β-Thalassaemia/haemoglobin E tissue ferritins

Summary Ferritins from liver and spleen of both-thalassaemia/haemoglobin E (HbE) and non-thalassaemic patients were purified by heating a methanol-treated homogenate, followed by molecular exclusion chromatography. The concentrations of ferritins in the-thalassaemia/HbE liver and spleen were calculated as 3.8 and 2.0 mg/g wet tissue. The-thalassaemia/HbE ferritin iron/protein ratios were higher than those of normal ferritins. On PAGE, all ferritins gave a single major monomeric band with only very small differences in their mobility. Ferritins from thalassaemic patients also possessed bands corresponding to oligomers. On SDS/PAGE, all ferritins were resolved into two major subunits: H and L with L subunit predominating. While the isoferritin profiles of ferritins from-thalassaemia/HbE liver and spleen were similar to each other and to those of normal liver and spleen, some extra bands were present in the acidic region. The microstructure of these pathological ferritins appears to result, to a large degree, from the particular nature and amount of iron loading present.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Calorimetric studies of the haemoglobin–haptoglobin reaction

Haptoglobin binds haemoglobin so firmly that there is practically no dissociation. It would be expected that the heat of the reaction would be relatively large. The development of the microcalorimeter by Benzinger offered the opportunity to measure the heat of reaction. The experiments were carried out in the Beckman 190B Microcalorimeter in two ways: (1) a constant amount of haptoglobin (Kabi; 65mg.) with different amounts of haemoglobin, and (2) a constant amount of haemoglobin (32.5mg.) with different amounts of haptoglobin. The proteins, each in 5ml. of 0.15m-phosphate buffer, pH7.4, were placed in equal-volume calorimeter cells. The heat produced/mg. of haemoglobin was calculated from the slope of the curve for a constant amount of haptoglobin and from the maximum heat for a constant amount of haemoglobin. This heat is about 70kcal./mole at 37 degrees . DeltaH varies with temperature, being -70.2 at 37 degrees , -29.7 at 20 degrees and 7.2 at 4 degrees . From the amount of haptoglobin required to attain maximum heat with 32.5mg. of haemoglobin and the amount of haemoglobin required to attain maximum heat with 65mg. of haptoglobin, it appears that at excess of haptoglobin there is competition between the reactions of 2moles of haptoglobin with 1mole of haemoglobin (or 2 alphabeta-chains) and 1mole of haptoglobin with 1mole of haemoglobin.     

A role for haemoglobin in all plant roots?

Abstract. We have found haemoglobin in plant roots whereas previously it has been recorded only in nitrogen fixing nodules of plants. Haemoglobin occurs not only in the roots of those plants that are capable of nodulation but also in the roots of species that are not known to nodulate. We suggest that a haemoglobin gene may be a component of the genome of all plants. The gene structure and sequence in two unrelated families of plants suggests that the plant haemoglobins have had a single origin and that this origin relates to the haemoglobin gene of the animal kingdom. At present we cannot completely rule out the possibility of a horizontal transfer of the gene from the animal kingdom to a progenitor of the dicotyledonous angiosperms but we favour a single origin of the gene from a progenitor organism to both the plant and animal kingdoms. We speculate about the possible functions of haemoglobin in plant roots and put the case that it is unlikely to have a function in facilitating oxygen diffusion. We suggest that haemoglobin may act as a signal molecule indicating oxygen deficit and the consequent need to shift plant metabolism from an oxidative to a fermentative pathway of energy generation.     

Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

Characterization of haemoglobin from Actinorhizal plants – An in silico approach

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.     

Hb Riesa or β93 (F9) Cys→Ser, a new electrophoretically silent haemoglobin variant interfering with haemoglobin A1c measurement

A new β variant was found in a German diabetic patient whose blood samples appeared to contain 45% Hb A(1c) using Bio-Rad Variant V-II A1c-analyzer but 7.6% on boronate affinity chromatography. Structural studies using, HPLC, mass spectrometry, and the genomic DNA analysis revealed a new substitution in which the cysteine residue at position β93 was replaced by serine. The variant was named Hb Riesa or β93 (F9) Cys→Ser and accounted for 54.3% of the total haemoglobin. This suggests that the protein-synthesis processes for the mutant could be slightly more promoted than those of the wild-type. Hb Riesa is clinically and electrophoretically silent.     

