JOURNAL OF

Inorganic

Biochemistry
Journal of Inorganic Biochemistry 98 (2004) 11511159
www.elsevier.com/locate/jinorgbio

Equilibrium characterization of the As(III)cysteine and the As(III)

glutathione systems in aqueous solution
a

Nicol_as A. Rey , Oliver W. Howarth , Elene C. Pereira-Maia

a

Departamento de Qu_mica ICEx, Universidade Federal de Minas Gerais 31.270-901 Belo Horizonte MG, Brazil
University of Warwick, Coventry CV4 7AL, UK

a,*

Centre for NMR, Department of Chemistry,

Received 27 November 2003; received in revised form 19 March 2004; accepted 24 March 2004
Available online 21 April 2004

Abstract
Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of
infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be
ecient in the treatment of acute promye-locytic leukemia. The exact mechanism of action has not been
elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins.
Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism.
This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in
aqueous solution.
The behavior observed for the As(III)glutathione system is very similar to that of As(III)cysteine. In both cases, the formation of two complexes in
aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry.

The formation constants of the cysteine complexes [As(H _1Cys)3], log K 29:846, and [As(H_2Cys)(OH)2] , log K
3_

2_

12:019, and of the glutathione complexes [As(H _2GS)3] , log K 32:06, and [As(H_3GS)(OH)2] , log K 103
were calculated from potentiometric and spectroscopic data.

In both cases, the [As(HL)3] species, in which the amine groups are protonated, predominate from acidic to
neutral media, and the [As(L)(OH)2] species appear in basic medium (the charges were omitted for the sake of
simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom
of cysteine. In the [As(L)(OH)2] species, the coordination sphere is completed by two hydroxyl groups. In both
_

cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH) 2O]
excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present
as [As(HL)3].

2004 Elsevier Inc. All rights reserved.

Keywords: Arsenic(III); Glutathione; Cysteine; Equilibrium studies; Spectroscopy

1. Introduction
The discovery of an organoarsenic compound
by Ehrlich and co-workers in 1909, salvarsan

(arsphena-mine), proven to be eective in the

treatment of syphilis, stimulated the
development of a wide range of arsenicals for
the treatment of infectious diseases [1]. More
re-cently, As2O3 has been reported to induce
complete re-mission in patients with acute
promyelocytic leukemia

Corresponding author. Tel.: +55-31-34226506; fax: +55-3134995700.

E-mail address: elene@apolo.qui.ufmg.br (E.C. Pereira-Maia).

0162-0134/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2004.03.010

[2]. Like other antitumoral drugs, arsenic has

been used in the clinical practice before its
mechanism of action being completely

elucidated. Arsenic trioxide was shown to

inhibit cell growth and induce apoptosis in
several cell lines [36]. However, the
mechanism of arsenic-induced apoptosis in
tumor cells remains unclear. Arsenite in-duces
the breaking of DNA strands, stimulates poly(ADP-ribosylation), induces an increase in
cellular levels of nitric oxide and superoxide,
and aects protein phosphorylation by binding
to vicinal thiols [7].
The eects of arsenic can be modulated by
combining it with other compounds, such as thiolcontaining mol-ecules. For example, Watson et al.
[8] found that some

1152

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

thiols protect cells from the toxic

eects of arsenite. Accordingly,
Dai and co-workers [4] observed
that the As2O3-induced apoptosis
was inversely related to intracellular glutathione (GSH)
concentration. In con-trast, Gurr et
al. [7] found that dithiothreitol
enhances arsenic trioxide-induced
apoptosis. The authors pro-posed
that arsenite can complex
dithiothreitol produc-ing a new
compound that has a higher
potency in inducing apoptosis.
St_yblo et al. [9] have shown that
arsenothiols were more potent
inhibitors of the gluta-thione
reductase than arsenicals.
The biometabolism of arsenic
involves a redox cycle between
As(V) and As(III) with subsequent
methylation of As(III), giving rise
to the mono, di- and trimethylated
derivatives. The reduction of
arsenate to arsenite can be
promoted nonenzymatically by
glutathione, however, in
physiological conditions, the
reduction by As(V) re-ductases
may predominate. Arsenic(III) is
methylated in the liver during its
hepato-enteric circulation by
meth-yltransferases [10]. Arsenic
is mostly excreted in urine in the
form of methylated metabolites,
being dimethylated arsenic the
major form [11]. Because of this
fact, bio-transformation was
during a long time considered a
detoxification mechanism. But
recently, it was proposed that, in
fact, methylation is a pathway for
arsenic acti-vation because
methylated forms can persist in
tissues and are more toxic than
inorganic arsenic(III) [12].
It is well known that arsenic(III)
has a high anity for sulfur
containing molecules such as
dithiothreitol [13] and glutathione
[14]. Scott et al. [15] isolated and
characterized an arsenite complex
of glutathione as As(SG)3 by mass
spectrometry. Delnomdedieu et al.

