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Protein Science (2003), 12:690-695.
Copyright © 2003 The Protein Society

Stable octameric structure of recombinant hemoglobin {alpha}2ß283 Gly->Cys

Christophe Fablet1, Michael C. Marden1, Brian N. Green2, Chien Ho3, Josée Pagnier1 and Véronique Baudin-Creuza1

1 INSERM U 473, 94276 Le Kremlin-Bicêtre Cedex, France
2 Micromass UK Ltd., Altrincham, Cheshire WA14 5RZ, UK
3 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-2683, USA

Reprint requests to: Véronique Baudin-Creuza, INSERM U 473 84, rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France; e-mail: baudin{at}kb.inserm.fr; fax: 33-1-4959-5662.

(RECEIVED October 1, 2002; FINAL REVISION December 18, 2002; ACCEPTED December 19, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0234403.


   Abstract
TOP
 Abstract
Introduction
 Results and discussion
 Materials and methods
 References
 
We have engineered a recombinant hemoglobin (rHb ßG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb ßG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric ßG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric ßG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer ßG83C was not modified after incubation with fresh plasma.

Keywords: Hemoglobin; disulfide bridge; oligomerization; octamer; blood substitute

Abbreviations: DTT, dithiothreitol • DCL-Hb, diaspirin cross-linked hemoglobin • GSH, reduced glutathione • ESI-MS, electrospray ionization mass spectrometry • Hb, hemoglobin • Hb A, natural human hemoglobin • Hb-CO, carbonmonoxyhemoglobin • MetHbCN, cyanmethemoglobin


   Introduction
TOP
 Abstract
 Introduction
Results and discussion
 Materials and methods
 References
 
Many of the designed hemoglobin (Hb)-based blood substitutes can fulfill the essential functions of transfused blood, such as expanding plasma volume and transporting oxygen. However, among other problems, the question of the duration of action remains. This may be due to short residence times and/or to oxidation. In fact, free native Hb in plasma is rapidly dissociated into dimers which escape from circulation and may damage renal tubular cells. Another potential problem is the vasoactivity of free Hb in plasma (Stowell et al. 2001). The study of DCL-Hb (HemAssistTM, Baxter Healthcare), an intramolecularly crosslinked tetramer, has shown that this product with a molecular weight of 64 kD possesses a half-life of between 12 and 20 h in humans but induces vasoconstriction. Recent studies showed that the use of polymerized or polyethyleneglycol-Hb allows a further increase in vascular retention time and a decrease in extravasion (Haney et al. 2000). With the objective of improving the retention time and the vasoactivity, we have introduced by site-directed mutagenesis a cysteine residue at the ß83 position, based on the natural variant, Hb Ta-Li, ß83(EF7)Gly->Cys, which has been described as forming polymers via S-S bonds (Blackwell et al. 1971).

We report here the properties of the recombinant Hb ß83Gly->Cys (rHb ßG83C). Using size exclusion chromatography and electrospray ionization mass spectrometry (ESI-MS), we determined the size of the major form of rHb ßG83C.


   Results and discussion
TOP
 Abstract
 Introduction
 Results and discussion
Materials and methods
 References
 
Structural studies
The effects of the mutation ßG83C on the assembly of the Hb molecule were investigated by size exclusion chromatography at different stages of the purification. Three different size molecular species were present. From the calibration curve, the elution volumes of the three peaks correspond to 245.8, 127.9, and 64.5 kD. These values are consistent with molecular complexes formed by 4, 2, and 1 tetramers, respectively. Just after treatment with polyethyleneimine and centrifugation, the supernatant exhibited only 6% of a molecular complex formed by two tetramers. After concentration of the supernatant, the proportion of the complex increased to about 50%. After the first chromatographic separation (Q-Sepharose), the rHb ßG83C exhibited more than 80% octamers according to the preparation.

The gel filtration profile of the rHb ßG83C obtained after final purification on mono S cation exchanger column showed 88% to 90% of the molecular complex comprising two tetramers (Fig. 1Go). Two minor peaks were observed eluting at the expected volumes for a tetramer and for four tetramers (not shown). This last fraction occurs as a very small population which varies slightly from one preparation to another. Further analysis shows that it was not homogenous, consisting of two species of molecular mass around 320 and 240 kD, corresponding to five and four tetramers, respectively. The polymeric fraction can also be reduced by DTT to form smaller species. It may thus be the result of a series of singly bridged tetramers, which are less stable than the doubly bridged octamer.



