Major Hemoglobin Research Articles

Structural and functional characterization of a canine minor glycosylated hemoglobin, Hb Can AIc.

Structural and functional studies of minor glycosylated hemoglobins in non-human mammals have been very few. Therefore, we attempted further characterization of a canine glycosylated hemoglobin (tentatively designated as Hb Can AIc) which had been isolated by Bio-Rex 70 chromatography (Enoki, Y. et al. (1982) Hemoglobin 6, 143-151). After isolation of the constituent subunits by hybridization-mercuration technique, we found by peptide mapping that the structural modification was confined in amino-termini of the beta subunits in which glycosyl ketoamine linkage was also shown to be localized by thiobarbituric acid reaction. Compared with the major hemoglobin (Hb Can A0), Hb Can AIc was characterized by such functional properties as a slightly lower oxygen affinity and a markedly reduced response to the allosteric effects of carbon dioxide and organic phosphates. The anion and H+ Bohr effects were not different from those in Hb Can A0. All these facts support that this hemoglobin is a canine counterpart of Hb AIc.

The primary structure of sperm whale hemoglobin (Physeter catodon, cetacea).

The complete primary structure of the two major hemoglobin components of sperm whale (Physeter catodon) is presented. The major components A and B account for 55% and 40% respectively whereas the minor component constitutes for 5% of the total hemoglobin. The globin chains were separated on CM-Cellulose in 8M urea buffer. The sequence was determined by automatic Edman degradation of tryptic and hydrolytic peptides in a liquid phase sequencer. Alignment of the sequence with human hemoglobin shows 22 exchanges each for the alpha I and alpha II and 21 exchanges for the beta I and beta II chains. Within the two beta-chains three differences have been located, beta NA2 His/Gln, beta A2 Gly/Ala and beta A8 Leu/Val. The two alpha-chains are characterized by heterogeneities at position alpha A8 Val/Ile or Ala/Ile (ratio of the phenylthiohydantoin derivatives of the amino acids 1:1) and alpha AB1 Asn/Ser (ratio of the phenylthiohydantoin derivatives of the amino acids 6:4). The role of these exchanges in modulating oxygen affinity is discussed.

Genetic Variation of the Dimeric Haemoglobin of the Bivalve Mollusc Anadara trapezia

The bivalve mollusc A. trapezia has two haemoglobins, a tetrameric major haemoglobin, and a dimeric minor haemoglobin, the latter having two identical chains that are different from the chains in the tetramer. Genetic variation in the dimer results in two different haemoglobins, HbIIa and HbIIb, and it is known that the relative proportions of these two polymorphic forms vary with latitude along the eastern coastline of Australia. The HbIIb variant is more common at higher latitudes where water temperature may act as selecting agent. Comparative peptide mapping and amino acid analysis of peptides have shown that in HbIIa an aspartyl residue replaces the seryl residue found in HbIIb at residue 64, position E2 in the E helix. The E and F helices have recently been shown to be highly conserved in arcid globins and to be involved in subunit contacts in the cooperative dimers.

Heme orientation in the major monomeric hemoglobins of Glycera dibranchiata

Resonances of heme substituents and distal amino acid protons have been assigned for the three major and one of the minor monomeric hemoglobins from Glycera dibranchiata. The NMR spectra show that each hemoglobin has a unique heme environment. For each of the major hemoglobins, the vinyl group orientation differs from that depicted in the X-ray crystal structure (Padlan, E.A. and Love, W.E. (1974) J. Biol. Chem. 249, 4067–4078). In addition, the predominant heme orientation in solution is the reverse of that reported in the crystal structure. For two of the major Glycera hemoglobins, there is a significant degree of heme rotational disorder.

Hb Izu (Macaca)β83 (EF 7) Gly→Cys: The major hemoglobin of the Japanese monkey (Macaca fuscata) in a troop at Shimokita, the northernmost limit of its habitat

A hemoglobin variant was discovered in Japanese monkeys (Macaca fuscata) of the Wakinosawa A-1 troop of the Shimokita peninsula, the northernmost limit of this species' habitat. The variant was characterized by slow migration on starch gel electrophoresis due to its polymerizing nature. Structural analysis revealed that Gly at the 83rd amino acid residue of theβ chain was substituted by Cys, and the variant was identified as Hb Izu (Macaca). The gene frequency of the variant was extremely high, at 85.7%, and much higher than the 21.3% obtained for the troops of the Izu peninsula. The genotypic frequencies in the troops of both the Izu and Shimokita peninsulas agreed with expectations as calculated from the Hardy-Weinberg equilibrium.