[16] found that glutathione

reduces arsenate to arsenite and
forms a (glutathione)3arsenite
complex.
Another point of interest when
considering the clin-ical use of
arsenicals is the resistance to
metalloid salts found in bacteria,
fungi, parasites and animals. In
bac-teria, the resistance system
responsible for detoxification of
metalloids transports arsenite out
of the cell. This arsenite-eux
system can be conferred by a
carrier protein (ArsB) or an aniontranslocating ATP-ase (Ar-sAB)
[17]. If arsenic is present as
arsenate, it must be reduced to
arsenite prior to extrusion. The
reduction is catalized by thiollinked reductases that use glutaredoxin, glutathione or thioredoxin
as reductants [18]. In eukaryotes,
another transmembrane protein,
MRP1, functions as an eux
pump. It has been shown that
tumor cells overexpressing MRP1
exhibit cross-resis-tance to
arsenite [19]. Resistance mediated
by MRP1 requires intracellular GSH
[20], suggesting that either GSH is
a co-transported substrate or the
transported substrate is an
arsenite complex of GSH. There is
a controversy in current literature
about the formation of a complex
between arsenic and GSH at
physiological pH values. The
formation of an As(glutathione)3
com-plex has already been
evidenced, but there is a lack of
information about the stability of
this species. So there is a need for
a better quantitative
understanding of the equilibrium
between As(III) and glutathione.
Once the stability constants of all
the relevant complexes formed
have been evaluated, one could
simulate spe-cies distribution
under physiological pH and GSH
concentrations.

2. Experimental

2.1. Reagents
Oxidized and reduced forms of
glutathione and DL-cysteine
hydrochloride were used as
obtained from Sigma. Stock
solutions of glutathione and
cysteine were prepared just before
use under nitrogen atmosphere to
prevent ligand oxidation. For NMR
experiments, the ligands were
dissolved in D2O. Stock solutions
of so-dium metaarsenite, also
from Sigma, contained 0.1 M of
perchloric acid.
2.2. Spectroscopic measurements
A Diode Array Hewlett Packard 8451
A spectrometer equipped with a
Masterline 2095 thermostat at 25 LC
was used for UV and visible
absorption measurements. Re-sults
are expressed in terms of the molar
absorption co-ecient e related to
the total concentration of the ligand.
The concentration of ligand used in
the spectroscopic measurements
_4

_2

varied from 2.0 _ 10 to 1.5 _ 10

M. The stability constants were
calculated from the spectrophotometric data with the SQUAD
algorithm [21].

NMR spectra were obtained using

a Bruker Avance DRX 400

spectrometer with
tetramethylsilane as an internal
standard. A small quantity of
acetone was ad-ded to the
samples and used as a secondary
reference (d 2.05). The
concentration of ligand used in the
_2
mea-surements was 1.5 _ 10 M.
2.3. Potentiometric studies
Potentiometric titrations were
performed by mea-suring the
electromotive force (emf) using
the Metrohm glass electrode
6.0102.100 (PB) as indicator and
the Metrohm calomel electrode
6.0702.100 (0.1 M NaCl solution)
as reference. A titroprocessor
Metrohm 670, coupled to a
Metrohm Dosimat 665
autoburette, was used to measure
the emf. Experiments were carried
out under nitrogen atmosphere
and the temperature was kept
constant at 25 LC. The ionic
strength was main-tained at 0.1 M
with sodium perchlorate. The
ligand concentration lay in the
_ 3
_2
range 4 _ 10 M to 1 _ 10 M and
the ligand-to-metal molar ratios
were in the range 50.5. The
electrode system was calibrated
for hydrogen ion concentration by
perchloric acid titration (or sodium

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

1153

hydroxide) with a standardized

sodium hydroxide so-lution (or a
perchloric acid solution) at ionic
strength of 0.1 M. The standard
L

electrode potential E and the

calculated water dissociation
constant (Kw) under the
experimental conditions employed
were used to calcu-late hydrogen

ion concentration (pH _ logH &)

from measured potentials. Stability
constants were calculated from the
potentiometric titration data with
the SU-PERQUAD [22] algorithm.

