies from 1.4210
–8
to 1.9610
–7
,respec-
tively. The activation energy of this process as deter-
mined from the Arrhenius plot is 21 kcal/mol (Fig. 2).
The half-life of H
2
O
2
at a concentration of 10
–7
Mand
atemperature of 25°Cinseawater is about 2 h. Addi-
tion of superoxide dismutase [5–7] after heating for
4hat50°C increases the H
2
O
2
content by half. This
suggests the formation of superoxide radicals on heat-
ing of seawater. As one would expect, incubation of
heated seawater with catalase decreases the H
2
O
2
con
-
tent to background values.
The results of studying of the effect of individ
-
ual salts on the formation of H
2
O
2
are presented in
Fig. 3. Both 0.53 M NaCl (Fig. 3b)and2.33 mM
NaHCO
3
(Fig. 3c)atpH8.5 increase the hydrogen
peroxide production on heating under standard condi
-
tions by a factor of 2.5–2.6, whereas the joint action
of these salts (Fig. 3d)causes a superadditive increase
in the hydrogen peroxide production up to 80% of the
H
2
O
2
production in seawater (Fig. 3e). Sulfate anions
at a concentration of 28.87 mM, which is characteris
-
tic of seawater [13], at the pH value corresponding to
seawater significantly decrease the standard hydrogen
peroxide production in water down to zero.
It is known that hydrogen peroxide can be re
-
duced to form hydroxyl radicals in the presence of
transition metal ions, such as Fe(II) and Cu(I), which
944 BRUSKOV et al.
Fig. 1. Effect of anions Cl
and
HCO
3
contained in a
50 µM aqueous solution of 5,5-dithiobis(2-nitrobenzoic
acid) (pH 8.75) on the formation of 5-thio-2-nitrobenzoic
acid (TNA) as determined from the absorption A
412
after
heating at 50°C for 20 min: (a) reference sample, (b)
0.53 M NaCl, (c) 2.33 mM NaHCO
3
,(d) 0.53 M NaCl
and 2.33 mM NaHCO
3
, and (e) seawater (pH 8.7). Mean
values and their standard errors are presented (n = 4–6).
are electron donors, in the Fenton reaction. Hydroxyl
radicals can also, in principle, be produced by the re-
duction of hydrogen peroxide using the electron do-
nor properties of bicarbonate and chloride anions in
the reaction
HO =OH+OH
22 aq
+
−−
e
. (1)
To explore such a possibility, we used the highly
efficient OH radical trap coumarin-3-carboxylic acid.
7-Hydroxycoumarin-3-carboxylic acid, a product of
hydroxylation of coumarin-3-carboxylic acid, is a
specific fluorescent probe for these radicals [8]. The
samples studied contained 2.33 mM sodium bicarbon
-
ate and 0.53 M NaCl. Figure 4 illustrates the kinetics
of the reaction in water and solutions containing chlo
-
ride and bicarbonate anions at concentrations charac
-
teristic of seawater at the pH value corresponding to
seawater. Figure 4 shows that, in the presence of chlo
-
ride and bicarbonate anions, the production of hydro
-
xyl radicals significantly (by a factor of 1.75) in
-
creases. Addition of exogenous H
2
O
2
at a concentra
-
tion of 1 mM to a coumarin-3-carboxylic acid solu
-
tion containing NaCl and NaHCO
3
at concentrations
characteristic of seawater at the pH value correspond
-
ing to seawater on heating at 80°C for up to 1 h leads
to an additional increase in the rate of thermal genera-
tion of OH radicals, on the average, by a factor of 1.5.
DISCUSSION
Thus, along with hydroxyl ions [7–10], Cl
and
HCO
3
anions activated on heating of the solution act
as reducers and their joint effect exceeds the effect
characteristic of seawater. Heating activates the re
-
ducing properties of seawater anions Cl
and
HCO
3
,
and this increases the production of hydrogen perox
-
ide and hydroxyl radicals. The reducing properties of
the bicarbonate anion is 250 times higher than those
of the chloride anion, as indicated by the strength of
the effect and the ratio between the concentrations of
chloride and bicarbonate anions.
Note the nonequilibrium character of the pro
-
cesses studied. On heating, an additional energy is
supplied, and then, as the system is restored to the ini
-
tial temperature, it relaxes to the initial energy state.
As we showed earlier [8], the thermally activated re
-
ducing properties of bicarbonate and chloride anions
are rather brief and has a half-life of about 4 min.
The reducing activity of seawater anions on
heating is high and ranges from 5 to 20 µM (Fig. 1).
