The effect of non-binding molecules on the gelation of HbS.

The influence of an inert globular macromolecule upon the solubility of sickle cell hemoglobin has been determined as a function of the degree of oxygenation. The thermodynamic theory required to treat this and related problems is derived starting with the Gibbs-Duhem equation and introducing the effect of specific binding (oxygen) by use of the binding partition function. The treatment includes non-ideal solution behaviour as measured by osmotic pressure of highly concentrated macromolecular solutions. Application of the theoretical equation demonstrates how the solubility of hemoglobin is influenced by the presence of the binding ligand (oxygen) and the inert macromolecule, bovine serum albumin (BSA). Good agreement is obtained between experimentally determined and theoretically calculated solubilities using 1) oxygen binding curves to solution and gel phases, 2) activity coefficients from osmotic pressure data, 3) one solubility under the condition where oxygen and BSA are absent, and 4) the value of the water content of the gel phase. Examination of theoretical equations suggests that inert molecules of intermediate size, that are partially excluded from crystalline or gel phases, have the potential of generally increasing the solubility when non-ideal solution effects are small.