Avian Hemoglobins Research Articles

Parakeet Hemoglobin - Its Crystal Structure and Oxygen Affinity in Relation to Some Avian Hemoglobins.

"Avians" often show efficient oxygen management to meet the demands of their metabolism. Hemoglobin, a transporter protein consists of four non-covalently linked subunits contain haem binding hydrophobic pocket serves as a site of allosteric cooperativity. The physiology and anatomy of both mammals and avian are functionally different, in birds, the respiratory system formed by small air sacs that serve as tidal ventilation for the lungs and have no significant exchange across their cells. Parakeet (Psittacula krameri) a tropical and non-migrating species and it is easily adapted to living in disturbed habitat. The sequence analysis reveals that α and β chain of parakeet hemoglobin highly similar grey lag goose and bar headed goose hemoglobin respectively. Thus it has been tempted us to study in to analyzing the sequence and structural comparison of this hemoglobin to find out the physiological capabilities of parakeet hemoglobin. The structure determination studies of parakeet hemoglobin by X-ray diffraction. The sequence and structure are compared with goose, chicken and human Hb, emphasizing the role of amino acids in the subunit contacts that facilitate survival by low oxygen demand. The Hb was purified and crystallized by hanging drop vapor diffusion method using poly ethylene glycol (PEG) 3350 and sodium phosphate buffer. X-ray diffracted data set was collected at 3Å resolution, the data was processed in Automar and molecular replacement, refinements, model building was carried out in CCP4i program package. The final refined model was deposited in protein data bank with accession id 2zfb. The tertiary structure of Parakeet Hb is compared with the met form of BHG Hb (1c40) and oxy form of GLG (1faw) and oxy form of human Hbs (1hho). Superimposing parakeet Hb α1β1 subunit with 'R' state human Hb shows an r.m.s.d of 0.98 Å and for BHG and GLG Hb, the r.m.s.d shows 0.72 and 0.61 Å. The replacement of α115Asp in parakeet Hb as against the α115Glu in human Hb results in the movement of GH corners. The amino acid proline at α50 present only in Parakeet Hb and Chicken HbD and not present in any other avian family which includes human Hb. The residue α78Thr located in EF corner loop region, which slightly diverge when superimposing with human and BHG Hb and also replacement of α113Asn present only in Parakeet Hb placed near the FG helix corner. The present study describes the structure determination of parakeet hemoglobin and its structural features to understand its oxygen affinity characteristics. The crystals were obtained by buffered low-salt conditions, like those of chicken HbD, carbonmonoxy and cyanomet human Hb. The present study reveals several interesting and unique modifications in the finer aspects of the quaternary structure of parakeet Hb, which are involved in oxygen affinity characteristics and the α1β1 subunit contacts. Crystallization of parakeet Hb with allosteric effectors like Inositol pentaphosphate may bring further understanding of the influence of physiological and environmental factors on the quaternary structure.

R and R2 quaternary structures of carbonmonoxyhemoglobins: Differential effect of inositol hexakisphosphate on their affinity for Ellman's reagent

The reaction of 5,5′-dithiobis(2-nitrobenzoate), DTNB, with hemoglobin sulfhydryl groups is linked to three negatively contributing Bohr effect groups: His2β is present in all avian hemoglobins but absent in some mammalian hemoglobins; His77β and His143β are absent in avian but present in nearly all mammalian hemoglobins. To probe the consequences of these differences, we determined the influence of inositol hexakisphosphate (inositol-P6) on the DTNB affinities of avian and mammalian carbonmonoxyhemoglobins. Inositol-P6decreases by two orders of magnitude the DTNB affinity of guinea pig hemoglobin, which has His2β and the R2 quaternary structure. It decreases, or has no effect on, the DTNB affinities of hemoglobins that have His2β and whose structures lie along the R2 ⇌ R quaternary equilibrium. Finally, inositol-P6increases by one to two orders of magnitude the DTNB affinities of hemoglobins that lack His2β. Thus His2β, DTNB and inositol-P6, in combination, distinguish the R2 from the R quaternary structure.

