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Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure

¹ INSERM, Unité 473, 94276 Le Kremlin-Bicêtre Cedex, France
² Equipe Ecophysiologie, CNRS-UPCM-INSU, Station Biologique, 29682 Roscoff Cedex, France
³ Micromass UK Ltd., Altrincham, Cheshire, WA14 5RZ, United Kingdom
⁴ Institut de Chimie Moléculaire Paul Sabatier, 31062 Toulouse Cedex 4, France
⁵ INSERM, Unité 468, Hopital Henri Mondor, 94010 Créteil Cedex, France

Reprint requests to: V. Baudin-Creuza, INSERM U 473, 84 rue du Général Leclerc, 94276 Le Kremlin-Bicêtre, France; e-mail: baudin{at}kb.inserm.fr; fax: ++33-1-49-59-56-61.

(RECEIVED August 21, 2001; FINAL REVISION October 15, 2001; ACCEPTED October 16, 2001)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.35702.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods References

 
The effects of the mutation ß9(A6)Ser Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the ß9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher-molecular-weight oligomeric fraction (30%). Reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ESI-MS) under denaturing conditions showed that the tetrameric fraction contained only normal - and ß-chains, whereas the oligomeric fraction contained only normal -chain and disulfide-linked ß^PA dimer. Under native conditions, ESI-MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 Å diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA-CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer–dimer allosteric interface. None of the other variants investigated formed such a large oligomer.

Keywords: Hemoglobin; disulfide bridge; structure oligomer; oxygen transport

Abbreviations: DTT, dithiothreitol • ESI-MS, electrospray ionization mass spectrometry • Hb, hemoglobin • Hb A, human normal adult hemoglobin • Hb PA, hemoglobin Porto Alegre • Hb-CN, cyanmethemoglobin • Hb-CO, carbonmonoxyhemoglobin • DCL-Hb, diaspirin cross-linked hemoglobin • RP-HPLC, reversed-phase high-performance liquid chromatography • T, Hb tetramer • T_i, molecule of i tetramers • TEM, transmission electron microscopy • TFA, trifluoroacetic acid.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Hemoglobin Porto Alegre (Hb PA), ß9(A6)Ser Cys, carries an extra thiol group oriented towards the exterior of the Hb molecule (Tondo et al. 1963). During storage of the hemolysate, Hb PA spontaneously polymerizes by forming intermolecular disulfide bridges involving this cysteine residue (Tondo 1971). Other substitutions introducing a cysteine residue have been described in human Hb, but only Hb PA, Hb Mississippi ß44(CD3)Ser Cys (Adams et al. 1987), and Hb Ta-Li ß83(EF7)Gly Cys (Blackwell et al. 1971) are known to form polymers.

Tondo reported that in the fresh hemolysate only tetrameric Hb PA was found. In contrast, polymers were found in the aged hemolysate from homozygous and heterozygous patients (Tondo 1971, 1972). Using ultracentrifugation and osmotic pressure measurements, Tondo found that the polymer from a homozygote was composed of three tetramers of Hb PA, possibly with a closed ring structure (Tondo 1971). Surprisingly, when studying the aged hemolysate from a heterozygote, the same author described a polymer consisting of two Hb tetramers with one abnormal and one normal ß-chain per tetramer (Tondo 1972). Likewise, animal Hbs have been reported that are able to polymerize in vitro such as mouse Hb (BALB/cj) (Bonaventura and Riggs 1967). In all cases, the cysteine residues were in an external position and the polymerization did not appear to alter the functional properties of the Hb.

Here we report on an investigation into the effects of the Cys substitution in Hb PA on the interactions within the oligomer which involve both covalent and noncovalent bonding. We also studied other Hb variants in which the substitution resulted in the introduction of an additional cysteine. Using gel permeation chromatography, electrospray ionization mass spectrometry (ESI-MS) and transmission electron microscopy (TEM), we determined the size and structure of the principal PA oligomer and propose a model for its spatial arrangement.

	Results

TOP Abstract Introduction Results Discussion Materials and methods References

 
Characterization of the Porto Alegre mutation
Hb PA was from a 30-year-old woman of Portuguese origin found to be heterozygous for this variant during routine antenatal screening. Table 1 shows the pattern of electrophoretic mobility observed under the various experimental conditions used for presumptive diagnosis in the Hb laboratory of the Henri Mondor hospital. The presence, in isoelectric focusing gel, of a small component with decreased pI, and, in urea-Triton PAGE, of an abnormal chain with increased hydrophobicity were the most striking features. The hematological parameters of the propositus were normal.