BIOPHYSICS Vol. 48 No. 6 2003
THERMAL ACTIVATION OF REDUCING PROPERTIES OF SEAWATER ANIONS 945
Fig. 2. Arrhenius plot of –logk versus inverse tempera
-
ture for determining the activation energy of the early
formation of H
2
O
2
in seawater on heating. k is the
pseudofirst-order reaction rate constant, s
–1
; T is temper-
ature, K.
Fig. 3. Thermally activated formation of H
2
O
2
on heating
solutions for3hat40°CatpHofwaterand the solutions
of 8.5: (a)water (n = 8), (b) 0.53 M NaCl (n = 2), (c)
2.33 mM NaHCO
3
(n = 2), (d)0.53MNaCl and 2.33 mM
NaHCO
3
(n = 3), and (e) seawater (n = 5).
This suggests that this activity cannot be caused by
transition metal impurities in water and the salt solu-
tions we used. Moreover, additional treatment of wa-
ter and the salt solutions with the chelator Chelex-100
did not influence the experimental results.
The previous data on the thermal generation of
hydrogen peroxide in double distilled water are indic
-
ative of the formation of a hydroxyl radical and a hy
-
drated electron from a hydroxyl ion by the reaction
OH
OH
.
+
e
aq
,and also suggest two interrelated
pathways of formation of H
2
O
2
on heating [7]. The
first pathway is the recombination of OH radicals.
This reaction is favored by the presence of electron
acceptors in water, one of which is usually oxygen
dissolved in water. A necessary step of the formation
of H
2
O
2
on heating is the transition of oxygen into the
singlet state [5–7]. Addition of electron to singlet ox
-
ygen yields superoxide anion radicals, whose dismut
-
ation gives rise to H
2
O
2
. The kinetics of the reaction
of formation of H
2
O
2
has a quasi-oscillating character
[5, 7]. Along with singlet oxygen, another most essen
-
tial acceptor of hydrated electron in water and aque
-
ous solutions is hydrogen ions H
+
in the reaction
2H = 2H H
aq 2
+−
+
2e=
, (2)
which yields a hydrogen molecule.
It is known that, under the action of light quanta,
a number of anions in polar solutions go into an ex
-
cited state and then dissociate to form a solvated elec
-
tron and anion radicals [16]. It was previously estab
-
lished that the photoexcited state (
A
aq
)* of anions
A
aq
can lead to thermally activated dissociation to
form an electron–radical pair comprising a hydrated
electron
e
aq
and an oxidized (radical) product
A
aq
[16]:
AA A
aq aq aq aq
−−
++
heν ()
*
. (3)
It was shown that a wide variety of anions, such
as Cl
,Br
,I
,OH
,
PO
4
2
,and
CO
3
2
, produce a hy
-
drated electron in process (3) in their flash photolysis
[17, 18]. Since matter at a given temperature emits
electromagnetic radiation similar to electromagnetic
blackbody radiation throughout the electromagnetic
radiation frequency spectrum [19, 20], one can as
-
sume that heat, much as light quanta, also causes a
similar process:
AAA
aq aq aq aq
−−
++
kT e()
, (4)
where kT means thermal electromagnetic radiation. In
their turn, radicals recombine to yield molecular
forms by the reaction
A
aq
+
A
aq
=A
2
, and if there are
a number of various radicals, cross recombination
products additionally form. Process (4) of formation
of an electron–radical pair on heating was previously
established for hydroxyl ions [8].
Thus, anions can act as reducers in the reaction
of formation of electron–radical pairs and the subse
-
quent recombination of radicals. The cross recombi
-
nation of radicals of different anions can be the cause
of the synergic superadditive effect observed in our
experiments.
Our results suggest that the formation of radical
products on heating has a universal character and is
qualitatively similar to the effects of UV and ionizing
radiation. We previously [7] demonstrated the simi
-
larity between the effects of heat and ionizing radia
-
tion on water, including the “oxygen effect” in both
cases. It was concluded that water thermolysis gives
rise to the same radicals and molecular products as
radiolysis by ionizing radiation does. Hyperthermia
with allowance for its features can, in some cases, be
regarded as a model of oxidative stress similar to the
effect of ionizing radiation. Note that it is water
thermolysis that causes the formation of ions OH
and
H
+
, which is accompanied by the rupture of a covalent
bond in the water molecule: this is suggested by the
BIOPHYSICS Vol. 48 No. 6 2003
946 BRUSKOV et al.
Fig. 4. Kinetics of formation of hydroxyl radicals in
0.5 mM coumarin-3-carboxylic acid solutions (pH 8.7)
obtained by dissolving the acid in (1) double distilled wa
-
ter and (2)asalt solution containing 0.53 M NaCl and
2.33 mM NaHCO
3
on heating at 80°Casdetermined
from the concentration of 7-hydroxycoumarin-3-carbo-
xylic acid (7-OH-3-CCA). Mean values and their stan-
dard errors are presented (n = 3).
strong temperature dependence of this process [21].
