Báo cáo khoa học: Rhodanese–thioredoxin system and allyl sulfur compounds Implications in apoptosis induction docx

Rhodanese–thioredoxin system and allyl sulfur
compounds
Implications in apoptosis induction
Renato Sabelli
1
, Egidio Iorio
2
, Angelo De Martino
3
, Franca Podo
2
, Alessandro Ricci
2
,
Giuditta Viticchie
`
1
, Giuseppe Rotilio
3
, Maurizio Paci
1
and Sonia Melino
1
1 Department of Sciences and Chemical Technologies, University of Rome ‘‘Tor Vergata’’, Italy
2 Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanita
`
, Rome, Italy
3 Department of Biological Sciences, University of Rome ‘‘Tor Vergata’’, Italy
The induction of programmed cell death by sulfane
sulfur compounds [alk(en)yl thiosulfate, selenodigluta-
thione, allyl disulfide, etc.] poses significant questions

concerning their metabolism and on the role of the
enzymes involved in the cancerogenesis. Toohey [1]
suggested that uncontrolled proliferation of neoplastic
cells is a result of sulfane sulfur deficiency or overacti-
vity of the enzymes involved in their metabolism.
Several organosulfur compounds (OSCs) such as
diallyl disulfide, diallyl trisulfide, allicin and (more
Keywords
garlic; mobile lipids; sodium 2-propenyl
thiosulfate (2-PTS); sulfane sulfur;
sulfurtransferase
Correspondence
S. Melino, Dipartimento di Scienze e
Tecnologie Chimiche, University of Rome
‘‘Tor Vergata’’, via della Ricerca Scientifica
00133-Rome, Italy
Fax: +39 0672594328
Tel: +39 0672594449
E-mail:
E. lorio, Department of Cellular Biology and
Neurosciences, Istituto Superiore di Sanita
´
,
Viale Regina Elena, 299 00161 Rome, Italy
Fax: +39 06 49387143
Tel: +39 06 49902548
E-mail:
(Received 7 March 2008, revised 30 May
2008, accepted 3 June 2008)
doi:10.1111/j.1742-4658.2008.06535.x

Sodium 2-propenyl thiosulfate, a water-soluble organo-sulfane sulfur com-
pound isolated from garlic, induces apoptosis in a number of cancer cells.
The molecular mechanism of action of sodium 2-propenyl thiosulfate has
not been completely clarified. In this work we investigated, by in vivo and
in vitro experiments, the effects of this compound on the expression and
activity of rhodanese. Rhodanese is a protein belonging to a family of
enzymes widely present in all phyla and reputed to play a number of dis-
tinct biological roles, such as cyanide detoxification, regeneration of iron–
sulfur clusters and metabolism of sulfur sulfane compounds. The cytotoxic
effects of sodium 2-propenyl thiosulfate on HuT 78 cells were evaluated by
flow cytometry and DNA fragmentation and by monitoring the progressive
formation of mobile lipids by NMR spectroscopy. Sodium 2-propenyl thio-
sulfate was also found to induce inhibition of the sulfurtransferase activity
in tumor cells. Interestingly, in vitro experiments using fluorescence spec-
troscopy, kinetic studies and MS analysis showed that sodium 2-propenyl
thiosulfate was able to bind the sulfur-free form of the rhodanese, inhibit-
ing its thiosulfate:cyanide-sulfurtransferase activity by thiolation of the
catalytic cysteine. The activity of the enzyme was restored by thioredoxin
in a concentration-dependent and time-dependent manner. Our results sug-
gest an important involvement of the essential thioredoxin–thioredoxin
reductase system in cancer cell cytotoxicity by organo-sulfane sulfur com-
pounds and highlight the correlation between apoptosis induced by these
compounds and the damage to the mitochondrial enzymes involved in the
repair of the Fe–S cluster and in the detoxification system.
Abbreviations
DCF-DA, 2’, 7’-dichlorodihydrofluorescein diacetate dye; 2-PTS, sodium 2-propenyl thiosulfate; ESI, electrospray ionization; GSH, reduced
glutathione; GSSG, oxidized glutathione; OSC, organosulfur compound; PCho, phosphocholine; RhdA(E), sulfur-free form of rhodanese;
RhdA(ES), sulfur-loaded rhodanese; RhdA, recombinant rhodanese from Azotobacter vinelandii; RhdA-PS, propexylsulfide-form of rhodanese;
ROS, reactive oxygen species; Trd, thioredoxin reductase; Trx, thioredoxin; TST, thiosulfate:cyanide sulfurtransferase.
3884 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS

recently), n-propyl thiosulfate and sodium 2-propenyl
thiosulfate (2-PTS) have been shown to suppress the
proliferation of tumor cells in vitro through the induc-
tion of apoptosis [2–7]. The biochemical mechanisms
underlying the antitumorigenic and antiproliferative
effects of garlic-derived OSCs are not yet fully under-
stood, although it seems likely that the rate of clear-
ance of allyl sulfur groups from cells is a determinant
of the overall response. It has also been hypothesized
that a reduced or incorrect functionality of enzymes
involved in the metabolism of OSCs may cause an
excess of seleno or sulfane sulfur compounds in the
cell, inducing the onset of degenerative states and
apoptosis. The in vitro study of their interaction with
enzymes probably involved in the metabolism of sulfur
may explain their in vivo effects. Sulfurtransferases,
enzymes that act at the borderline between sulfur and
selenium metabolism, may have an important role in
the processes induced by these sulfur compounds. In
particular, rhodanese displays a sulfurtransferase activ-
ity in vitro, transferring the sulfane sulfur atom from
thiosulfate to cyanide to produce thiocyanate by a
double-displacement mechanism [thiosulfate:cyanide
sulfurtransferase EC 2.8.1.1, (TST)] [8]. In this activity,
rhodanese cycles between two stable intermediates,
namely a sulfur-loaded form (ES) and a sulfur-free
form (E). The observed abundance of potentially func-
tional rhodanese-like proteins suggests that members
of this homology superfamily (accession number:
PF00581; may play

distinct biological roles [9,10]. Rhodanese modules,
either alone or in combination with other proteins, can
perform a variety of roles, ranging from transport
mechanisms of sulfur ⁄ selenium in a biologically avail-
able form [11–13], to the modulation of general
detoxification processes [8] and to the restoration of
iron–sulfur centres in Fe–S proteins, such as ferredoxin
[14]. In the last few years, many different studies have
supported the hypothesis that the rhodanese-like pro-
teins have roles in ‘managing’ stress tolerance and in
maintaining redox homeostasis [15–18]. It is also note-
worthy that a study, using microarray to examine the
gene expression in colonic mucosa from cancerous and
normal tissues, hypothesized that a possible cause of
colorectal cancer carcinogenesis might be located in
the mitochondria and identified the rhodanese gene as
one out of three mitochondrial genes that had a statis-
tically significant decrease in expression from normal
tissue to tumor at every Dukes’ stage A–D [19,20].
More recently an increase in TST expression was
observed in colonocyte differentiation. In a human
colon cancer cell line, TST activity and expression were
significantly increased by butyrate and by histone-
deacetylase inhibitors, which promote the differentia-
tion of these cells [21]. Thus, butyrate could protect
the colonocytes from sulfide-induced cytotoxicity, thus
promoting TST expression. [21]. Down-regulation of
rhodanese gene expression has also been observed in
diseases such as Friedreich’s ataxia [22]. The associa-
tion between the expression of rhodanese and degener-

