Covalent fixation of hemoglobin to dextran phosphates decreases its oxygen affinity.

The interactions between various dextran phosphates and Hb (hemoglobin) were studied by measuring the oxygen-binding parameters of the mixtures. The effector properties of polymers were found to depend on the concentration of monoalkylmonophosphate groups on the polymers and also on their molecular weights. The covalent fixation of dextran phosphates bearing aldehydic groups to oxyHb and deoxyHb was carried out. The oxygen-binding properties of the conjugates thus obtained depended upon the initial form of the protein. Thus, only the conjugates synthesized from deoxyHb exhibited a low oxygen affinity, which means that, in this case, the linkages between the dextran phosphate and the protein allow a permanent interaction of the phosphate groups with amines of the 2,3-diphosphoglycerate binding site. The Hill coefficient values of these conjugates were smaller than that of free Hb, corresponding to a loss of the cooperativity of the protein upon fixation of polymers. However, as these new conjugates are capable of unloading more O2 than blood when subjected to oxygen pressures corresponding to physiological conditions, they can be regarded as potential erythrocyte substitutes.     

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.     

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 quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO₂ of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO₂ of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO₂, methemoglobin (metHb) level, molecular weight distribution, O₂ affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross‐link density. T‐state PolyHb solutions exhibited lower O₂ affinity compared to unmodified Hb, whereas R‐state PolyHb solutions exhibited higher O₂ affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross‐link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

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.     

Age related changes in the affinity of hemoglobin to oxygen in rabbits]

It has been demonstrated that the affinity of hemoglobin to oxygen decreases after birth due to changes in the saline composition of erythrocytes. It is suggested that this decrease is mainly due to accumulation of 2.3-diphosphoglycerate in erythrocytes of the growing rabbits.     

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

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

Building quantitative relationship between changed sequence and changed oxygen affinity in human hemoglobin beta-chain

244 point mutations have been recorded in human hemoglobin beta-chain, of which some change the oxygen affinity of human hemoglobin beta-chain. We use the amino-acid distribution probability to quantify these mutations, and use the cross-impact analysis with Bayes' law to determine the probability that changes the oxygen affinity of human hemoglobin beta-chain under mutations.     

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

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

Semihemoglobins, high oxygen affinity dimeric forms of human hemoglobin respond efficiently to allosteric effectors without forming tetramers

Significant reduction in oxygen affinity resulting from interactions between heterotropic allosteric effectors and hemoglobin in not only the unligated derivative but also the fully ligated form has been reported (Tsuneshige, A., Park, S. I., and Yonetani, T. (2002) Biophys. Chem. 98, 49-63; Yonetani, T., Park, S. I., Tsuneshige, A., Imai, K., and Kanaori, K. (2002) J. Biol. Chem. 277, 34508-34520). To further investigate this effect in more detail, alpha- and beta-semihemoglobins, namely, alpha(heme)beta(apo) and alpha(apo)beta(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60- and 130-fold for alpha- and beta-semihemoglobins, respectively. 1H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as single-heme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated.     

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.     

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.     

Three-state combinatorial switch models as applied to the binding of oxygen by human hemoglobin

We have generated a series of all 6561 unique, discrete three-state combinatorial switch models to describe the partitioning of the cooperative oxygen-binding free change among the 10 variously ligated forms of human hemoglobin tetramers. These models were inspired by the experimental observation of Smith and Ackers that the cooperative free energy of the intersubunit contact regions of the 10 possible ligated forms of human hemoglobin tetramers can be represented by a particular distribution of three distinct energy levels [Smith, F. R., & Ackers, G. K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5347-5351]. A statistical thermodynamic formulation accounting for both dimer-tetramer equilibria and ligand binding properties of hemoglobin solutions as a function of oxygen and protein concentrations was utilized to exhaustively test these thermodynamic models. In this series of models each of the 10 ligated forms of the hemoglobin tetramer can exist in one, and only one, of three possible energy levels; i.e., each ligated form was assumed to be associated with a discrete energy state. This series of models includes all possible ways that the 10 ligation states of hemoglobin can be distributed into three distinct cooperative energy levels. The mathematical models, as presented here, do not permit equilibria between energy states to exist for any of the 10 unique ligated forms of hemoglobin tetramers. These models were analyzed by nonlinear least-squares estimation of the free energy parameters characteristic of this statistical thermodynamic development.(ABSTRACT TRUNCATED AT 250 WORDS)