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Completely Different Effects of Desferrioxamine on Hemin/Nitrite/ H2O2-Induced Bovine Serum Albumin Nitration and Oxidation Naihao Lu,#,†,‡ Mingyi Zhang,#,†,‡ Hailing Li,†,‡ and Zhonghong Gao*,†,‡,§ Department of Chemistry and Chemical Engineering, Huazhong UniVersity of Science & Technology, Wuhan 430074, People’s Republic of China, Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, Huazhong UniVersity of Science & Technology, Wuhan 430074, People’s Republic of China, and E-Institutes of Shanghai UniVersities, DiVision of Nitric Oxide and Inflammatory Medicine, Shanghai UniVersity of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China ReceiVed January 9, 2008
Protein tyrosine nitration is becoming increasingly recognized as a prevalent post-translational modification that could serve as a biomarker of nitric oxide (NO)-mediated oxidative stress. One received protein nitration model is the heme- or hemoprotein-dependent pathway that involves the contribution of iron and the formation of free radicals. Therefore, the iron chelating agent desferrioxamine (DFO) can affect the development of oxidative and nitrative stress. In the hemin/nitrite/H2O2 system-induced bovine serum albumin (BSA) nitration and oxidation model, BSA was analyzed for 3-nitrotyosine and carbonyl groups measured by spectrophotometry, SDS-PAGE, and Western blotting upon exposure to DFO. The results showed a significant dose-dependent inhibitory effect of DFO on BSA nitration, while an enhancement on oxidation was surprisingly observed. The promotion on protein oxidation could not be the result of the formation of ferrioxamine since the antinitration and prooxidant effect of DFO was abolished when it combined with Fe3+ to form ferrioxamine. Our studies also indicated that the abnormal effect of DFO on promoting protein oxidation probably originated from the hemin-DFO complex, which needs further study. The completely different effects of DFO on hemin-induced protein tyrosine nitration and protein oxidation should be taken into account when DFO is used in experimental and clinical applications. Introduction Oxidative injury has been implicated in the pathogenesis of numerous diseases including cardiovascular diseases (1, 2), neurodegenerative diseases, and inflammation in which reactive oxygen species (ROS1) (3) and reactive nitrogen species (RNS) (4, 5) are involved. Excessive generation of ROS/RNS can damage protein, DNA, and polyunsaturated fatty acids, and may lead to oxidative stress and a variety of diseases (1, 2, 5–7). The production of 3-nitrotyrosine (3-NT) has been used as a biomarker of pathological disease and oxidative stress process (2, 8–11). Over the past several years, substantial evidence has been accumulated that the major pathways of protein tyrosine nitration in vivo include peroxynitrite (ONOO-) and heme peroxidase (hemoprotein)-dependent reaction in which free radicals and iron catalysis are involved (2, 12, 13). The nitration of proteins modulates catalytic activity, cell signaling, and cytoskeletal organization (14). Desferrioxamine (DFO, a natural siderophore isolated from Streptomyces pilosus, as shown in Figure 1), is a powerful iron * Corresponding author. Tel: +86-27-87543532. Fax: +86-27-87543632. E-mail:
[email protected]. † Department of Chemistry and Chemical Engineering, Huazhong University of Science & Technology. ‡ Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, Huazhong University of Science & Technology. § Shanghai University of Traditional Chinese Medicine. # These authors contributed equally to this work. 1 Abbreviations: BSA, bovine serum albumin; DFO, desferrioxamine; DNPH, 2,4-dinitrophenylhydrazine; L-GDH, L-glutamic dehydrogenase; 3-NT, 3-nitrotyrosine; ONOO-, peroxynitrite; Pro, protoporphyrin; RNS, reactive nitrogen species; ROS, reactive oxygen species.
