Báo cáo khoa học: Recombinant hemoglobinbG83C-F41Y An octameric protein pdf

Recombinant hemoglobin bG83C-F41Y An octameric protein Corinne Vasseur-Godbillon 1 , Sarata C. Sahu 2 , Elisa Domingues 1 , Christophe Fablet 1 , Janel L. Giovannelli 2 , Tsuey Chyi Tam 2 , Nancy T. Ho 2 , Chien Ho 2 , Michael C. Marden 1 and Ve ´ ronique Baudin-Creuza 1 1 INSERM Unite ´ 473, Le Kremlin-Bice ˆ tre, France 2 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Considerable progress has been made in the develop- ment of red blood cell substitutes, in particular with hemoglobin (Hb) based oxygen carriers designed to correct oxygen deficiency. Different problems are encountered with acellular Hb in the plasma. The two major problems that have been very well investigated in the past years are the optimum oxygen affinity for adequate oxygen delivery to tissues and the dissoci- ation of Hb tetramers into dimers. Different molecules have been developed to decrease the oxygen affinity and to prevent tetramer dissociation, either by chem- ical modification (HemAssist TM , Baxter Healthcare, Deerfield, IL, USA) 1 [1] or protein engineering technology (Optro TM , rHb1.1) [2]. These solutions theoretically Keywords blood substitute; disulfide bridge; hemoglobin; octamer; oligomerization kinetics Correspondence V. Baudin-Creuza, INSERM U 473, 78 rue du Ge ´ ne ´ ral Leclerc, 94275 Le Kremlin-Bice ˆ tre Cedex, France Fax: +33 1 49 59 56 61 Tel: +33 1 49 59 56 84 E-mail: veronique.baudin-creuza@ kb.inserm.fr (Received 4 July 2005, revised 20 September 2005, accepted 16 November 2005) doi:10.1111/j.1742-4658.2005.05063.x We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb bG83C-F41Y) tetra- mers joined by disulfide bonds at the b83 position. One of the major prob- lems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the mole- cular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb bG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinet- ics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb bG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduc- tion of the disulfide bridge by reduced glutathione showed a rate of 1000 m )1 Æh )1 (observed time coefficient of 1 h at 1 mm glutathione) at 25 °C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 lm Hb and 25 °C. In addition, the octameric rHb bG83C-F41Y was resistant to potential redu- cing agents present in fresh plasma. Abbreviations DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; GSH, reduced glutathione; HbA, adult human hemoglobin; rHb, recombinant hemoglobin; DCL-Hb, diaspirin cross-linked hemoglobin; Hp, haptoglobin; SEC, size exclusion chromatography; Tm, melting temperature. 230 FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS allow correct oxygen delivery; however, clinical trials have shown a vasoactivity of these molecules in plasma. Increasing the molecular size of the carrier has been proposed to reduce the undesirable vasoactive properties. Different approaches have been developed, such as surface modification of different Hbs by poly(ethylene) glycol 2 conjugation [3–5]. These different Hb derivatives, chemically modified by polymerization or coupling to macromolecules, may overcome the ex- travasation and vasoactive effects of acellular Hb. We have produced the recombinant Hb b83Glyfi Cys, b41PhefiTyr (rHb bG83C-F41Y), where the first mutation provides an intertetramer disulfide bridge and the second mutation [6] decreases the oxygen affin- ity of the rHb. In this study, in addition to the basic ligand binding properties and the thermal stability, we explore the oxido-reduction kinetics of the disulfide bridge; once formed, the octameric form (dimer of tetramers) is stable whatever the protein concentration, and does not interact with haptoglobin (Hp), a plasma glycoprotein. Results The elution profile of rHb bG83C-F41Y obtained by size exclusion chromatography (SEC) after purification on Q-Sepharose XL anion exchanger, shows the pres- ence of a major fraction eluted at a volume corres- ponding to 129 kDa, as previously observed for the singly mutated rHb bG83C [7]. This value is consistent with a dimer of tetramers or an octamer [a 2 b 2 G83C- F41Y] 2 . A minor peak eluting at the expected volume for a tetramer was observed; this tetramer fraction evolves to the octameric fraction over several days at 4 °C. In this study, we show that under specific experi- mental conditions, the process of forming the octamers can be much faster. Stability of the bG83C-F41Y oligomers Concentration dependent dissociation equilibrium We have studied the concentration dependence of the oligomers by SEC on Superose TM 12 HR 10 ⁄ 300 GL (Amersham Biosciences, Uppsala, Sweden). For the control adult human hemoglobin (HbA), the eluted peak profile shifts with decreasing concentration from the tetrameric to dimeric form (Fig. 1A); the peak position and width of the predominantly dimeric form occurred at about 4 lm concentration (on a heme basis) applied to the column, with typically a 60-fold dilution of sample in the column. In contrast, irres- pective of the applied concentration (from 150 to 6 lm), the octameric rHb bG83C-F41Y eluted at the same volume (Ve ¼ 12.19 ± 0.02 mL), corresponding to a molecular complex formed by 2 tetramers (2.08 ± 0.055) 3 (Fig. 1B). The peak width at half