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  		Figure 1. Gel filtration profiles of rHb ßG83C and two controls. Ten-µL aliquots were applied at a Hb concentration of 335 µM (heme basis), and eluted at 0.4 mL/min flow rate. DCL-Hb and Hb Rothschild [ß37Trp->Arg] were used as controls for undissociated tetrameric Hb and dimeric Hb, respectively. The Gilson system was monitored at 415 and 280 nm using Unipoint 1.8 software.

 
The natural mutants Hb Porto Alegre (Tondo 1971) and rHb Prisca (Fronticelli et al 2001) also oligomerize through inter-tetramer S-S bonds due to the presence of a cysteinyl residue at the ß9 position (Ser->Cys mutation). In both cases the oligomerization process was not observed immediately, either after lysis of the red cells or after purification of the rHb. In the case of rHb Prisca, ß [S9C + C93A + C112G], the maximum oligomer size was obtained after 110 d at 25°C (Fronticelli et al 2001). In contrast, in the case of the rHb ßG83C, more than 80% oligomer was present immediately after the first purification step. The size of the major fraction remained the same for incubation times up to 4 mo.

As expected, addition of a large (100-fold) excess of the reducing agent DTT to a solution of rHb ßG83C (100 µM on a heme basis) resulted in the loss of the octameric form, confirming the reversibility of the S-S bonds in the oligomerization process.

These results were corroborated by ESI-MS studies. Under noncovalent conditions the major fraction gave a spectrum (Fig. 2AGo) that showed a series of multiply charged ions whose mass is consistent with an octamer comprising four {alpha}-chains, four ß-chains, and eight heme groups ({alpha}4ßG83C4h8). At low declustering potentials, these ions dominated the spectrum. The only other Hb-related ions present at significant but low levels correspond to heme and {alpha}-chain plus heme. A spectrum from normal human Hb (Fig. 2BGo) shows tetramer ions as the dominant species with dimer ions at low levels. Supporting evidence for the identity of the chains comprising the major fraction was obtained by analyzing the reduced major fraction under denaturing conditions. These data showed major species at 15,126.4 and 15,913.4 Da corresponding to the normal {alpha}-chain (sequence mass 15,126.4 Da) and ßG83C-chain (sequence mass 15913.3 Da), respectively.



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  		Figure 2. ESI mass spectra obtained under noncovalent conditions for (A) the major rHb ßG83C fraction (8 µM) in aqueous 10 mM ammonium acetate, pH 7.0 at 60 V declustering potential, and (B) Hb A. The octamer (O) mass (130,770 Da) was determined from ions with 26–29 charges assuming multiple protonation. It is 0.67% higher than the mass predicted for {alpha}4ßG83C4h8 from the intensity-weighted mean masses of the {alpha}- and ß-chains determined from the reduced Hb and is in accord with the mass excesses observed from various invertebrate Hbs, determined under similar conditions (Green et al. 2001). Ions T and D correspond to the Hb A tetramer ({alpha}2ß2h4) and dimer ({alpha}ßh2), respectively. The numbers after the colons indicate the number of charges on the ions; h represents the heme group.

 
Auto-oxidation and thermal stability
At a concentration of 10 µM (on a heme basis), the auto-oxidation rate of the rHb ßG83C, of the undissociable tetramer DCL-Hb, and of Hb A were similar. The kinetics were monophasic with rates 0.013 h-1, 0.015 h-1, and 0.014 h-1, respectively. Note that at a lower concentration (5 µM) the auto-oxidation rate for rHb ßG83C was not significantly modified (0.015 h-1). Because dimers in Hb A oxidize much more rapidly than tetramers (Griffon et al 1998), the oxidation rate is sensitive to the Hb concentration. The solutions of rHb ßG83C did not show this effect, implying that the oligomers are more stable than Hb A tetramers.

After 20-min incubation at 65°C, the CO form of rHb ßG83C exhibited 4% denaturation, similar to Hb-DCL and slightly less than that observed for Hb A (10%).

Kinetics studies
The CO rebinding kinetics for rHb ßG83C were typical of Hb A, showing two phases corresponding to the two allosteric states (Fig. 3Go). The ßG83C mutation did not influence the kinetics. Unlike Hb A, the kinetics do not switch to the rapid form, typical of dimers, at low concentration. Over the range 0.5 to 20 µM, the CO rebinding remained typical only of tetramers, that indicate the bi-tetramer form remains intact, even at the lowest protein concentration.



