Human Normal Adult Hemoglobin Research Articles

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(beta)->Lys mutation-induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the alpha1beta1 and alpha1beta1 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(alpha) of Tg-HbP, a residue of the alpha1beta1 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 alpha1beta2 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(beta). In short, Lys-108(beta) 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.

Site mutations disrupt inter-helical H-bonds (α14W–α67T and β15W–β72S) involved in kinetic steps in the hemoglobin R→T transition without altering the free energies of oxygenation

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (αT67V), rHb (βS72A), and rHb (αT67V, βS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between α67T and α14W and between β72S and β15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 °C show that rHb (αT67V), rHb (βS72A), and rHb (αT67V, βS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (αT67V), rHb (βS72A), rHb (αT67V, βS72A), and Hb A have similar quaternary structures in the α1β2 subunit interfaces. In particular, the inter-subunit H-bonds between α42Tyr and β99Asp and between β37Trp and α94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the α- and β-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that α67T and β72S are H-bonded to α14W and β15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at αT67V and βS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.

Ligand binding properties and structural studies of recombinant and chemically modified hemoglobins altered at beta 93 cysteine.

To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.

NMR Investigation of the Dynamics of Tryptophan Side-chains in Hemoglobins

NMR relaxation measurements of 15N spin–lattice relaxation rate ( R 1), spin–spin relaxation rate ( R 2), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7 T and 14.1 T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or ( 2H, 15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (βD99N), rHb (αY42D/βD99N), and rHb (αV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (α14, β15, and β37) in Hb A for each αβ dimer. These Trp residues are located in important regions of the Hb molecule, i.e. α14Trp and β15Trp are located in the α 1β 1 subunit interface and β37Trp is located in the α 1β 2 subunit interface. The relaxation experiments show that amino acid substitutions in the α 1β 2 subunit interface can alter the dynamics of β37Trp. The transverse relaxation rate ( R 2) for β37Trp can serve as a marker for the dynamics of the α 1β 2 subunit interface. The relaxation parameters of deoxy-rHb Kemspey (βD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (αY42D/βD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY–CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of β37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure–function relationship in hemoglobin.

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 interfaces.

Substitutions of Asn, Glu, and Leu for Gln at the beta131 position of the hemoglobin molecule result in recombinant hemoglobins (rHbs) with moderately lowered oxygen affinity and high cooperativity compared to human normal adult hemoglobin (Hb A). The mutation site affects the hydrogen bonds present at the alpha(1)beta(1)-subunit interface between alpha103His and beta131Gln as well as that between alpha122His and beta35Tyr. NMR spectroscopy shows that the hydrogen bonds are indeed perturbed; in the case of rHb (beta131Gln --> Asn) and rHb (beta131Gln --> Leu), the perturbations are propagated to the other alpha(1)beta(1)-interface H-bond involving alpha122His and beta35Tyr. Proton exchange measurements also detect faster exchange rates for both alpha(1)beta(1)-interface histidine side chains of the mutant rHbs in 0.1 M sodium phosphate buffer at pH 7.0 than for those of Hb A under the same conditions. In addition, the same measurements in 0.1 M Tris buffer at pH 7.0 show a much slower exchange rate for mutant rHbs and Hb A. One of the mutants, rHb (beta131Gln --> Asn), shows the conformational exchange of its interface histidines, and exchange rate measurements have been attempted. We have also conducted studies on the reactivity of the SH group of beta93Cys (a residue located in the region of the alpha(1)beta(2)-subunit interface) toward p-mercuribenzoate, and our results show that low-oxygen-affinity rHbs have a more reactive beta93Cys than Hb A in the CO form. Our results indicate that there is communication between the alpha(1)beta(1)- and alpha(1)beta(2)-subunit interfaces, and a possible communication pathway for the cooperative oxygenation of Hb A that allows the alpha(1)beta(1)-subunit interface to modulate the functional properties in conjunction with the alpha(1)beta(2) interface is proposed.