Figure 1. Schematic structure of desferrioxamine (DFO).
chelator for the treatment of iron overload diseases (15–17). It can react with iron to form ferrioxamine (the 1:1 DFO-Fe complex), which is kinetically and thermodynamically stable (15, 18). DFO exhibited protective effect on several oxidative stress models in vivo (19–21), such as cold-induced brain edema (20) and hypoxic-ischemic brain injury in mice (21). Although DFO has been viewed primarily as a metal chelating agent that blocks iron-dependent hydroxyl radical (•OH) formation via Fenton or Haber-Weiss reactions (16, 22), additional or alternative antioxidant mechanisms may operate in some of the models (23–26). Among these, DFO is capable of scavenging free radicals, such as superoxide (O2•-) (24), •OH (25), •NO2 radicals (23), as well as oxo-ferryl compounds (26). DFO can also inhibit peroxynitrite-mediated oxidation and nitration, which is independent of iron chelation but reacts with peroxynitritederived radicals (23). Because of the ubiquitous nature of hemoprotein, nitrite, and H2O2, it seems likely that the metal- and free radical-mediated protein tyrosine nitration is important in physiology and pathobiology of various diseases (12, 13). To further elucidate the biochemical basis of DFO therapies on hemin/nitrite/H2O2-
10.1021/tx800013e CCC: $40.75 2008 American Chemical Society Published on Web 05/07/2008
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Figure 2. Effect of DFO on protein nitration and oxidation in the hemin/ NO2-/H2O2 system. Protein nitration was detected spectrophotometrically by monitoring pH-dependent NO2-Tyr absorbance and Western blotting. Protein oxidation was determined spectrophotometrically, and with SDS-PAGE and Western blotting. (A) Protein nitration and oxidation determination by spectrophotometry. The reaction mixtures containing BSA (0.5 mg/mL), hemin (25 µM), NaNO2 (1 mM), H2O2 (1 mM), and different concentrations of DFO were incubated at 37 °C for 30 min. (Blank represents the group of reaction mixture only containing BSA (0.5 mg/mL), Control represents the group of reaction mixtures containing BSA (0.5 mg/mL), hemin (25 µM), NaNO2 (1 mM), and H2O2 (1 mM), DFO groups represent the control group plus DFO (5, 10, and 20 µM).) The values are the mean ( SD of three determinations. (*P < 0.05, **P < 0.01, and ##P < 0.01 represent the comparison between the reaction group and the control group.) The SDS-PAGE (B) and Western blotting with anti-3-NT antibody (C) or anti-DNP antibody (D) analysis corresponding to the above reaction mixtures.
Figure 3. Effect of DFO on nitrite level in the hemin/NO2-/H2O2 system. After incubating the reaction mixtures at 37 °C for 30 min, the nitrite level in solution was determined by the Griess method. (Blank represents the group of the reaction mixture containing BSA (0.5 mg/ mL), NaNO2 (1 mM), and H2O2 (1 mM), Control represents the blank group plus hemin (25 µM), and DFO group represents the control group plus DFO (25 µM).) The values are the mean ( SD of three determinations. (*P < 0.05 and **P < 0.01 represent the comparison between the reaction group and blank group.)
induced oxidative and nitrative stress, we examined the effects of DFO on the protein nitration and oxidation process.
Experimental Procedures Materials. Bovine serum albumin (BSA), L-glutamic dehydrogenase (L-GDH), protoporphyrin (Pro), ferriprotoporphyrin IX (hemin), 3-morpholinosydnonimine (SIN-1), desferrioxamine (DFO), 2,4-dinitrophenylhydrazine (DNPH), 3-nitrotyrosine (3NT), rabbit polyclonal antibody against 3-NT, and dinitrophenol (DNP) were purchased from Sigma. All solvents and other reagents were of the highest purity commercially available.
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Figure 4. Effect of ferrioxamine (DFO-Fe3+) on protein nitration and oxidation in the hemin/NO2-/H2O2 system. The process of determining protein nitration and oxidation is described in Experimental Procedures. (A) Protein nitration and oxidation determination by spectrophotometry. (Blank represents the group of reaction mixture only containing BSA (0.5 mg/mL), Control represents the group of reaction mixtures containing BSA (0.5 mg/mL), hemin (25 µM), NaNO2 (1 mM), and H2O2 (1 mM), DFO group represents the control group plus DFO (25 µM), and the DFO-Fe3+ group represents the control group plus DFOFe3+ (25 µM).) The values are the mean ( SD of three determinations. (**P < 0.01 and #P < 0.05 represent the comparison between the reaction group and control group.) SDS-PAGE (B) and Western blotting with anti-3-NT antibody (C) or anti-DNP antibody (D) analysis corresponding to the above reaction mixitures. (E) Effect of DFO and ferrioxamine on scavenging the ABTS+ radical. The detailed procedures are described in ref 31.
