Activation of the low oxygen affinity-inducing potential of the Asn108(beta)-->Lys mutation of Hb-Presbyterian on intramolecular alpha alpha-fumaryl cross-bridging.

The Asn108 beta-->Lys mutation in hemoglobin (HbPresbyterian mutation) endows a low O(2) affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two alpha 99 lysine residues also induces a low O(2) affinity into HbA. We have now engineered an alpha alpha-fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O(2) affinity-inducing structural perturbations. Despite the presence of the additional epsilon-amino group of Lys108(beta) within the central cavity, the epsilon-amino group of Lys99(alpha alpha) of deoxy Hb-Presbyterian retained high selectivity for alpha alpha-fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The alpha alpha-fumaryl cross-linking of Hb-Presbyterian reduced its O(2) affinity much more significantly than that observed with HbA, indicating a synergy between the two low O(2) affinity-inducing structural perturbations. Apparently, the alpha alpha-fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O(2) affinity-inducing potential of Lys108(beta) that is generally activated in the presence of chloride. The synergy between the Asn108(beta)-->Lys mutation and the alpha alpha-fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, alpha alpha-fumaryl Hb-Presbyterian accessed a low O(2) affinity T-state that is accessed by HbA, alpha alpha-HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the alpha alpha-fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99(alpha) and Lys108(beta) [presumably His103(alpha) and/or Glu101(beta)] to compensate for the extra positive charge of Lys108(beta). Molecular modeling studies identified two potential chloride binding sites per alpha beta dimer within the middle of the central cavity of alphaalpha-fumaryl HbA involving residues His103(alpha), Arg104(beta) and Asn108(beta). The affinity of these sites is increased in alpha alpha-fumaryl Hb-Presbyterian as a result of the Asn108(beta)-->Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the alpha alpha-fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the alpha alpha- fumaryl-Hb Presbyterian to access a low O(2) affinity T-state structure much more readily than either Hb-Presbyterian or alpha alpha-fumaryl HbA.     

Combining the influence of two low O2 affinity-inducing chemical modifications of the central cavity of hemoglobin

HexaPEGylated hemoglobin (Hb), a non-hypertensive Hb, exhibits high O2 affinity, which makes it difficult for it to deliver the desired levels of oxygen to tissues. The PEGylation of very low O2 affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2 affinity. Toward this goal, a doubly modified Hb with very low O2 affinity has been generated. The amino terminal of the beta-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans beta-chain. To further modulate the oxygen affinity of Hb, the alpha alpha-fumaryl cross-bridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (alpha alpha-fumaryl-HPPr-HbA) exhibits an O2 affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of alpha alpha-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(beta). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2 affinity comparable to that of erythrocytes for developing blood substitutes.     

Perturbation of the intermolecular contact regions (molecular surface) of hemoglobin S by intramolecular low-O2-affinity-inducing central cavity cross-bridges

The general assumption among researchers on hemoglobin is that the intramolecular central cavity cross-bridging of Hb does not result in any generalized perturbations at the protein surface. A corollary of this is that central cavity cross-bridges are unlikely to influence the polymerization of deoxy HbS, since polymerization is a protein surface phenomenon involving the participation of multiple protein surface amino acid residues. In an attempt to evaluate this experimentally, we have introduced two low-O₂-affinity-inducing central cavity cross-bridges into HbS, -sebacyl [between the two Lys-82() residues] and -fumaryl [between the two Lys-99() residues], and investigated their influence on the polymerization of the deoxy protein. The O₂ affinities of the cross-bridged HbS exhibited sensitivity toward the buffer ions and pH in a cross-link-specific fashion. The modulation of the O₂ affinity of these cross-bridged HbS in the presence of allosteric effectors, DPG and L-35, is also very distinct, reflecting the differences in the conformational features these two cross-bridges induce within the central cavity at the respective effector-binding domains. In addition, the -fumaryl cross bridge inhibited the polymerization, reflecting the perturbation of the microenvironment of one or more intermolecular contact residues, protein surface residues, as a consequence of the central cavity cross-bridge. On the other hand, the -sebacyl cross-bridge exerted a slight potentiating effect on the polymerization of HbS. This reflects the fact that the perturbations at the protein surface are limited and favor polymerization. The results presented demonstrate that the structural changes induced by the central cavity cross-bridges are very specific and not simply restricted to the sites of modification, but are propagated to distant sites/domains, both within and outside the central cavity. It is conceivable that other surface regions that are not involved in the polymerization could also experience similar structural/conformational consequences. These results should be taken into consideration in designing intramolecularly cross-bridged asymmetric hybrid HbS for mapping the contribution of the intermolecular contact residues in the cis and trans dimers of deoxy HbS during polymerization.     

Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.     

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2)

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.     

Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).

HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.     

Liganded hemoglobin structural perturbations by the allosteric effector L35

Effector binding to liganded hemoglobin (Hb) provides a new understanding of structural determinants of Hb function. L35, a bezafibrate-related compound, is one of the more potent synthetic regulators of Hb oxygen (O(2)) affinity. In the presence of inositol hexaphosphate and bezafibrate (or derivatives), liganded Hb at low pH (pH approximately 6.5) exhibits extremely low O(2) affinity and very low cooperativity. In this study, the nature of L35 binding to COHbA at pH 6.35, an altered R-state, is presented. Solution-active site-specific spectroscopic probings by front-face fluorescence and circular dichroism reveal that L35 induces a global heterogeneous conformation in COHbA at pH 6.35 that includes: a T-like structural feature at the alpha1beta2 interface; an R-like structural feature within the heme environment; and an intermediate-like state at the central cavity. These long-range structural perturbations appear to stem from L35 binding to two classes of binding sites: the central cavity (primarily at the alphaalpha cleft) and the surface. These results indicate that L35 induces an allosteric transition species, characterized by domain-specific tertiary and quaternary-like conformation within a global R-quaternary structure.     

O-raffinose crosslinked hemoglobin lacks site-specific chemistry in the central cavity: structural and functional consequences of beta93Cys modification

Reacting human deoxyHbA0 with oxidized raffinose (O-raffinose), a trisaccharide, results in a low oxygen affinity "blood substitute," stabilized in a noncooperative T-conformation and possesses readily oxidizable rhombic heme. In this study, we fractionated the O-raffinose-modified HbA0 heterogeneous polymer (O-R-PolyHbA0) into six distinct fractions with a molecular weight distribution ranging from 64 to approximately 600 kDa using size-exclusion chromatography (SEC). Oxygen equilibrium and kinetics binding parameters of all fractions were nearly identical, reflecting a lack of heterogeneity in ligand binding properties among O-R-PolyHbA0 species (Hill coefficient n equal to 1.0). Several mass spectrometry techniques were used to evaluate undigested and digested HbA0, O-R-PolyHbA0, and O-R-PolyHbA0 fractions. Proposed sites of intramolecular crosslinking (i.e., beta1Lys82, beta2Lys82, and beta1Val1) were not found to be the predominant site of crosslinking within the central cavity. Intermolecular crosslinking with O-raffinose results in no discernible site of amino acids modifications with the exception of beta93Cys and alpha104Cys. Based on accessible surface area (ASA) calculations in intact deoxyHbA0, slight conformational changes are required to allow for the S on alpha104Cys to be modified during the reaction with O-raffinose or its partially oxidized product(s). The stabilization of HbA0 in the T-conformation may not be a direct correlate of O-raffinose induced changes, but an indirect consequence of changing hydration in the water-filled central cavity and/or the distal heme pocket leading in the latter case to accelerated iron oxidation. Structural data presented here when taken together with the oxidative instability of O-R-PolyHbA0 may provide some basis for the reported toxicity of this oxygen carrier.     

Crystal structures of unliganded and half-liganded human hemoglobin derivatives cross-linked between Lys 82beta1 and Lys 82beta2

A number of ligand binding studies of human adult hemoglobin (HbA) cross-linked between Lys 82beta(1) and Lys 82beta(2) with bis(3,5-dibromosalicyl)fumarate have been reported. The oxygen binding properties of native HbA, including the cooperativity and Bohr effect, are not substantially changed by the modification, provided care is taken to remove electrophoretically silent impurities arising from side reactions. We have refined the high-resolution structure of this modified Hb and found it adopts the T state when crystallized in the absence of heme ligands, contrary to a previously published structure. These results suggest the slightly altered crystal form determined previously may be due to unremoved side products of the cross-linking reaction with high oxygen affinity. Two nickel-substituted Hbs cross-linked in the same way have also been crystallized in the presence of carbon monoxide, which binds only to the ferrous heme. In the case of the nickel-substituted alpha subunit, the absence of a covalent link between the central metal of the heme and the proximal histidine leads to a new conformation of the histidine stabilized by a water molecule. This structure may mimic that of partially NO-liganded species of HbA; however, overall, the changes are highly localized, and both doubly ligated species are in the T conformation.     