ative states might make rhodanese a potential
tumor ⁄ disease biomarker and treatment target. Not all
cells are equally susceptible to the deleterious effects of
the garlic sulfur compounds and, in particular, non-
neoplastic cells are less susceptible [3,23]. Therefore,
the greater sensitivity of tumor cells to these com-
pounds may be related to a down-regulated expression
of TST, which leads to a reduced rate of clearance of
these compounds; notably, epidemiological investiga-
tion has revealed that increased consumption of garlic
diminishes the risk of stomach and colorectal cancers
[24–27]. To explore the importance of rhodanese in the
metabolism of the allyl-sulfur compounds, we investi-
gated, in the present work, the effect of some natural
sulfur constituents of garlic on TST activity. In
particular, we studied the interaction of 2-PTS with
rhodanese and investigated the effect of this OSC on
the expression and activity of rhodanese. Sodium
2-propenyl thiosulfate is present in aqueous garlic
extract [28] and has an anti-aggregator effect in vitro
on both canine and human platelets as a result of the
inhibition of cyclooxygenase activity [29]. The anti-
tumor effect of 2-PTS, resulting from induction of the
apoptosis process, has been recently investigated [7].
The data reported here show that 2-PTS is able to
bind to the active site of rhodanese, resulting in TST
inhibition. We also investigated the role of reduced
thioredoxin (Trx) as a possible sulfur acceptor in this
reaction to restore TST activity. Moreover, the cyto-
toxic effect of 2-PTS on HuT 78 cells was also evalu-

ated using flow cytometry analysis after treatment and
by monitoring the progressive formation of mobile
lipids by NMR spectroscopy. All results obtained
provide evidence that highlights the possible role
of rhodanese in the management of the cytotoxicity
of reactive OSCs in tumor cells and may contribute to
the design of a scheme of the mechanism of action of
2-PTS as an apoptosis inducer.
Results
Inhibition of cell cycle progression and induction
of apoptosis by 2-PTS
The effects of 2-PTS on the human T-lymphoblastoid
cell line, HuT 78, were analysed and a typical time-
R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds
FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3885
dependent and dose-dependent inhibition of cell growth
of these cells as a result of the presence of 2-PTS was
observed. In fact, a statistically significant decrease in
the number of viable cells, at 0.25, 0.5 and 1 mm concen-
trations of 2-PTS and at different time-points, was
found when comparing control and thiosulfate-treated
cells (data not shown). About 75 and 10% of the HuT
78 cells were viable following exposure to 0.5 mm 2-PTS
for 24 and 48 h respectively (Fig. 1A). The growth-
inhibitory effects of 2-PTS were determined by using the
Trypan Blue dye-exclusion assay. Flow cytometric anal-
ysis of HuT 78 cells after 24 and 48 h of treatment with
0.5 mm 2-PTS, following staining with propidium
iodide, resulted in a statistically significant increase in
the fraction of subG1 compared with the control

(Fig. 1B), showing a characteristic feature of apoptosis.
Sodium 2-propenyl thiosulfate induced an increase in
the fraction of HuT 78 cells in the G
2
⁄ M phase after
24 h of treatment, reaching a value of 67.43% (Fig. 1B).
After 48 h, the percentage of the cell population that
showed apoptotic features (subG
1
region) was $ 39%,
suggesting that an antiproliferative activity of 2-PTS
against HuT 78 cells (blockage in G
2
⁄ M phase) results
in the triggering of apoptotsis.
The effect of treatment with 2-PTS on DNA frag-
mentation was assessed by analysis of the agarose-gel
electrophoretic pattern of HuT 78 cells (see supplemen-
tary Fig. S1). A typical DNA-fragmentation pattern was
observed as early as at 24 h after the addition of 2-PTS,
thus confirming the apoptosis-inducing effects of 2-PTS.
The role of reactive oxygen species (ROS) as potential
mediators of 2-PTS-induced cytotoxicity in HuT 78 cells
was also investigated. Figure 1C shows the production
of ROS during the first 3 h of treatment with 2-PTS.
HuT 78 cells underwent an increase of ROS flux as early
as 1 h after addition of 2-PTS. These results implied a
strict association between 2-PTS effects and oxidative
imbalance. Reduced glutathione (GSH) is the most
important physiological antioxidant, both by directly

reacting with ROS and indirectly by preserving cysteine
residues of proteins from irreversible oxidations, so giv-
ing rise to GS-R. The intracellular reduced (GSH) and
oxidized (GSSG) glutathione levels were measured using
HPLC chromatography in order to show the potential
involvement of this redox system in the resistance of
HuT 78 cells to treatment with 2-PTS. Untreated cells
showed an average intracellular GSH concentration of
$ 55 nmolÆ mg
)1
of protein, whereas 2-PTS-treated cells
showed a rapid and sustained increase of GSH levels
(118.5 nmolÆmg
)1
protein) up to 12 h, probably as a
result of to the detoxification process. The intracellular
GSSG content was not significantly affected by treat-
ment with 2-PTS and remained at very low levels.
Formation of
1
H-NMR-visible mobile lipids during
2-PTS-induced apoptosis
The apoptotic parameters were quantitatively moni-
tored by NMR spectroscopy carried out on intact HuT
78 cells and on their aqueous cell extracts after cell expo-
sure to 2-PTS. Detection of the progressive formation of
mobile lipids in intact cells indicated the induction of
apoptosis after treatment with 2-PTS (from 6 to 48 h).
Resonances centered at d = 1.3 p.p.m., as a result of
the saturated fatty acyl chain methylene segments

-(CH
2
)
n
-, and at d = 0.9 p.p.m., arising from methyl
A
B
C
Fig. 1. In vitro effect of 2-PTS on the growth of HuT 78 cells. (A)
Viability of HuT 78 cells in culture after treatment with 0.5 m
M
2-PTS. Trypan Blue staining was used to differentiate viable from
nonviable cells. Data are expressed as mean ± SD. *P < 0.005,
**P < 0.001 (n = 8). (B) Percentage of the cell cycle distribution of
HuT 78 cells after 24 and 48 h of treatment with 0.5 m
M 2-PTS. (C)
Intracellular production of ROS in HuT 78 cells after 1 and 3 h of
treatment with 0.5 m
M 2-PTS, detected by measurement of DCF
fluorescence using a FACSCalibur flow cytometer. Data are
expressed as mean ± SD. *P < 0.001, **P < 0.05 (n = 3).
Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.
3886 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS
groups, were followed. Quantitative analysis of mobile
lipid spectral profiles (see the supplementary material)
was obtained by measuring the peak area (a) ratio
R
chains
= a[(CH
2

