Asunto(s)
Eritroblastos/patología , Enfermedad de la Hemoglobina C/patología , Hemoglobina C/química , Colecistectomía , Cálculos Biliares/complicaciones , Cálculos Biliares/patología , Cálculos Biliares/cirugía , Enfermedad de la Hemoglobina C/complicaciones , Enfermedad de la Hemoglobina C/diagnóstico , Humanos , Masculino , Persona de Mediana EdadRESUMEN
An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson-Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG-IEF and non-IPG-IEF (opposite to IPG-IEF) were developed to unveil stability mechanism of IPG-IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG-IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG-IEF system, (ii) there existed high diffusional current in non-IPG-IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG-IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG-IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG-IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique.
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Algoritmos , Focalización Isoeléctrica/métodos , Modelos Químicos , Proteínas/química , Fuerza Protón-Motriz , Animales , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/aislamiento & purificación , Bovinos , Citocromos c/química , Citocromos c/aislamiento & purificación , Hemoglobina A/química , Hemoglobina A/aislamiento & purificación , Hemoglobina C/química , Hemoglobina C/aislamiento & purificación , Caballos , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/instrumentación , Lactoglobulinas/química , Lactoglobulinas/aislamiento & purificación , Mioglobina/química , Mioglobina/aislamiento & purificación , Ficocianina/química , Ficocianina/aislamiento & purificación , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Proteínas/aislamiento & purificación , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (ß6 GluâLys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K(4)) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.
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Sustitución de Aminoácidos/genética , Hemoglobina C/química , Hemoglobina C/metabolismo , Oxígeno/metabolismo , Absorción , Carboxihemoglobina/química , Carboxihemoglobina/metabolismo , Cristalografía por Rayos X , Hemo/química , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Análisis Espectral , EstereoisomerismoRESUMEN
Individuals expressing hemoglobin C (beta6 Glu-->Lys) present red blood cells (RBC) with intraerythrocytic crystals that form when hemoglobin (Hb) is oxygenated. Our earlier in vitro liquid-liquid (L-L) phase separation studies demonstrated that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS or HbA, and that L-L phase separation preceded and enhanced crystallization. We now present evidence for the role of phase separation in HbC crystallization in the RBC, and the role of the RBC membrane as a nucleation center. RBC obtained from both human homozygous HbC patients and transgenic mice expressing only human HbC were studied by bright-field and differential interference contrast video-enhanced microscopy. RBC were exposed to hypertonic NaCl solution (1.5-3%) to induce crystallization within an appropriate experimental time frame. L-L phase separation occurred inside the RBC, which in turn enhanced the formation of intraerythrocytic crystals. RBC L-L phase separation and crystallization comply with the thermodynamic and kinetics laws established through in vitro studies of phase transformations. This is the first report, to the best of our knowledge, to capture a temporal view of intraerythrocytic HbC phase separation, crystal formation, and dissolution.
Asunto(s)
Eritrocitos/química , Hemoglobina C/química , Hemoglobina C/aislamiento & purificación , Animales , Cristalización , Citosol , Membrana Eritrocítica/metabolismo , Humanos , Ratones , Temperatura , Factores de TiempoRESUMEN
A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.
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Antígenos de Protozoos/química , Hemoglobina C/fisiología , Hemoglobina Falciforme/fisiología , Malaria Falciparum/inmunología , Plasmodium falciparum/metabolismo , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genotipo , Hemoglobina C/química , Hemoglobina Falciforme/química , Humanos , Sistema Inmunológico , Inmunoglobulina G/química , Vacunas contra la Malaria/química , Malaria Falciparum/prevención & control , Proteínas Protozoarias/químicaRESUMEN
Crystallization of the mutated hemoglobin, HbC, which occurs inside red blood cells of patients expressing betaC-globin and exhibiting the homozygous CC and the heterozygous SC (in which two mutant beta-globins, S and C, are expressed) diseases, is a convenient model for processes underlying numerous condensation diseases. As a first step, we investigated the molecular-level mechanisms of crystallization of this protein from high-concentration phosphate buffer in its stable carbomonoxy form using high-resolution atomic force microscopy. We found that in conditions of equilibrium with the solution, the crystals' surface reconstructs into four-molecule-wide strands along the crystallographic a (or b) axis. However, the crystals do not grow by the alignment of such preformed strands. We found that the crystals grow by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation or by screw dislocations. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force. These results show that the crystallization mechanisms of HbC are similar to those found for other proteins. Therefore, strategies developed to control protein crystallization in vitro may be applicable to pathology-related crystallization systems.