height remained small and constant (653 ± 10 lL), indicating that the oligomer has a high degree of size homogeneity and stability, and does not dissociate into smaller species. The same results were obtained with the singly mutated rHb bG83C [7]. Auto-oxidation The sensitivity of the octamers in relation to heme oxidation was determined by measuring the auto- oxidation rates. The octameric rHb bG83C-F41Y and octameric rHb bG83C were studied at 37 °C in phos- phate buffer at low protein concentrations of 24 lm and 10 lm, on a heme basis, respectively. The diaspirin cross-linked hemoglobin (DCL-Hb) at 38 lm was used A B Fig. 1. SEC profiles of HbA (A) and octameric rHb bG83C-F41Y (B) at protein concentrations ranging from 150 to 6 l M, on a heme basis. Aliquots of 10 lL were applied on Superose TM 12 HR 10 ⁄ 300 GL column and eluted at 0.4 mLÆmin )1 flow rate. C. Vasseur-Godbillon et al. Tetramer–octamer transition of Hb bG83C-F41Y FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS 231 as a tetrameric Hb control. The time coefficient of auto-oxidation kinetics was  17 h for DCL-Hb, 12 h for rHb bG83C-F41Y and 15 h for rHb bG83C sam- ples. In all cases, there was a small fraction having an auto-oxidation rate nearly 10 times higher; note that Hb dimers oxidize about 10 times more rapidly than tetramers [9]; a polymer formed of a series of dimers would probably show an enhanced oxidation rate. The time coefficient of auto-oxidation of octameric bG83C- F41Y was slightly decreased compared to that of DCL-Hb but was much longer than other polymeric mutants described recently [8]. Secondary structure and thermal stability The far-UV CD spectrum (results not shown) deconvo- luted using cdnn software (Bohm, Halle, Germany, http://bioinformatik.biochemtech.uni-halle.de/cdnn) 4 , revealed that octameric rHb bG83C-F41Y and rHb bG83C contained 72% and 77% a-helix, respectively. These values were similar that those observed for HbA and DCL-Hb (74% a-helix), suggesting that the disul- fide bridge does not modify the secondary structure of the molecule. The stability of octameric rHb bG83C-F41Y was investigated as a function of temperature. Figure 2 shows the first derivative of the ellipticity as a function of temperature for HbA, DCL-Hb, and the octameric forms of rHb bG83C and rHb bG83C-F41Y. The calculated melting temperature (Tm) values are 72, 79, 78 and 77 °C, respectively. The Tm values for the octamers exceed that for HbA, indicating that the oc- tamers maintain good conformational stability. Reaction with Hp Hp binds rapidly to Hb dimers but not to tetramers [10] according to the reaction scheme: a 2 b 2 þ Hp $ 2ab þ Hp ! HpðabÞ 2 We have studied the possible interaction between octa- meric rHb bG83C-F41Y and Hp that would indicate whether dimers might dissociate from the octamers. In Fig. 3A, we show the elution profile by SEC of Hp Fig. 2. First derivative of the fraction unfolded (f u ) vs. temperature, of native HbA (s), octameric rHb bG83C (–), octameric rHb bG83C- F41Y (h) and DCL-Hb (m). Protein concentration was 18 l M (on a heme basis) in 2.5 m M Na 2 HPO 4 , 37.5 mM NaCl buffer at pH 7.4. The change in ellipticity was recorded at 222.6 nm from 25 to 100 °C with a heating rate of 1 °CÆmin )1 . The peaks correspond to the median melting temperature (Tm). Fig. 3. SEC profiles after reaction of Hp with HbA (A) or octameric rHb bG83C-F41Y (B). The reactions were achieved at 25 °Cin 150 m M Tris ⁄ acetate buffer at pH 7.5 and after 15 min incubation, the different species were analyzed on Superose TM 12 HR 10 ⁄ 300 GL column. The mixtures of Hp with HbA or octameric rHb bG83C-F41Y are represented by a solid line. The Hp is represented with a dotted line. The control Hb samples are represented by dash-dot line. Tetramer–octamer transition of Hb bG83C-F41Y C. Vasseur-Godbillon et al. 232 FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS (Ve ¼ 10.44 mL), HbA (Ve ¼ 13.28 mL) and the mix- ture of HbA and Hp after a 15 min incubation at room temperature. With the mixture, only one peak was observed with Ve ¼ 10.06 mL corresponding to the elution volume of the Hp:(dimer) 2 . When the same experiment was performed by mixing octameric rHb bG83C-F41Y with Hp (Fig. 3B), the elution profile of this mixture shows the presence of two species (at elution volumes of 10.45 mL and 11.63 mL) corres- ponding to the elution volumes of Hp and octameric rHb bG83C-F41Y, respectively. The same result was obtained for Hp with the octameric rHb bG83C. The same type of result (no interaction with Hp) was also obtained with DCL-Hb, which does not dissociate into dimers (data not shown). The lack of interaction with Hp indicates that the two octamers do not dissociate into dimers. Stability in fresh plasma The stability of octameric rHb bG83C-F41Y was tes- ted in the presence of the reducing agents present in blood. The octameric rHb bG83C-F41Y was incubated in fresh human plasma at 37 °C. The analysis of the relative populations of the disulfide species by SEC showed only the octameric species for incubation times as long as 24 h (data not shown). These different results indicate a stable octameric form for rHb bG83C-F41Y vs. temperature, protein dilution, or in a physiological environment. Study of disulfide bridge kinetics It is important to determine if the formation