The increase in the degree of water dissociation into
ions with an increase in temperature should favor re
-
action (4).
Physically, water thermolysis can be explained
as follows. Thermal electromagnetic radiation, along
with components with averaged energies on the order
of kT, contains small numbers of high-energy quanta
whose energies significantly exceed kT. Since such
quanta are few, their induced high-energy processes,
which can lead to by the rupture of covalent bonds,
are slow. Besides, highly sensitive methods for de
-
tecting products of such reactions are necessary.
Note the well-established existence of a phys
-
icochemical mechanism for energy transformation of
weak influences into high-energy processes using at
-
mospheric air bubbles in water in sonoluminescence
[22–24]. Natural waters contain a significant number
of air microbubbles about 1–30 µmindiameter [2]
because of the hydrophobicity of air bubbles in such a
polar liquid as water.
In sonoluminescence in air bubbles, the ultra-
sonic energy is accumulated, increases by several or-
ders of magnitude, and is released through emission
of visible or UV radiation. Gas bubbles are sensors
and transducers (amplifiers and transformers) of rela-
tively low ultrasonic energy into giant fluctua-
tionselectromagnetic field energy bundles. The in-
stability of air bubbles manifests itself in the initial
expansion and the subsequent implosive compres
-
sioncollapse accompanied by a sharp increase in
the temperature (up to 10
5
K) and pressure within
the bubble. In this case, the gas bubble not only emits
photons but also acts as a microreactor, in which radi
-
cal products, such as hydroxyl radicals, form and
there are chemical processes accompanied by the for
-
mation of nitrite ions [24]. Moreover, under certain
conditions, gas bubble collapse leads to such a local
increase in temperature that a thermonuclear reaction
becomes possible [25].
One can assume that a small part of natural gas
microbubbles in solutions are capable of similarly
collapsing on heating to yield the same radical and
molecular products in solution as those produced by
ionizing radiation [7]. Previously, Domrachev et al.
[26] detected mechanochemically activated water de
-
composition by sonication to form hydrogen peroxide
in the absence of cavitation and in pumping air
through a filter and thin capillaries. Potselueva et al.
[27] found that extremely high frequency electromag
-
netic radiation causes the formation of hydrogen per
-
oxide in 50 mM carbonate buffer. It was established
that this process is characterized by the oxygen effect,
which consists in the participation of dissolved atmo
-
spheric oxygen in the formation of H
2
O
2
. Ikeda et al.
[28] showed that, in the presence of simple metal ox
-
ide catalysts, water molecules decompose to form ox
-
ygen and hydrogen on exposure to visible light and
even in the dark under vigorous stirring.
Hodgson and Fridovich [29] demonstrated that
OH radicals interact with carbonate ions to form car
-
bonate radicals. In their turn, carbonate radicals re
-
combine, emitting light quanta. This is indicated by
the quadratic dependence of the intensity of this emis
-
sion on the concentration of carbonate ions [29]. One
can suppose that the products of recombination of
(bi)carbonate radicals are peroxocarbonates [30]. Pre
-
viously, Klimov and Baranov [31] showed that bicar
-
bonate can act as an electron donor in water oxidation
in the water-oxidizing complex of photosystem II of
chloroplasts. It was also supposed bicarbonate is a
more preferable reducer than water and is an integral
component of the evolution of oxygen photosynthesis
in the Archean period [32].
The product of recombination of chloride radi-
cals is molecular chlorine, and the product of cross re-
combination of chloride and hydroxyl radicals is
hypochlorite. The possibility of the oxidation of the
chloride ion by hydrogen peroxide to form hypo
-
chlorite has recently been shown by Voeikov and
Khimich [33]. The electrochemical oxidation of chlo
-
ride ions in NaCl solutions to form molecular chlo
-
rine, hypochlorous acid, and sodium hypochlorite was
shown by Miroshnikov [34]. When considering the
formation of radical products from chloride and bicar
-
bonate anions on heating, account should also be
taken of the possibility of their reaction with super
-
oxide radicals forming by the addition of hydrated
electron to singlet oxygen [5–7].
