Macromolecules such as hemoglobin exert both kinetic and matrix effects on osmotic pressure. The kinetic osmotic pressure of sickle-cell hemoglobin is lost upon deoxygenation at physiological erythrocyte concentrations. The nonkinetic or matrix component of osmotic pressure remains relatively unchanged. Loss of thermal-osmotic activity during deoxygenation occurs throughout a hemoglobin concentration range between 25 and 35 g/100 ml. Deoxygenation of sickle-cell hemoglobin causes aggregation such that the matrix effect is unchanged but the kinetic (van't Hoff) effect nearly vanishes. A loss of intracellular osmotic pressure during deoxygenation could dehydrate the erythrocyte sufficiently to promote more rapid sickl ell hemoglobin aggregation. Subsequently, comlete gelation of these agegates could cause additional water loss and thrust the sickled cell into an irreversible cycle. The osmotic pressure of normal hemoglobin does not change appreciably during deoxygenation and is essentially the same as the osmotic pressure of oxygenated sickle-cell hemoglobin.Sickling of deoxygenated erythrocytes containing hemoglobin S (Hb S) results from an aggregation of Hb S tetramers into a gel of long, multistranded polymers (1)(2)(3)(4)(5) METHODSThe osmotic effect on Hb S was measured after oxygenation and deoxygenation in a wide range of concentrations obtained by a simple dilution procedure, going from 35 to 2.5 g of hemoglobin per 100 ml. Similarly, the osmotic behavior of normal hemoglobin (Hb A) was studied in its oxygenated and deoxygenated states in the same range of concentration.Venous blood was drawn from subjects homozygous for Hb S or Hb A. The anticoagulant-treated samples were centrifuged at 1300 X g and washed three times with 0.9% NaCl. The packed cells were lysed in an equal volume of distilled water, followed by extraction of membrane lipids with an equal volume of chloroform. Cell debris were removed by centrifugation at 1300 X g for 15 min, and the hemolysate was carefully removed by aspiration. The resulting hemoglobin solution was dialyzed and concentrated, using a membrane with an exclusion limit of 25,000 daltons. A sample was converted to cyanomethemoglobin and the concentration was determined by absorbance at 450 nm, using a Cary 118 C spectrophotometer. These purified hemoglobin solutions were concentrated to slightly above 35 g/100 ml and subsequently diluted to produce a series of hemoglobin concentrations from 35 to 2.5 g/100 ml.Each hemoglobin sample was placed in a glass tonometer and equilibrated at 220C with humidified gas [p2 was 100 mm Hg and 0 mm Hg for oxy and deoxy forms, respectively; the remaining gas was N2 (1 mm Hg = 133 Pa)]. During each measurement of osmotic pressure, the respective gases were continuously passed over the sample.Osmotic pressure in the purified hemoglobin solution was measured in a simple Plexiglas colloid osmometer (18), which combined membrane rigidity with accuracy to within 1%. A dialysis membrane with a molecular-weight retention of 10,000 was str...