RESUMO

Sickle cell anemia is a genetic disease characterized byan increase in generation of reactive oxygen species, abnormal iron release and low antioxidant activity which can lead to cell injury. Several therapies have been used to decrease the oxidative damage in these patients. In this study, we investigated the effect of flavonoids (quercetin and rutin) on the oxidation of red blood cells (RBC) from sickle cell anemia patients following exposure of the cells to tert-butyl hydroperoxide (t-BOOH). Quercetin provided greater protection against Hb oxidation, the binding of Hb to membrane and lipid peroxidation than did rutin. Quercetin (150 microM) reduced Hb oxidation by 30% and increased the level of oxyHb from 17.5 to 29 microM. Rutin prevented Hb oxidation only at concentrations higher than 200 microM and did not prevent the binding of Hb to RBC membrane. These distinct effects of the flavonoids probably reflect their structural characteristics. Thus, quercetin, which possesses a suitable structure for free-radical scavenging and ion quelation, was a more effective antioxidant than rutin. The presence of rutinose at position C(3) in rutin may impair its antioxidant effect. The presence of ascorbic acid enhanced the protective effect of quercetin and rutin against oxidative stress in sickle Hb and lipid peroxidation. This synergistic action helped to maintain a constant supply of flavonoids and thus, rescue the cells from the injury caused by free radicals and iron ions.

Assuntos

RESUMO

The hemolysate from Geochelone denticulata contains two main hemoglobin components, as shown by ion exchange chromatography and polyacrylamide gel electrophoresis (PAGE). Electrophoresis under dissociating conditions showed three types of globin chains. The apparent molecular mass, as determined by gel filtration on Sephadex G-200, was compatible with tetrameric Hb, which was unable to polymerize. The G. denticulata Hb has a P50 value of 9.56 mm Hg at pH 7.4. The Hb oxygenation appears to be under the control of organic phosphates and hydrogen ion since it is strongly affected by those species. In the presence ATP or IHP the P50 values increased to 29.51 mm Hg and 54.95 mm Hg, respectively, at pH 7.4. The n50 was generally lower than 1.5 in stripped Hb, suggesting a dissociation of tetramers. In the presence of organic phosphates n50 values increased to approximately 2.5. The Bohr effect was evident in oxygen equilibrium experiments. The hematocrit (32 percent) and Hb concentration (5.7 mM as heme) of G. denticulata blood were substantially larger than those of G. carbonaria, but the methemoglobin levels were similar in both species, approximately 1 percent. Thus, the oxygen capacity of blood appears to be higher in G. denticulata than in G. carbonaria, particularly considering the functional properties of their Hbs, which would guarantee the survival of animals

Assuntos

RESUMO

The hemolysate from Geochelone denticulata contains two main hemoglobin components, as shown by ion exchange chromatography and polyacrylamide gel electrophoresis (PAGE). Electrophoresis under dissociating conditions showed three types of globin chains. The apparent molecular mass, as determined by gel filtration on Sephadex G-200, was compatible with tetrameric Hb, which was unable to polymerize. The G. denticulata Hb has a P50 value of 9.56 mm Hg at pH 7.4. The Hb oxygenation appears to be under the control of organic phosphates and hydrogen ion since it is strongly affected by those species. In the presence ATP or IHP the P50 values increased to 29.51 mm Hg and 54.95 mm Hg, respectively, at pH 7.4. The n50 was generally lower than 1.5 in stripped Hb, suggesting a dissociation of tetramers. In the presence of organic phosphates n50 values increased to approximately 2.5. The Bohr effect was evident in oxygen equilibrium experiments. The hematocrit (32%) and Hb concentration (5.7 mM as heme) of G. denticulata blood were substantially larger than those of G. carbonaria, but the methemoglobin levels were similar in both species, approximately 1%. Thus, the oxygen capacity of blood appears to be higher in G. denticulata than in G. carbonaria, particularly considering the functional properties of their Hbs, which would guarantee the survival of animals.

