## 9 Discussion  
Cellular life is reliant upon rapid and efficient responses to internal and external conditions. The basic molecular events associated with these processes are the structural transitions of the proteins involved. Therefore, a deeper understanding of the structural bases of protein allostery is of paramount importance to characterize these processes [26]. The human Hb molecule (\(\alpha_{2}\beta_{2}\); alternatively, a dimer of \(\alpha\beta\) protomers), whose \(a\) and \(\beta\) chains contain 141 and 146 amino-acid residues, respectively, hold a special position in this field. This is due to the achievements of [PERSON], whose pioneering studies led to the identification of two distinct Hb structures, the T (tense) and the R (relaxed) state, which are associated with the deoxygenated and oxygenated form of the protein, respectively [49-51]. Regarding human HbO\({}_{2}\), a representative set of successive O\({}_{2}\)-binding constants is given in terms of mmHg\({}^{-1}\) as follows: \(K_{1}=0.0188\), \(K_{2}=0.0566\), \(K_{3}=0.407\), \(K_{4}=4.28\) in 0.1 M buffer (pH 7.4) containing 0.1 M KCl at 25 \({}^{\circ}\)C [52]. In this reaction, by comparing their X-ray crystal structures, major differences have been defined between the deoxygenated and oxygenated forms. These include movement of the iron atom into the heme plane with simultaneous change in the orientation of the proximal (F8) His, rotation of the \(\alpha_{1}\beta_{1}\) dimer relative to the other \(\alpha_{2}\beta_{2}\) dimer about an axis \(P\) by 12-15\({}^{\circ}\), and a translation of one dimer relative to the other along the \(P\) axis by \(\sim\)1 A (10\({}^{-1}\) nm). These changes are accompanied with sequential breaking of \"salt bridges\" by C-terminal residues [53-57]. When HbO\({}_{2}\) goes from the deoxygenated to the oxygenated quaternary structure, the \(\alpha_{1}\)-\(\beta_{2}\) (and \(\alpha_{2}\)-\(\beta_{1}\)) interface undergoes the principal changes associated with the cooperative oxygen binding, so it is named as the \"sliding contact\" that involves 19 residues, including mainly helices C and H and the FG corner [55,58]. Conversely, negligible changes are found with respect to the crystal structure examined for the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface that associates 35 residues including B, G, and H helices as well as the GH corner. As mentioned, understanding of subunit interactions between the four Hb chains (and how these explain cooperative O\({}_{2}\) binding) has been the primary focus in Hb research. This has been coupled with a tendency for structural analyses to focus on the changes at the proximal side of the heme and at the \(\alpha_{1}\)-\(\beta_{2}\) (and \(\alpha_{2}\)-\(\beta_{1}\)) interface. This is despite the fact that the configuration of the residues lining the distal side of the heme pocket (where molecular O\({}_{2}\) binds) are also altered by \begin{table}
\begin{tabular}{c|c c c} \hline \multicolumn{4}{c}{Transit blood sample volume (\(\mu\)L)} \\ \cline{2-4}  & 20 \(\mu\)L & 40 \(\mu\)L & 60 \(\mu\)L \\ \hline Temperature-untreated reference & 8.0 \(\pm\) 1.08 & 16.3 \(\pm\) 2.24 & 25.1 \(\pm\) 3.50 \\ Temperature-treated at 37 \({}^{\circ}\)C & 7.8 \(\pm\) 1.35 & 16.4 \(\pm\) 2.57 & 26.4 \(\pm\) 3.42 \\ Temperature-treated at 42 \({}^{\circ}\)C & 8.5 \(\pm\) 1.38 ** & 18.3 \(\pm\) 2.94 ** & 36.3 \(\pm\) 6.32 ** \\ Temperature-treated at 45 \({}^{\circ}\)C & 9.3 \(\pm\) 2.46 ** & 21.2 \(\pm\) 5.99 ** & 41.8 \(\pm\) 13.23 ** \\ \hline \end{tabular}
\end{table} 
Table 2: Summary of the mean \(\pm\) standard deviation of transit time of blood samples (s) for a given transit sample volume (\(\mu\)L). Prior to application to MC-FAN, blood samples were subjected to mild heating at 37 \({}^{\circ}\)C, 42 \({}^{\circ}\)C and 45 \({}^{\circ}\)C for 30 min, respectively. The number of subjects was 10. Significant differences (\(p<0.001\)) of the temperature-treated samples against the temperature-untreated reference were shown as **.