Studies of haemoglobin functions by site-directed mutagenesis

Abstract
Human (3-globin was synthesized in Escherichia coli as a cleavable fusion protein by using the expression vector pLcIIFX|3-globin(nic - ). The authentic (3-globin was liberated by digestion with blood coagulation factor X a and a 2 (3 2 tetramers were reconstituted. The oxygen-binding properties of reconstituted haemoglobin (Hb) were essentially the same as those of human native Hb. Two mutant haemoglobins were constructed by site-directed mutagenesis. HbNymphéas (Cys-93(3->Ser) showed a slightly increased oxygen affinity and diminished co-operativity with normal DPG (2,3-diphosphoglycerate) effect and slightly reduced alkaline Bohr effects. Hb Daphne (Cys-93(3->-Ser, His-143(3->- Arg) showed low co-operativity with high oxygen affinity. The alkaline Bohr effect was slightly reduced, but the DPG effect was enhanced by 50% by the His-143(3^ Arg mutation.