)
n
]
lip
⁄ a[CH
3
]
tot
, according to our previ-
ous work [30], where a[(CH
2
)
n
]
lip
is the integral of the
mobile lipids, (CH
2
)
n
is the resonance and a(CH
3
)
tot
is
the integral of the total CH
3
resonance at 0.9 p.p.m.
caused by amino acids and lipids. The value R
chains

in
untreated control cells was 0.20 ± 0.1, not significantly
different from that measured in HuT 78 cells exposed to
0.25 mm 2-PTS at any time-point. The average R
chains
value at 24 h of exposure to 2-PTS increased with the
apoptotic fraction from 1.25 to 1.56 in cells treated with
0.5 and 1.0 mm 2-PTS, respectively. The spectral profiles
of cells treated with either 0.5 or 1.0 mm 2-PTS for 48 h
were mainly characterized by signals from mobile lipids
(R
chains
= 4.45 ± 1.1), while all resonances caused by
small aqueous metabolites decreased to very low levels
as a result of the massive apoptosis of treated HuT 78
cells, probably associated with loss of cell integrity.
Quantitative analysis on
1
H-NMR spectra of cell
extracts after exposure to 0.5 mm 2-PTS for an interme-
diate time interval (24 h, not yet associated with late
apoptosis) showed that the intracellular level of taurine
(measured from the triplet centred at d = 3.45 p.p.m.)
decreased about two-fold from a basal control level of
13.0 ± 2.4 to 6.5 ± 1.0 nmol per 10
6
cells). Under the
same experimental conditions, individual choline-
containing metabolites (tCho, detected under a reso-
nance band centered at a chemical shift of

$ 3.22 p.p.m.) underwent differential changes; in fact
phosphocholine (PCho) decreased by 45% (from
15.1 ± 1.4 to 8.1 ± 0.8 nmol per 10
6
cells), while free
choline and glycerophosphocholine both increased by
about three-fold. The changes in the levels of water-
soluble choline-containing metabolites observed in cells
treated with 2-PTS for 24 h probably reflect the progres-
sive activation of phospholipases and phosphodiester-
ases. On the other hand, a complex network of
pathways may, in principle, contribute to the measured
decrease in PCho, a metabolite particularly sensitive to
conditions determining a block in cell proliferation
and ⁄ or to the activation of enzymes involved in choline–
phospholipid degradation. In fact, both increases and
decreases in PCho have been reported to occur in differ-
ent systems of apoptotic induction, according to partic-
ular experimental conditions [30].
Effect of 2-PTS on TST expression and activity in
HuT 78 cells
The effects of 2-PTS on the expression and activity of
TST in HuT 78 cells were analyzed. HuT 78 cells were
treated without and with 2-PTS at various concentra-
tions (0.25, 0.5 and 1 mm). Figure 2A shows the
western blot of the HuT 78 lysates after 8 and 24 h of
treatment with 0.5 mm 2-PTS. Densitometry measure-
ments of western blots, corrected for actin or for
glyceraldehyde-3-phosphate dehydrogenase (see the
supplementary material) expression, show that no sig-

nificant variation of the expression of TST with respect
to the control was induced by treatment with 2-PTS
(see Fig. 2A). By contrast, a reduction of TST activity
was observed after 24 h, as shown in Fig. 2B.
Interaction of 2-PTS and allyl compounds with
rhodanese
In vitro interaction and kinetic studies were performed
using the recombinant Azotobacter vinelandii rhoda-
nese (RhdA) [31–33]. RhdA has similar properties and
kinetic behaviour and a high structural homology with
bovine rhodanese [34], which is the rhodanese consid-
ered as an appropriate model for using to study
human rhodanese [32,33,35]. The A. vinelandii rhoda-
A
B
Fig. 2. Effect of 2-PTS on TST expression and activity in HuT 78
cells. (A) Western immunoblotting showing the expression of TST
in HuT 78 cell lysates after treatment with 2-PTS. Thirty micro-
grams of protein lysates from untreated cells (CTRL) incubated for
8 and 24 h, and from cells treated with 0.5 m
M 2-PTS for 8 and
24 h, were analysed. A monoclonal anti-actin Ig was used as a con-
trol of the protein concentrations; and densitometry measurements
of western immunoblotting were calculated by comparison with
the intensity of actin expression. (B) Percentage the TST activity of
HuT 78 cellular extracts [untreated cells (CTRL) and cells treated
with 0.5 m
M 2-PTS (2-PTS) after 8, 24 and 48 h of incubation]. The
control after 24 h was used as the reference value. *P < 0.05.
R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds

FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3887
nese has been shown to contain only one cysteine resi-
due, which is essential for the catalytic reaction, and
therefore it represents a good model for using to study
the possible interaction of TST proteins with other
proteins or molecules involved in sulfur and selenium
metabolism [35,36].
In order to investigate the action of 2-PTS on sulfur-
transferase activity, we analyzed the effect of 2-PTS by
monitoring the changes of intrinsic fluorescence that
occur when the rhodanese cycles between the sulfur-
free [RhdA(E)] form and the sulfur-loaded [RhdA(ES)]
form and that are caused by long-range energy trans-
fer and local conformational changes in the protein
[36–40].
No fluorescence changes were observed when 2-PTS
was added to RhdA(ES) (Fig. 3A). By contrast, the
addition of 2-PTS induced an evident quenching of
intrinsic fluorescence of RhdA(E) (Fig. 3B), in a con-
centration-dependent manner, indicating a specific
interaction of 2-PTS with the active site. Moreover,
the quantum yield of intrinsic fluorescence of RhdA(E)
after the addition of 2-PTS was lower than that
obtained following the addition of thiosulfate
(DF% = 27.2 compared with thiosulfate DF% =
15.7) (Fig. 3B). This major fluorescence quenching was
probably a result of the closeness of the allyl group to
the Trp residues present in the protein active site, and
is probably responsible for the differential quenching
of the intrinsic fluorescence in the two states of the

enzyme [37,39,41]. Interestingly, cyanide did not
restore the unloaded form of the enzyme (Fig. 3C) and
a significant loss of the TST activity of the enzyme
was observed.
Inactivation of sulfur-free rhodanese by 2-PTS
and characterization of the derivative protein
In order to study chemical modifications of sulfur-free
rhodanese, it is necessary first to remove the persulfide
from the enzyme by adding an excess of cyanide to the
protein, thus forming the thiocyanate product and
sulfur-free rhodanese. As sulfur-free rhodanese is
somewhat unstable, it is customary to add the modify-
ing reagent immediately after cyanide treatment. We
performed the experiment in this way to analyze the
effect of the 2-PTS on the sulfur-free form of the
enzyme. Figure 4 shows the time course of inactivation
of RhdA(E) caused by an interaction with 2-PTS. Pre-
incubation of RhdA in the presence of a three-fold
molar excess of cyanide and a 200-fold molar excess of
2-PTS induced a decrease of sulfurtransferase activity
over time, and a complete loss of sulfurtransferase
activity was observed after 90 min at 37 °C (Fig. 4A).
The activity of treated RhdA was not restored after
dialysis, indicating that stable binding occurs between
2-PTS and RhdA(E). By contrast, no inhibition of the
TST activity was observed after pre-incubation of
RhdA(ES) in the presence of the same concentration
of 2-PTS without CN
-
. TST activity of the propenyl-

sulfide-form of rhodanese, RhdA-PS, was restored by
treatment with dithiothreitol. In fact, 53.6% of the
TST activity, with respect to the TST activity of
A
B
C
Fig. 3. Intrinsic fluorescence changes of RhdA following the addi-
tion of substrates. (A) RhdA(ES) (2.3 l
M)(
____
), after the addition of
460 l
M 2-PTS (-
__
-) and after the addition of 460 lM CN
-
(- - -);
(B) RhdA(E) (4.6 l
M)(
_____
), with a molar ratio E: thiosulfate 1 : 400
(- - - -), with a molar ratio E:2-PTS 1 : 200 (
___
) and E:2-PTS
1 : 400 (
__
-
__
); and (C) RhdA(E) (5 lM)(
_____

