Oxygen Binding Curves Research Articles

Human Erythrocyte Membrane Band 3 Protein Influences Hemoglobin Cooperativity: POSSIBLE EFFECT ON OXYGEN TRANSPORT

Hemoglobin function can be modulated by the red cell membrane but some mechanistic details are incomplete. For example, the 43-kDa chymotryptic fragment of the cytoplasmic portion of red cell membrane Band 3 protein and its corresponding N-terminal 11-residue synthetic peptide lower the oxygen affinity of hemoglobin but effects on cooperativity are unclear. Using highly purified preparations, we also find a lowered Hill coefficient (n values <2) at subequivalent ratios of Band 3 fragment or of synthetic peptide to Hb, resulting in an oxygen affinity that is moderately decreased and a partially hyperbolic shape for the O2 binding curve. Both normal HbA and sickle HbS display this property. Thus, the determinant responsible for the Hb cooperativity decreases by the 43-kDa fragment resides within its first 11 N-terminal residues. This effect is observed in the absence of chloride and is reversed by its addition. As effector to Hb ratios approach equivalence or with saturating chloride normal cooperativity is restored, and oxygen affinity is further lowered because the shape of the oxygen binding curve becomes completely sigmoidal. The relative efficiencies of 2,3-diphosphoglycerate (DPG), the 43-kDa Band 3 fragment, and the 11-residue synthetic peptide in lowering cooperativity are very similar. The findings are explained based on the stereochemical mechanism of cooperativity because of two populations of T-state hemoglobin tetramers, one with bound effector and the other with free (Perutz, M. F. (1989) Q. Rev. Biophys. 22, 139-237). As a result of this property, hemoglobin at the membrane inner surface in contact with the N-terminal region of Band 3 could preferentially bind O2 at low oxygen tension and then release it upon saturation with 2,3-diphosphoglycerate in the interior of the red cell. Membrane modulation of hemoglobin oxygen affinity has particularly interesting implications for the polymerization of hemoglobin S in the sickle red cell.

A potential role for water in the modulation of oxygen-binding by tarantula hemocyanin

Hemocyanin from the tarantula Eurypelma californicum is a large respiratory protein with an exceptional high cooperativity. In contrast to hemocyanins from other species, no physiological allosteric effectors other than protons have been identified so far for this 24-meric oligomer. Here we report for the first time the mediating effects of water activity on the oxygen binding properties of a hemocyanin. Oxygen binding curves were measured in presence of several concentrations of glycine and sucrose since both substances reduce water activity. A pronounced shift of the p 50 was observed in both cases but in different directions: adding sucrose shifts the p 50 towards lower values whereas presence of glycine shows the same tendency as for human hemoglobin. Furthermore, prolonged incubation in sucrose slightly distorts the oxygen binding characteristics of spider hemocyanin. Therefore, only the influence of glycine was further analysed. An analysis based on the nested MWC model indicates, that presence of glycine leads to a preferential population of the two states with lower oxygen affinity (tR and tT) compared to the high affinity states rT and rR. The results corroborate the presence of hierarchically organized interactions in this hemocyanin.

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.

Oxygen binding by single red blood cells from the red-eared turtle Trachemys scripta.

Oxygen-binding properties of single red blood cells from the red-eared turtle Trachemys scripta were measured by microspectrophotometry to describe the variation in oxygen affinity of red blood cells and to gain insight into the distribution of functionally different hemoglobins among red blood cells. Methodologically, this study represents the first report on the cell-to-cell variation in oxygen-binding properties based on oxygen-binding curves of single vertebrate red blood cells. The cells differed significantly with respect to oxygen affinity. Mean oxygen pressure at half saturation of the cells in a blood sample was found to be 20.1 +/- 3.3 (SD) Torr. The distribution of oxygen affinities among red blood cells is unimodal, indicating that the two hemoglobins found in turtle blood are not segregated in distinct cells. Therefore, the functional interaction shown by these hemoglobins in vitro is likely to take place in vivo. The considerable variation in oxygen affinity between individual red blood cells calls for its incorporation in models of tissue oxygenation.

