A spin-label study of the correlation between stomatocyte formation and membrane fluidization of erythrocytes

Change in the membrane fluidity of human erythrocytes on transformation to stomatocytes was observed by ESR spectroscopy using 12-doxyl stearic acid or its methyl ester as a probe. When the transformation to stomatocytes was induced by four qualitatively different methods, i.e. (a) addition of cationic amphiphilic agents such as chloropromazine, tetracaine, chloroquine or primaquine, (b) addition of Triton X-100, a non-ionic detergent, (c) lowering the pH, and (d) depleting membrane cholesterol, membrane fluidization was always observed. This indicates the existence of a close correlation between stomatocyte formation and increase in the membrane fluidity. Furthermore, since the stomatocytes fixed by diamide treatment exhibited membrane fluidization only in the presence of the reagent, the enhanced membrane fluidity was a direct consequence of the reagent interacting with, and changing the state of, the lipid bilayer itself, and not through the influence of some structural alteration of spectrin. These results provide experimental support for the theoretical prediction made by Brailsford et al. [J. theoret. Biol. 86, 531 (1980)]. Plausible mechanisms for the discocyte-stomatocyte transformation are discussed.