of the disulfide bridge is a reversible process. To test this hypothesis, the octameric rHb bG83C-F41Y was reduced by 100 molar excess of reduced glutathione (GSH) or dithiothreitol (DTT), and then checked along with time for the tetrameric species apparition from the octameric ones. These experiments demonstrated that the disulfide bond could be reduced, leading to the dissociation into tetramers on the order of hours; the process of forming the octamer could be repeated by stripping the reducing agents and simply incubating the sample under air, an oxidizing condition. Octamer to tetramer transition The disulfide bridge can be reduced by GSH provo- king loss of the octameric form. In the first experi- ment, we studied the reduction of octamers at various concentrations of GSH during a 2 h incubation at 25 °C (Fig. 4). Two concentrations of octamers were tested: 6 and 13 lm on a heme basis. For both concen- trations, the curves are biphasic. Treating the rate coefficients as a second-order reaction, the rapid phase has a time coefficient of about 1 h at 1 mm GSH whereas the second phase was nearly an order of mag- nitude slower and concerns about 25% of octamers. In the second experiment, we studied the disulfide bridge reduction kinetics of octamers for fixed (1 or 25 mm) GSH concentrations (Fig. 5). The kinetics show an initial phase, followed by a plateau; the rate of the initial phase is 1000 m )1 Æh )1 for 1 mm of GSH. At 25 mm GSH, the rapid phase was not fully resolved. The experiment was achieved in the presence of air, which explains why the octamers were not com- pletely dissociated, because equilibrium is established between air oxidation and GSH reduction. The reduc- tion reaction is more complete at higher GSH concen- trations or for samples under a nitrogen atmosphere. Disulfide bridge formation between rHb bG83C-F41Y tetramers The octameric rHb bG83C-F41Y was first reduced by a 100 molar excess of GSH, taken up on Superose TM 12 HR 10 ⁄ 300 GL, and then the tetrameric rHb bG83C-F41Y at 11 lm on a heme basis was incubated at 4 °Cor25°C. The kinetics of octamer formation at 4 °C and 25 °C are shown in Fig. 6. The kinetic curves are not simple exponential, but show a form with Fig. 4. Reduction of disulfide bridge in octameric rHb bG83C-F41Y by GSH. The fraction of octamers vs. GSH concentration is shown for samples after an incubation of 2 h at 25 °C. The initial concentra- tions of bG83C-F41Y octamers were 6 l M (d) and 13 lM (m)ona heme basis. The experiments were performed at 25 °C in 150 m M Tris ⁄ acetate buffer at pH 7.5. The solution of GSH was prepared in the same buffer. After incubation, a 70 lL aliquot of the mixture was analyzed on Superose TM 12 HR 10 ⁄ 300 GL column. C. Vasseur-Godbillon et al. Tetramer–octamer transition of Hb bG83C-F41Y FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS 233 decreasing rate vs. time, as expected for a dimerization reaction. As the source of reactants (tetramers in this case) is depleted, the overall bimolecular rate coeffi- cient decreases. The curves were therefore simulated with a dimerization model and a single bimolecular rate coefficient of 2000 m )1 Æh )1 at 25 °C. The kinetics showed observed rates of 0.0055 h )1 and 0.025 h )1 at 4 °C and 25 °C, respectively. A reduction ⁄ oxidation cycle can thus be repeated to form the disulfide bridges in rHb bG83C-F41Y. For both the reduction and the oxidation processes, the transition was faster at 25 °C than at 4 °C. The octamer formation kinetics were slower at lower protein concentrations; the kinetics at 0.3 lm (on a heme basis) were too slow to be observed on the time- scale used. These results confirm the bimolecular char- acter of the kinetics of octamer formation. Hybridization with HbA HbA does not form disulfide bonds via the b93 cys- teine residues; however, there was a question as to whether Hb Tali could form octamers via a b93–b83 interaction. The experiments for the tetramer to octamer transition of rHb bG83C-F41Y were per- formed at 25 °C in the presence of DCL-Hb or HbA to determine if the b93 cysteine residues could partici- pate in the disulfide bridge formation (Fig. 7). For a fixed concentration of rHb bG83C-F41Y, the initial velocity of octamer formation and the amount of octamer were similar in the presence or absence of the other Hb (HbA or DCL-Hb), meaning that the quantity of octamers formed was reduced by half, Fig. 6. Disulfide bridge formation kinetics of tetrameric rHb bG83C- F41Y. A stock of tetramers was prepared by incubating rHb bG83C-F41Y in a 100 molar excess of GSH at 25 °C for 2 h. After purification on Superose TM 12 HR 10 ⁄ 300 GL column in 150 mM Tris ⁄ acetate buffer at pH 7.5, the tetrameric fraction rHb bG83C- F41Y (11 l M on a heme basis) was incubated under air at 4 °C(d) or at 25 °C(m). At different times, a 70 lL aliquot of mixture was analyzed on Superose TM 12 HR 10 ⁄ 300 GL column. The kinetics show the shape typical of dimerization (lines are simulations), where the effective rate constant decreases as the source of reac- tants is depleted. Fig. 7. Oxidation disulfide bridge kinetics of tetrameric rHb bG83C- F41Y in the presence of HbA or DCL-Hb. The experiments were performed at 25 °Cin150m M Tris ⁄ acetate buffer at pH 7.5. The tetrameric fraction of rHb bG83C-F41Y was purified