), in the presence of
2-PTS (E: 2-PTS 1 : 250 c ⁄ c) (
___
), E: 2-PTS 1 : 500 c ⁄ c(-
__
-) and
after the addition of CN
-
(E: 2-PTS: CN 1 : 500 : 1000 c ⁄ c ⁄ c) (- - - -).
a.u., arbitrary units.
Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.
3888 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS
RhdA(ES) before treatment, was recovered after
30 min of incubation at room temperature (23°C) (see
Fig. 4B). These results were in agreement with an oxi-
dation state of the catalytic Cys. Thiosulfate sulfur-
transferase activity of RhdA-PS was also restored
when Trx protein was added to the solution and this
activity was dependent on the Trx concentration
(Fig. 5A). Thioredoxin was able to re-establish 66% of
the TST activity of RhdA-PS when was incubated with
Trx at an RhdA-PS ⁄ Trx molar ratio of 1 : 2. The yield
of the recovery was faster and higher when thioredoxin
reductase (Trd) and NADPH were also present in the
solution. Figure 5B shows the recovery of the TST
activity of RhdA-PS in the presence of Trd, NADPH,
and different molar concentrations of reduced Trx. A
total recovery of the TST activity was obtained after
70 min of incubation with 0.5 lm Trx. No increase of
thiocyanate production was observed in the presence

of Trx, Trd and NADPH without rhodanese. These
results, together with the fluorescence spectra, indicate
an interaction of 2-PTS with the catalytic Cys. Thus,
either the reduced Trx or dithiothreitol can reduce a
disulfide bond between the OSC and the catalytic cys-
teine of RhdA, and thereby restore the enzyme to its
active state. This hypothesis was confirmed by MS
analysis of the new form of the enzyme. RP-HPLC
chromatography of RhdA-PS and RhdA(E) was per-
formed, followed by electrospray ionization (ESI) MS
analysis. Although the two forms of the protein
showed the same retention times in RP-HPLC (see the
supplementary material), they showed different molec-
ular mass peaks. In fact, RhdA-PS and RhdA(E) had
molecular mass values that corresponded to
m ⁄ z 31138.9 ± 3.19 and m ⁄ z 31063.8 ± 3, respec-
tively. These results are consistent with thiolation of
A
B
Fig. 4. Inhibition of the sulfur-free form of RhdA by 2-PTS. (A)
Time-dependent decrease of the TST activity of 48.3 l
M RhdA(E) in
50 m
M Tris–HCl buffer, pH 8.0, 0.3 M NaCl. RhdA(E) was treated
with a three-fold molar excess of cyanide and a 200-fold molar
excess of 2-PTS at 37 °C. (B) Effects of dithiothreitol on the TST
activity of RhdA(ES) and RhdA-PS. The proteins were incubated
with 4 m
M dithiothreitol at room temperature and the TST activity
was assayed after 0, 15 and 30 min. RhdA(ES) and RhdA-PS were

treated identically, except that cyanide and 2-PTS were absent dur-
ing the treatment of RhdA(ES). The TST activity of the enzyme
before treatment was taken to represent 100% activity. Each value
represents the average of three independent determinations. DTT,
dithiothreitol.
A
B
Fig. 5. Recovery of the TST activity of 17 lM RhdA-PS detected
using the So
¨
rbo assay. (A) Recovery of TST activity after 2 h of
incubation at 25 °C in the absence and in the presence of thiore-
doxin at molar ratios 1 : 0, 1 : 0.5, 1 : 1 and 1 : 2 c ⁄ c RhdA-PS ⁄ Trx.
All values are expressed as a percentage of the TST activity value
of RhdA(ES). (B) Recovery of the TST activity of 8.1 l
M RhdA-PS
after incubation in the presence of 0.1 U Trd, 50 l
M NADPH and
different concentrations of Trx (0, 0.05, 0.15, 0.25 and 0.5 l
M).
R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds
FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3889
the catalytic cysteine of the enzyme with a propenylsul-
fide (m ⁄ z 73), confirming modification of the protein at
the catalytic site.
Sensitivity of RhdA-PS to proteolysis
To characterize the RhdA-PS form in greater detail,
limited proteolysis of RhdA-PS was performed. Lim-
ited proteolysis of globular proteins generally occurs at
sites that contain the most flexible regions of the poly-

peptide chain within a domain or at the flexible hinges
between domains. Therefore, a limited trypsin diges-
tion of RhdA-PS was performed to investigate the flex-
ibility of this modified enzyme. As previously
observed, the sulfur-loaded form of RhdA [RhdA(ES)]
[42] appeared to be quite resistant to limited proteoly-
sis. In fact, Rhd(ES), which was treated in the same
manner as for RhdA-PS, but in the absence of cyanide
and 2-PTS, was not proteolyzed by trypsin and
remained intact, even after incubation overnight
(Fig. 6A). By contrast, RhdA-PS showed a higher sen-
sitivity to proteolysis than RhdA(ES), as shown in
Fig. 6B,and a band rapid digestion was observed. A
stable daughter band (b band), of about 17.3 kDa,
appeared after a few minutes of proteolysis and was
present also after many hours of digestion. These data
suggest that RhdA-PS is more flexible than RhdA(ES),
probably as a result of local conformational differ-
ences. RhdA-PS showed behaviour very similar to that
previously observed for the alkylated and oxidated
forms of the bovine rhodanese [43,44]. In fact, limited
proteolysis of the alkylated and oxidized forms yielded
fragments that were about half of the apparent molec-
ular mass of the protein, as a result of cleavage at the
interdomain tether that connects the two domains into
which the single polypeptide chain protein is folded
[42,43,45]. Thus, the inactivation of the rhodanese by
2-PTS induces local conformational changes that make
the enzyme much more sensitive to proteolytic degra-
dation.

