Báo cáo khoa học: A superoxide dismutase–human hemoglobin fusion protein showing enhanced antioxidative properties pptx

A superoxide dismutase–human hemoglobin fusion protein showing enhanced antioxidative properties Marie Grey 1 , Sakda Yainoy 1,2 , Virapong Prachayasittikul 2 and Leif Bu ¨ low 1 1 Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, Sweden 2 Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand Introduction The toxicity of Hb outside of its natural protective red blood cell environment has been linked partly to the redox activity of the Hb molecule. Consequently, Hb can react with, and generate, reactive oxygen species such as the superoxide anion. Hb, both inside and out- side of the red blood cell, readily undergoes autoxida- tion, but the degree of oxidation is normally restricted to approximately 3% of the total Hb by the metHb reductase system. However, if damage occurs to the red blood cells, Hb may be released and, as the normal pro- tection systems, involving also superoxide dismutase (SOD) and catalase, are no longer associated with the Hb, Hb is exposed to oxidative damage. Autoxidation leads to nonfunctional metHb (HbFe 3+ ) and superox- ide ions (Eqns 1–4). Furthermore, reoxygenation of a tissue previously deprived of oxygen will produce super- oxide, a phenomenon known as reperfusion injury [1,2]. HbFe 2þ O 2 þ H 2 O 2 ! HbFe 4þ @O þ H 2 O þ O 2 ð1Þ HbFe 2þ O 2 ! HbFe 3þ þ O À 2 ð2Þ HbFe 3þ þ H 2 O 2 !  HbFe 4þ @ O þ H 2 O ð3Þ HbFe 4þ @ O þ H 2 O 2 ! HbFe 3þ þ O À 2 þ H 2 O ! heme degradation ð4Þ Superoxide can be reduced either enzymatically by SOD, or spontaneously by dismutation to H 2 O 2 . Both superoxide and H 2 O 2 increase the rate of Keywords antioxidation; fusion protein; hemoglobin; superoxide dismutase Correspondence L. Bu ¨ low, Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-211 00, Lund, Sweden Fax: +46 46 222 4611 Tel: +46 46 222 9594 E-mail: Leif.Bulow@tbiokem.lth.se (Received 13 May 2009, revised 27 July 2009, accepted 24 August 2009) doi:10.1111/j.1742-4658.2009.07323.x Much of the toxicity of Hb has been linked to its redox activity; Hb may generate reactive oxygen species, such as the superoxide anion. Superoxide is intrinsically toxic, and superoxide dismutase (SOD) provides important cellular protection. However, if the Hb molecule is located outside the red blood cell, the normal protection systems involving SOD and catalase are no longer closely associated with it, exposing Hb and its cellular surround- ings to oxidative damage. In order to produce less toxic Hb molecules, we have explored gene fusion to obtain homogeneous SOD–Hb conjugates. The chimeric protein was generated by coexpressing the human Hb a-chain ⁄ manganese SOD gene together with the b-chain gene in Escherichia coli. We show that the engineered SOD–Hb fusion protein retains the oxygen-binding capacity and, moreover, decreases cytotoxic ferrylHb (HbFe 4+ ) formation when challenged with superoxide radicals. The SOD– Hb fusion protein also exhibits a 44% lower autoxidation rate and higher thermal stability than Hb alone. Abbreviations DSC, differential scanning calorimetry; ROS, reactive oxygen species; SOD, superoxide dismutase. FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS 6195 metHb formation [3] and eventually lead to heme degradation. The catalase present intracellularly may be sufficient to remove H 2 O 2 by conversion to water [4]; however, there is not sufficient SOD to eliminate the superoxide. Chang [2], Alagic [4] and Tarasov [5] have characterized Hb chemically conjugated to SOD alone or SOD combined with catalase, i.e. Hb–SOD and polyHb–SOD–catalase, respectively. The latter Hb conjugate has been shown to have antioxidative properties and offer protection against reperfusion injury [6]. In order to create proximity between SOD and Hb, we consequently explored gene fusion. Such closeness generates a favorable microenvironment in which the formed superoxide ions can be directly taken care of. The SOD–Hb protein was prepared by linking the human Hb a-chain gene with the human manganese SOD gene. Manganese SOD was chosen because it shows lower product inhibition by H 2 O 2 and has a much longer half-life in serum, 5–6 h as compared to 6–10 min for copper and zinc SOD [7,8]. This approach enabled us to produce the SOD–Hb in Escherichia coli and to obtain a homogeneous product. We characterized this novel enzyme by analyzing the stability, activity and antioxidative properties of the fused SOD–Hb, and we then compared it with human Hb, also produced in E. coli. Results Protein