A chromatographic study of the reaction sequence and effect of ligand on the reaction of human hemoglobin with negatively charged isothiocyanates: characterization of an intermediate modified only on the amino termini of the alpha chains

A HPLC investigation of the reaction of 4-isothiocyanatobenzoic acid and 4-isothiocyanatobenzenesulfonic acid with oxy- and deoxyhemoglobin was carried out. The initial reaction of aromatic isothiocyanato sulfonates and benzoates with either oxy- or deoxyhemoglobin is with the amino termini of the alpha chains followed by a much slower reaction with the amino termini of the beta chains. Both reactions are much faster with deoxyhemoglobin than with oxyhemoglobin. An intermediate reacted only at the termini of the alpha chains with 4-isothiocyanatobenzoic acid was isolated and purified and its functional properties determined. The intermediate showed a reduced oxygen affinity over a wide pH range and a reduced alkaline Bohr effect in the absence of chloride. The oxygen affinity of the intermediate showed a reduced but still significant response to chloride.     

The effect of 4-isothiocyanatobenzene sulfonic acid on the oxygen affinity of human hemoglobin

Human hemoglobin reacts with 4-Isothiocyanatobenzene sulfonic acid at the four amino groups of the N-terminal valines. The modified protein shows a decreased oxygen affinity over a wide pH range, a reduced alkaline Bohr effect, decreased co-operativity, and a reduced effect of inositol hexasulfate on the oxygen affinity.     

The effect of potassium chloride on the Bohr effect of human hemoglobin.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.     

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.     

On the oxygen-linked anion-binding sites in human hemoglobin. Functional properties of human hemoglobin reacted with 4-isothiocyanatobenzenesulphonic acid and its hybrids

Human hemoglobin, reacted at the four amino termini with 4-isothiocyanatobenzenesulphonic acid (Hb-ICBS), was separated into its constituent chains. Recombination of the ICBS-reacted chains with the unmodified mate chains produced the hybrid tetramers modified at either the beta or the alpha chains: alpha 2 beta 2ICBS and alpha 2ICBS beta 2. All of the modified tetramers show a reduced oxygen affinity and reduced cooperativity; furthermore the oxygen affinity of the Hb-ICBS and alpha 2 beta 2ICBS is unaffected by 2,3-bisphosphoglycerate while the oxygen affinity of alpha 2ICBS beta 2 is decreased in the presence of this organic phosphate. The oxygen affinity of Hb-ICBS and alpha 2ICBS beta 2 is independent of chloride concentration, while the alpha 2 beta 2ICBS hybrid shows a reduced response to this anion. The tetramers alpha 2ICBS beta 2 and alpha 2ICBS beta 2ICBS show a decreased alkaline Bohr effect, which can be rationalized as being due to disruption of the oxygen-linked chloride-binding sites; in the case of alpha 2 beta 2ICBS the Bohr effect is instead (partially) maintained. The functional properties of artificial tetramers have been studied also from a kinetic point of view by CO combination and the results obtained compare satisfactorily with equilibrium data. The possibility of obtaining selectively modified hemoglobins promises to provide further insight into the properties of the oxygen-linked anion-binding sites in hemoglobin.     

On the identity of the dissociation-linked chloride binding sites in human hemoglobin. Studies with hemoglobin modified with 4-isothiocyanatobenzenesulfonic acid and 4-isothiocyanatobenzenesulfonamide

The binding of chloride ions to specific sites on the human hemoglobin molecule has well-known effects on the oxygen equilibrium and on the stability of the tetrameric structure. Several lines of evidence suggest that the oxygen-linked and the dissociation-linked chloride binding sites differ. Direct evidence for this difference has been obtained from the chloride dependence of the dimer-tetramer equilibrium of oxyhemoglobin modified with 4-isothiocyanatobenzenesulfonic acid, in which all the oxygen-linked chloride binding sites are blocked, or with 4-isothiocyanatobenzenesulfonamide, in which the linkage between chloride and oxygen is unperturbed. Thus, the chloride dependence of the dimer-tetramer assembly is unaffected by the chemical modification in both proteins and resembles that of unreacted hemoglobin. It is suggested that histidines alpha-103, alpha-122 and beta-97 may constitute, at least in part, the dissociation-linked chloride binding sites.     

The effect of 2-methoxy-5-nitrotropone on the oxygen affinity of human erythrocytes and hemoglobin.

Human hemoglobin reacted with 2-methoxy-5-nitrotropone at pH 7.4 undergoes modification of the four N-terminal amino groups. The modified protein shows increased oxygen affinity with complete abolition of the effect of K-glycerate 2, 3-bisphosphate. The Bohr effect is abolished in the acid range and drastically reduced at alkaline pH values. Changes in the kinetics of ligand reactions parallel the oxygen equilibrium results. Cooperative effects are still present. Human erythrocytes treated with 2-methoxy-5-nitrotropone show increased oxygen affinity and some decrease in methemoglobin reductase efficiency but no change in resistance to hemolysis.     

Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions

Under physiological conditions of pH (7.4) and chloride concentration (0.15 M), the oxygen affinity of bovine hemoglobin is substantially lower than that of human hemoglobin. Also, the Bohr effect is much more pronounced in bovine hemoglobin. Numerical simulations indicate that both phenomena can be explained by a larger preferential binding of chloride ions to deoxyhemoglobin in the bovine system. Also, they show that the larger preferential binding may be produced by a decreased affinity of the anions for oxyhemoglobin, thereby stressing the potential relevance of the oxy conformation in regulating the functional properties of the protein. The conformation of the amino-terminal end of the beta subunits appears to regulate the interaction of hemoglobin with solvent components. The pronounced sensitivity of the oxygen affinity of bovine hemoglobin to chloride concentration and to pH suggests that in bovine species these are the modulators of oxygen transport in vivo.     

Analysis of oxygen equilibria of the giant hemoglobin from the earthworm Eisenia foetida using the Adair model

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.     

Modulation of oxygen affinity in hemoglobin by solvent components. Interaction of bovine hemoglobin with 2,3-diphosphoglycerate and monatomic anions

In the absence of Cl- in Hepes buffer at pH 7.4, the oxygen affinity of bovine and human hemoglobin is equally sensitive to 2,3-diphosphoglyceric acid. The low oxygen affinity measured for bovine hemoglobin at physiological salt concentration can be explained by the high affinity of Cl- anions for oxygen-linked sites that are absent in human hemoglobin. Bovine hemoglobin can discriminate between the different halogens in the sense that different halide concentrations are necessary to produce the same P50. Competition experiments indicate that the halogens interact with the same oxygen-linked sites. In agreement with the different affinities for halides, the Bohr effect of bovine hemoglobin is larger in the presence of Cl- than in that of Br- and there is good agreement between the number of protons and anions exchanged with the solvent upon oxygenation of bovine hemoglobin.     

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10^?3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum.The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li⁺, Ca²⁺ and Mg²⁺.The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10^?2 m to 2 m.The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be ?14·6 kcal/mol of O₂.     

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.     

Studies on the interaction of zinc with human hemoglobin

Zn has previously been shown to increase the oxygen affinity of both normal and sickle red blood cells. Experiments are presented which demonstrate that the oxygen affinity effect of Zn is due to a Zn-hemoglobin binding mechanism rather than a Zn-2,3 diphosphoglycerate binding mechanism. Further a large shift (6 mm Hg) in the oxygen affinity of a red cell-saline suspension occurs with a low Zn/hemoglobin (tetramer) molar ratio (0.4). Zn had no influence on the Bohr effect of hemoglobin but it did decrease the Hill coefficient. Hemoglobin binding experiments using partially purified hemoglobin indicated that Zn can bind to more than one amino acid residue but it appears that the amino acid residue with the highest binding capacity for Zn is also the residue involved in the oxygen affinity effect of Zn. Hydrogen ion concentration (pH 5–8) had no influence on the Zn/hemoglobin ratios obtained in these binding experiments. The possible (and the improbable) Zn binding sites on the hemoglobin molecule are discussed.     

Effect of Cl- and H+ on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes

Bovine hemoglobin was cross-linked with glutaraldehyde, resulting in high oxygen affinity polymeric hemoglobin dispersions of varying molecular weight distributions. High oxygen affinity acellular oxygen carriers were designed in order to exhibit oxygen release profiles closer to that of human red blood cells (RBCs), without exhibiting the inherent increased vasoactivity that occurs with low oxygen affinity acellular oxygen carriers (1, 2). Oxygen dissociation curves were measured for polymerized hemoglobin dispersions at various pH values (7.0, 7.4, and 8.0) and chloride ion concentrations. Unmodified hemoglobin showed an increase in oxygen affinity with increased chloride ion concentration and a decrease in oxygen affinity with increased pH, as was previously demonstrated in the literature (3). For glutaraldehyde-polymerized hemoglobin dispersions, the ability of the oxygen affinity to respond to changes in Bohr H+ and Cl- concentration was weakened. However, at acidic physiological pH (pH = 7), the Bohr effect was still present at high Cl- concentrations. Thus, the Bohr effect maintained some dependency on the Cl- concentration.     

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2)

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Ligand binding properties of hemoglobin 3 of the trout, Salmo gairdneri. The occurrence of an acid Bohr effect in the absence of heme-heme interaction.