Assuntos

RESUMO

The hemolysate from Geochelone denticulata contains two main hemoglobin components, as shown by ion exchange chromatography and polyacrylamide gel electrophoresis (PAGE). Electrophoresis under dissociating conditions showed three types of globin chains. The apparent molecular mass, as determined by gel filtration on Sephadex G-200, was compatible with tetrameric Hb, which was unable to polymerize. The G. denticulata Hb has a P50 value of 9.56 mm Hg at pH 7.4. The Hb oxygenation appears to be under the control of organic phosphates and hydrogen ion since it is strongly affected by those species. In the presence ATP or IHP the P50 values increased to 29.51 mm Hg and 54.95 mm Hg, respectively, at pH 7.4. The n50 was generally lower than 1.5 in stripped Hb, suggesting a dissociation of tetramers. In the presence of organic phosphates n50 values increased to approximately 2.5. The Bohr effect was evident in oxygen equilibrium experiments. The hematocrit (32%) and Hb concentration (5.7 mM as heme) of G. denticulata blood were substantially larger than those of G. carbonaria, but the methemoglobin levels were similar in both species, approximately 1%. Thus, the oxygen capacity of blood appears to be higher in G. denticulata than in G. carbonaria, particularly considering the functional properties of their Hbs, which would guarantee the survival of animals.

O hemolisado de Geochelone denticulata contém dois componentes principais, de acordo com a cromatografia de troca iônica e PAGE. Eletroforese sob condições dissociantes mostrou 3 tipos de cadeias de globina. A massa molecular aparente, determinada pela filtração em gel sobre Sephadex G-200, foi compatível com Hb tetramérica que foi incapaz de polimerizar. A Hb de G. denticulata tem valor de P50 de 9,56 mm Hg em pH 7,4. A oxigenação da Hb parece estar sob controle de fosfatos orgânicos e íons hidrogênio, uma vez que ela é fortemente afetada por essas espécies. Na presença de ATP ou IHP, os valores de P50 aumentaram para 29,51 mm Hg e 54,95 mm Hg, respectivamente, a pH 7,4. O n50 foi geralmente menor que 1,5 na Hb stripped, sugerindo dissociação de tetrâmeros. Na presença de fosfatos orgânicos, os valores de n50 aumentaram para aproximadamente 2,5. O efeito Bohr foi evidente nos experimentos de equilíbrio com oxigênio. O hematócrito de 32% e a concentração de Hb de 5,7 mM em heme no sangue de G. denticulata foram substancialmente maiores do que da G. carbonaria, mas os níveis de metahemoglobina foram similares em ambas as espécies, aproximadamente 1%. Portanto, a capacidade de oxigenação do sangue parece ser maior na G. denticulata, particularmente considerando as propriedades funcionais da Hb, que garantiria a sobrevivência dos animais.

RESUMO

The hemolysate from Geochelone denticulata contains two main hemoglobin components, as shown by ion exchange chromatography and polyacrylamide gel electrophoresis (PAGE). Electrophoresis under dissociating conditions showed three types of globin chains. The apparent molecular mass, as determined by gel filtration on Sephadex G-200, was compatible with tetrameric Hb, which was unable to polymerize. The G. denticulata Hb has a P50 value of 9.56 mm Hg at pH 7.4. The Hb oxygenation appears to be under the control of organic phosphates and hydrogen ion since it is strongly affected by those species. In the presence ATP or IHP the P50 values increased to 29.51 mm Hg and 54.95 mm Hg, respectively, at pH 7.4. The n50 was generally lower than 1.5 in stripped Hb, suggesting a dissociation of tetramers. In the presence of organic phosphates n50 values increased to approximately 2.5. The Bohr effect was evident in oxygen equilibrium experiments. The hematocrit (32%) and Hb concentration (5.7 mM as heme) of G. denticulata blood were substantially larger than those of G. carbonaria, but the methemoglobin levels were similar in both species, approximately 1%. Thus, the oxygen capacity of blood appears to be higher in G. denticulata than in G. carbonaria, particularly considering the functional properties of their Hbs, which would guarantee the survival of animals.