expression and purification It is notoriously difficult to produce human Hb in microorganisms, because the a-chains and b-chains must be produced in equal amounts to give functional Hb molecules. The a-chains are especially prone to precipitation and degradation if expression is unbal- anced [9]. By linking the SOD gene to the a-chain gene, we could stabilize fusion protein expression. The proteins were purified using affinity chromatography; details can be found in Doc. S1. After optimization of the expression protocol, SOD–Hb was expressed well, with a yield of 8 mgÆL )1 , which is four times higher than the yields obtained with other Hb fusion proteins [10]. The expression level was lower for SOD–Hb than for wild-type Hb, but may be further optimized by introducing compensatory mutations to increase expression and improve heme transport into the cell [11]. Denaturation SDS ⁄ PAGE analysis (Fig. 1) showed bands of the expected sizes: 16 kDa for b-chain Hb, and 38 kDa for SOD–a-chain Hb. In addition, SOD–Hb showed the characteristic red color of Hb. Molecular mass determination Molecular masses were determined by gel filtration on a Superdex 200 prep grade column, which gave four components. Two of these, corresponding to almost 50% of the peak area (Fig. 2), were complexes larger than 160 kDa. The remaining peaks corresponded to SOD–Hb in the monomer and dimer forms at equal concentrations (where the Hb b-chain is included; Fig. 2). Activity measurements The SOD activity assay showed a SOD activity of 1.3 · 10 5 UÆlmol )1 for SOD–Hb, which means that the fusion protein retained more than half (52%) of the native SOD activity. This corresponds closely to the amount of high molecular mass SOD–Hb. As the native SOD is functional as a tetramer, it is possi- ble that the dimer and monomer fractions of SOD–Hb exhibit much lower activity, or may even be nonfunc- tional [7,12]. In the CO and O 2 binding assays, the protein sam- ples were reduced with sodium dithionite and then gently bubbled with CO or O 2 (Fig. 3). SOD–Hb showed the typical absorption spectrum characteristics of human Hb, with peaks located at wavelengths of 417, 536 and 566 nm, which are almost identical to those of HbCO (417, 537 and 567 nm). An O 2 spec- trum was also recorded, and SOD–Hb again showed the typical peaks at 414, 538 and 573 nm, similar to HbO 2 (413, 539 and 574 nm). In particular, the O 2 peaks of the fusion protein in the visible region appear to be slightly broader than those of Hb, and there also appears to be a minor peak at 630 nm on the CO spectrum. These could be explained by very low amounts of ferric protein in both the SOD–Hb spectra [13]. Fig. 1. The results of SDS ⁄ PAGE on crude extract and purified sample. Lane 1: molecular mass marker. Lane 2: crude extract of SOD–Hb. Lane 3: purified SOD–Hb. An Hb fusion protein with antioxidative properties M. Grey et al. 6196 FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS Autoxidation studies were performed at room tem- perature (20–22 °C) for 48 h, using an Hb concentration of 8 lm, and followed using visible spectrophotometry. The rate constant for Hb (0.18 h )1 ) was almost twice as high as that for SOD–Hb (0.10 h )1 ). Stability Thermal stability was investigated using differential scanning calorimetry (DSC). The asymmetric shape of the DSC curve (Fig. 4) suggests a complex denatur- ation path, which is irreversible under the present con- ditions. Thermal denaturation of Hb begins with the dissociation of the tetramer into monomers [14], and ends with a certain degree of aggregation. The Fig. 2. Schematic representation of SOD–Hb showing possible conformations resulting in (from top to bottom): the high molecular mass complex, an SOD–Hb dimer, and finally an SOD–Hb monomer. A B Fig. 3. Reduced SOD–Hb and Hb were bubbled with O 2 (A) or CO (B). SOD–Hb shows the absorption spectrum characteristics typical of human Hb for both O 2 and CO. Critical regions of the protein spectra have been enlarged. The scale on the y-axis is offset for clarity. Fig. 4. DSC was performed on 60 lM Hb (dashed line) or 30 lM SOD–Hb (solid line). Cp, heat capacity. M. Grey et al. An Hb fusion protein with antioxidative properties FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS 6197 broader, less well-defined peak of SOD–Hb indicates contributions from more than one thermal process, and thus a more intricate mixture of complexes (e.g. higher-order ‘aggregates’), in agreement with the results of the gel filtration experiments showing several species. In addition, both Hb and SOD are normally tetramers, which