on Superose TM 12 HR 10 ⁄ 300 GL column, then the tetrameric rHb bG83C-F41Y, at 11 l M on a heme basis, was incubated in the presence of the same concentration of HbA (d) or of DCL-Hb (n) or in Tris ⁄ acetate buffer as control sample (m). At different times, a 70 lL aliquot of the mix- ture was analyzed on a Superose TM 12 HR 10 ⁄ 300 GL column. Fig. 5. Disulfide bridge reduction kinetics of octameric rHb bG83C- F41Y by 1 m M (m)or25mM (d) GSH. The experiments were per- formed at 25 °C in 150 m M Tris ⁄ acetate buffer at pH 7.5. The solution of GSH was prepared in the same buffer. The final concen- tration of octameric rHb bG83C-F41Y was 5 l M on a heme basis. At different times, a 70 lL aliquot of the mixture was analyzed on Superose TM 12 HR 10 ⁄ 300 GL column. Tetramer–octamer transition of Hb bG83C-F41Y C. Vasseur-Godbillon et al. 234 FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS considering the total Hb concentration. This indicates that neither HbA nor DCL-Hb participated in the intermolecular S–S cross-linking between the b93 and b83 cysteine residues. Functional studies CO rebinding kinetics The CO rebinding kinetics for rHb bG83C and the double mutant rHb bG83C-F41Y were typical of HbA, showing two phases characteristic of the oxy (R-state) and deoxy (T-state) conformations of tetra- meric Hb (Fig. 8). Unlike HbA, the octameric rHbs did not dissociate into component dimers at low pro- tein concentration. At low Hb concentration, HbA shows a higher percentage of the rapid CO recombina- tion, because dimers display kinetics that are similar to the rapidly reacting R-state tetramer conformation. However, the rHb bG83C-F41Y octamers did not show a change in kinetics over the range 0.2–10 lm. The CO rebinding kinetics for rHb bG83C-F41Y showed more of the slow CO recombination, relative to HbA and rHb bG83C (Fig. 8). This same effect occurs for rHb bF41Y relative to HbA, indicating that the shift towards the T-state conformation occurs for both the tetrameric and octameric form. This mutation at the b41 site can thus be useful to modulate the over- all oxygen affinity [6]. The main difference between the octameric and tetra- meric forms is in the ligand cooperativity. The octamers do not show a full transition. At high CO photodissocia- tion levels (50%), the CO recombination kinetics of octameric rHb bG83C-F41Y were similar to HbA, except for the increase in the amount of slow phase mentioned above. As the laser photolysis energy is decreased, the kinetics of HbA tend to show only the rapid phase, because the main photoproduct is triply liganded Hb which is predominantly R-like. However, the octameric rHbs maintain a significant fraction slow phase, even at low photodissociation levels. Oxygen-binding properties of octameric rHbs The oxygen-binding properties of rHb bG83C-F41Y, rHb bG83C, and HbA are summarized in Fig. 9, for Fig. 8. Recombination kinetics of CO to rHb bG83C-F41Y. At a 50% photodissociation level, the rHb bG83C-F41Y (n) shows two phases, corresponding to two Hb allosteric states, as for HbA (d). The rHb bG83C-F41Y shows more slow phase than rHb bG83C (m), due to the b41 mutation. Fig. 9. pH-dependence of the oxygen affinity (A) and the maximum Hill coefficient (n max )(B):(d) HbA; (m)rHbbG83C; (n)rHbbG83C- F41Y. Oxygen dissociation data were obtained at a concentration of 0.1 m M Hb (in terms of heme) in 0.1 M 14 sodium phosphate buffer in the pH range 5.8–8.4 at 29 °C. P 50 (mmHg) and n max were deter- mined from each curve. C. Vasseur-Godbillon et al. Tetramer–octamer transition of Hb bG83C-F41Y FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS 235 samples in 0.1 m sodium phosphate buffer at 29 °C. Oxygen affinity (or P 50 ) corresponds to the oxygen partial pressure at 50% saturation of the hemes. The rHb bG83C-F41Y has an oxygen affinity, and pH dependence of P 50 , similar to that of HbA, although there is a somewhat higher oxygen affinity at pH < 7 compared to that of HbA. The rHb bG83C has a higher oxygen affinity than that of HbA (e.g. at pH 7.4, P 50 ¼ 4.6 mmHg vs. 9.3), and displays a wea- ker pH dependence. Both octamers show a cooperativity of oxygen bind- ing, but there is a decrease in cooperativity relative to the control HbA. The Hill coefficient for the rHbs is slightly lower than that of HbA over the pH (e.g. n max  2.6 for the double mutant or 2.3 for the single mutant vs. 3.0 for HbA). There is not much difference in the Bohr effect (–Dlog P 50 ⁄ log pH) between HbA and rHb bG83C- F41Y over the pH range from 6.5 to 8.2. The rHb bG83C shows a much lower Bohr effect. 