O hemolisado de Geochelone denticulata contém dois componentes principais, de acordo com a cromatografia de troca iônica e PAGE. Eletroforese sob condições dissociantes mostrou 3 tipos de cadeias de globina. A massa molecular aparente, determinada pela filtração em gel sobre Sephadex G-200, foi compatível com Hb tetramérica que foi incapaz de polimerizar. A Hb de G. denticulata tem valor de P50 de 9,56 mm Hg em pH 7,4. A oxigenação da Hb parece estar sob controle de fosfatos orgânicos e íons hidrogênio, uma vez que ela é fortemente afetada por essas espécies. Na presença de ATP ou IHP, os valores de P50 aumentaram para 29,51 mm Hg e 54,95 mm Hg, respectivamente, a pH 7,4. O n50 foi geralmente menor que 1,5 na Hb stripped, sugerindo dissociação de tetrâmeros. Na presença de fosfatos orgânicos, os valores de n50 aumentaram para aproximadamente 2,5. O efeito Bohr foi evidente nos experimentos de equilíbrio com oxigênio. O hematócrito de 32% e a concentração de Hb de 5,7 mM em heme no sangue de G. denticulata foram substancialmente maiores do que da G. carbonaria, mas os níveis de metahemoglobina foram similares em ambas as espécies, aproximadamente 1%. Portanto, a capacidade de oxigenação do sangue parece ser maior na G. denticulata, particularmente considerando as propriedades funcionais da Hb, que garantiria a sobrevivência dos animais.

RESUMO

Sulfhydryl groups are important to avoid oxidative damage to the cell. In RBC, tert-butyl hydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2) are capable of oxidizing heme and promoting lipid peroxidation. H2O2 caused greater oxidation of heme than tert-BOOH, although the oxidation of sulfhydryl groups was similar. Geochelone carbonaria Hb, a rich sulfhydryl protein, inhibited the TBA-reactive substances formation of human erythrocytes exposed to tert-BOOH by about 30%; this decrease was smaller with Geochelone denticulata Hb. Sulfhydryl reagents diminished the number of reactive sulfhydryl groups in the G. carbonaria Hb resulting in a decrease of its antioxidant power, suggesting the involvement of sulfhydryls of Hb in the protection against lipid peroxidation.

Assuntos

Assuntos

RESUMO

Thiol groups of hemoglobin and blood glutathione are higher in Geochelone carbonaria than in Geochelone denticulata. Exposure of stripped hemolysate of both tortoises to terc-butyl hydroperoxide, resulted in a higher ferroheme oxidation of G. denticulata hemoglobin. In this example glutathione reductase and glutathione peroxidase, were not active due to the absence of GSH and NADPH, suggesting that the thiol groups of G. carbonaria hemoglobin act as antioxidant, similar to GSH. In the total hemolysate, however, where the antioxidant enzymes are active, both species showed similar levels of hemoglobin oxidation, suggesting that the protective effect of thiol groups of hemoglobin are less effective for heme protection. The activity of glutathione reductase and glutathione peroxidase was higher in erythrocytes of G. denticulata and the activity of catalase and superoxide dismutase was higher in erythrocytes of G. carbonaria.

Assuntos

RESUMO

Geochelone carbonaria hemoglobin (Hb) was analyzed by polyacrylamide gel electrophoresis (PAGE) and purified by ion exchange chromatography on CM-cellulose. Seven fractions were obtained using fresh Hb preparations. CM-cellulose chromatography of Hb reacted with iodoacetamide, showed one minor (HbI) and one major band (HbII). Analysis of the molecular masses of recently collected Hb and of aged solutions determined by gel filtration showed that polymerization increased with the duration of storage. The reaction with oxidized glutathione changed the electrophoretic pattern of Hb, and highlighted the bands corresponding to glutathionyl-Hb. The presence of these bands in fresh Hb solutions and in alkylated preparations suggests that they may occur in vivo. PAGE under dissociating conditions showed that the hemolysate contained 3 different polypeptide chains (G1, G2 and G3). Both Hb components shared the G1 globin chain with HbI containing G1 and G2 and HbII, G1 and G3 chains.

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RESUMO

The cDNA sequence encoding the turtle Geochelone carbonaria beta-chain was determinated. The isolation of hemoglobin mRNA was based on degenerate primers' PCR in combination with 5'- and 3'-RACE protocol. The full length cDNA is 615 bp with the ATG start codon at position 53 and TGA stop codon at position 495; The AATAAA polyadenylation signal is found at position 599. The deduced polypeptyde contains 146 amino-acid residues. The predicted amino acid sequence shares 83% identity with the beta-globin of a related specie, the aquatic turtle C. p. belli. Otherwise, identity is higher when compared with chicken beta-Hb (80%) than with other reptilian orders (Squamata, 69%, and Crocodilia, 61%). Compared with human HbA, there is 67% identity, and at least three amino acid substitutions could be of some functional significance (Glu43 beta-->Ser, His116 beta-->Thr and His143 beta-->Leu). To our knowledge this represents the first cDNA sequence of a reptile globin gene described.