Covalent binding of glutathione to hemoglobin. II. Functional consequences and structural changes reflected in NMR spectra

Binding of glutathione by disulfide linkage to Cys-beta 93 of hemoglobin tetramers within sickle cells increases the oxygen affinity and significantly inhibits sickling at low partial oxygen pressure (Garel, M-C., Domenget, C., Caburi-Martin, J., Prehu, C., Galacteros, F., and Beuzard, Y. (1986) J. Biol. Chem. 261, 14704-14709). This article reports a characterization of the oxygen-binding properties of glutathionyl hemoglobin (G-Hb) in solution in the presence or absence of allosteric effectors. The studies reveal a nearly 6-fold increase in oxygen affinity compared to native HbA and a Hill coefficient at half-saturation (n50) of 1.50 compared to n50 of approximately 2.9 for HbA. The oxygen Bohr effect measured in the alkaline pH range is reduced by 38%. Addition of 2,3-diphosphoglycerate decreases the oxygen affinity of G-Hb and HbA to a similar extent and increases the Bohr effect, indicating that the binding sites for organic phosphates are not perturbed in G-Hb. The rate of autooxidation of G-HbO2 is slower than of HbAO2. Oxidation by ferricyanide of G-HbCO is also reduced and is biphasic, demonstrating a heterogeneous susceptibility of the hemes in G-Hb. Flash photolysis experiments indicate that the tetramer-dimer dissociation constant is 1 order of magnitude greater for G-HbCO than for HbACO. High resolution NMR spectra at 400 MHz show that in G-Hb: the tertiary structure of the beta heme pocket is significantly perturbed, particularly in the F helix and the EF corner; the formation of the salt bridge between His-beta 146 and Asp-beta 94, a feature of the deoxy state, is precluded; and a deoxy interchain (alpha 1 beta 2) contact between Asp beta 2 99 and Tyr alpha 1 42 is appreciably destabilized. The NMR data provide a structural basis for interpreting the high oxygen affinity, reduced cooperativity, and diminished polymerization of G-HbS.     

Molecular basis of bird respiration: primary hemoglobin structure component from Tufted duck (Aythya fuligula, Anseriformes)--role of alpha99Arg in formation of a complex salt bridge network

The primary structure of the major hemoglobin component, HbA (alpha(A)- and beta-chain), from Tufted duck (Aythya fuligula) is presented. The separation of the globin subunits was achieved by ion exchange chromatography on CM-cellulose in 8 M urea. The amino acid sequence was determined by automatic Edman degradation of native chains as well as tryptic and hydrolytic peptides in a gas-phase sequencer. The automated homology model was generated by the protein structure modeling package WHAT IF using the crystal structure coordinates of Bar-headed goose hemoglobin. The 3D structure prediction enables alpha99Arg and beta101Glu to emerge as a new intersubunit contact site not found in the hemoglobin structure of any other species. alpha99Arg forms a complex salt bridge network involving alpha99Arg-beta101Glu-beta104Arg-beta108Asp. Also the substitution at alpha34 --> Ile, alpha38 --> Gln and beta55 --> Leu serves to stabilize the oxy-structure, leading to higher oxygen affinity.     

