Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).     

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.     

Spectroscopic properties of the nitric oxide derivative of ferrous man, horse, and ruminant hemoglobins: a comparative study.

The spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous man, horse, buffalo, deer, mouflon, musk ox, ox, and reindeer hemoglobin (HbNO) have been investigated in the absence of any allosteric effector at pH 6.5 (in 0.1 M 2-[N-morpholino]ethanesulphonic acid/NaOH chloride-free buffer system), as well as at 100 K and/or 20 degrees C. Man and horse HbNO show spectroscopic properties that are generally taken as typical of the high affinity state of ferrous tetrameric Hb's; on the other hand, the spectroscopic properties of ruminant (i.e., buffalo, deer, mouflon, musk ox, ox, and reindeer) HbNO are characteristic of the low affinity conformation. These results are in keeping with the functional properties of the mammalian Hb's considered and have been related to the peculiar low oxygen affinity of ruminant Hb's.     

Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c (HbA1cNO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA1cNO show the same basic characteristics described for the nitrosyl derivative of ferrous HbA0, the nonglycated major component of human hemoglobin (HbA0NO); and 2) HbA1cNO binds IHP with an apparent dissociation equilibrium constant (upsilon = 1.8 x 10(-2) M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with HbA0NO (less than or equal to 3 x 10(-6) M). These data provide further independent evidence that interaction(s) of polyphosphates at the specific cleft between beta-chains along the dyad-axis is sterically hindered in HbA1c by the presence of the two glucose residues covalently bound to the N-termini of beta-chains, this finding being in agreement with the reduced effect of polyanions on HbA1c spectral and ligand-binding properties.     

Effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin. Evidence for two polyanion binding sites

The effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin has been investigated at 110 K. In the absence of inositol hexakisphosphate, the nitrosyl derivative of dromedary hemoglobin shows an EPR spectrum with a rhombic shape and a weak hyperfine splitting in the gz region, a feature that is generally taken as characteristic of the high-affinity state of tetrameric hemoproteins. On addition of 1 mole of inositol hexakisphosphate/tetramer, three new hyperfine lines (Az = 1.7 mT), centered at gz = 2.01, appear; this type of spectrum is indicative of the low-affinity state of hemoglobins. A further addition of inositol hexakisphosphate, corresponding to a 20-fold molar excess, completely reverses the polyphosphate-dependent transition, giving an EPR spectrum that is exactly superimposable to that observed in the absence of the allosteric effector, i.e., is typical of the high-affinity state of the macromolecule. Both in the absence and presence of inositol hexakisphosphate, the EPR spectra are virtually independent of pH in the range explored (from 5.5 to 7.5). These results, taken together with the behavior of the nitric oxide derivative of human hemoglobin, provide further evidence for the existance in dromedary hemoglobin of two polyanion binding sites that affect in an opposite way the conformational equilibrium of the macromolecule.     

Different structural effects of allosteric modulators on subunits of tetrameric ferrous nitrosylated human hemoglobin: an EPR spectroscopic study

 The X-band EPR spectroscopic features of the ferrous nitrosylated derivative of α(Fe)₂β(Co)₂ and of α(Co)₂β(Fe)₂ metal hybrids of human hemoglobin (Hb) have been investigated at pH 7.0 and analyzed in parallel with those of the native nitrosylated tetramer (HbNO). The effect of 2,3-biphosphoglycerate (BPG), inositol hexakisphosphate (IHP) and bezafibrate (BZF) has been investigated in order to understand the perturbations induced on α and β subunits in the tetramer by the binding of allosteric effectors. A large perturbation is observed in both subunits upon BZF binding, while in the case of IHP only α-chains are affected; on the other hand, BPG leaves both chains essentially unperturbed. Thus, different binding modes of allosteric effectors to HbNO may occur, and the simultaneous addition of two effector molecules, namely BPG and BZF or IHP and BZF to HbNO, brings about different alterations of the X-band EPR spectroscopic properties. This behavior indicates that the intramolecular communication pathway(s) between the heme and the binding pockets of the heterotropic ligands (i.e., IHP and BZF, or BPG and BZF) are different, leading to distinct structural perturbations. Received: 19 September 1997 / Accepted: 16 December 1997     

NMR studies of the quaternary structure and heterogeneity of nitrosyl- and methemoglobin.

