Alpha Chains Of Hemoglobin Research Articles

5'-deoxypyridoxal as a potential anti-sickling agent.

5'-Deoxypyridoxal, which reacts specifically with the terminal amino groups of the alpha chains of hemoglobin, increases the oxygen affinity of hemoglobin solutions as well as of dilute suspensions of normal and sickle red cells and whole blood. As a result, the proportion of deoxyhemoglobin (which is responsible for sickling) is decreased at venous oxygen tensions and this is reflected by a sharply reduced sickle cell count in this range of oxygen pressures.

Hemoglobin alpha chain deficiency in black children with variable quantities of hemoglobin Bart's at birth.

Hematologic and globin chain synthesis studies have been made in 21 children, aged 2 to 6 years, many of their parents, and several normal adults and alpha-thalassemia heterozygotes. At birth, 11 children had about 5% hemoglobin (Hb) Bart's, 5 had about 2% Hb Bart's, and 5 had no trace of Hb Bart's. A significant decrease in mean corpuscular volume. (MCV) and mean corpuscular hemoglobin (MCH) values and an increase in the beta/alpha ratio was observed in the first group; microcytosis and hypochromia were absent in the children of the second group although the beta/alpha ratio was significantly increased. The alpha chain deficiency is familial. Increased alpha/alpha ratios were present in many parents although only two parents of children with 5% Hb Bart's at birth had hematologic findings suggestive of the presence of the same type of defect as observed in the children with the larger amount of Hb Bart's at birth.

Multiple structural genes for the alpha chain of canine (Canis familiaris) hemoglobin.

There are two forms of the alpha chain of canine hemoglobin differing only at residue 130. One form (taualpha) contains threonine at this position, the other (Aalpha) contains alanine. Studies of two Labrador dog families strongly support the existance of multiple alpha-chain structural genes as the basis of the alpha-chain heterogeneity. There must be at least one gene locus for Talpha and one for Aalpha; the exact number of loci has not been determined. Two other dog breeds, the Basenji and the Beagle, also have both Talpha and Aalpha chains.

Influence of carbon monoxide on hemoglobin-oxygen binding.

The oxygen dissociation curve and Bohr effect were measured in normal whole blood as a function of carboxyhemoglobin concentration [HbCO]. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HCl at constant PCO2 (fixed acid Bohr effect). As [HbCO] varied through the range of 2, 25, 50, and 75%, P50 was 26.3, 18.0, 11.6, and 6.5 mmHg, respectively. CO2 Bohr effect was highest at low oxygen saturations. This effect did not change as [HbCO] was increased. However, as [HbCO] was increased from 2 to 75%, the fixed acid Bohr factor increased in magnitude from -0.20 to -0.80 at very low oxygen saturations. The effect of molecular CO2 binding (carbamino) on oxygen affinity was eliminated at high [HbCO]. These results are consistent with the initial binding of O2 or CO to the alpha-chain of hemoglobin. The results also suggest that heme-heme interaction is different for oxygen than for carbon monoxide.

Detection of a diffusible factor controlling the synthesis of a single hemoglobin chain in mice

The synthesis of hemoglobin alpha-chains was investigated in splenic colonies derived from syngenic colony-forming units injected in lethally irradiated C3H mice. Whereas normal adult mice of this strain synthesize two structurally distinct alpha-chains, the "alphaC57" and the "alpha NB", chains, it was found that erythrocytes from splenic colonies of irradiated recipient mice contained no alpha chain of the C57 type. Daily injection of serum from adult C3H mice into irradiated recipient mice of the same strain stimulates the synthesis of alphaC57 chain, whereas the injection of serum of lethally irradiated mice, drawn 8 days after irradiation, does not. It is concluded that the serum of adult C3H mice contains one or several factors that are responsible for the differential activation of the synthesis of the alphaC57 globin chain. This factor is shown to be associated exclusively with a high molecular weight fraction of serum.

Trimodality in the proportion of hemoglobin G Philadelphia in heterozygotes: evidence for heterogeneity in the number of human alpha chain loci.

