## 9 Discussion
Such bis-histidyl coordination was also discovered in the crystals of horse metHb exposed to acidic pH (where the proximal His [His87(F8)\(\alpha\)] and a water molecule are the axial heme ligands) to the hemichrome (bis-histidine) form (in which the proximal His and the distal His [His58(E7)\(\alpha\)] are the axial heme ligands) [79]. In the case of horse metHb, the bis-histidyl coordination was seen in \(\alpha\) chains, and not in \(\beta\) chains. These crystal structures suggest a different binding state of \(\alpha\) and \(\beta\) chains as well as a different pathway to hemichrome. As described above, the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface seemed to have dual faces. One is for stabilizing the HbO\({}_{2}\) tetramer against the acidic autoxidation and the other is for controlling the fate (removal) of its own erythrocyte from the blood circulation. The \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface produces a conformational constraint in the constructed chains of the Hb molecule via tilting of the distal (E7) His residues, _i.e._, \(\alpha\)58 His (E7) in the \(\alpha\) chain and \(\beta\)63 His (E7) in the \(\beta\) chain. It was also shown that the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface appears to have a pH-sensor and a temperature-sensor. By virtue of these endowments, it seemed that Hb molecules can accomplish their roles not only as particularly suitable O\({}_{2}\) carriers between the lungs and tissues but also as equipped \"molecular sensors\" within the erythrocyte to control the fate (removal) of their own erythrocytes from the blood circulation depending on internal and extraneous circumstances, including pH and temperature (Figure 21). Regarding the latter issue, normal erythrocytes develop Heinz bodies late in their lifespan. A wide variety of biochemical changes have been reported to accompany the aging of red cells [80]. These include carboxymethylylation of proteins, activation of proteases, glycosylation of proteins, loss of membrane area, decline in changes in the ratio of bands 4.1a to 4.1b, increases in oxidized lipids and proteins, changes in the rheology and fragility of cells, changes in the exposure of cell-surface sugars, and gradual accumulation of Ca\({}^{2+}\) Low [81] demonstrated that band-3 clustering might warrant closer scrutiny as a possible transducer of distress signals from the cytoplasm to the external surface of the cell because it can be caused by hemichrome binding, ATP depletion, malondialdehydedehyde formation, Ca\({}^{2+}\) accumulation, oxidative crosslinking, or weakening of the skeletal membrane. It is known that hemichromes formed within erythrocytes bind to the cytoplasmic portion of band 3 in the membrane, then rapidly copolymerize with the soluble cytoplasmic domain of membrane band 3, forming an insoluble copolymer, followed by other changes involved in the pathogenesis of red-cell destruction [82-86]. The dominant role of band-3 clustering suggests that hemichrome formation-induced band-3 clustering could also provide a key to the control of the fate of senescent and damaged red cells in the blood circulation. Figure 21 also shed light on some clinical aspects of hemoglobinopathies and hemolytic anemia and its associated acute blood loss. Quite a large number of unstable Hbs have been reported [21,22]. Among these, many of the mutants which occur at the \(\alpha_{1}\)-\(\beta_{2}\) (and \(\alpha_{2}\)-\(\beta_{1}\)) interface have altered oxygen affinity, but the bulk of evidence suggests that the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface is much more important in maintaining the molecular stability of Hb than the \(\alpha_{1}\)-\(\beta_{2}\) (and \(\alpha_{2}\)-\(\beta_{1}\)) interface. Hemolytic anemia results from substitutions affecting the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface or the heme pocket.