Probing the importance of the amino-terminal sequence of the beta- and gamma-chains to the properties of normal adult and fetal hemoglobins.

A recombinant mutant of human fetal hemoglobin (Hb F), named rHb Oscar, has been constructed to explore the importance of the sequence of the amino-terminal region of the gamma-chain to the structural and functional properties of Hb F as compared to human normal adult hemoglobin (Hb A). Substitutions in the N-terminal region of Hb A have shown this region to be important to its structural and functional properties. Recent studies of recombinant mutants of Hb A with gamma-chain mutations have been used to probe the significance of the N-terminal sequence to the properties of Hb F. One of these mutants of Hb A, called rHb Felix, contains eight substitutions in the N-terminal region of the beta-chain corresponding to the sequence of the gamma-chain in that region [Dumoulin et al. (1998) J. Biol. Chem. 273, 35032-35038]. rHb Felix exhibits a 2,3-bisphosphoglycerate (2,3-BPG) response like that of Hb A, but its tetramer-dimer dissociation constant is similar to that of Hb F. In contrast, rHb Oscar contains a gamma-chain with eight mutations at the N-terminal end corresponding to the sequence of the beta-chain of Hb A in that region. (1)H NMR studies of rHb Oscar indicate a global structure like that of Hb F. rHb Oscar is not as stable against alkaline denaturation as Hb F but is more stable than Hb A, and it exhibits a stronger response to 2,3-BPG and inositol hexaphosphate as compared to Hb F. The 2,3-BPG effect in rHb Oscar also appears to be slightly enhanced compared to that in Hb A. Subzero isoelectric focusing experiments suggest that rHb Oscar does not have dissociation properties like those of Hb A. These results along with those of rHb Felix illustrate that the effects of the N-terminal region on structure and function of the Hb molecule are complicated by interactions with the rest of the molecule that are not yet well defined. However, studies of complementary mutations of Hb A and Hb F may eventually help to define such interactions and lead to a better understanding of the relationship between the amino acid sequence and the properties of the Hb molecule.

Distal ligand reactivity and quaternary structure studies of proximally detached hemoglobins.

The linkage between the proximal histidines and the proximal polypeptide in normal adult human hemoglobin (Hb A) has been proposed to play a major role in transmitting allosteric effects between oxygen binding sites [Perutz, M. F. (1970) Nature 228, 726-734]. Here we present circular dichroism (CD), (1)H NMR, analytical ultracentrifugation, and stopped-flow kinetic data to better define the quaternary structure of hemoglobins in which the linkage between the proximal histidines and the polypeptide backbone has been broken [Barrick et al. Nat. Struct. Biol. 4, 78-83 (1997)] and to characterize the distal ligand binding properties of these proximally detached Hbs. CD spectroscopy indicates that rHb (alphaH87G) and rHb (alphaH87G/betaH92G) retain at least partial T-quaternary structure with distal ligand bound, whereas rHb (betaH92G) does not, consistent with (1)H NMR spectra. Analytical ultracentrifugation reveals significant tetramer dissociation in rHb (betaH92G) to be the likely cause of loss of T-state markers. These quaternary structure studies indicate that in distally liganded Hb, the T-state is compatible with proximal linkages in the beta- but not the alpha-chains. (1)H NMR titrations of rHb (alphaH87G) with n-butyl isocyanide demonstrate the alpha-chains to be of high affinity as compared with the beta-chains. Comparing ligand association and dissociation rates between the rHb (alphaH87G) variant with the T- and R-states of wild-type Hb A indicates that at the alpha-chains, carbon monoxide affinity is modulated entirely by the proximal linkage, rather than from distal interactions. Some residual allosteric interactions may remain operative at the beta-chains of rHb (alphaH87G).