The extent of variability in the number of human hemoglobin (Hb) alpha chain loci has not yet been conclusively determined. There is evidence that in some populations individuals may possess two alpha-chain loci, while in other populations only one locus is present. Electrophoresis of peripheral blood from 53 heterozygotes for Hb G Philadelphia (alpha 68 Asn leads to Lys) revealed that the proportion of Hb G is trimodally distributed, with modes at approximately 20, 30, and 40% Hb G. Familial, hematologic, and statistical studies suggest that hte proportion of Hb G is not random but is genetically controlled and inversely correlated with mean cell volume. Two alternative genetic models are proposed to explain these findings: one assums alpha-thalassemia, while the other postulates variability in the number of alpha-chain loci in the American Black population. Biosynthetic studies of blood from 15 subjects revealed balanced synthesis of alpha and beta globin chains in heterozygotes from all three classes, strongly supporting variable gene dosage rather than alpha-thalassemia as the mechanism underlying the observed trimodality in the proportion of Hb G. Incompatibilities between out results and current concepts of alpha-thalassemia are discussed in the context of differences between Black compared with Oriental and Italian forms of Hb H disease.

Association of hemoglobin Saint Etienne (alpha2beta295F8 His replaced by G1n) with hemoglobins A and F. Synthesis and subunit exchange in vitro.

The unstable hemoglobin (Hb) Saint Etienne (alpha2beta295F8 His replaced by G1n) (betaSE) was found in the red blood cells of an 8-year-old boy. The composition of this hemoglobin was 26% Saint Etienne, 52% A, 3% A2 and 19% HbF. Studies of hemoglobin synthesis indicate: a) a balanced synthesis of alpha and non-alpha chains (alpha=betaA + betaSE + gamma), b) an increased pool of free alpha hemoglobin chains, and c) a rapid exchange of alpha chains between this pool and HbSE. The alpha chain pool resulted from the dissociation of HbSE and the greater instability of betaSE chains than alpha chains upon heating. Hemoglobin F is of the fetal type and is heterogeneously distributed among the red cells. Furthermore, two populations of red blood cells could be separated according to their i antigen content. Analysis of the hemoglobins revealed a heterogeneous distribution. Thus, F hemoglobin was preferentially associated with cells having low i antigen level, while Saint Etienne hemoglobin was increased in cells having a high i antigen level. HbF and HbSE were not present in the parents of the propositus. Study of the genetic markers confirmed the filiation. The parents were normal upon clinical and hematological examination; they exhibited a normal pattern and synthesis of hemoglobin. The Hb Saint Etienne case is compared with Hb Istanbul, which in spite of the same amino acid substitution is not associated with increased HbF level.

Unequivalence between hemoglobin subunits. The effects of inositol hexakisphosphate on the absorption spectrum of liganded valency hybrids.

The effects of inositol hexakisphosphate on the visible absorption spectrum of liganded valency hybrids alphaCO2 beta+H20, alpha+H2ObetaCO2 and alphaCO2beta+CN2, alpha+CN2beta CO2 (where alphaCO, alpha+H2O, alpha+CN and betaCO, beta+H2O, beta+CN represent the alpha and beta chain of hemoglobin, respectively, in their carbon monoxide, aquomet and cyanomet forms) have been examined in an attempt to determine if their alpha and beta chains are equally sensitive to the action of the anion. The difference spectra induced by inositol hexakisphosphate, are different for the two chains. The beta+H2O subunit contributes chiefly to the absorbance changes, rather than alpha+H2O. In contrast, it is the alphaCO chains which is more sensitive to inositol hexakisphosphate than betaCO. The question of whether such a selective response would also occur in aquomethemoglobin and carboxyhemoglobin is discussed. Criteria of changes in the quaternary structure of hybrids has been studied by measuring the effects of inositol hexakisphosphate on their ultraviolet absorption spectrum.

Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin.

The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.

High Resolution Polyacrylamide Gradient Gel Electrophoresis

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e.g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%) and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickettsii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.

Structure primaire de la chaîne α du composant majeur de l'hémoglobine d'oie ( Anser anser.)

