Direct measurement of the internal viscosity of sickle erythrocytes as a function of cell density

Abstract The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37°C = 1; human plasma at 37°C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37°C = 4.92 ± 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density > 1.128 g/ml is 15–40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity ( η μ ) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) η μ increases with increasing cell density; (2) η μ obtained from sickle cells is higher than η μ obtained from normal cells at a given density, and this effect is greater at 37°C than at 20°C; (3) η μ is proportional to MCHC, but η μ in erythrocytes is higher than η μ obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.