Chloride ions in oxygenated aqueous solutions
are known to protect DNA from the destructive action
of ionizing radiation [35]. These data are consistent
with recent results [6] showing that the presence of
NaCl in solution decreases the oxidative damage of
guanine in DNA by reactyive oxygen species on heat
-
ing. It was also established that carbon dioxide at a
partial pressure close to the tension in the blood of
homoiothermal animals significantly inhibits the
BIOPHYSICS Vol. 48 No. 6 2003
THERMAL ACTIVATION OF REDUCING PROPERTIES OF SEAWATER ANIONS 947
generation of superoxide anion radicals by cells of
homoiothermal, poikilothermal, and unicellular or
-
ganisms [36]. Among other previously studied anions,
phosphate should be noted. Goncharova et al. [37]
demonstrated the possibility of the participation of in
-
organic phosphate as an electron donor in primary
photosynthetic reactions.
Thermal processes giving rise to radical prod
-
ucts in seawater and the blood plasma of homo
-
iothermal animals can be similar because the compo
-
sitions of these media are quite close. Therefore, pos
-
sible biological consequences of the thermal genera
-
tion of reactive oxygen species, and the effect of vari
-
ous anions on the generation of hydroxyl radicals and
hydrogen peroxide can be significant and diversified.
First of all, these are processes caused by intracellular
oxidative stressenhanced production of reactive ox
-
ygen species, which are associated with many patho
-
physiological consequences for the organism [4], in
-
cluding aging [38]. The thermal generation of reactive
oxygen species is a new convincing argument for the
free-radical theory of aging [6, 38], since for poikilo-
thermal organisms, ambient temperature is the most
significant factor determining the lifetime. The bio-
regulatory, bioenergetic, and information role of reac-
tive oxygen species in a number of biological pro-
cesses and the origin and evolution of life on the
Earth was considered by Voeikov et al. [39]. Hyper-
thermia causes damage to DNA and other cellular
structures and processes, which are mediated by reac-
tive oxygen species [6, 40], in particular, hydroxyl
radicals [41].
It seems topical to study further the reducing
properties of anions contained in seawater, biological
fluids, and intracellular space in the context of their
possible role in various biologically significant redox
processes. On this way, it will probably be possible to
arrive at scientifically substantiated understanding of
the therapeutic and preventive effect of natural min
-
eral waters. The results of this work predicted that
heating of salt solutions gives rise to such molecular
products as chlorine, hypochlorite, peroxocarbonate,
molecular hydrogen, and others. Experimental confor
-
mation of their formation will be the subject of further
studies.
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THERMAL ACTIVATION OF REDUCING PROPERTIES OF SEAWATER ANIONS 949
... Environmental factors, such as ionizing radiation, UV-light, and xenobiotics, stimulate ROS production in living organisms [1][2][3]. It has been demonstrated that heating can generate ROS in aqueous solutions [4][5][6][7]. Elevation of intracellular ROS can result in oxidative stress, which damages important biological molecules: DNA, lipids, proteins etc. [1][2][3]. All organisms possess systems of antioxidant defense, enzymes and low-molecular antioxidants, which control the level of ROS. ...
... Detection of hydrogen peroxide. Hydrogen peroxide was quantitatively assessed by using a sensitive method of enhanced chemiluminescence with luminol/p-iodophenol/peroxidase in 1 mM solutions of amino acids in phosphate buffer (5 mM, pH 7.4) after X-ray irradiation to 7 Gy and after heating for 200 min at 40°C [7]. Solutions were irradiated in Eppendorf tubes using an X-ray machine RUT-15 (Mosrentgen, Russia) and the following instrumental settings: power of 1 Gy/min, focal distance of 0.375 m, current of 20 mA, and voltage of 200 kV. ...
... It has been demonstrated that both ionizing irradiation and heating can induce reactive oxygen species [3][4][5][6][7]. Hydrogen peroxide is the most long-lived among ROS and is able to damage intracellular structures [1,2]. ...
Article
Full-text available
The action of 1 mM solutions of L-amino acids in 5 mM phosphate buffer, pH 7.4, on the production of hydrogen peroxide and hydroxyl radicals under the action of X-rays and heating has been studied. Hydrogen peroxide was estimated by the method of enhanced luminescence in a system luminol-paraiodophenol-peroxidase and hydroxyl radicals were determined by using the fluorescence probe coumarin-3-carboxylic acid. It was shown that amino acids can be divided by their influence on H202 formation into three groups: those that reduce the yield of H202, that do not influence it, and that increase it. A similar action of amino acids was observed upon heating, but the composition of the groups was different. All amino acids lowered the formation of hydroxyl radicals under the action of X-rays, and the most effective among them were Cys > His > Phe = Met = Trp > Tyr. Met, His and Phe lowered the amount of hydroxyl radicals by heating, Ser raised it, whereas Tyr and Pro did not change it. Thus, amino acids differently influence the formation of reactive oxygen species by the action of X-rays and heat, and some of amino acids reveal themselves as effective natural antioxidants.