1 H-NMR Studies Figure 10 shows the 300 MHz NMR spectra of rHb bG83C-F41Y and HbA in the CO and deoxy forms in 0.1 m sodium phosphate buffer at 29 °C. A general feature of the NMR spectra of this rHb is that the line-width is much broader than that of HbA due to the oligomerization of this rHb. Figure 10A shows the exchangeable proton resonance region of a Hb mole- cule in the CO form. The exchangeable resonances at 12.9 and 12.1 p.p.m. from 2,2-dimethyl-2-silapentane- 5-sulfonate (DSS) are excellent markers for the a 1 b 1 subunit interface arising from the H-bonds between a122His and b35Tyr and between a103His and b131Gln, respectively [11–13]. There is a very slight downfield shift ( 0.1 p.p.m.) for the resonance at 12.9 p.p.m. for the rHb compared to HbA and no observ- able change in the resonance at 12.1 p.p.m. These results indicate that the a 1 b 1 subunit interface remains essentially intact in the rHb. The resonance at 10.7 p.p.m. has been assigned to the side chain of b37Trp in the a 1 b 2 subunit interface of HbA in CO form [12,14]. There is a very slight downfield shift ( 0.1 p.p.m.) for this resonance in rHb bG83C-F41Y. Figure 10B shows the hyperfine-shifted and exchange- able proton resonances for rHb bG83C-F41Y and HbA in the deoxy form in 0.1 m sodium phosphate buffer at 29 °C. The line-widths for the observed reso- nances for the rHb are broader than those of HbA due to the oligomerization of the rHb. The exchange- able resonance at 14.2 p.p.m. has been assigned as the H-bond between a42Tyr and b99Asp in the a 1 b 2 subunit interface of deoxy HbA [15], an important quaternary structural marker of deoxy HbA [16]. There is a downfield shift of  0.2 p.p.m. in this reson- ance in the rHb, indicating that there is a slight pertur- bation in the a 1 b 2 subunit interface compared to that in deoxy HbA. This is consistent with the observed slight shift of the resonance assigned to b37Trp in the a 1 b 2 subunit interface of the rHb in the CO form men- tioned above. It is noted that the hyperfine-shifted pro- ton resonances over the spectral region from 14 to 24 p.p.m. are different between rHb bG83C-F41Y and HbA in the deoxy form. These resonances arise from the protons of amino acid residues situated in the vicinity of the heme groups and of the porphyrins of both the a- and b-chains of hemoglobin. The amino acid substitutions in this rHb could perturb the heme environment, resulting in an alteration of the hyper- fine-shifted proton resonances as observed. In addition, Fig. 10. 1 H-NMR spectra (300 MHz) of 5% HbA and rHb bG83C- F41Y in 0.1 M sodium phosphate 15 at pH 7.0 in H 2 O and at 29 °C: exchangeable proton resonances in the CO form (A) and hyperfine- shifted and exchangeable proton resonances in the deoxy form (B). Tetramer–octamer transition of Hb bG83C-F41Y C. Vasseur-Godbillon et al. 236 FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS the much broader resonances in the region from 14 to 24 p.p.m. are probably due to formation of met-Hb in the rHb. In general, these broad resonances due to the hyperfine-shifted proton resonances of met-Hb can be removed by the addition of dithionite. However, we have found that the presence of dithionite can break S–S bonds in hemoglobin (results not shown) and other proteins as reported in the literature [17,18]. Thus, we did not add dithionite to our deoxy-rHb bG83C-F41Y for NMR measurements resulting from the formation of met-Hb in the sample. Discussion In vivo and in vitro disulfide formation is catalyzed by specialized enzymes. In vivo, disulfide formation was achieved in the endoplasmic reticulum by enzymes belonging to a thioredoxin superfamily, such as protein disulfide isomerase in eukaryotes and disulfide bond proteins in prokaryotes [19]. It has been shown that the oxidative folding of different proteins in vitro is accelerated by protein disulfide isomerase [20]. In the case of the natural mutant Hb Porto Alegre (Hb bS9C) [21,22] or the rHb Prisca (rHb bS9C + C93A + C112G) [23] both of which carry in position b9 an extra thiol group oriented towards the exterior of the Hb molecule, the oligomerization pro- cess was not observed immediately, either after lysis of the red cells or after purification of the rHb. In the case of rHb Prisca, the maximum oligomer was obtained after a 110 day incubation at 25 °C [23]. Recently, another recombinant polymeric Hb was described, the rHb Minotaur containing a-human and b-bovine in which the b9Ala was replaced by Cys and the b93Cys was replaced by Ala. The polymer of puri- fied rHb Minotaur was obtained after 2 days at 30 °C or 30 days at 4 °C [8]. The present study of rHb bG83C-F41Y shows that the disulfide bridge forma- tion in this recombinant mutant is a relatively fast pro- cess that does not require any external reagents such as the glutathione redox system. Contrary to the other polymeric Hbs, the oligomerization process of rHb bG83C-F41Y and of the single mutant rHb bG83C was observed immediately at 4 °C after purification. Number of disulfide bonds In rHb bG83C-F41Y, as for rHb bG83C, disulfide bonds between the b83 cysteine residues stabilize the octameric structure. Once formed the octameric frac- tion rHb bG83C-F41Y remained stable for several months at 4 °C. There is still a question as to whether the octamer is formed with one or two disulfide bonds. A single disulfide bond would correspond to tethered tetramers, each relatively free to make the allosteric transition or tetramer-dimer reaction. By symmetry, if each beta chain participates in the formation of a disulfide bond (2 per octamer), the tetramers would be more constrained. The octamer would be more stable as well; even if one tetramer dissociated into dimers, each dimer would be held via an S–S bond to the other tetra- mer (see Fig. 5 in [7]). After intravascular hemolysis, Hp binds the free Hb, allowing the clearance of Hb from the plasma. The monocyte ⁄ macrophage specific glycoprotein CD136 was recently described as a receptor that scavenges Hb by mediating endocytosis of the Hp–Hb