RhdA-PS form catalysed the Trx oxidation
Thioredoxin oxidation analyses were performed with
the aim of clarifying the mechanism involved in restor-
ing the TST activity of RhdA-PS. The oxidation of
Trx by RhdA was observed by NADPH oxidation.
The change in NADPH concentration was caused by
oxidation of the reduced Trx to its disulfide form,
which was reduced by NADPH in the reaction cata-
lyzed by Trd. The presence of RhdA(ES) at an equi-
molar concentration of Trx caused oxidation of the
reduced Trx at equilibrium with NADPH, as previ-
ously observed for the bovine rhodanese [46,47] even
in the absence of a sulfur donor (Fig. 7A). Thus, also
in this case, reduced Trx behaves as sulfur-acceptor
substrate. Control experiments showed that no oxida-
tion of NADPH occurred in the presence of rhodanese
when reduced Trx was absent. The addition of 2-PTS
(as a substrate of RhdA) to the solution resulted in
further oxidation of NADPH (Fig. 7A). 2-PTS was
also able to oxidize Trx, but the presence of RhdA
caused an increase in the NADPH oxidation rate
(Fig. 7A). In addition, RhdA-PS was able to catalyze
Trx oxidation. A rapid decrease in the concentration
of NADPH was observed when an equimolar
A
B
Fig. 6. Time course of trypsin digestion of RhdA-PS and RhdA(ES).
Three-hundred micrograms of enzyme was subjected to limited
digestion with 1% (w ⁄ w) trypsin in 1 mL of 50 m
M Tris–HCl buffer,

pH 8, at room temperature. After the reaction the samples were
subjected to SDS-PAGE. (A) Lanes 2–7, proteolysis products of
RhdA(ES) (lane 1) at 0, 5, 10, 15, 20 and 30 min and overnight incu-
bation. (B) Lanes 2–8, proteolysis products of RhdA-PS (lane 1) at
0, 5, 10, 15, 20, 30, 60 min and overnight incubation, respectively.
‘a’ and ‘b’ bands are the parent and daughter bands, at about 29.7
and 17.3 kDa respectively. Molecular markers are on the left. STDs,
molecular mass standards.
Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.
3890 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS
concentration of RhdA-PS was added to the stabilized
solution containing Trx, 0.1 U Trd and 50 lm NADPH
(Fig. 7B). Moreover, the subsequent addition of 2-PTS
to the solution led to a further increase in NADPH
oxidation. These results indicate that the rhodanese–
Trx–Trd system is not inhibited by 2-PTS and that this
system has an antioxidant action against OSCs as well
a sulfane sulfur detoxification system.
Discussion
Natural compounds, which improve detoxification
enzymes and ⁄ or reduce the expression and activity of
the carcinogen activating enzymes, are good candidates
for cancer chemoprevention. The controlled prolifera-
tion of the cell may be a result of the presence of a sul-
fane sulfur compound that has the ability to reduce or
promote the activity of important proteins implicated
in the cellular process. Many OSCs present in allium
vegetables have been shown to be able to inhibit the
proliferation of cancer cells [4,48–51]. The induction of
programmed cell death by sulfane sulfur compounds

raises relevant questions about the role of enzymes
involved in their metabolism. A feature of a neoplastic
cell is the residual activity of cysteine aminotransfer-
ase, 3-mercaptopyruvate sulfurtransferase and rhoda-
nese, as well as the total lack of cystathionase activity
[52]; in fact, the biosynthesis and transport of com-
pounds from the sulfane sulfur pool does not occur in
these cells [53].
Our studies presented here, on HuT 78 cells, showed
that the viability of these cells is reduced significantly
upon 24 h of exposure to 2-PTS and that this reduced
growth rate was related to a blockage in the G
2
⁄ M
phase of the cell cycle.
The detection, by NMR, of increasing amounts of
mobile lipids in 2-PTS-treated HuT 78 cells, further
supports mitochondrial dysfunction in these cells. In
fact, several studies have reported the appearance of
mobile lipids in a variety of cells induced to apoptosis
[33,54–56], in which the characteristic apoptotic pheno-
type generally results in loss of the mitochondrial
membrane potential, release of cytochrome c and mito-
chondrial-dependent activation of effector caspases.
Recently, alterations of mitochondrial functions by
different uncouplers of the respiratory chain have been
found to be responsible for the accumulation of intra-
cellular lipid bodies and therefore for the detection of
NMR-visible mobile lipids in intact HuT 78 cells (E.
lorio, C. Testa, A. Stringaro, M. Condello, G. Ara-

ncia, E. Lococo, R. Carnevale, R. Strom, L. Lenti &
F. Podo, unpublished data). Furthermore, mobile lipid
signals have been reported in tumor cells exposed to
lipophilic cationic compounds that cause mitochon-
drial damage [57]. Also the observed decrease of intra-
cellular taurine in cells exposed to 2-PTS for 24 h (i.e.
before the occurrence of massive cell death at 48 h)
seems to be in general agreement with the view of pro-
gressive mitochondrial impairment in these cells. In
fact, Hansen et al. [58] recently suggested a new role of
taurine in mitochondrial function by acting as a modu-
lator of pH in the mitochondrial matrix and therefore
altering the overall oxidative capacity of this subcellu-
lar organelle, including a reduction in fatty acyl b-oxi-
dation. According to this hypothesis, the simultaneous
decrease in taurine and an increase in NMR-detected
mobile lipids suggest a substantial 2-PTS-induced loss
of mitochondrial function with subsequent accumula-
tion of long fatty acyl chains in triglycerides in cyto-
plasmatic lipid bodies. In recent years, it has become
A
B
Fig. 7. Trx oxidation by 2-PTS, in the presence and in the absence
of rhodanese, by measurement of NADPH (50 l
M) consumption
(absorbance at 340 nm), in 50 m
M Tris–HCl buffer, pH 8.0, 1 mM
EDTA, with 4 lM Trx, 0.1 U Trd and in the presence of 0.5 mM
2-PTS. (A) Tris–HCl buffer (a) or 4 lM RhdA(ES) (b) was added to
the solution after 30 min at 37 °C, and, after stabilization, 2-PTS

was added. The data were normalized against an A
340
of 0.093
(which represents 100%). (B) Tris–HCl buffer (a) or 4 l
M RhdA-PS
(b) was added to the solution after 15 min of incubation at 37 °C
and, after stabilization (about 10 min), 2-PTS was added. The data
were normalized against an A
340
of 0.085 (which represents
100%).
R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds
FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3891
apparent that mitochondria are integrally involved in
the mechanism of cell death, and anticancer drugs,
which inhibit the functions of mitochondria can sensi-
tize the cells to undergo apoptosis [59,60]. It is impor-
tant to consider that rhodanese is an important
mitochondrial enzyme in mammalian cells [61] and,
certainly, the rhodanese–Trx system is involved in the
integrity of this main energy-generator organelle.
It is noteworthy that recently it has also been dem-
onstrated that rhodanese deficiency affects the activity
of Fe–S enzymes of the tricarboxylic acid cycle, such
as the aconitase in A. vinelandii [15]. These studies sug-
gest that in tumor cells in which there is a decrease in
expression of the TST gene [19,20], the involvement of
rhodanese in the detoxification of OSCs could lead to
an inhibition of the normal function of the cyanide
detoxification and Fe–S repair activities of this

enzyme, thus inhibiting the normal functions of the
mitochondria. The elucidation of interactions between
OSCs and proteins, such as rhodanese or Trx that are
involved in maintaining the redox homeostasis of the
cell, could help to explain the mechanism whereby they
can induce programmed cell death in tumor cells. In
the present work we investigated the effect of 2-PTS
on the activity and expression of TST. We performed
our experiments in vitro using the A. vinelandii rhoda-
nese. The data reported here demonstrate that 2-PTS
is able to interact with the Cys of RhdA(E), inducing
a covalent modified form (RhdA-PS). 2-PTS interacts
with the active site of the rhodanese enzyme by thiola-
tion of the catalytic cysteine, therefore inhibiting its
TST activity. A peculiar characteristic of the A. vine-
landii enzyme is the presence of only one cysteine resi-
due, which is the catalytic site. The structural
similarity of RhdA with the bovine rhodanese led us
to speculate that 2-PTS can induce a thiolation at the
level of the catalytic Cys residue of the human rhoda-
nese. The ability of this garlic compound to thiolate an
internal Cys, such as that of the active site of rhoda-
nese, is an important observation that should be borne
in mind when considering the mechanism of action of
OSCs. We showed that the thiolation of a mitochon-
drial enzyme, which is involved in ‘managing’ the
redox state of the cell, could be a relevant event in the
mechanism of action of this compound.
The major sensitivity of RhdA-PS to degradation by
proteolysis is an important factor that should be con-

