Báo cáo khoa học: Roles of 1-Cys peroxiredoxin in haem detoxification in the human malaria parasite Plasmodium falciparum potx

Roles of 1-Cys peroxiredoxin in haem detoxification
in the human malaria parasite Plasmodium falciparum
Shin-ichiro Kawazu
1,2
, Nozomu Ikenoue
1
, Hitoshi Takemae
1,2
, Kanako Komaki-Yasuda
1,2
and Shigeyuki Kano
1
1 Research Institute, International Medical Center of Japan, Tokyo, Japan
2 Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan
Plasmodium falciparum is the parasite that causes falci-
parum malaria, one of the most debilitating and life-
threatening diseases in tropical regions of the world.
The trophozoite of the malaria parasite digests host
haemoglobin to obtain amino acids for metabolism
[1,2]. This process produces a large quantity of haem
(ferriprotoporphyrin IX; FP) in the parasite’s food
vacuole, but the parasite does not possess a haem-
oxygenase in the vacuole or cytosol. The parasite pro-
tects itself from noxious FP through two major
mechanisms. Most FP is polymerized into harmless
haemozoin (malaria pigment) in the food vacuole
[3–5], and the remainder is decomposed by glutathione
(GSH) in the cytosol [5–7]. The latter process produces
free iron, which can enter redox cycling and generate
O
2

–
in the parasite cytosol [5,7–9]. This O
2
–
can be dis-
mutated either spontaneously or by parasite superoxide
dismutase [8–10] into H
2
O
2
. A hydroxyl radical (OHÆ)
is then produced in the presence of H
2
O
2
and Fe
2+
through the Fenton reaction [6,8,9]. OHÆ is a highly
reactive radical that can injure parasite proteins and
membranes. Therefore, the malaria parasite must have
an effective antioxidant mechanism to cope with such
oxidative burdens. However, the parasite lacks catalase
and genuine GSH peroxidase, and thus the major part
of the peroxide-detoxifying capacity in the cytosol
Keywords
glutathione; haem; malaria; peroxiredoxin;
Plasmodium falciparum
Correspondence
S i. Kawazu, Research Institute,
International Medical Center of Japan,

1-21-1 Toyama, Shinjuku-ku, Tokyo
162-8655, Japan
Fax: +81 3 3202 7364
Tel: +81 3 3202 7181, extn 2878
E-mail:
(Received 30 December 2004, revised 9
February 2005, accepted 14 February 2005)
doi:10.1111/j.1742-4658.2005.04611.x
In the present study, we investigated whether Plasmodium falciparum 1-Cys
peroxiredoxin (Prx) (Pf1-Cys-Prx), a cytosolic protein expressed at high lev-
els during the haem-digesting stage, can act as an antioxidant to cope with
the oxidative burden of haem (ferriprotoporphyrin IX; FP). Recombinant
Pf1-Cys-Prx protein (rPf1-Cys-Prx) competed with glutathione (GSH) for
FP and inhibited FP degradation by GSH. When rPf1-Cys-Prx was added
to GSH-mediated FP degradation, the amount of iron released was
reduced to 23% of the reaction without the protein (P<0.01). The rPf1-
Cys-Prx bound to FP–agarose at pH 7.4, which is the pH of the parasite
cytosol. The rPf1-Cys-Prx could completely protect glutamine synthetase
from inactivation by the dithiothreitol–Fe
3+
-dependent mixed-function oxi-
dation system, and it also protected enolase from inactivation by coincuba-
tion with FP ⁄ GSH. Incubation of white ghosts of human red blood cells
and FP with rPf1-Cys-Prx reduced formation of membrane associations
with FP to 75% of the incubation without the protein (P<0.01). The
findings of the present study suggest that Pf1-Cys-Prx protects the parasite
against oxidative stresses by binding to FP, slowing the rate of GSH-medi-
ated FP degradation and consequent iron generation, protecting proteins
from iron-derived reactive oxygen species, and interfering with formation
of membrane-associated FP.