Structural basis of peroxide-mediated changes in human hemoglobin: a novel oxidative pathway

Hydrogen peroxide (H(2)O(2)) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H(2)O(2) to highly purified human hemoglobin (HbA(0)) induced structural changes that primarily resided within beta subunits followed by the internalization of the heme moiety within alpha subunits. These modifications were observed when an equal molar concentration of H(2)O(2) was added to HbA(0) yet became more abundant with greater concentrations of H(2)O(2). Mass spectrometric and amino acid analysis revealed for the first time that betaCys-93 and betaCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA(0) was treated with H(2)O(2). Oxidation of further amino acids in HbA(0) exclusive to the beta-globin chain included modification of betaTrp-15 to oxyindolyl and kynureninyl products as well as betaMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the beta subunits as a result of the H(2)O(2) attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two alpha-peptide fragments (alpha128-alpha139) and a heme moiety with the loss of iron, cross-linked between alphaSer-138 and the porphyrin ring. The novel oxidative pathway of HbA(0) modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.     

Perturbation of the Intermolecular Contact Regions (Molecular Surface) of Hemoglobin S by Intramolecular, Low-O2-Affinity-Inducing Central Cavity Cross-Bridges

The general assumption among researchers on hemoglobin is that the intramolecular central cavity cross-bridging of Hb does not result in any generalized perturbations at the protein surface. A corollary of this is that central cavity cross-bridges are unlikely to influence the polymerization of deoxy HbS, since polymerization is a protein surface phenomenon involving the participation of multiple protein surface amino acid residues. In an attempt to evaluate this experimentally, we have introduced two low-O₂-affinity-inducing central cavity cross-bridges into HbS, ββ-sebacyl [between the two Lys-82(β) residues] and αα-fumaryl [between the two Lys-99(α) residues], and investigated their influence on the polymerization of the deoxy protein. The O₂ affinities of the cross-bridged HbS exhibited sensitivity toward the buffer ions and pH in a cross-link-specific fashion. The modulation of the O₂ affinity of these cross-bridged HbS in the presence of allosteric effectors, DPG and L-35, is also very distinct, reflecting the differences in the conformational features these two cross-bridges induce within the central cavity at the respective effector-binding domains. In addition, the αα-fumaryl cross bridge inhibited the polymerization, reflecting the perturbation of the microenvironment of one or more intermolecular contact residues, protein surface residues, as a consequence of the central cavity cross-bridge. On the other hand, the ββ-sebacyl cross-bridge exerted a slight potentiating effect on the polymerization of HbS. This reflects the fact that the perturbations at the protein surface are limited and favor polymerization. The results presented demonstrate that the structural changes induced by the central cavity cross-bridges are very specific and not simply restricted to the sites of modification, but are propagated to distant sites/domains, both within and outside the central cavity. It is conceivable that other surface regions that are not involved in the polymerization could also experience similar structural/conformational consequences. These results should be taken into consideration in designing intramolecularly cross-bridged asymmetric hybrid HbS for mapping the contribution of the intermolecular contact residues in the cis and trans dimers of deoxy HbS during polymerization.     

Probing the conformation of hemoglobin presbyterian in the R-state

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108()->Lys mutation–induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the ₁₁ and ₁₂ interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103() of Tg-HbP, a residue of the ₁₁ interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the ₁₂ interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(). In short, Lys-108() in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbA's affinity to oxygen.     

Introduction of a new regulatory mechanism into human hemoglobin

Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl(-). Accordingly, we have constructed and characterized a multiple mutant, PB5, [beta(V1M + H2 Delta + T4I + P5A + A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, beta T4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P(50) of HbA, PB5 and HbBv in the absence of Cl(-) are 1.6, 4.6 and 4.8 torr, respectively, and in 100 mM Cl(-) are 3.7, 10.5 and 12 torr, respectively. Moreover, PB5 has 3-fold slower autoxidation rate compared to HbA and HbBv. These are desirable characteristics for a human hemoglobin to be considered for use as a clinical artificial oxygen carrier. Although the functional properties of PB5 and HbBv are similar, van't Hoff plots indicate that the two hemoglobins interact differently with water, suggesting that factors regulating the R to T equilibrium are not the same in the two proteins. A further indication that PB5 is not a functional mimic of HbBv derives from PB5(control), a human hemoglobin with the same substitutions as PB5, except the beta T4I replacement. PB5(control) has a high oxygen affinity (P(50)=2.3 torr) in the absence of Cl(-), but retains the Cl(-) effect of PB5. The Cl(-) regulation of oxygen affinity in PB5 involves lysine residues at beta 8 and beta 76. PB4, which has the same substitutions as PB5 except beta A76K, and PB6, which has all the substitutions of PB5 plus beta K8Q, both have a low intrinsic oxygen affinity, like HbBv and PB5, but exhibit a decreased sensitivity to Cl(-). Since HbBv has lysine residues at both beta 8 and beta 76, these results imply that Cl(-) regulation in HbBv likewise involves these two residues. The mechanism responsible for the low intrinsic oxygen affinity of HbBv remains unclear. It is suggested that residues peculiar to HbBv at the alpha(1)beta(1) interface may play a role.     