could account for the numerous ther- mal processes as they dissociate into dimers and mono- mers. In addition to dissociation into dimers, the complexity of the DSC curve is increased by the ther- mal inactivation of the SOD enzymatic activity, result- ing in smaller conformational changes [15], which probably contribute to the broadness of the peak. Apparent T m values were calculated using the Microcal software origin, and found to be 48.7 °C (Hb) and 55.1 °C (SOD–Hb), corresponding to the T m value of 53.2 °C reported by Olsen [16] (HbACO). SOD–Hb thus exhibits greater heat stability than Hb, although lower stability than SOD alone (T m of 68–94 °C) [12,17]. Heme reactivity Heme loss is dependent on the geometry of the protein and, possibly, also water shielding. Oxidation of the heme to the met form (Fe 3+ ) increases the probability of heme loss as the fifth coordination bond to the proximal histidine is weakened [18]. The fast phase has been associated with heme loss from the b-chains, whereas the slow phase is attributed to the a-chains [19]. SOD–Hb showed a heme loss rate of 0.19 ± 0.05 min )1 for the fast phase, whereas the loss rate of Hb was almost three times lower (0.069 ± 0.008 min )1 ). Although the b-chains do not take part directly in the fusion, the effect is more profound here, suggesting that the b-chains are less protected during fusion than Hb. As expected, for the slow phase both Hb and SOD–Hb a-chains exhi- bited lower heme loss rates (0.011 ± 0.001 and 0.017 ± 0.003 min )1 , respectively), indicative of higher heme affinity. SOD–Hb and Hb were also incubated together with a xanthine ⁄ xanthine oxidase superoxide-generating sys- tem. After 30 min of incubation, sodium sulfide, which reacts with ferrylHb to form sulfHb, was added. As shown in Fig. 5, SOD–Hb was significantly more effec- tive in reducing the amount of ferrylHb formed (P = 0.0018) than Hb. Heme degradation of Hb and SOD–Hb by H 2 O 2 was measured by monitoring the fluorescence of the degradation products (Fig. 6). For all H 2 O 2 concentra- tions used (1, 2 and 4 mm), the amount of heme degra- dation for SOD–Hb was much lower than for Hb alone. For both proteins, increasing the H 2 O 2 concen- tration increased the denaturation, as indicated by flu- orescence measurements. Blank measurements without H 2 O 2 gave no contribution to the fluorescence signal (data not shown). Discussion The proximity between two proteins is often impor- tant. For instance, the physical closeness between two or several enzymes catalyzing sequential reac- tions is a feature of many metabolic pathways. The frequently observed improved kinetic behavior of such associated proteins has been explained by the formation of a favorable ‘microenvironment’ in Fig. 5. FerrylHb formation was measured with 10 lM Hb or SOD– Hb. *Significantly different from Hb (P = 0.0018, Student’s t-test). Fig. 6. The formation of fluorescent heme degradation products was measured at a fixed concentration of oxyHb (15 l M) and vary- ing concentrations of H 2 O 2 : e, Hb with 4 mM H 2 O 2 ; D, Hb with 2m M H 2 O 2 ; h, Hb with 1 mM H 2 O 2 ; ¤, SOD–Hb with 4 mM H 2 O 2 ; , SOD–Hb with 2 mM H 2 O 2 ; , SOD–Hb with 1 mM H 2 O 2 . An Hb fusion protein with antioxidative properties M. Grey et al. 6198 FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS which the local concentration of intermediates is higher. Similarly, a second protein in an aggregate can provide protection against a toxic compound or intermediate. There are several ways of creating proximity between proteins. A frequently used technique involves chemical cross-linking of the relevant proteins. This normally generates protein conjugates with random linking and random orientation of the active centers. Proximity between two or more proteins may also be achieved by performing in-frame gene fusion of the corresponding structural genes. The chimeric gene then encodes a polypeptide chain carrying two or more active centers. This strategy resembles the proposed model for the evolution of naturally occurring multi- functional proteins [20]. Through comparison of DNA sequences from different species, it has been demon- strated that such proteins probably evolved through gene translocation followed by gene fusion. Extensive studies of such naturally occurring protein aggregates have enabled the three-dimensional structures to be determined for some of them, notably phosphoribosyl anthranilate isomerase ⁄ indole glycerol phosphate syn- thase and tryptophan synthase. Besides generating homogeneous conjugates, this method also allows