ATP-dependent modulation and autophosphorylation
of rapeseed 2-Cys peroxiredoxin
Martin Aran
1
, Daniel Caporaletti
1
, Alejandro M. Senn
1
, Marı
´a
T. Tellez de In
˜
on
2
, Marı
´a
R. Girotti
1
,
Andrea S. Llera
1
and Ricardo A. Wolosiuk
1
1 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina
2 INGEBI-CONICET, Buenos Aires, Argentina
Rather than viewing reactive oxygen species (ROS) as
toxic by-products of aerobic metabolism we now
know them to be members of signaling networks
that modulate important physiological processes [1,2].
Germane to the homeostatic regulation of ROS con-
centrations, a large group of peroxidases devoid of

Fax: +54 11 5238 7501
Tel: +54 11 5238 7500
E-mail:
(Received 12 November 2007, revised
14 January 2008, accepted 16 January
2008)
doi:10.1111/j.1742-4658.2008.06299.x
2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidas-
es that have been implicated in antioxidant defense and signal transduction.
Although their biochemical features have been extensively studied, little is
known about the mechanisms that link the redox activity and non-redox
processes. Here we report that the concerted action of a nucleoside triphos-
phate and Mg
2+
on rapeseed 2-Cys Prx reversibly impairs the peroxidase
activity and promotes the formation of high molecular mass species. Using
protein intrinsic fluorescence in the analysis of site-directed mutants, we
demonstrate that ATP quenches the emission intensity of Trp179, a residue
close to the conserved Cys175. More importantly, we found that ATP
facilitates the autophosphorylation of 2-Cys Prx when the protein is succes-
sively reduced with thiol-bearing compounds and oxidized with hydroper-
oxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the
phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl
[Prx-(Cys175)-SO
2
PO
3
2)
] and the sulfonic-phosphoryl [Prx-(Cys175)-SO
3

tures (kinetic control) have been less clear. Therefore,
the reactions leading to ROS generation and detoxifi-
cation have been elucidated, but little is known about
how oxidative stress is linked to non-redox processes
in the signaling networks that modulate cellular func-
tions. Studies addressing this issue have found signi-
ficant changes in the quaternary structure and dual
functions when human 2-Cys Prx is phosphorylated on
Thr90 by cyclin-dependent protein kinases, preferably
CDK1 (formerly Cdc2) [17,18]. A putative intermedi-
ate at the peroxidatic cysteine (-Cys-S(=O)-O-PO
3
2)
)
has recently been suggested in the multiple-step pro-
cess underlying sulfiredoxin-mediated reduction of
2-Cys Prx-SO
2
H, however, experimental evidence is lack-
ing [19–21]. As with many proteins, phosphorylation
of 2-Cys Prx via these two mechanisms requires the
participation of additional catalysts, i.e. protein kinase
and sulfiredoxin. Despite numerous studies showing
the close association between ATP and chaperone
activity [22], with the exception of serving as the phos-
phoryl donor for CDK1 and sulfiredoxin, the direct
interaction of a nucleotide with 2-Cys Prx has not been
previously addressed. Here, we report that the con-
certed action of a nucleoside triphosphate and Mg
2+

dent manner, purine nucleotides being more potent
than pyrimidine derivatives. In particular, the response
of the peroxidase activity to increasing concentrations
of ATP exhibited three well-defined stages: (a) monot-
onous decay (I
0.5
= 0.25 mm), (b) stabilization at half
of the maximal activity from 0.9 to 1.2 mm, and (c) a
sharp decrease to undetectable levels beyond 1.5 mm
(I
0.5
= 1.40 mm). Interestingly, inhibition mediated by
the other purine nucleotide, GTP, was significantly
similar in the first two stages, but lacked the third.
Following these initial experiments, we investigated
whether other phosphorylated compounds and bivalent
cations exhibited similar capacity. In the presence of
2mm Mg
2+
, the rate of H
2
O
2
removal was inhibited
by 60, 5 and 5% when it was assayed with 2 mm
ADP, AMP or orthophosphate, respectively (not
shown). By contrast, Mg
2+
was the most efficient
cation in assisting nucleotide-dependent inhibition

