## 3 Chain Separation and Rate Measurements for the Separated Chains  
To not only elucidate the biphasic characteristics of human HbO\({}_{2}\) autoxidation but also to clarify how the Hb molecule can prompt the range of fast (\(k_{\mathrm{A}}\)) and slow (\(k_{\mathrm{A}}\)) components against pH values of the solutions, chain separation of the constituted chains from the parent molecules is essential. In this section, we discuss the results of our study investigating chain separation of \(\alpha\) and \(\beta\) chains from the HbO\({}_{2}\) tetramer and their rate measurements. Isolation of \(\alpha\) and \(\beta\) chains from the parent molecules was made using sodium _p_-hydroxymercurbenzoate (_p_-MB) by a one-column method using carboxymethyl (CM)-cellulose and alternatively by a two-column method. The following procedure was for two-column method; details for the one-column method can be found in [PERSON] _et al._[20]. One-hundred milligrams of _p_-MB were dissolved in 2 mL of 0.1 M NaCl and neutralized with 1 M CH\({}_{3}\)COOH. This was reacted with 10 mL of HbO\({}_{2}\) solution in 30 mM phosphate buffer, pH 6.0, in the presence of 0.1 M NaCl. To obtain the \(\alpha_{p\text{-}\text{MB}}\) chains, the mercurated HbO\({}_{2}\) solution was adjusted to pH 8.2 by filtration through a Sephadex G-25 column (\(25\times 40\) cm) equilibrated with 15 mM HEPES buffer (pH 8.2). The resultant solution was then passed through a DEAE-cellulose column (\(3\times 12\) cm) equilibrated with the same buffer. Under these chromatographic conditions, the \(\alpha_{p\text{-}\text{MB}}\) chains were readily eluted out because the unreacted HbO\({}_{2}\) and mercurated \(\beta\) chains were retained on top of the column at this pH. With respect to separation of the \(\beta_{p\text{-}\text{MB}}\) chains, the pH of the mercurated HbO\({}_{2}\) solution was adjusted to 6.7 using a Sephadex G-25 column (\(25\times 40\) cm) equilibrated with 10 mM phosphate buffer (pH 6.7) and the solution applied on a CM-cellulose column (\(3\times 12\) cm) equilibrated with the same buffer. Under these conditions, only the \(\beta_{p\text{-}\text{MB}}\) chains were eluted completely. Measurement of the autoxidation rate for the isolated chains was identical to the procedure of rate measurements for HbO\({}_{2}\). Figure 4 shows the pH profiles for the autoxidation rate of the isolated \(\alpha_{p\text{-}\text{MB}}\) and \(\beta_{p\text{-}\text{MB}}\) chains. With reference to the rates (\(k_{\mathrm{A}}\)/ and \(k_{\mathrm{A}}\)') of the parent molecules calculated from biphasic autoxidation curves, the isolated chains were shown to be oxidized much more rapidly to the ferric met-form over the entire pH range (5-10.5) because the reaction followed simple, first-order kinetics as follows:  
\[k_{\mathrm{A}}\,^{\mathrm{sub}} \tag{5}\] \[\mathrm{Hb}_{\mathrm{sub}}(\mathrm{II})\mathrm{O}_{2}\to \mathrm{metHb}_{\mathrm{sub}}\,(\mathrm{III})+4(\mathrm{O_{2}}\text{${}^{-}$}) \tag{6}\]  
where Hb\({}_{\mathrm{sub}}\) represents each subunit of the Hb molecule. It was also shown that the rate of the initial first component (\(k_{\mathrm{A}}\)/) of the parent molecules was found to lie closer to the values of \(k_{\mathrm{A}}\) for the isolated \(\alpha_{p\text{-}\text{MB}}\) and \(\beta_{p\text{-}\text{MB}}\) chains if the pH of solutions was \(<\)6. Similar pH profiles were obtained for \(p\)-MB-removed chains, _i.e._, the \(a_{\rm SH}\) and \(\beta_{\rm SH}\) chains. \(p\)-MB can be removed from the \(a_{p\text{-}\text{MB}}\) and \(\beta_{p\text{-}\text{MB}}\) chains by incubation with 2-mercaptoethanolhanol, as described elsewhere [17]. In separated-chain solutions, the protein is known to exist in equilibrium of \(a\leftrightarrow a_{2}\) or \(\beta\leftrightarrow\beta_{4}\), respectively. Under our experimental conditions, the monomeric form (87%) was present in the separated \(a\) chain, whereas the tetrameric form (99%) was predominant in the \(\beta\) chain. This estimation was made on the basis of the results by [PERSON] _et al._[29].