8.78a
10.71a

8.33b
10.50b
1.88c
8.15c
10.29c
1.69(2)d
8.17(1)d
10.30(1)d
(b) As(III)Cys

3. Results and discussion

3.1. Proton complexes

Species
log K

3.1.1. Potentiometric studies

Cysteine possesses three ionizable
protons: one in the carboxylic acid,
another in the thiol group and the
other in the ammonium group. The
pK of the carboxyl group can be
easily determined and identified
because it is much more acid than
the other groups. Inasmuch as the
acid strengths of the thiol and
ammonium groups are similar, it is
possible that there is an overlap
be-tween their dissociation. Thus,
the macroscopic con-stants
determined (Table 1) are
composite and cannot be assigned
to individual groups. Our
potentiometric
Table 1
Macroscopic dissociation constants (pKa) of
cysteine and glutathione and complex stability
constants for the As(III)Cys and As(III)GSH
systems
(a) Cys (H3Cys)

pK1
pK2
pK3
2.44a

[As(H_1Cys)3]
29.84(6)d

[As(H_2Cys)(OH)2]_
12.01(9)d

(c) GSH (H4GS)

pK1
pK2
pK3
pK4
2.60e
3.82e
9.16e
9.88e

3.59f
8.75f
9.65f
2.04g
3.54g
8.54g
9.42g
2.10(3)d
3.53(3)d
8.65(2)d
9.52(2)d
(d) As(III)GSH

obtained by other authors [2628].

3.1.2. Spectroscopic measurements
Species
log K

The H NMR spectrum of cysteine at

pH 7.0 consists of three doublets of
doublets: one of them due to the amethine proton at d 3.79, and the
two others to the non-equivalent bmethylene protons at d 2.84 and
2.90. The resonance frequencies,
their assignments and the cou-pling
constants are shown in Table 2.

[As(H_2GS)3]3_
32.0(6)d

[As(H_3GS)(OH)2]2_
10(3)d

Standard deviations are given in parentheses.

Ref. [27].
b
Ref. [23].
c

Ref. [24]. This work.

Ref. [28].

Ref. [26].

Ref. [27].

data are in a good agreement with

other values re-ported in the
literature [2325]. Clement and
Hartz [25] also did
spectrophotometric
measurements aiming the
calculation of the microscopic
constants. They con-cluded that
both the ionization of the thiol and
the ammonium groups contribute
to the second deproto-nation.
Since the thiol is more acidic it
makes a larger contribution.
The ionization pattern of
glutathione is more com-plex than
that of cysteine. Glutathione (c-Lglutamyl-L-cysteinylglycine)
contains four ionizable protons:
two in the carboxylic acids of the
L-glutamyl and the glycyl groups,
one in the L-cysteinyl sulfhydryl
group and the other in the Lglutamyl ammonium group. Since
the ionizations of both carboxylic
acid protons are close together
and so are the ionizations of the
sulfhydryl and the ammonium
groups, the macroconstants
determined are composite of the
microscopic constants for ionization from the individual groups.
Our macroscopic con-stants (Table
1) are in accordance with those

For GSH at pH 7, the two-cysteinyl

methylene pro-tons give rise to
closely spaced doublets of
doublets at d 2.74 and 2.80. At pH
9.5 these doublets are shifted towards lower frequencies and are
more separated, d 2.61 and 2.72.
The deprotonation, which occurs
mainly at the thiol group, produces
a shielding eect on the protons.
At pH 7.0, the cysteinyl a-methine
proton is overlapped by the
solvent signal around d 4.4, but at
pH 9.5 it ap-pears as a doublet of
doublets at d 4.09. At pH 7.0, the
glycyl methylene protons give rise
to a single signal at d 3.59, but at
pH 9.5 the signal splits into two
doublets at d 3.55 and 3.60. The
assignments shown on Table 2
were confirmed by 2D COSY
correlation (data not shown). The
1
analysis of the H NMR spectrum
of the glutathi-one molecule has
already been reported [15,16,29]
but the analysis presented here is
more complete.
The ionization of the sulfhydryl
proton can also be followed by
ultraviolet absorption. Until pH
6.5, a so-lution of cysteine
practically does not absorb in the
in-vestigated region 220290 nm.
From pH 7, the appearance of a
broad band centered at 230 nm
indi-cates the ionization of the
thiol. As the pH is increased, this
_
absorption, assigned to an n ! r
transition of thiolate, is intensified
and undergoes a bathochromic
shift to 236 nm. Above pH 11, the
absorbance is maxi-mal (e 3:95 _
3
_1
_1
10 M cm ) indicating the

complete deprotonation of the

thiol. For glutathione the same

1154
N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159
Table 2

H NMR data for cysteine and glutathione in D2O at pD 7

d
Multiplicity
J (Hz)

Cysteine

Cys Ha
3.79
dd
HaHb(1) 5.60; HaHb(2) 4.20
Cys Hb(1)
2.90
dd
Hb(1)Hb(2) 14.8; Hb(1)Ha 5.60
Cys Hb(2)
2.84
dd
Hb(2)Hb(1) 14.8; Hb(2)Ha 4.20
Glutathione

