X-ray crystallographic studies of the deoxy form of human adult hemoglobin (Hb A) have shown that ${eta}99Asp$ is hydrogen bonded to both ${alpha}42Tyr$ and ${alpha}97Asn$ in the ${alpha}_1{eta}_2$ subunit interface, suggesting that the essential role of ${eta}99Asp$ is to stabilize the deoxy-Hb by creating the intersubunit hydrogen bond. In particular, for Hb Kempsey (${eta}99Asp{ ightarrow}Asn$), molecular dynamics simulation indicated that a new hydrogen bond involving ${eta}99Asn$ can be induced by replacing ${alpha}42Tyr$ with a strong hydrogen-bond acceptor such as Asp. Designed mutant recombinant (r) Hb (${eta}99Asp{ ightarrow}Asn$, ${alpha}42Tyr{ ightarrow}Asp$) have been produced in the Escherichia coli expression system and have shown that functional defects of Hb Kempsey could be compensated by the ${alpha}42Tyr{ ightarrow}Asp$ substitution. However, as the ${alpha}42 Tyr{ ightarrow}Asp$ mutation has never been reported before, it is still possible that the functional properties of r Hb (${eta}99Asp{ ightarrow}Asn$, ${alpha}42Tyr{ ightarrow}Asp$) may be due to the mutation itself. Thus, it is required to produce r Hb (${alpha}42Tyr{ ightarrow}Asp$) and r Hb Kempsey (${eta}99Asp{ ightarrow}AsnX$( as controls, and to compare their properties with those of r Hb (${eta}99Asp{ ightarrow}Asn$, ${alpha}42Tyr{ ightarrow}Asp$). r Hb (${alpha}42Tyr{ ightarrow}Asp$) could not be purified because it is an unstable hemoglobin which forms Heinz bodies. r Hb Kempsey (${eta}99Asp{ ightarrow}Asn$) exhibits very high oxygen affinity and greatly reduced cooperativity. Thus, r Hb (${eta}99Asp{ ightarrow}Asn$) and r Hb (${alpha}42Tyr{ ightarrow}Asp)$ compensate each other.