... On the one hand, they lead to oxidative damage to biomolecules [1][2][3]; on the other, come to be important signal regulatory molecules [4,5]. Earlier in our works it has been shown that such physical factors as thermal impact [6,7], visible light [8,9], infrared radiation [10], electromagnetic radia tion of extremely high frequencies (EMR EHF) [11] lead to formation of ROS. A key step in processes of ROS formation under action of physical factors pre sents as transition of oxygen into singlet state and reduction of singlet oxygen to superoxide anion radi cal. ...
... Determination of hydroxyl radical concentration was done with the aid of a reaction of these radicals with CCA, the hydroxylation product of which-7 OH CCA-comes to be a fluorescent probe convenient for determining the formation of these radicals [7]. The fluorescence of the product of reaction of CCA with hydroxyl radical-7 OH CCA-was measured on a Cary Eclipse spectrofluo rimeter (Varian, Australia) with λ ex = 400 nm, λ em = 450 nm [22]. ...
... Chloride anion at a concentra tion of 1 mM practically does not influence the forma tion of ROS in aqueous solutions (Tables 1-3), although at concentrations close to physiological (about 120 mM) one observes an increase in ROS gen eration roughly three times. Earlier it has been shown that in the presence of chloride anions at a concentra tion of 530 mM one observes almost a four time more intense formation of hydrogen peroxide and hydroxyl radicals under action of heat [7], which agrees with data presented in the present article. Bicarbonate anion at all investigated concentrations 1-50 mM leads to additional generation of ROS, i.e. possesses essential prooxidant properties, these being the most pronounced as compared with the other investigated anions. ...
Article
Full-text available
The influence of biologically relevant anions (succinate, acetate, citrate, chloride, bicarbonate, hydroorthophosphate, dihydroorthophosphate, nitrite, nitrate) on the formation of hydrogen peroxide and hydroxyl radicals in water was studied under the effect of non-ionizing radiation: heat, laser light with a wavelength of 632.8 nm, corresponding to the maximum absorption of molecular oxygen, and electromagnetic radiation of extremely high frequencies. It has been established that various anions may both inhibit the formation of reactive oxygen species and increase it. Bicarbonate and sulfate anions included in the biological fluids and medicinal mineral waters have significant, but opposite effects on reactive oxygen species production. Different molecular mechanisms of reactive oxygen species formation are considered under the action of the investigated physical factors involving these anions, which may influence the biological processes by signal�regulatory manner and provide a healing effect in physical therapy.
... Hydrogen peroxide was assayed by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system as described previously [14], using a Beta-1 liquid scintillation counter (USSR) in the single photon counting mode (without the coincidence circuit). Figure 1 displays the dependence of the OG content in γ-irradiated double-helical (1) and denatured (2) DNA on the uranyl concentration. ...
... We have earlier demonstrated that heat causes ROS production in water [14,[33][34][35], and these ROS cause OG formation in DNA [34]. Here we show that these processes are enhanced in the presence of uranyl ions. ...
Article
Full-text available
Enzyme-linked immunosorbent assay with monoclonal antibodies against 8-oxoguanine was used to assess the formation of the latter in native and denatured DNA under the action of uranyl ions, γ-radiation, heating (37°C), and combinations thereof, in view of environmental pollution with uranium oxides resulting from the use of armor piercing shells with depleted uranium. The 8-oxoguanine content was found to change in a complicated multiphase manner suggesting that uranyl ions cause additional generation of reactive oxygen species leading both to formation of oxoguanine and to its further oxidation. Uranyl (5 μM) increased several times the thermal deamination of cytosine but did not influence the thermal apurination of DNA. It also increased the production of hydrogen peroxide and hydroxyl radicals in water at 37°C. These results are indicative of the pronounced chemical genotoxicity of uranyl ions and aggravation of DNA damage by heating and γ-irradiation.
... Rate constants and activation energy of this process have been determined, and the data extrapolated to a physiological temperature of 37°C [4]. Again, evidence has been obtained that ROS are formed in heated water and aqueous solutions [2][3][4][5][6] and cause formation of OG in DNA [4]. In the present work, direct experimental assessment of OG production in DNA at 37°C has given a paradoxical result. ...