Glu Ha
3.62
appt
Glu HaGlu Hb 6.30
Glu Hb
2.021.96
m

Glu Hc
2.452.31
m

Cys Ha
#
#
#
Cys Hb(1)
2.80
dd
Cys Hb(1)Cys Hb(2) 14.2

Cys Hb(1)Cys Ha 5.12

Cys Hb(2)
2.75
dd

Cys Hb(2)Cys Hb(1) 14.2

Cys Hb(2)Cys Ha 7.24

Gly Ha
3.59
s

holds, being the maximal value of e 5:80 _

3
_1
_1
10 M cm at 232 nm.
3.2. Arsenic complexes
3.2.1. Potentiometric studies
Acidified mixtures of cysteine and sodium
metaarse-nite were titrated with NaOH. Data
ranging from pH 2.0 to 11.0, shown in Fig. 1,
correspond to solutions with four metal-toligand molar ratios, 1:5, 1:2, 1:1 and 2:1. This
set of data was used to calculate, simultaneously, the acid dissociation constants of
cysteine and the metalcomplex formation
constants.
Ligand protonation, arsenic(III) hydrolysis and
complexation can all be represented by the
following general equation:

2_

pH & qAsOH3& rH_2Cys &

Hp_r AsOH3_r qH_2Cys2_r &p_2r rH2O& 1

where p, q and r represent the stoichiometric

2_
coecients and H_2Cys represents the completely
deprotonated

cysteine. According to this notation,

protonation of this form of cysteine leads to
the species H_1Cys_, Cys, and HCys. The
corresponding formation constants of these
species are shown in Table 1.
The titration curves were divided in two sections,
which were separately analysed: the first one
from pH 2.0 to 8.4 and the second one from pH
8.5 to 11.0.
Up to pH 8.4, the best fit between experimental
and calculated titration curves was attained
assuming the formation of the complex
[As(H_1Cys)3], where the carboxylic group is
deprotonated, according to:

AsOH3 3Cys AsH_1Cys3& 3H2O

Above pH 8.5, calculations from potentiometric
data lead to the proposition of another
_

complex species [As(Cys)(OH)2] , where Cys is

completely deproto-nated. The following
equation represents its formation AsOH3
H_1Cys_ AsH_2CysOH2&_ H2O
In these species, the p, q and r coecients of Eq. (1)
are 6, 1 and 3 in the first case and 1, 1 and 1, in the
second one. The species [As(H_1Cys)3] should be
written more accurately as [As(HCys)3] and the
species

Fig. 1. Titration curves of solutions containing: 1, [Cys] 10 mM and [As(III)] 2 mM; 2, [Cys] 10 mM and [As(III)] 5 mM; 3, [Cys] 5
mM and [As(III)] 5 mM; 4, [Cys] 5 mM and [As(III)] 10 mM at 25 LC (I 0:1 M) with a solution of NaOH (0.1 M).

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

1155

[As(H_2Cys)(OH)2] , as [As(Cys)
_

(OH)2] . Thus, during the formation

_

of [As(Cys)(OH)2] from
[As(HCys)3] the number of protons
lost depends on the ligand excess.
The formation constants of the
arsenic(III) hydroxo complexes
_

2_

[As(OH)2O] and [As(OH)O2] were

deter-mined in a separate
experiment and were fixed during
the calculations of the equilibrium
constants involving cysteine or
glutathione.
The solution properties of
arsenous acid resemble that of
boric acid. From acid to neutral
media, ar-senic(III) exists in
solution mainly as As(OH)3. When
a solution of arsenic at pH 7.0 is
added to a solution of cysteine,
also at pH 7.0, the pH of the
resultant solution does not change
after complexation. Thus, the
forma-tion of the species
[As(HCys)3] does not involve the
liberation of protons to the
medium. Evidently arsenic
coordination to cysteine occurs
concomitantly with the
deprotonation of the thiol group
and the binding of the dissociated
proton to a hydroxyl group of
As(OH)3, releasing a water
molecule. The assignment of the
co-ordination site was made from
spectroscopic data and not from
potentiometry.
Several other putative species such
as [As(H-Cys)2(OH)] and its
deprotonated forms were tested for
the equilibrium model but the
SUPERQUAD algorithm rejected all.