sidered. Limited proteolysis of RhdA-PS shows that,
as well the alkylation of bovine rhodanese [43], local
conformational changes occur when the enzyme is
modified by 2-PTS interactions, and high flexibility of
the enzyme with respect to RhdA(ES) is induced.
These data were also in agreement with a significant
decrease in the expression of TST after 48 h of treat-
ment with 2-PTS (data not shown). Therefore, the
major flexibility of RhdA-PS may also explain its
ability to interact with and oxidize the Trx compared
to RhdA(ES).
Our results highlight a direct interaction between
OSCs from garlic and rhodanese. The bond of the
propenyl sulfide with the catalytic cysteine shows the
characteristic of a disulfide bond (i.e. it is not cleavable
by nucleophilic attack of the cyanide). The propenyl-
sulfur protein has a low redox potential, so Trx and
dithiothreitol reduce it to restore the TST activity.
These results provide evidence that, in vitro, reduced
Trx also regulates TST activity via redox regulation.
2-PTS alone was also able to oxidize the reduced Trx,
but in the presence of RhdA(ES) or RhdA-PS Trx was
oxidized more rapidly. The results presented here sup-
port a possible mechanism (Fig. 8) where the reduced
Trx could be the sulfide acceptor of the rhodanese in the
intracellular system. Trd was able to reduce the oxidized
Trx, and thus this process may be a potential sulfane
sulfur detoxification system present in the cell. This
result highlights the antioxidant action of a rhodanese–
Trx–Trd system against OSCs. As described in previous

work, reduced Trx is a sulfur-acceptor substrate for rho-
danese. It has been also hypothesized that a primary
function of the rhodanese could be Trx-linked oxygen
radical detoxification [46,47]. The data presented here
shed new light on the role of this enzyme in the sulfane
sulfur detoxification system and on the involvement of
the Trx–Trd system in this process. Recently, evidence
for the down-regulation of TST expression in some
cancer cells has been reported [20,21]. Our data show
that no change in the expression of rhodanese occurs
after 8 and 24h of treatment with 2-PTS. By contrast,
O
|
|
H
C
2
HC-
H
C
=
2
O
-S
-S
-
-
a
N
+

dhR
-
S
_
_
||
O
STP-2
S
_
_
S
_
_
|
HC
2
dhR
H
S53
xrT
S
23
-
S2
3
__
S
_
_

H
C
2
__
HC=HC
2
xrT
S5
3
__
H
dhR
-
S
__
2
|
HC
|
|
HC
2
HP
DAN
dT
xrT
S53
S23
|
SH

__
HC
2
__
HC
=HC
2
T
r
d
Fig. 8. Scheme of the proposed reactions of the interaction
between rhodanese (Rhd) and 2-PTS, and of the restoration of rho-
danese activity by the thioredoxin (Trx)–thioredoxin reductase (Trd)
system in the cell.
Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.
3892 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS
a significant reduction of TST activity was observed dur-
ing treatment, indicating that cyanide detoxification of
rhodanese was reduced by the presence of 2-PTS. Thus,
the data suggest that rhodanese could be a target
enzyme of the garlic OSCs and that the reduced TST
activity could be caused by an increase of the sulfur-
detoxification activity of the enzyme, which also
involves the Trx system.
The ability of 2-PTS to inhibit, either in vitro or in
the cell, the TST activity of the rhodanese and to oxi-
dize the Trx in vitro, both in the absence and in the
presence of rhodanese, can be related to its ability to
induce apoptosis in the cell. Our studies showed that
the viability of HuT 78 cells is reduced significantly

after 24 h of exposure to 2-PTS and this reduced
growth rate is related to a blockage in the G
2
⁄ M phase
of the cell cycle. HuT 78 cells underwent an early
increase of ROS flux after the addition of 2-PTS, and
2-PTS-treated cells showed a rapid and sustained
increase of GSH levels up to 12 h, most probably
because of a detoxification process. These results
implied a strict correlation between the apoptotic
effects of 2-PTS and oxidative imbalance, and this can
also be linked to a reduction in the ability of the rhoda-
nese–Trx system to detoxify by oxygen radicals in vivo.
Interestingly, the blockage in the G
2
⁄ M phase of the
cell cycle was linked to an early increase of ROS flux.
Cells treated with 2-PTS showed a rapid and sustained
increase in GSH levels up to 12 h; this was attributable
to a detoxification process. These results imply a strict
correlation between 2-PTS apoptotic effects and oxida-
tive unbalance, and this can be also linked to a reduc-
tion in the activity of the oxygen radicals detoxification
of the rhodanese–thioredoxin system.
These results are in agreement with previous studies
reported by Chang et al. [7], where it was observed
that 2-PTS suppresses, in a dose-dependent manner,
the growth of HL-60 cells through the induction of
apoptosis initiated by oxidative stress. Although there
might be several mechanisms involved in the apoptosis

of cancer cells, we believe that the effects of this sulfur
compound, and probably also of other OSCs, may be
linked to mitochondrial expression levels and activity
both of rhodanese and of Trx in the cancer cells and
that the RNA interference technique could be used to
demonstrate this hypothesis.
Interestingly, high levels of Trx have also been associ-
ated with cancer that is resistant to therapy, and low
Trx levels have been associated with apoptosis in gastric
carcinomas [62–64]. The Trx ⁄ Trd system is considered
to act as an endogenous antioxidant system in all living
cells, in addition to the glutathione system, so the mal-
function of this antioxidant system in mitochondria can
lead to an increase of intracellular ROS [65]. Mitochon-
drial rhodanese–Trx ⁄ Trd-system oxidation by OSCs
could lower the normal reducing activity of Trx and thus
inhibit the activity of important enzymes, such as perox-
iredoxin 3, an enzyme involved in H
2
O
2
metabolism in
mitochondria, whose oxidation plays an important role
in the promotion of apoptosis [66].
In conclusion, these studies contribute to extend the
knowledge on the physiological role of rhodanese and
may represent a relevant starting point to elucidate the
implication of the rhodanese–Trx ⁄ Trd system in che-
moprevention therapy approaches using sulfane sulfur
compounds, whose biochemical metabolism and

