Solution-active structural alterations in liganded hemoglobins C (beta6 Glu --> Lys) and S (beta6 Glu --> Val).
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Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the alpha and beta chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the alpha1beta2 interface (beta37), and in the A-helix (alpha14/beta15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC approximately HbS > HbA. (+info)
Serum transferrin receptor levels are increased in asymptomatic and mild Plasmodium falciparum-infection.
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BACKGROUND AND OBJECTIVE: The serum transferrin receptor (sTfR) concentration in an individual reflects the extent of erythropoietic activity and is considered a useful marker of iron deficiency independent of concurrent inflammation or infection. However, data on the impact of malaria on this parameter are ambiguous. We have examined potential associations of asymptomatic and mild Plasmodium falciparum-infections and of several erythrocyte variants with sTfR values in South West Nigeria. DESIGN AND METHODS: In a cross-sectional study among 161 non-hospitalized children, sTfR concentrations and P. falciparum parasitemia were assessed. In addition, hemoglobin (Hb) and serum ferritin values, Hb-types, glucose-6-phosphate dehydrogenase (G6PD)deficiency and a-globin genotypes were determined and the effects of these factors on sTfR levels were analyzed by univariate and multivariate statistical methods. RESULTS: P. falciparum-infection was present in 77% of the children. Mean sTfR levels were higher in infected than in non-infected children (geometric mean, 3.68, 95% confidence interval [3.5-3.9] vs. 2.99 [2.7-3.3] mg/L; p = 0.0009). There was a significant trend for higher sTfR values with increasing parasite density. sTfR values decreased continuously with age. Hb-types, G6PD-, and a-globin genotypes did not correlate with sTfR levels. In the multivariate analysis, age, Hb and log ferritin values, and parasite density of P. falciparum were independently associated with log sTfR values. INTERPRETATION AND CONCLUSIONS: sTfR concentrations are increased in asymptomatic and mild P. falciparum-infections suggesting adequate bone marrow response in this condition. The diagnostic value of sTfR levels for iron deficiency may be impaired in areas where stable malaria occurs. (+info)
"In vivo" and "in vitro" demonstration of hemoglobin C crystals in non-splenectomized patients.
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We studied 12 Hb C carriers: 4 homozygotic Hb CC and 8 heterozygotic. We observed the presence of free crystals in the peripheral blood of the homozygotes but in none of the heterozygotes. However, after incubation with 3% NaCl we were able to detect crystals in the heterozygotes (Hb AC and Hb SC), and in the homozygotes (Hb CC). In patient 04 (P04) less crystals formation occurred due to inhibition of the process by the presence of elevated levels of Hb F (12.2%). All the homozygotic patients had a splenomegaly of 3 to 6 fingerbreadths. We believe that the spleen wears off with time, thus allowing the passage of crystals to the peripheral blood. This finding might be associated with splenic insufficiency without a reduction of its dimensions. Finally, the finding of crystals in the peripheral blood permitted the diagnosis of Hb C obviating the need for electrophoresis. (+info)
Hemoglobin C associated with protection from severe malaria in the Dogon of Mali, a West African population with a low prevalence of hemoglobin S.
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The malaria hypothesis proposes a survival advantage for individuals with hemoglobin variants in areas of endemic Plasmodium falciparum malaria. Hemoglobin C (HbC) is a possible example in West Africa, where this hemoglobin has a centric distribution with high frequencies among certain populations including the Dogon ethnic group. To test whether HbC is associated with protection from malaria, we performed a case-control study in the Dogon of Bandiagara, Mali. HbC was present in 68 of 391 (17.4%) of uncomplicated malaria control cases, whereas it was detected in only 3 of 67 cases (4.5%) of severe malaria (odds ratio [OR], 0.22; P =. 01). Further, HbC was present in only 1 of 34 cases (2.9%) with cerebral manifestations, the most common presentation of severe malaria in this population (OR, 0.14; P =.03). Episodes of uncomplicated malaria and parasitemias (4800-205 050/microL) were identified in cases of homozygous HbC (HbCC), which indicates that P falciparum parasites are able to efficiently replicate within HbCC erythrocytes in vivo. These findings suggest that HbC does not protect against infection or uncomplicated malaria but can protect against severe malaria in the Dogon population of Bandiagara, Mali. The data also suggest that the protective effect associated with HbC may be greater than that of HbS in this population. (+info)
Comparison of electrophoresis on citrate agar, cellulose acetate, or starch for hemoglobin identification.
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We compare and discuss three electrophoretic methods for identifying hemoglobins S, A, C, F, and D or G. Electrophoresis on citrate agar gel was more sensitive than electrophoresis on cellulose acetate for detecting hemoglobins S and F, a fundamental consideration in designing cord-blood screening programs for detecting hemoglobin S carriers. Electrophoresis on starch gel is evidently an acceptable method for subtyping hemoglobins AA, CC, AS, SS, AC, and SC, and is more sensitive than cellulose acetate for identifying hemoglobin A1A2. Costs for the citrate agar gel, cellulose acetate, and starch gel procedures are presented. (+info)
The presence of hemoglobin S and C Harlem in an individual in the United States.
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The first reported case of hemoglobin S and C Harlem in an individual is described. The patient, a 35-yr-old female, had numerous crises during adolescence and early adulthood, but these occurred more infrequently as she grew older. Chemical evidence is presented for the characterization of both variant hemoglobins. The clinical course of this individual with Hb S in combination with Hb C Harlem appears to be similar to that for persons with sickle cell anemia. (+info)
Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis.
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Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important. (+info)
Intermolecular interactions, nucleation, and thermodynamics of crystallization of hemoglobin C.
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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. (+info)