... Here we have observed quasi-oscillations in the hydrogen peroxide content over prolonged incubation, probably due to formation of hydrated electrons, hydroxyl radicals, singlet oxygen, and superoxide radicals [2][3][4][5][6]. Oscillatory formation of OG during DNA oxidation in the Fenton reaction at 37°C has been noted [37]. ...
Article
Full-text available
The content of 8-oxoguanine, a most important marker of DNA damage by reactive oxygen species, in native and denatured DNA exposed at 37°C was examined by the enzyme-linked immunosorbent assay with monoclonal antibodies against 8-oxoguanine, and was found to change as much as twofold in a complicated multiphase manner. Much the same pattern was observed for the production of hydrogen peroxide in water or 1 mM phosphate buffer pH 6.8 over 50 h at 37°C as assayed by enhanced chemiluminescence in the luminol-p-iodophenol-peroxidase system. After heating the DNA at 80°C for 24 h and excising the guanine oxidation products with 8-oxoguanine-DNA glycosylase, the material was resolved by liquid chromatography on Sephadex LH-20 and Toyopearl HW-40 and the components were identified by their UV absorption spectra. The results suggested generation of reactive oxygen species at 37°C, with ensuing formation of 8-oxoguanine in DNA and its elimination by further oxidation to a number of unstable products, including imidazolone, spiroiminodihydantoin, and diiminoimidazole. The appearance of such products explains the occurrence of G:C→C:G transversions under the action of reactive oxygen species.
... Concentrations of hydroxyl-radicals were measured by fluorescence of 7-hydroxycoumarin-3-carboxylic acid (7-OH-CCA) [56]. 7-OH-CC is a product of coumarin-3-carboxylic acid (CCA) hydroxylation in presence of OH-radicals. ...
Article
Full-text available
A method for obtaining a stable colloidal solution of silver oxide nanoparticles has been developed using laser ablation. The method allows one to obtain nanoparticles with a monomodal size distribution and a concentration of more than 108 nanoparticles per mL. On the basis of the obtained nanoparticles and the PLGA polymer, a nanocomposite material was manufactured. The manufacturing technology allows one to obtain a nanocomposite material without significant defects. Nanoparticles are not evenly distributed in the material and form domains in the composite. Reactive oxygen species (hydrogen peroxide and hydroxyl radical) are intensively generated on the surfaces of the nanocomposite. Additionally, on the surface of the composite material, an intensive formation of protein long-lived active forms is observed. The ELISA method was used to demonstrate the generation of 8-oxoguanine in DNA on the developed nanocomposite material. It was found that the multiplication of microorganisms on the developed nanocomposite material is significantly decreased. At the same time, the nanocomposite does not inhibit proliferation of mammalian cells. The developed nanocomposite material can be used as an affordable and non-toxic nanomaterial to create bacteriostatic coatings that are safe for humans.
... In some cases, the equilibrium can be mixed, the process of decomposition of the formed hydrogen peroxide will be observed. Obviously, this process depends both on the type of the selected salt and on the storage method of the prepared solution [23]. ...
Article
Full-text available
In this work, we, for the first time, manufactured a plasma-chemical reactor operating at a frequency of 0.11 MHz. The reactor allows for the activation of large volumes of liquids in a short time. The physicochemical properties of activated liquids (concentration of hydrogen peroxide, nitrate anions, redox potential, electrical conductivity, pH, concentration of dissolved gases) are characterized in detail. Antifungal activity of aqueous solutions activated by a glow discharge has been investigated. It was shown that aqueous solutions activated by a glow discharge significantly reduce the degree of presence of phytopathogens and their effect on the germination of such seeds. Seeds of cereals (sorghum and barley) and fruit (strawberries) crops were studied. The greatest positive effect was found in the treatment of sorghum seeds. Moreover, laboratory tests have shown a significant increase in sorghum drought tolerance. The effectiveness of the use of glowdischarge-activated aqueous solutions was shown during a field experiment, which was set up in the saline semi-desert of the Northern Caspian region. Thus, the technology developed by us makes it possible to carry out the activation of aqueous solutions on an industrial scale. Water activated by a glow discharge exhibits antifungicidal activity and significantly accelerates the development of the grain and fruit crops we studied. In the case of sorghum culture, glow-discharge-activated water significantly increases drought resistance.
... Earlier it was found that thermolysis and photolysis of water result in formation of products typical of radiolysis of water [1]. Similarity of the radiolysis and thermolysis processes for sea water was first shown in [4]. These are paradoxical results, since quantum energy in thermolysis and photolysis of water induced by some laser radiations is insufficient for breaking the covalent bond in the water molecule and giving rise to and [1]. ...