2-Cys Prx are molecular chaperones [10,11]. Therefore,
it was important to examine 2-Cys Prx beyond a single
activity and establish whether the regulation described
above previously had wider implications. We found
that the rapeseed orthologue efficiently prevents the
thermal aggregation of citrate synthase indicating that
the chaperone activity is likely to be a general function
of typical 2-Cys Prx (Fig. 1D). Remarkably, incorpo-
ration of increasing amounts of Mg
2+
into the incuba-
tion milieu led to a concomitant reduction in the
chaperone capacity which, at variance with the peroxi-
dase activity, was not affected by the presence of
2.5 mm ATP. These data provide the first evidence
that the dual functions of 2-Cys Prx can be differen-
tially regulated by ATP and Mg
2+
.
The interaction with ATP modifies structural
features of 2-Cys Prx
Given the essential role of ATP in the peroxidase
activity, we evaluated changes in the structure of
2-Cys Prx brought about by the concerted action of
the nucleotide and the bivalent cation. To accurately
determine the molecular mass of our 2-Cys Prx prepa-
rations, static light-scattering measurements were per-
formed, because this spectroscopic technique allows
direct estimation of the species in solution [23]. In
the absence of perturbants, the predominant form of

,Ca
2+
,Mn
2+
,Zn
2+
) were both fixed at 3 mM. (C) EGTA-mediated reversal of (ATP ⁄ Mg
2+
)-dependent inhibition of peroxidase activity.
2-Cys Prx (3 l
M) was incubated for 3 min with 2 mM ATP and 2 mM MgCl
2
. After the addition of EGTA to a final concentration of 5 mM, the
protein solution was further incubated for 5 min and the peroxidase activity was assayed as in (A). (D) Effect of ATP and Mg
2+
on the chap-
erone activity. 2-Cys Prx (5 l
M) was incubated in 25 mM Tris ⁄ HCl (pH 8) containing, as indicated, different concentrations of MgCl
2
and
2.5 m
M ATP. After 10 min at 25 °C followed by 10 min at 45 °C, the assay was started by the addition of citrate synthase and measured as
described in Experimental procedures [25].
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1452 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
rapeseed 2-Cys Prx (polypeptide: 22 316 kDa) had a
molecular mass of 260 kDa indicating that it was
essentially similar to counterparts from other sources,
wherein covalently linked dimers (a
2

protein. In line with this prediction, positive and nega-
tive differences in absorbance appeared following incu-
bation of 2-Cys Prx with ATP in the absence and
presence of Mg
2+
, respectively (supplementary
Fig. S1). Although these experiments confirmed an
interaction between the nucleotide ⁄ Me
2+
couple and
the protein, the differential response could not be
attributed specifically to any of the interacting species.
Therefore, we turned our attention to fluorescence
emission spectroscopy which provides information
about the polarity of local environments surrounding
either extrinsic probes that bind to proteins or intrinsic
fluorophores buried in the protein interior. In a first
set of experiments, we relied on a biophysical probe
commonly used to study the characteristics of protein
surfaces, 8-anilinonaphthalene-1-sulfonate (ANS),
which, as expected, exhibited an emission maximum
wavelength at 512 nm that was not modified by the
presence of 3 mm ATP or 3 mm Mg
2+
(Fig. 2A). At
variance, reflecting the affinity of this extrinsic probe
towards exposed protein hydrophobic surfaces,
2-Cys Prx led to a marked enhancement of the emis-
sion intensity with a concurrent displacement of the
A

filter effect, data were fitted to the saturation curve equation using
nonlinear least-squares regression analyses. The difference in fluo-
rescence (DF) between 2-Cys Prx (F
o
) and 2-Cys Prx-ATP-Mg
2+
complex (F) at 340 nm was plotted according to Lehrer [28] (inset).
(C) Quenching of emission intensity in W88F and W179F 2-Cys Prx.
Fluorescence measurements were performed as described in (B),
except that W88F and W179F mutants replaced for the wild-type
2-Cys Prx.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1453
spectrum to a maximum at 480 nm. At this stage, the
addition of 3 mm ATP and 3 mm Mg
2+
did not shift
the maximum emission wavelength, but progressively
increased the emission intensity, indicating that the
nucleotide and the bivalent cation significantly
enhanced the proportion of protein hydrophobic
patches.
Although these experiments were informative regard-
ing the ability of 2-Cys Prx to interact with ATP, it
was imperative to determine the nucleotide binding
site. This information could be gained from the intrin-
sic fluorescence because the constituent polypeptide
held two conserved tryptophan residues that exhibited
a maximum emission wavelength centered at 343 nm,
suggesting a rather polar environment around the in-

