Stabilization of the T-state of hemoglobin

The effect of inositol hexaphosphate and bezafibrate on binding of O2 and CO to HbAO at high concentrations (1 mM) has been evaluated using thin layer optical techniques. Data analysis shows 1) the occurrence of greatly reduced ligand dependent cooperativity (Hill slope of 2.23 for CO and 1.51 for O2), and 2) the presence of significant triply ligated species. The data fits a nested allosteric two-state MWC model in which the T state consists of two allosteric substrates, Tt and Tr, where Tt binds only to the alpha chains and Tr binds to both alpha and beta chains. The model indicates that the triply ligated species consists of a predominant amount of T form, agreeing with kinetic observations of CO ligated hemoglobin. The maximum amount of triply ligated R molecules (CO or O2) implicated is less than 1%, a result similar to that found previously for binding studies made in the absence of BZF and IHP.