Abbreviations
FP, ferriprotoporphyrin IX; GSH, glutathione; GPx, GSH peroxidase; GR, GSH reductase; GS, glutamine synthetase; MFO, mixed-function
oxidation; Prx, peroxiredoxin; RBC, red blood cell; ROS, reactive oxygen species; RT, room temperature.
1784 FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS
appears to be provided by peroxiredoxins (Prxs) [8,9].
As an additional protective mechanism against FP,
P. falciparum produces histidine-rich protein 2, which
binds FP and participates in detoxification of FP and
H
2
O
2
[11,12].
Pf1-Cys-Prx, a cytoplasmic Prx in P. falciparum,
shows stage-specific expression; it is expressed during
the trophozoite and early schizont stages [13,14].
Trophozoite-specific expression of Prx has also been
observed in the blood and insect stages of the homo-
logous molecule in the rodent malaria parasite P. yoelii
[15]. Prx is expressed at high levels in the parasite cyto-
plasm during the trophozoite stage and represents
approximately 0.5% of the total cellular protein [13].
Although the physiological functions of Pf1-Cys-Prx
are not known, the limited and abundant expression of
this Prx during the trophozoite stage suggests that it
may be associated with FP metabolism.
In the present study, we examined the role of Pf1-
Cys-Prx in detoxifying FP, protecting parasite proteins
from FP-derived reactive oxygen species (ROS), and
interfering with the FP dissolution in the membrane.

Results and Discussion
Effect of Pf1-Cys-Prx on FP degradation by GSH
When FP was mixed with GSH, we observed a shift in
the maximal absorbance of FP from 390 to 370 nm
and a rapid decline in this peak absorbance, which
may be due to formation of GSH ⁄ FP complexes and
degradation of FP [6,16] (Fig. 1A). A similar shift in
the maximal absorbance was observed when FP was
degraded by GSH in the presence of recombinant
(r)Pf1-Cys-Prx (Fig. 1B); however, the rate of FP deg-
radation, as evaluated by decline in absorbance at
370 nm, was slower than that observed in the reaction
without recombinant protein (Fig. 1C). The rate of FP
degradation was [FP]-dependent with an apparent K
m
of 71 lm and a V
max
of 2.4 nmolÆmin
)1
(Fig. 1D). The
rate in the presence of rPf1-Cys-Prx was also [FP]-
dependent with an apparent K
m
of 100 lm (Fig. 1D).
The double-reciprocal plot showed the effect of com-
petitive inhibition of GSH by recombinant protein
(data not shown).
This modification of FP degradation was not due to
oxidation of GSH by recombinant Prx because the
recombinant protein did not oxidize GSH in either the

presence or absence of hydroperoxides (Fig. 2). Previ-
ously reported weak GSH peroxidase (GPx) activity
for rPf1-Cys-Prx [13] was evaluated based on measure-
ments of H
2
O
2
consumption with the ABTS ⁄ H
2
O
2
⁄
peroxidase system, and therefore, this might not be
direct evidence for GSH-dependent reduction of the
substrate.
Binding of Pf1-Cys-Prx to FP
The interaction between Pf1-Cys-Prx and FP was
examined with the FP–agarose binding assay
(Fig. 3A). When rPf1-Cys-Prx was incubated with
FP-agarose in NaCl ⁄ P
i
pH 7.4 most of the added
Fig. 1. Effect of Pf1-Cys-Prx on FP degrada-
tion by GSH. (A) FP was mixed with GSH in
Hepes buffer pH 7.0 at RT. Absorption spec-
tra (300–500 nm) were taken at 100-s inter-
vals starting immediately after mixing (0 s)
to 300 s (four upper continuous traces) and
then at 600 s and 1200 s. The traces went
downward according to time (0, 100, 200,

