Spectrofluorometric and microcalorimetric study of the thermal poration relevant to the mechanism of thermohaemolysis.

This study sheds light on the structural changes in erythrocyte membrane during thermally induced poration, an event involved in thermohaemolysis. Two major membrane disturbing events can be induced during transient heating, the denaturation of spectrin and thermoporation. The first one precedes the latter but is not involved in it. Ethanol linearly reduces the onset temperature of both events but with different efficiencies. Thermoporation efficiency exceeds by 3.5 fold that of spectrin denaturation. Thus, at a specific concentration of ethanol (18% v/v), the poration occurs at 39.5 degrees C, which precedes the denaturation of spectrin by 6 degrees C. To induce and study the poration avoiding spectrin denaturation, cells were put in contact with preheated (39 degrees C) isotonic (60mM) NaCl) media containing 18% v/v ethanol and sucrose as an osmotic protectant. After 3 min heating, the porated cells were washed, their membranes isolated and studied. The control cells were processed similarly except that they were incubated at 23 degrees C, thus avoiding thermoporation. Using scanning microcalorimetry, the enthalpy and the temperature of denaturation of spectrin were found to be the same in control as well as in porated membranes which indicates similar spectrin structure in both membranes. While the enthalpy of denaturation of the anion channel was preserved, its denaturation temperature was lowered by 2.5 degrees C after poration. These results confirmed that the heat denaturation of the main membrane proteins was not needed and not involved in thermoporation and, hence, in thermohaemolysis. Analysis of the fluorescence of membrane bound ANS gave an apparent increase in the number of binding sites for ANS in membranes after poration. In relation to the control, the eximerization of pyrene in porated membranes changed, depending on the location of the probe: in the domain of free lipids it decreased by 18% but it increased by 60% in the lipid milieu proximal to membrane proteins. Likewise, the eximerization of N-(3-Pyrene) maleimide bound to membrane proteins increased by 67% after poration, which proves increased intramolecular mobility of proteins following poration. The maximal efficiency for transferring energy from tryptophans to neighbouring pyrene was determined to be 0.93 in control, which is almost the same as in intact membranes, and 0.70 in porated membranes, indicating a strong decrease in the lipid/protein contact zone. This data suggests a mild conformational change, possibly an irreversible perturbance of the transbilayer distribution of membrane proteins in porated membranes in comparison to the control and intact ones.