Amidation of basic carboxyl groups of hemoglobin

Crystallographic studies of human hemoglobin A (Hb A) and solution studies on the Bohr effect of the protein have implicated unique roles for one or more side-chain carboxyl groups (the β-carboxyl of Asp and/or the γ-carboxyl of Glu residues) in the structure and function of the protein. The formation and breakage of a salt bridge between the imidazole of His-146(β) and the β-carboxylate of Asp-94(α) have been suggested to play a major role in the alkaline Bohr effect of Hb. The side-chain carboxylates of Glu-22(β), Asp-23(α), Glu-43(β), Asp-73(β), and Glu-121(β) of sickle cell hemoglobin (Hb S) have been implicated as part of the intermolecular contact regions of the polymer of the deoxy protein. The contribution of some of these acidic amino acid residues in the quinary interaction of Hb S polymer may be simply a consequence of the higher pK_a as compared to those of other side-chain carboxyl groups of the protein. This chapter discusses the development of procedures for the site-selective amidation of carboxyl groups of Hb A and Hb S and the use of this chemical reaction as an in situ probe of the oxy-deoxy conformational changes at the α₁β₂ interface of mutant hemoglobins as compared with that of Hb A. The chapter also describes amidation chemistry.