Æ[Q]})] [28,29].
As shown in Fig. 2B (inset), the straight line was con-
gruent with a unique tryptophan residue of 2-Cys Prx
accessible to ATP⁄ Mg (f
a
= 0.26; K
SV
= 9.7 · 10
)3
Æ
m
)1
). To unambiguously define the indol ring sensitive
to ATP ⁄ Mg, we examined the intrinsic emission fluo-
rescence in variants of 2-Cys Prx where Trp88 and
Trp179 were replaced conservatively by phenylalanine
via site-directed mutagenesis. The results in Fig. 2C
clearly illustrate that the marked reduction in emission
intensity caused by the quencher in W88F 2-Cys Prx
was similar to its wild-type counterpart, whereas the
W179F variant was insensitive to ATP ⁄ Mg. These
findings demonstrated that the ATP binds to 2-Cys
Prx close to Trp179 and, as a consequence, to the
resolving Cys175. In this study, two complementary
experiments indicated that the conservative replace-
ment of tryptophan residues did not lead to gross
alterations in the structure of 2-Cys Prx. First, the
emission spectrum of W88F 2-Cys Prx was similar to
its wild-type counterpart (k
max

remained in the solution. To our surprise, a 23 kDa-
labeled band appeared when the recombinant protein
was (a) incubated successively with 10 mm dithiothrei-
tol, 10 mm cumene hydroperoxide and [ c
32
P]ATP ⁄
Mg
2+
, (b) subjected to non-reducing SDS ⁄ PAGE, and
(c) characterized by Ponceau Red staining and autora-
diography (Fig. 3B). Although not shown, four control
experiments carried out under comparable conditions
were consistent with the specific covalent binding of
the phosphoryl moiety to 2-Cys Prx. First,
32
P-labeled
bands did not appear in the autoradiography when
chloroplast thioredoxin-m, chloroplast fructose-1,6-
bisphosphatase or a-lactalbumin were used in place
of 2-Cys Prx. Second, the autophosphorylation of
2-Cys Prx could not be attributed to artifacts linked to
the unspecific binding of the nucleotide, as neither the
presence of ADP, AMP or GTP, nor pulse and chase
experiments with 3 mm nonradioactive ATP affected
the incorporation of the
32
P-label into the protein.
Third, supporting the formation of a covalent link as
opposed to a protein highly resistant to SDS denatur-
ation [30], the radioactive label remained linked to

and W179F 2-Cys Prx to incorporate the
32
P-label
after successive incubations with dithiothreitol and
cumene hydroperoxide. As shown in Fig. 3C, the for-
mer variant was indistinguishable from wild-type
2-Cys Prx, whereas the latter was not functional. Near
Trp179, the resolving cysteine is an additional con-
served residue that can be predicted to interact with
ATP. Supporting this view, we estimated in modeling
work on 2-Cys Prx that the nitrogen atom in the indol
ring of Trp179 is located 1.571 and 0.401 nm from the
sulfur atoms of the peroxidatic and resolving cysteines,
respectively [31]. Taken together, the close proximity
to Trp179 and the requirement for sequential reduc-
tion fi oxidation raised the possibility that Cys175
was actively involved in incorporation of the phos-
phoryl moiety. Consistent with this, Fig. 3C shows
that a serine in place of Cys53 and Cys175 retained
and abrogated, respectively, the ability to incorporate
the
32
P-label into 2-Cys Prx. Notably, this active par-
ticipation of the resolving cysteine in the autophospho-
rylation uncovered a new function that departed
markedly from the known role in the peroxidase
activity.
Surprisingly, autophosphorylation of C53S
2-Cys Prx did not require successive incubation with
dithiothreitol and the hydroperoxide but it was extre-

