Distribution of shape-changing compounds across the red cell membrane.

The effects of two oppositely charged pyrene derivatives, 1-pyrenebutyrylcholine (PBC) and 1-pyrenebutyric acid (PBA), on red blood cell shape have been examined. Both compounds convert normal biconcave erythrocytes into echinocytes. However, with extended incubation time at elevated temperature, the morphology of PBC-induced echinocytes is reversed. Examination of probe uptake confirmed that, in contrast to PBA, equilibration of PBC with intact cells occurs very slowly. For PBA-induced echinocytes, it was possible to quantitate the fraction of probe bound in each half of the bilayer from nanosecond fluorescence measurements. Analysis of the heterogeneous decay showed that 71% of the bound PBA was associated with a lifetime (tau) of 102 ns and 29% with tau = 8 ns. It is likely that the later, highly quenched, component corresponds to fluorophores bound at the cytoplasmic surface because of efficient energy transfer to hemoglobin and that the long component corresponds to probe bound exclusively at the outer surface. Evidence in support of this interpretation was obtained by showing that when the paramagnetic cation Mn2+ bound at the extracellular surface the 102-ns component is quenched. The excimer fluorescence of PBC bound to red cells was examined and found to show time and temperature dependencies which correlate with morphological effects. These results indicate that red cells become crenated with PBC molecules are highly concentrated in the outer bilayer half and that shape reversal is subsequently brought about as PBC permeates and accumulates in the inner bilayer half. Finally, hemolysis protection due to PBC or PBA binding was observed also to show striking correlations with cell shape, In summary, these findings support the hypothesis [Sheetz, M. P., & Singer, S. J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4457] that shape changes are induced in red cells by amphiphilic molecules as a consequence of their relative partitioning between the two halves of the bilayer.