Utilising the homology between goose alpha chain hemoglobin and chicken, the primary arrangement of the amino acid residues of the alpha chain (major component) of goose hemoglobin is presented. Data were obtained from amino acid analysis of the isolated alpha chain and of tryptic peptides of the chain. Their chemical structure was established by Edman degradation, carboxypeptidase A and B and leucineaminopeptidase digestion.

A heterozygote for HbβS, HbβC and HbαG Philadelphia in a family presenting evidence for heterogeneity of hemoglobin alpha chain loci

A child heterozygous for the genes for hemoglobins S, C and Gα Philadelphia presented with a clinical picture similar to sickle cell anemia. Her hemoglobin electrophoretic pattern contained three components with the mobilities of hemoglobins S (35 per cent), C (47 per cent) and a more slowly migrating hybrid solG C molecule (15 per cent). Seven relatives were heterozygous for Hb α G and Hb β S and five were heterozygous only for Hb α G Among the latter, three had approximately 30 per cent and two had 40 per cent of Hb G. These proportions are consistent with the hypothesis that the American Negro genome contains two types of chromosomes bearing structural loci for alpha chains, some possessing one Hb α locus, others having two loci. Hb Gα-Philadelphia presumably arose as a mutation on a chromosome with a single locus. Those heterozygotes having 30 per cent and 40 per cent Hb G presumably have two loci and only one locus, respectively, on the homologous chromosome.

A heterozygote for [formula omitted], [formula omitted] and [formula omitted] in a family presenting with evidence for heterogeneity of hemoglobin alpha chain loci : Rucknagel DL, Rising JA: Am J Med 59: 53–60, 1975

A heterozygote for [formula omitted], [formula omitted] and [formula omitted] in a family presenting with evidence for heterogeneity of hemoglobin alpha chain loci : Rucknagel DL, Rising JA: Am J Med 59: 53–60, 1975

Saturation dependency of the Bohr effect: interactions among H-+, CO2, and DPG

The Bohr effect was measured in normal whole blood and in blood with low DPG concentration as a function of oxygen saturation. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HC1 at constant PCO2 (fixed acid Bohr effect). At nornal DPG concentration CO2 Bohr effect was -0.52 at 50% blood oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. In DPG depleted blood with base excess (BE) similar to 0 meq/1, there was similar dependence of CO2 Bohr effect on oxygen saturation. At BE similar to -10 meq/1, influence of saturation was comparable, but the magnitude of the Bohr effect was markedly increased at all saturations. Fixed acid Bohr effect at normal DPG concentration was -0.45 at saturations of 50-90% but decreased at lower saturations. In DPG-depleted blood fixed acid Bohr effect averaged about -0.33 with minimal variation with saturation. Influence of DPG on oxygen affinity was greater at intermediate saturations and less at saturations below 20% and above 80%. Effect of CO2, independent of pH, was many fold greater at lower oxygen saturations than at higher saturations. These results support the suggestion that the alpha chain of hemoglobin is the site of the initial oxygenation reaction. Physiologically they indicate that the relative contribution of CO2 and fixed acid, as well as the level of oxygen saturation and DPG concentration, may be important in determining PO2 of capillary blood and resulting oxygen delivery.

Oxidation-reduction potentials of human fetal hemoglobin and gamma chains. Effects of blocking sulfhydryl groups.