Assuntos

RESUMO

The reaction of thiol reagents with G. carbonaria hemoglobin was studied, and the oxygen equilibrium and kinetic of oxidation of derivatives determined. The oxygen affinity and kinetic of oxidation of hemoglobin derivatives were modified to various extents depending on the nature of thiol reagents used. Diamide yielded approximately 80% polymeric hemoglobin, although the oxidation kinetic, and the functional properties, were practically invariant (T1/2 = 10.0 min.; P50 = 5.0 mm Hg at pH 7.4; alkaline Bohr effect = -0.64). Iodoacetamide did not modify the electrophoretic pattern significantly, although all the free SH groups of hemoglobin were alkylated. A P50 of 2.5 mmHg at pH 7.4 and the Bohr effect of -0.15 were obtained; the T1/2 of about 6.4 min. was shorter than that for un-modified Hb. Similar T1/2 were obtained for Hb treated with oxidized glutathione, which produced polymeric Hb and glutathionyl-Hb. The oxygen binding characteristics showed that both of Hb derivatives, glutathionyl-Hb and polymeric Hb, maintain the capacity to transport the gas.

Assuntos

RESUMO

The oxygen-binding properties of hemoglobin (Hb) from the adult terrestrial turtle Geochelone carbonaria are described. Turtle hemoglobins have a low intrinsic oxygen affinity and a low sensitivity to an endogenous cofactor (ATP) usually present at high concentrations in the reptile erythrocytes. The amplitude of the Bohr effect for O2 binding was virtually the same in the absence and presence of saturating ATP concentrations (deltalogP50/deltapH, about -0.60) and increased in the total hemolysate (-0.83). The large Bohr effect found in G. carbonaria Hb may be important for 02 delivery to the tissue. The degree of cooperativity displayed by Hb for 02 binding ranged between 1.5 and 2.0 in stripped solution and total hemolysate. These observations suggest the stability of the low affinity conformation, which needs to be confirmed by additional experiments.

Assuntos

RESUMO

The oxygen-binding properties of hemoglobin (Hb) from the adult terrestrial turtle Geochelone carbonaria are described. Turtle hemoglobins have a low intrinsic oxygen affinity and a low sensitivity to an endogenous cofactor (ATP) usually present at high concentrations in the reptile erythrocytes. The amplitude of the Bohr effect for O2 binding was virtually the same in the absence and presence of saturating ATP concentrations (delta logP50/delta pH, about -0.60) and increased in the total hemolysate (-0.83). The large Bohr effect found in G. carbonaria Hb may be important for O2 delivery to the tissue. The degree of cooperativity displayed by Hb for O2 binding ranged between 1.5 and 2.0 in stripped solution and total hemolysate. These observations suggest that stability of the low affinity conformation, which needs to be confirmed by additional experiments.

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RESUMO

We describe the combination of polymorphic restriction-enzyme sites in the beta globin gene cluster (haplotypes) for 74 chromosomes from Brazilian Blacks bearing the sickle hemoglobin gene (beta s). The three most common African beta s haplotypes account for 67 chromosomes: 49/74 (66.2%) were identified as Central African Republic (CAR or Bantu) type, 17 (23.0%) as Benin, and one as Senegal; seven chromosomes (9.5%) had minor atypical haplotypes. This distribution is different from that observed in the United States or Jamaica, where the Benin haplotype predominates, and results from different patterns of slave trades to North and South Americas. Since the beta s gene cluster polymorphisms modulate the severity of sickle cell anemia, this heterogeneity may explain differences of the clinical behavior of the disease in the United States and South America, and should also be considered in relation to other features and diseases.

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RESUMO

The water-snake Liophis miliaris presents hemoglobin which binds organic polyphosphate through a simple single-site per tetramer (Mol. Wt. 64500) as judged by titration curves of reduced nicotinamide adenine dinucleotide phosphate either in the presence or absence of inositol hexaphosphate. The site seems to have the same structural nature of that found on other hemoglobins and is able to strongly bind most of the known protein effectors such as inositol hexaphosphate, adenosine triphosphate or 2,3-diphosphoglicerate. The high association constant at pH 7 of reduced nicotinamide for the deoxy hemoglobin of about K(D) = 7 x 10(6) M-1 compared to human hemoglobin (K(D) = 7 x 10(5) M-1), and to that of adenosine triphosphate (its natural erythrocytic polyphosphate) still higher of about K(D) = 10(11) M-1, shows clearly the very high affinity of this snake hemoglobin for such allosteric effector. The results besides corroborating the dimer-tetramer transition mechanism proposed to describe the oxygen transport by the hemoglobin of Liophis miliaris--may explain the difficulties to obtain the oxy dimeric conformation of the protein by usual hemolysis and stripped off procedures.