Sample Treatment. DFO-mediated protein nitration and oxidation experiments were carried out by using reactions catalyzed by hemin as an iron complex. Samples (BSA (0.5 mg/mL, final concentration, the same below)) in phosphate buffered saline (0.1 M, pH 7.4) were incubated at 37 °C with NaNO2 (1 mM), H2O2 (1 mM), hemin (25 µM), and different concentrations of DFO or ferrioxamine (DFO-Fe3+) for 30 min. To further investigate the mechanism, samples containing BSA (0.5 mg/mL), NaNO2 (1 mM), and H2O2 (1 mM), with or without protoporphyrin, DFO, and Fe3+ were also incubated at 37 °C for the same period. The reaction mixtures obtained were used in later assays. Detection of Protein Tyrosine Nitration (3-NT) with Spectrophotometer. NO2-Tyr formation in BSA could be detected by a spectrophotometer (27). NO2-Tyr absorbance was monitored at 430 nm (ε430 nm ) 4,400 M-1 cm-1, pH 12) using a spectrophotometer. Briefly, the reaction mixtures (in 0.1 M phosphate buffered saline, pH 7.4) containing BSA (0.5 mg/ mL), hemin (25 µM), NaNO2 (1 mM), H2O2 (1 mM), and different concentrations of DFO or ferrioxamine (DFO-Fe3+) were incubated at 37 °C for 30 min. Then, 280 µL of mixture was mixed with NaOH (20 µL, 1 M), and the absorbance was read immediately. Because the absorbance of hemin itself also exists in 430 nm, the spectral results were determined by
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Figure 5. Effect of ferrioxamine, protoporphyrin and Fe3+ with or without DFO on BSA oxidation. (A) Effect of ferrioxamine (DFO-Fe3+) on BSA oxidation in H2O2 system determined by spectrophotometry. (Blank represents the group of reaction mixture only containing BSA (0.5 mg/ mL), Control represents the group of reaction mixtures containing BSA (0.5 mg/mL) and H2O2 (1 mΜ), and DFO-Fe3+ groups represent the control group plus different concentrations of DFO-Fe3+ (10, 20, 40 µΜ).) (B) Effect of protoporphyrin and Fe3+ on BSA oxidation in the NO2-/ H2O2 system determined by spectrophotometry. (Blank represents the group of reaction mixture only containing BSA (0.5 mg/mL), Control represents the group of reaction mixtures containing BSA (0.5 mg/mL), hemin (25 µM), NaNO2 (1 mM), and H2O2 (1 mM), and Pro represents the group of reaction mixtures containing BSA (0.5 mg/mL) with protoporphyrin (25 µM), NaNO2 (1 mM), and H2O2 (1 mM).) Fe represents the group of reaction mixtures containing BSA (0.5 mg/mL) with Fe3+ (25 µM), NaNO2 (1 mM), and H2O2 (1 mM). Pro-Fe represents the Fe group plus protoporphyrin (25 µM). Pro-Fe-DFO represents the Pro-Fe group plus DFO (25 µM). (C) and (D) are the SDS-PAGE analysis corresponding to the above reaction mixtures (A) and (B), respectively.