It should be emphasized that the

same set of titration data was
used to calculate both the acid
dissociation constants of cysteine
and the arseniccysteine complex
formation constants. Furthermore,
the pKa values of the ligand

measured in the presence of

arsenic are in agreement with the
data given in the literature
indicating the validity of the
model. In the absence of arsenic,
but keeping the same physical
chemical conditions, the estimated pKa values are 1.69, 8.17
and 10.30 for the first, second and

third deprotonation of HCys ,

respectively. These values may be
compared with those estimated in
the presence of arsenic. From the
section of the titration
curves up to pH 8.4, we calculated
the first and second pKa values of
1.69 and 8.16, respectively. From
the data above pH 8.5, we
obtained the values of 8.17 and
10.30 for the second and third pKa.
All values agreed closely with
those determined in the absence
of arsenic. Fig. 2(a) shows the
species distribution of the AsCys
system calculated with the
program SCECS [30]. The
[As(HCys)3] species appears in the
range of pH 1.58.5, competing
with the As(OH)3 species. From pH
_

6.0, the [As(Cys)(OH)2] appears

and predominates between pH 8.0
and 9.5. With the increase of the
pH, the hydroxide ions exclude
two cysteine molecules from the
arsenic coordination sphere.
Above pH 10, the formation of
_

[As(OH)2O] leads to ligand

dissociation from the ar-senic
coordination sphere.
The co-ordination pattern of As(III)
to glutathione is similar to that of
cysteine, in spite of the complexity
of glutathione molecule.
Glutathione contains four ionizable protons instead of three in
cysteine. At pH 7, glu-tathione is
negatively charged, being
deprotonated at the two carboxyl
oxygens and protonated at the
thiol and ammonium groups.
As done for cysteine, acidified

mixtures of glutathione and

sodium metaarsenite were titrated
with NaOH. Data ranging from pH
2.0 to 8.4 were used to calculate,
simultaneously, the first and
second ligand acid disso-ciation
constants and the metalloid
complex formation constant. From
the part of the titration curves
above pH 8.5, the second and
third ligand deprotonations and
the metalloid-complex formation
constant were calculated (data not
shown). Despite the dierences

between glu-tathione and

cysteine, their interactions with
arsenic follow the same
coordination patterns and the
conclu-sions will be presented
concisely.
The best fit between experimental and
calculated ti-tration curves was
attained assuming the formation of
3
two complexes [As(HGS)3] _ and
2_

[As(GS)(OH)2] . In the former, the

ammonium group remains protonated

Fig. 2. Species distribution curves for the systems: (a) AsCys (1) As(OH)3; (2) As(OH)2O ; (3)
2 _
As(OH)O 2 ;
2 _
As(OH)O 2 ;

(4) [As(HCys)3]; (5) As(Cys)(OH)2] ; and (b) AsGSH (1) As(OH)3; (2) As(OH)2O ; (3)
3_

2_

(6) [As(HGS)3] ; (7) [As(GS)(OH)2] . In both cases the total ligand concentration is 15 mM
and the total As(III) concentration is 5 mM.

1156

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

and in the later, it is deprotonated.

In both cases, GSH is
deprotonated in the carboxylic
groups and in the thiol. Once
again, other species such as

concentration indicates a
stoichiometry equal to 1:3
metalloid-to-ligand (inset Fig. 3).
The curve reaches a plateau at
arsenic-to-cysteine molar ratio
equal to 2.

2 _

[As(HGS)2-(OH)] and its

deprotonated forms were also
included in the calculations but
rejected by the computer program. The pKa values of GSH
determined in the absence of
arsenic (2.10, 3.53 and 8.65) are
in good agreement with those
determined in its presence (2.10,
3.59 and 8.68). The similarities
with the AsCys system indicate
that the binding site is the
cysteinyl sulfur atom, in accordance with the spectroscopic
data. The species dis-tribution
curves for this system were
calculated by the SCECS program
and are shown in Fig. 2(b).
3.3. Spectrometric studies
The interactions of As(III) and the
two ligands were followed by UV
1

visible absorption and H nuclear

magnetic resonance spectroscopies
at two fixed pH values: 7.0 and 9.5.
1

H NMR spectra were used to assign the binding

site and follow the binding ability of the ligands
studied.

H NMR spectra of solutions

_2

containing 1.5 _ 10 M of
cysteine and various
concentrations of As(III) at pH 7.0
have been recorded (Fig. 3). By
increasing the concentration of
As(III) the b-methylene doublets of
doublets overlap, become broad
and deshielded, indi-cating
coordination to the sulfur atom.
The a-methine proton is also
deshielded but to a lesser extent.
The broadening of the peaks
probably results from the
75

quadrupole moment of the As

nucleus (I 3=2). The presence of
quadrupolar nuclei can result in
very eec-tive relaxation of any
spins to which they couple. A plot
of the methylene proton chemical
shift in function of the arsenic

A similar experiment was carried

out with GSH, and Fig. 4 shows the
variation of the cysteinyl bmethylene resonance frequencies
_2
of solutions containing 1.5 _ 10
M of GSH and dierent
concentrations of As(III), at pH 7.0.
As the amount of As(III) is
increased, all peak po-sitions are
shifted towards higher
frequencies, the most aected
signals being those of the
cysteinyl b-methylene protons,
indicating coordination of arsenic
via sulfhy-dryl group. A plot of the
methylene chemical shift against
metal-to-ligand molar ratio
indicates a stoichi-ometry of 1:3.
At pH 9.5, arsenic addition causes
a cysteinyl protons shift to higher
frequencies indicating
complexation (data not shown). At
this pH value, it was necessary to
add an excess of arsenic over the
ligand in order to attain saturation. The results suggest a
stoichiometry of 1:1.
In the UVvisible range, both ligands
practically do not absorb when the
thiol group is protonated. The
coordination of arsenic(III) to Cys or
to GSH strongly

