Oxygen Equilibrium and EPR Studies on α1β1 Hemoglobin Dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp→Glu at β37 using our Escherichia coli expression system at the α1β2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the α or β subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [αN(Fe-NO)βM(Mg)] hybrid showed some α1β1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.