12] Detection of hemoglobin hybrid formation at subzero temperature

Abstract

This chapter discusses the detection of hemoglobin hybrid formation at subzero temperature. The study of hybrid species (asymmetric hybrids) in mixtures of unlike tetrameric hemoglobins yields significant information on the structure–function relationship of hemoglobin. The formation of hybrid molecules between hemoglobin S and minor hemoglobin components present in the red cells of sickle cell blood—such as hemoglobin A, hemoglobin A2, and hemoglobin F—is recognized as an important factor in the inhibition of hemoglobin S gelling promoted by these minor components. Intermediates in the reactions of hemoglobin with ligands or with oxidants can also be considered hybrid molecules. In this case, the dimers forming the tetrameric hybrid belong to the same hemoglobin species but exist in different liganded or valency states (ligand and valency hybrids). Hybridization reactions that occur via the dimerization of tetramers and subsequent reassociation of dimers are one possible source of instability of the asymmetric hybrid molecules. Standard chromatographic and electrophoretic methods are used for the isolation of stable valency and asymmetric hybrids. The ability to isolate hybrids by these methods depends on a favorable ratio between the rate of tetramer dissociation and the rate of protein separation.