The aim of this project is to use osmotic stress as a tool in extending the thermodynamic model of hemoglobin assembly into the region of gel structure and compression, and as a method for the evaluation of the factors which influence phase behavior. Hemoglobin solubility has been the most useful molecular parameter for evaluating the tendency of deoxy sickle cell hemoglobin to polymerize in solutions and in red cells, under the conditions of the experiment. Changes in solubility have been used to evaluate the effect on the solution to gel phase transition of ligands, additives and other hemoglobins, and to explore polymer contact sites by using mutant hemoglobins. Relative solubilities, determined under one set of conditions have been inferred to be valid in others, even when the form of the aggregated phase differs. Until recently, experimental studies of condensed state thermodynamic behavior have not been available. Using the osmotic stress technique, it is now possible to describe hemoglobin phase behavior as a function of solvent activity over the range of solution, polymerization and gelation, and condensed phase (gel). The thermodynamic data thus obtained permit comparison of solubilities obtained in different solvent systems, and comparisons of aggregated state behavior as well. The effect of additives or solvent components which act specifically on hemoglobin should be distinguishable from those effects caused by changes in solvent activity. Osmotic stress data provide a unique experimental test of theoretical treatments which predict polymerization and phase behavior based on intermolecular forces, polymer form and assembly process. The purpose of the research is to use osmotic stress studies (1) on hemoglobin in high ionic strength phosphate buffer, to establish relevance to physiological conditions; (2) on hemoglobin in 0.15M phosphate buffer with high ionic strength achieved by addition of chloride or sulfate salts, to determine specific ion effects; (3) on nickel sickle hemoglobin in low and high ionic strength phosphate buffers to help determine its usefulness as a non-liganding substitute for deoxy sickle hemoglobin under aerobic conditions. The results will be used to judge the relevance of conditions that are usually enforced in in vitro analysis of protein gelation. The phase diagrams derived will be compared with predictions of current theories of polymer formation.