Crystal Structure Analysis of Great Cormorant (Phalacrocorax carbo) Hemoglobin to Understand its High Oxygen Affinity Characteristics by Special Structural Features.

Hemoglobin (Hb) subunits are composed of the specific functional prosthetic group "heme'' and a protein moiety "globin". Bird Hbs are functionally similar to mammalian Hbs but they are structurally dissimilar with mammalian. The insufficient structural studies on avian Hbs limit us to understand their degree of adaptation to such critical environments. The Great Cormorant (GCT) can fly and swim, the dual characteristic of GCT leads to study the sturcture of hemoglobin. To determine the crystal structure of Great Cormorant Hemoglobin and to compare its three dimensional structure with other high and low oxygen affinity hemoglobin species to understand its characteristic features of high oxygen affinity. The GCT hemoglobin has been purified, crystallized and data sets were processed using iMosflm. The integrated data has been solved using Molecular replacement method using Graylag hemoglobin (1FAW) as the template. The structure has been deposited in Protein Data Bank with PDB code: 3WR1. In order to characterize the tertiary and quaternary structural differences, the structure of cormorant hemoglobin is compared with GLG, BHG and human Hb. The larger variation observed between GCT and human Hb indicates that GCT Hb differs remarkably from human. The α1β1 interface of Great cormorant Hb is similar to bar-headed goose Hb with few amino acid substitutions. It has been found that the interaction which is common among avian hemoglobins (α119 Pro- β55Leu) is altered by Ala 119 in GCT. This intra-dimer contact (α119 Pro - β 55 Leu) disruption leads to high oxygen affinity in BGH Hb. In cormorant, GLG and human the proline is unchanged but interestingly, in cormorant Hb, the β55 position was found to be Thr instead of Leu. Similar kind of substitutions (β 55 Leu - Ser) observed in Andean goose Hb structure leads to elevated oxygen affinity between Hb-O2. To our surprise, such type of substitution at β 55 (Thr) in cormorant Hb confirms that it is comparable with Andean goose Hb structure. Thus the sequence, structural differences at alpha, beta heme pocket and interface contacts confirms that GCT adopts high oxygen affinity conformation. The three dimensional structure of Great cormorant hemoglobin has been investigated to understand its unique structural features to adopt during hypoxia condition. By comparing the sequence and overall structural similarities with high and low oxygen affinity species, it appears that GCT has more possibilities to subsist with low oxygen demand.

Bohr effect of avian hemoglobins: Quantitative analyses based on the Wyman equation

The Bohr effect data for bar-headed goose, greylag goose and pheasant hemoglobins can be fitted with the Wyman equation for the Bohr effect, but under one proviso: that the pKa of His146β does not change following the T→R quaternary transition. This assumption is based on the x-ray structure of bar-headed goose hemoglobin, which shows that the salt-bridge formed between His146β and Asp94β in human deoxyhemoglobin is not formed in goose deoxyhemoglobin. When the Bohr data for chicken hemoglobin were fitted by making the same assumption, the pKa of the NH3+ terminal group of Val1α decreased from 7.76 to 6.48 following the T→R transition. When the data were fitted without making any assumption, the pKa of the NH3+ terminal group increased from 7.57 to 7.77 following the T→R transition. We demonstrate that avian hemoglobin Bohr data are readily fitted with the Wyman equation because avian hemoglobins lack His77β. From curve-fitting to Bohr data we estimate the pKas of the NH3+ terminal group of Val1α in the R and T states to be 6.33±0.1 and 7.22±0.1, respectively. We provide evidence indicating that these pKas are more accurate than estimates from kinetic studies.

The crystal structure of oxy hemoglobin from high oxygen affinity bird emu (Dromaius novaehollandiae).

