Hemoglobin Components Research Articles

Hemoglobin components from trout ( Salmo irideus): determination of their peroxidative activity

The peroxidative activity of trout hemoglobins, HbI and HbIV, which differ in their conformation, was compared with that of HbA. Artificial substrates (guaiacol and dopamine) and more physiological substrates such as model lipid membranes containing unsaturated fatty acids were used. The results indicate that all the hemoglobin molecules assayed show different levels of peroxidative activity. The capability to act as peroxidases is greater in HbIV than in HbI and HbA. In contrast, native globins did not show peroxidase activity. The different peroxidative activity of the Hbs is discussed in relation to stability both vs. protein oxidation and protein dissociation. The results confirm the view that hemoglobin may be of importance in establishing the life span of the erythrocyte itself.

Amino acid sequence of Kalinowaski's Tinamou (Nothoprocta kalinowskii) hemoglobin and the rate of evolution of bird alphaD-globin.

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou alphaD-, alphaA-, and beta-globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the alphaD-globin chain. This has not been reported in the literature, except in pigeon embryonic alphaD-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, alphaD = alphaA > beta, was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.

Changes in the G Gamma- and A Gamma-Globin mRNA Components of Fetal Hemoglobin during Human Development

To determine the postnatal developmental changes in ^Gγ- and ^Aγ-globin mRNAs of HbF, blood samples were obtained from 28 pre-term infants born at ≤ 33 weeks of gestation (27.9 ± 2.5), 9 term-born infants and 26 adults. Adult samples were used to determine the relationship between the levels ^Gγ- and ^Aγ-mRNAs of HbF and the–158 (C→T) variation in the promoter region of the ^Gγ-globin gene. The results showed that ^Gγ-globin mRNA to total γ-globin mRNAs remained around 66% (66.2 ± 4) until the 44 week of postconceptual age when a change in the ^Gγ- and ^Aγ-globin mRNA proportions occurred. The immature red cells of adults had a range of ^Gγ-globin mRNA to total γ-globin mRNAs varying from 20 to 74% (53.1 ± 16.9). The high levels of ^Gγ-globin mRNA to total γ-globin mRNAs are associated with the presence of a –158 (C→T) polymorphism. The gene frequency of polymorphism was 0.32.

Hagfish Hemoglobins: STRUCTURE, FUNCTION, AND OXYGEN-LINKED ASSOCIATION

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528).

The influence of two concentrations of sodium hypochlorite on human blood: changes in haemolysis, pH and protein.

To evaluate the in vitro effect of 2.5% and 5.0% sodium hypochlorite (NaOCl) on human blood. Each concentration of NaOCl was reacted with human blood for 5 min at volume ratios of 1 : 1, 1 : 6, 1 : 12, each creating changes in colour, pH and temperature. Reaction suspensions were separated by centrifugation, and absorption measurements made for separated bilirubin, Fe, and protein supernatants. Each supernatant was desalted, lyophilized, and treated by SDS polyacrylamide gel electrophoresis (SOS-PAGE). Increased ratios and concentrations of NaOCl caused an increase in both pH and temperature. Protein supernatants tended to decompose on SDS-PAGE. Supernatants showed increased decolourisation with 5.0% NaOCl. Concentrations of bilirubin, Ferrum and protein in supernatants decreased with increased NaOCl concentration. NaOCl had an effect on the protein component in blood. These data suggest that changes in molecular structure are due to the chemical effects of NaOCl. Protein bands tended to show low molecular weight, suggesting that haemoglobin components effect the oxidation-reduction reaction.

Different functional modulation by heterotropic ligands (2,3-diphosphoglycerate and chlorides) of the two haemoglobins from fallow-deer (Dama dama).

Two haemoglobin components have been identified and purified from fallow-deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same alpha chain, while two different beta chains are detected in the two components. The beta chains differ by 14 residues, even though they both have 145 amino-acid residues, which account for a molecular mass of 16,023 and 16,064 Da, respectively, while alpha chain has 141 residues, yielding a molecular mass of 15,142 Da. Compared with human Hb, the N-terminal region of both beta chains shows deletion of Val beta 1 and the replacement of His beta 2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3-diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between beta chains of the two haemoglobin components.

Detection of haemoglobin variants and inference of their functional properties using complete oxygen dissociation curve measurements.