The four components of hemoglobin from the rainbow trout (Salmo gairdneri) have been isolated. The oxygen affinities of the first two components eluted from the DEAE-cellulose column have much smaller pH dependencies than the last two components. These components have very low O2 affinities at low pH. The effect of pH on the equilibrium and kinetics of ligand binding to the third fraction, the pH-dependent component present in greatest amounts, has been studied. Measurements of ligand binding equilibria demonstrate the presence of both an alkaline and an acid Bohr effect. In the region of the alkaline Bohr effect the value of n in the Hill equation is a function of ligand affinity. For CO binding n decreases as the pH is decreased until at pH 6, the minimum ligand affinity is reached. At this pH there is also a complete loss of cooperative ligand binding. Decreasing the pH further results in an increase of ligand affinity, but this acid Bohr effect is not associated with a reappearance of cooperativity. This suggests that Fraction 3 of S. gairdneri is frozen in the low affinity, deoxygenated conformation at low pH and that the quaternary structure does not change even when fully liganded. However, the properties of the low affinity conformation of this hemoglobin are pH-dependent.     

The sensitivity of hemoglobin oxygen affinity to diphosphoglycerate and the characteristic pH of methemoglobin.

The sensitivity of the oxygen affinity of a hemoglobin to 2,3-diphosphoglyceric acid concentration has been defined as the change in log1/2O2 (deltalogp1/2O2) which results from saturating the hemoglobin with 2,3-diphosphoglyceric acid. The sensitivity varies from one hemoglobin species to another and is linearly rated to the difference in the logarithm of the binding constants of 2,3-diphosphoglyceric acid to deoxy- and oxyhemoglobin, the characteristic pH (pHch), and inversely proportional to the magnitude of the alkaline Bohr effect measured in a saturating amount of 2,3-diphosphoglyceric acid. Its magnitude is higher in large animals than in small animals and varies linearly with the charged amino acid composition of the hemoglobin. The charged amino acid residues must have been selected for in mammals with high metabolic needs and against in animals with low metabolic needs. Variability in the effect of 2,3-diphosphoglyceric acid on the oxygen transport in the different animal hemoglobins must therefore be the result of a positive Darwinian Selection of the charged amino acid residues in their hemoglobins. Furthermore, all the charged groups and not those at the binding site alone, affect the 2,3-diphosphoglyceric acid binding constant of a hemoglobin.     

Saturation dependency of the Bohr effect: interactions among H-+, CO2, and DPG.

The Bohr effect was measured in normal whole blood and in blood with low DPG concentration as a function of oxygen saturation. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HC1 at constant PCO2 (fixed acid Bohr effect). At nornal DPG concentration CO2 Bohr effect was -0.52 at 50% blood oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. In DPG depleted blood with base excess (BE) similar to 0 meq/1, there was similar dependence of CO2 Bohr effect on oxygen saturation. At BE similar to -10 meq/1, influence of saturation was comparable, but the magnitude of the Bohr effect was markedly increased at all saturations. Fixed acid Bohr effect at normal DPG concentration was -0.45 at saturations of 50-90% but decreased at lower saturations. In DPG-depleted blood fixed acid Bohr effect averaged about -0.33 with minimal variation with saturation. Influence of DPG on oxygen affinity was greater at intermediate saturations and less at saturations below 20% and above 80%. Effect of CO2, independent of pH, was many fold greater at lower oxygen saturations than at higher saturations. These results support the suggestion that the alpha chain of hemoglobin is the site of the initial oxygenation reaction. Physiologically they indicate that the relative contribution of CO2 and fixed acid, as well as the level of oxygen saturation and DPG concentration, may be important in determining PO2 of capillary blood and resulting oxygen delivery.     

Functional characterization of the single hemoglobin of the migratory bird Ciconia ciconia

Hemolysate from white stork displayed a single hemoglobin component, thus resulting into two bands and two globin peaks in dissociating PAGE and reversed phase-HPLC, respectively. Stripped hemoglobin showed an oxygen affinity higher than that of human HbA, a small Bohr effect, and a cooperative oxygen binding. A small decrease of oxygen affinity, of the same extent in all the pH range examined, was observed by addition of chloride, thus indicating an unusual chloride-independent Bohr effect (DeltalogP50/Deltalog pH=-0.24). Saturating amounts of inositol hexakisphosphate, largely decreased hemoglobin-oxygen affinity (DeltalogP(50)=1.17 at pH 7.0), and increased the extent of its Bohr effect (DeltalogP50/DeltalogpH=-0.45). The phosphate binding curve allowed to measure a very high overall binding constant (K=1.18 x 10(5) M(-1)). The effect of temperature on the oxygen affinity was measured, and the enthalpy change of oxygenation resulted almost independent on pH. Structural-functional relationships are discussed by considering some amino acid residues situated at alpha1/beta1 and alpha1/beta2 interfaces, such as alpha38 and alpha89 positions. The presence of only one hemoglobin component, a rare event among birds, and its functional properties have been related to the physiological oxygen requirements of this soaring migrant bird and to its technique of flight during migration.