Characterization of the acid–alkaline transition in the individual subunits of human adult and foetal methaemoglobins

Abstract

Human adult haemoglobin (Hb A), a tetrameric oxygen transfer haemoprotein, has been recognized as an excellent model for investigating the structure-function relationships in allosteric proteins, and has been characterized exhaustively from both experimental and theoretical aspects. Despite the detailed structural and spectroscopic information available for the protein, functional properties have not been as fully elucidated as expected, and hence have remained unexplored. A major drawback for the functional characterization of Hb A is the lack of experimental techniques which enable quantitative characterization of functional properties of the individual subunits of the intact protein. In this study, we have developed techniques for determining the equilibrium constant of the acid-alkaline transition, usually represented as the 'pK(a)' value, in the individual subunits of the met-forms of Hb A (metHb A) and human foetal haemoglobin (metHb F). The pK(a) values of the individual subunits of metHb A and metHb F have been shown to constitute novel and highly sensitive probes for characterizing the effects of structural changes of not only the interfaces between the subunits within the protein, but also the contact between haem and the protein in the haem pocket. In addition, haem replacement studies of the proteins revealed that the contact between the haem peripheral vinyl side chain and the protein in the haem pocket is important for maintaining the non-equivalence in the haem environment between the subunits of Hb A and Hb F, which could be relevant to the cooperative ligand binding of the proteins.