Acetylphenylhydrazine induced haemoglobin oxidation in erythrocytes studied by Mössbauer spectroscopy

The oxidative action of acetylphenylhydrazine (APH) on red blood cells obtained from healthy donors and from patients with breast cancer has been investigated by Mössbauer spectroscopy. Whole blood was incubated with APH for different time periods and the Mössbauer spectra of the packed red cells were recorded and compared. The evolution with time of the oxidation products has been followed. The largest difference in red cells analysis between healthy persons and patients was found after about 50 min of treatment where Mössbauer spectra of patient samples show a much broader spectral pattern due to an advanced haemoglobin oxidation.     

The distribution and interaction of haemoglobin variants and the β thalassaemia gene in Liberia

In a population survey in Liberia, West Africa, 12 major tribes were examined for the prevalence of Hb S, Hb C, and the beta thalassaemia (beta Thal) gene. Hb C is rare; Hb S and beta Thal occur in polymorphic frequencies. The distribution of both genes shows an inverse correlation. The beta Thal trait was diagnosed by quantitation of Hb A2 on DE 52-microchromatography. This method proved to be reliable and useful for mass screening.     

High activity antioxidant enzymes protect flying-fox haemoglobin against damage: an evolutionary adaptation for flight?

Flying-foxes are better able to defend haemoglobin against autoxidation than non-volant mammals such as sheep. When challenged with the common physiological oxidant, hydrogen peroxide, haemolysates of flying-fox red blood cells (RBC) were far less susceptible to methaemoglobin formation than sheep. Challenge with 1-acetyl-2-phenylhydrazine (APH) caused only half as much methaemoglobin formation in flying-fox as in ovine haemolysates. When intact cells were challenged with phenazine methosulfate (PMS), flying-fox RBC partially reversed the oxidant damage, and reduced methaemoglobin from 40 to 20% over 2 h incubation, while ovine methaemoglobin remained at 40%. This reflected flying-fox cells’ capacity to replenish GSH fast enough that it did not deplete beyond 50%, while ovine RBC GSH was depleted to around 20%. The greater capacity of flying-foxes to defend haemoglobin against oxidant damage may be explained in part by antioxidant enzymes catalase, superoxide dismutase and cytochrome-b ₅ reductase having two- to four-fold higher activity than in sheep (P < 0.001). Further, their capacity to limit GSH depletion to 50% and reduce methaemoglobin (in the presence of glucose), despite ongoing exposure to PMS may result from having ten-fold higher activity of G6PD and 6PGD than sheep (P < 0.001), indicating the presence of a very efficient pentose phosphate pathway in flying-foxes.     

Glutathione metabolism of the erythrocyte. The enzymic cleavage of glutathione–haemoglobin preparations by glutathione reductase

A complex of haemoglobin and GSH was prepared by incubating haemoglobin with GSH and acetylphenylhydrazine. GSH could be released from the crude preparation by incubation with NADPH. However, when the haemoglobin preparation was separated from glutathione reductase by DEAE-Sephadex chromatography, NADPH no longer released GSH. Rather, the addition of a combination of either partially purified human erythrocyte or crystalline glutathione reductase and NADPH was required to release GSH from the haemoglobin-GSH complex. This complex is commonly believed to represent a mixed disulphide of GSH and the cysteine-beta-93 thiol group. This interpretation was supported by the finding that prior alkylation of available haemoglobin thiol groups prevented the formation of the complex. By using haemoglobin-[(35)S]GSH complex as a substrate, it was shown that GSH itself released the radioactivity from the complex only very slowly. In contrast, the release of [(35)S]GSH was very rapid in the presence of NADPH and glutathione reductase. This suggests that the cleavage of the haemoglobin-GSH complex is not mediated by GSH with cyclic reduction of GSSG formed, but rather proceeds enzymically through glutathione reductase.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Non-equilibrium state of a monomeric insect haemoglobin induced by γ-irradiation and detected by Mössbauer spectroscopy

