Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.     

Proton nuclear magnetic resonance studies of hemoglobin Providence (beta82EF6 Lys replaced by Asn or Asp): a residue involved in anion binding

High-resolution proton nuclear magnetic resonance studies of hemoglobins Providence-Asn (beta82EF6 Lys replaced by Asn) and Providence-Asp (beta82EF6 Lys replaced by Asp) show that different amino acid substitutions at the same position in the hemoglobin molecule have different effects on the structure of the protein molecule. Hemoglobin Providence-Asp appears to be in a low-affinity tertiary structure in both the deoxy and carbonmonoxy forms. Deoxyhemoglobin Providence-Asn has its beta heme resonance shifted downfield slightly from its position in normal adult hemoglobin; however, the tertiary structures of the heme pocket of hemoglobins A and Providence-Asn are very similar when both proteins are in the carbonmonoxy form. These results are consistent with the oxygen equilibrium measurements of Bonaventura, J., et al. [(1976) J. Biol. Chem. 251, 7563] which show that both Hb Providence-Asn and Hb Providence-Asp have oxygen affinities lower than normal adult hemoglobin, with Hb Providence-Asp having the lowest. Our studies of the effects of sodium chloride on the hyperfine shifted proton resonances of deoxyhemoglobins A, Providence-Asn, and Providence-Asp indicate that the beta82EF6 lysine is probably one, but not the only binding site for chloride ions.     

Novel recombinant hemoglobin, rHb (beta N108Q), 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.     

Effects of crosslinking on the thermal stability of hemoglobin. I. The use of bis(3,5-dibromosalicyl) fumarate

The double-headed aspirin, bis(3,5-dibromosalicyl) fumarate, has been used to crosslink hemoglobin A between Lys 82 beta 1 and Lys 82 beta 2 (J. A. Walder et al. (1979) Biochemistry 18,4265). Denaturation experiments were used to compare the stability of this crosslinked protein to that of hemoglobin A. Thermal denaturations, done in 0.01 M 4-morpholine-propanesulfonic acid, pH 7, containing 0.9 M guanidine to prevent precipitation at high temperatures, were monitored by changes in absorbance between 190 and 650 nm using a diode array spectrophotometer. The sample was heated from 25 to 70 degrees C at 0.3 degrees C/min. The data were analyzed by using both a two-state model and a novel first derivative method. As expected, methemoglobin A had a single, broad transition with a midpoint of 40.7 degrees C. The crosslinked methemoglobin showed a transition at 57.1 degrees C. Two minor transitions, one of which was apparently due to residual unmodified hemoglobin, were also observed in the crosslinked sample. Thus, a single crosslink between only two of the four subunits can lead to a significantly more stable molecule. These results can be explained by Le Chatelier's principle, since crosslinking prevents dissociation of the beta-subunits and, thereby, holds the entire tetramer together.     

Crystal structure of Lysbeta(1)82-Lysbeta(2)82 crosslinked hemoglobin: a possible allosteric intermediate

The crystal structure of human hemoglobin crosslinked between the Lysbeta82 residues has been determined at 2.30 A resolution. The crosslinking reaction was performed under oxy conditions using bis(3, 5-dibromosalicyl) fumarate; the modified hemoglobin has increased oxygen affinity and lacks cooperativity. Since the crystallization occurred under deoxy conditions, the resulting structure displays conformational characteristics of both the (oxy) R and the (deoxy) T-states. beta82XLHbA does not fully reach its T-state conformation due to the presence of the crosslink. The R-state-like characteristics of deoxy beta82XLHbA include the position of the distal Hisbeta63 (E7) residue, indicating a possible reason for the high oxygen affinity of this derivative. Other areas of the molecule, particularly those thought to be important in the allosteric transition, such as Tyrbeta145 (HC2) and the switch region involving Proalpha(1)44 (CD2), Thralpha(1)41 (C6) and Hisbeta(2)97 (FG4), are in intermediate positions between the R and T-states. Thus, the structure may represent a stabilized intermediate in the allosteric transition of hemoglobin.     

Effects of crosslinking on the thermal stability of hemoglobins. II. The stabilization of met-, cyanomet-, and carbonmonoxyhemoglobins A and S with bis(3,5-dibromosalicyl) fumarate

