Membrane alterations in irreversibly sickled cells: Hemoglobin–membrane interaction

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Abstract
Irreversibly sickled cells (ISCs) are sickle erythrocytes which retain bipolar enlongated shapes despite reoxygenation and owe their biophysical abnormalities to acquired membrane alterations. Freeze-etched membranes both of ISCs produced in vitro and ISCs isolated in vivo reveal microbodies fixed to the internal (PS) surface which obscure spectrin filaments. Intramembranous particles (IMPs) on the intramembrane (PF) surface aggregate over regions of subsurface microbodies. Electron microscopy of diaminobenzidine-treated ISC ghosts show the microbodies to contain hemoglobin and/or hemoglobin derivatives. Scanning electron microscopy and freeze-etching demonstrate that membrane–hemoglobin S interaction in ISCs enhances the membrane loss by microspherulation. Membrane-bound hemoglobin is five times greater in in vivo ISCs than non-ISCs, and increases during ISC production, paralleling depletion of adenosine triphosphate. Polyacrylamide gel electrophoresis of ISC membranes shows the presence of high-molecular-weight heteropolymers in the pre–band 1 region, a decrease in band 4.1 and an increase in bands 7, 8, and globin. The role of cross-linked membrane protein polymers in the generation of ISCs is discussed and is synthesized in terms of a unified concept for the determinants of the genesis of ISCs.