Identifying the conformational state of bi-liganded haemoglobin

While most researchers agree on the global features of cooperative ligand b inding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations o f the partially liganded states and makes observation of the transitions be tween these substates more difficult. For example, the equilibrium populati on of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxyg en equilib-rium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilib ria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predic ts a shift in the allosteric equilibrium based on the number of ligands bou nd, but not on their distribution within the tetramer. From studies of dime r-tetramer equilibria of various pure and hybrid forms, it was concluded th at a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded te tramers which resemble the triply liganded forms [Ackers et al. (1992), Sci ence 255: 54-63]. The implications of this model and the relevant experimen ts will be reviewed here.