The oxidation-reduction equilibrium of the gamma chains of human fetal hemoglobin (Hb F) has been studied and compared with that of the alpha and beta chains of human adult hemoglobin (Hb A). The effects of the sulfhydryl (--SH) reagents, iodoacetate, iodoacetamide, and p-mercuribenzoate (PMB), on the three kinds of chains and on Hb F have been compared. The midpoint potentials (E-m) of all three sorts of chains are lower than those of tetrameric hemoglobin A or F. The E-m values of alpha chains are the lowest, E-m = 0.049 volt at 6 degrees, and are unaffected by pH change or by PMB treatment, at least from pH 6 to 8. The E-m values of beta-SH chains are higher; E-m = 0.102 volt at pH 7, decreasing to 0.050 volt at pH 8, both at 6 degrees. These results agree with those of Banerjee and Cassoly ((1969) J. Mol. Biol. 42, 337-349). They reported no effect of PMB on beta chains, but we find that 2 eq of PMB/chain raise E--M to 0.139 volt at pH 7 at 6 degrees, chiefly as the result of reaction at beta-93, not at beta-112. Carboxymethylation at beta-93 has an insignificant effect compared with that of PMB. The oxidation-reduction potential of gamma chains is similar to that of beta chains. E-m = 0.098 volt at pH 7 at 6 degrees, decreasing to 0.064 at pH 8 and 0.010 at pH 9. The effects of --SH reagents, reacting at position gamma-93 (the only --SH group present in gamma chains), are essentially the same as those seen with beta chains. The oxidation-reduction potential of Hb F is almost identical with that of Hb A, except for being 0.008 volt lower at pH 6 at 6 degrees. This agrees with the results reported by Flohe and Uehleke ((1966) Life Sci. 5, 1041-1045). PMB or iodoacetamide treatment lowers E-m by 0.02 to 0.03 volt, depending on the pH, from 6 to 9, in much the same way as previously reported for Hb A(Brunori, M., Taylor, J.F., Antonini, E., Wyman, J., and Rossi-Fanelli, A. (1967) J. Biol. Chem. 242, 2295-2300). The "residual oxidation Bohr effect" noted in Hb F can be attributed to the oxidation Bohr effect of the gamma chains. The apparent pK of the heme-linked water molecule was found at 25 degrees to be, for Hb F, 8.1; for gamma-SH chains, 7.85; for gamma-PMB chains, 8.35; and for gamma chains treated with iodoacetate, 7.80. Sedimentation coefficients, s-20, w, at a protein concentration of 5 mg/ml, were found to be, for fetal hemoglobin 4.09, for iodoacetamide-treated fetal hemoglobin 4.04, for PMB-treated fetal hemoglobin 3.41, for fetal gamma-SH chains 4.25, and for fetal gamma-PMB chains 3.08.

Formation of Superoxide in the Autoxidation of the Isolated alpha and beta Chains of Human Hemoglobin and Its Involvement in Hemichrome Precipitation

The purpose of the work reported here is to look at O−2 production during autoxidation of the isolated α and β chains of human hemoglobin. The formation of superoxide by isolated chains. in fact, may be involved in pathological phenomena observed in red blood cell diseases where excess of either α or β chains is produced because of inherited defects of synthesis (thalassemus). The autoxidation of the oxygenated chains of human hemoglobin is followed by the transformation of the oxidized molecule (high-spin Fe3+) into a species absorbing as a low-spin Fe3+ compound, that is a hemichrome, which tends to precipitate. The overall process may be described by the following reactions: Depending on conditions the different steps proceed with different relative rates and therefore may overlap to a greater or lesser extent. The results obtained indicate that superoxide is produced during autoxidation of the isolated α and β chains of human hemoglobin. This conclusion has been reached on the basis of an experimental approach based on cooxidation of epinephrine to adrenochrome during O−2 production and inhibition of this effects by superoxide dismutase. In addition the rate of precipitation is, in all cases, very much reduced upon addition of either superoxide dismutase or catalase. These facts seem to imply very strongly that oxygen radicals are involved also in the precipitation following oxidation and hemichrome formation in the chains. In particular the effect of superoxide dismutase implies the involvement of O−2. Thus production of O−2 could well be one of the important events leading to alteration of the red cell membrane and consequently to reduced life span of thalassemic erythrocytes.

Kinetics of oxygen and carbon monoxide binding to synthetic analogs of the myoglobin and hemoglobin active sites.

Kinetics of reversible oxygenation and carbon monoxide complex formation of the simple heme compounds pyrroheme-N-[3-(1-imidazolyl)propyl]amide and pyrroheme-3-(3-pyridyl)propyl ester have been measured in different solvent environments. The oxygen on and off rates and equilibria of these compounds can be made to closely match those of myoglobin, of hemoglobin alpha chains, or of the various steps for hemoglobin by varying solvent environment or the basicity of the proximal base. These results suggest that the protein could alter oxygen on rates by varying the basicity of the proximal base and the off rates by changing the polarity of the distal environment.