subtracting the absorbance of the reaction mixture, which contained hemin, but with no sodium nitrite added. Detection of Protein Oxidation with Spectrophotometer. Oxidative damage of protein is accompanied by the formation of protein carbonyl group, which has been used widely as a marker of protein oxidation (28). Protein carbonyls were quantitated by spectrophotometric measurement of 2,4-dinitrophenylhydrazone derivatives (DNPH, ε370 nm ) 22,000 M-1 cm-1) as described by Levine et al. (29). The spectral contributions of nitrotyrosine and hemin at 370 nm were determined in samples not treated with DNPH, and this value was subtracted from samples treated with DNPH. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). After treatment, protein samples were subjected to SDS-PAGE to semiquantify protein damage and separate target protein (30). Following incubation, 80 µL aliquots were mixed with 20 µL of 5× sample loading buffer, heated at 100 °C for 3 min, then subjected to SDS-PAGE. The gels were stained with Coomassie Brilliant Blue and analyzed by AlphaImager 2200 software. Detection of Protein Nitration and Oxidation with Western Blotting. For detection of protein oxidation, the carbonyl groups in proteins were first derivatized with DNPH in the presence of SDS (3%). After 1 h of incubation at room temperature, the reaction was stopped with neutralization solution, and then samples were mixed with loading buffer and subjected to SDS-PAGE. For detection of protein tyrosine nitration, samples were directly mixed with loading buffer and subjected to SDS-PAGE. After electrophoresis, proteins were transferred to the nitrocellulose membrane and stained with Ponceau S to ensure equal loading, then immunoblotted with a rabbit polyclonal antibody against 3-NT or DNP. The antibody was detected using an anti-rabbit secondary antibody conjugated with horseradish peroxidase. Chemiluminescence was used to identify specific proteins according to the ECL system (Pierce). Antioxidant Activity Assay on DFO and Ferrioxamine (DFO-Fe3+). To compare the antioxidant activity of DFO and DFO-Fe3+ in oxidative damage, the ABTS•+ scavenging activities were determined as previously described (31). UV-vis Spectroscopy Analysis. The UV-vis spectrum from 240 to 700 nm of different compounds and their combination
were recorded in phosphate buffered saline (pH 7.4, except the spectrum of FeCl3 at pH 2.0). Statistical Analysis. All data shown were from at least three different experiments. Experimental values are the mean ( SD of the number of experiments indicated in the figure legends. Significance was assessed by using the Student’s t-test (P < 0.05 as significant).
Results Effect of DFO on Protein Nitration and Oxidation in the Hemin/NO2-/H2O2 System. As shown in Figure 2A and C, incubation of BSA with DFO in the hemin/NO2-/H2O2 system for 30 min resulted in a dose-dependent decrease of 3-nitrotyrosine detected by spectrophotometry and Western blotting. The inhibitory efficiency was up to 70% versus that of the control when the concentration of DFO was 20 µM. Meanwhile, the decrease of nitrite concentration also was inhibited after DFO addition, which was detected by the Griess method (32) (Figure 3). In contrast to the effect on protein nitration, DFO enhanced protein oxidation dose-dependently detected by spectrophotometry and SDS-PAGE, as well as Western blotting in parallel (Figure 2A, B, and D). Enhancing efficiency was up to nearly 30% versus that of the control when the concentration of DFO was up to 20 µM. Effect of Ferrioxamine (DFO-Fe3+) on Protein Nitration and Oxidation. We also evaluated the effect of ferrioxamine on protein nitration and oxidation. Unlike DFO treatment, ferrioxamine had less inhibitory effect on BSA nitration and did not exhibit prooxidant effect on protein oxidation in nitrative/ oxidative damage (Figure 4A, B, and D, and Figure 5A and C). Also, the different concentrations of DFO did not promote H2O2 /Fe3+-induced BSA oxidation (data not shown). Ferrioxamine could be observed and detected at 425 nm after adding DFO to Fe3+ solution (Figure 6C), which was also reported previously (33). Effect of Protoporphyrin and Fe3+ with or without DFO on BSA Oxidation. To investigate the possible mechanisms of action in the hemin-induced oxidation system, the effects of protoporphyrin, protoporphyrin-Fe3+, and protoporphyrin-Fe3+-
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Figure 7. Effect of adding hemin and DFO modes on protein oxidation in the hemin/NO2-/H2O2 system. Protein oxidation was determined by spectrophotometry, SDS-PAGE, and Western blotting. (A) Protein oxidation determination by spectrophotometry. The reaction mixtures were incubated at 37 °C for 30 min (Blank represents the group of reaction mixture only containing BSA (0.5 mg/mL), Control represents the group of reaction mixtures containing BSA (0.5 mg/mL), hemin (25 µM), NaNO2 (1 mM), and H2O2 (1 mM), Separately represents the control group plus DFO (25 µM), and the Premixed group represents BSA (0.5 mg/mL), NaNO2 (1 mM), H2O2 (1 mM), plus prereaction hemin-DFO (25 µM) (t ) 10min)). The values are the mean ( SD of three determinations. (*P < 0.05 and **P < 0.01 represent the comparison between the reaction group and Control group.) The SDS-PAGE (B) and Western blotting with anti-DNP antibody (C) analysis corresponding to the above reaction mixtures.