Intrinsic fluorometric determination of the stable state of aggregation in hemoglobins

Front-face fluorometry can detect steady-state intrinsic fluorescence of hemoglobins (R. E. Hirsch, R. S. Zukin, and R. L. Nagel, 1980, Biochem. Biophys. Res. Commun., 93, 432–439), a property that can be used to study the dimerization of human hemoglobins (R. E. Hirsch, N. A. Squires, C. Discepola, and R. L. Nagel, 1983, Biochem. Biophys. Res. Commun., 116, 712–718). We report that the stable dimeric hemoglobin components of the arcid clams Noetia ponderosa and Anadara ovalis exhibit fluorescence emission maxima shifted to longer wavelengths compared to tetrameric human hemoglobin. Conversely, the tetrameric major hemoglobin (Hb) component of A. ovalis exhibits an emission maximum similar to that of tetrameric Hb A. Hence, stable dimeric hemoglobins can be detected by emission maxima at longer wavelengths relative to Hb A. Fluorescence studies of ligand binding to these clam hemoglobins indicate structural and functional differences among these components and compared to Hb A. We conclude that different stable aggregation states of hemoglobins may be determined by intrinsic fluorescence when studied with front-face optics.

Amino-acid sequences of the two major components of adult hemoglobins from the stump-tail monkey, Macaca speciosa.

The adult Stump-Tail Monkey (Macaca speciosa) was found to have two major hemoglobin components (Hb 1 and Hb 2) which were separated by carboxymethyl cellulose column chromatography. The tryptic peptides of the alpha and beta chains from the two components were isolated and sequenced. The peptides were aligned based on the homology of their sequences with that of human adult hemoglobin. Only one amino-acid difference was found between the alpha chains from Hb 1 and Hb 2 at the 15th position from the N-terminus. On the other hand, the beta chains from the two hemoglobin components were considered to be identical.

The amino terminal acetylation of cat globin chains and their tryptic peptides

Cat blood contains two major hemoglobins, HbA and HbB, both of which are synthesized within the same red cell. They contain identical α chains but different β chains. HbB is somewhat unusual in that it contains β chains that are modified by N α-acetylation. Although the selective amino terminal acetylation of the β chain of cat HbB occurs during nascent peptide chain growth, it can also occur after completion of globin chain synthesis and its assembly into hemoglobin. However, the latter process is not as efficient as the former, and it occurs non-enzymatically. Two peptides in a mixture of tryptic peptides from cat HbA and non-acetylated cat HbB contain serine at their amino terminus. One is the octapeptide, non-acetylated B-βT-1, and the other is the tetrapeptide, αT-2, derived from both HbA and HbB. We provide evidence here that in a mixture of soluble tryptic peptides, acetylation of αT-2 takes place in the presence of acetyl-CoA and ribosomal acetyltransferase at pH 7.0. The amino terminal acetylation requires the presence of an acetyltransferase.

Biosynthesis of cat hemoglobins: translation of poly(A)-RNA from animals of various HbA/HbB phenotypes.

The molecular basis for the genetic control of variable proportions of the two hemoglobins in domestic cat blood was investigated. Both major hemoglobins of cat blood, HbA (alpha 2 beta A2) and HbB (alpha 2 beta B2), were synthesized in an mRNA-dependent rabbit reticulocyte system using poly(A)-RNA from cat reticulocyte polysomes as the source of the message. The relative amounts of HbA and HbB synthesized in the system were a function of the HbA/HbB phenotype of the cat from which the reticulocytes and poly(A)-RNA were obtained. Higher ratios of HbA/HbB synthesis were found when the source of poly(A)-RNA was the polysomes from a 90/10 (HbA/HbB) phenotype than when it was from a 50/50 (HbA/HbB) phenotype. These results indicate that the variable proportions of HbA and HbB found in the blood of different members of the cat population result from the genetic control of the relative amounts of functional beta A and beta B mRNA.

A two-state thermodynamic and kinetic analysis of the allosteric functioning of the haemoglobin of an extreme poikilotherm.