Fig. 3. H NMR spectra of solutions containing

15 mM Cys and dierent As(III)
concentrations. Metalloid-to-ligand ratios of 0;
0.2; 0.33; 1.0 and 2.0 in D2O (I 1:5 M) at pH
7.0. Inset: Methylene protons chemical shift
against arsenic concentration.

modifies their spectra. As

observed in the potentiometric
study, the results are very similar
for both ligands and only the
spectrophotometric study of the
interactions with glutathione is
_2
shown in Fig. 5. A 1.5 _ 10 M
aqueous solution of GSH at pH 7.0
practically does not absorb in the
range 250280 nm, indicating a
low degree of ionization of the
_2

thiol group. In a 1.5 _ 10 M

aqueous solution of GSH at pH 7.0,
97% of the ligand exists as H2GS

2_

and 3% as HGS . The addition of

increasing concentrations of As(III)
induces the ap-pearance of a
shoulder around 280 nm, assigned
to a sulfur-to-arsenic charge
transfer transition. The satura-tion
is attained at arsenic
concentration equal to 3.2 _ 10
M. These data and also that
obtained for the

_2

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

1157

used and fixed

during the calculations. We tested
several equilibrium models and
the best fit allowed us to calculate
the stability constants (log b) of
3_
the complex species [As(HGS)3]
as equal to 33.28 _ 0.01 and
[As(HCys)3] as equal to 30.92 _
0.02. The dierences with respect
to that estimated from
potentiometric experiments (Table
1) are 1.28 and 1.08, respectively.
The estimated molar absorptivities
3_
of [As(HGS)3] can be seen in the
inset of Fig. 5(a). The molar
absorptivities of [As(HCys)3] and
_
Hcys calculated by the program
are also in a good agreement with
earlier data and are not shown.

Fig. 4. H NMR spectra of solutions

containing 15 mM GSH and dierent As(III)
concentrations showing the variation of the
cysteinyl b-methylene protons resonance
frequencies. Metalloid-to-ligand ratios of 0;
0.2; 0.33; 0.5; 1.0 and 3.0 in D2O (I 1:5
M) at pH 7.0. Inset: Cysteinyl b-methylene
protons chemical shift against arsenic
concentration.

As(III)cysteine system were

treated by means of the SQUAD
program, which searches for the
best combi-nation of stability
constants of the species to fit the
data and simultaneously
calculates the molar absorptivities
based on the current value of bpqr .
Not all stability constants and
molar absorptivities relevant to
the sys-tem need to be varied at
once. The constants of ligand
ionization and of arsenic
hydrolysis determined sepa-rately
by potentiometric titration were

As already said, As(III) in acid to

neutral aqueous solution is already
strongly complexed by hydroxide
ions, existing as As(OH)3.
Coordination to the SH group
occurs without liberation of
protons to the me-dium.
Therefore, the three protons of the
thiol groups bind to three hydroxyl
groups liberating three water
molecules, with subsequent
binding of arsenic to thio-late. As a
consequence, the calculated
stability constant does not
correspond to the formation of the
complex from free arsenic(III)
concentrations but from As(OH)3
concentration.
Similar experiments were carried
out at pH 9.5 (Fig. 5(b)) with both
cysteine and glutathione. As in the
previous case, due to the similarity
of the results, only those obtained
_2
for GSH are shown. In a 1.5 _ 10
M aqueous solution of GSH at pH
9.5, 6% of the ligand exists as
_

H2GS , 48% as HGS

3_

2_

and 46% as

GS . As described for pH 7.0,

As(III) addition induces the appearance of a shoulder around 280
nm, assigned to a sulfur-to-arsenic
charge transfer transition.

However, the absorption spectra

change further on adding an
excess of As(III). A molar ratio of
metalloid-to-ligand of 8:1 is
required to attain saturation. The
absorption data in the range 260
320 nm were used in the

calculation of the complex

formation constant by means of
the SQUAD program. The best fit
between experimental and
calculated spectra were obtained
assuming the

Fig. 5. UVVis spectra of solutions containing 15 mM GSH and As(III) concentrations varying
from 3 to 120 mM at 25 LC l 0:1 cm (I 1:5 M).
3_

(a) pH 7.0. Inset: Molar absorptivities of the complex species [As(HGS) 3] . (b) pH 9.5. Inset:
2_

Molar absorptivities of the complex species [As(GS)(OH)2] .