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  		Figure 3. Recombination kinetics of CO to Hb. As for Hb A (not shown), the rHb ßG83C shows two phases corresponding to the two Hb allosteric states. The amount of slow phase, characteristic of the deoxy conformation, decreases at low photodissociation levels (15% vs. 54% shown here) because less of the deoxy state is generated. Similarly, mixing metHbACN with HbACO leads to less slow phase, because the CN ligands are not photodissociable. In contrast to Hb A (not shown), the rHb ßG83C did not show an interaction with metHbA-CN (open circles).

 
Stability of the oligomer
The concentration-dependent dissociation equilibrium of the oligomers was studied by gel filtration. For Hb A, the eluted peak profile shifts with decreasing concentration from tetrameric to dimeric form; the peak position and width of the predominantly dimeric form occurred at about 4 µM concentration (heme basis) applied to the column, with typically 60-fold dilution of sample in the column. In contrast and irrespective of applied concentration (from 500 to 4 µM), the rHb ßG83C oligomer eluted at the same volume, 12.044 ± 0.025 mL, corresponding to a molecular complex formed by two tetramers (2.1 ± 0.04). The peak width at half height remained small and constant (667.4 ± 6.9 µL), indicating that the oligomer has a high degree of size homogeneity and stability, and does not dissociate into smaller species. These results are consistent with the kinetics studies.

Another test of the stability of the oligomer is to mix the rHbCO sample with metHbA-CN. HbA-CO and metHbA-CN normally exchange dimers within seconds, producing CO/CN hybrids that display different CO rebinding kinetics: less of the slow (deoxy-like) phase is observed, because only two of the ligands can be photodissociated. Crosslinked tetramers such as DCL-Hb do not show an interaction with metHbA-CN. rHb ßG83C-CO samples did not show an interaction with metHbA-CN, indicating a stable oligomeric form that does not exchange dimers (Fig. 3Go).

Action of GSH on rHb G83C octamers
The SS bond can be reduced by GSH, provoking loss of the octameric form. This reaction was studied in two ways: the kinetics of the octameric fraction was measured at fixed GSH concentration, or the octameric fraction was determined versus GSH concentration after a fixed incubation time (Fig. 4Go). In these two experiments, the curves are biphasic; treating the rate coefficients as a second-order reaction, the rapid phase has a time coefficient of about 2 h at 1 mM GSH (kobs = 0.45/mM/h), whereas the second phase was nearly an order of magnitude slower. At low GSH concentrations, the reaction did not go to completion, but seemed to go into equilibrium with competing oxidation reactions. This suggests two types of environment for the SS bonds, with a difference of over an order of magnitude in the relative rate of reduction.



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  		Figure 4. Reduction of disulfide bridges in rHb ßG83C octamers by glutathione (GSH). The fraction of octamers vs. GSH concentration is shown for samples after an incubation of 2 h. The initial concentration of ßG83C octamers in the CO form was from 7 to 12.5 µM on a heme basis, in tris-acetate pH 7.5 buffer; the solution of GSH was prepared in the same buffer. The incubation and experiments were performed at 25°C. After incubation, a 70-µL aliquot of the mixture was analyzed on Superose 12 HR 10/30.

 
Reduction by plasma
It is also of interest for a potential blood substitute to consider the oxidation-reduction reactions in fresh plasma. Normally a reducing environment would be welcome to maintain the hemes in the functional ferrous form. However, reduction of the SS bonds would lead to the undesired tetramer and dimer species.

Incubation of G83C octamers with whole blood or plasma at 37°C to simulate the physiological conditions and separation of different species by size exclusion chromatography on a SuperoseR 12 HR 10/30 showed that the rHb ßG83C remains in octameric form. Although some reduction might occur, the slow reduction by GSH and the lack of an observed effect by fresh plasma indicate that the plasma would not have the potential to reduce the large Hb concentrations needed for use as a blood substitute. The resistance of the dimer of tetramers G83C to reducing agents present in the plasma is particularly interesting in the development of a hemoglobin-based oxygen carrier.