Hemoglobins A and S were crosslinked between Lys 82 beta 1 and Lys 82 beta 2 using bis (3,5-dibromosalicyl) fumarate (J. A. Walder et al. (1979) Biochemistry 18, 4265). Thermal denaturation experiments were used to compare the stabilities of the met, cyanomet, and carbonmonoxy forms of these crosslinked hemoglobins to the corresponding uncrosslinked proteins. Uncrosslinked carbonmonoxy- and cyanomethemoglobins had transition temperatures about 11 degrees C higher than the corresponding met samples. The increase in denaturation temperature (Tm) due to crosslinking was 15 degrees C for the methemoglobins, 10 degrees C for the cyanomethemoglobins, and 4 degrees C for the carbonmonoxy ones. There was no significant difference in stability between the met and carbonmonoxy crosslinked proteins. In order of increasing stability the samples were: met Hb S less than met Hb A less than CO Hb S less than CO Hb A = CN-met Hb A less than met XL-Hb S = CO XL-Hb S less than met XL-Hb A = CO XL-Hb A less than CN-met XL-Hb A. The slight decrease in the stability of Hb S (beta 6 Glu----Val) compared to Hb A can be explained by the replacement of an external ionic group by a hydrophobic residue in Hb S. In mixtures of crosslinked and normal Hb A, the Tm of the uncrosslinked material was slightly increased by the presence of the more stable crosslinked hemoglobin. The effects of both crosslinking and cyanide or carbon monoxide binding can be explained by Le Chatelier's principle since both would favor the native form of the protein.     

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative ligand binding of crosslinked hemoglobins at very high temperatures

Human hemoglobin was reacted with the bifunctional reagent bis(3,5-dibromosalicyl) fumarate to yield a derivative (Hb alpha alpha) crosslinked between the two alpha-chains; when the reaction was carried out with HbA already crosslinked between the two beta-chains by 2-nor-2-formylpyridoxal 5'-phosphate, a doubly crosslinked derivative (Hb alpha alpha beta beta) was obtained. We have observed that both modified hemoglobins are extremely stable up to temperatures of at least 85 degrees C. The carbon monoxide binding kinetics of both crosslinked hemoglobins, studied at temperatures between 15 and 85 degrees C, by means of stopped flow and flash photolysis techniques, show that the ligand-linked allosteric transition is maintained even at the highest temperatures. These results are also relevant to the mechanism of thermal unfolding of human hemoglobin, since they show that dissociation into alpha beta dimers (and exposure of the relatively hydrophobic dimer-dimer interfaces) is an obligatory step in the irreversible denaturation of deoxy and carbon monoxy hemoglobin.     

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.     

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.     

Differential pathways in oxy and deoxy HbC aggregation/crystallization

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.     

Specifically carboxymethylated hemoglobin as an analogue of carbamino hemoglobin. Solution and X-ray studies of carboxymethylated hemoglobin and X-ray studies of carbamino hemoglobin

Hemoglobin can be specifically carboxymethylated at its NH2-terminal amino groups (i.e. HbNHCH2COO-) to form the derivatives alpha 2Cm beta 2, alpha 2 beta 2Cm, and alpha 2Cm beta 2Cm, where Cm represents carboxymethyl. Previous studies (DiDonato, A., Fantl, W. J., Acharya, A. S., and Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895) suggested that these derivatives could be used as stable analogues of the corresponding carbamino (Hb-NHCOO-) forms of hemoglobin, adducts that are generated reversibly in vivo when CO2 combines with alpha-amino groups. In this paper we present x-ray diffraction studies of both carbamino hemoglobin and carboxymethylated hemoglobin that verify this proposal and we use the carboxymethylated derivatives to study the functional consequences of placing a covalently bound carboxyl group at the NH2 terminus of each hemoglobin subunit. Our studies also provide additional information concerning the oxygen-linked binding of anions and protons to Val-1 alpha. Difference electron density analysis of deoxy alpha 2Cm beta 2Cm versus the unmodified deoxyhemoglobin tetramer (deoxy alpha 2 beta 2) shows that the covalently bound carboxyl moieties replace inorganic anions that are normally bound to the free NH2-terminal amino groups in crystals of native deoxyhemoglobin grown from solutions of concentrated (2.3 M) ammonium sulfate. In the case of the beta-subunits, the carboxymethyl group replaces an inorganic anion normally bound between the alpha-amino group of Val-1 beta, the epsilon-amino group of Lys-82 beta, and backbone NH groups at the NH2-terminal end of the F'-helix. In the case of the alpha-subunits, the carboxymethyl group replaces an anion that is normally bound between the alpha-amino group of Val-1 alpha and the beta-OH group of Ser-131 alpha. A corresponding difference electron map of carbamino deoxyhemoglobin in low-salt (50 mM KCl) crystals shows that CO2 bound in the form of carbamate occupies the same two anion binding sites. The alkaline Bohr effect of alpha 2Cm beta 2 is only marginally lower (approximately 7%) than that of alpha 2 beta 2. Previous studies (Kilmartin, J. V., 1977) have shown that about 30% of the alkaline Bohr effect is the result of an oxygen-linked change in the pK alpha of Val-1 alpha, and O'Donnell et al., 1979, found that this portion of the Bohr effect is the result of the oxygen-linked binding of chloride to Val-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temperature dependent spectral changes of iron and nickel hemoglobins and their derivatives

Temperature dependent absolute and difference spectra for deoxy and oxy human hemoglobin, alpha and beta subunits, NiHbA, carboxypeptidase A treated deoxy HbA and NiHbA have been investigated. It is shown for the first time that the alpha subunits are mainly responsible for the temperature dependent spectral changes in the absorption spectra of Hb in the range from 0 degrees C to 40 degrees C. It has also been found that in the R state the spectral alterations caused by temperature variation are about 85% of those found for the T state of Hb. The value of following the temperature dependence of the porphyrin bands of hemoproteins, as a sensitive probe for subtle changes in the region of the heme, is demonstrated.     

