Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Functional properties of carboxypeptidase-digested hemoglobins

Hemoglobin molecules after digestion with carboxypeptidase A or B show large differences in their ligand affinity, heme-heme interaction, Bohr effect and response to organic phosphates. Removal of β146 histidine and β145 tyrosine produces a molecule of high oxygen affinity, low Bohr effect and no heme-heme interaction. Removal of α141 arginine produces similar, but less drastic, changes. In both digestion products normal functional behavior may be partially restored by addition of inositol hexaphosphate. Doubly digested hemoglobins (carboxypeptidase A followed by B or vice versa) have properties similar to the initial digestion products but are no longer responsive to organic phosphates. The equilibrium and kinetic properties of these proteins are presented and are consistent with the idea that various conformations of the unliganded molecules may be in equilibrium.     

Gelation of sickle hemoglobin. III. Nitrosyl hemoglobin.

Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl &; Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974).Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl &; Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.     

Structural studies on a low oxygen affinity hemoglobin from mammalian species: Sheep (Ovis aries)

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express ‘high’ and ‘low’ oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7 Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the “joint region”, shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1β2/α2β1 interfaces of sheep metHb structure. In addition, the constrained β subunits heme environment and the contraction of N-termini and A-helices of β subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.     

Recombinant hemoglobins with low oxygen affinity and high cooperativity

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in 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.     

The interaction of inositol hexaphosphate with methaemoglobin

1. Inositol hexaphosphate causes the shape of the oxidation-reduction equilibrium curve to become hyberbolic at acid pH values. 2. Inositol hexaphosphate also causes a decrease in the alkaline oxidation Bohr effect at these same pH values. 3. These results support the idea that inositol hexaphosphate causes methaemoglobin to take up the deoxyhaemoglobin quaternary structure at pH6.5.     

Structure of deoxyhaemoglobin Yakima: a high-affinity mutant form exhibiting oxy-like 1 2 subunit interactions

Crystals of deoxyhaemoglobin Yakima (Asp Gl(99)β → His) are isomorphous with those of deoxyhaemoglobin A, even though the mutation produces disturbances in both the tertiary structure of the subunits and the quaternary structure of the tetramer. Asp Gl(99)β₂ lies at the α₁β₂ subunit interface, and in deoxyhaemoglobin A forms a crucial hydrogen bond with Tyr C7(42) α₁. The histidine residue that replaces the aspartate results in the removal of this single important intersubunit bond, and it further acts as a wedge between the α₁ and β₂ subunits, so that they are pushed apart and displaced part of the way towards the oxy structure. These disturbances are accompanied by the formation of a new intersubunit hydrogen bond, which is usually only observed in the oxy quaternary structure of haemoglobin. The disturbances at the α₁β₂ contact affect the stereochemistry of the entire molecule and are transmitted to the α and β haems. The X-ray structure of deoxy Yakima therefore provides a stereochemical explanation for its abnormal function; this being an abnormally high affinity for oxygen and vastly diminished haem-haem interactions.     

The oxygen affinity of hemoglobin betaSH chains is concentration dependent.

Oxygen binding to isolated hemoglobin β^SH chains exhibits heterotropic interactions with H⁺, inositol hexaphosphate and CO₂ which implies different structures of the liganded and unliganded β chains. In order to find out if the dissociation behaviour of $\text{β}{}_4{}^{\mathrm{SH}}$ homotetramers is likewise linked to oxygenation, we have measured the oxygen affinity of the pigment as a function of the protein concentration at different pH values. We found that a decrease in protein concentration is associated with a decrease in oxygen affinity. This result accords with predictions reached from studies on the self-association of liganded and unliganded β chains. Furthermore, it was established that both at high and low protein concentrations the oxygen affinity of the β chains is pH dependent.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran

Human hemoglobin (Hb) conjugated to benzene tetracarboxylate substituted dextran produces a polymeric Hb (Dex-BTC-Hb) with similar oxygen affinity to that of red blood cells (P(50)=28-29 mm Hg). Under physiological conditions, the oxygen affinity (P(50)) of Dex-BTC-Hb is 26 mm Hg, while that of native purified human HbA(0) is 14 mm Hg, but it exhibits a slight reduction in cooperativity (n(50)), Bohr effect, and lacks sensitivity to inositol hexaphosphate (IHP), when compared to HbA(0). Oxygen-binding kinetics, measured by rapid mixing stopped-flow method showed comparable oxygen dissociation and association rates for both HbA(0) and Dex-BTC-Hb. The rate constant for NO-mediated oxidation of the oxy form of Dex-BTC-Hb, which is governed by NO entry to the heme pocket, was reduced to half of the value obtained for HbA(0). Moreover, Dex-BTC-Hb is only slightly more sensitive to oxidative reactions than HbA(0), as shown by about 2-fold increase in autoxidation, and slightly higher H(2)O(2) reaction and heme degradation rates. Dextran-BTC-based modification of Hb produced an oxygen-carrying compound with increased oxygen release rates, decreased oxygen affinity and reduced nitric oxide scavenging, desirable properties for a viable blood substitute. However, the reduction in the allosteric function of this protein and the lack of apparent quaternary T-->R transition may hinder its physiological role as an oxygen transporter.     

Intrinsic fluorescence of carp hemoglobin: a study of the R----T transition

The intrinsic fluorescence of hemoglobins is known to respond to ligand-induced changes in the quaternary structure of the protein. Carp hemoglobin is an interesting model to study the quaternary transition since its R----T equilibrium is pH-dependent and at low pH, in the presence of organic phosphate, it remains in the T or 'deoxy' quaternary structure, even when saturated with ligand. In this study, using front-face fluorometry, we show that the intrinsic fluorescence intensity exhibited by carp carboxyhemoglobin increases as the pH is lowered below 6.5 in the presence of inositol hexaphosphate. At low pH, carp methemoglobin is less affected by the addition of inositol hexaphosphate than is the CO derivative, while little or no change is observed in the met-azide derivative. We conclude: (1) the exact nature of the R to T state transition induced by inositol hexaphosphate differs for carp carboxy-, met- and met-azide hemoglobin derivatives; (2) the chromophores responsible for the changes observed with absorption spectroscopy may not be the same as those chromophores responsible for the fluorescence differences; and (3) alpha 46-Trp is tentatively assigned as one source of fluorescence emission. Furthermore, fluorescence properties of carp hemoglobin are compared to those of human hemoglobin.     

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).     

High and low oxygen affinity conformations of T state hemoglobin

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.     

The binding of hemoglobin to red cell membrane lowers its oxygen affinity

The mode of interaction of human hemoglobin (Hb) with the red cell membrane was investigated with special reference to the effect on oxygen binding properties and Hb-membrane binding constants. Compared to free native Hb, the membrane-bound native Hb showed a strikingly lowered oxygen affinity and smaller response to organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate. Similar effects of membrane binding were also observed for intermediately cooperative Hbs such as N-ethylmaleimide-treated Hb (NES-Hb) and iodoacetamide-treated Hb (AA-Hb), but very small effects were observed for non-cooperative Hb, i.e., carboxypeptidase A-treated Hb (des-His-Tyr Hb). The magnitude of the affinity lowering was in the order: NES-Hb greater than native Hb greater than AA-Hb much greater than des-His-Tyr Hb. In the presence of inositol hexaphosphate, the three chemically modified Hbs showed an increased oxygen affinity when bound to the red cell membrane, probably due to partial replacement of bound inositol hexaphosphate by membrane. The binding to membrane caused a slight decrease in cooperativity for native Hb, but no distinct change in cooperativity was observed for the three modified Hbs. These results imply: a) the red cell membrane binds to deoxyHb more strongly than to oxyHb; b) the difference in membrane binding affinity between oxyHb and deoxyHb is closely related to the quaternary structure change in the Hb molecule occurring upon oxygenation. The higher affinity of the membrane for deoxyHb than for oxyHb apparently disagrees with the conclusion drawn by earlier investigators. However, the present binding experiments by means of ultrafiltration proved that the red cell membrane actually binds to deoxyHb much more strongly than to oxyHb, validating the present conclusion based on oxygenation experiments. Our results are consistent with those obtained recently by other investigators using a synthetic peptide or the cytoplasmic fragment of red cell membrane band 3.     

Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering

The influence of quaternary structure on the low frequency molecular vibrations of the haem within deoxyhaemoglobin (deoxy Hb) and Oxyhaemoglobin (oxy Hb) was studied by resonance Raman scattering. The FeO₂ stretching frequency was essentially identical between the high affinity (R) state (Hb A) and low affinity (T) state (Hb Kansas and Hb M Milwaukee with inositol hexaphosphate). However in deoxy Hb, only one of the polarized lines showed an appreciable frequency shift upon switch of quaternary structure, i.e. 215 to 218 cm^?1 for the T state (Hb A, des-His(146β) Hb, and des-Arg(141α) Hb (pH 6.5)) and 220 to 221 cm^?1 for the R state (des-Arg(141α) Hb (pH 9.0), des-His(146β)-Arg(141α) Hb and NES des-Arg(141α) Hb). Based on the observed ⁵⁴Fe isotopic frequency shift of the corresponding Raman lines of deoxy Hb A (214 → 217 cm^?1), of deoxy NES des-Arg Hb (220 → 223 cm^?1), of the protoporphyrinato-Fe(II)-(2-methylimidazole) complex in the ferrous high spin state (207 → 211 cm^?1) and of deoxymyoglobin (220 → 222 cm^?1) (Kitagawa et al., 1979), and on substitution of perdeuterated for protonated 2-methylimidazole in the deoxygenated picket fence complex (T_pivPP)Fe²⁺ (2-MeIm) (209 → 206 cm^?1), and on the results of normal co-ordinates calculation carried out previously, we proposed that the 216 cm^?1 line of deoxy Hb is associated primarily with the FeN_ε(HisF8) stretching mode and accordingly that the FeN_ε(HisF8) bond is stretched in the T state due to a strain exerted by globin.     

Functional properties of the glycosylated minor hemoglobins A1a-1,A1a-2 and A1b. EPR evidence for increased stability of the low affinity quaternary structure and decreased susceptibility to organic phosphate

Electron paramagnetic resonance (EPR) spectra of the glycosylated minor hemoglobins A1a-1, A1a-2, A1b and A1c and the major hemoglobin A0 in the nitrosyl form have been obtained in the absence and presence of inositol hexaphosphate. In the absence of inositol hexaphosphate, nitrosyl hemoglobins A1a-1, A1a-2 and A1b exhibited a triplet hyperfine structure centered at g = 2.009 which has been shown to be diagnostic of the low affinity (T) quaternary structure. Addition of inositol hexaphosphate to nitrosyl hemoglobins A0, A1c, A1b and A1a-2 developed a triplet hyperfine structure of the EPR spectra but the magnitude of the hyperfine was decreased in the order of hemoglobins A0, A1c, A1b and A1a-2. However, inositol hexaphosphate had essentially no effect on the EPR spectrum of nitrosyl hemoglobin A1a-1. The present results account qualitatively for the oxygen binding properties of these glycosylated minor hemoglobins in the framework of a two-state allosteric model.     

Structures and oxygen affinities of crystalline human hemoglobin C (β6 Glu->Lys) in the R and R2 quaternary structures

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (β6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K₄) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2–3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.     

Some functional properties of hemoglobin Leiden

Hemoglobin Leiden, a human mutant that contains a deletion of a glutamic residue at position 6 or 7 (A3 or A4) of the #x03B2; chains, has a slightly higher oxygen affinity in 0.1 M Phosphate, a normal Bohr effect but an abnormal response to 2,3 diphosphoglycerate and inositol hexaphosphate. Hb Leiden participates to the same extent as does Hb A in gelation with deoxy Hb S.     

The influence of organic phosphates on the Bohr effect of human hemoglobin valency hybrids.

The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaphosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the alpha chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized beta chains is slighly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species.