Figure 6. UV-vis spectra of solutions containing hemin and DFO (A), protoporphyrin and Fe3+ with or without DFO (B), and Fe3+ and DFO (C). (A) The mixture of hemin and DFO for different time. Curves: 1, hemin (25 µM); 2-5, hemin (25 µM) plus DFO (25 µM) (t ) 0, 10, 20, 30 min). (B) The mixture of protoporphyrin and Fe3+ with or without DFO. Curves: 1, protoporphyrin (25 µM); 2-3, protoporphyrin (25 µM) plus Fe3+ (25 µM) (t ) 10, 20 min); 4-5, protoporphyrin (25 µM) and Fe3+ (25 µM) plus DFO (25 µM) (t ) 10, 20 min). (C) The reaction of Fe3+ and DFO. Curves: 1, Fe3+ (500 µM, pH 2.0); 2-3, Fe3+ (500 µM) plus DFO (500 µM) (pH 7.4, t ) 0, 10 min). -
DFO on NO2 /H2O2 system-induced BSA oxidation were studied. As shown in Figure 5B and D, the promoting effect on protein oxidation was not observed in these conditions, which meant that only hemin-DFO significantly promoted BSA oxidation. Together with the UV-vis spectra in Figure 6A, the spectra of DFO:hemin with a ratio of 1:1 showed the presence of the hemin-DFO complex by the shift of the flat peak to a sharp peak at 370 nm with the time increased. Additionally, the mixture of protoporphyrin-Fe3+ and protoporphyrin-Fe3+DFO did not simply form the specific spectra of hemin or the hemin-DFO complex (Figure 6B), and the related intermediates were not easily observed (34). Effect of Adding Hemin and DFO Modes on Protein Oxidation. To further study the role of the hemin-DFO complex in protein oxidation, we changed the ways of adding hemin and DFO, and examined their effects on protein oxidation. The premix of hemin and DFO for 10 min before adding to the reaction mixture aggravated more protein oxidation than when adding hemin and DFO to the reaction mixture one by one (Figure 7). Effect of DFO on L-Glutamic Dehydrogenase (L-GDH) Nitration and Oxidation in the Hemin/NO2-/H2O2 System
Figure 8. Effect of DFO on L-GDH nitration and oxidation in the hemin/NO2-/H2O2 system (A) and SIN-1 (B). The Western blotting with anti-3-NT antibody or anti-DNP antibody analysis of the L-GDH in different protein nitration models. The reaction mixtures containing L-GDH, hemin/NO2-/H2O2 or SIN-1, and DFO were incubated at 37 °C for 30 min. (Blank represents the group of reaction mixture only containing L-GDH (1 mg/mL), Control in hemin/NO2-/H2O2 system represents the group of reaction mixtures containing L-GDH (1 mg/ mL), hemin (10 µM), NaNO2 (1 mM), and H2O2 (0.5 mM), Control in SIN-1 treatment represents the group of the reaction mixtures containing L-GDH (1 mg/mL) and SIN-1 (0.25 mM), and DFO group represents the corresponding Control group plus DFO (10 µM).)
and SIN-1. To verify the widespread effect of DFO on hemin/ NO2-/H2O2-induced protein oxidation, another protein, L-GDH was used as the target protein. Similar to the effect on BSA oxidation, DFO also promoted L-GDH oxidation while inhibiting protein nitration (Figure 8A). Moreover, the prooxidant effect was not observed when the nitrating agent was changed to SIN-1 (a peroxynitrite-generating substance) (Figure 8B), in which both protein oxidation and protein nitration were inhibited.