for in vivo testing of the fusion proteins, as opposed to chemical approaches. Superoxide is intrinsically toxic, and SOD provides important cellular protection. Superoxide radicals and ⁄ or impaired SOD functionality have been impli- cated in diabetes, cancer and neurodegenerative dis- eases, in addition to cellular damage such as lipid peroxidation, protein oxidation, and DNA damage [21,22]. Hb may generate reactive oxygen species, which can be particularly deleterious in clinical appli- cations, e.g. in blood substitutes. When a gene fusion approach is used, the link between the SOD and Hb is always present between the same amino acid resi- dues, which precludes heterogeneity. Conversely, the chemically linked Hb–SOD products described by Alagic [4] and Tarasov [5] have cross-links introduced at different residues, as well as reactive groups, which must be blocked in a separate step. In these chemical linking approaches, usually both intra-tetramer and inter-tetramer bonds are introduced, with the involve- ment of multiple side chains [23]. In addition, it has been shown that shorter cross-links may provide better stabilized Hb [24]. The SOD–Hb was designed with a very short linker of only one alanine, in order to promote proximity, as well as to reduce proteo- lytic degradation during production. However, our SOD–Hb shows the presence of different aggregation forms, with large complexes as well as dimers and monomers. The gel filtration step described could, however, be used to remove these fractions. As it is likely that the monomer and dimer forms are inac- tive, this simple step could increase the homogeneity of the product as well as removing the less active fractions. SOD–Hb retains about half of the specific activity of native SOD. This lower activity may be due to the steric difficulties encountered in forming the necessary SOD tetramers [7,12]. The tetrameric conformation is important for the formation of an active site suitable for Mn ligation [12]. An alternative strategy could be to coexpress native SOD with our fusion protein, which may facilitate tetramer formation. Similarly, we can coexpress native Hb a-chains together with SOD– Hb to generate a free N-terminal end of this polypep- tide chain. It is therefore essential to consider subunit interactions when engineering fusion proteins, particu- larly when producing larger Hb conjugates. As indi- cated earlier, catalase is a key component when generating functional Hb-based blood substitutes. We have prepared an SOD–catalase fusion protein that can be coexpressed with SOD–Hb in E. coli.By exploring the natural SOD subunit interactions, we can then form a SOD–catalase–Hb protein. The poly- Hb–SOD–catalase developed by Chang [2] also showed lower activity, 85–90% of that of native SOD, depend- ing on the conjugation ratio, implying that this prepa- ration also suffers from steric hindrance. However, the chemically linked Hb–SOD [4] showed identical activ- ity for both conjugated and free SOD, probably owing to locking of the SOD in the tetrameric form during conjugation. The genetic fusion of Hb and SOD does not seem to perturb the globin structure noticeably, as both the O 2 and CO spectra are essentially identical to those of Hb. All peaks in the Soret region correspond well to that of Hb, indicative of full functionality. The autoxi- dation rate constants were determined to be 0.18 h )1 for Hb and 0.10 h )1 for SOD–Hb at room tempera- ture. The rate constant for SOD–Hb is thus approxi- mately half of that for Hb, although higher than the values reported by Vandegriff (0.007–0.0021 h )1 ) [18]. This may partly be explained by the residual catalase activity, as the Hb in their study was derived from outdated donated blood. An in vitro test was performed to evaluate the antioxidative properties of SOD–Hb. SOD–Hb and Hb were incubated together with a superoxide ⁄ H 2 O 2 - generating system. The formation of ferrylhemoglobin with SOD–Hb after 30 min was significantly lower (P = 0.0018) than with Hb (Fig. 5). FerrylHb is cytotoxic and has been implicated in oxidative stress M. Grey et al. An Hb fusion protein with antioxidative properties FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS 6199 situations in a range of diseases [25,26]. The ability to reduce the formation of this toxic species means that SOD–Hb has considerable promise for the future development of a clinical product. Interestingly, extra- cellular Hb polymers with SOD activity, with a molec- ular mass of over 3800 kDa, occur naturally in the blood of earthworms [32,33]. Heme degradation of Hb