300, 600, 1200 s). (B) FP degradation by
GSH in (A) was observed in the presence of
rPf1-Cys-Prx. The absorption spectra were
taken as described in (A) except for an addi-
tional measurement at 2400 s (C) Decrea-
ses in the absorbance at 370 nm in (A, d)
and (B, s). (D) [FP]-dependent rates of FP
degradation by GSH (decrease in absorb-
ance at 370 nmÆmin
)1
) were measured at
RT without (d) or with addition of rPf1-
Cys-Prx (s). Data are representative (A–C)
or means (D) of three experiments.
S i. Kawazu et al. Roles of Prx in heme degradation of P. falciparum
FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS 1785
protein bound to the agarose. This binding was con-
firmed by the observation that only a trace of the pro-
tein was left in the unbound fraction. Pre-incubation of
the recombinant protein with FP abolished most of this
binding, suggesting that binding of rPf1-Cys-Prx to the
agarose was FP specific. Recombinant PfTPx-1 (2-Cys
Prx) protein (rPfTPx-1), which was included as a negat-
ive control, also bound to FP–agarose, but the binding
was not affected by preincubation of the protein with
FP. These results also indicate that Pf1-Cys-Prx can
bind to FP at pH 7.4, which is the pH of the parasite
cytosol. The fact that the majority of Pf1-Cys-Prx is pre-
sent in the cytosolic fraction of the parasite lysate sug-
gests that Prx is localized in the cytosol (Fig. 3B).

Abundant expression of Prx during the trophozoite
stage, which is the haemoglobin-digesting stage of the
parasite, may facilitate formation of FP–Prx complex.
Pf1-Cys-Prx reduces levels of iron released
by FP degradation by GSH
The effect of Pf1-Cys-Prx on GSH-mediated FP degra-
dation was examined with the recombinant protein.
We evaluated the effect of rPf1-Cys-Prx on the release
of iron from the degradation reaction. Thirty micro-
moles of FP released 0.36 ± 0.03 lgÆmL
)1
(6.5 lm)of
iron when degraded by GSH for 5 min at 37 °C and
pH 7.0. When rPf1-Cys-Prx (4 lm) was added to the
reaction, the amount of iron released was reduced sig-
nificantly to 0.08 ± 0.03 lgÆmL
)1
(1.5 lm, P < 0.01)
as compared with the reaction without the Prx protein.
Addition of autoclaved protein to the reaction reduced
iron release (0.29 ± 0.04 lgÆmL
)1
) slightly, but this
change was not statistically significant. To rule out the
possibility that the recombinant protein does not affect
the FP degradation reaction but instead forms a
Fig. 2. GPx activity of Pf1-Cys-Prx. GPx activity of rPf1-Cys-Prx (–)
as NADPH oxidation (decrease in absorbance at 340 nm) was mon-
itored after addition of NADPH (0-s), t-butylhydroperoxide (t-BOOH)
(180 s) and H

2
O
2
(300 s). Bovine erythrocyte GPx ( ) was used as
a positive control. The rates of substrate reductions by rPf1-Cys-Prx
were equivalent to those of nonenzymatic reaction (data not
shown). Data are representative of three experiments.
A
B
Fig. 3. Binding of Pf1-Cys-Prx to FP. (A) rPf1-Cys-Prx (r1-Cys Prx)
and rPfTPx-1 (r2-Cys Prx) were bound to FP-agarose in NaCl ⁄ P
i
pH 7.4 at RT. The protein was mixed with agarose directly or after
preincubation with FP. Bound and unbound proteins to agarose
were separated by SDS ⁄ PAGE (15% acrylamide), and the gel was
stained with Coomassie brilliant blue. Molecular mass markers
(kDa) are indicated on the left. Lane 1, 10% (1 lg each) of the pro-
teins mixed with agarose (input); lane 2, proteins bound to the
agarose without preincubation; lane 3, proteins bound to agarose
after preincubation; lane 4, unbound proteins recovered from
agarose when binding was performed without preincubation; lane
5, unbound proteins recovered from the agarose when binding was
performed after preincubation. (B) A homogenate of parasite cells
was centrifuged for separation of the parasite cytosol as a soluble
fraction. Homogenate, soluble fraction and pellet were separated
by SDS ⁄ PAGE (12.5% acrylamide), and the proteins were probed
with rabbit anti-[rPf1-Cys-Prx (1-Cys Prx)] IgG. Molecular mass
markers (kDa) are indicated on the left. Lane 1, homogenate
(25 lg); lane 2, pellet corresponding to 25 lg homogenate; lane 3,
soluble fraction corresponding to 25 lg homogenate.