The blood of the extreme poikilotherm Trematomus borchgrevinki contains one major haemoglobin component, which may be separated from the minor species by ion-exchange chromatography. This haemoglobin shows co-operative CO-binding isotherms at pH 8.2. An analysis of the temperature-dependence of the binding curves has allowed the thermodynamic constants associated with the two-state allosteric parameters L, KR and KT to be measured. The binding of CO at lower pH (6.2) is characterized by the maintenance of the T-state to relatively high degrees of saturation. Kinetic investigations with the use of flash photolysis of the haemoglobin-CO complex under various conditions has allowed the determination of the thermodynamic parameters association with the T-state and R-state rate constants. An amalgamation of these data has allowed the mathematical simulation of predicted time courses for CO binding to this haemoglobin, by using the two-state model, which very closely represents those obtained experimentally. The overall findings show that this haemoglobin closely follows the two-state model of co-operative interaction.

Amino-acid Sequence of β-chain of hemoglobin IV from trout ( Salmo irideus)

The complete amino-acid sequence of the β-chain of trout Hb IV, the hemoglobin component functionally characterized by the occurence of the Root effect, has been determined; the framentation strategy included cleavage of the carboxymethylated chain with trypsin and chymotrypsin. Comparison of the sequence with the corresponding chain from human hemoglobin shows a difference of 49.7%; differences of 27.2% and 21.8% are found with the β-chains of carp and goldfish hemoglobins, respectively, two hemoglobins functionally similar to component IV from trout, whereas a greater difference (40.8%) is found with the β-chain of trout Hb 1, the other major hemoglobin component of trout blood, devoid of heterotropic phenomena. Inspection of amino-acid substitutions, already shown to be extremely useful in providing structural explanations of specific functional properties of fish hemoglobins, suggests a simple consideration relevant to the molecular evolution of oxygen carriers.

Organic phosphates increase the solubility of avian haemoglobin D and embryonic chicken haemoglobin.

The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.

The amino-acid sequence of alpha A- and beta-chains from the major hemoglobin component of American flamingo (Phoenicopterus ruber ruber).

The complete amino-acid sequence of alpha A- and beta-chains from the major hemoglobin component (HbA) of American Flamingo ( Phoenicopterus ruber ruber) is presented. The minor component (HbD) with alpha D-chains was detected in similar amounts (25%) as in chicken and pheasant hemoglobins. The comparison of American Flamingo and Greylag Goose (Anser anser) hemoglobins shows that alpha A-chains differ by 22 exchanges and beta-chains by only 4 exchanges. Two substitutions modify alpha 1 beta 1-contacts. Amino-acid replacements between American Flamingo and other bird hemoglobins are discussed.

The Globin Genes of Rabbit and Man

The past 5 years have witnessed a revolution in our understanding of the structure and expression of eukaryotic genes. This has been made possible by the advent of recombinant DNA technology and other more specific techniques, such as Southern blotting (Southern, 1975) which made it possible to study single-copy mammalian genes directly in total DNA (Jeffreys & Flavell, 1977a). In about 1975 I switched my attention to the genes that encode the major blood protein haemoglobin; this choice was made for a number of reasons. First, much background information on the gene products was available. For example, at that time the sequence of the aand &globin proteins was available and most of the sequence of &globin mRNA had been determined. Secondly, the gene system exhibits many of the properties that intrigue us about the genes of higher organisms, that is, they are only expressed in certain tissues of the animal, they are only expressed at certain times during development and, at any given time, the expression of at least two genes (e.g. aand 8-globin) has to be co-ordinated. Finally, a specific point of great interest is the fact that genetic disorders of globin gene expression are common in the human population-these provide both interesting models for genetic diseases and the hope of applying molecular biology to clinical medicine.

Interference of fetal hemoglobin and labile glycosylated hemoglobin with measurements of glycosylated hemoglobin.

We examined the effect of fetal hemoglobin and labile glycosylated hemoglobin on a number of diverse methods used to measure glycosylated hemoglobin. Samples were supplemented with various amounts of cord blood to give proportions of fetal hemoglobin ranging from 1 to 20% of total hemoglobin concentration. Procedures in which the separation of hemoglobin A1 from the major hemoglobin A fraction is based on differences in ionic properties (cation-exchange chromatography and electrophoresis) are subject to interference by fetal hemoglobin, whereas procedures that base the quantitation on other properties (colorimetry and affinity column chromatography) are not. The same procedures that are affected by the presence of fetal hemoglobin are also subject to interference by labile glycosylated hemoglobin. We conclude that the affinity chromatographic and colorimetric methods may give a more nearly accurate determination of glycosylated hemoglobin.