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Cristalización/métodos , Hemoglobina C/química , Hemoglobina C/ultraestructura , Microscopía de Fuerza Atómica , Hemoglobina C/análisis , Complejos Multiproteicos/química , Conformación ProteicaRESUMEN
Pancreatic islets and insulinoma cells are particularly vulnerable to serious damage by cytotoxic nitric oxide (NO) and/or oxidative stress, most probably due to their low expression levels of antioxidant enzymes. This cellular damage has been regarded as one of major obstacles to success of encapsulated islet transplantation for the treatment of type 1 diabetes. As an approach to preventing NO induced damage, crosslinked hemoglobin (Hb-C) with poly(ethylene glycol) was co-encapsulated with rat islets or insulinoma cells (RINm5F) in alginate/poly(L-lysine)/alginate microcapsules. Hb-C effectively protected the cells from NO damage, generated by treating the cell microcapsules with S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide donor) at concentrations up to 400 microM, preserving higher viability and insulin secretion than a control group (no SNAP and no Hb-C). When the cells were incubated with SNAP without Hb, there was SNAP concentration dependent cellular damage, and a colorimetric TUNEL assay revealed a typical cell apoptosis sign, indicating DNA damages.
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Materiales Biocompatibles Revestidos/metabolismo , Hemoglobina C/metabolismo , Insulinoma/patología , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Óxido Nítrico/metabolismo , Penicilamina/análogos & derivados , Penicilamina/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Materiales Biocompatibles Revestidos/síntesis química , Materiales Biocompatibles Revestidos/química , Reactivos de Enlaces Cruzados , Hemoglobina C/química , Masculino , Óxido Nítrico/química , Neoplasias Pancreáticas/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Typically certain features of red cell morphology predict the results of osmotic fragility testing. Microspherocytes generally have increased and target cells decreased fragility. Blood smears in homozygous hemoglobin C disease show an interesting admixture of microspherocytes and target cells. Yet osmotic fragility studies generally show only reduced fragility and no population of fragile cells to correspond with the spherocytes. The present study demonstrates that the red cells of patients with hemoglobin C-beta thalassemia share many characteristics with hemoglobin C red cells, including the decreased osmotic fragility of all cells despite the presence of both spherocytes and target cells. These paradoxically osmotically resistant spherocytes probably arise because of cellular dehydration due to a K-Cl transport system which may be activated by binding of hemoglobin C to the red cell membrane.
Asunto(s)
Enfermedad de la Hemoglobina C/sangre , Soluciones Hipotónicas/farmacología , Fragilidad Osmótica , Esferocitos/efectos de los fármacos , Talasemia beta/sangre , Adulto , Índices de Eritrocitos , Femenino , Hemoglobina C/química , Enfermedad de la Hemoglobina C/genética , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Talasemia beta/genéticaRESUMEN
Prompted by the reported lack of solvation effects on the oxygen affinity of fish (trout I) hemoglobin that questioned allosteric water binding in human hemoglobin A (Bellelli, A., Brancaccio, A., and Brunori, M. (1993) J. Biol. Chem. 268, 4742-4744), we have investigated solvation effects in fish and human hemoglobins by means of the osmotic stress method and allosteric analysis. In contrast to the earlier report, we demonstrate that water potential does affect oxygen affinity of trout hemoglobin I in the presence of inert solutes like betaine. Moreover, we show that upon oxygenation electrophoretically anodic hemoglobin from trout and eel bind a similar number of water molecules as does human hemoglobin A, whereas the cathodic hemoglobins of trout and eel bind smaller, but mutually similar, numbers of water molecules. Addition of cofactors strongly increases the number of water molecules bound to eel hemoglobin A (as in human hemoglobin) but only weakly affects water binding to eel hemoglobin C.
