The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, ␤Glu 7 -␤Lys 132 , are dependent on which residue of the pair is modified. The ␤K132A mutants exhibit the spectroscopic signatures of the R 3 T state transition in both the "hinge" and "switch" regions of the ␣ 1 ␤ 2 interface. Both singly and doubly mutated hemoglobin (Hb) ␤⌭7〈 exhibit the switch region signature for the R 3 T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb ␤⌭7〈 mutants. The observed large decrease in the W3 ␣14␤15 band intensity for doubly mutated Hb ␤⌭7〈 is attributed to an enhanced separation in the A helix-E helix tertiary contact of the ␤ subunits. The results for the Hb A ␤Glu 7 -␤Lys 132 salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the ␣ 1 ␤ 2 dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.Sickle cell hemoglobin (Hb S, 1 ␤6(A3) Glu 3 Val) exhibits the property of anomalous and pathologic self-assembly. DeoxyHb S forms polymers in the erythrocyte, which leads to microvascular blockage, organ damage, and often premature death. Structure-based drug design requires knowledge of optimal polymer disruption sites. The specific interaction in the Hb S polymer involves the steric fit of the mutated hydrophobic ␤6 donor site in a hydrophobic acceptor site located in an adjacent Hb S tetramer. It is well known that the hydrophobicity and the stereospecificity of the donor site are essential to the initiation of the polymerization.Lesecq et al.(1, 2) have been investigating whether modification of the polarity close to the ␤6 site could influence the packing of the donor and acceptor sites, thus modifying the polymerization process. Replacing the hydrophilic Glu ␤7(A4) residue with a hydrophobic Ala residue resulted in a decreased polymerization of the doubly mutated rHb ␤E6V/E7A. It was postulated (1, 2) that the loss of the normal salt bridge between ␤Glu 7 (A4) and ␤Lys 132 (H10) in the rHb ␤E7A mutants might lead to an alteration in both the position and the mobility of the A helix, illustrated in Fig. 1. These alterations of the A helix might result in a misfit between the donor and acceptor sites, which could explain the observed diminution in polymerization. It follows from this hypothesis that modifying the other partner of the salt bridge, ␤Lys 132 (H10), should have similar consequences on polymerization (2).Visible resonance Raman spectroscopy is very useful in providing detailed information relating to the influence of tertiary and quaternary structure upon specific heme-related vibrational degr...

We have engineered a recombinant hemoglobin (rHb ␤G83C) 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 ␤G83C 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, autooxidation rate, and gel filtration experiments on the oligomeric ␤G83C 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 ␤G83C 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 ␤G83C was not modified after incubation with fresh plasma.

The effects of the mutation ␤9(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 ␤9 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 ␤-chains, whereas the oligomeric fraction contained only normal ␣-chain and disulfide-linked ␤ 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.

We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb betaG83C-F41Y) tetramers joined by disulfide bonds at the beta83 position. One of the major problems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb betaG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, 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 betaG83C-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 reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M(-1) x h(-1) (observed time coefficient of 1 h at 1 mM glutathione) at 25 degrees 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 microM Hb and 25 degrees C. In addition, the octameric rHb betaG83C-F41Y was resistant to potential reducing agents present in fresh plasma.

Summary. The liganded (R-state) form of sickle cell haemoglobin (HbS) is of particular relevance at non-polymerizing concentrations as oxy HbS exhibits unusual properties compared with oxy HbA: mechanical precipitability (resulting from surface denaturation), greater unfolding at an air-water interface and a tendency to oxidize more readily. In human haemoglobins, the b7 (A4) Glu residue forms an intrachain salt bridge with b132 (H10) Lys in both liganded and deoxy structures. In the present study, recombinant haemoglobins with substitutions in the b7 and b132 sites were studied in order to determine the role of the b7-b132 salt bridge on Hb conformational integrity and stability. The elimination of this interhelix bridge correlates with enhanced surface denaturation and conformational alterations in the central cavity 2,3-diphosphoglycerate (DPG) cleft and a1b2 interface. The A-helix b7 Ala substitution generates a class of conformational change at the DPG pocket and a1b2 interface that is distinct from that dictated by the H-helix b132 Ala substitution. These results are significant with regard to the communication pathway between the a1b1 and a1b2 interfaces, and the new understanding of Hb allostery dependent upon tertiary structural constraints caused by effector binding to the R-state.