Roles of Prx in heme degradation of P. falciparum S i. Kawazu et al.
1786 FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS
complex with iron, the recombinant protein after FP
degradation was precipitated with trichloroacetic acid,
and the iron concentration of the precipitate was meas-
ured. The fact that iron was not detected in the preci-
pitated fraction indicates that iron ⁄ Prx complexes do
not form. These results suggest that Pf1-Cys-Prx can
interfere with GSH-mediated FP degradation and iron
release. Continuation of the reaction with intact Prx
protein also yielded FP degradation equal to that of
the control reaction (Fig. 1A and B). This finding sug-
gests that Prx slows GSH-mediated FP degradation
rather than irreversibly inhibiting degradation. It was
previously reported that GSH-mediated FP degrada-
tion occurs under physiological conditions, even when
FP is bound nonspecifically to protein, and that the
rate of degradation of protein-bound FP was some-
what lower than that of free FP [6,7]. The present
results suggest that Pf1-Cys-Prx binds FP, reduces de-
gradation by GSH, and slows release of the free iron
that consequently generates intracellular ROS. Prx
may help to keep levels of FP-derived ROS below that
which the parasite antioxidant system can manage.
Pf1-Cys-Prx protects enzymes from the
inactivation systems
Degradation of FP by GSH releases iron, which can
participate in redox cycling and produce ROS. To
examine whether Pf1-Cys-Prx can protect parasite pro-
teins from ROS, the protective action of rPf1-Cys-Prx

in the inactivation of glutamine synthetase (GS) by the
mixed-function oxidation (MFO) system, which gener-
ates ROS by auto-oxidation of thiol in the presence of
iron, was evaluated (Fig. 4A). The dithiothreitol ⁄ Fe
3+
-
dependent MFO system reduced GS activity to
8.6 ± 2.5% of the initial value. When rPf1-Cys-Prx
was added to the system, complete protection against
inactivation was observed and the initial GS activity
was maintained (116.5 ± 7.3% of the initial activity).
The protective effect was abolished when the recom-
binant protein was heat-inactivated by autoclaving,
suggesting that the native structure is required for the
protective effect. The ability of recombinant Pf1-Cys-
Prx to reduce H
2
O
2
in vitro has been reported [13,17],
and therefore, this peroxidase activity may contribute
to protection of GS from the MFO-derived radicals.
In this system, dithiothreitol in the MFO could act as
a donor for rPf1-Cys-Prx.
Pf1-Cys-Prx could protect yeast enolase from inacti-
vation by coincubation with FP ⁄ GSH (Fig. 4B). The
FP ⁄ GSH coincubation reduced enolase activity to
4.6 ± 5.0% of the initial value. When rPf1-Cys-Prx
was added to the system, complete protection against
inactivation was observed (124.4 ± 29% of the initial

