## 1 Introduction  
In erythrocytes _in situ_, the reaction dynamics of human hemoglobin (Hb) with molecular dioxygen (O\({}_{2}\)) make them a particularly suitable O\({}_{2}\) carrier. Hb can bind O\({}_{2}\) in the ferrous form to carry out its physiological functions. During this reversible O\({}_{2}\) binding, the oxygenated form of Hb (HbO\({}_{2}\)) is known to be oxidized by the bound O\({}_{2}\) to the ferric met form (metHb), which cannot be oxygenated, and is thus physiologically inactive. However, the content of metHb in normal red cells _in situ_ has been reported to be \(\leq\)1% [1,2]. An intra-erythrocytic nicotinamide adenine dinucleotide (NADH)-dependent reducing system can reduce metHb to deoxy-ferrous Hb and prevent the continued accumulation of metHb resulting from oxidation of the bound O\({}_{2}\) to the ferrous heme iron(II) [3-5]. Even so, it has been suggested that its oxidation (autoxidation) process can be followed by transformation of the oxidized molecule (high-spin Fe\({}^{3+}\)) into a species absorbing as a low-spin compound, _i.e._, hemichrome, the formation of which can result in the accumulation of soluble and insoluble hemichromes and precipitation [6-11]. Despite these findings, direct evidence of hemichrome formation in normal erythrocytes is lacking. Since the early works of [PERSON] [12,13], the oxidative process (autoxidation) of human Hb (HbO\({}_{2}\)) has been investigated by several workers. Among others, [PERSON] [14] and [PERSON] _et al._[15] observed that the rate of autoxidation is strongly dependent upon the pH values of solutions. The study of [PERSON] and [PERSON] [14] also showed that autoxidation of human HbO\({}_{2}\) exhibited a biphasic nature with a initial fast (\(k_{\rm A}^{\prime}\)) reaction, followed by a second slower (\(k_{\rm A}^{\rm s}\)) reaction, when observed in acidic solutions. Following the method of [PERSON] and [PERSON] [14], our previous ultraviolet-visible (UV/VIS) spectroscopic study [16] indicated that this biphasic behavior seen in acidic solutions disappeared if the pH of solutions was \(>\)8, and became \(k_{\rm A}^{\rm s}\)\(=\)\(k_{\rm A}^{\rm s}\). This finding leads to certain questions: how does the Hb molecule prompt the range of fast (\(k_{\rm A}^{\rm s}\)) and slow (\(k_{\rm A}^{\rm s}\)) components in acidic solutions? How does the molecule provoke \(k_{\rm A}^{\rm s}\)\(=\)\(k_{\rm A}^{\rm s}\) if the pH of solutions is \(>\)8? Does the biphasic autoxidation seen in acidic pH regions reflect a different rate due to the individual Hb chains or the presence of valency hybrid intermediates such as (\(\alpha^{2+}\beta^{3+}\))\({}_{2}\) and (\(\alpha^{3+}\beta^{2+}\))\({}_{2}\)? Through attempts to locate a site integrating these puzzling problems behind the phenomenon, a pH-sensed molecular device was suggested to exist in the HbO\({}_{2}\) tetramer. Consequently, it was shown that this mechanism produces a tilting of the distal (E7) histidine (His) residue in the \(\beta\) chains at position 63,thereby preventing the proton-catalyzed displacement of O2' by a solvent water molecule [17, 18]. The \(\beta\) chains thus acquire pH-dependent delayed autoxidation in the HbO2 tetramer. Similar phenomena suggesting participation of an inherent tilting capability of the distal (E7) His residues of the Hb molecule was also very interesting. Following the method of [PERSON] _et al._[19], our previous UV/VIS spectroscopic study [20] showed that human HbO2 obtained from healthy donors tended to degrade to produce hemichrome even at close to physiological temperatures and pH because its occurrence was a function of pH, temperature and the progress of autoxidation of ferrous HbO2 to the ferric met form. It is known that Heinz bodies are intra-erythrocytic inclusions of hemichrome formed from oxidized or denatured Hb. They have been found and characterized in drug-induced hemolytic anemia, defects in the intra-erythrocytic reducing system (e.g., glucose 6-phosphate dehydrogenase [G-6-PD] deficiency) and in unstable Hb disease [21, 22].