Hemoglobin is an honorary enzyme, a two-way respiratory carrier, transporting oxygen from the lungs to the tissues and facilitating the return transport of carbon dioxide. Hemoglobin has high affinity for oxygen and low affinity for carbon dioxide and other substances in the arterial circulation, whereas in the venous circulation these relative affinities are upturned. The oxygen affinity of hemoglobin increases with the fall in temperature and decreases with the increase in pH and 2, 3-bisphosphoglycerate; point mutations also affect the tetrameric arrangement and alter the oxygen affinity. Though several studies have revealed the specific reasons for the adaptation of increased oxygen affinity of avian hemoglobins at high-altitudes, further structural insights on hemoglobins from high oxygen affinity species are required to understand the detailed oxygen adaptation at the molecular level. Herein, we describe the structural investigation of hemoglobin from emu (Dromaius novaehollandiae), a high oxygen affinity bird. Hemoglobin from emu was purified using anion-exchange chromatography, crystallized and determined the structure in the oxy form at a resolution of 2.3 Å; the R-factor of the model was 19.2%. The structure was compared with other oxy hemoglobins of high oxygen affinity avian species; significant changes are noted at intra-subunit contacts which provide the clues for increased oxygen affinity of emu hemoglobin.

Molecular mechanism of high altitude respiration: primary structure of a minor hemoglobin component from Tufted duck ( Aythya fuligula, Anseriformes)

Avian hemoglobins have attracted much attention in view of the unique oxygen transport characteristics. The present study describes the primary structure of minor hemoglobin component HbD from Tufted duck ( Aythya fuligula), a migratory bird seen in Pakistan during the winter season. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. Molecular masses of the intact protein as well as peptides obtained from chemical and enzymatic cleavages were determined by electrospray ionization mass spectrometry. The sequence was studied by automatic Edman degradation of the native chains and their tryptic/hydrolytic fragments in a gas-phase sequencer. Comparison of the hemoglobin sequence with the corresponding sequences of Anseriform representatives and other avian species shows residues like α D23 Asp, α D120 Asp as being specific to Tufted duck. The three-dimensional structure analyzed with the protein structure modeling package, WHAT IF, using the crystal structure coordinates of chicken hemoglobin (PDB code = 1hbr) shows α D34 Val, α D38 Gln, and α D94 Asp as possible mediators offering alternate pathway for oxygen uptake and release thereby leading to distinct hypoxia tolerance in the Tufted ducks. Results are discussed with reference to function and evolution in the Anseriform representatives.

Comparative structural and functional studies of avian and mammalian hemoglobins.

Thermal stabilities of chicken, grey lag goose (Anser anser), turkey as avian hemoglobins (Hbs); and human, bovine, sheep and horse as mammalian Hbs in hemolysate form were investigated and compared with oxygen affinities taken from literature. The thermal stability was obtained from thermal profiles using temperature scanning spectrophotometry. The buffer conditions were 50 mM Tris, pH 7.2, and 1 mM EDTA. The average of the inverse temperature transitions, average hydrophobicity, total van der Waals volume, partial molal volume and hydration potential were calculated by computational methods. The hemolysed avian Hbs have a lower oxygen affinity, higher thermal stability and higher self association than the mammalian Hbs. These observations are based on amino-acid composition, influence of ionic effectors, and the presence of Hb D in several avian Hbs. The results indicate that the avian Hbs have a more tense (T) conformation than the mammalian Hbs.

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin α and β chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

Amino-acid sequence of the alpha D- and beta-polypeptide chains of the Japanese quail hemoglobin.

Two hemoglobin components are recognized in the erythrocytes of the adult Japanese quail: a major (Q-II) and a minor (Q-I) component. We have determined the amino-acid sequence of the alpha D-globin of the minor component and the beta-globin which is common to both components by conventional protein sequence analysis. The sequences of both the alpha D- and the beta-globins showed close homology with those of their counterpart constituents in other avian hemoglobins. Proline at position 119 in the alpha D-globin which is known to be critical for the altitude respiration by the alpha 1 beta 1 interface is conserved in the Japanese quail hemoglobin.