This substitution was confirmed by further structure analysis. Chromatography of the globin on the Partisil column led to the isolation of an abnormal peak significantly more hydrophobic than the normal ß-chain. RP-HPLC separation of the peptides resulting from aminoethylation, and tryptic digestion of this fraction, showed that the normal ßT-2 peptide was missing and replaced by a large abnormal peak eluting earlier. When this abnormal peptide was analyzed by tandem mass spectrometry (m/z 845.2), it was demonstrated to correspond to a ßT-2 peptide (sequence ß10–17) lacking the serine residue (ß9) located at its N-terminus. This situation results from the formation of a new tryptic cleavage site following replacement of Ser ß9 by Cys and aminoethylation.

Purification, size and structure of the oligomers
Gel filtration of the patient's hemolysate (2 mM on a heme basis) revealed the presence of two peaks (Fig. 1). One peak, amounting to about 70%, eluted at the expected volume for Hb tetramers (molecular weight 64,500 Da). Analysis of this fraction by RP-HPLC yielded only normal - and ß-chains (Fig. 2A). When analyzed under denaturing conditions by ESI-MS, this fraction showed only normal - and ß-chains. The other peak eluted from the gel filtration column over a volume range corresponding to molecular weights ranging from 120 to 350 kD. In the range of loaded volume, we estimated, from the top of the peak, a mean molecular weight of 288 ± 0.5 kD for this species. This value is consistent with a mixture of polymers assembled from four or five tetramers (4.5 ± 0.1). This high-molecular-weight peak showed a shoulder towards lower molecular weights, indicating an equilibrium with smaller species.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Gel filtration profile of the hemolysate from a patient heterozygous for Hb PA. Experiments were performed at 25°C in 150 mM tris-acetate pH 7.5 as described by Manning et al. (1996). The flow rate was 0.4mL/min, and the elution was monitored at 280 nm and 415 nm; 10–60 µL aliquots were applied at an Hb concentration of 2 mM on a heme basis, and eluted at a 0.4mL/min flow rate. DCL-Hb (diaspirin cross-linked hemoglobin) was used as a control for undissociated tetrameric Hb. Hb Rothschild [ß37(C3)Trp Arg] was used as a control for dimeric Hb.

View larger version (17K): [in this window] [in a new window]   Fig. 2. RP-HPLC profile of chain separation for (A) tetrameric Hb and (B) oligomeric Hb. The chromatograms were obtained before (upper) and after (lower) treatment with DTT, a thiol reducing agent. Each sample was applied to an Aquapore RP300 column, using an isopropanol-trifluoroacetic acid 0.2% gradient (Wajcman et al. 2001).

View larger version (27K): [in this window] [in a new window]   Fig. 3. ESI mass spectra of oligomeric Hb PA. (A) Under denaturing conditions, the sample (2.5 µM) was analyzed in 1:1 acetonitrile:water containing 0.2% formic acid and introduced into the electrospray source at 5 µL/min. Data were acquired for 3 min. (B) Under noncovalent conditions, the sample (5 µM) was analyzed in water plus 10 mM ammonium acetate. The flow rate into the source was 4 µL/min, and data were acquired for 10 min. In (B), T1–T5 correspond to multiply charged species comprising 1–5 tetramers [2(ßPA-PAß)h4], respectively. The figures after the colons indicate the number of charges on the ions. "F" indicates tetramer ions that have lost an -chain, probably in the ESI source, because their intensity increased with declustering potential.

The study of oligomeric PA by TEM shows that it appears as spheres of 140 Å diameter with a central cavity (Fig. 4). Some molecules have a crescent shape, suggesting a cleavage between two adjacent subunits (inset in Fig. 4).