The amino-acid sequence of Northern Mallard (Anas platyrhynchos platyrhynchos) hemoglobin.

The complete amino-acid sequence of the major hemoglobin component (HbA) of the adult Northern Mallard (Anas platyrhynchos platyrhynchos) is presented. A minor component HbD was also detected but in low concentrations. The sequences of alpha A- and beta-chains were established by automatic Edman degradation on tryptic peptides and peptides obtained by specific chemical cleavages. The alignment of the peptides was performed by comparison with the alpha A- and beta-chains of Greylag Goose hemoglobin (Anser anser). Thereby an exchange of five positions in the alpha A-chains and three in the beta-chains was observed. No exchanges were found in the surroundings of the heme, in alpha 1 beta 2-contact points, or allosteric regulatory sites. In the alpha 1 beta 1-subunit interface one amino-acid residue in alpha A-chains and one in beta-chains are exchanged. By comparison with the amino-acid sequence derived from globin genes of Domestic Duck (Anas platyrhynchos), the alpha A-chains differ by two exchanges in the N-terminal region and the beta-chains by five exchanges the in C-terminal region. The comparison of the amino-acid sequence derived from alpha A-globin gene of the Moscovy Duck (Cairina moschata) and alpha A-chains of the Northern Mallard, shows only one replacement.

Primary structure of hemoglobin from trout ( salmo irideus) : Amino acid sequence of the β chain of trout Hb I

The amino acid sequence of the β chain of trout lib I is presented; it adds to the previously reported sequence of the α chain (Bossa et al. (1978) Biochim. Biophys. Acta 536, 298–305), thus completing the primary structure of the hemoglobin component of trout's blood devoid of heterotropic phenomena. Comparison of β chain from trout Hb I with the corresponding sequences from human and carp shows differences of 46.6% and 34.7%, respectively; the sequence (almost completed) of the β chain from the other major hemoglobin component of trout, i.e., trout Hb IV, displays more differences (41.6%) from β trout Hb I than from the corresponding chain of other fishes, such as carp or goldfish.

Structural comparison of the haemoglobin components of the armoured catfish Pterygoplichthys pardalis. Evolutionary considerations.

Amino acid analyses and peptide mapping were performed for the four main haemoglobins from the armoured catfish Pterygoplichthys pardalis; component I, which is functionally distinct from the others, is structurally unique, whereas components II, III and IV, functionally indistinguishable, are closely related in structure. Compositional difference indices are calculated for the four components and for the two major haemoglobins from the trout Salmo irideus, and the results are discussed in terms of structural relationships and evolutionary history of fish haemoglobins.

Synthesis of the minor fetal hemoglobin Fic in colonies of erythropoietic precursors isolated from human umbilical cord blood.

The biosynthesis of the minor hemoglobin FIc, which contains acetylated gamma chains, and the major hemoglobin Fo was studied during erythroid cell differentiation and maturation in cultures of erythroid precursors isolated from five human umbilical cord blood samples. A gradual decrease in the synthesis of Hb FIc was observed during the maturation of the erythroid cells when cultured from 7 to 15 days. The synthesis of Hb Fo did not show a consistent change; however, the relative synthesis of Hb FIc was the highest in the 7-day-old colonies and decreased at day 15 to levels observed in the reticulocytes. The addition of sodium butyrate, which is known to promote histone acetylation, significantly increased the synthesis of Hb FIc in 7-day-old colonies.

Characterization and Quantitation of Glycosylated Hemoglobins in Canine Blood

Canine hemolysates exhibited two minor and one major hemoglobins when chromatographed on Bio Rex-70 column with 0.05 M phosphate-0.01 M KCN (pH 6.5) containing 0.015 M NaCl. The first minor fraction was electrophoretically heterogeneous, constituting at least two hemoglobins. The second was completely homogeneous with somewhat faster anodal mobility than the major one and the difference was shown to be localized solely in the beta subunits. Thiobarbituric acid reaction was strongly positive for this hemoglobin, indicating the presence of glycosyl ketoamine linkage. We describe a simple and reliable procedure for quantitation of the canine glycosylated hemoglobins with a home-made Bio Rex-70 mini-column.