NMR was used to study the quaternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the alpha and beta subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the alpha subunit 10 times more strongly than it does to the beta subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5 x 10(-3) s-1 from the beta subunit and at 5 x 10(-4) s-1 from alpha subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the alpha than the beta subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both alpha and beta subunits were oxidized at the same rate.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids.

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays a high bimolecular quantum yield and a large fraction of slow (deoxy-like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably reflecting the differences in the alpha and beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion- and pH-linked effects on the heme-iron geometry in ferrous nitrosylated monomeric myoglobins

 The cooperative effect of anions and proton concentration on the EPR spectroscopic properties of the ferrous nitrosylated derivative of monomeric Mb from loggerhead sea turtle (Caretta caretta), sperm whale (Physeter catodon), and horse (Caballus caballus) has been investigated between pH 4.5 and 9.0, at 100 K. In the absence of anions, an EPR spectrum characteristic of the hexa-coordinated species of ferrous nitrosylated Mb with an axial geometry is observed, which is unaffected by pH. On the other hand, a transition toward a species characterized by an EPR spectrum corresponding to a hexa-coordinated rhombic geometry takes place in the presence of phosphate, acetate, citrate, sulfate, and chloride. Only the hexa-coordinated form characterized by the rhombic EPR spectrum appears then to undergo a pH-dependent transition toward the penta-coordinated species. Present results show clear-cut evidence for the spectroscopic coupling of proton and anion binding sites with the Mb reactive center, indicating that an allosteric mechanism might modulate the proximal HisF8-heme-NO geometry in monomeric hemoproteins. Received: 15 December 1997 / Accepted: 15 June 1998     

Effect of polyanions on the spectroscopic properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin

The effect of inositol hexakisphosphate, 2,3-diphosphoglycerate, dextran sulphate, and heparin on the spectroscopic (absorbance, circular dichroism, EPR) properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin was investigated. The results obtained show that: (i) all polyanions bind to the protein at the same sites, but with different affinities; (ii) polyanions affect the protein conformation of the ferrous nitrosyl derivative in a different way with respect to aquo-ferric and ferrous oxy dromedary hemoglobin; and (iii) the data obtained provide further independent evidence for the existence in dromedary hemoglobin of two functionally distinct polyanion binding sites that affect the conformational equilibrium of the protein in opposite ways.     

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.     

Rates of release of nitric oxide from HbSNO and internal electron transfer

The discovery that hemoglobin (Hb) in erythrocytes contains a fraction of beta-Cys-93 thiols as the nitrosylated derivative (HbSNO) led to the suggestion that this species is involved in transporting and releasing nitric oxide, which is the signal for local vasodilation. The release of NO from HbSNO requires an electron transfer to facilitate release and to regenerate the cysteine thiol via one-electron reduction in the absence of added thiols. An alternative mechanism, which has received much attention, transfers the nitrosyl group to an external thiol, which in turn would have to be reduced. The observed first order rate constant for the spontaneous oxidation of the ferrous heme of deoxy HbSNO is 1.0 x 10(-4)s(-1) in the absence of thiols. Under the same conditions, native Hb is stable. The oxidation of HbSNO occurs with the same rate constant that can be derived for the rate reported for the formation of HbNO from HbSNO. These similarities suggest that both processes involve the same reaction: internal electron transfer and direct release of nitric oxide.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

Cooperative cyanide dissociation from ferrous hemoglobin.

Rapid reduction of cyano-met hemoglobin (Hb+CN-) leads to the formation of an intermediate species, the cyanide derivative of ferrous hemoglobin, which dissociates to unliganded hemoglobin because of the extremely low affinity of the ligand for the ferrous heme iron. The properties of the intermediate were studied by transient spectroscopy in human hemoglobin and its isolated alpha and beta chains, in the presence and absence of CO. When mixing with dithionite, the time courses of reduction of the heme iron and dissociation of cyanide overlap considerably; addition to the reaction mixture of the redox indicator methyl viologen considerably increases the rate of reduction and allows unequivocal determination of the spectroscopic and kinetic properties of the intermediate. The results show that (i) the dissociation of cyanide from the isolated alpha and beta chains (as well as the (alpha CO)2(beta + CN-)2 hybrid) is a simple process; (ii) the two chains display similar rate parameters, but show spectroscopic inequivalence, both in the Soret and the visible regions; (iii) cooperative effects are shown to control the rate of dissociation of cyanide from hemoglobin, similarly to what happens for oxygen; and (iv) allosteric effectors (typically inositol hexaphosphate) increase the overall rate of dissociation by stabilization of the T state. We have, therefore, shown for the first time that the dissociation of cyanide from ferrous hemoglobin is controlled by the quaternary state, thereby adding one more ligand to the analysis of the structure-function relationships in hemoglobin.     