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Hemoglobinas/química , Sitio Alostérico , Animales , Betaína/química , Anguilas , Hemoglobina A/química , Hemoglobina C/química , Hemoglobinas/metabolismo , Humanos , Modelos Químicos , Ósmosis , Oxígeno/metabolismo , Unión Proteica , Conformación Proteica , Especificidad de la Especie , Temperatura , Trucha , Agua/químicaRESUMEN
Homozygous HbC gene results only in mild hemolytic anemia. In HbSC disease red cells contain equal levels of HbS and HbC. It is a paradox that HbSC exhibit a moderately severe phenotype in spite of being a mixture of HbS trait and HbC trait, neither of which has significant pathology. Why does the combination of these two Hbs result in a serious disease? The short answer is that HbC enhances, by dehydrating the SC red cell, the pathogenic properties of HbS, resulting in a clinically significant disorder, but somewhat milder that sickle cell anemia (SCA). Nevertheless, retinnitis proliferans, osteonecrosis, and acute chest syndrome have equal or higher incidence in HbSC disease compared to SCA. This pathogenic trick is accomplished by HbC inducing, by mechanisms not fully understood, an increase in the activity of K:Cl cotransport that induces the lost of K(+) and consequently of intracellular water. This event creates a sufficient increase of MCHC, so that the lower levels of HbS found in SC red cells can polymerize rapidly and effectively. This situation offers a unique opportunity: if we could inhibit the effect of HbC on K(+) transport we can cure the disease.
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Enfermedad de la Hemoglobina SC/sangre , Enfermedad de la Hemoglobina SC/fisiopatología , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/fisiopatología , Cristalización , Eritrocitos Anormales/metabolismo , Eritrocitos Anormales/ultraestructura , Hemoglobina C/química , Hemoglobina C/genética , Enfermedad de la Hemoglobina SC/genética , Hemoglobina Falciforme/química , Hemoglobina Falciforme/genética , Humanos , Microscopía Electrónica de Rastreo , Fenotipo , Potasio/metabolismoRESUMEN
Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.
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Hemoglobina C/química , Hemoglobina Falciforme/química , Mutación , Cristalización , Hemoglobina C/genética , Hemoglobina Falciforme/genética , Humanos , Cinética , Microscopía de InterferenciaRESUMEN
Anionic regulation of hemoglobin (Hb) is of increasing interest for the design of Hb-based oxygen carriers. Even "external" amino-acid substitutions can alter the nature and extent of anionic control. This was shown by evaluation of the anion sensitivities of liganded, R-state, forms of HbA, HbC (beta6 Glu --> Lys) and HbS (beta6 Glu --> Val). The beta6 mutants differ in the anion-sensitivity of their central cavities, alpha1beta2 interfaces, and heme and beta93 Cys environments. The mutant Hbs also exhibit increased anion-dependent oxidation and surface denaturation. Moreover, differential chloride effects on oxygen binding by Hbs C, S compared to HbA occur after R-state stabilization by fluoresceination of beta93 Cys. It is concluded that the "external" substitutions in the mutant Hbs have structural consequences that are propagated to varying extents to other domains as a result of anion binding, and that these anion-dependent changes may underlie mechanisms leading to the observed increase in oxidation propensity and surface denaturation.
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Hemoglobinas/química , Hemoglobinas/genética , Aniones/farmacología , Sitios de Unión , Precipitación Química , Cloruros/farmacología , Hemoglobina A/química , Hemoglobina A/genética , Hemoglobina C/química , Hemoglobina C/genética , Hemoglobina Falciforme/química , Hemoglobina Falciforme/genética , Humanos , Mutación , Oxígeno , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
We show with three proteins that trapping and release of the water molecules upon crystallization is a determinant of the crystallization thermodynamics. With HbC, a strong retrograde solubility dependence on temperature yields a high positive enthalpy of 155 kJ mol(-1), i.e., crystallization is only possible because of the huge entropy gain of 610 J mol(-1) x K(-1), stemming from the release of up to 10 water molecules per protein intermolecular contact. With apoferritin, the enthalpy of crystallization is close to zero. The main component in the crystallization driving force is the entropy gain due to the release upon crystallization of two water molecules bound to one protein molecules in solution. With both proteins, the density of the growth sites imaged by AFM is in excellent agreement with a calculation using the crystallization free energy. With lysozyme, the entropy effect due to the restructuring of the water molecules is negative. This leads to higher solubility.
