## 4 Innate Instability of the Hb Molecule and Its Degradation to Hemichrome
}}<\) [HbO\({}_{2}\)]\({}_{t\,=\,E.P}\)/[HbO\({}_{2}\)]\({}_{0}=0.75\);  
(2) [HbO\({}_{2}\)]\({}_{t\,=\,E.P}\)/[HbO\({}_{2}\)]\({}_{0}=0.75\leq\) t\({}_{\text{E.P}}<\) [HbO\({}_{2}\)]\({}_{t\,=\,E.P}\)/[HbO\({}_{2}\)]\({}_{0}=0.25\);  
(3) t\({}_{\text{E.P. }}\leq\) [HbO\({}_{2}\)]\({}_{t\,=\,E.P}\)/[HbO\({}_{2}\)]\({}_{0}=0.25\);  
(4) no hemichrome formation during the entire process. E.P. is the observed emergence point of hemichrome formation in each run. [HbO\({}_{2\,t\,=\,E.P}\)/[HbO\({}_{2}\)]\({}_{0}\) is the ratio of HbO\({}_{2}\) concentration after time \(t=E.P.\) to that at time \(t=0\) and can be monitored by the absorbance ratio of (A\({}_{t}-\) A\({}_{\infty}\))/(A\({}_{0}-\) A\({}_{\infty}\)) at 576 nm (\(\alpha\)-peak of HbO\({}_{2}\)). [HbO\({}_{2}\)]\({}_{t\,=\,E.P}\)/[HbO\({}_{2}\)]\({}_{0}=0.5\) represents equal mixtures of HbO\({}_{2}\) and metHb, _i.e._, the midpoint of the autoxidation reaction. Hence, case (1) means that hemichrome formation was noticeable at the initial stage of autoxidation. Accordingly, case (2) indicates its occurrence at the intermediate stage, and case (3) at the final stage. In Figure 5(b), the symbols used correspond to: \(\bullet\) for case (1), \(\blacktriangle\) for case (2), \(\blacktriangle\) for case (3) and \(\circ\) for case (4). To determine if the phenomenon was represented by case (3) or case (4), the reaction mixture was converted to metHb by the addition of small amounts of ferricyanide and maintained at the given temperature for 2 days to see if hemichrome precipitation occurred. Thus, the findings suggested that HbO\({}_{2}\) was highly susceptible to hemichrome formation, even under physiological pH and temperature. The broken lines in Figure 5(b) show that the threshold for this susceptibility in relation to pH and temperature. When compared with the tetrameric parent molecule, the isolated \(\alpha\) (\(\alpha_{p\text{-MB}}\)) and \(\beta\) (\(\beta_{p\text{-MB}}\)) chains were found to have much higher susceptibilities to hemichrome formation, and showed individual pH-temperature diagrams (Figure 6). The occurrence of hemichrome can be described as follows:  
\[k_{\text{A}}\] \[\text{Hb(II)O}_{2}\rightarrow\text{metHb(III)}+4(\text{O}_{2}^{-}) \rightarrow\text{hemichrome} \tag{6}\]  
Figure 5: Hemichrome formation associated with human HbO\({}_{2}\) autoxidation, redrawn from [PERSON] _et al_. [20]. (**a**) Spectral changes over time for hemichrome formation associated with human HbO\({}_{2}\) autoxidation in which the reaction was monitored in 0.1 M MES buffer (pH 5.0) at 37 \({}^{\circ}\)C and scanning interval = 15 min; (**b**) pH-temperature diagram for hemichrome formation of HbO\({}_{2}\). \(\bullet\)—hemichrome formation noticeable at the initial stage during the course of autoxidation; \(\blacktriangle\)—at the intermediate stage; \(\blacktriangle\)—at the final stage; \(\circ\)—autoxidation reaction proceeded with no hemichrome formation during the entire process. We must be careful when evaluating the data of the isolated chains shown in Figure 4. Because of the instability of Hb molecules and their relationship to hemichrome, the first-order rate constant for the isolated chains was established in each experimental run using only the initial slope of the -ln([HbsubO2]/[HbsubO2]0) _versus_ time plot. Once hemichrome precipitation arose, one could not get the endpoint spectrum (the ferric met form) of the reaction, whereby Hb molecules in the reaction mixture should be all completely converted to the ferric met form by addition of potassium ferricyanide. Hence, measurement must be confined within the initial range, in which existence of isosbestic points of the spectra that means non-occurrence of hemichrome formation during the entire reaction process was surely guaranteed.