Primary structure of hemoglobin α-chain from Cuckoo (Eudynamys scolopaceae, Cuculiformes)

The complete amino acid sequence of the alpha A-chain of major hemoglobin component from Cuckoo (Eudynamys scolopaceae) is presented. Separation of the polypeptide subunits was achieved by ion exchange chromatography in the presence of 8 M urea. The sequence was studied by automatic Edman degradation of the native chain and its tryptic fragments in a gas-phase sequencer. Comparison with other avian hemoglobins shows residues alpha 21, alpha 30, alpha 96, alpha 110, and alpha 114 as being specific to Cuckoo. The functional significance of these is discussed.

Hemoglobin of the adult white stork (Ciconia ciconia, ciconiiformes). The primary structure of alpha A- and beta-chains from the only present hemoglobin component.

The hemolysate obtained from erythrocytes of the adult White Stork (Ciconia ciconia) contains only one hemoglobin component, identified to be HbA. The complete primary structures of alpha A- and beta-chains are presented. The minor hemoglobin component HbD with alpha D-chains usually present in adult avian species was not detected by the White Stork. The sequence was determined by automatic Edman degradation of tryptic peptides and in the case of beta-chains additionally of the C-terminal peptide obtained by chemical cleavage at the Asp-Pro bond. Homologous comparison with the Greylag Goose (Anser anser) hemoglobin showed that the alpha A-chains differ by 23 amino-acid exchanges, the beta-chains by 17. Four of the substitutions in the alpha A-chains are in the alpha 1 beta 1-contact points, one in the alpha 1 beta 2-contacts and one in the amino acids near the heme. The amino-acid substitutions of the White Stork hemoglobin as compared to the other avian hemoglobins are discussed. We suggest that alpha D-chain is persistence of an embryonic gene.

Electrophoretic studies on hemoglobins of Brewer's blackbird Euphagus cyanocephalus

1. 1. Two populations of Brewer blackbirds were sampled for possible polymorphisms in hemoglobins. Unlike other proteins in this species (e.g. ovotransferrin) none was found, but the hemoglobins were present as a multiple molecular system. 2. 2. Electrophoretic, chemical and spectral studies were performed to determine the nature of the multiple hemoglobin system. Structural analysis by comparison of monomers of the different tetramers indicated that avian hemoglobins are similar structurally to those of mammals.

The Effects of Buffer pH and Storage Time on the Electrophoretic Properties of Embryonic Hemoglobin

THERE have been conflicting reports in the literature concerning the number of adult avian hemoglobins and the number of molecular forms of hemoglobin found in developing chick embryos of various ages (Fraser, 1964; Hashimoto and Wilt, 1966; Manwell et al., 1963; Simons, 1966; and Washburn, 1968a). It has been demonstrated that genetic changes in the adult hemoglobin type influence the embryonic hemoglobin types (Denmark and Washburn, 1968). The effect of adult hemoglobin genotype on embryonic hemoglobin could explain some of the disagreement found in the literature pertaining to the hemoglobin types of chick embryos at various ages. In addition, various other factors such as storage, denaturation of the hemoglobin sample and the pH of different buffers used in electrophoretic analyses may also have an effect on the migration and separation of hemoglobin components found in the blood of chick embryos (Manwell et al., 1963, 1966; Fraser, 1964). Using starch gel…

Comparative studies on avian hemoglobins

1. 1. Studies on paper electrophoretic mobility of hemoglobins obtained from avian species representing thirteen natural orders show that avian erythrocytes possess one, two or three hemoglobins. 2. 2. The avian hemoglobins may be categorized into five groups depending on the degree of mobility towards anode. Although the ratio of hemoglobin-1 to other hemoglobins varies considerably, hemoglobin-1 was found to be the major component. 3. 3. The avian hemoglobins are relatively resistant to alkaline denaturation and the resistance characteristics depend on individual species. 4. 4. Duck, chick, pigeon and dove show a comparable amino acid composition in hemoglobin-1 whereas a widely divergent amino acid composition has been found in hemoglobin-2 of duck and of chick. 5. 5. Wide differences in amino acid composition of α 2 1- and β 2 1-chains of duck, chick and pigeon have been observed.

Paper electrophoresis of avian and mammalian hemoglobins.

Paper electrophoresis of avian and mammalian hemoglobins.