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Cristalización/métodos , Proteínas/química , Animales , Apoferritinas/química , Cristalización/estadística & datos numéricos , Entropía , Hemoglobina C/química , Humanos , Muramidasa/química , Solventes , Termodinámica , AguaRESUMEN
The mutated hemoglobin HbC (beta 6 Glu-->Lys), in the oxygenated (R) liganded state, forms crystals inside red blood cells of patients with CC and SC diseases. Static and dynamic light scattering characterization of the interactions between the R-state (CO) HbC, HbA, and HbS molecules in low-ionic-strength solutions showed that electrostatics is unimportant and that the interactions are dominated by the specific binding of solutions' ions to the proteins. Microscopic observations and determinations of the nucleation statistics showed that the crystals of HbC nucleate and grow by the attachment of native molecules from the solution and that concurrent amorphous phases, spherulites, and microfibers are not building blocks for the crystal. Using a novel miniaturized light-scintillation technique, we quantified a strong retrograde solubility dependence on temperature. Thermodynamic analyses of HbC crystallization yielded a high positive enthalpy of 155 kJ mol(-1), i.e., the specific interactions favor HbC molecules in the solute state. Then, HbC crystallization is only possible because of the huge entropy gain of 610 J mol(-1) K(-1), likely stemming from the release of up to 10 water molecules per protein intermolecular contact-hydrophobic interaction. Thus, the higher crystallization propensity of R-state HbC is attributable to increased hydrophobicity resulting from the conformational changes that accompany the HbC beta 6 mutation.
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Hemoglobina C/química , Algoritmos , Fenómenos Biofísicos , Biofisica , Humanos , Cinética , Luz , Mutación , Oxihemoglobinas/metabolismo , Unión Proteica , Conformación Proteica , Dispersión de Radiación , Cloruro de Sodio/farmacología , Temperatura , TermodinámicaAsunto(s)
Anemia de Células Falciformes/complicaciones , Hemoglobina C/fisiología , Hemoglobina Falciforme/química , Malaria/genética , Anemia de Células Falciformes/parasitología , Animales , Femenino , Hemoglobina C/química , Hemoglobina C/genética , Humanos , Malaria/epidemiología , Masculino , Factores de RiesgoRESUMEN
We have studied the self-assembly of Hemoglobin C-Harlem (HbC-Harlem), a double mutant of hemoglobin that possesses the beta6 Glu-->Val mutation of sickle hemoglobin (HbS) plus beta73 Asp-->Asn. By electron microscopy we find it forms crystals, rather than the wrapped multistranded fibers seen in HbS. Fourier transforms of the crystals yield unit cell parameters indistinguishable from crystals of HbS. Differential interference contrast (DIC) microscopy and birefringence also show crystal formation rather than the polymers or domains seen for HbS, while the growth patterns showed radiating crystal structures rather than simple linear crystalline forms. The solubility of the assembly was measured using a photolytic micromethod over a temperature range of 17-31 degrees C in 0.15 M phosphate buffer and found to be essentially the same as that of fibers of HbS. The assembly kinetics were observed by photolysis of the carbon monoxide derivative, and the mass of assembled hemoglobin was found to grow exponentially, with onset times that were stochastically distributed for small volumes. The stochastic onset of assembly showed strong concentration dependence, similar to but slightly greater than that seen in sickle hemoglobin nucleation. These observations suggest that like HbS, HbC-Harlem assembly proceeds by a homogeneous nucleation process, followed by heterogeneous nucleation. However, relative to HbS, both homogeneous and heterogeneous nucleation are suppressed by almost 11 orders of magnitude. The slowness of nucleation can be reconciled with the similarity of the solubility to HbS by an increase in contact energy coupled with a decrease in vibrational entropy recovered on assembly. This also explains the linearity of the double-strands, and agrees with the chemical nature of the structural replacement.
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Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Hemoglobina C/química , Hemoglobina C/ultraestructura , Biopolímeros/química , Biopolímeros/metabolismo , Cristalización , Entropía , Eritrocitos/química , Análisis de Fourier , Hemoglobina C/genética , Hemoglobina C/metabolismo , Humanos , Cinética , Microscopía Electrónica , Mutación/genética , Estructura Cuaternaria de Proteína , Solubilidad , Procesos Estocásticos , Temperatura , VibraciónRESUMEN
CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.