The oxidation and functional studies did not show the presence of dimers, indicating that the octamer was not composed of tethered dimers. We propose a model (Fig. 5Go) in which both ß-chains of one tetramer are linked to the ß-chains of the second tetramer via S-S bonds. This structure would prevent the simultaneous dissociation of both allosteric interfaces and confer a high degree of stability to the oligomeric structure. Based on the crystallographic structure of Hb A, the distance between the ß83 {alpha}-carbons of the two ß-subunits of the tetramer changes from about 19 Å in the oxy form to 24.1 Å for deoxy Hb. This implies that the two tetramers forming the octamer (Fig. 5Go) must simultaneously make the allosteric transition.



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  		Figure 5. Proposed model for structure of octameric rHb ßG83C. Two disulfide bonds between neighboring tetramers would explain the stability of the octamer. Although one tetramer may separate into dimers (lower structure), rupture of both interfaces would be required for full dissociation.

 
In conclusion, the octameric rHb ßG83C appears to be a good model for an artificial oxygen carrier, due to the size homogeneity, the stability of the oligomer in vitro and in the physiological environment such as plasma. In addition to a sufficient residence time, the efficacy of an artificial Hb-based blood substitute in terms of oxygen delivery needs the introduction of a further mutation that reduces the oxygen affinity; this can be obtained for rHb ßG83C by the additional substitution of Phe for Tyr at the ß41 position (Baudin et al 1992).


   Materials and methods
TOP
 Abstract
 Introduction
 Results and discussion
 Materials and methods
References
 
Hemoglobin expression and purification
The mutated Hb was produced in Escherichia coli using the expression plasmid pHE7 containing human {alpha}- and ß-globin cDNAs, and an E. coli methionine aminopeptidase cDNA (Shen et al. 1993, 1997), after introduction of the ß83Gly->Cys mutation (Quick changeTM site directed-mutagenesis kit, Stratagene Europe) and verification of the {alpha}- and ß-globin coding sequences. The cells were harvested by centrifugation and stored frozen at -80°C until needed for purification. The rHb was isolated and purified as described by Shen et al. (1993, 1997) with minor modifications. Briefly, the E.coli cells were suspended in lysis buffer, sonicated, saturated with CO gas, and centrifuged to eliminate the cell membranes. The supernatant was treated with polyethyleneimine to precipitate the nucleic acids. After centrifugation, the supernatant was concentrated and equilibrated in 20 mM Tris-HCl, 0.5 mM triethylenetetraamine. The rHb was purified using the AKTApurifier10 system (Amersham Biosciences) on a Q-Sepharose XL anion exchanger column, and followed by a mono-S cation exchanger column. The oligomeric and tetrameric fractions were then separated by size exclusion chromatography on a SuperoseR 12 HR 10/30 column (Amersham Biosciences) equilibrated at 25°C with 150 mM tris-acetate buffer at pH 7.5 (Manning et al. 1996).

Structural and functional studies
ESI-MS analyses were performed on an LCT time-of-flight instrument (Micromass UK) under denatured and native conditions as described (Green et al. 2001).

Kinetics of CO recombination were obtained after flash photolysis using 10-nsec YAG laser pulses (Quantel) providing 160 mJ at 532 nm. Samples were in 1- or 10-mm cuvettes, with observation at 436 nm. Measurements were made at 25°C, 150 mM tris-acetate, pH 7.5, 100 µM CO (Marden et al. 1988).

Interaction of the oligomers with Hb A dimers was tested by mixing rHb-CO and metHbA-CN. This method has previously been used to study hybrid Hb tetramers: the two parent forms, Hb-CO and metHbA-CN, can be mixed to produce the dimerCO-dimerCN hybrid. These hybrid molecules show less of the slow (deoxy or T-state) CO rebinding, because only two of the ligands can be photodissociated (Marden et al. 1996).

The kinetics of oxidation for liganded Hb samples were followed by absorption spectrophotometry at 37°C for samples under air (Griffon et al. 1998). Hb solutions were 10 µM (on a heme basis), in 20 mM potassium phosphate at pH 7.0.

The heat stability of the Hb was determined by incubating the rHb ßG83C, at 65°C in 10 mM phosphate buffer at pH 7.0 (Wajcman et al. 1973). Samples were 0.1 mM on a heme basis, under 1 atm of CO. Hb A and Hb-DCL served as controls.