Kinetics of carbon monoxide binding to monomeric hemoproteins. Role of the proximal histidine

The effect of pH on (i) the second-order rate constant for CO binding and (ii) the spectral properties of the deoxygenated derivative of several monomeric hemoproteins has been investigated in the pH range between 2.3 and 9.0. As in the case of 3-[1-imidazolyl]-propylamide monomethyl ester mesoheme, the rate constant for CO binding to sperm whale, horse, Dermochelys coriacea, Coryphaena hippurus, and Aplysia limacina myoglobins (the latter only in the presence of acetate/acetic acid mixture) increases, as the pH is lowered, to a value at least 1 order of magnitude higher than at pH 7.0. Such an effect is not observed in A. limacina myoglobin (in the absence of the acetate/acetic acid mixture) and Chironomus thummi thummi erythrocruorin. Moreover, the absorption spectrum, in the visible region, of the deoxy derivative of all these monomeric hemoproteins (with the exception of A. limacina myoglobin in the absence of the acetate/acetic acid mixture) undergoes a transition as the pH is lowered, an effect observed previously with 3-[1-imidazolyl]-propylamide monomethyl ester protoheme. On the basis of analogous spectroscopic and kinetic properties of chelated heme model compounds we attribute this behavior to the protonation of the N epsilon of the proximal imidazole involved in the bond with the iron atom. On the basis of this model the movement of the iron atom to the heme plane appears as a crucial step for CO binding, the activation free energy of the process amounting to approximately 2 kcal/mol.     

Mutations of the betaN102 residue of HbA not only inhibit the ligand-linked T to Re state transition, but also profoundly affect the properties of the T state itself

The properties of three HbA variants with different mutations at the beta102 position, betaN102Q, betaN102T, and betaN102A, have been examined. All three are inhibited in their ligand-linked transition from the low affinity T quaternary state to the high affinity Re quaternary state. In the presence of inositol hexaphosphate, IHP, none of them exhibits cooperativity in the binding of oxygen. This is consistent with the destabilization of the Re state as a result of the disruption of the hydrogen bond that normally forms between the beta102 asparagine residue and the alpha94 aspartate residue in the Re state. However, these three substitutions also alter the properties of the T state of the hemoglobin tetramer. In the presence of IHP, the first two substitutions result in large increases in the ligand affinities of the beta-subunits within the T state structure. The betaN102A variant, however, greatly reduces the pH dependencies of the affinities of the alpha and beta subunits, K1(alpha) and K1(beta), respectively, for the binding of the first oxygen molecule in the absence of IHP. In the presence of IHP, the T state of this variant is strikingly similar to that of HbA under the same conditions. For both hemoglobins, K1(alpha) and K1(beta) exhibit only small Bohr effects. In the absence of IHP, the affinities of the alpha and beta subunits of HbA for the first oxygen are increased, and both exhibit greatly increased Bohr effects. However, in contrast to the behavior of HbA, the ligand-binding properties of the T state tetramer of the betaN102A variant are little affected by the addition or removal of IHP. It appears that along with its effect on the stability of the liganded Re state, this mutation has an effect on the T state that mimics the effect of adding IHP to HbA. It inhibits the set of conformational changes, which are coupled to the K1 Bohr effects and normally accompany the binding of the first ligand to the HbA tetramer in the absence of organic phosphates.     

Oxygen binding to partially nitrosylated hemoglobin

Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb–NO). Many aspects of the formation and persistence of Hb–NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O₂. Partially nitrosylated Hb samples had predominantly hexacoordinate NO–heme geometry and resisted oxidation when exposed to O₂ in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO–heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO–heme geometry also affected O₂ binding equilibria. O₂-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O₂ affinity T-state of the Hb by increasing percentages of NO–heme, much as occurs with increasing levels of CO–heme. Samples containing IHP showed small decreases in O₂ affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O₂-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO–heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O₂ transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl-), 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adair constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O2)j(j=0,1,...,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of Cl- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i.e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O2)2 were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i.e., the binding constants in the presence of Cl- for Hb and Hb(O2)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O2)j(J=0,1,...,4), the number of anions bound to Hb(O2)J, And the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.