Direct intracellular measurement of deoxygenated hemoglobin S solubility

The solubility of deoxygenated hemoglobin S (HbS), which is the concentration of fully deoxygenated HbS in equilibrium with polymer (C_SAT), is a factor that determines in vivo polymer formation. However, measurement of C_SAT is usually performed under conditions that are far from physiological. In solution studies of HbS by Benesch et al, it was demonstrated that p50, the point at which hemoglobin is half-saturated with oxygen, is proportional to the amount of polymer formed and that it may be used to measure C_SAT. This method has been extended to measure C_SAT in intact red cells by varying extracellular osmolarity, which, in turn, varies intracellular hemoglobin concentration. This method measures intracellular C_SAT under physiological conditions with intact red cell contents and can be applied to human and transgenic mouse red cells. The principle is demonstrated by measuring p50 as a function of extracellular osmolarity for AA, SS, and AS red cells.