complex [24]. Accordingly, the Hp–Hb complex is eliminated from the circulation. Neither of the octameric rHbs (bG83C-F41Y and rHb bG83C) react with Hp, confirming that these octamers do not dissociate easily into dimers; the absence of the formation of complexes of Hp with these octamers would increase their useful lifetime for oxygen delivery. The lack of any interaction with Hp indicates that there are no free dimers and would sup- port the model with two disulfide bonds. A hypothetical diagram is shown in Fig. 11, based on the crystallographic structure of the Hb tetramer. By symmetry, both beta chains can form a disulfide bond, provided there is no steric hindrance of other protein residues. The b83 glycine (in red) would have two additional atoms (C–S) for the bG83C mutation. A disulfide bond is typically about 6 A ˚ between the C a atoms. As can be seen, the intervening residues (b79 and 80) would not require a greater distance. On the other hand, forming two b83–b93 bonds would be more difficult as the helix A would cause a larger hin- drance. As an alternative analysis, one could consider the two tetramers, in a first approximation, as tangent spheres. Based on the angle between vectors from the center to each b83 residue, one can calculate the dis- tance between the b83 C a of the opposing spheres. For two disulfide bonds via the b83 residues, the distance would be 6 A ˚ , which is quite compatible with a typical disulfide bond. For a b83–b93 bonding, the distance required would be 8 A ˚ and therefore not possible. Note that the distance between certain residues such as b83 between the b chains of the same tetra- mer depend on the allosteric state. In going from the deoxy to oxy conformation the b 1 83–b 2 83 dis- tance would decrease from 24 to 19 A ˚ (for b 1 83– b 2 93, the change would be from 31 to 25 A ˚ ). This would require the two tetramers forming the octamer to make the allosteric transition together. This could C. Vasseur-Godbillon et al. Tetramer–octamer transition of Hb bG83C-F41Y FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS 237 explain the decreased cooperativity observed for the octamer in the equilibrium and especially the kinetic experiments. Finally the experiments detecting the number of tit- ratable cysteines confirm the hypothesis of two disul- fide bonds. For unfolding condition, the mutant and native Hb samples show the same signal amplitude, indicating the same number of marked cysteines; this implies that the new cysteines are not marked and must therefore be part of a disulfide bond. There is still the possibility of a b83–b93 bond, but the hybrid (mutant + HbA) experiments, and the steric hin- drance considerations, indicate that b83 is the most probable site for formation of the two disulfide bonds. Heme oxidation It has been shown that cross-linked or polymerized Hb may show an acceleration of the auto-oxidation process [25]. The study of auto-oxidation of octa- meric rHb bG83C-F41Y shows that this octamer does not modify the half time of heme auto-oxida- tion, which remains close to that of DCL-Hb or HbA, contrary to the other polymers such as rHb Minotaur, which has a half time of auto-oxidation of 3.2 h [8]. The oxygen equilibrium curves of the rHb bG83C display a higher average affinity, and a lower cooper- ativity (Fig. 9). Addition of the second mutation decreases the oxygen affinity, but induces a further decrease in the Hill coefficient. The results of the lig- and binding kinetics and oxygen equilibrium curves indicate some limitations in the allosteric transition. One factor to consider is the double bridging of the tetramers; that is, each beta chain might form a disul- fide bond with the opposing tetramer, unless some steric hindrance prevents the formation of the second bond. With both bonds present, the two tetramers must make the allosteric transition together, because the distance between the b83 residues changes. This could lead to new constraints on the synchronized allosteric transition of both tetramers within the octamer. With their stabilization in dilute solution, the octameric rHb bG83C-F41Y and the octameric rHb bG83C are both good model molecules to develop hemoglobin-based oxygen carriers. The octa- meric form of both recombinant Hbs (with or with- out the additional mutation at the b41 site) showed a high stability; there was no interaction with hapto- globin and no dissociation provoked by incubation in fresh plasma. These octameric Hbs are thus poten- tially useful as blood substitutes. The clinical trial of HemeAssist TM (Baxter Healthcare) revealed some escape of the tetrameric Hb from the blood vessels [26]; an octameric form is thus the logical extension of research for a blood substitute based on Hb solu- tions. The best choice between the two molecules tes- ted here is not obvious. There was initially an attempt to mimic the physiological oxygen affinity, which is better approximated by the mutant b41. However, lower oxygen affinities lead to higher oxidation rates, and in the present case the double mutant displayed less cooperativity. Current ideas suggest that a higher oxygen affinity may still be useful and provide a better oxygenation of the capillaries [27]. The single mutant may thus be the better candidate molecule. Experimental procedures Hemoglobin