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: skawazu@ri.imcj.go.jp (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). References 1 Olliaro PL & Goldberg DE (1995) The Plasmodium digestive vacuole: Metabolic headquarters and choice drug target. Parasitol Today 11 , 294–297. 2 Lew VL, Tiffert T & Ginsburg H (2003) Excess hemoglo- bin digestion and osmotic stability of Plasmodium falci- parum-infected red blood cells. Blood 101, 4189–4194. 3 Yamada KA & Sherman IW (1979) Plasmodium lophurae: composition and properties of hemozoin, the malarial pigment. Exp Parasitol 48, 61–74. 4 Slater AF, Swiggard WJ, Orton BR, Flitter WD, Gold- berg DE, Cerami A & Henderson GB (1991) An iron- carboxylate bond links the heme units of malaria pigment. 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