37] Measurement and analysis of ligand-linked subunit dissociation equilibria in human hemoglobins

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This chapter describes methods that are useful in the studies of reversible association–dissociation equilibria in human hemoglobin and focuses primarily on the problems of category. The methods described are successfully applied to the study of (1) the linkage between dimer–tetramer association and oxygen binding, (2) the effects of temperature, pH, and chloride on the oxygenation linked dimer–tetramer reactions, and (3) the ligand-linked self-association of isolated α and β chains. The chapter presents basic definitions and concepts of the ligand-linked dissociation scheme for human hemoglobin. The chapter also describes some experimental techniques, such as equilibrium gel permeation methods for determining equilibrium constants of subunit association and the haptoglobin-binding method for determining tetramer to dimer dissociation rates in unliganded hemoglobins. The reversible dissociation of human hemoglobin tetramers occurs in the region of neutral pH leading to the formation of dimers.