View larger version (123K): [in this window] [in a new window]   Fig. 4. Electron micrographs of oligomeric Hb PA negatively stained with 2% uranyl acetate. Scale bar 260 Å. Inset shows cleavage between two adjacent subunits.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Dimer exchange between HbPA-CO oligomers and HbA-CN. (A) Schema for the interaction between HbPA-CO oligomers (T4 ) and Hb A-CN. Only one of the possible products is shown. (B) CO recombination kinetics after flash photolysis indicate an interaction resulting in less slow phase, characteristic of the deoxy Hb conformation. This is probably due to formation of [dimer-CN/dimer-CO] hybrids; because only the CO is photodissociable, less deoxy (or singly liganded) form can be produced.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods References

We report here the study of a hemolysate from a heterozygote for Hb PA. The red cells were stored at -80°C for several months before analysis. Just after thawing and hemolysis, the gel filtration profile showed the presence of tetrameric and oligomeric Hbs. Thus the kinetics of oligomer formation could not be compared to those observed by Tondo, because the preparation and storage conditions were different. In contrast to Tondo's results, both RP-HPLC and ESI-MS analysis show that all the PA ß-chains are involved in the oligomer (30%). The tetrameric Hb fraction (70%) is free of the abnormal subunit. Moreover, the major form of the oligomer has a structure comprising four tetramers of composition [₂ (ß^PA-S-S-PAß)h₄]₄, according to the ESI-MS results under native conditions and TEM analysis. The species of mass 258,400 Da observed by ESI-MS appears to be the most stable form. Its mass is about 0.2% higher than the calculated mass of four tetramers (257,934 Da). This is consistent with measurements that have been made on several invertebrate Hb subunits (200–210 kD), where the mass excess ranged between 0.1 and 0.5%. Presumably, the higher than expected mass is due to additional water molecules and alkali metal ions (Green et al. 1999,2001). The ESI-MS results also indicate the presence of an oligomer incorporating a fifth tetramer (T₅ in Fig. 3B); this species is present at a relatively low level probably because of additional constraints.

Several results suggest that the PA oligomer structure is not frozen. First, minor groups of peaks of size T₁–T₃ were observed by ESI-MS, indicating that different structures exist. Second, the filtration studies showed that, when the PA oligomer concentration was greatly decreased, the elution volume increased, indicating a decreased oligomer size. The width of the oligomer peak at half height also increased, indicating a higher heterogeneity of species (Table 2). Indeed, the minimum peak width occurred when one species was predominant (Dumoulin et al. 1997). The third piece of evidence is that the oligomer interacts with HbA, probably through incorporation of ^Aß^A dimers. These results can be compared to those of Tondo when mixing Hb A and Hb PA tetramers (Tondo and Reishl 1979), even though we did not find any oligomers with ß^A subunits in the hemolysate from a heterozygote.

Using ESI-MS and TEM results, we propose the model presented in Figure 6. The oligomer is composed of four mutated tetramers. In this model, the formation of the fourth S-S bridge with the first tetramer would lead to a closed ring. Our TEM results for the PA oligomer support this hypothetical model. The dimensions of tetrameric hemoglobin are 50 x 55 x 65 Å corresponding to a mean diameter of 57 Å. The calculated diameter of the oligomer central cavity (64 Å) is near to the experimental value (40 Å) measured on electron micrographs (Fig. 4).

View larger version (167K): [in this window] [in a new window]   Fig. 6. Three-dimensional model of the structure of HbPA oligomers. The Hb A crystallographic coordinates for the deoxy quaternary structure were taken from the file 4HHB (Protein Data Bank). The mutation ß9(A6)Ser Cys was introduced into the deoxy HbA using Hyperchem software. Four Hb PA molecules were imported using WebLab Viewer Pro (MSI) software, and positioned to form intermolecular disulfide bridges between the ß9(A6)Cys residues belonging to different tetramers. The -subunits are shown in yellow, and the ß-subunits are blue and green.

To our knowledge, Hb PA is the only variant of human Hb able to assemble into such high-molecular-weight oligomers through covalent bonds. This model is particularly interesting, because the oligomer PA exhibits functional properties similar to those observed for Hb A. In some primitive vertebrates and invertebrates such as annelids, the Hb exhibits deoxygenation-dependent self-association of monomers (Riggs 1998).

The Hb PA oligomer appears to be an interesting model for the realization of a blood substitute. Even at very low concentration, the size of PA remains higher than that observed for Hb A. Nevertheless additional constraints, such as crosslinking, would be needed to avoid separation at the dimer–dimer interfaces that do not involve the disulfide bonds.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

 
Hb variants
The structural abnormality was characterized as previously described (Wajcman et al. 2001). In brief, globin was prepared from whole hemolysate by the acid acetone method, and the polypeptide chains separated by RP-HPLC on a Partisil column (Whatman). After aminoethylation, the variant ß-globin chain was digested with trypsin, and the resulting peptides were separated by RP-HPLC using a Vydac C8 column (The Separations Group). The substitution was characterized by tandem mass spectrometric analysis of the ßT2 tryptic peptide.