and SOD–Hb by H 2 O 2 was measured by monitoring the fluorescence of the degradation products. The oxyHb first reacts with one molecule of H 2 O 2 , forming oxyferrylHb (HbFe 4+ =O). When a second molecule of H 2 O 2 reacts with the oxy- ferrylHb, metHb and a superoxide radical (Eqns 2–4) are produced, initiating the degradation process. The damage is irreversible, and leads to a cascade of reac- tions that ultimately result in iron release and fluores- cent degradation products. The superoxide radical, which has a lifetime of 0.2 s [27], is perfectly located to react with the heme group. Moreover, the superoxide exhibits higher reactivity, owing to the heme pocket environment [28,29]. As can be seen in Fig. 6, heme degradation in SOD–Hb was much lower than in Hb, and both showed an increase in denaturation when the H 2 O 2 concentration was increased. The combination of SOD and Hb thus protects the heme molecule from denaturation during oxidative stress in vitro, probably via a proximity effect. The heme degradation is dependent on the lifetime of ferrylHb and the susceptibility of the heme to super- oxide-induced damage. The close association of SOD with Hb can allow removal of the superoxide radicals quickly, leading to less degradation, as shown in this study. Additional modifications of SOD–Hb, e.g. by introducing mutations affecting ferryl reduction kinet- ics, may further reduce the Hb toxicity. We have previ- ously shown that either introducing or removing suitably located tyrosines affects ferryl reduction kinet- ics in human Hb [26]. Introducing the same mutations in SOD–Hb could result in a SOD–Hb molecule that is even more suitable for practical use. Additionally, to allow further in vivo studies of our construct, suitable protein surface protection, such as pegylation or encapsulation, needs to be developed. In conclusion, the engineered SOD–Hb exhibits a lower autoxidation rate and higher thermal stability than Hb alone, and the process creates a homogeneous link between the Hb and the SOD, which chemical conjugation does not. Additionally, in vitro tests show that cytotoxic ferrylHb formation is significantly decreased in the presence of superoxide radicals. Con- sequently, the combination of SOD and Hb protects the Hb molecule from denaturation during oxidative stress. Experimental procedures Construction of human manganese SOD and human SOD–Hb Please see the Supporting information. Primers used for site-directed mutagenesis or cloning can be found in Table S1. Protein expression and purification Protein expression and purification were performed essen- tially as described previously [26]. Details can be found in Doc. S1. Molecular mass determination Gel filtration chromatography was used for molecular mass determination on an A ¨ KTA purifier system controlled by unicorn software (GE Healthcare, Uppsala, Sweden). Highly purified protein was loaded on a HiLoad 16 ⁄ 60 Superdex 200 column (also GE Healthcare), equilibrated with 50 mm phosphate buffer (pH 7.2) containing 0.15 m EDTA, and eluted with the same buffer at a flow rate of 1.0 mLÆmin )1 . Standard proteins with molecular masses ranging from 6.5 to 158.0 kDa (GE Healthcare) were used to produce standard curves. SOD activity assay The assay used to determine SOD activity was a slightly modified version of that described by Ewing [30]. In prin- ciple, the assay is based on the ability of SOD to inhibit nitroblue tetrazolium reduction by an aerobic mixture of NADH and prenazine methosulfate, which produces superoxide at nonacidic pH. Details can be found in Doc. S1. DSC DSC measurements were performed with a Microcal differ- ential scanning calorimeter (Microcal, Northampton, MA, USA) with a cell volume of 0.5072 mL. All samples were degassed for 15 min at room temperature prior to scanning. Baseline scans were obtained with buffer in both the refer- ence and sample cells, and these were later subtracted from sample scans. Protein samples (60 lm Hb or 30 lm SOD– Hb) in 70 mm sodium phosphate (pH 7.2) were scanned in the temperature range 20–90 °C at a rate of 60 °CÆh )1 . Heme loss rates The heme exchange rate between metHb and human serum albumin was determined as described by Benesch [19] and An Hb fusion protein with antioxidative properties M. Grey et al. 6200 FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS Jeong [31], with slight modifications. Potassium ferricyanide was added in excess in order to oxidize the proteins to the ferric form, and this was followed by removal of ferricya- nide and ferrocyanide with a Sephadex G-25 column (GE