Effects of inositol hexasulfate on the oxygen affinity of hemoglobin: verification of the integral function theory of thermodynamic linkage.

A detailed series of experimental measurements have been carried out to investigate the effects of inositol hexasulfate (IHS) on the oxygen binding curves of human hemoglobin. The data provide a critical test of the integral function theory for the mutual interaction of two ligands binding to a nondissociating macromolecule [Ackers, G. K. (1979) Biochemistry 18, 3372-3380]. This theory, which is required for cases where the fractions of bound and unbound ligands are of comparable magnitude, was found to predict quantitatively the observed effects. The experimentally determined variation of the median oxygen concentration with IHS concentration was analyzed by least-squares methods to determine the IHS binding constants for unliganded and fully oxygenated hemoglobin. The derived constants are in good agreement with independent estimates of their values, providing further verification of the theoretical treatment. General aspects of the integral function approach to thermodynamic linkage are briefly outlined. The importance of this approach for treating physiological situations is discussed.