On the mechanism of loss of deformability in human erythrocytes due to Heinz body formation: a flow EPR study.

The effects of phenylhydrazine (PHZ) and monomethylhydrazine (MMH) on the deformability of human erythrocytes and ghosts in relation to Heinz body formation are investigated using the sensitive flow EPR (electron paramagnetic resonance) method. The decrease in deformability starts even before Heinz body formation is recognized and is enhanced with PHZ as Heinz bodies are attached on the inner surface of the membrane, but remains at a low level in MMH-treated cells in which Heinz bodies are formed mostly away from the membrane surface. Dithioerythritol recovers part of the lost deformability by MMH but has no effect in PHZ-treated cells or in the ghosts. The membrane fluidity measurements by 12-doxyl stearate indicate that there are two distinct modes of spin-label interaction in PHZ-treated cell membrane, the one corresponding to more immobilization gaining with the PHZ concentration, while MMH has only minor effects on fluidity. The motional freedom of membrane proteins probed with maleimide label shows that the membrane binding of hemoglobin denatured with PHZ causes more immobilization than that with MMH. These observations and Fe(III) EPR absorptions of the treated cells and ghosts are interpreted on the basis of the difference in reaction products (denatured hemoglobin) by the two reagents: the ones with PHZ preferentially bind to the membrane proteins, such as band 3, resulting in a perturbation of the membrane viscoelastic properties, while MMH-denatured hemoglobins remain mostly in the cytosol as Heinz bodies, or in the polymeric form contributing to the internal viscosity.