Incorporation of L-azetidine-2-carboxylic acid into hemoglobin in rabbit reticulocytes in vitro.

L-Azetidine-2-carboxylic acid is the naturally occurring lower homologue of L-proline. Reticulocytes from anemic rabbits incubated with DL-[14-C]azetidine-2-carboxylic acid synthesized radiolabeled hemoglobin, which when isolated from cell lysates co-chromatographed with unlabeled hemoglobin on Sephadex G-100 columns. Amino acid analysis of hemoglobin from reticulocytes incubated with DL-[14-C]-azetidine-2-carboxylic acid suggested that the homologue was incorporated into hemoglobin intact and unaltered. Alternatively, another amino acid analogue, 1-aminocyclopentane-[1-14-C]carboxylic acid, which is purported to be a valine antagonist, was not incorporated into hemoglobin under these conditions. Incubation of reticulocytes with 1, 5, and 10 mM L-azetidine-2-carboxylic acid reduced L-[U-14-C]proline (0.10 mM) incorporation into hemoglobin by 25, 58, and 72%, respectively. Conversely, 1.45 and 145 muM L-proline reduced radiolabeled azetidine-2-carboxylic acid (0.8 mM) in corporation into hemoglobin by 45 and 92%, respectively. Incorporation of L-[U-14-C]leucine and L-[U-14-C]lysine (0.1 mM each) into hemoglobin was unaffected at these concentrations of L-azetidine-2-carboxylic acid. These results suggest that L-azetidine-2-carboxylic acid is incorporated into hemoglobin without reducing the rate of globin synthesis in rabbit reticulocytes in vitro. The alpha and beta chains of hemoglobin into which [14-C]azetidine-2-carboxylic acid had been incorporated in rabbit reticulocytes in vitro were resolved electrophoretically on sodium dodecyl sulfate-polyacrylamide gels. The ratio of total radioactivity in the alpha and beta chains separately extracted from gels was in good agreement with the known 7:4 ratio of prolyl residues in the respective chains. Autoradiograms of two-dimensional tryptic peptide maps of rabbit globin into which either [14-C]azetidine-2-carboxylic acid or [14-C]proline had been incorporated showed nearly identical patterns of radioactivity. These results suggest that azetidine-2-carboxylic acid substitutes specifically for prolyl residues during in vitro hemoglobin synthesis in rabbit reticulocytes.

Differences in spectra of alpha and beta chains of hemoglobin between isolated state and in tetramer.

The absorption spectrum of deoxygenated human hemoglobin differs from that of the isolated alpha and beta chains, and the difference has been ascribed to the conformational changes on which depends the cooperative ligand binding. Studies on hybrid-heme hemoglobins, in which one chain contains protoheme and the other chain mesoheme, showed that the prominent peak in the Soret region of the difference spectrum for deoxyhemoglobin is due to the spectral change in alpha chains and that beta chains show a small trough in the Soret region at the wavelength 10 nm longer than that for the absorption peak. Difference spectra of oxygenated hybrid-heme hemoglobin showed only small peaks which seem to be due to the spectral changes in beta chains. Difference spectra of alpha-NO-beta-unliganded hemoglobin showed a large negative band in the Soret region at the wavelength of the absorption peak for alpha-NO chains, and beta-unliganded chains in this half-liganded hemoglobin showed a small trough corresponding to that of beta chains in fully unliganded hemoglobin. Alpha-Unliganded-beta-NO chains, however, showed only small peaks in the difference spectra. The peaks in difference spectra ascribed to each chain did not completely correlate with the kinetic results reported for hybrid-heme hemoglobin (Nakamura, T., Sugita, Y., Bannai, S. (1973) J. Biol. Chem. 248, 4119-4122) and for half-liganded hemoglobin (Antonini, E., Brunori, H., Wyman, J., and Noble, R.W. (1966) J. Biol. Chem. 241, 3236-3238). Spectral change when alpha and beta chains were mixed was a second order reaction for deoxygenated hemoglobin and was a first order reaction for oxygenated and alpha-NO-beta-unliganded hemoglobin, suggesting the different rate-limiting steps.

Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.