1158

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

showed by NMR that glutathione

initially reduces arsenate to
arsenite and then that an

Fig. 6. Proposed molecular structures of the As(III)

GSH complexes.

formation of only one complex of

1:1 stoichiometry. We found log b
equal to 11.54 _ 0.02 for [As(GS)
2_

(OH)2]

and 12.79 _ 0.02 for

_

[As(Cys)(OH)2] . The dierences

with respect to that estimated
from potentiometric ex-periments
(Table 1) are 1.54 and 0.78,
respectively. The species
distribution curves as a function of
arsenic concentration at pH 7.0
show that, even in the presence of
an excess of arsenic,
approximately 10% of the ligand
exists under the uncomplexed
_

form H2GS .
The proposed molecular structures
of the arsenic(III) complexes of
glutathione are represented in Fig.
6. In both cases the geometry
around arsenic is pyramidal. The
main dierences between these
complexes and the corresponding
ones formed by cysteine are the
net charge and the bulk. The
larger volume of glutathione does
not hinder the formation of these
complexes, be-cause of its
flexibility.
The equilibrium model proposed
explains the system arsenicglutathione consistently with some
previous lit-erature results and
clarifies some remaining
controver-sial points. It is already
known that arsenic(III) coordinates
to glutathione at low pH values
[16,31]. Delnomdedieu et al.

As(glutathione)3 complex is
formed. Although the au-thors did
not calculate the stability
constants for the species formed
in solution, they observed that the
complex was stable from pH 1.5 to
7.0. When the pH was raised, they
observed the appearance of free
gluta-thione in the solution
indicating the dissociation of the
complex, which led to the
proposition that at higher pH
values no complexation would
occur [16]. Here we identify for
the first time the presence of a
complex species in weakly basic
medium. According to our re-sults,
in the pH range 37 the main
species is the [As(HL)3] but above
pH 7.5, hydroxo ions displace two
ligand molecules from the arsenic
coordination sphere leading to the
formation of an [AsL] species.
Therefore, our results can explain
those of Delnomdedieu et al.
because when the pH of a solution
containing the As(glutathione)3
complex is raised above 7.5, the
pres-ence of free GSH is detected
due to the liberation of two ligand
molecules per complex to form the
1:1 complex. Our results indicate
that, under physiological conditions, arsenic(III) interactions with
glutathione should
also occur. Indeed, Kazuo et al.
[32] have identified by HPLCICPMS (high performance liquid
chromatog-raphyinductively
coupled argon plasma mass spectrometry) the presence of free and
GSH-conjugated arsenite
metabolites in the bile and urine
of rats.
Despite the importance of the
interactions with cys-teine side
chains in proteins for the
biochemistry of ar-senic, we did
not find equilibrium reports for this
system in the literature. However,
very early studies of the synthesis
of compounds for the treatment of

trypano-somiasis describe an
As(cysteine)3 complex that was
characterized by elemental
analysis [33]. Our results
demonstrate that at physiological
pH arsenic(III) forms a quite stable
complex with cysteine and
glutathione suggesting that
arsenic interactions with cysteine
resi-dues in polypeptides or
proteins in vivo may not be
neglected.
Despite the increasing interest in
arsenic chemistry in biological
systems, this work is the first to
present values for equilibrium
constants of the species formed
between the metalloid and the
amino acid cysteine or the intracellular tripeptide glutathione. The
knowledge of these complexes
stability is very important to the
evaluation of the role of thiolcontaining molecules in arsenic
biochemistry.

4. Abbreviations
GSH glutathione
Cys

cysteine

Acknowledgements
This work was supported by grants
of CNPq (Con-selho Nacional de
Desenvolvimento Cient_fico e Tecnologico,_ Brazil) and FAPEMIG
(Fundac~ao de Amparo _a
Pesquisa de Minas Gerais, Brazil).
Nicol_as A. Rey is grateful to CNPq
for the fellowship. The authors
thank Prof. Jos_e D. Souza Filho
and Ivana S. Lula for the
assistance during the NMR
experiments and Prof. H_elio A.
Duarte for helpful discussions.

References
Christiansen, J. Am. Chem. Soc. 42 (1920)
24022405.
Z.X. Shen, G.Q. Chen, J.H. Ni, X.S. Li, S.M.
Xiong, Q.Y. Qiu, J. Zhu, W. Tang, G.L. Sun, K.Q.
Yang, Y. Chen, L. Zhou, Z.W. Fang, Y.T. Wang, J.
Ma, P. Zhang, T.D. Zhang, S.J. Chen, Z.Y. Wang,
Blood 89 (1997) 33543360.
A. Bode, Z. Dong, Drug Resist. Update 3 (2000)
2129.
X.H. Zhu, Y.L. Shen, Y.K. Jing, X. Cai, P.M. Jia, Y.
Huang, W. Tang, G.Y. Shi, J. Dai, Z.Y. Wang, S.J.
Chen, T.D. Zhang, S. Waxman, Z. Chen, G.Q.
Chen, J. Natl. Cancer Inst. 91 (9) (1999) 772
778.

