## 4 Innate Instability of the Hb Molecule and Its Degradation to Hemichrome
With respect to chain separation, _p_-MB (100 mg) was dissolved in 2 mL of 0.1 M NaOH in the presence of 0.1 M NaCl and neutralized with 1 M CH\({}_{3}\)COOH. Ten microliters of this solution were then mixed with each 100 \(\mu\)L of a series of collected Hb A samples. Chain separation was continued overnight (\(\sim\)16 h) at 4 \({}^{\circ}\)C in 30 mM phosphate buffer (pH 6.0) containing 0.1 M NaCl and 20% (_v/v_) glycerol. Thereafter, electrophoresis was then carried out for 6 h on a 7.5% polyacrylamide slab gel in Tris-glycine buffer (pH 8.6) at a constant current of 12 mA and constant voltage of 150 V. The buffer contained 25 mM Tris and 192 mM glycine. Fixation and staining were undertaken in 7.5% acetic acid containing 1% Coomassieie blue. Densitometry was identical to that described for the ampholine plate gel. The _a_SH and _b_SH chains were prepared by removal of _p_-MB with 2-mercaptoethanol from the _a_\({}_{p\text{-}\text{MB}}\) and _b_\({}_{p\text{-}\text{MB}}\) chains. According to our specifications as described elsewhere [17], the mixture with 15 mM 2-mercaptoethanol was applied on a CM-cellulose column for isolation of the _a_SH chains, whereas it was on a DEAE-cellulose column for the _b_SH chains. Each column was then washed with buffer solution containing 2-mercaptoethanol. Thereafter the mercaptoethanol was removed by washing the column with buffer alone. The buffers used were 10 mM piperazine-_N_, _N_'-bis(2-ethanesulfonic acid) (pH 6.5) for the \(\alpha_{\text{SH}}\) chains and 10 mM HEPES (pH 8.2) for the \(\beta_{\text{SH}}\) chains. Glycerol was unaffected not only with respect to spectrophotometric changes but also with regard to values of \(k_{\text{A}}\)\({}^{f}\) and \(k_{\text{A}}\)\({}^{s}\). Similarly, there were no quantitative differences between the resulting isoelectric-focusing profiles in the presence or absence of glycerol, whereas the presence of glycerol had a good effect in preventing hemichrome formation during the time course of the experiment. Figure 7 demonstrates UV/VIS spectrophotometric, isoelectric-focusing electrophoretic and polyacrylamide gel electrophoretic measurements in which the reaction proceeded under high HbO\({}_{2}\) concentration (1.25 mM in heme content) in 0.05 M phosphate buffer (pH 5.0) at 37 \({}^{\circ}\)C in the presence of 20% (_v/v_) glycerol. Figure 7(a) shows the spectrophotometric changes every 10 min from 450 nm to 650 nm in the monitoring cell. Simultaneously, sample collection was carried out from the sampling cell at 0, 1.2, 2.5, 4.5, 7, 10.5, 16, 25, 42.5, and 76 h. In terms of percentage decrease of HbO\({}_{2}\) concentration in the reaction mixture, the sample picked up at time (_t_) = 0 h corresponded to 100% [HbO\({}_{2}\)], and that at: \(t\) = 1.2 h to 90%; \(t\) = 2.5 h to 80%; \(t\) = 4.5 h to 70%; \(t\) = 7 h to 60%; \(t\) = 10.5 h to 50%; \(t\) = 16 h to 40%; \(t\) = 25 h to 30%; \(t\) = 42.5 h to 20%; \(t\) = 76 h to 10%. As mentioned above, these were used for valency hybrid analyses (Figure 7(b)) as well as detection of the oxidized ratio of the constituted chains (Figure 7(c)). Figure 8 describes the densitometric profiles of the ampholine plate gel shown in Figure 7(b). Four components appeared during the entire reaction process. These had satisfactory-to-good consistency with those reported by [PERSON] _et al._[31-36]. On the basis of the arguments and assignments of [PERSON] _et al._, the first peak must be (\(\alpha^{2^{+}}\beta^{2^{+}}\))\({}_{2}\), the second can be (\(\alpha^{3^{+}}\beta^{2^{+}}\))\({}_{2}\), the third can be (\(\alpha^{2^{+}}\beta^{3^{+}}\))\({}_{2}\), whereas the fourth must be (\(\alpha^{3^{+}}\beta^{3^{+}}\))\({}_{2}\) as an endpoint.