Hemoglobin A(2) (alpha(2)delta(2)) is an important hemoglobin variant which is a minor component (2-3%) in the circulating red blood cells, and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is beta-chain production failure, HbA(2) acts as the predominant oxygen deliverer. HbA(2) has two more important features. (1) It is more resistant to thermal denaturation than HbA, and (2) it inhibits the polymerization of deoxy sickle hemoglobin (HbS). Hemoglobin E (E26K(beta)), formed as a result of the splice site mutation on exon 1 of the beta-globin gene, is another important hemoglobin variant which is known to be unstable at high temperatures. Both heterozygous HbE (HbAE) and homozygous HbE (HbEE) are benign disorders, but when HbE combines with beta-thalassemia, it causes E/beta-thalassemia which has severe clinical consequences. In this paper, we present the crystal structures of HbA(2) and HbE at 2.20 and 1.74 A resolution, respectively, in their R2 states, which have been used here to provide the probable explanations of the thermal stability and instability of HbA(2) and HbE. Using the coordinates of R2 state HbA(2), we modeled the structure of T state HbA(2) which allowed us to address the structural basis of the antisickling property of HbA(2). Using the coordinates of the delta-chain of HbA(2) (R2 state), we also modeled the structure of hemoglobin homotetramer delta(4) that occurs in the case of rare HbH disease. From the differences in intersubunit contacts among beta(4), gamma(4), and delta(4), we formed a hypothesis regarding the possible tetramerization pathway of delta(4). The crystal structure of a ferrocyanide-bound HbA(2) at 1.88 A resolution is also presented here, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in DPG binding. The pH dependence of ferrocyanide binding with hemoglobin has also been investigated.

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We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for alpha-globin over that for beta-globin and intact HbA in an adenosine 5'-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the alpha-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in alpha-globin compared with intact haemoglobin, HbA and the beta-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the alpha-globin chain over that of the beta-globin in the haemoglobin disorder beta-thalassaemia.

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane-protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-thalassemia.

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