Consequences of chemical modifications on the free radical reactions of human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.     

Conformational changes in hemoglobin S (betaE6V) imposed by mutation of the beta Glu7-beta Lys132 salt bridge and detected by UV resonance Raman spectroscopy

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, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> 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 betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(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.     

Hemoglobins of reptiles. The primary structure of the major and minor hemoglobin component of adult Western Painted Turtle (Chrysemys picta bellii)

Red blood cells of adult Western Painted Turtles (Chrysemys picta bellii) contain two hemoglobin components: HbA (alpha A2 beta 2) and HbD (alpha D2 beta 2). We present the complete amino-acid sequences of the alpha A-chains from the major component and of the beta-chains common to both components. Structural features are discussed with respect to the animals extreme tolerance of severe hypoxic conditions during hibernation which is accompanied by a high oxygen affinity of the hemoglobin. The strong ATP dependence of Western Painted Turtle hemoglobin oxygen affinity is contrasted by the loss of one ATP-binding site, beta 143(H21)-Arg----Leu. The primary structure of the beta-chains excludes an allosteric control mechanism by hydrogencarbonate as it was found in crocodiles. Except in turtles a hemoglobin pattern with HbA and HbD sharing the same beta-subunits has been found only in birds. In comparison to other vertebrate hemoglobins there is a surprising similarity of the sequences to those of bird hemoglobins. alpha A- as well as alpha D-chains show larger homologies to chains of the same type in different species than alpha A- and alpha D-chains to each other in the same species. This indicates a duplication of the alpha-gene preceding the divergence of turtles and birds.     

R-state hemoglobin bound to heterotropic effectors: models of the DPG, IHP and RSR13 binding sites

We performed a docking study followed by a 500-ps molecular dynamics simulation of R-state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3-diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2-[4-[(3,5-dichlorophenylcarbamoyl)-]methyl]-phenoxy]-2-methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T-state HbA, the effector binding sites are also located in the central cavity of the R-state and differ depending on effector anionic character. DPG and IHP bind between the alpha-subunits and the RSR13 site spans the alpha1-, alpha2- and beta2-subunits. The generated models provide the first report of the molecular details of R-state HbA bound to heterotropic effectors.     

Kinetic investigations of the quaternary enhancement effect and alpha/beta differences in binding the last oxygen to hemoglobin tetramers and dimers

Analysis of O2 binding equilibria by two independent groups has suggested that the affinity for binding the fourth O2 to Hb tetramers is very high, about 800-1200 cal/mol higher than that of dimers (Chu, A. H., Turner, B. W., and Ackers, G. K. (1984) Biochemistry 23, 604-167; Di Cera, E., Robert, C. H., and Gill, S. J. (1987) Biochemistry 26, 4003-4008). Recently, Gibson and Edelstein challenged the reality of the quaternary enhancement effect, based on kinetic data (Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516-519). However, these studies failed to directly address the key issue of the relative affinities of dimers and alpha 2 beta 2(O2)3. Furthermore, the extent to which alpha/beta differences influence these results remains an open question. Using partial laser photolysis and O2/CO replacement techniques we have, for the first time, resolved the rates of O2 association and dissociation to both alpha and beta chains within "R state" tetramers and dimers. We find that the beta chains are faster than alpha for both O2 binding (approximately 2-fold) and release (approximately 3-fold). The kinetically determined O2 affinities derived from these data are essentially identical for dimers and alpha 2 beta 2(O2)3. That is, the data do not show significant quaternary enhancement and suggest that the equilibrium data have both overestimated the affinity of alpha 2 beta 2(O2)3 and underestimated the affinity of dimers. The significance of and possible origins for the discrepancy between equilibrium and kinetic data are discussed.