32
P]ATP, (b) subjected to
non-reducing SDS ⁄ PAGE, and (c) transferred to nitrocellulose
membranes for protein estimation and autoradiography, as
described in Experimental procedures. (C) Role of conserved
tryptophan and cysteine residues. W88F, W179F, C53S and
C175S 2-Cys Prx were incubated, as indicated, with 10 m
M
dithiothreitol, 10 mM cumene hydroperoxide and [c
32
P]ATP prior to
non-reducing SDS ⁄ PAGE and autoradiography, as outlined in (A).
(D) Autophosphorylation of C53S 2-Cys Prx. C53S 2-Cys Prx was
incubated for 10 min only in the presence and absence of 10 m
M
dithiothreitol prior to the addition of [c
32
P]ATP, non-reducing
SDS ⁄ PAGE and autoradiography.
M. Aran et al. ATP modulates 2-Cys peroxiredoxin
FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS 1455
oxygen atoms to the respective 160–184 tryptic pep-
tide, we further analyzed the sequence of informative
ions to confirm the presence of a sulfinic group at
Cys175. Accordingly, fragment ions from y1 to y9
showed the expected mass for residues spanning
from Lys184 to Pro176, whereas trapped ions
beyond y10 exhibited a mass shift of 32. The
unequivocal assignment of two oxygen atoms to
the Cys175 residue of C53S 2-Cys Prx revealed the

masses matched the monosodium adducts [M + Na]
+
of the phosphorylated 160–184 peptide bearing sulfinic
and sulfonic groups, respectively (Fig. 5B) [32–34]. As
illustrated for the latter signal, sequence informative
y-ions from m ⁄ z 0 to 970 were identical to those
obtained in the spectra of m ⁄ z 2800.36 (Fig. 5A) and
2832.36 (see Fig. 4), thus proving that they originated
from the 160–184 peptide. But more importantly, the
absence of ions from y10 to y19 and the presence of
Fig. 4. MS ⁄ MS spectra of the 160–184 tryptic peptide from C53S 2-Cys Prx. Expanded view of peaks at m ⁄ z 2800.36 and 2832.35 and the
fragmentation of the peak at m ⁄ z 2832.35. 2-Cys Prx was digested with trypsin and prepared for MALDI-TOF MS as described in Experi-
mental procedures. Data were first collected, smoothed and calculated the centroid using the software
FLEXANALYSIS, and then plotted in
GRAPHPAD PRISM. All labeled peaks were at least three times above background. The amino acid sequence of the 160–184 tryptic peptide
bearing the sulfinic group is displayed above the spectrum. The fragmentation patterns that generate ions y and b are illustrated along the
peptide sequence wherein (*) are fragment ions bearing –SO
2
H.
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1456 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
shifted ions from y10* to y17* revealed that the sul-
fonic form of Cys175 held the monosodium adduct of
one phosphoryl group (-SO
3
PO
3
2)
) thereby providing
the first direct evidence for the phosphorylation of an

activity, whereas only Mg
2+
lowers the chaperone
capacity [10,11]. In addition to the differential regula-
tion of the dual functions, inhibition of the peroxi-
dase activity is highly specific because, of the
nucleotides presented here, purine derivatives are
markedly more effective than pyrimidine bases. Given
that nucleotides do not participate directly in the
reduction of hydroperoxides, it follows that the
observed loss of activity is almost certainly due to a
local effect on the structure of the protein (see
below). These findings are important for understand-
ing the fundamental question of how 2-Cys Prx uti-
lizes non-redox compounds to regulate the associated
functions and, in so doing, to cope with situations of
oxidative stress. This extremely rapid and reversible
association with low molecular mass compounds
devoid of redox capacity may have wide applicability
because we recently reported that 2-Cys Prx in con-
certed action with fructose-1,6-bisphosphate and
Ca
2+
stimulates the activity of chloroplast fructose-
1,6-bisphosphatase [25].
2-Cys Prx is an obligate homodimer (a
2
) whose con-
version to doughnut-shaped (a
2

