Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids.
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As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer. (+info)
Assembly of human hemoglobin (Hb) beta- and gamma-globin chains expressed in a cell-free system with alpha-globin chains to form Hb A and Hb F.
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Rates of in vitro synthesis of radiolabeled gamma and beta chains made in a cell-free transcription/translation system were similar, but expressed globin chains were unstable. The addition of unlabeled beta or gamma chains at the start of chain synthesis generated radiolabeled beta(4) or gamma(2) and gamma(4) chains, respectively. If unlabeled alpha-globin chains were added at the start of chain synthesis, then approximately equal amounts of radiolabeled alphabeta or alphagamma bands were generated. If unlabeled Hb A or Hb F was added to reactions containing radiolabeled alphabeta or alphagamma prior to electrophoresis, then radiolabeled Hb A or Hb F tetramers, respectively, were generated. If alpha chains were added after synthesis of radiolabeled gamma chains made in the presence of unlabeled gamma chains, then little radiolabeled alphagamma formed. In contrast, if alpha chains were added after synthesis of radiolabeled beta chains made in the presence of unlabeled beta chains, then radiolabeled alpha(2)beta(2) formed. These findings suggest that beta and gamma chains associate with alpha chains during or soon after translation. This would prevent the formation of unstable monomers as well as stable gamma(2) dimers and suggests that alpha chains may bind to nascent non-alpha chains, acting as folding catalysts to promote functional tetrameric hemoglobin formation in vivo. (+info)
Hemoglobin F synthesis is not restricted to fetal erythropoietic organs during extramedullary hematopoiesis.
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We investigated whether the anatomic distribution of hematopoietic cells determines the type of hemoglobin produced in patients with extramedullary hematopoiesis (EMH). Fetal hemoglobin (HbF) production is not restricted to fetal erythropoietic organs during EMH. A shift of erythropoiesis to fetal hematopoietic organs in EMH does not necessarily induce HbF synthesis in adulthood. (+info)
Partitioning of benzene in blood: influence of hemoglobin type in humans and animals.
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Earlier studies have shown that air/blood partition coefficients (PCs) for many volatile organic chemicals (VOCs) are much higher in rat blood than in human blood. It has been suggested that the discrepancy could be attributed to the fact that hemoglobin (Hb) in rat blood exists in a quasi-crystalline form of hydrophobicity greater than that of normal human Hb (HbA) and thus has a higher carrying capacity for VOCs. In the present study, we used benzene as a prototypic VOC to examine its relative partitioning into human and animal blood. Additionally, we sought to ascertain whether the water-insoluble form of hemoglobin (HbS) found in subjects with homozygous sickle cell (SC) disease has a greater VOC-carrying capacity than does HbA blood. At a low-O(2) tension, HbS switches to water-insoluble polymers, which physically deforms the red blood cells (RBCs) to the sickle shape. We equilibrated HbA, HbS, Hartley guinea pig, CD1 mouse, and rat (F-344, Wistar, and Sprague-Dawley) blood and their respective fractions with benzene vapor (80 or 400 ppm) for 3 hr at 37 degrees C in air-tight vials. We introduced benzene vapor into the vial head space that contained air or respiratory mixtures of venous-type (low-O(2)) or arterial-type (high-O(2)) gases. The blood measurements included the PC, Hb, partial pressures of O(2)(pO(2)) and CO(2)(pCO(2), pH, and percentage of SCs. The benzene concentration had no effect on these parameters, and the high- and low-O(2) gas mixtures produced the expected changes in pO(2), pCO(2), and pH. At equilibrium, the low-O(2) HbS blood had approximately 85% SCs compared with roughly 15% with air or high-O(2) gas. PCs for rat and mouse blood were about 100% higher than those for human and guinea pig blood, but the PC for deoxygenated HbS blood was only slightly higher than that for HbA or oxygenated HbS blood. Benzene showed higher affinities for RBCs in the deoxygenated HbS, rat, and mouse blood and higher affinity for plasma in the guinea pig blood. There was no evidence of disproportionate partitioning of benzene into oxygenated HbS or into HbA blood forms. These data suggest that the water solubility of Hb alone appears to have little effect on the VOC-carrying capacity of blood and that the influence of species is large in comparison. These latter differences in partitioning may depend on the number of hydrophobic sites on the surface of the plasma/heme proteins and thus be unique to the species. (+info)
Dehydration response of sickle cells to sickling-induced Ca(++) permeabilization.