Complete oxygen dissociation curves for red cell suspensions of three haemoglobinopathies, namely haemoglobin (Hb) H, Hb Köln and Hb Tak/beta thalassaemia diseases, were measured using automatic recording methods. These curves were left-shifted compared with the normal red cell curve and showed a biphasic shape as a result of co-existence of the high and normal affinity haemoglobin components. Computer-assisted simulation of these biphasic curves enabled us to infer the curves for the pure abnormal haemoglobins and their fraction in the total haemoglobin of the red cell. The inferred values of fraction agreed with those determined by haemoglobin type analysis or the literature values. The curve for Hb Köln red cells deviated from the normal red cell curve in the whole range of oxygen saturation, whereas the curve for Hb H was close to the normal curve at the middle and upper portions. This difference in deviation was ascribed to a possible interaction between Hb Köln and Hb A through subunit exchange, and its absence between Hb H and Hb A. The present results indicate that measurement of the complete oxygen dissociation curve is important for the detection of non-interacting variants such as Hb H and is useful for inferring the functional properties of haemoglobin components that are not easily isolated.

Functional and structural properties of the hemoglobin components from Italian sturgeon ( Acipenser naccarii )

The structural and oxygen binding properties of Acipenser naccarii blood have been investigated. The electrophoretic analysis of the hemolysate of this sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. Constituent globin chains were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography. N-terminal amino acid sequence analysis revealed the presence of a N-terminal proline in two of the three α-chains present in the globin pattern, and the presence of a histidine residue in 2βposition. Oxygen equilibria reveal a very low sensitivity of the individual hemoglobins to chloride ions and temperature; however, in the presence of organic phosphates the oxygen affinity of the hemoglobin components decreases strongly. In particular, when Guanosine-5-triphosphate (GTP) is added, the reduction of the oxygen affinity, at pH 7.4 and 20 °C, is 60% and 50%, respectively, for HbI (anodic component) and HbII (cathodic component). As the effect of protons is concerned, the small Root effect shown by total hemolysate at physiological conditions, seems to be due mainly to the cathodic component. On the whole, the functional properties shown by sturgeon hemoglobin components seem to be related to the particular physiological needs dictated by the environmental characteristics.

Studies of the genetic relationship between deep-sea and oceanic Sebastes mentella in the Irminger Sea

A joint venture between Iceland and Norway to study the beaked redfish, Sebastes mentella, in the Irminger Sea was undertaken in 1995. The genetic relationship of deep-sea and oceanic S. mentella in the Irminger Sea and their relationship to redfish groups in adjacent waters were studied by gel electrophoresis and isoelectric focusing of haemoglobin and allozymes. The oceanic type of redfish in the Irminger Sea occurred as a homogenous group, but differed from the deep-sea type. This result was mainly based on frequency distributions of the enzyme loci MEP-1* and IDHP-2* and minor haemoglobin components. Heterogeneity was found within the deep-sea type, which also showed similarities with beaked redfish from the Icelandic shelf area. One sample of “untyped” S. mentella found below 600 m resembled the oceanic type in morphology and gene frequencies. Both types of S. mentella differed from S. mentella from Canada and Norway, but they could not be distinguished from immature S. mentella from Greenland as far as could be judged from previous studies.

Biochemical genetic markers to identify two morphologically similar South African Mastomys species (Rodentia: Muridae).

The two common southern African mice species (Mastomys coucha and M. natalensis) are morphologically almost identical, making field identification impossible at present. Specimens from two localities were collected and tissue and blood samples taken. The habitat type of each locality was studied, and a distribution map compiled. A definite correlation between biome-type and species range was found to be present. Three isozyme markers were identified: glucose phosphate isomerase in liver, and two general (non-specific) protein coding loci in muscle. In addition, we also identified species characteristic haemoglobin components in both species. This is the first study to report genetic variation within, and differentiation between these species. Our results are of medical importance because Mastomys coucha carries bubonic plague and M. natalensis carries Lassa Fever.

Amino acid sequences of hemoglobin from guinea fowl (Numida meleagri) and California quail (Lophortyx californica) with phylogenetic analysis of major groups of Galliformes.

We determined the complete amino acid sequences of the hemoglobin of two species, guinea fowl and California quail, in Galliformes from intact globin chain and chemical cleavage fragments in order to analyze the molecular evolution of hemoglobin for the classification of Galliformes. Galliformes have two types of hemoglobin components, HbA and HbD, which consist of identical beta chain and different alpha chains. The sequences are similar to globin chains of Galliformes reported previously. These sequences were compared with those of other Galliformes (Phasianidae, Meleagrididae) using duck and goshawk as out-groups. The phylogenetic tree of major groups of Galliformes based on hemoglobin was similar to the tree model produced based on the amino acid sequence of lysozyme c.

