Abstract
This chapter discusses the rapid-rate equilibrium analysis of the interactions between organic phosphates and hemoglobins. The linkage relationships between the binding of protons, organic phosphate, chloride, bicarbonate, and oxygen to hemoglobin are well documented. While the major organic phosphate in mammalian erythrocytes is 2,3-diphosphoglycerate (DPG), different organic phosphates are employed by other species. This chapter also presents the general principle of the method. The method involves the use of a dialysis cell with an upper chamber, containing the macromolecule and radioactive ligand, separated by a suitable membrane from a lower chamber. Buffer is pumped through the lower chamber at a constant rate, and the effluent is collected and sampled for radioactivity measurements. The equilibrium concentration of free ligand can be measured by determining the rate of dialysis of the radioactive ligand across the membrane. The chapter presents the preparation and characterization of hemoglobin.
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