activity). Inactivation of the recombinant protein by
autoclaving also abolished the protector activity. The
fact that this system contained FP, GSH and rPf1-Cys-
Prx in an enzyme inactivation reaction suggests that the
manner in which the Prx could protect the enzyme is in
an FP ⁄ Prx complex. However, the mechanism by which
Pf1-Cys-Prx protected the enzyme in this system was
unclear because the system did not contain any possible
electron donor for the Prx.
These results suggest that Pf1-Cys-Prx can protect
parasite proteins from ROS generated by FP degrada-
tion by complex mechanisms which may include a
Fig. 4. Enzyme protection activity of Pf1-Cys-Prx. (A) GS was inacti-
vated by preincubation with FeCl
3
, dithiothreitol and rPf1-Cys-Prx.
Remaining GS activity was measured by adding c-glutamyltrans-
ferase assay mixture to the inactivation reaction and is expressed
as a percentage of the original activity (–). F, D, FD indicate inacti-
vation reactions containing FeCl
3
, dithiothreitol and both, respect-
ively. FDP and FDaP indicate inactivation reactions containing
FD + rPf1-Cys-Prx and FD + autoclaved rPf1-Cys-Prx, respectively.
(B) Enolase was inactivated by preincubation with FP, GSH and
rPf1-Cys-Prx. Remaining enolase activity was measured by adding
2-phosphoglyceric acid assay mixture to the inactivation reaction
and is expressed as a percentage of the original activity (–). HG
indicates inactivation reactions containing FP and GSH. HGP and
HGaP indicate inactivation reactions containing HG + rPf1-Cys-Prx

and HG + autoclaved rPf1-Cys-Prx, respectively. Data are mean +
SD of three experiments.
S i. Kawazu et al. Roles of Prx in heme degradation of P. falciparum
FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS 1787
peroxidase activity. The identity of the physiological
electron donor for 1-Cys Prx remains controversial in
general [18,19] and for P. falciparum 1-Cys-Prx in par-
ticular [13,17,20]. The thiol dependency of Pf1-Cys-Prx
requires further study [8,9,20], and such information
will provide further insights into the physiological
functions of this protein. On the other hand, Prx is
known to be multifunctional, and its molecular chaper-
one function has recently been demonstrated in yeast
2-Cys Prx [21]. This function of Pf1-Cys-Prx and its
contribution to the protector protein activity should be
investigated, and experiments are in progress in our
laboratory.
Pf1-Cys-Prx interferes with membrane-associated
FP formation
If Pf1-Cys-Prx slows FP degradation by GSH, free FP
in the parasite cytosol during haemoglobin digestion
may readily move into the cell membrane and alter
permeability. To examine the possibility that Pf1-Cys-
Prx affects membrane association of FP, white ghosts
of human red blood cell (RBC) and FP were coincu-
bated with or without recombinant Prx. When white
ghosts and 10 lm FP were incubated at 37 °C for
7 min at pH 7.0, more than half (6.3 ± 0.15 lm)of
the FP was contained in the ghost fraction. Incubation
of ghosts and FP with recombinant Prx protein

(1.3 lm) reduced formation of membrane associations
with FP (4.7 ± 0.16 lm). Although the reduction was
not remarkable ( 25%), it was significant (P > 0.01)
in comparison to the reaction without Prx protein.
Incubation of ghosts and FP with autoclaved recom-
binant protein did not affect membrane associations
with FP (6.6 ± 0.29 lm), suggesting that this activity
requires intact Prx. These results indicate that Pf1-Cys-
Prx can interfere with membrane association of FP.
FP can be degraded by GSH even after it associates
with membranes, although such degradation was
slower than that of FP in solution [6,7]. The delay of
free FP in reaching the membrane would benefit the
parasite, because it gains time for FP degradation by
GSH in the cytosol.
Conclusions
The findings presented here suggest that Pf1-Cys-Prx
may help to protect the parasite against the oxidative
stresses resulting from metabolism of haemoglobin.
This may occur in multiple steps: binding to FP, slow-
ing GSH-mediated FP degradation in a competitive
inhibitory manner and reducing Fe-derived ROS, pro-
tecting the proteins in the cytosol from ROS and,
finally, interfering with formation of the membrane-
associated FP. Further studies of such functions of Prx
will clarify the mechanism underlying detoxification of
FP by P. falciparum and may facilitate development of
alternative therapies for malaria.
Experimental procedures
Parasite culture