Hb Ilmenau [ß41(C7)Phe Cys], Hb Arta [ß45(CD4) Phe Cys], Hb Harrow [ß118(GH1)Phe Cys], and Hb Montfermeil [ß130(H8)Tyr Cys] had been previously studied and were stored at -80°C. Hb Mississippi [ß44(CD3)Ser Cys] and Hb Nunobiki [141(HC3)Arg Cys] were from the reference collection of samples at the Hemoglobin Laboratory of Henri Mondor Hospital (Créteil, France).

Purification of Hb oligomers
The oligomeric and tetrameric fractions were separated by gel filtration of the hemolysate on a Superose^R 12 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated at 25°C with 150 mM tris-acetate pH 7.5 buffer as described (Manning et al. 1996). The chromatographic fractions were concentrated with 10 kD cutoff microconcentrators (Microcon YM-10, Millipore).

Studies of oligomeric and tetrameric Hbs
The subunit composition of the fractions obtained by gel filtration was determined by RP-HPLC using an Aquapore RP300 column (Brownlee) eluted with a gradient of isopropanol in 0.2% trifluoroacetic acid (TFA) (Wajcman et al. 2001).

Electrospray ionization mass spectrometry (ESI-MS)
Denatured conditions
Aliquots of the fractions isolated by gel filtration were diluted with 50% aqueous acetonitrile containing 0.2% formic acid. These solutions (2.5 µM based on heme content) were introduced at 5 µL/min into the Z-Spray source of a Quattro Ultima mass spectrometer (Micromass) scanning over m/z 930–1180 (8 sec/scan). Data were accumulated for 3 min. Mass scale calibration employed the multiply charged normal -chain peaks present in each spectrum. The m/z spectra were deconvoluted (m/z 970–1180) to present the data on a molecular mass scale using the Maximum Entropy (MaxEnt)-based software supplied with the instrument.

Native conditions
Initially, aliquots of the fractions isolated by gel filtration were diluted with HPLC-grade water. These solutions (200 µL, 5 µM) were then desalted by manually shaking with 10 mg of previously washed (twice with water) mixed bed ion exchange resin beads (AG 501-X8, BioRad) for 1 min. After pipetting the solutions from the beads, ammonium acetate was added to a 10 mM concentration and the pH was adjusted from 6.7 to 7.0 by adding a trace of dilute ammonia solution. These solutions were introduced at 4 µL/min into the Z-Spray source of a time-of-flight instrument (LCT, Micromass). The source temperature was 110°C, and the desolvation gas was turned off. The pressure in the intermediate region between atmospheric pressure and high vacuum was increased to 7.0 mbar (normally 2 mbar) by throttling the backing line to the rotary pump. Data were accumulated over m/z 600–16000 for 10 min at several declustering potentials (cone voltages) between 50 and 100 V. Masses of the noncovalently assembled species were determined from smoothed m/z spectra using peak top values by assuming the ions were protonated; i.e., mass = n(m/z-H), where n is the number of charges on an ion and H = 1.00794. Mass scale calibration employed the CS_(n+1)I_n peaks from a separate introduction of CsI (2 mg/mL in 50% aqueous 2-propanol).

Transmission electron microscopy (TEM)
TEM was performed on purified Hb PA prepared as described above. Hb PA was diluted (1:900, final concentration 5 µM on a heme basis) in distilled water, and applied to a very thin carbon substrate supported on a microgrid stained with 2% (w/v) uranyl acetate solution as described by Valentine et al. (1968). The specimens were observed with an electron energy of 80 keV, using a Jeol JEM-1200EX microscope (Jeol).

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

Interaction of Hb PA oligomers with Hb A dimers was tested by mixing Hb PA-CO oligomers and HbA-CN. This method has previously been used to produce and study hybrid Hb tetramers: the two parent forms Hb-CO and HbA-CN can be mixed to produce the dimer-CO*dimer-CN hybrid (Marden et al. 1996). The dimer exchange occurs on the order of 1 sec for liganded Hb. These hybrid molecules show less of the slow (deoxy or T-state) CO rebinding, because only two of the ligands can be photodissociated.

	Acknowledgments

 
We thank E. Domingues, G. Caron, and J. Riou for skillful technical assistance and J. Sourimant for transmission electron micrographs. 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 Centre National de la Recherche Scientifique, and the Association Recherche et Transfusion (contract n°21–2000). C.F. was supported by the Délégation Génerale pour l'Armement (Ministére 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.

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