Investigation of surface and shape changes accompanying the membrane alteration responsible for the heat-induced lysis of human erythrocytes

Abstract

Heat induces several successive events in erythrocyte membrane; the denaturation of spectrin at about 50°C, thermoporation at 62°C and denaturation of the anion channel at 67°C. The heat denaturation of major membrane proteins, spectrin and the anion channel, is not needed for the thermoporation which is involved in thermohemolysis. This study reports about the surface and shape changes which are specific for thermoporated membranes with spectrin and anion channel preserved intact. Thermoporation was produced exposing human erythrocytes to 39.5°C for 3 min in isotonic medium containing 18% (v/v) ethanol as membrane fluidizer and sucrose as osmotic protectant which prevents hemolysis (Ivanov, J. Therm. Biol. 1996). The control cells were processed similarly except that they were incubated at 23°C, thus avoiding thermoporation. In control and porated membranes the overall structure of spectrin and the anion channel was retained inasmuch as their enthalpies and denaturation temperatures were microcalorimetrically found preserved. Nevertheless, irregular shape, grainy surface and asymmetric spicules were apparent in porated cells through scanning electron microscopy. A decrease in the number of binding sites for Alcian blue and an increased binding of eosine was established in the membranes of porated cells. After poration the hexane/aqueous partition coefficient K d of cells increased from 5 to about 220 and the electrophoretic mobility of cells decreased by about 25% indicating marked increase in cell surface hydrophobicity and a decrease in surface charge, respectively. In addition, adhesivity to hydrophobic interfaces and aggregability in low ionic media strongly increased after poration. In contrast to intact and control cells, the porated ones (all prefixed with 0.2% glutaraldehyde) made molecular contacts with inclined hydrophobic interfaces at low (5 mM NaCl) but not at high (150 mM NaCl) ionic media. Thus, the microtopological shape changes and exposure of new hydrophobic and charge groups over the outer cell surface, without major thermal unfolding, possibly indicates an irreversible redistribution of membrane material and disturbed lipid–protein complementation during thermoporation.