The Allosteric Effector l-Lactate Induces a Conformational Change of 2×6-meric Lobster Hemocyanin in the Oxy State as Revealed by Small Angle X-ray Scattering

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.

Respiratory responses to short term hypoxia in the snapping turtle, Chelydra serpentina

Among vertebrates, turtles are able to tolerate exceptionally low oxygen tensions. We have investigated the compensatory mechanisms that regulate respiration and blood oxygen transport in snapping turtles during short exposure to hypoxia. Snapping turtles started to hyperventilate when oxygen levels dropped below 10% O 2. Total ventilation increased 1.75-fold, essentially related to an increase in respiration frequency. During normoxia, respiration occurred in bouts of four to five breaths, whereas at 5% O 2, the ventilation pattern was more regular with breathing bouts consisting of a single breath. The increase in the heart rate between breaths during hypoxia suggests that a high pulmonary blood flow may be maintained during non-ventilatory periods to improve arterial blood oxygenation. After 4 days of hypoxia at 5% O 2, hematocrit, hemoglobin concentration and multiplicity and intraerythrocytic organic phosphate concentration remained unaltered. Accordingly, oxygen binding curves at constant P CO 2 showed no changes in oxygen affinity and cooperativity. However, blood pH increased significantly from 7.50±0.05 under normoxia to 7.72±0.03 under hypoxia. The respiratory alkalosis will produce a pronounced in vivo left-shift of the blood oxygen dissociation curve due to the large Bohr effect and this is shown to be critical for arterial oxygen saturation.

Characteristics and function of human hemoglobin vesicles as an oxygen carrier

A liposome-encapsulated human hemoglobin (Neo Red Cell, (NRC) has been developed and evaluated as an artificial oxygen carrier. The NRC is a liposome-encapsulated highly concentrated (>45%) stroma-free human hemoglobin with inositol hexaphosphate (IHP as an allosteric effector), a coenzyme and substrates for reducing methemoglobin (metHb). The NRC's surface was coated with polyethylene glycol to prevent aggregation in plasma and to prolong their retention time in the blood stream. The oxygen binding behavior of the NRC in vitro was investigated and it was found that it effectively transports oxygen in vivo as an oxygen carrier. The oxygen binding behavior and kinetics were studied by the stopped-flow method and the oxygen binding curve of the NRC was determined. The oxygen binding speed and binding coefficient (Kon) of NRC, washed human red blood cells (WRBC) and stroma-free human hemoglobin (SFHb) were measured by stopped-flow method. The oxygen binding speed of SFHb was the highest, while that of RBC was the lowest and that of NRC was intermediate. The oxygen binding of NRC ended within 60 msec when deoxy-NRC was mixed with oxygen. The Kon of NRC was 2.9 × 105, 10 times faster than that of RBC. The oxygen binding curve and P50O2 of NRC that contained various IHP concentrations were measured. The oxygen-binding curve of the NRC sequentially shifted to the right as the IHP content was increased. Exchange transfusion of 70% was carried out for rats with NRC containing various concentrations of IHP and of Hb, and investigated the optimum concentration of NRC in vivo. The lactate value after exchange transfusion was three times higher than before exchange transfusion, when rats were subjected to exchange transfused with NRC that did not contain IHP. But the increase of lactate was suppressed when rats were transfused with NRC that contained IHP. When the Hb concentration of NRC was 5 and 6%, exchange transfused rats recovered to normality just like rats transfused with RBC. Copyright © 2000 John Wiley & Sons, Ltd.

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.