The met-aquo form of the monomeric insect haemoglobin CTT III has been investigated by Mössbauer spectroscopy before and after reduction with thermolyzed electrons at low temperature. The native met haemoglobin dissolved in water and water/glycerol mixtures, respectively, exhibits in the range of pH 5.8 to 9.0 high-spin iron(III). The electronic state of the haemoglobin is not affected by the solvent conditions. In water/glycerol γ-irradiation at 77 K results in the reduction of the haem iron by thermolyzed electrons. Due to this process, the hexacoordinated high-spin iron(III) is transformed into a hexacoordinated low-spin iron(II). This latter complex is a transition state which changes into the high-spin iron(II) state of the deoxyhaemoglobin when increasing the temperature. Thus, a kinetically stabilized non-equilibrium state of the haemoglobin exists at low temperature which relaxes with increasing temperature and finally reaches the equilibrium state to form deoxyhaemoglobin. This transition occurs at T > 190 K and corresponds with drastic changes in the temperature dependence of the Lamb-Mössbauer factor. Both effects indicate an alteration of the intramolecular flexibility of the haemoglobin.     

Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent?

The ability of oxyhaemoglobin and methaemoglobin to generate hydroxyl radicals (OH.) from H2O2 has been investigated using deoxyribose and phenylalanine as 'detector molecules' for OH.. An excess of H2O2 degrades methaemoglobin, releasing iron ions that react with H2O2 to form a species that appears to be OH.. Oxyhaemoglobin reacts with low concentrations of H2O2 to form a 'reactive species' that degrades deoxyribose but does not hydroxylate phenylalanine. This 'reactive species' is less amenable to scavenging by certain scavengers (salicylate, phenylalanine, arginine) than is OH., but it appears more reactive than OH. is to others (Hepes, urea). The ability of haemoglobin to generate not only this 'reactive species', but also OH. in the presence of H2O2 may account for the damaging effects of free haemoglobin in the brain, the eye, and at sites of inflammation.     

Stabilization of pepsin on duolite for the continuous hydrolysis of bovine haemoglobin at pH2 and 40°C

Summary Several methods of pepsin immobilization have been applied in order to achieve the continuous hydrolysis of a 2.5% haemoglobin solution at pH 2 and 40°C. Methods using glutaraldehyde were unsuccesful because of the unstability of the derived enzyme at low pH. Pepsin covalently bounded to a Duolite amine resin by a carbodiimide showed a half life of 15 days during the hydrolysis of haemoglobin in a column reactor. No enzyme activity was detected in the hydrolysates. No accumulation of haem in the column was noticed which could have limited long term studies by plugging the system. Modulation of the degree of hydrolysis was also performed by changing the feeding flow rate of the reactor.     

Does temperature preference relate to the anaerobic capacity of Atlantic cod (Gadus morhua L.) with different haemoglobin phenotype?

The effect of Hb-I* phenotype on white muscle lactate dehydrogenease (LDH, E. C. 1.1.1.27) activity and buffering capacity was studied in Atlantic cod (Gadus morhua), acclimated and measured at temperatures near their behavioral temperature preference. It was hypothesized that these conditions would optimize biochemical processes but no difference was found in LDH activity between the Hb-I* phenotype after 56 d of acclimation to 6 and 14°C. However, LDH activity was both mass- and temperature-dependent; mean activity was 162.2±5.0 and 275.9±6.4 IU g^-1 wet mass (mean±SEM) at 6 and 14°C respectively and larger fish had the highest rate of enzyme activity. White muscle buffer capacity was unaffected by Hb-I* phenotype but higher in cod held at 14°C.     

Could cholesterol bound to haemoglobin be a missing link for the occasional inverse relationship between superoxide dismutase and glutathione peroxidase activities?

The concept of an anti-oxidant defence system as a means to prevent oxidative cell damage implies balanced activities of anti-oxidant defence enzymes. As well as positive correlations between anti-oxidant enzyme activities in human erythrocytes, it has been observed that sometimes when glutathione peroxidase activity is increased, CuZn-superoxide dismutase activity is decreased. In our current study we have examined the plasma lipid profile and the anti-oxidant defence enzymes in erythrocytes from humans, pigs, and bulls. We found that a negative correlation existed between CuZn-superoxide dismutase and glutathione peroxidase activities in human erythrocytes when the concentrations of both plasma triglycerides and total cholesterol were high. This correlation was also found in pig erythrocytes, but not in bull erythrocytes. We propose that cholesterol could affect membrane lipid peroxidation and superoxide generation in erythrocytes via the recently found fraction of cholesterol bound to haemoglobin, termed haemoglobin-cholesterol.