## 4 Innate Instability of the Hb Molecule and Its Degradation to Hemichrome
In theory, the following seven species of valency hybrids can be in existence during the entire process: (\(\alpha^{2^{+}}\alpha^{2^{+}}\beta^{3^{+}}\beta^{2^{+}}\)), (\(\alpha^{3^{+}}\alpha^{2^{+}}\beta^{2^{+}}\)), (\(\alpha^{2^{+}}\beta^{3^{+}}\))\({}_{2}\), (\(\alpha^{3^{+}}\beta^{2^{+}}\))\({}_{2}\), (\(\alpha^{2^{+}}\alpha^{3^{+}}\beta^{3^{+}}\)), (\(\alpha^{2^{+}}\alpha^{3^{+}}\beta^{3^{+}}\)) and (\(\alpha^{3^{+}}\alpha^{3^{+}}\beta^{3^{+}}\))\({}^{2}\)). Among these, however, it was evident that only two valency hybrids, _i.e._, (\(\alpha^{3^{+}}\beta^{2^{+}}\))\({}_{2}\) and (\(\alpha^{2^{+}}\beta^{3^{+}}\))\({}_{2}\), emerged in the human HbO\({}_{2}\) autoxidation as demonstrated by [PERSON] _et al._[31,32,35,36]. It was also shown that the appearance (or disappearance) of (\(\alpha^{3^{+}}\beta^{2^{+}}\))\({}_{2}\) (the second peak) seemed to be preceded by (\(\alpha^{2^{+}}\beta^{3^{+}}\))\({}_{2}\) (the third peak). Figure 9 illustrates the densitometric changes of the 7.5% polyacrylamide slab gel (Figure 7(c)) resulting from oxidized ratio analyses of the constituted chains. In contrast to the ampholine plate gel, the obtained electrophoretic migration profiles were complex. Hence, the initial sample (HbO\({}_{2}\)) and partially (50%) oxidized sample just after the chain separation using _p_-MB were applied on a 7.5% polyacrylamide slab gel as well as the isolated chains (\(\alpha_{\text{SH}}\) and \(\beta_{\text{SH}}\)) as an endpoint species. These were completely converted to the ferric met form by addition of potassium ferricyanide and thereafter the chain separation procedure was carried out. The resulting densitometric profiles are shown in Figure 10.  
Even under these qualified conditions, the profiles were complex. However, it was shown that two bands reflecting fast mobility could be attributed to the \(\alpha\) chain, whereas those with medium mobility seemed to be unreacted Hbs with _p_-MB. Two bands found to have relatively slower mobility could have originated from the \(\beta\) chain. From this point of view, designation was made in Figures 9 and 10 as follows: I and II originated from the \(\alpha\) chains, III and IV were attributed to unreacted Hbs with _p_-MB, and V and VI derived from the \(\beta\) chains. We could not ascertain which band corresponded to \(\alpha_{\text{O}}\): and \(\alpha_{\text{met}}\) or \(\beta_{\text{O}}\): and \(\beta_{\text{met}}\). However, we made an attempt by plotting the ratios of II/I and VI/V against time (Figure 11). It was obvious that a decrease in II/I (as a reflection of \(\alpha\)-chain oxidation)was much faster than a decrease in VI/V (as a reflection due to \(\beta\)-chain oxidation). On the basis of these findings, the reaction can be written as follows:  
\[ \begin{split}\text{fast}\\ (\alpha^{2+}\beta^{2+})_{2}&\rightarrow(\alpha^{3+}\beta^{2 +})_{2}\rightarrow(\alpha^{3+}\beta^{3+})_{2}\\ \text{slow}\\ (\alpha^{2+}\beta^{2+})_{2}&\rightarrow(\alpha^{2+} \beta^{3+})_{2}\rightarrow(\alpha^{3+}\beta^{3+})_{2}\end{split}  \tag{7}\]  
**Figure 7.** The available data resulting from UV/VIS spectrophotometric, isoelectric-focusing electrophoretic and polyacrylamide gel electrophoretic measurements for autoxidation of human HbO\({}_{2}\). The reaction proceeded in the presence of 20% (_v/v_) glycerol in 0.05 M phosphate buffer (pH 5.0) at 37 \({}^{\circ}\)C. **(a)** Spectrophotometric changes from 450 nm to 650 nm with scanning interval = 10 min; **(b)** isoelectric-focusing electrophoretic profiles; and **(c)** polyacrylamide gel electrophoretic profiles. Figure 8: Densitometric profiles resulting from isoelectric-focusing electrophoresis by use of an image scanner. Each curve corresponds to the relevant column shown in Figure 7(b). Figure 9: Densitometric profiles resulting from polyacrylamide gel electrophoresis by use of an image scanner. Each curve corresponds to the relevant column shown in Figure 7(c).