certain biological effects warrant further investigation.
Materials and methods
2-PTS synthesis
Sodium 2-propenyl thiosulfate was synthesized according to
a method described by Chapelet et al. [67]. The product
was dried in vacuo , extracted with methanol and the extract
was purified by silica gel chromatography (methanol ⁄
chloroform; 45 : 55, v ⁄ v). The purity and structure of the
compound were evaluated by RP-HPLC, LC-MS and
1
H-NMR.
Cell proliferation assay
HuT 78 human T-lymphoblastoid cells were purchased
from the ISS (Istituto Superiore di Sanita
`
, Rome, Italy).
Approximately 0.2 · 10
6
HuT 78 cells were pre-incubated
for 24 h in RPMI 1640 (GIBCO, Milan, Italy) in the pres-
ence of 1% glutamine, 10% heat-inactivated fetal bovine
serum and antibiotics (1% penicillin and streptomycin
sulfate) at 37 °C in air supplemented with 5% CO
2
and
were then exposed to 2-PTS for 24 and 48 h. The cells were
collected and counted, after staining with Trypan Blue
(0.4% Tripan blu solution; Sigma-Aldrich, Milan, Italy), by
optical microscopy using a Thoma chamber. The rate of
growth inhibition by 2-PTS was calculated with respect to

the control culture taken as 100% growth.
Cell cycle analysis
The cell cycle distribution of HuT 78 cells was measured by
flow cytometry. Approximately 0.5 · 10
6
harvested cells
were stained with 50 lgÆmL
)1
of propidium iodide (Sigma-
Aldrich) in NaCl ⁄ P
i
with 0.1% Triton X-100 and
1mgÆmL
)1
of sodium citrate. Then, the cells were imme-
diately analysed using a flow cytometer (FACSCalibur;
Becton Dickinson, San Jose
`
, CA, USA) and the percentage
of cells in each phase of the cell cycle was evaluated accord-
ing to Nicoletti et al. [68].
R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds
FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3893
DNA fragmentation
For detection of apoptosis by the DNA fragmentation
method, 1 day prior to treatment 1.2 · 10
6
cells were
resuspended in 0.5 mL of lysis buffer (20 mm EDTA,
10 mm Tris, pH 8.0, 200 mm NaCl, 0.2% Triton X-100,

0.01% RNAse and 100 lgÆmL
)1
of proteinase K), incu-
bated for 1 h at 37 °C and, after a second addition of
100 lgÆmL
)1
of proteinase K, were incubated for 1.5 h at
37 °C. Samples were then centrifuged (11 500 g) for
5 min at room temperature. Supernatants were transferred
to a new microfuge and equal volumes of isopropanol
and 25 lLof4m NaCl (100 mm final concentration)
were added, followed by overnight incubation of the sam-
ples at )20 °C. DNA was acquired by centrifugation of
the samples, washed with 70% and 90% cold ethanol,
and finally dried and dissolved in 20 lLof10mm Tris–
HCl, 1 mm EDTA, pH 8.0 (TE buffer). DNA (about
1 lg) was loaded onto each lane of a 1.5% agarose gel
and electrophoresed at 60 V.
Measurement of glutathione, ROS and oxidative
damage
Intracellular reduced (GSH) and oxidized (GSSG) forms of
glutathione were assayed upon formation of S-carboxym-
ethyl derivatives of free thiols with iodoacetic acid, followed
by the conversion of free amino groups to 2,4-dinitrophenyl
derivatives by the reaction with 1-fluoro-2,4-dinitrobenzene,
as described previously [69]. Data are expressed as nanomo-
les of GSH equivalents per mg of protein. ROS measure-
ments were determined as described by Ruefli et al. [70].
Cells were incubated with 20 lm 2¢,7¢-dichlorodihydrofluo-
rescein diacetate dye (DCF-DA) (Invitrogen-Molecular

Probes) for 30 min at 37 °C before the addition of 2-PTS.
After treatment with 2-PTS, the cells were immediately
washed and resuspended in ice-cold NaCl ⁄ P
i
, and then
analysed by flow cytometry. Detection was performed using
a FACSCalibur fluorescence-activated cell sorter using an
excitation wavelength of 480 nm and an emission wave-
length of 525 nm. The cellquest
TM
software was used for
data analysis.
Preparation of cell extracts in aqueous phase
Hut 78 cells were washed twice with ice-cold NaCl/P
i
solu-
tion and the cell pellets were resuspended in 0.5 mL of ice-
cold double-distilled water. Aqueous extracts (20–30 · 10
6
cells ⁄ sample) were prepared in EtOH ⁄ H
2
O (70 : 30, v ⁄ v).
Samples were ultrasonicated at 20 kHz with an exponential
probe, 8 lm peak-to-peak, using an MSE ultrasonic disinte-
grator Mk2 (Crawley, Sussex, UK) and centrifuged at
14 000 g for 30 min. Supernatants were lyophilized twice in
an RVT 4104 Savant lyophilizer (Mildford, ME, USA),
and the residue was resuspended in 0.7 mL of D
2
O (Sigma-

Aldrich) containing 0.1 mm 3-(trimethylsilyl)-propionic-
2,2,3,3-d4 acid sodium salt (TSP) as the internal standard
(Merck & Co, Montreal, Canada).
NMR spectroscopy
High-resolution NMR experiments (25 °C) were performed
at 400 MHz (Bruker AVANCE spectrometer, Karlsruhe,
Germany). Intact cells were counted, washed three times in
NaCl ⁄ P
i
, centrifuged at 600 g and resuspended in 700 lL
of NaCl ⁄ P
i
in D
2
O, before being transferred to a 5 mm
NMR tube (20 · 10
6
cells). NMR analyses on intact cells
were carried out using 90° flip angle pulses preceded by
2.50 s presaturation for water signal suppression (interpulse
delay 2.50 s, acquisition time 1.86 s, spectral width
11 p.p.m., 32 K data points, 128 scans). These conditions
ensured that the fatty chain (CH
2
)
n
⁄ CH
3
ratio was deter-
mined at the magnetization equilibrium (as verified by a

series of preliminary experiments). Quantification of indi-
vidual metabolites in cell extracts was obtained from peak
areas using correction factors determined by experiments at
the equilibrium of magnetization (90° pulses, 30.00 s inter-
pulse delay). Metabolite quantification was expressed as
nanomoles and normalized to the number of extracted cells.
Deconvolution of signals under the 1D
1
H-NMR spectral
profiles was performed using a Bruker win-nmr software
package.
Protein extraction and western blot analysis
Proteins were extracted from HuT 78 cells in 200 lLof
50 mm Tris–HCl, pH 7.09, containing a protease inhibitors
cocktail (Sigma-Aldrich) and sonicated over four steps of
5 s and 1 min of pause in ice. The samples were centrifuged
(10 min, 5000 g,4°C). The protein contents were
determined using the bicinchoninic acid protein assay
(Sigma-Aldrich), cell extracts (30 lg of protein) were elec-
trophoresed on a 15% polyacrylamide gel, electro-blotted
onto poly(vinylidene difluoride) (PVDF) membrane
(Applied Biosystem, Milan, Italy) and the TST levels were
analysed using polyclonal anti-(rabbit-TST) Ig (1 : 250)
(ATLAS Antibodies, Stockolm, Sweden). The immunoblot
was also probed with monoclonal anti-(glyceraldehyde-
3-phosphate dehydrogenase) Ig (Sigma-Aldrich) or
anti-actin Ig (Santa-Cruz D.B.A. Italia, Milan, Italy) as
protein-loading controls. The protein complex formed upon
incubation with specific secondary antibodies (dilution
1 : 10 000) (Sigma-Aldrich) was identified using a Fluor-