Site-directed mutagenesis in haemoglobin. Functional role of tyrosine-42(C7) alpha at the alpha 1-beta 2 interface

To clarify the functional role of Tyr-42(C7) alpha, which forms a hydrogen bond with Asp-99(G1) beta at the alpha 1-beta 2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42 alpha was replaced by Phe (Hb Phe-42 alpha) or His (Hb His-42 alpha), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99 beta mutants, Hb Phe-42 alpha showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42 alpha, the deoxy T quaternary structure is highly destabilized and the strain imposed on the Fe-N epsilon (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42 alpha, Hb His-42 alpha showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42 alpha and Asp-99 beta. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42 alpha and Asp-99 beta plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Hemoglobin Saint Mandé [beta 102 (G4) Asn----Tyr]. Functional studies and structural modeling reveal an altered T state

Oxygen equilibrium studies of purified hemoglobin Saint Mandé (Hb SM) [beta 102 (G4) Asn----Tyr] reveal a decreased oxygen affinity and cooperativity but to a lesser extent than found for Hb Kansas (beta 102 Thr). The low affinity of Hb SM depends on environmental conditions: eliminating chloride or raising the pH greatly elevated the ratio of p50 of Hb SM to that of Hb A. The alkaline Bohr effect is reduced by about 40%. The effects of anions (chloride, organophosphates) binding to deoxy Hb SM are also reduced. These data indicate that the functional properties of Hb SM are intermediary between Hb A and Hb Kansas. In addition, molecular graphics modeling of Hb SM in the oxy and deoxy structures indicate the possibility of a new hydrogen bond in the T state between beta(1)102 Tyr and alpha(2)42 Tyr. Stabilisation of the T state in this manner is a plausible explanation for several of the effects observed.     

Hemoglobin Fort Gordon or alpha2beta2145 Tyr replaced by Asp, a new high-oxygen-affinity hemoglobin variant.

Hemoglobin Fort Gordon, alpha2beta2145 Tyr replaced by Asp (HC2), has been observed in a 20-year-old black male with compensatory erythrocytosis. The variant was readily identified by electrophoresis and chromatography, and comprised about 30% of the red cell hemoglobin. The substitution was identified through analyses of tryptic peptides of various digests of the isolated beta chain. The oxygen affinity of whole blood was increased; two components were observed one of which had a greatly increased affinity for oxygen and a markedly reduced subunit cooperativity. It appears that the Tyr replaced by Asp substitution resembles the Tyr replaced by His substitution in hemoglobin Bethesda (Bunn, H. F. et al. (1972) J. Clin. Invest. 51, 2299-2309; Olson, J. S. and Gibson, G. H. (1972) J Biol. Chem. 247, 3662-3670; Adamson et al. (1972) J. Clin. Invest. 51, 2883-2888) in that both inhibit the quarternary change of the oxy to the deoxy conformation, resulting in greatly altered functional properties. Studies of a few members of the family were negative.     

The crystal structure of bar-headed goose hemoglobin in deoxy form: the allosteric mechanism of a hemoglobin species with high oxygen affinity

The crystal structure of a high oxygen affinity species of hemoglobin, bar-headed goose hemoglobin in deoxy form, has been determined to a resolution of 2.8 A. The R and R(free) factor of the model are 0.197 and 0.243, respectively. The structure reported here is a special deoxy state of hemoglobin and indicates the differences in allosteric mechanisms between the goose and human hemoglobins. The quaternary structure of the goose deoxy hemoglobin shows obvious differences from that of human deoxy hemoglobin. The rotation angle of one alphabeta dimer relative to its partner in a tetramer molecule from the goose oxy to deoxy hemoglobin is only 4.6 degrees, and the translation is only 0.3 A, which are much smaller than those in human hemoglobin. In the alpha(1)beta(2) switch region of the goose deoxy hemoglobin, the imidazole ring of His beta(2)97 does not span the side-chain of Thr alpha(1)41 relative to the oxy hemoglobin as in human hemoglobin. And the tertiary structure changes of heme pocket and FG corner are also smaller than that in human hemoglobin. A unique mutation among avian and mammalian Hbs of alpha119 from proline to alanine at the alpha(1)beta(1 )interface in bar-headed goose hemoglobin brings a gap between Ala alpha119 and Leu beta55, the minimum distance between the two residues is 4.66 A. At the entrance to the central cavity around the molecular dyad, some residues of two beta chains form a positively charged groove where the inositol pentaphosphate binds to the hemoglobin. The His beta146 is at the inositol pentaphosphate binding site and the salt-bridge between His beta146 and Asp beta94 does not exist in the deoxy hemoglobin, which brings the weak chloride-independent Bohr effect to bar-headed goose hemoglobin.     

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.