T State Hemoglobin Binds Oxygen Noncooperatively with Allosteric Effects of Protons, Inositol Hexaphosphate, and Chloride

Hemoglobin is the paradigm of allosteric proteins. Over the years, cooperative oxygen binding has been explained by different models predicting that the T state of hemoglobin binds oxygen either noncooperatively or with some degree of cooperativity or with strong cooperativity. Therefore, a critical test that discriminates among models is to determine the oxygen binding by the T state of hemoglobin. Fixation of hemoglobin in the T state has been achieved either by crystallization from polyethylene glycol solutions or by encapsulation in wet porous silica gels. Hemoglobin crystals bind oxygen noncooperatively with reduced affinity compared with solution, with no Bohr effect and with no influence of other allosteric effectors. In this study, we have determined accurate oxygen-binding curves to the T state of hemoglobin in silica gels with the same microspectrophotometric apparatus and multiwavelengths analysis used in crystal experiments. The T state of hemoglobin in silica gels binds oxygen noncooperatively with an affinity and a Bohr effect similar to those observed in solution for the binding of the first oxygen molecule. Other allosteric effectors such as inositol hexaphosphate, bezafibrate, and chloride significantly affect oxygen affinity. Therefore, T state hemoglobins that are characterized by strikingly different functional properties share the absence of cooperativity in the binding of oxygen. These findings are fully consistent with the Monod, Wyman, and Changeux model and with most features of Perutz's stereochemical model, but they are not consistent with models of both Koshland and Ackers.

A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins.

The binding of oxygen to the three human embryonic haemoglobins, at pH 7.4, has been shown to occur as a co-operative process. Analysis of oxygen-binding curves obtained in the absence of organic phosphate allosteric effectors shows that the process can be described quite accurately by the two-state model of allosteric action. In the presence of organic phosphates, the binding affinity for oxygen to the T-state of the alpha 2 epsilon 2 and zeta 2 epsilon 2 haemoglobins is significantly lowered. The values of the best-fit two-state parameters determined for each of the embryonic haemoglobins together with the temperature-dependence of the overall equilibrium binding process are discussed in terms of oxygen transfer from the maternal blood supply. Fast-reaction studies have been used to determine the rate constants of the oxygen association and dissociation processes occurring in the R-state and the rate of the allosteric R > T conformational transition. Analysis of these data suggests a likely reason for the high affinity and low co-operativity of the embryonic proteins and identifies the origins of the inability of equilibrium measurements to identify chain non-equivalence in the R-state.

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).

A Recombinant Sickle Hemoglobin Triple Mutant with Independent Inhibitory Effects on Polymerization

As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(beta) and Lys-95(beta), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(beta), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the beta-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(beta)/K95I(beta) mutant, i.e. when the K95I(beta) substitution was present on the same tetramer together with the naturally occurring E6V(beta) substitution, the L88A(beta) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(beta) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent.

Oxygen binding by single crystals of hemoglobin: the problem of cooperativity and inequivalence of alpha and beta subunits.

Oxygen binding by the human hemoglobin tetramer in the T quaternary structure is apparently noncooperative in the crystalline state (Hill n = 1.0), as predicted by the two-state allosteric model of Monod, Wyman, and Changeux (MWC) (Mozzarelli et al., Nature 351:416-419, 1991; Rivetti et al., Biochemistry 32:2888-2906, 1993). However, cooperativity within the tetramer can be masked by a difference in affinity between the alpha and beta subunits. Indeed, analysis of the binding curves derived from absorption of light polarized along two different crystal directions, for which the projections of the alpha and beta hemes are slightly different, revealed an inequivalence in the intrinsic oxygen affinity of the alpha and beta subunits (p50(alpha) approximately 80 torr, p50(beta) approximately 370 torr at 15 degrees C) that compensates a small amount of cooperativity (Rivetti et al., Biochemistry 32:2888-2906, 1993). To further investigate this problem, we have measured oxygen binding curves of single crystals of hemoglobin (in a different lattice) in which the iron in the alpha subunits has been replaced by the non-oxygen-binding nickel(II). The Hill n is 0.90 +/- 0.06, and the p50 is slightly different for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystallographically inequivalent beta subunits. The average crystal p50 is 110 +/- 20 torr at 15 degrees C, close to the p50 of 80 torr observed in solution, but about threefold less than the p50 calculated by Rivetti et al. (Biochemistry 32:2888-2906, 1993) for the beta subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the alpha/beta inequivalence. They therefore did not underestimate the cooperativity within the T quaternary structure, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our conclusion of nearly perfect MWC behavior for binding to the T state of unmodified hemoglobin raises the question of the relevance of the large T-state cooperativity inferred for cyanide binding to partially oxidized hemoglobin (Ackers et al., Science 255:54-63, 1992).

