Hemoglobin Richmond. Subunit dissociation and oxygen equilibrium properties.

In hemoglobin Richmond (beta102 leads to Lys), amino acid substitution has occurred at the same site as the mutation in hemoglobin Kansas (beta102 Asn leads to Thr), a variant with very low oxygen affinity. Although hemoglobin Richmond has been shown to have increased tetramer-dimer dissociation, its oxygen affinity has been inferred to be normal from studies on hemolysates of carriers. We have isolated hemoglobin Richmond and have further studied its properties. We confirm that the oxygen affinity of pure hemoglobin Richmond under conditions similar to those found in vivo is normal. However, the Bohr effect of the variant hemoglobin is markedly abnormal. Its oxygen affinity is low at high pH and high at low pH, relative to hemoglobin A. The tetramer-dimer equilibrium displays a strong pH dependence such that protons promote dissociation. A model is presented in which the structural change in hemoglobin Richmond results in low oxygen affinity, like hemoglobin Kansas. However, the close linkage between tetramer-dimer dissociation and proton concentration seen with hemoglobin Richmond results in normal oxygen affinity at intracellular pH and hemoglobin concentration, and carriers display no hematological abnormalities.     

Thermodynamics of anti-sickling agents with hemoglobin S

The effects of oxygen and a second ligand, the anti-sickling agent butylurea, on the hemoglobin S gel-solution phase equilibrium have been studied. The results have been analyzed using thermodynamic properties of the system. In particular, the solubility of deoxy hemoglobin S as a function of butylurea concentration was determined and the thermodynamic analysis shows that there are at least two cooperatively linked butylurea binding sites. Liquid phase oxygen binding studies at various butylurea concentrations show that the linkage between oxygen and butylurea binding is small. The influence of oxygen and butylurea on hemoglobin S solubility was determined by birefringence measurements. The results were interpreted by use of the Gibbs-Duhem equation which combined ligand binding expressions with the non-ideal solution properties and properties of the gel phase. The predicted influence of oxygen and butylurea upon the solubilities of hemoglobin S agrees with experimentally determined values.     

Orally administrated cerium chloride induces the conformational changes of rat hemoglobin, the hydrolysis of 2,3-DPG and the oxidation of heme-Fe(II), leading to changes of oxygen affinity

The structure and oxygen affinity of hemoglobin from erythrocytes of CeCl(3) fed Wistar rats in the dose range of 0.2-20.0 mg/kg body weight/day were investigated by means of various spectroscopic methods. The changes in oxygen saturation curves of hemoglobin are dependent upon both feeding dose and feeding time. After 40 days feeding with 20 mg CeCl(3)/kg body weight/day, the curve changed to a double sigmoid shape and the oxygen affinity in low oxygen pressure increases. It regained the sigmoid form after 80 days feeding, but the degree of oxygen saturation in higher oxygen pressure became higher than that in the control. These results indicate that CeCl(3) can increase the oxygen affinity of hemoglobin of rat erythrocytes. This effect is further demonstrated by the analysis of M?ssbauer spectra of erythrocytes. Increase of hemoglobin content in erythrocytes was found in rats fed with CeCl(3). It might be the offset response to the poor oxygen-releasing capability of the hemoglobin. CD and FT-IR deconvoluted spectra indicate that secondary structures of hemoglobin have remarkable changes, characterized by a gradual decrease of alpha-helix content, in a dose- and feeding time-dependent fashion. Meanwhile, the 31P NMR spectra demonstrate that the level of 2,3-diphosphoglyceric acid (2,3-DPG) in erythrocytes, an allosteric regulator of oxygen release from hemoglobin, decreases due to its hydrolysis. In addition, the M?ssbauer and ESR spectra show clearly that a fraction of the heme-iron changes from Fe (II) to Fe (III) in CeCl(3) fed rats. The results indicate that the oral administration of CeCl(3) leads to a microenvironment changes of heme in intracellular hemoglobin. Oxygen affinity changes might be attributed to a series of events triggered by the binding of Ce (III) to hemoglobin and 2,3-DPG, including conformational changes of hemoglobin and 2,3-DPG hydrolysis, respectively and also the partial transformation from heme-Fe (II) to heme-Fe (III).     