Novel Recombinant Hemoglobin, rHb (βN108Q), with Low Oxygen Affinity, High Cooperativity, and Stability against Autoxidation

Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule. rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K). It has been reported by Olson and co-workers [Carver et al. (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin. Hence, we have further introduced this mutation, alpha L29F, into beta N108Q. rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A. The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F. (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature. On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system.

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.

Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: chemical, conformational and functional equivalence with the human-derived protein.

Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human alpha- and beta-globins have been carried out to authenticate its correct expression, post-translational processing and assembly. The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column. The chemical identity of the alpha- and beta-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing. The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA. The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies. Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery. Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the alpha- and beta-globin transgenes, translates the respective alpha- and beta-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the alpha- and beta-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effectors identical with that of human-derived HbA. Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed. An additional advantage of the transgenic swine system is the stability of the transgenes over many generations. Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Contribution of surface histidyl residues in the alpha-chain to the Bohr effect of human normal adult hemoglobin: roles of global electrostatic effects.

We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(alpha20His-->Gln or alpha:H20Q); r Hb(alpha:H50Q); r Hb(alpha:H72Q); r Hb(alpha:H89Q); and r Hb(alpha:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the alpha-chain to the alkaline Bohr effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the alpha-chain, alpha20, alpha50, alpha72, alpha89, and alpha112, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance spectra of Hb A with those of the r Hbs. For the assignment of the C2 proton resonances of alpha20His and alpha112His, we have used combinations of mutations to compensate for the spectral perturbations resulting from the single mutations, which obscure the resonance assignment. On the basis of the new findings, in solvent containing 0.1 M chloride, the overall contributions from surface histidyl residues of both the alpha- and beta-chain and from other previously identified alkaline Bohr groups account for approximately 75% of the observed Bohr effect at pH 7.3 (the maximum Bohr effect under the prescribed solvent conditions). Our results show that some histidyl residues contribute to the Bohr effect and some oppose the net Bohr effect. In some cases, the addition of anions can diminish or reverse the contributions of specific histidyl residues to the overall Bohr effect. Thus, the Bohr effect, a heterotropic effect, depends on the intricate arrangement and interactions of all hydrogen and anion binding sites in the hemoglobin molecule. It is an excellent example of global electrostatic effects in proteins.

Assembly of Human Hemoglobin: STUDIES WITH ESCHERICHIA COLI-EXPRESSED α-GLOBIN

The alpha-globin of human hemoglobin was expressed in Escherichia coli and was refolded with heme in the presence and in the absence of native beta-chains. The functional and structural properties of the expressed alpha-chains were assessed in the isolated state and after assembly into a functional hemoglobin tetramer. The recombinant and native hemoglobins were essentially identical on the basis of sensitivity to effectors (Cl- and 2,3-diphosphoglycerate), Bohr effect, CO binding kinetics, dimer-tetramer association constants, circular dichroism spectra of the heme region, and nuclear magnetic resonance of the residues in the alpha1beta1 and alpha1beta2 interfaces. However, the nuclear magnetic resonance revealed subtle differences in the heme region of the expressed alpha-chain, and the recombinant human normal adult hemoglobin (HbA) exhibited a slightly decreased cooperativity relative to native HbA. These results indicate that subtle conformational changes in the heme pocket can alter hemoglobin cooperativity in the absence of modifications of quaternary interface contacts or protein dynamics. In addition to incorporation into a HbA tetramer, the alpha-globin refolds and incorporates heme in the absence of the partner beta-chain. Although the CO binding kinetics of recombinant alpha-chains were the same as that of native alpha-chains, the ellipticity of the Soret circular dichroism spectrum was decreased and CO binding kinetics revealed an additional faster component. These results show that recombinant alpha-chain assumes alternating conformations in the absence of beta-chain and indicate that the isolated alpha-chain exhibits a higher degree of conformational flexibility than the alpha-chain incorporated into the hemoglobin tetramer. These findings demonstrate the utility of the expressed alpha-globin as a tool for elucidating the role of this chain in hemoglobin structure-function relationships.