expression and purification The mutated Hbs were produced in JM 109 strains of Escherichia coli using the expression plasmid pHE7 contain- ing human a-, b-globin cDNAs and an E. coli methionine aminopeptidase cDNA [28], after introduction of the b41Phe fi Tyr from the pHE7 template containing the b83Gly fi Cys mutation (Quick change TM site Fig. 11. Proposed scheme for an octamer formed by disulfide bonds between Hb tetramers via the b83 site (glycine in HbA; shown as red spheres), where only residues b79 (orange) and b80 (blue) might interfere. The mutation bG83C would introduce addi- tional atoms (–C–S) to bridge the distance (shown as a dotted yellow line) between b83 sites of the opposing (top vs. bottom) tetramers. Within a tetramer the b 1 83 to b 2 83 distance is 24 A ˚ 16 for the deoxy Hb conformation (but only 19 A ˚ for the oxy form; where the analogous position for the b83 glycine is shown in green). This large change in distance would imply that the two tetramers must make the allosteric transition together. Note that the corresponding distances for a b83 to b93 (yellow residue) bonding are higher and would involve more steric hindrance. Tetramer–octamer transition of Hb bG83C-F41Y C. Vasseur-Godbillon et al. 238 FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS directed-mutagenesis kit, Stratagene Europe, Amsterdam, the Netherlands) and verification of the a- and b-globin coding sequences (MWG Biotech, Courtaboeuf, France). The cells were harvested by centrifugation at 6000 g for 10 min at 4 °C 7 and stored frozen at )80 °C until needed for purification. The rHb was isolated and purified as des- cribed by Shen et al. [28,29] with minor modifications [7]. The oligomeric and tetrameric fractions were then separated by SEC on a Superose TM 12 HR 10 ⁄ 300 GL column 8 (Amersham Biosciences, Uppsala, Sweden) equilibrated at 25 °C with 150 mm Tris ⁄ acetate buffer at pH 7.5 [7]. Auto-oxidation kinetics The kinetics of auto-oxidation of rHb bG83C-F41Y and rHb bG83C were followed by absorption spectrophoto- metry at 37 °C, for samples under air [9]. Hb solutions were in 100 mm potassium phosphate at pH 7.0. Thermal denaturation Thermal denaturation was achieved with a Jasco J810 spectropolarimeter (Jasco, Tokyo, Japan), using a 0.5 mm path-length cell, and the temperature in the cell was pro- grammed using a Jasco PTC-423S thermoelectric tempera- ture controller. The ellipticity at 222.6 nm was monitored over a temperature range of 25–100 °C, using a bandwidth of 1 nm, and a temperature gradient of 1 °C per min. The Hb samples were in the CO form and at a concentration of 18 lm (on a heme basis) in 2.5 mm Na 2 HPO 4 , 37.5 mm NaCl buffer at pH 7.4. The Tm corresponds to 50% unfolded molecule. The CD signal was normalized to obtain the unfolded fraction: f u ¼ (y N –y obs ) ⁄ (y N –y u ), where y obs is the observed CD signal and y N and y u the CD signal of the native and unfolded protein, respectively. Interaction of octamers with haptoglobin Reaction with Hp (Sigma Aldrich, Saint Quentin Fallavier, France) was achieved at room temperature in 150 mm Tris ⁄ acetate buffer at pH 7.5 containing Hp at 2.9 lm and either 7 lm of HbA (control reaction) (on a heme basis), or 6 lm octameric rHb bG83C or rHb bG83C-F41Y (on a heme basis). After a 15 min incubation, the presence of different species was analyzed by SEC on a Superose TM 12 HR 10 ⁄ 300 GL column. Stability of octamers in fresh plasma The octameric rHb bG83C-F41Y was mixed with fresh human plasma in a ratio of 7 g Hb to 500 mL plasma at 37 °C. At different times, an aliquot was withdrawn, centrifuged at 3000 g at room temperature for 2 min 9 and analyzed on Superose TM 12 HR 10 ⁄ 300 GL column. Disulfide reduction kinetics of the oligomeric rHb bG83C-F41Y In the first experiment, 100 lL aliquots of purified oligo- meric fraction at 6 and 13 lm (heme basis) were incubated at 25 °C in the presence of increased concentration of GSH during 2 h and the relative populations of the disulfide spe- cies obtained were analyzed by SEC on a Superose TM 12 HR 10 ⁄ 300 GL column. In the second experiment, the puri- fied oligomeric fraction at 6 lm (heme basis) was incubated at 25 °C in the presence of 1 or 25 mm GSH. Aliquots of 100 lL were withdrawn at various times, and the relative populations of the disulfide species of the mixture were ana- lyzed by SEC [7]. Disulfide bridge formation of rHb bG83C-F41Y The disulfide bridge of rHb bG83C-F41Y was first reduced with a 100 molar excess of GSH for 2 h at 25 °C; the GSH was removed by SEC. Then the tetramer to oc- tamer formation of rHb bG83C-F41Y (11 lm on a heme basis) was achieved at 25 °C and 4 °C. At various times, an aliquot was withdrawn and the relative populations of the disulfide species of the mixture were analyzed by SEC. In the second experiment the re-oxidation kinetics of the tetrameric rHb bG83C-F41Y (11 lm on a heme basis) were achieved in the presence of the same concentration of DCL-Hb or HbA. CO recombination kinetics Kinetics of CO recombination were obtained after flash photolysis using 10 ns YAG laser pulses (Quantel 10 , Les Ulis, France) providing 160 mJ at 532 nm. Samples were in 1 or 10 mm cuvettes equilibrated under 0.1 atm (100 lm) CO, with observation at 436 nm. Measurements were made at 25 °C in 150 mm Tris ⁄ acetate