Functional Defects of Hb Kempsey ( ${\beta}99Asp{\rightarrow}Asn$ ) Can be Compensated by Insertion of a New Intersubunit Hydrogen Bond at the ${\alpha}_1{\beta}_2$ Subunit Interface

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