Oxygen binding by single crystals of hemoglobin: The problem of cooperativity and inequivalence of alpha and beta subunits

Oxygen binding by the human hemoglobin tetramer in the T quaternary structure is apparently noncooperative in the crystalline state (Hill n = 1.0), as predicted by the two-state allosteric model of Monod, Wyman, and Changeux (MWC) (Mozzarelli et al., Nature 351:416–419, 1991; Rivetti et al., Biochemistry 32:2888–2906, 1993). However, cooperativity within the tetramer can be masked by a difference in affinity between the α and β subunits. Indeed, analysis of the binding curves derived from absorption of light polarized along two different crystal directions, for which the projections of the α and β hemes are slightly different, revealed an inequivalence in the intrinsic oxygen affinity of the α and β subunits (p50(α) ≅ 80 torr, p50(β) ≅ 370 torr at 15°C) that compensates a small amount of cooperativity (Rivetti et al., Biochemistry 32:2888–2906, 1993). To further investigate this problem, we have measured oxygen binding curves of single crystals of hemoglobin (in a different lattice) in which the iron in the α subunits has been replaced by the non-oxygen-binding nickel(II). The Hill n is 0.90 ± 0.06, and the p50 is slightly different for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystallographically inequivalent β subunits. The average crystal p50 is 110 ± 20 torr at 15°C, close to the p50 of 80 torr observed in solution, but about threefold less than the p50 calculated by Rivetti et al. (Biochemistry 32:2888–2906, 1993) for the β subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the α/β inequivalence. They therefore did not underestimate the cooperativity within the T quaternary structure, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our conclusion of nearly perfect MWC behavior for binding to the T state of unmodified hemoglobin raises the question of the relevance of the large T-state cooperativity inferred for cyanide binding to partially oxidized hemoglobin (Ackers et al., Science 255:54–63, 1992). © 1996 Wiley-Liss, Inc.

Effect of reduced p50actual on mixed venous PO2 in critically ill patients

The amount of oxygen delivered to the oxygen-consuming mitochondria within a tissue is critically dependent on the oxygen tension gradient between the capillary lumen and the tissue. Leftward shift of oxygen binding curve decreases the magnitude of this gradient. In our patients total oxygen supply was not compromised sufficiently to enable us to demonstrate any actual disadvantage caused by the decreased p50act, however we found an unsafe drop in PvO2, below 32 mmHg, in 13 patients. p50act has a strong correlation with mixed venous pH and pCO2. The maintenance of a mild acidosis and hypercapnia could prevent unsafe drops in PvO2 values.

Systematically Cross-Linked Human Hemoglobin: Functional Effects of 10 Å Spans between Beta Subunits at Lysine-82

The structure and properties of hemoglobin are altered by the introduction of cross-links of defined structure between specific residues. The bis methyl phosphates and bis 3,5-dibromosalicylates of 4-carboxy-trans-cinnamic acid as well as the bis methyl phosphate of 2,6-naphthalenedicarboxylic acid produce a 10 A cross-link between the e-amino groups of each β-lys-82 of human hemoglobin. The oxygen affinity of the modified proteins fits the correlation interpolated from those for shorter and longer cross-links. The oxygen binding curve shows a high degree of cooperativity. These results support the idea that the length of the semirigid cross-link in a structurally homogeneous series constrains the relaxation of the protein upon oxygen binding by a mechanism that is specifically reflected in the oxygen affinity, while interactions between hemes that affect cooperativity are not diminished.

The influence of patent blue V on pulse oximetry and haemoximetry.