Sequential changes in glutamate transporter protein levels during Fe 3+-induced epileptogenesis

Severe head injury in humans causes recurrent seizures; this form of epilepsy appears to correlate with occurrence of parenchymal hemorrhage. Injection of ferric cations, one component of hemoglobin, into rat amygdala, causes lipid peroxidation, and recurrent spontaneous seizures. We wondered whether regulation of extracellular glutamate might be perturbed as a mechanism of chronic epileptogenesis, therefore levels of glutamate transporter proteins GLT-1, GLAST and EAAC-1 were measured in ipsilateral and contralateral hippocampi removed from rats having spontaneous iron-induced limbic seizures. The neuronal transporter EAAC-1 was elevated bilaterally up to 30 days following the microinjection that initiated seizures. The neuronal transporter EAAC-1 was elevated bilaterally up to 30 days following the microinjection that initiated seizures. The glial transporter GLT-1 increased 5 and 15 days after iron injection on the side contralateral to the injection then returned to basal levels 30 days after the lesion. GLAST also showed an initial increase but at 15 and 30 days after injection, when experimental animals were experiencing spontaneous limbic behavioral seizures, this protein was down-regulated. The results suggest that iron-induced epileptogenesis involves alteration in glial glutamate transport that may lead to enhanced excitation within the hippocampus.

Sequential changes in glutamate transporter mRNA levels during Fe 3+-induced epileptogenesis

Severe head injury in humans can cause recurrent seizures; this form of epilepsy appears to correlate with the occurrence of parenchymal hemorrhage. The injection of ferric cations, one component of hemoglobin, into rat amygdala, causes lipid peroxidation, and recurrent spontaneous seizures. We wondered whether the regulation of glutamate might be perturbed as a result of severe head injury, which might then act as a mechanism of chronic epileptogenesis. Levels of glutamate transporter glutamate-aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), and excitatory amino-acid carrier (EAAC-1) mRNA were measured in ipsilateral and contralateral hippocampi and cerebral cortex removed from rats at 60 min, 24 h, and 5, 15 and 30 days after FeCl 3 injection into the amygdaloid body. While the neuronal transporter EAAC-1 mRNA was elevated bilaterally for up to 30 days following the microinjection that initiated seizures, GLT-1 mRNA, derived from glial cells, returned to basal levels. At 15 and 30 days after injection, however, when the experimental animals were experiencing spontaneous limbic behavioral seizures, GLAST mRNA was down-regulated. Epileptogenesis may correlate with the impairment of glial glutamate transport, leading to an excitation and imbalance of transmitter influences within the hippocampi and cerebral cortex.

Purification and characterization of the hemoglobin components of Adriatic sturgeon (Acipenser naccarii) blood

The oxygen binding properties of Acipenser naccarii hemoglobins have been investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes and hemolysate in the presence of the physiological cofactors GTP and chloride ions. In particular, the oxygen equilibrium reveal a very low sensitivity of the hemolysate to the chloride ions and temperature while in the presence of organic phosphates their oxygen affinity decreased strongly. The electrophoretic analysis of the hemolysate of the sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. The partial amino acid sequence of the a and P chain of the single hemoglobins was also established.

Population genetic analysis of the haemoglobins of the black‐chinned tilapia

Three highly heterogeneous haemoglobin phenotypes, each composed of 22 different haemoglobin components, were identified among 17 West African populations of Sarotherodon melanotheron. Natural populations from (1) Senegal, (2) Ivory Coast/Ghana/Togo/Benin, and (3) Congo were distinguished. The heterogeneity and specificity of these respiratory pigments was based on genetic variations at the globin chain coding loci. In total, five different α‐chains and four different β‐chains were detected by acidic urea polyacrylamide gel electrophoresis (PAGE). Combinations of α‐chains were characteristic for populations in (1) Senegal, (2) Ivory Coast, (3) Ghana/Togo/Benin, and (4) Congo. Pronounced variations at the β‐globin chain cluster were found by acidic urea triton PAGE. Cladistic analyses of the globin chain characteristics confirmed the validity of the following taxonomic units previously ranked as sub‐species: (1) populations from Ivory Coast, Ghana, Togo and Benin belong to the sub‐species S. m. melanotheron; (2) populations from Senegal form genetically a separate cluster representing the sub‐species S. m. heudelotii; (3) the Congo population, morphologically considered to represent the sub‐species S. m. nigripinnis, forms another distinct unit; but there was no evidence of S. m. paludinosus within the samples from Senegal.