binding of ATP ⁄ Mg
2+
halts the catalytic cycle via ste-
ric perturbation of the resolving cysteine. However, we
can not exclude the possibility that the reduction of
hydroperoxides is inhibited by an allosteric effect of
ATP ⁄ Mg
2+
on the peroxidatic cysteine. Although fur-
ther studies are required to clarify this issue, our data
definitively identify the region surrounding the resolv-
ing cysteine of typical 2-Cys Prx as the target for
nucleotides.
The main outcome of our study is, however, the
importance of oxyacid groups at the resolving Cys175
for the in vitro autophosphorylation of 2-Cys Prx. A
combination of evidence from the lack of a similar
capacity in other proteins to the behavior of site-direc-
ted mutants clearly dismiss the possibility that trace
quantities of contaminating bacterial kinases may co-
purify with the recombinant protein [38]. The finding
that the successive addition of a reductant and an oxi-
dant promotes incorporation of the c-phosphoryl moi-
ety of ATP indicates that, like other events mediated
by 2-Cys Prx, autophosphorylation depends on a spe-
cific redox state. The 23 kDa subunit contains two
cysteines conserved throughout evolution, and analyses
of site-directed mutants show that Cys175 holds the
unique reactive thiol involved in autophosphorylation.
Moreover, MS detection of over-oxidized sulfur atoms

the finding that the thiol of mammalian sulfiredoxin
[Srx-SH] recruits the c-phosphoryl moiety of ATP
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1458 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
yielding a thiophosphate [Srx-S-PO
3
2)
] led to the
proposal that sulfiredoxin subsequently transfers the
phosphoryl group to the sulfinic form of the peroxid-
atic cysteine in human PrxI [-Cys-S(=O)-OH] [19–21].
At this stage, the sulfinic–phosphoric mixed anhydride
[-Cys-S(=O)-O-PO
3
2)
] would be cleaved by a thiol
reductant [R-S-H] yielding a disulfide-S-monoxide
[-Cys-S(=O)-S-R] that would be finally reduced back
to thiol [-Cys-SH]. In this context, the strategy of our
phosphorylation of 2-Cys Prx diverges markedly from
previous studies in two important aspects: neither
requires a complementary catalyst, like cyclin-depen-
dent kinases or sulfiredoxin, nor proceeds via Thr91
or the peroxidatic cysteine. Indeed, our data provide
entry into a previously unsuspected mechanism by
which the successive reductionfioxidation of 2-Cys
Prx generates oxyacid groups at Cys175 for the subse-
quent formation of the sulfinic-phosphoryl [-(Cys175)-
SO
2

intercatenary disulfide bond with the sulfenic acid of
the peroxidatic cysteine. Moreover, in line with the
current paradigm on the mechanism for the reduction
of hydroperoxides [8], the resolving cysteines of try-
paredoxin peroxidase and AhpC from Trypanosoma
brucei brucei and Salmonella typhymurium have been
identified as targets in the reduction of the disulfide
bond for the reactivation of peroxidase activity
[41,42]. Against this background, we put forward a
new scenario wherein ATP interacts actively with
2-Cys Prx and, in so doing, modifies the quaternary
structure and associated functions. Moreover, the
unusual phosphorylation of Cys175 oxyacid groups
brings together the redox chemistry of the sulfur atom
and the phosphorylating capacity of ATP, thereby
providing a versatile mechanism wherein Cys175
appears as dual sensor able to perceive changes in the
redox and energy status of the cell. By virtue of the
flexibility of using redox and nonredox chemistries at
a single cysteine residue, the possibilities to process a
wide spectrum of stimuli into different cellular
responses greatly extend the prevalent view circum-
scribed to redox transformations of sulfhydryl groups
[43].
Experimental procedures
Materials
Recombinant rapeseed 2-Cys Prx was prepared as described
previously [25]. Biochemicals were purchased from Sigma-
Aldrich (St Louis, MO, USA).
Construction of 2-Cys Prx mutants