The FCR-3 strain of P. falciparum was cultured according
to the modified method of Trager and Jensen [22]. Parasites
in the trophozoite ⁄ schizont stages of development were
obtained from sorbitol-synchronized cultures by treating
cultures with 5% d-sorbitol [23].
Preparation and purification of recombinant
protein
The coding sequence for Pf1-Cys-Prx was amplified from
cDNA of the blood-stage P. falciparum with primers
5¢-GCGAATTC
ATGGCTTACCATTTAGGAGC-3¢ and
5¢-GCGAATTC
TTACATTTGAACAAATCTTA-3¢. The
primers, which contain EcoRI sites (italics) adjacent to the
initiation and the termination codons (underline), were
designed on the basis of the sequence reported previously
[13]. PCR products were digested with EcoRI to create
cohesive ends for ligation into the pGEX-6P-1 expression
vector (Amersham Biosciences, Piscataway, NJ, USA). The
recombinant plasmid, with the cDNA inserted in the cor-
rect orientation, was transformed into Escherichia coli
strain BL21. The fusion protein with N-terminal GST was
expressed by induction of the bacterial culture with 0.3 mm
isopropyl-b-d-thiogalactoside. The protein was purified by
Glutathione Sepharose
TM
4B column chromatography
(GST-Glutathione Affinity System, Amersham Biosciences).
The GST-tag of the fusion protein was removed with Pre-
Scission

TM
protease (Amersham Biosciences) and the GST-
Glutathione Affinity System. Column chromatography was
performed either manually or with the A
¨
KTA
TM
Prime
Liquid Chromatography System (Amersham Biosciences)
according to the manufacturer’s instructions. The rPfTPx-1
was prepared in the same manner as rPf1-Cys-Prx, with the
exception of the PCR primers. The primers were 5¢-GCGA
ATTC
ATGGCATCATATGTAGGA-3¢ and 5¢-CGGA
ATTC
TTACAACTTTGATAAATATT-3¢ (EcoRI sites in
italics; initiation and termination codons underlined).
Degradation of FP by GSH and modification
by Pf1-Cys-Prx
FP (hemin chloride; ICN, Costa Mesa, CA, USA) and
GSH (Sigma-Aldrich, St. Louis, MO, USA) stock solutions
were freshly prepared prior to experiments as described
Roles of Prx in heme degradation of P. falciparum S i. Kawazu et al.
1788 FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS
previously [6] and were kept on ice in the dark. FP (10 lm)
degradation by GSH (2 mm) was observed in the presence
or absence of rPf1-Cys-Prx (4 lm) in 0.2 m Hepes buffer
pH 7.0 at room temperature (RT). Spectral changes
between 300 and 500 nm were measured immediately after
addition of FP to the assay solution and thereafter at 100–

600-s intervals with an Ultrospec 3000 spectrophotometer
(Amersham Biosciences). The [FP]-dependent degradation
rate was measured at 370 nm for 50 s.
GPx activity assays
GPx activity of rPf1-Cys-Prx was examined by monitoring
oxidation of NADPH in a GPx ⁄ GSH ⁄ GSH reductase (GR)
system at 340 nm at RT as described previously [24].
Briefly, assay solution containing 1 mm GSH, 4 lm rPf1-
Cys-Prx and 5 U yeast GR (Oriental Yeast, Tokyo, Japan)
was preincubated for 10 min at RT. After addition of
NADPH (0.3 mm), hydroperoxide-independent oxidation
was monitored for 3 min, and GPx activity was examined
with 75 lm t-butylhydroperoxide (Sigma-Aldrich) and
75 lm H
2
O
2
as substrates. The assay system was checked
with bovine erythrocyte GPx (0.5 U; Sigma-Aldrich) as a
positive control, and the nonenzymatic reaction rate was
observed by replacing the enzyme with buffer (0.1 m potas-
sium phosphate, 1 mm EDTA, pH 7.0).
Binding of Pf1-Cys-Prx to FP-agarose
FP-agarose binding was performed as described by Cam-
panale et al. [25] but with minor modifications. Briefly,
hemin–agarose (20 lL; Sigma-Aldrich) was washed three
times with NaCl ⁄ P
i
by centrifugation (5000 g, 5 min, 4 °C).
For competition binding assay, FP was prepared as a