Haemoglobin components and oxygen transport in relation to habitat distribution in triplefin fishes (Tripterygiidae)

Haemoglobin components were analysed for nine species of New Zealand triplefins and their isoelectric points (pI) ranged from 5.1 to 7.0. The number of well-expressed isohaemoglobins was larger in shallow-water and tidal pool species, ranging from four in Grahamina signata to eight in Grahamina capito, and were relatively cathodal. Two strongly anodal isohaemoglobins were expressed in the mid-depth species Ruanoho decemdigitatus and Ruanoho whero, and one in the deeper water species Karalepis stewarti and Forsterygion malcolmi. The red blood cell oxygen-binding properties were determined at 15 °C and 25 °C in the pH range 6.7–7.9 for the shallow-water species G. capito, the shallow to mid-depth species Forsterygion varium, and the deep-water species F. malcolmi. Oxygen affinity was highest for G. capito and the magnitude of the Bohr effect lower (Δlog P50/ΔpH = −0.37 at 25 °C, where P50 is the half-saturation coefficient) compared to the two Forsterygion species (Δlog P50/ΔpH = −0.52 to −0.59). Further, the cooperativity factor, n50, was lower in G. capito thus maintaining oxygen transport over a wide range of environmental oxygen pressures. Oxygen binding was similarly influenced by temperature in both G. capito and F. malcolmi (maximum heat of oxygenation ΔHmax = −27 kJ mol−1 and −37 kJ mol−1, respectively). Thus, triplefin fishes living in shallow, thermally unstable habitats possess a greater number of cathodally migrating isohaemoglobins, and their red blood cells have a higher oxygen affinity and reduced cooperativity which is less sensitive to changes in pH than do species occurring in more stable, deeper water habitats. Our analysis of an assemblage of closely related species circumvents some of the difficulties inherent in studies where interpretation of experimental results is confounded by phylogeny.

Copper(II) Schiff-base complexes and apoglobin stability

N,N′-Propylene-bis-( N-salicylidene)copper(II) (Cu(Salprn)) explicitly stabilizes apomyoglobin. The optical spectrum of this copper(II) Schiff-base complex of apomyoglobin arises from the electronic excitations of π *- O-Salprn→d x 2-y 2 and N-Salprn→d x 2-y 2 . Shifts of these transitions with respect to those of the parent complex may be a consequence of hydrophobic solvatochromism or binding of an additional ligand. ESR parameters imply no change in the identity of the first coordination sphere around the copper, while hydrophobic solvatochromism cannot be excluded. Combination of copper(II) Schiff-base complex with apomyoglobin does not inhibit the ability of apomyoglobin to extract hemin from the main component of Glycera dibranchiata hemoglobin. Hemin replaces the copper complex, and the value of the apparent first-order rate constant varies with time. The mechanism involves dissociative and associative interchange pathways. Values of rate constants for transfer of hemin to copper(II) Schiff-base apomyoglobin complex, as well as the change of concentration with time are evaluated.

近縁ユスリカ 2 種, Polypedilum nubeculosum と P. nubifer の対応ヘモグロビン成分の N 末端アミノ酸配列の比較

近縁ユスリカ 2 種, Polypedilum nubeculosum と P. nubifer の対応ヘモグロビン成分の N 末端アミノ酸配列の比較

Fish haemoglobins: the order Clupeiformes

The haemoglobin systems of the order Clupeiformes have been studied by several researchers in 41 species belonging to three out of its five families. Most of them were investigated in the native form using electrophoretic methods, and a few were also examined from the functional point of view. Both approaches corroborate the widespread view that acidic and basic haemoglobin components, which are structurally and functionally distinct, may be present in teleost fish. However, the former are always present, whereas the latter are often lacking, depending on the taxonomic group. Both kinds of components are found in families Clupeidae and Pristigasteridae, but only acidic ones in Engraulidae.

An ecophysiological interpretation of hemoglobin multiplicity in three herbivorous marine teleost species from New Zealand

Hemoglobin components (isohemoglobins) and their isoelectric points (pI) were analysed for three herbivorous marine teleosts, and the functional properties of their unfractionated, stripped hemolysates were determined. Kyphosus sydneyanus and Girella tricuspidata possess six isohemoglobins and share a dominant cathodic band (pI=8.61). Odax pullus possesses four isohemoglobins characterised by two strongly anodic bands (pI=5.80, 4.89). Equilibrium binding studies of the hemolysate complexes from K. sydneyanus and G. tricuspidata revealed high oxygen affinities which were relatively insensitive to pH, whereas oxygen affinity was markedly lower and more pH sensitive in O. pullus. Moreover, hemoglobin-oxygen affinity was less temperature-sensitive in K. sydneyanus than in O. pullus. These observations support the hypothesis that hemoglobin oxygen loading in the cool-temperate species ( O. pullus) may be compromised in the most northerly (warm) limits of its distribution, and that ecologically equivalent species in warmer and thermally variable habitats ( K. sydneyanus and G. tricuspidata) possess more cathodic isohemoglobins which are less influenced by pH and temperature.