Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation.

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Abstract
13C/1H magnetic double-resonance spectroscopy was used to measure the amount of sickle Hb polymer within [human] sickle erythrocytes as a function of O2 saturation. The methods of cross-polarization and scalar decoupling can be used to accurately measure the polymer fraction in deoxygenated sickle Hb solutions [Noguchi et al. (1979)]. The amount of intracellular deoxyhemoglobin S polymer increases monotonically with decreasing O2 saturation. Polymer can be detected at O2 saturation values > 90%. This result can be theoretically explained by the excluded volume effect of the oxyhemoglobin S in the cell. The very high total intracellular Hb concentration (34 g/dl) reduces the amount of soluble deoxyhemoglobin S to about 3 g/dl at 90% O2 saturation. The agreement between theory and experiment indicates that the equilibrium properties of intracellular polymerization can be described by the analyses resulting from studies of concentrated sickle Hb solutions. The curve for polymer formation as a function of O2 saturation is roughly hyperbolic whereas that for cell sickling is sigmoidal; the difference is most apparent for measurements at pH 7.65. Intracellular polymer formation may in general have a different relationship to O2 saturation than cell sickling and may be a more meaningful parameter of the pathophysiological process in sickle cell anemia than cell morphology. In addition, measurements of intracellular polymer should be useful in evaluating potential therapeutic agents.