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RESUMO

Spectrofluorometric techniques were used to quantify NADPH-hemoglobin interactions based on the quenching of NADPH fluorescence upon binding to hemoglobin. Fluorometric titrations were carried out with hemoglobin in varied states and with hemoglobins in which the beta-chain anion site is altered. At pH 6.5 in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, NADPH binds with high affinity, Kd = 1.03 microM, to deoxy human hemoglobin tetramers. Lower affinity binding of NADPH occurs as the beta-chain anion-binding site is discharged by increasing the pH. Moreover, the cofactor binds in a 1:1 ratio to deoxy tetramers, inositol hexaphosphate binds competitively, and binding is decreased in hemoglobins whose structural alterations result in decreased effects of 2,3-diphosphoglycerate. The cofactor binds to oxidized (met) hemoglobin with an estimated Kd of 33.3 microM but has little or no affinity for the oxy form. These results indicate that NADPH binds at the beta-chain anion-binding site and can be considered as a fluorescent analog of 2,3-diphosphoglycerate. Fluorescence measurements gave no indication of NADPH binding to deoxygenated ferrous or ferric myoglobin. Reductive processes within the erythrocyte, such as reduction of met hemoglobin and hemoglobin-catalyzed enzymatic reactions, may be affected by the significant binding of the reduced cofactor to both deoxygenated and oxidized hemoglobin. Cofactor-hemoglobin interactions predict a shift in redox potential as red cells become oxygenated, which may account for unexplained oxygen-linked shifts in red cell metabolism.

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RESUMO

The affinity constants for the binding of NADPH to human hemoglobin A were directly determined by fluorescence analysis since nucleotide fluorescence is quenched on binding to the protein. The binding constants 6.1 x 10(5), 5.02 x 10(5) and 1.2 x 10(5) were found for deoxyhemoglobin at pH 6.5, 7.0 and 7.5, respectively. Oxyhemoglobin does not bind NADPH significantly. These results are consistent with those found in oxygen-hemoglobin equilibrium experiments. The human hemoglobin variant, Providence-Asp, which has a marked decrease in 2,3 DPG affinity was also investigated. NADPH does not bind to the variant suggesting that the Lys B 82 residue is of fundamental importance to nucleotide binding and showing that the binding site is the same as that of 2,3 DPG or other organic polyphosphate, allosteric modulators of hemoglobins. Experiments of inositol hexaphosphate (IHP)-NADPH site competition corroborate these results.

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RESUMO

Liophis miliaris hemoglobins in the stripped form exhibit a high oxygen affinity, a small alkaline Bohr effect, a pronounced organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The oxygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. beta-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alpha 1-beta 2 interface by valine in snake hemoglobin.

Assuntos

RESUMO

The affinity constants for the binding of NADPH to human hemoglobin A were directly determined by fluorescence analyssis since nucleotide fluorescence is quenched on binding to the protein. The binding constants 6.1 x 10**5, 5.02 x 10**5 and 1.2 x 10**5 were found for deosyhemoglobin at pH 6.5, 7.0,respectively. Oxyhemoglobin does not bind NADPH significantly. These results are consistent with those found in oxygen-hemoglobin equilibrium experiments. The human hemoglobin variant, Providence-Asp, which has a marked decrease in 2,3 DPG affinity was also investigated. NADPH does not bind to the variant suggesting that the Lys B 82 residues is of fundamental importance to nucleotide binding and showing that the binding site is the same as that of 2,3 DPG or other organic polyphosphate, aloosteric modulators of hemoglobins. Experiments of inositol hexaphosphate (IHP)-NADPH site competition corroborate these results

Assuntos

RESUMO

Liophis miliaris hemoglobins in the stripped form exhibit a high oxigen affinity, a small alkaline Bohr effect, a pronounce organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The osygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. ß-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alfa1-ß2 interface by valine in snake hemoglobin

Assuntos