Discussion The iron chelating agent DFO could form a stable octahedral coordination complex with Fe(III), thus preventing iron-
Effects of DFO on Protein Nitration and Oxidation Scheme 1. Different Effects of DFO on Protein Nitration and Oxidation
catalyzed free radical formation (35–38). In this regard, chelation therapy with DFO has attracted much attention as a potential treatment of several diseases that relate to the metal and free radical mechanism (39, 40). However, DFO administration has been shown to lead to prooxidant action for paraquat (41), ferrous iron (42), and ascorbate (43, 44). In the presence of ascorbate, DFO can enhance alkaline phosphatase inactivation in which DFO acts as an electron transfer carrier and then produces cytotoxic radicals. However, the promoting effect is abolished by shielding DFO with the iron donor hemin (43, 44). As an iron-containing complex, hemin has been shown to be an oxidant in several systems involving the catalytic and prooxidant action of iron (45–48). Thomas et al. (12) and Bian et al. (13) reported the catalytic effect of hemin on H2O2/NO2-dependent BSA nitration, which is closely related to the iron and free radical mechanism. Originally, we used the nitrative model to investigate the effects of DFO on metal-catalyzed nitration and concomitant oxidation. The research of DFO on the new biomarker of nitrative/oxidative stress can expand the clinical application of the metal chelator. Bovine serum albumin incubated with the hemin/NO2-/H2O2 system resulted in a significant increase on protein nitration, which was detected by spectrophotometry and Western blotting respectively (Figure 2A and C). After the addition of DFO, it could inhibit the hemin-induced nitration dose-dependently. The inhibitive effect was lost when DFO was blocked by ferric to form ferrioxamine. The antinitration effect may be related to metal chelation and free radical scavenging with regard to DFO contribution. As previously shown, DFO was reported to be capable of inhibiting ONOO--mediated nitration and oxidation by a mechanism independent of metal chelation, but could react with ONOO--derived free radicals (23, 49), such as O2•- (24), • OH (25), •NO2, CO3•-, and the tyrosyl radical (23, 49). However, in hemin/NO2-/H2O2-induced protein nitration, metal chelation may play a more important role, which can be supported by two results. First, when this chelator was blocked by ferric ion, most of its antinitration effect was lost, though the ferrioxamine still had the ability to scavenge radicals (Figure 4C and E), and second, in the presence of the chelator, more nitrite remained in the mixture (Figure 3), which meant that less nitrite was oxidized to •NO2 or other nitrating species, implying that DFO had less contribution to scavenge the intermediate •NO2 radical. The increased carbonyl group, as well as protein degradation to fragment and cross-linkage, has been used as an important marker of oxidative damage (28, 29). In contrast to its role in protein tyrosine nitration, the addition of DFO, a well-known antioxidant, enhanced BSA oxidation dose-dependently at the same time. In addition, the promoting effect of DFO on hemin/ NO2-/H2O2-induced protein oxidation was also observed in some other proteins, such as L-GDH (Figure 8A) and lysozyme (unpublished data), which meant that this effect was not
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exclusive to BSA. This promoting role of DFO on protein oxidation was only observed for hemin/NO2-/H2O2 systeminducded modification but not peroxynitrite-induced protein oxidation (Figure 8B). Usually, DFO carried out inhibitory effects on hemin-induced injury that was mainly related to iron chelation (45–48) and in which hemin was found to be bound as the hemin-DFO complex (46). However, in our study, the abnormal effect of DFO on promoting protein oxidation probably originated from the hemin-DFO complex. This conclusion was supported by the following facts: First, prooxidation was not carried out by the sole DFO because of its inertia in the oxidation process (data not shown). The promoting effect of ferrioxamine or protoporphyrin on oxidation was not observed (Figure 5A-D). Second, DFO could directly interact with hemin to form a novel intermediate, which was confirmed from the shift of the hemin spectrum after adding DFO (Figure 6), and this intermediate was reported by Sullivan et al. (46). Furthermore, the premix of hemin and DFO for 10 min before adding to the reaction mixture increased more protein oxidation than when adding hemin and DFO to the reaction mixture one by one (Figure 7), which meant that the formation of the DFOhemin complex promoted protein oxidation. All these results indicate that the prooxidation of DFO on hemin/nitrite/H2O2induced BSA oxidation may be closely related to the formation of the hemin-DFO complex. The abnormal action for the heminDFO complex may involve the iron pool to accept or donate electrons, which was convenient to promote protein oxidative damage (43, 44, 50). In conclusion, the completely different effects of DFO on hemin/NO2-/H2O2-induced protein nitration and oxidation are revealed in Scheme 1. The administration of DFO into the hemin/NO2-/H2O2 system can form the hemin-DFO complex, which will promote protein oxidation while inhibiting the hemincatalyzed •NO2 production and subsequent protein tyrosine nitration. However, the detailed mechanisms of action, such as the formation and structure determination of the hemin-DFO intermediate, or other possible contributions independent of the hemin-DFO complex need further study. Acknowledgment. This work was supported by grants from the National Natural Science Foundation of China (No. 30300073, 30670481), the Natural Science Foundation of Hubei Province (No. 2005ABB008), the Program for New Century Excellent Talents in University (No. NCET-05-0649), and E-Institutes of Shanghai Municipal Education Commission (No.E-04010).
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