Reduction of the disulfide bridge of the rHb ßG83C oligomer
In a first experiment, 100-µL aliquots of purified oligomeric fraction were incubated in the presence of GSH, varying in concentration from 0.01 to 70 mM in 150 mM tris-acetate pH 7.5 buffer. After 2 h at 25°C, the relative populations of the disulfide species of the mixture were analyzed by size exclusion chromatography on a SuperoseR 12 HR 10/30 as described above. In a second experiment, the purified oligomeric fraction was incubated in the presence of 1 or 25 mM GSH in 150 mM tris-acetate buffer at pH 7.5, 25°C. At various times, 100-µL aliquots were withdrawn and analyzed on SuperoseR 12 HR 10/30.


   Acknowledgments
 
We thank E. Domingues for skillful technical assistance. We are grateful to the Baxter Healthcare Company for supplying DCL-Hb. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Association Recherche et Transfusion (contract no. 21-2000), and the U.S. NIH (research grant HL-24525). C.F. was supported by the Délégation Génerale pour l’ Armement (Ministère de la Défense).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 References
 
Baudin, V., Pagnier, J., Lacaze, N., Bihoreau, M.T., Kister, J., Marden, M.C., Kiger, L., and Poyart, C. 1992. Allosteric properties of haemoglobin ß41(C7) Phe->Tyr: A stable, low oxygen affinity variant synthesized in Escherichia coli. Biochim. Biophys. Acta 1159: 223–226.[CrossRef][Medline]

Blackwell, R.Q., Liu, C.S., and Wang, C.L. 1971. Hemoglobin Ta-Li: 83 Gly leads to Cys. Biochim. Biophys. Acta 243: 467–474.[Medline]

Fronticelli, C., Arosio, D., Bobofchak, K.M., and Vasquez, G.B. 2001. Molecular engineering of a polymer of tetrameric hemoglobins. Proteins 44: 212–222.[CrossRef][Medline]

Green, B.N., Gotoh, T., Suzuki, T., Zal, F., Lallier, F.H., Toulmond, A., and Vinogradov, S.N. 2001. Observation of large, noncovalent globin subassemblies in the ~3600k Da hexagonal bilayer hemoglobin by electrospray ionization time-of-flight mass spectrometry. J. Mol. Biol. 309: 553–560.[CrossRef][Medline]

Griffon, N., Baudin, V., Dieryck, W., Dumoulin, A., Pagnier, J., Poyart, C., and Marden, M.C. 1998. Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation-oxidation rates. Protein Sci. 7: 673–680.[Abstract]

Haney, C.R., Buehler, P.W., and Gulati, A. 2000. Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers. Adv. Drug Deliv. Rev. 40: 153–169.[CrossRef][Medline]

Manning, L.R., Jenkins, W.T., Hess, J.R., Vandegriff, K., Winslow, R.M., and Manning, J.M. 1996. Subunit dissociations in natural and recombinant hemoglobins. Protein Sci. 5: 775–781.[Abstract]

Marden, M.C., Kister, J., Bohn, B., and Poyart, C. 1988. T-state hemoglobin with four ligands bound. Biochemistry 27: 1659–1664.[CrossRef][Medline]

Marden, M.C., Griffon, N., and Poyart, C. 1996. Asymmetric hemoglobin hybrids. J. Mol. Biol. 263: 90–97.[CrossRef][Medline]

Shen, T.-J., Ho, N.T., Simplaceanu, V., Zou, M., Green, B.N., Tam, M.F., and Ho, C. 1993. Production of unmodified human adult hemoglobin in Escherichia coli. Proc. Natl. Acad. Sci. 90: 8108–8112.[Abstract/Free Full Text]

Shen, T.-J., Ho, N.T., Zou, M., Sun, D.P., Cottam, P. F., Simplaceanu, V., Tam, M.F., Bell, D.A., and Ho, C. 1997. Production of human normal adult and fetal hemoglobins in Escherichia coli. Protein Eng. 10: 1085–1097.[Abstract/Free Full Text]

Stowell, C.P., Levin, J., Spiess, B.D., and Winslow, R.M. 2001. Progress in the development of RBC substitutes. Transfusion 41: 287–299.[CrossRef][Medline]

Tondo, C.V. 1971. Study of the polymerization of hemoglobin Porto Alegre tetramers by disulfide bridge formation. An. Acad. Bras. Cienc. 43: 651–669.

Wajcman, H., Elion, J., and Labie, D. 1973. Heat stability of hemoglobin: Theoretical and practical value of a kinetic method of study. Nouv. Rev. Fr. Hematol. 13: 89–93.


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