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Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability ("P(sickle)") to (at least) Na(+), K(+), Ca(2+), and Mg(2+). Resulting changes in [Ca(2+)] and [H(+)] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca(2+)-sensitive K(+) ("Gardos") channel (K(Ca)) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of P(sickle) expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca(2+) pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca(2+))-induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their P(sickle) values. P(sickle)-mediated dehydration was inhibited by clotrimazole, verifying the role of K(Ca), and also by elevated [Ca(2+)](o), above 2 mM. Very high P(sickle) values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of P(sickle) documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in P(sickle)-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations. (+info)
Heme nitrosylation of deoxyhemoglobin by s-nitrosoglutathione requires copper.
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NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions. (+info)
Control of the allosteric equilibrium of hemoglobin by cross-linking agents.
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The kinetics of ligand rebinding have been studied for modified or cross-linked hemoglobins (Hbs). Several compounds were tested that interact with alpha Val 1 or involve a cross-link between alpha Val 1 and alpha Lys 99 of the opposite dimer. By varying the length of certain cross-linking molecules, a wide range in the allosteric equilibrium could be obtained. Several of the mono-aldehyde modified Hbs show a shift toward the high affinity conformation of Hb. At the other extreme, for certain di-aldehyde cross-linked Hbs, the CO kinetics are typical of binding to deoxy Hb, even at low photodissociation levels, with which the dominant photoproduct is the triply liganded species; in these cases the hemoglobin does not switch from the low to high affinity state until after the fourth ligand is bound. Although each modified Hb shows only two distinct rates, the kinetic data as a function of dissociation level cannot be simulated with a simple two-state model. A critical length is observed for the maximum shift toward the low affinity T-state. Longer or shorter lengths of the cross-linker yielded more high affinity R-state. Unlike native Hb, which is in equilibrium with free dimers, the cross-linked Hbs maintain the fraction slow kinetics, which is unique to Hb tetramers, even at 0.5 microM (total heme). Addition of HbCN to unmodified HbCO solutions results in dimer exchange, which decreases the relative fraction of slow bimolecular kinetics; the cross-linked Hbs did not show such an effect, indicating that they do not participate in dimer exchange. (+info)
Product-conformation-driven ligation of peptides by V8 protease.
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Organic co-solvent-induced secondary conformation of alpha(17-40) of human hemoglobin facilitates the splicing of E30-R31 in a mixture of its complementary segments by V8 protease. The amino acid sequence of alpha(17-40) has been conceptualized by the general structure FR(I)-EALER-FR(II) and the pentapeptide sequence EALER playing a major role in inducing the alpha-helical conformation. The primary structure of alpha(17-40) has been engineered in multiple ways to perturb one, two, or all three regions and the influence of the organic co-solvent-induced conformation and the concomitant resistance of E30-R31 peptide bond to V8 protease digestion has been investigated. The central pentapeptide (EALER), referred to here as splicedon,(3) appears to dictate a primary role in facilitating the splicing reaction. When the same flanking regions are used, (1) splicedons that carry amino acid residues of low alpha-helical potential, for example G at position 2 or 3 of the splicedon, generate a conformational trap of very low thermodynamic stability, giving an equilibrium yield of only 3%-5%; (2) splicedons with amino acid residues of good alpha-helical potential generate a conformational trap of medium thermodynamic stability and give an equilibrium yield of 20%-25%; (3) the splicedons with amino residues of good alpha-helical potential and also an amino acid that can generate an i, i + 4 side-chain carboxylate-guanidino (amino) interaction, a conformational trap of maximum thermodynamic stability is generated, giving an equilibrium yield of 45%-50%; and (4) the thermodynamic stability of the conformational trap of the spliced peptide is also influenced by the amino acid composition of the flanking regions. The V8 protease resistance of the spliced peptide bond is not a direct correlate of the amount of alpha-helical conformation induced into the product. The results of this study reflect the unique role of the splicedon in translating the organic co-solvent-induced product conformation as a site-specific stabilization of the spliced peptide bond. It is speculated that the splicedon with higher alpha-helical potential as compared to either one of the flanking regions achieves this by integrating its potential with that of the flanking region(s). Exchange of flanking regions with the products of other V8 protease-catalyzed splicing reactions will help to establish the general primary structural requirements of this class of splicing reactions and facilitate their application in modular construction of proteins. (+info)