Patent Blue V (PBV) is a blue dye solution which is used to visualize lymphatic vessels for surgical procedures. There has been some conflicting reports about the influence of this dye solution on pulse oximetry and haemoximetry, why we decided to: 1) produce a spectrum of PBV in plasma, 2) measure absorbances of full blood before and after addition of PBV and 3) to record the effect on the pulse oximeter and haemoximeter of an intradermal injection of PBV to a patient. Computer analysis of the blood gas measurements were carried out by the Oxygen Status Algorithm (OSA). The spectrum of PBV demonstrated a peak absorption at 640 nm and correspondingly the absorbances of the haemoximeter increased most significantly at 622 and 636 nm. These changes invalidated the determination of haemoglobin pigments, and the results should not be used. Computer analysis interpreted the measurements as a shift of the haemoglobin oxygen binding curve.

A recombinant human hemoglobin with asparagine‐102(β) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride

A recombinant (r) mutant hemoglobin (Hb) with Asn-102(beta) replaced by an Ala (N102A(beta)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn-102(beta) is part of an important region of the alpha 1 beta 2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn-102(beta) and Asp-94(alpha) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(beta); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated beta-globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS-PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen binding properties of blood and hemoglobin from the pigeon Columba livia

Oxygen binding curves were measured on pigeon red blood cells (RBCs) suspensions, at the temperatures of 25, 30, 37 and 41°C, from pH 7.0 to 8.2, and on pigeon hemoglobin solutions, in the absence and presence of organic phosphates. Data were analysed on the basis of the MWC model. P 50 for pigeon RBCs is 46.9 mmHg, at pH 7.4 and 41°C. This low O 2 affinity is due, in part, to the existence of a large pH gradient across the membrane of the RBCs. The overall Bohr effect ( Δ logP m Δ pH ) is –0.44 in the 7.0–8.2 extracellular pH range and increases to –0.70 if intracellular pH is considered (pHi range, 6.8–7.5). A significant pH dependence for K T and K R is observed. The maximal Hill coefficient ( n max) for pigeon RBCs never exceeds 3.35 in all the conditions of pH and temperature examined, n max is slightly higher for RBCs than for hemoglobin dilute solutions. This might denote a diminished solubility of hemoglobin molecule in the physiological milieu, and is not observed for the oxygen equilibrium of human RBCs and hemoglobin solutions. Apart from this, the functional properties of pigeon RBCs are close to those of hemoglobin solutions, under comparable concentrations of protons, Cl – and organic phosphates, and for the same temperature. The enthalpy change for overall oxygenation of pigeon RBCs is rather small (about – 7 kcal/mol).

Inhibition of hemoglobin S polymerization by N‐terminal band 3 peptides: New class of inhibitors: Solubility studies

Two synthetic peptides corresponding to the N-terminal amino acids (AA) of band 3 were designed to inhibit deoxyhemoglobin S (deoxy S) polymerization through two different mechanisms. Peptide I, an N:1-15AA fragment, was employed to bind to the 2,3-diphosphoglycerate (2,3-DPG) receptor locus of single deoxy S molecules with 5-7 AA extending internally and the remaining 10-8 AA projecting external to hemoglobin (Hb) S, thereby inhibiting polymerization by steric hindrance. Peptide II consisted of two N:1-8AA + K (lysine) sequences linked by a coupler through the lysine, and it was employed to bind to the 2,3-DPG loci of two deoxy S molecules, tethering them together to form "binary hemoglobin complexes" incapable of entering the polymer chains. Decreased polymerization would result from reduction in effective concentration of deoxy S. Binding of peptides to the 2,3-DPG receptor loci was demonstrated by a progressive rightward shift in the hemoglobin oxygen binding curves as a function of increasing peptide concentrations. Inhibition of deoxy S polymerization was studied by equilibrium solubility measurements of purified, stripped solutions of Hb S. Physiologically significant inhibition was demonstrated for both peptides with near-maximum increases in solubility achieved by Peptide II at 1:1 peptide:Hb S ratios. These peptides represent a new class of inhibitors of deoxy S polymerization.