Healthcare) equilibrated with 0.05 m bis-Tris and 0.1 m NaCl (pH 7.5). Each Hb sample was then mixed with human serum albumin (5 lm final concentration of each), and 0.5 m Tris buffer (pH 9.05) was added to a total vol- ume of 2 mL. The absorbance (A) at 578 and 620 nm was then recorded every minute for 90 min, using a Beckman Coulter DU-800 spectrophotometer. The amounts of metHb and methemalbumin were calculated using the following two equations: metHb½¼146:03  A 578 À 134:48  A 620 and methemalbumin½¼À61:95  A 578 þ 220:01  A 620 The data were then fitted to a double-exponential decay curve, essentially as described by Vandegriff [18]: Y ¼ A  e Àk fast Ât þ e Àk slow Ât ÀÁ þ C: Autoxidation The autoxidation analysis was performed as described by Jeong [31], but with a lower Hb concentration of 8 lm.CO was removed by shining light on the sample, which was kept on ice, while gently oxygenating the solution using a stream of oxygen gas. The reaction was carried out in 0.1 m sodium phosphate (pH 7.0) at room temperature (20–22 °C). Spectra from 400 to 700 nm were collected at specific times, using a Beckman Coulter DU-800 spectro- photometer. The baseline was adjusted by setting the absor- bance at 700 nm to zero. The experimental data were then fitted to a single-exponential equation of the form [18]: Y ¼ DY max  1 À e kt ÀÁ þ Y 0 where Y is the relative metHb concentration (%), DY max is the total relative change in metHb at the end of the reac- tion, k is the rate constant, t is time, and Y 0 is the relative metHb concentration at t =0. Measurement of ferrylHb formation FerrylHb formation was measured using xanthine ⁄ xanthine oxidase as the oxidation system, based on a method described by D’Agnillo [2,3]. Ten micromolar Hb or SOD–Hb in 70 mm sodium phosphate (pH 7.2) was reacted with 100 l m xanthine and 10 mUÆmL )1 xanthine oxidase, both from Sigma-Aldrich (Stockholm, Sweden). The Hb concentration was calculated on the basis of heme, using the relation: e 523 = 7.12 mm )1 Æcm )1 [32]. At given times, an excess of catalase (Roche, Mannheim, Germany) was added to remove residual H 2 O 2 , after which 2 mm sodium sulfide was added; finally, the absorbance at 620 nm (A 620 nm ) was measured. Measurement of heme degradation by fluorescence Fluorescence measurements originally described by Nagababu [33] were slightly modified. OxyHb (15 lm) was incubated with H 2 O 2 (1, 2 or 4 mm)in50mm potas- sium phosphate buffer (pH 7.4) in a total volume of 2mL at 25°C. Reagent-grade H 2 O 2 (30% v ⁄ v) was obtained from Sigma-Aldrich, and standardized using an extinction coefficient of 72.8 m )1 Æcm )1 at 230 nm [34]. The fluorescence signal was recorded for 30 min at excita- tion and emission wavelengths of 460 and 525 nm, respec- tively, using a fluorimeter (PTI Photon Technology International, London, Canada). Acknowledgements This study was supported by European Union Frame- work VI Eurobloodsubstitutes project and the Staff Development Project by the Ministry of Education, Thailand (SY). References 1 Alayash AI (2004) Oxygen therapeutics: can we tame haemoglobin? 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Grey et al. 6202 FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS anine, alpha 96valine -> tryptophan, beta 108aspara- gine -> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation. Biochemistry 38, 13433–13442. 32 Snell SM & Marini MA (1988) A convenient spectro- scopic method for the estimation of hemoglobin concen- trations in cell-free solutions. J Biochem Biophys Methods 17, 25–34. 33 Nagababu E & Rifkind JM (1998) Formation of fluo- rescent heme degradation products during the oxidation of hemoglobin by hydrogen peroxide. Biochem Biophys Res Commun 247, 592–596. 34 George P (1953) The chemical nature of the second hydrogen peroxide compound formed by cytochrome c peroxidase and horseradish peroxidase. I. Titration with reducing agents. Biochem J 54, 267–276. Supporting information The following supplementary material is available: Doc. S1. Experimental procedures. Table S1. Primer sequences. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. M. Grey et al. An Hb fusion protein with antioxidative properties FEBS Journal 276 (2009) 6195–6203 ª 2009 The Authors Journal compilation ª 2009 FEBS 6203 . A superoxide dismutase–human hemoglobin fusion protein showing enhanced antioxidative properties Marie Grey 1 , Sakda Yainoy 1,2 , Virapong Prachayasittikul 2 and. indi- cated earlier, catalase is a key component when generating functional Hb-based blood substitutes. We have prepared an SOD–catalase fusion protein that can