Comparison of human and shirbot (Cyprinidae: Barbus grypus) hemoglobin: a structure-function prospective

Hemoglobin is a tetrameric protein with two alpha and two beta subunits binds oxygen in a cooperative manner. In dominant tetrameric form of fish hemoglobin carry more than 90 percent of oxygen from gill to tissues at 20° C. The tetrameric form of fish hemoglobin is changed to monomeric form at low oxygen pressure in order to increase its oxygen affinity. This is one of adaptive mechanisms used by different kinds of fish. The major aim of this paper is to study the molecular basis of shirbot hemoglobin adaptation mechanism to various environmental conditions. Using different methods such as ion exchange chromatography, UV-Vis, fluorescence and circular dichroism spectroscopy, we extracted the main tetrameric fraction of shirbot hemoglobin and studied the structural characteristics of shirbot and human hemoglobins in a comparative way. Our results showed that tetrameric form of shirbot hemoglobin has less stable and loosely folded structure in contrast to human hemoglobin. Our data also indicate, in case of exposure to life-threatening environmental factors such as low oxygen level, acidic pH, oxidizing chemicals and other water pollutants especially detergents (surfactants) triggering tetramer to monomer dissociation in shirbot hemoglobin is more prominently than in human hemoglobin. The resulting monomer of hemoglobin has more oxygen affinity and could take up oxygen more strongly even at low pressure. We hypothesize that this mechanism helps shirbot to adapt and to survive at such harsh environment. The mechanism that is may be adapted by other fish species.     

Reduced hemoglobin affinity for oxygen in venous blood following hemodilution, independent of changes in pH or PCO2.

A rapidly induced and readily reversible shift in the affinity of hemoglobin for oxygen has been demonstrated. The shift, similar to the Bohr effect, is independent of PCO2 or pH changes. It occurred within 30 min of hemodilution and was seen in portal venous blood but not arterial blood. A hypothesis is suggested involving a phasic alteration in levels of 2,3-diphosphoglycerate (DPG) or ATP binding to hemoglobin. It is proposed that, following hemodilution, the degree of these phosphates to hemoglobin increases on passage through the intestinal vascular bed. The increased DPG binding to hemoglobin results in displacement of additional oxygen. As the blood becomes reoxygenated, the levels of DPG-hemoglobin binding decline and DPG is displaced from the hemoglobin by oxygen.     

Nonequilibrium-Facilitated Oxygen Transport in Hemoglobin Solution

We have used the quasi-linearization method to obtain numerical solutions to the equations which describe steady-state diffusion of oxygen through layers of hemoglobin solution. The numerical solutions show how the facilitated flux of oxygen depends upon the layer thickness, reaction-rate coefficients, and other parameters of the system. The results indicate that steady-state oxygen diffusion in layers of hemoglobin solution, similar to those studied by Scholander, should be adequately described by the models which assume chemical equilibrium exists throughout the layer, but for layers of concentrated hemoglobin solution about the thickness of a human erythrocyte, the facilitation of oxygen diffusion should be much less than the equilibrium models predict.     

Nitric oxide binding to oxygenated hemoglobin under physiological conditions.

We have added nitric oxide (NO) to hemoglobin in 0.1 M and 0.01 M phosphate buffers as well as to whole blood, all as a function of hemoglobin oxygen saturation. We found that in all these conditions, the amount of nitrosyl hemoglobin (HbNO) formed follows a model where the rates of HbNO formation and methemoglobin (metHb) formation (via hemoglobin oxidation) are independent of oxygen saturation. These results contradict those of an earlier report where, at least in 0.01 M phosphate, an elevated amount of HbNO was formed at high oxygen saturations. A radical rethink of the reaction of oxyhemoglobin with NO under physiological conditions was called for based on this previous proposition that the primary product is HbNO rather than metHb and nitrate. Our results indicate that no such radical rethink is called for.     

Nitric oxide binding to oxygenated hemoglobin under physiological conditions

We have added nitric oxide (NO) to hemoglobin in 0.1 M and 0.01 M phosphate buffers as well as to whole blood, all as a function of hemoglobin oxygen saturation. We found that in all these conditions, the amount of nitrosyl hemoglobin (HbNO) formed follows a model where the rates of HbNO formation and methemoglobin (metHb) formation (via hemoglobin oxidation) are independent of oxygen saturation. These results contradict those of an earlier report where, at least in 0.01 M phosphate, an elevated amount of HbNO was formed at high oxygen saturations. A radical rethink of the reaction of oxyhemoglobin with NO under physiological conditions was called for based on this previous proposition that the primary product is HbNO rather than metHb and nitrate. Our results indicate that no such radical rethink is called for.     

Oxygen equilibrium of emulsified solutions of normal and sickle hemoglobin.

Emulsions containing microdroplets of concentrated solutions of normal or sickle hemoglobin dispersed in a continous oil phase have been prepared, and the aggregation of sickle hemoglobin within microdroplets in a deoxygenated emulsion demonstrated. The equilibrium oxygen saturation of hemoglobin in the emulsions has been measured as a function of the partial pressure of oxygen by a novel spectrophotometric technique which corrects for the scattering of light in the emulsion. The half-saturation oxygen pressure and cooperativity of oxygen binding are substantially greater in concentrated (27 g/dl) solutions of sickle hemoglobin than in solutions of non-aggregating hemoglobin. The shape of the oxygen equilibrium curve of concentrated sickle hemoglobin is qualitatively discussed in terms of a previously proposed model (1).     