10 mm stock solution in 0.1 m NaOH. One hundred micro-
liters of protein mixture in NaCl ⁄ P
i
pH 7.4 containing
10 lg rPf1-Cys-Prx or rPfTPx-1 was preincubated both
with and without 0.5 mm FP at RT for 15 min. After pre-
incubation, hemin–agarose was added, and the reaction
mixture was incubated for 60 min at RT with gentle
mixing. The suspension was separated into agarose and
supernatant by centrifugation (5000 g, 5 min, 4 °C). The
agarose was washed three times with 0.5 mL NaCl ⁄ P
i
con-
taining 0.5 m NaCl. Agarose-bound recombinant protein
and free recombinant protein in the supernatant were boiled
in SDS ⁄ PAGE sample buffer containing 2-mercaptoethanol
[26] and analysed by SDS ⁄ PAGE (15% acrylamide).
Western blot analysis
The parasite-infected erythrocytes were lysed with NaCl ⁄ P
i
containing 0.05% saponin. The pellet was collected by cen-
trifugation (25 000 g, 15 min, 4 °C), washed several times
with NaCl ⁄ P
i
and stored in liquid nitrogen until used. The
pellet (wet weight 100 mg) was suspended in 1 mL ice-cold
NaCl ⁄ P
i
containing 5 lL protease inhibitor cocktail
(Sigma-Aldrich) and homogenized on ice in a glass tissue

grinder. The homogenate was cleared of cell debris (500 g,
5 min, 4 °C) and then centrifuged (100 000 g, 1 h, 4 °C) in
an Optima
TM
TLX Ultracentrifuge (Beckman, Palo Alto,
CA, USA). The supernatant was used as the soluble frac-
tion, and the pellet was resuspended in the original volume
of NaCl ⁄ P
i
. Homogenate, soluble fraction and pellet were
mixed with SDS ⁄ PAGE sample buffer [26]. After separation
by SDS ⁄ PAGE (12.5% acrylamide), the proteins were
transferred electrophoretically to polyvinylidene difluoride
sheets (Immobilon
TM
-P; Millipore, Billerica, MA, USA)
and reacted with the IgG fraction of rabbit antisera to rPf1-
Cys-Prx (25 lgÆmL
)1
) [14]. The blot was developed with
horseradish peroxidase-conjugated antirabbit IgG antibody
(1 : 1250; Cappel, Aurora, OH, USA) and ECL
TM
detec-
tion reagents (Amersham Biosciences).
Assay for iron release from FP
FP decomposition mixture containing 30 lm FP, 3 mm
GSH and 0.2 m Hepes pH 7.0 was incubated both with and
without rPf1-Cys-Prx (4 lm)at37°C for 5 min. Free iron
was then measured by the Ferrozine method [6,27]. Briefly,

the reaction mixture (300 lL) was mixed with 33 lL 100%
(w ⁄ v) trichloroacetic acid, and the supernatant was collec-
ted by centrifugation (18 000 g, 3 min). Three-hundred
microlitres of supernatant was mixed with 333 lL of redu-
cing reagent (0.02% ascorbic acid in 0.2 m HCl) and kept
at RT for 5 min. Then, 226 lL of buffer solution (10%
ammonium acetate) and 66 lL of ferrozine reagent (9 mg
ferrozine, 9 mg neocuproin in 3 mL water plus a drop of
10 m HCl) were added, and the reaction was incubated at
RT for 5 min. After centrifugation, the supernatant was
removed and colour development was measured at 562 nm.
The iron concentration was calculated from a calibra-
tion curve generated from reactions with standard iron
solutions that had been prepared by dissolving Fe
(NH
4
)
2
(SO
4
)
2
Æ6H
2
O at 0.2–1.0 mgÆmL
)1
.
Assay for protective activity
Protective activity of recombinant protein was assayed with
the MFO system according to the method described by

