t-BOOH-induced oxidative damage in sickle red blood cells and the role of flavonoids.

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.

Functional behavior of tortoise hemoglobin Geochelone denticulata

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

Functional behavior of tortoise hemoglobin Geochelone denticulata.

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.

Hemoglobin-sulfhydryls from tortoise (Geochelone carbonaria) can reduce oxidative damage induced by organic hydroperoxide in erythrocyte membrane.

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.

Sulphydryl groups and their relation to the antioxidant enzymes of chelonian red blood cells.

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.

Involvement of available SH groups in the heterogeneity of hemoglobin from the tortoise Geochelone carbonaria.

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.

cDNA-derived amino-acid sequence of a land turtle (Geochelone carbonaria) beta-chain hemoglobin.

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.

Effect of thiol reagents on functional properties and heme oxidation in the hemoglobin of Geochelone carbonaria.

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.

Oxygenation properties of hemoglobin from the turtle Geochelone carbonaria

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.

Oxygenation properties of hemoglobin from the turtle Geochelone carbonaria.

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.

Bantu beta s cluster haplotype predominates among Brazilian blacks.

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.

Polyphosphate binding sites in Liophis miliaris hemoglobin. Evidence with reduced nicotinamide adenine dinucleotide phosphate.

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.

Dimer-tetramer transition in hemoglobins from Liophis miliaris--I. Effect of organic polyphosphates.

Hemoglobin from the water-snake Liophis miliaris in the stripped form presents high oxygen affinity of about P50 = 1 mmHg and Hill coefficient of about 1.0 at pH from 6.8 to 8.5. In the presence of ATP such values become P50 = 20 mmHg and nH about 2.0, respectively, at low pH from 6.5 to 7.5. When the pH increases an abrupt decrease of both P50 and nH values occurs falling close to those found for the stripped hemoglobin. Gel-filtration in Sephadex G-100 equilibrated with 0.05 M Tris-HCl buffer containing 1 mM EDTA of the stripped hemoglobin show the presence of only one component of mol. wt of about 32,500 dt similar to the dimer of human hemoglobin A. The deoxy form of the dimer previously treated with ATP and placed on Sephadex column in the same condition but containing 1 mM IHP emerges as tetramer with mol. wt similar to that found for human hemoglobin, i.e. of about 65,000 dt. Results of the multiplicity of the snake hemoglobin, as well as the large alkaline Bohr effect in the presence of ATP previously reported, seems to be inconsistent due to the dimer-tetramer transition that occurs when ATP is bound to the stripped hemoglobin. A molecular mechanism involving the dimer-tetramer transition is proposed to described the oxygen transport in these animals.

Fluorescence studies on the binding of reduced nicotinamide adenine dinucleotide phosphate to human hemoglobin A and its variant hemoglobin Providence.

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.

Further evidence of dimer-tetramer transition in hemoglobin from Liophis miliaris.

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.

Fluorescence studies on the binding of reduced nicotinamide adenine dinucleotide phosphate to human hemoglobin A and its variant hemoglobin providence

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

Further evidence of dimer-tetramer transition in hemoglobin from Liophis miliaris

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

Haemoglobin Bohr effect and lactic acid content of the blood of 2 water-snakes with different degrees of aquatic adaptation.

H. modestus, a water-snake with morphological respiratory adaptation to its habitat, presents haemoglobins with a lower Bohr effect than those of L. miliaris, an aquatic snake without such respiratory adaptations. The difference in blood lactic acid content of the 2 snakes submitted to mechanical stimuli appears to be compatible with the properties of their haemoglobins.