Roles of α114 and β87 Amino Acid Residues in the Polymerization of Hemoglobin S: Implications for Gene Therapy

Three novel recombinant mutants of sickle hemoglobin (Hb S, β6Glu → Val) have been constructed to assess the role of proline at α114 and threonine at β87 in the polymerization of deoxygenated Hb S. Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia colito produce the four recombinant hemoglobins: r Hb S (β6Glu → Val); r Hb S-Chiapas (β6Glu → Val, α114Pro → Arg); r Hb S-D-Ibadan (β6Glu → Val, β87Thr → Lys); and r Hb S-Chiapas-D-Ibadan (β6Glu → Val, α114Pro → Arg, β87Thr → Lys). The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions. The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A. Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4°C to 30°C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S. A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility ( C sat) in 0.1 M potassium phosphate (pH 7.35) at 25°C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb F) and Hb A with Hb S. The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues α114Pro and β87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential. Of particular interest is the inhibitory effect of α114Pro → Arg, which offers a novel opportunity to use an α-chain construct, in addition to a β-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression.

Magnetic resonance investigations of blood: From hemoglobin to lymphocyte migration

We have applied various nuclear magnetic resonance (NMR) techniques, from high-resolution solution-state NMR spectroscopy to magnetic resonance imaging MRI), to investigate blood components such as hemoglobin and T-cells. We are interested in the structure-function relationship in human normal adult hemoglobin (Hb A) and in tracking the movement of T-cells in live rats. In this presentation, we shall give a brief summary of our approach and recent experimental results.We have recently developed an expression system to produce authentic Hb A in Escherichia coli. With our Hb A expression plasmid (pHE2) (Fig. 1), we are in a position to produce any desired hemoglobin in E. coli. We are carrying out a study to learn the rules governing oxygen affinity and cooperativity of the oxygen binding process in hemoglobin. We have constructed several recombinant hemoglobins which exhibit varying degrees of oxygen affinity and cooperativity (as measured by the Hill coefficient).

A Novel Low Oxygen Affinity Recombinant Hemoglobin (α96Val→Trp): Switching Quaternary Structure Without Changing the Ligation State

Using ourEscherichia coliexpression plasmid (pHE2) in which synthetic human α and β-globin genes are coexpressed with theE. colimethionine aminopeptidase gene under the control of separatetacpromoters, we have constructed a new artificial hemoglobin in which the valine residue at position 96 of the α chain, located in the α1β2subunit interface, has been replaced by a tryptophan residue using site-directed mutagenesis. We have determined the oxygen-binding properties of this recombinant hemoglobin, r Hb (α96Val→Trp), and have used proton nuclear magnetic resonance spectroscopy to investigate its tertiary structure around the heme group and the quaternary structure in the α1β2subunit interface. This artificial hemoglobin shows a low oxygen affinity, but high cooperativity in oxygen binding, and exhibits no unusual subunit dissociation when ligated. Molecular dynamics simulations suggest that the unique oxygen-binding property of r Hb (α96Val→Trp) may be due to an extra hydrogen bond between α96Trp and β99Asp in the α1β2subunit interface in the deoxy form. Despite the replacement of a small amino acid residue, valine, by a large tryptophan residue in the α1β2subunit interface, this artificial hemoglobin shows very similar tertiary structure around the heme pockets and quaternary structure in the α1β2subunit interface compared to those of human normal adult hemoglobin. Another unique feature of this artificial hemoglobin is that the ligated form, e.g. carbonmonoxy form, of this hemoglobin in the oxy-quaternary structure can be converted to the deoxy-like quaternary structure by the addition of an allosteric effector, inositol hexaphosphate, as well as by lowering the temperature in the absence of inositol hexaphosphate, without changing its ligation state. Thus, this recombinant hemoglobin can be used to gain new insights regarding the nature of subunit interactions in the α1β2interface and the molecular basis for the allosteric mechanism of hemoglobin.

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.