... It is known that heat and visible light also induces generation of reactive oxygen species [49]. Hydrogen peroxide in the main end product in heat-or light-induced ROS generation in water in the absence of transition metals [50]. Hydrogen peroxide is a moderate oxidant that not only has a damaging effect on biomolecules, but also plays an important signaling and regulatory role in the organism [51]. ...
Article
Full-text available
A technology for obtaining materials from nanostructured nitinol with titanium- or tantalum-enriched surface layers was developed. Surface layers enriched with titanium or tantalum were shown to provide a decrease in the formation of reactive oxygen species and long-lived protein radicals in comparison to untreated nitinol. It was determined that human peripheral vessel myofibroblasts and human bone marrow mesenchymal stromal cells grown on nitinol bases coated with titanium or tantalum-enriched surface layers exhibit a nearly two times higher mitotic index. Response to implantation of pure nitinol, as well as nano-structure nitinol with titanium or tantalum-enriched surface layers, was expressed though formation of a mature uniform fibrous capsule peripherally to the fragment. The thickness of this capsule in the group of animals subjected to implantation of pure nitinol was 1.5 and 3.0-fold greater than that of the capsule in the groups implanted with nitinol fragments with titanium- or tantalum-enriched layers. No signs of calcinosis in the tissues surrounding implants with coatings were observed. The nature and structure of the formed capsules testify bioinertia of the implanted samples. It was shown that the morphology and composition of the surface of metal samples does not alter following biological tests. The obtained results indicate that nano-structure nitinol with titanium or tantalum enriched surface layers is a biocompatible material potentially suitable for medical applications.
... As known [42], uranyl ions are highly selective acceptors of hydrated electron which is produced in the water-air system by a number of anions, including hydroxyl anion, via formation of an electron-radical pair under the influence of various physical factors [43][44][45][46][47]. Uranyl ions were shown to significantly increase the yield of hydrogen peroxide and hydroxyl radicals in water under action of heat, light, and radiation [24,48], which is true of even the micromolar concentration range of uranyl nitrate [49]. ...
Article
Full-text available
Studies into the effects from environmental pollution by uranium compounds have been overviewed. Analysis of the impact of uranium oxides resulted from military operations using armor-piercing shells made of depleted uranium shows a predominance of chemical toxicity caused by the strong oxidizing power of uranyl ions. They induce oxidative stress through the generation of reactive oxygen species. As a result, oxidative damage to biomolecules and disruption of metabolic processes occur. Oxidative DNA damage causes long-term genotoxic effects in the form of mutagenesis, carcinogenesis, and other pathologies. The necessity of prohibiting the use of depleted uranium shells as chemical weapons of mass destruction has been substantiated.
... Известно, что ионы уранила являются высокоизбирательными акцепторами гидратированного электрона [42]. Установлено, что гидратированный электрон возникает в системе вода-воздух из ряда анионов, в том числе из гидроксил аниона, в результате образования электрон-радикальной пары при действии различных физических факторов [43][44][45][46][47]. Показано, что ионы уранила существенно увеличивают выход перекиси водорода и гидроксильных радикалов при тепловом, световом и радиационном воздействии на воду [24,48]. ...
Article
Full-text available
Результаты исследований, представленные в данном обзоре, касаются следующих тем: 1. Обедненный уран и токсичность при его использовании; 2. Токсичность урановых соединений (Химическая и радиационная компонента); 3. Образование активных форм кислорода под действием ионов уранила; 4. Эффекты ионов уранила на клеточном и организменном уравнях; 5. Защита организма от токсического воздействия урановых соединений.
Article
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The historical “radiation hypothesis” as a mechanism for activating unimolecular thermal dissociation of gas-phase molecules, long discredited on the authority of Langmuir, has been revitalized by the discovery and characterization of the process of thermal dissociation of trapped gas-phase ions by the ambient blackbody radiation field surrounding the ions. This development was made possible by improvements in Fourier transform ion cyclotron instrumentation that allowed long-time trapping of weakly bound cluster ions at extremely low pressures. Binding energies can be derived from measurements of these dissociation rate constants both by detailed kinetic modeling and by simpler Arrhenius temperature-dependence approaches, although the latter require special considerations for small molecules. These approaches have been applied to thermal dissociations of molecules, including cluster ions and large biomolecule ions.
Article
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We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-γ-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H2O2-mediated nitration of γ-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H2O2-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H2O2-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H2O2, suggest that SOD1/H2O2 oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H2O2 by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226–1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H2O2. However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.