Kim et al. [28] with slight modifications. Inactivation mix-
tures (50 lL) containing 50 mm Hepes pH 7.0, 10 mm
dithiothreitol, 3 lm FeCl
3
and 0.5 lg GS were preincubated
with or without rPf1-Cys-Prx (4 lm)at30°C for 30 min.
Remaining GS activity was measured by adding 1 mL of
assay solution containing 0.4 mm ADP, 150 mm glutamine,
10 mm K-ASO
4
,20mm NH
2
OH, 0.4 mm MnCl
2
and
100 mm Hepes pH 7.4. The reaction was incubated at
30 °C for 30 min and then terminated by addition of
0.45 mL stop solution (25 mL stop solution contained
1.375 g FeCl
3
Æ6H
2
O, 0.5 g trichloroacetic acid, 10 mL
S i. Kawazu et al. Roles of Prx in heme degradation of P. falciparum
FEBS Journal 272 (2005) 1784–1791 ª 2005 FEBS 1789
HCl). Absorbance of c-glutamylhydroxamate-Fe
3+
com-
plex was measured at 540 nm. The GSH ⁄ FP-mediated
inactivation was performed as follows using yeast enolase

as the test enzyme. Inactivation mixtures (60 lL) containing
83 mm Hepes pH 7.0, 17 mm GSH, 5 lm FP were preincu-
bated with or without 6.7 lm rPf1-Cys-Prx at 30 °C for
1 h. After preincubation, 0.05 U yeast enolase (40 lL, Ori-
ental Yeast) was added, and the inactivation mixture was
incubated on ice for another 30 min. The remaining enolase
activity in the reaction mixture was assayed in 1.0 mL of
assay mixture containing 50 mm Tris ⁄ HCl pH 7.5, 1 mm
MgCl
2
, and 1 mm 2-phosphoglyceric acid (Sigma-Aldrich).
The production of phoshoenolpyruvate was monitored as
the increase in absorbance at 240 nm at RT for 100 s.
Assay for membrane-associated FP
Human RBC ghosts were prepared as described previously
[7]. A known number of human RBCs were diluted 1 : 10
into an ice-cold solution of 5 mm NaHPO
4
pH 8.0. The
suspension was incubated on ice for 5 min, and ghosts were
collected by centrifugation (27 000 g, 10 min, 4 °C). The
pellet was washed four more times with 5 mm NaHPO
4
pH 8.0 followed by centrifugation until it became white.
White ghosts (equivalent to 10
8
RBCs) were incubated with
10 lm FP as previously described [7] with or without rPf1-
Cys-Prx (1.3 nmol) at 37 °C for 7 min in 1 mL 0.2 m Hepes
pH 7.0. After incubation, ghosts were collected by centrifu-

gation (15 000 g, 20 min, 4 °C), washed once with 0.2 m
Hepes pH 7.0 and dissolved in 1 mL 0.2 m Hepes pH 7.0
containing 1% (w ⁄ v) SDS. The absorbance was measured
at 400 nm. FP concentration was calculated from a calibra-
tion curve generated with standard FP solutions that had
been prepared by dissolving FP at 1–10 lm in 0.2 m Hepes
pH 7.0 containing 1% (w ⁄ v) SDS.
Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
tific Research on Priority Areas (2) (16017318 to
S.I.K) from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan and
by a Grant for Precursory Research for Embryonic
Science and Technology, Japan Science and Technol-
ogy Agency (to S.I.K).
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