chem Imaging system (Alpha Innotech Corporation-Analiti-
ca De Mori, Milan, Italy) after incubation with the
ChemiGlow chemiluminescence substrate. Densitometry
analysis of western blots was performed using
quantity one Software (Bio-Rad, Milan, Italy).
Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.
3894 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS
Preparation of RhdA and RhdA-PS proteins
The His-tag–RhdA protein was overexpressed in Escherichia
coli BL-21(DE3)[pREP4] after induction with isopropyl
thio-b-d-galactoside (IPTG) [32]. RhdA was purified by
chromatography on a Ni-nitrilotriacetic acid agarose
column (Qiagen, Milan, Italy) and eluted by addition of
200 mm imidazole. The protein concentration was deter-
mined using A
0.1%
280nm
= 1.3 [37] and the molecular mass
of RhdA(E) was estimated to be 31063.8 ± 6 Da by ESI-
MS. RhdA(E) was prepared by adding a 10-fold molar
excess of cyanide to RhdA(ES) in 50 mm Tris–HCl buffer,
pH 7.4, and 0.3 m NaCl, followed by incubation for 30 min
at room temperature. Excess cyanide and thiocyanate were
removed using centrifugal filter units Centricon-3
(3000 MW cut-off; Amicon, Milan, Italy) or by dialysis.
The conversion of RhdA(ES) fi RhdA(E) was monitored
by measuring the increase in the fluorescence quantum yield,
which is accompanied by removal of the persulfide sulfur
[37]. RhdA-PS was obtained by incubating 1 mL of 45 lm
RhdA in 50 mm Tris–HCl, pH 8, 0.3M NaCl, for 2 h at

37 °C in the presence of a three-fold molar excess of cyanide
and a 200-fold molar excess of 2-PTS. Excess cyanide and
2-PTS were removed by dialysis or by using centrifugal filter
units Viva-spin (18 000 MW cut-off; Vivascience, Sartorius,
Florence, Italy).
Fluorescence measurements
All fluorescence measurements were made using a LS50
Perkin-Elmer spectrofluorimeter equipped with a thermo-
stated stirrer cell holder. The temperature was always main-
tained at 23 °C. The excitation and emission bandwidths
were 5 and 3 nm respectively. The excitation wavelength
was set at 286 nm and the spectra were recorded from 300
to 400 nm. The changes in fluorescence intensity at 340 nm
(F
obs
) were given as DF%:
DF% ¼½ðF
obs
ÀF
0
Þ=F
0
Â100;
where F
0
is the original fluorescence intensity of RhdA. The
fluorescence measurements were performed in the presence
of different concentrations of the allyl compound (2-PTS)
with 3–5 lm enzyme in 50 m m Tris–HCl buffer, pH 7.2.
Kinetic analysis

Rhodanese activity was measured by the discontinuous
colorimetric assay described by So
¨
rbo [71], where the pro-
duction of thiocyanate from thiosulfate and cyanide was fol-
lowed at 460 nm using a Perkin-Elmer spectrometer. Briefly,
0.5 lg of enzyme (RhdA) was incubated in a reaction mix-
ture containing 58 mm KCN and 58 mm sodium thiosulfate
in 50 mm Tris–HCl buffer, pH 8.0, 0.3 m NaCl. The reaction
was stopped after 1 min by the addition of 100 lL of 15%
formaldehyde. Colour was developed by the addition of
250 lLofSo
¨
rbo reagent (100 g of ferric nitrate and 200 mL
of 65% nitric acid per 1500 mL) and the product was moni-
tored by recording the absorbance at 460 nm.
RP-HPLC analysis of RhdA-PS
The RP-HPLC analysis of RhdA-PS and RhdA(E) was per-
formed using RP-HPLC equipment (LC-10AVP; Shimadzu,
Milan, Italy) with a solvent B gradient (0–40 min, 0–60%;
40–45 min, 60%; and 45–70 min, 60–90%), using 0.1% triflu-
oroacetic acid as solvent A and 80% CH
3
CN, 0.1% trifluoro-
acetic acid as solvent B and a Brouwnlee C-18 column
(OD-300, 250 · 4.6mm, 7 lm). The eluate was monitored at
220 nm using a UV detector (Shimadzu). The samples were
then analyzed by ESI-MS (CIENGE Advanced Biotechnolo-
gies, Naples, Italy) using a Quattro Micro spectrometer
(Waters, Milford, MA, USA) with positive modality acquisi-

tion from 700 to 1400 m ⁄ z. The instrument was calibrated
using myoglobin.
Proteolysis of RhdA-PS
Limited proteolysis of RhdA was performed using 1%
(w ⁄ w) trypsin (Sigma-Aldrich) in 50 mm Tris–HCl, pH 8,
0.3 m NaCl. The reaction was stopped at different time-
points by the addition of SDS-PAGE sample buffer and
boiling the samples for 2 min. Subsequently, the samples
were analysed using SDS-PAGE with a 15% resolving gel.
Thioredoxin oxidase activity
The Trx oxidase activity of rhodanese was assayed as
described by Nandi et al. [44], following NADPH oxidation
spectrophotometrically at 340 nm in the presence of recom-
binant Trd from E. coli (Sigma-Aldrich). The assay was
performed as follows: the absorbance of a solution contain-
ing 50 lm NADPH (Sigma-Aldrich), 16.6 lm Trx from
E. coli (Sigma-Aldrich) and 0.1 U Trd in 50 mm Tris–HCl
buffer, pH 8.0, in a final volume of 1.0 mL, was read at
37 °C against a blank containing no NADPH. After reach-
ing equilibrium, rhodanese (4 lm) and thiosulfate or 2-PTS
were added to the solution and the oxidation of NADPH
was followed. The stock solutions of NADPH, Trx and
Trd were prepared in 50 mm Tris–HCl buffer, pH 8.0,
containing 1.0 mm EDTA.
Statistical analysis
All experiments were carried out at least four times (n =4)
unless otherwise indicated. Data were expressed as mean ±
SD. Comparisons between control and treated cells were
made using the Student’s t-test. Statistical significance was
defined as P < 0.05.

R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds
FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3895
These studies may represent the starting point to extend
the knowledge of the physiological role of this class of
enzyme and of other proteins containing rhodanese
domains for which biochemical characterization is still lack-
ing. A dysregulation of TST expression and activity
induced by hydrogen sulfide (H
2
S) was observed, and this
could result in the inability to detoxify effectively and be a
factor in the cell loss and inflammation observed in ulcera-
tive colitis and in colorectal cancer [16].
Acknowledgements
We thank Professor P. Tagliatesta for his helpful sug-
gestions during the organic synthesis of the 2-PTS
compound, and Dr C. Capo and Dr V. Brunetti for
technical support in some experimental studies. We are
grateful to Professor Silvia Pagani for kindly giving us
the plasmid encoding the RhdA protein. This work
was supported, in part, by grants PRIN and FIRB of
Italian MIUR.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. DNA fragmentation.
Fig. S2. ESI mass spectra of RhdA(E) (A) and RhdA-
PS (B).

Fig. S3. Effect of 2-PTS on the expression of TST in
HuT 78 cells.
Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.
3898 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS
Fig. S4.
1
H-NMR spectra of intact HuT 78 cells
exposed to different concentrations of 2-PTS analysed
at 24 and 48 h of treatment.
Fig. S5. RP-HPLC of RhdA-PS.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compounds
FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3899