Distribution of hemoglobin E in several Mongoloid populations of northeast India

Nine-hundred seventy-eight subjects from eight Mongoloid tribes of northeastern India were investigated for the distribution of hemoglobin phenotypes by starch-gel electrophoresis. The sample included 157 Khasi and 24 Bodo from Cherrapunji (Meghalaya), 148, Rengma Naga and 81 Hmar of the Cachar district of Assam, 215 Adi from different subtribes, 216 Nishi, 79 Apatani, and a mixed group of 58 individuals from several other tribes of Arunachal Pradesh in northeast India. The frequency of HBB*E was found to be very low (0.01-0.02) in the Khasi, Naga, and Hmar tribes, whereas it varied from 0.06 to 0.18 among the tribes of Arunachal Pradesh. As expected, the Bodo group had a very high frequency of HBB*E (0.38), confirming earlier reports. It appears that the lack of HBB*E in the Austro-Asiatic (Khasi) and Naga-Kuki-Chin groups is probably due to the absence of malarial selection pressure as well as to isolation from their neighbors.     

Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection

The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.     

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E,isolated from the blood samples of beta-thalassemic patients

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.     

The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.     

Studies on the oxidation of hemoglobin Zurich (beta 63 E7 Arg)

Autoxidation and chemically-induced oxidation of hemoglobin Zurich (beta 63 E7 Arg) have been investigated by electron paramagnetic resonance and optical absorption spectroscopy. The results show that the replacement of the distal histidine of the hemoglobin beta chains by an arginine greatly enhances the susceptibility of the heme-iron to oxidative challenge. Both the kinetics and the products of the oxidation are pH dependent. Thus, at acidic and neutral pH, treatment of the protein with ferricyanide leads to a fast conversion of the oxy-protein to aquo-methemoglobin, which, eventually, is slowly converted to hemichromes. In contrast, the hydroxy-met derivative, formed upon chemical oxidation at high pH, is rapidly converted to hemichromes. The electron paramagnetic resonance features of the ferric derivatives of hemoglobin Zurich are somewhat singular, reflecting the modifications of the heme environment in the distal region of the abnormal chains. However, they can be related to heme complexes having their structural counterparts in oxidation products of hemoglobin A.     

Faster heme loss from hemoglobin E than HbS,in acidic pH: Effect of aminophospholipids

We report studies on loss of heme at or below pH 3.0 from two clinically important hemoglobin variants, HbE and HbS, in the presence and absence of phopholipid membranes. The kinetics of heme loss has been studied at pH 3.0 to simulate the same at a faster rate than at physiological pH, for spectroscopic investigation. Results obtained from the study clearly establish the probable fate of the lost heme to partition into the phospholipid bilayer independent of the pH range. This is also of particular importance to membranes containing the aminophospholipid and cholesterol which are predominantly localized in the inner leaflet of erythrocytes. Absorption measurements indicated such loss of heme when the Soret peak at 415 nm blue-shifted to 380 nm at pH 3.0. The extent of this blue shift decreased from 35 nm to ~15 nm in the presence of small unilammelar vesicles of both dimyristoyl- and dioleoyl-based phosphatidylcholine and phosphatidylethanolamine, indicating partitioning of the released heme in the membrane bilayer. The kinetics of heme loss was faster from HbE than HbA and HbS, obeying first-order reaction kinetics. Released heme could be involved in the premature destruction of erythrocytes in hemoglobin disorders.     

5-Azacytidine increases the synthesis of embryonic hemoglobin (E2) in murine erythroleukemic cells

The addition of 5-azacytidine to erythroleukemic cells which were induced to differentiate with DMSO or BA altered the expression of the hemoglobins. After the addition of 5-azacytidine there was an increase in hemoglobin synthesis especially in the embryonic E2 band. The beta-globin increased in synthesis after 5-azacytidine treatment. The level of hemoglobin synthesis in DMSO-induced cells is less than BA-induced cells while the effect of the 5-azacytidine stimulation was greater with DMSO induction than with BA induction.     

The genetic basis of the interaction between pyrimidine 5' nucleotidase I deficiency and hemoglobin E

We have previously described a family in which the interaction between pyrimidine 5' nucleotidase I (P5N-I) deficiency and hemoglobin E resulted in severe haemolytic anaemia. In this study we explored the genetic basis of the severe clinical phenotype and look for evidence of the interaction between these conditions. A P5N-I gene mutation (IVS8 + 1-2delGT) was found in the family, confirming that the severe phenotype results from the interaction between two genetic diseases.     

Oxidation-reduction reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park

The kinetics and equilibrium of the redox reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park using the iron (II) and iron (III) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA4-) as the reducing and oxidizing agents have been studied. With respect to the equilibrium it was found that hemoglobin M Iwate (where the beta chains were reduced) was more readily reduced than hemoglobin M Hyde Park (where the alpha chains are reduced). This difference was shown to be a result of a difference in the rate constant for reduction but not oxidation. The observed rate contants for the reduction of all three hemoglobins were shown to decrease with increasing pH. This was attributed to a decrease in the [T]/[R] ratio. The observed rate contants for the oxidation reaction were shown to increase with increasing pH. Accompanying this increase was a change in the kinetic profile for hemoglobin A from pseudo first order to one in which the rate increased as the extent of reaction increased. Inositol hexaphosphate had no effect on the rate of oxidation of deoxyhemoglobin A. This was a result of binding of FeCDTA2- or HCDTA3- to the protein. However, in the presence of inositol hexaphosphate, the reduction of methemoglobin A exhibited biphasic kinetics. This result was interpreted in terms of the production of a small amount of a conformation which was more readily reduced.