Crystal Structures of HbA₂ and HbE and Modeling of Hemoglobin δ₄:  Interpretation of the Thermal Stability and the Antisickling Effect of HbA₂ and Identification of the Ferrocyanide Binding Site in Hb

Abstract

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.

Analysis of Minor Hemoglobins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…It is well established that HbA 2 is increased in the presence of the ␤-chain variant HbS (5, 19 -21 ) when analyzed by chromatographic methods. Additionally, HbA 2 is reported to inhibit polymerization of deoxy-sickle hemoglobin (HbS) in vitro (31 ). In this study, we analyzed samples from HbS carriers and compared their HbA 2 values with the values for controls and confirmed ␤-thalassemia carriers.…”

Structural and Functional Studies Indicating Altered Redox Properties of Hemoglobin E

“…Because solvent structure varies in the three presented HbE structures and the crystal environment differs for different HbE subunits, the ␤Lys-26 side chain conformation as well as conformations of neighboring ␤His-117 and ␤Glu-22 all show a high degree of variability. The orientation of the ␤ 2 His-116 (different from that of a cyanmetHbE structure (75)) and the ␤ 2 His-117 side chains, as well as ␤ 2 Lys-22, are clearly altered in the COHbE structure (PDB 1YVQ) compared with COHbA (PDB 1BBB) (Fig. 1, bottom panel).…”

Incidence of the Hb E [β26(B8)Glu→Lys,GAG>AAG] Variant In Totos, One of the Smallest Primitive Tribes in the World

“…The Hb E variant is formed as a result of the splice site mutation on exon 1 of the β-globin gene (16). This results in the reduced production of β-globin mRNA and the gene behaves like a mild β-thal mutation (17).…”