• BMB Reports
• /
• v.31 no.6
• /
• pp.595-599
• /
• 1998

To investigate conformational information of a low oxygen affinity recombinant hemoglobin (rHb) containing $96Val{\rightarrow}Trp$ mutation at the ${\alpha}96$ position, we ave produced rHb (${\alpha}96Val{\rightarrow}Phe$) and rHb (${\alpha}96Val{\rightarrow}Tyr$), using the Escherichia coli expression system and site-directed mutagenesis. The oxygen affinity of rHb (${\alpha}96Val{\rightarrow}Phe$) is similar to that of human normal adult hemoglobin (Hb A). However, the oxygen affinity of rHb (${\alpha}96Val{\rightarrow}Tyr$) showed much lower oxygen affinity than Hb A which is similar to that of rHb (${\alpha}96Val{\rightarrow}Tyr$), providing an opportunity as a potential candidate for a hemoglobin-based blood substitute. Both rHb (${\alpha}96Val{\rightarrow}Phe$) and rHb (${\alpha}96Val{\rightarrow}Tyr)$ showed high cooperativity in oxygen binding. IH-NMR spectroscopy shows that both rHb (${\alpha}96Val{\rightarrow}Phe$) and rHb (${\alpha}96Val{\rightarrow}Tyr$) have very similar tertiary structure around the heme pockets and uaternary structure in the ${\alpha}_1/{\beta}_2$ subunit interface ompared to Hb A. The low oxygen affinity of rHb (${\alpha}96Val{\rightarrow}Tyr$) has been suggested to be due to a hydrogen bond caused by an extra hydroxyl group not present in rHb (${\alpha}96Val{\rightarrow}Phe$). However, investigation of the carbonmonoxy form of rHb (${\alpha}96Val{\rightarrow}Phe$) and (${\alpha}96Val{\rightarrow}Try$) in the presence of inositol hexaphosphate at low temperature suggests that low oxygen affinity of (${\alpha}96Val{\rightarrow}Try$) may arise from a mechanism different to that of rHb (${\alpha}96Val{\rightarrow}Trp$).

• Bulletin of the Korean Chemical Society
• /
• v.22 no.3
• /
• pp.253-254
• /
• 2001

• BMB Reports
• /
• v.38 no.1
• /
• pp.115-119
• /
• 2005

Replacement of valine by tryptophan or tyrosine at position $\alpha$96 of the $\alpha$ chain ($\alpha$96Val), located in the ${\alpha}_1{\beta}_2$ subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the $\alpha$96 position. The characteristic of aromatic amino acid substitution at the $\alpha$96 of hemoglobin has been further investigated by producing double mutant r Hb ($\alpha$42Tyr$\rightarrow$ Phe, $\alpha$96Val$\rightarrow$Trp). r Hb ($\alpha$42Tyr$\rightarrow$Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between $\alpha$42Tyr and $\beta$99Asp in the ${\alpha}_1{\beta}_2$ subunit interface of deoxy Hb A. The second mutation, $\alpha$96Val$\rightarrow$Trp, may compensate the functional defects of r Hb ($\alpha$42Tyr$\rightarrow$Phe), if the stability due to the introduction of trypophan at the $\alpha$96 position is strong enough to overcome the defect of r Hb ($\alpha$42Tyr$\rightarrow$Phe). Double mutant r Hb ($\alpha$42Tyr$\rightarrow$Phe, $\alpha$96Val$\rightarrow$Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb ($\alpha$42Tyr$\rightarrow$Phe). $^1$H NMR spectroscopic data of r Hb ($\alpha$42Tyr$\rightarrow$Phe, $\alpha$96Val$\rightarrow$Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between $\alpha$42Tyr and $\beta$99Asp is essential for the novel oxygen binding properties of deoxy Hb ($\alpha$96Val$\rightarrow$Trp).