primer and the 5¢-flanking for the T7 promoter. After
amplification, all DNA fragments were cloned at the
XbaI ⁄ XhoI restriction sites of the pET-22b(+) vector and
mutations were confirmed by DNA sequentiation.
Protein purification
JM109 (kDE3) Escherichia coli cells harboring 2-Cys Prx
expression plasmids were grown at 37 °C in Luria–Bertani
medium supplemented with 100 lgÆmL
)1
ampicillin. After
induction with 0.6 mm isopropyl b-d-thiogalactoside, bacte-
ria were harvested by centrifugation (3000 g, 10 min),
washed with 20 mm Tris ⁄ HCl (pH 8.0), resuspended in the
same buffer containing 0.5 m NaCl and subjected to sonica-
tion. After centrifugation (20 000 g, 30 min), the superna-
tant fraction was loaded onto a Ni
2+
-iminodiacetate–
Sepharose column that was washed successively with
20 mm Tris⁄ HCl (pH 8.0) containing 20 and 100 mm imid-
azole. The fusion protein released in the latter elution was
dialyzed against and stored in 20 mm Tris ⁄ HCl (pH 8.0). A
molar extinction coefficient of 23.555 m
)1
Æcm
)1
at 280 nm
was used to estimate the protein content of homogeneous
2-Cys Prx preparations.
Assay of peroxidase activity

0.4 mL of 25 mm Tris ⁄ HCl (pH 8) was incubated for
10 min at 25 °C and subsequently for 10 min at 45 °C. The
assay was started by the addition of porcine heart citrate
synthase to a final concentration of 150 nm. Thermal aggre-
gation of citrate synthase was measured for 60 min by
monitoring light scattering at 360 nm in a Jasco FP 770
spectrofluorometer (excitation and emission light-paths: 0.2
and 1 cm, respectively).
Static light scattering measurements
The average molecular mass of 2-Cys Prx was determined
at 25 °C on a Precision Detectors PD2010 light-scattering
instrument connected in tandem to a Sephadex G-50 col-
umn and a LKB 2142 differential refractometer. 2-Cys Prx
(43 lm) was incubated for 5 min in 50 mm Tris ⁄ HCl
(pH 7.8) in the presence and absence of 3 mm ATP ⁄ 3mm
Mg
2+
and subsequently applied to a Sephadex G-50 col-
umn which had been equilibrated beforehand with the incu-
bation solution. The 90° light scattering and refractive
index signals of the eluting material were transferred to a
PC and analyzed with the discovery32 software supplied
by the manufacturer. The 90° light scattering detector was
calibrated using bovine serum albumin (66.5 kDa) as a
standard. Molecular masses were determined from the ratio
of the two detectors, light-scattering and refractive index,
using the Rayleigh-Debye-Gans light-scattering model for
dilute polymer solutions.
Intrinsic fluorescence measurements
The steady-state fluorescence of 2 lm 2-Cys Prx in 20 mm

o
) F)=1⁄ f
a
+(1⁄ {f
a
.K
SV
.[Q]}), where f
a
is a quen-
ching factor due to the presence of multiple fluorophores
ATP modulates 2-Cys peroxiredoxin M. Aran et al.
1460 FEBS Journal 275 (2008) 1450–1463 ª 2008 The Authors Journal compilation ª 2008 FEBS
in different environments. Nonlinear least-squares regres-
sion analyses were performed with the program graphpad
prism.
Autophosphorylation of 2-Cys Prx
Protein phosphorylation was carried out at 25 °Cin
0.04 mL of 50 mm Tris ⁄ HCl (pH 7.9) containing 10 lm
2-Cys Prx. The protein was incubated for 10 min at each
stage with (a) 10 mm dithiothreitol, (b) 10 mm cumene
hydroperoxide and (c) 0.1 mm (2.5 lCi) [c
32
P]ATP plus
3mm MgCl
2
. After the addition of SDS⁄ PAGE sample
buffer, proteins were transferred to nitrocellulose mem-
branes and detected by staining with Ponceau Red followed
by autoradiography. In control experiments, dithiothreitol

ProteinProspector ( />4.0.7/html/msprod.htm).
Acknowledgements
We acknowledge Gaston Mayol for technical help.
The assistance of Dr Silvia Moreno de Colonna, Dr
Gonzalo Prat Gay and Dr Leonardo Alonso with the
mass spectroscopy is gratefully appreciated. This study
was supported by grants from the Agencia Nacional
de Promocio
´
n Cientı
´
fica y Tecnolo
´
gica and the Uni-
versidad de Buenos Aires, and doctoral fellowships
from the Consejo Nacional de Investigaciones Cientı
´
fi-
cas y Te
´
cnicas (MA, DC, AS and MRG). MTI, ASL
and RAW are Established Investigators of the latter
institution.
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