buffer at pH 7.5 [30]. Oxygen-binding measurements Oxygen-binding measurements were carried out using a Hemox Analyzer (TCS Medical Products, Huntington Valley, PA, USA). As previously described, the experi- ments were run at 29 °C as a function of pH in 0.1 m sodium phosphate buffer and contained 0.1 mm Hb (on a heme basis) [28,29]. The maximum Hill coefficient, n max , was determined from the maximum slope of the Hill plot as a measure of cooperativity in the oxygenation process. The P 50 values (in mmHg) are given with an accuracy of ±5%. The n max values are reported with an accuracy of ±7%. The Bohr effect was obtained from the P 50 values as a function of pH using the linkage equation (DH + ¼ –¶log P 50 ⁄¶pH), which gives the number of the Bohr pro- tons released upon oxygenation per heme [31,32]. C. Vasseur-Godbillon et al. Tetramer–octamer transition of Hb bG83C-F41Y FEBS Journal 273 (2006) 230–241 ª 2005 The Authors Journal compilation ª 2005 FEBS 239 [...]... & Baudin-Creuza V (2003) Stable octameric structure of recombinant hemoglobin alpha (2) beta 2, 83 Gly fi Cys Protein Sci 12, 690–695 Bobofchak KM, Mito T, Texel SJ, Bellelli A, Nemoto M, Traystman RJ, Koehler RC, Brinigar WS & Fronticelli C (2003) A recombinant polymeric hemoglobin with conformational, functional, and physiological characteristics of an in vivo O2 transporter Am J Physiol Heart Circ... Acknowledgements We thank G Caron for skilful technical assistance and Dr Ming F Tam for carrying out the Edman degradation measurements for our rHb sample We are grateful to the Baxter Healthcare Company for supplying DCL-Hb This work was supported by the Institut ´ ´ National de la Sante et de la Recherche Medicale, the Association Recherche et Transfusion (contract no 212000) and by research grants from the... formation An Acad Bras Cienc 43, 651–669 T-state hemoglobin with four ligands bound BiochemisBaudin-Creuza V, Fablet C, Zal F, Green BN, Prome try 27, 1659–1664 D, Marden MC, Pagnier J & Wajcman H (2002) Hemo31 Wyman J (1948) Heme Proteins Adv Protein Chem 4, globin Porto Alegre forms a tetramer of tetramers 13 407–531 superstructure Protein Sci 11, 129–136 32 Wyman J (1964) Linked functions and reciprocal... & and high cooperativity of a novel recombinant hemogloAlayash AI (2002) Site-specific cross-linking of human bin (beta L105W) Biochemistry 39, 13708–13718 and bovine hemoglobins differentially alters oxygen Fung LWM & Ho C (1975) A proton nuclear magnetic binding and redox side reactions producing rhombic resonance study of the quaternary structure of human heme and heme degradation Biochemistry 41,... Abbott-Brown D, Cozart P, Durfee S, Hoffman S, Mathews AJ, Miller-Roehrich J, Shoemaker S, Trimble S, Fermi G, Komiyama NH, Nagai K & Stetler 240 13 14 GL (1992) A human recombinant haemoglobin designed for use as a blood substitute Nature 35, 258–260 Vandegriff KD, Malavalli A, Wooldridge J, Lohman J & Winslow RM (2003) MP4, a new nonvasoactive PEG-Hb conjugate Transfusion 43, 509–516 Barnikol WK, Burkhard... at pH 7 in 100 mm potassium phosphate buffer and 100 lm DTNB, with a protein concentration of 5 lm, determined from the Soret band for the CO species using the extinction 12 coefficient of 190 mm)1Æcm)1 at 420 nm The absorption at 455 nm was monitored over 2 h and corrected against a blank to which no protein was added Under denaturing conditions (6 m guanidinium chloride) these samples showed the same... Burkhard O, Poetzschke H, Domack U, Dinkelmann S, Guth S, Fiedler B & Manz B (2002) New artificial oxygen carriers made of pegulated polymerised pyridoxylated porcine haemoglobin (P(4)Hb) Comp Biochem Physiol A Mol Integr Physiol 132, 185–191 Manjula BN, Tsai A, Upadhya R, Perumalsamy K, Smith PK, Malavalli A, Vandegriff K, Winslow RM, Intaglietta M, Prabhakaran M, Friedman JM & Acharya AS (2003) Site-Specific... Cells Blood Substit Immobil Biotechnol 33, Wang PF, Veine DM, Ahn SH & Williams CH Jr 1–12 (1996) A stable mixed disulfide between thioredoxin 28 Shen TJ, Ho NT, Zou M, Sun DP, Cottam PF, reductase and its substrate, thioredoxin: preparation Simplaceanu V, Tam MF, Bell DA & Ho C (1997) and characterization Biochemistry 35, 4812–4819 Production of human normal adult and fetal Noiva R (1994) Enzymatic catalysis... Simplaceanu V, Lukin JA, Fang TY, Zou M, Ho NT & Ho C (2000) Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to haemoglobin Biophys J 79, 1146–1154 Chang CK, Simplaceanu V & Ho C (2002) Effects of amino acid substitutions at beta 131 on the structure and properties of hemoglobin: evidence for communication between alpha 1 beta 1- and alpha 1 beta 2-subunit... oxyhemoglobin mutants determined from auto-oxidation rates Protein Sci 7, 673–680 Nagel RL & Gibson QH (1971) The binding of hemoglobin to haptoglobin and its relation to subunit dissociation of hemoglobin J Biol Chem 246, 69–73 Russu IM, Ho NT & Ho C (1987) A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult haemoglobin Biochim Biophys Acta 914, 40–48 Simplaceanu V, . phosphate 15 at pH 7.0 in H 2 O and at 29 °C: exchangeable proton resonances in the CO form (A) and hyperfine- shifted and exchangeable proton resonances in the deoxy. Recombinant hemoglobin bG83C-F41Y An octameric protein Corinne Vasseur-Godbillon 1 , Sarata C. Sahu 2 , Elisa Domingues 1 , Christophe Fablet 1 , Janel