Solution of the spatial structure of dimeric transmembrane domains of proteins by heteronuclear NMR spectroscopy and molecular modeling

The goal of the work was to asses the effect of peroxynitrite on the affinity of hemoglobin for oxygen in in vitro experiments. It was demonstrated that the incubation of whole venous blood with peroxynitrite increased the affinity of hemoglobin for oxygen. Presumably, this effect is realized through generation of various forms of hemoglobin: heme-oxidized and modified at amino acid residues of the protein. The dependence of the results of hemoglobin-peroxynitrite reactions on carbon dioxide tension and the degree of hemoglobin oxygenation is discussed.     

Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins

An allosteric modulator of oxygen release in human erythrocytes is 2,3-diphosphoglycerate, but bovine erythrocytes apparently utilize chloride for this purpose since they contain little, if any, 2,3-diphosphoglycerate. In order to identify the sites to which these anions bind, the site-specific acetylating agent, methyl acetyl phosphate, has been employed to compete with these allosteric modulators and to mimic their effects on hemoglobin function. With human hemoglobin A, methyl acetyl phosphate competes with 2,3-diphosphoglycerate and acetylates only Val-1(beta), Lys-82(beta), and Lys-144(beta) within or near the cleft that binds this organic phosphate (Ueno, H., Pospischil, M. A., Manning, J. M., and Kluger, R. (1986) Arch Biochem. Biophys. 244, 795). With bovine hemoglobin, the acetylation is competitive with chloride ion. The sites of acetylation in oxy bovine hemoglobin are Met-1(beta) and Lys-81(beta) and for deoxy bovine hemoglobin, they are Val-1(alpha) and Lys-81(beta). Thus, these sites are expected to be involved in the binding of chloride to bovine hemoglobin. Treatment of either human or bovine hemoglobins with methyl acetyl phosphate under anaerobic conditions leads to a lowering of their oxygen affinity and hence the covalent modifier has the same effect on hemoglobin function as the non-covalent regulators, 2,3-diphosphoglycerate and chloride. The Hill's coefficient of hemoglobin is unaffected by treatment with methyl acetyl phosphate. Under aerobic conditions, specifically acetylated bovine hemoglobin also has a lowered oxygen affinity, and human hemoglobin A shows a slight change in its oxygen affinity. In general, bovine hemoglobin is more responsive than human hemoglobin to both chloride and methyl acetyl phosphate; the latter agent results in a permanent covalent labeling of the protein. Therefore, the results support the idea that methyl acetyl phosphate may be a useful probe for deciphering the sites of binding of anions to proteins.     

Determination of hemoglobin affinity for oxygen by a polarographic method

The method of determination of hemoglobin affinity for oxygen based on polarographic analysis and the method of half-saturation values P50 calculation are described. A special design of a polarographic cell is proposed for measurements in a wide temperature range. The results of temperature and pH dependences of the hemoglobin affinity for oxygen and erythrocytes of the White-Sea cod are presented.     

Hemoglobin redox reactions and oxidative stress

The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions. The reduction of oxygen to superoxide starts a cascade of oxidative reactions, which are a source for red cell-induced oxidative stress. The reduction of nitrite to nitric oxide produces a labile form of nitric oxide that can be a source for oxidative stress, but can also have important physiological functions.     

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.     

Rapid Determination of Oxygen Saturation and Vascularity for Cancer Detection

A rapid heuristic ratiometric analysis for estimating tissue hemoglobin concentration and oxygen saturation from measured tissue diffuse reflectance spectra is presented. The analysis was validated in tissue-mimicking phantoms and applied to clinical measurements in head and neck, cervical and breast tissues. The analysis works in two steps. First, a linear equation that translates the ratio of the diffuse reflectance at 584 nm and 545 nm to estimate the tissue hemoglobin concentration using a Monte Carlo-based lookup table was developed. This equation is independent of tissue scattering and oxygen saturation. Second, the oxygen saturation was estimated using non-linear logistic equations that translate the ratio of the diffuse reflectance spectra at 539 nm to 545 nm into the tissue oxygen saturation. Correlations coefficients of 0.89 (0.86), 0.77 (0.71) and 0.69 (0.43) were obtained for the tissue hemoglobin concentration (oxygen saturation) values extracted using the full spectral Monte Carlo and the ratiometric analysis, for clinical measurements in head and neck, breast and cervical tissues, respectively. The ratiometric analysis was more than 4000 times faster than the inverse Monte Carlo analysis for estimating tissue hemoglobin concentration and oxygen saturation in simulated phantom experiments. In addition, the discriminatory power of the two analyses was similar. These results show the potential of such empirical tools to rapidly estimate tissue hemoglobin in real-time spectral imaging applications.     

Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.     

Acylation of human hemoglobin with polyoxyethylene derivatives

Monomethoxypolyoxyethylene (Mw = 5000) was covalently linked to human hemoglobin via an amide bond formed between amino groups of the protein and a carboxylic group introduced onto the polymer. The conjugates thus obtained have a molecular size corresponding to that of a globular protein with a molecular weight of about 190 000. Their oxygen-binding properties depend upon the initial conformation of the hemoglobin and reaction pH: hemoglobin modified in the deoxy state exhibited a lower oxygen affinity than that modified in the oxy state, and the lower the reaction pH, the lower the oxygen affinity of polymer-linked hemoglobin. However, the affinity of modified hemoglobin is always higher than that of native hemoglobin. On the other hand, when deoxyHb was complexed with organic phosphates during the condensation reaction, the resulting conjugates exhibited oxygen-binding characteristics quite similar to those of native hemoglobin, i.e., the same oxygen affinity, modified cooperativity and the same alkaline Bohr effect. Finally, in order to decrease the oxygen affinity of hemoglobin conjugates, the polymer was coupled to deoxy hemoglobin previously covalently modified with pyridoxal phosphate. The oxygen affinity of such conjugates was in fact as low as that of the initial pyridoxylated hemoglobin.     

Influence of ligation state and concentration of hemoglobin A on its cross-linking by glycolaldehyde: functional properties of cross-linked, carboxymethylated hemoglobin

The ligation state of hemoglobin during its cross-linking by glycolaldehyde influences the ultimate oxygen affinity of the cross-linked protein. Thus, if the cross-linking is performed with carbonmonoxy-hemoglobin, the oxygen affinity increases slightly to a P50 of 7 mmHg from a P50 of 9 mmHg for unmodified hemoglobin. In contrast, when deoxyhemoglobin is cross-linked with glycolaldehyde, the oxygen affinity of the product decreases (P50 = 15 mmHg). When deoxyhemoglobin is first carboxymethylated and then cross-linked with glycolaldehyde, an even lower oxygen affinity is achieved (P50 = 23 mmHg). Carboxymethylated hemoglobin is very responsive to the presence of 5% CO2 with a P50 of 33 mmHg, which is lowered further to 42 mmHg when chloride (0.1 M) is also present. Hemoglobin carboxymethylated and cross-linked under anaerobic conditions is also responsive to the modulators CO2 and chloride with a resultant oxygen affinity of 27 mmHg. The type of cross-linking of liganded hemoglobin by the mild reagent glycolaldehyde is dependent upon the initial hemoglobin concentration. Thus, with dilute hemoglobin (45 microM in tetramer), cross-linking by glycolaldehyde (50 mM) results in about 75% of 64,000 molecular weight species (some of which are cross-linked within tetramer) and 25% of intertetrameric cross-linked species with a range of molecular weights averaging 128,000-512,000. With hemoglobin solutions of higher concentration (360 microM), the amount of the higher molecular weight species increases to about 65% with a corresponding reduction to 35% in the 64,000 molecular weight component.     

Hemoglobin redox reactions and oxidative stress

Abstract

The role of hemoglobin in transporting oxygen is dependent on the reversible binding of oxygen to Fe(II) hemoglobin with molecular oxygen released at reduced oxygen pressures. The partially oxygenated hemoglobin formed with the release of oxygen from hemoglobin is susceptible to redox reactions where the functional Fe(II) heme is oxidized to Fe(III) and the substrate is reduced. In this article, we review two important redox reactions of hemoglobin and discuss the ramifications of these reactions. The reduction of oxygen to superoxide starts a cascade of oxidative reactions, which are a source for red cell-induced oxidative stress. The reduction of nitrite to nitric oxide produces a labile form of nitric oxide that can be a source for oxidative stress, but can also have important physiological functions.     

Calcium-dependent allosteric modulation of the giant hemoglobin from the terrestrial oligochaete, Eisenia foetida

The effect of calcium and magnesium ions on the oxygen equilibrium of Eisenia hemoglobin was investigated by using an automatic oxygenation apparatus. On addition of calcium chloride (20 mM, pH 7.5), oxygen affinity and cooperativity (nmax) of the hemoglobin increased markedly (p 50:3.82 mmHg, nmax :9.76). The effect of magnesium on the oxygen equilibrium was weaker than that of calcium. The top asymptotes of the oxygen equilibrium curve shifted to the left by adding cations whereas the bottom asymptotes remained almost unchanged. The free energy of heme-heme interaction (delta GR,T) also increased remarkably. These results imply the binding of calcium to Eisenia hemoglobin in the oxygenated form and its physiological role in modulating the oxygen affinity and cooperativity.