N.A. Rey et al. / Journal of Inorganic Biochemistry 98 (2004) 11511159

1159
[5] J. Dai, R.S. Weinberg, S. Waxman, Y. Jing, Blood 93 (1) (1999)
[19] P. Borst, A.H. Schinkel, Trends Genet. 13 (1997) 217222.
268277.

[20] G.J.R. Zaman, J. Lankelma, O. Vantellingen, J. Beijnen, H.

[6] X. Cai, Y.L. Shen, Q. Zhu, P.M. Jia, Y. Yu, L. Zhou, Y. Huang,
Deckker, C. Paulusma, R.P.J. Oudeelferink, F. Baas, P. Borst,
J.W. Zhang, S.M. Xiong, S.J. Chen, Z.Y. Wang, Z. Chen, G.Q.
Proc. Natl. Acad. Sci. USA 92 (1995) 76907694.
Chen, Leukemia 14 (2) (2000) 262270.
[21] D.J. Leggett, W.A.E. McBryde, Anal. Chem. 47 (1975) 1065
[7] J. Gurr, D. Bau, F. Liu, S. Lynn, K. Jan, Mol. Pharmacol. 56
1070.
(1999) 102109.
[22] P. Gans, A. Sabatini, A. Vacca, J. Chem. Soc., Dalton Trans. 6
[8] R.W. Watson, H.P. Redmond, J.H. Wang, D. Bouchier-Hayes, J.
(1985) 11951200.
Leukoc. Biol. 60 (1996) 625632.
[23] D.D. Perrin, I.G. Sayce, J. Chem. Soc. A 1 (1968) 5357.
[9] M. Styblo,_
S.V. Serves, W.R. Cullen, D.J. Thomas, Chem. Res.
[24] A.E. Martell, R.M. Smith, in: A.E. Martell, R.M. Smith (Eds.),
Toxicol. 10 (1997) 2733.
Critical Stability Constants, vol. 1, Plenum Press, New York,
[10] D.J. Thomas, M. Styblo,_
S. Lin, Toxicol. Appl. Pharmacol. 176
1974, p. 47.
(2001) 127144.
[25] G.E. Clement, T.P. Hartz, J. Chem. Educ. 48 (6) (1971)
[11] T.K. Suzuki, T. Tomita, Y. Ogra, M. Ohmichi, Chem. Res.
395397.
Toxicol. 14 (12) (2001) 16041611.
[26] D.D. Perrin, A.E. Watt, Biochim. Biophys. Acta 230 (1971) 96
[12] M. Styblo,_
Z. Drobna,_ I. Jaspers, S. Lin, D.J. Thomas, Environ.
104.
Health Perspect. 110 (2002) 767771.
[27] A.M. Corrie, M.D. Walker, D.R. Williams, J. Chem. Soc., Dalton
[13] W.B.T. Cruse, M.N.G. James, Acta Crystallogr. Sect. B 28 (1972)
Trans. 11 (1976) 10121015.
13251331.

[28] N.C. Li, O. Gawron, G. Bascuas, J. Am. Chem. Soc. 76 (1) (1954)
[14] M. Delnomdedieu, M.M. Basti, J.D. Otvos, D.J. Thomas, Chem.
225229.
Res. Toxicol. 6 (1993) 598602.
[29] D.L. Rabenstein, J. Am. Chem. Soc. 95 (1973) 27972803.
[15] N. Scott, K.M. Hatlelid, N.E. MacKenzie, D.E. Carter, Chem.
[30] H.A. Duarte, S. Carvalho, F.F. Campos Filho, E.B. Paniago,

Res. Toxicol. 6 (1993) 102106.

Qu_m. Nova 17 (5) (1994) 397404.
[16] M. Delnomdedieu, M.M. Basti, J.D. Otvos, D.J. Thomas, Chem.[31] M. Salerno, M. Petroutsa, A. Garnier-Suillerot, J. Bioenerg.
Biol. Interact. 90 (1994) 139155.
Biomem. 34 (2) (2002) 135145.
[17] B.P. Rosen, Trends Microbiol. 7 (5) (1999) 207212.
[32] T.S. Kazuo, K.M. Badal, O. Yasumitsu, Talanta 58 (2002) 111
[18] R. Mukhopadhyay, B.P. Rosen, Environ. Health Perspect. 110
119.
(2002) 745748.
[33] J.M. Johnson, C. Voegtlin, J. Biol. Chem. 89 (1930) 2731.