Article
Excitation of the Fe(CN)64− ion in aqueous solution into the 1T1u or 1T2g excited singlet state, below 313 nm, leads to hydrated electron formation in competition with internal conversion to the lowest excited singlet 1T1g state. The limiting quantum yield of hydrated electron formation is a linear function of quantum energy between 313 and 228 nm, reaching &phgr;e≃ 0.9 at 228 and 214 nm. Above 313 nm hydrated electron formation is not observed, but photoaquation is. Dependence of &PHgr;e on scavenger (N2O) concentration is observed. The involvement of CTTS character in the process, and the role of rapid solvent rearrangement in the dissociation of the excited state, are discussed.
Article
DNA in oxygenated aqueous solution is protected from the effects of ionizing radiation by chloride ions, as measured by chromophore destruction. The mechanism of this protection is probably a change in the mode of decay of the DNA-OH radicals and not a change in the primary attacking species. Chloride ions have no effect on the radiation-induced destruction of nucleosides and nucleotides in neutral solution. The chloride effects observed for these compounds in acid solution can be attributed to conversion of OH radicals to Cl2− ion radicals, and differences in the reaction of the latter species with solutes. There is no observable chloride effect on thymine or adenine compounds. The reaction of Cl2− with pyrimidine nucleosides and nucleotides results in destruction of the glycosidic bond and the release of undamaged base. This effect is not observed with the purine compounds or with the deoxyribose compounds. The radical which results from the reaction of Cl2− with guanine compounds reacts efficiently to reform the parent compound.
Article
The weak luminescence that accompanies the aerobic xanthine oxidase reaction is inhibited by superoxide dismutase, by catalase, and by scavengers of hydroxyl radicals. It is also entirely dependent upon the presence of carbonate. It thus appears that the O2 and H2O2 produced during the aerobic action of xanthine oxidase interact to generate OH which, in turn, reacts with carbonate to yield the carbonate radical (CO3−). The species that is directly responsible for light emission appears to be produced by a dimerization of carbonate radicals, since the light intensity was a function of the square of the carbonate concentration. The data provide no reason to suppose that the light-emitting species is singlet oxygen.
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Nitric oxide is an important vasodilator which can be biologically produced from leukocytes and endothelial cells. However, it is highly unstable, which is an obstacle to detection and quantitation. We have exploited the reactivity of nitric oxide with thiols to establish an assay based on oxidation of thionitrobenzoic acid (TNB). The oxidation of thionitrobenzoic acid and the reaction with oxygen, which was measured by employing an oxygen electrode, were examined after the addition of nitric oxide solutions. The inhibition of aggregation of human platelets after challenge with 2.5 microM adenosine diphosphate was also investigated. These studies show the following properties of nitric oxide in aqueous solutions. (i) Nitric oxide is highly reactive to oxygen. (ii) Thiols react with a labile, highly reactive nitric oxide-oxygen product. (iii) Medium with very low oxygen content increases the life span of nitric oxide in aqueous solution. We also used the nitric oxide quantitation using TNB to study the metabolism of nitric oxide by porcine aortic endothelial cells and the results show that nitric oxide added to these cells in low oxygen content solution is stable. From these studies, we conclude that deoxygenated solutions stabilize nitric oxide. An important consequence of low oxygen content at localized tissue sites may be to augment biological effects mediated by nitric oxide.
Article
A methodology is described for the analysis of tissue SH components of a single tissue sample in terms of soluble and insoluble, reactive and unreactive, SH. The procedures utilize the SH reagent DTNB. GSH was estimated in the soluble reactive fraction by a kinetic analysis; the distinction of the other components is achieved using Millipore filters to partition soluble from insoluble, and detergent to render unreactive groups reactive. The method is illustrated with a set of data obtained from anesthetized rat cerebral cortex and single results obtained from other tissues.
Article
It has been hypothesized that hyperthermia promotes oxygen-centered free radical formation in cells; however, to date there is no direct evidence of this heat-induced increase in oxygen free radical flux. Using electron paramagnetic resonance (EPR) spin trapping, we sought direct evidence for free radical generation during hyperthermia in intact, functioning cells. Rat intestinal epithelial cell monolayers were exposed to 45 degrees C for 20 min, after which the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added. Compared to control cells at 37 degrees C, heat-exposed cells had increased free radical EPR signals, consistent with the formation of DMPO/.OH (aN = aH = 14.9 G). These findings indicate that heat increases the flux of cellular free radicals and support the hypothesis that increased generation of oxygen-centered free radicals and the resultant oxidative stress may mediate in part, heat-induced cellular damage.
Article
Living in an oxygenated environment has required the evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen known as reactive oxygen species. Here we review evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.
Article
It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.