Influence of temperature on hemolysis of erythrocytes by digitonin

The kinetics of hemolysis of pig erythrocytes by digitonin was continuously monitored by a potassium selective electrode. The following minimal mechanism of hemolysis was postulated, based upon kinetic measurements: 2 D + E (1)in equilibrium E x D2 (2)leads to (E x D2) (3)leads to (E x D2) where D denotes a digitonin molecule and E a specific digitonin binding-site on the membrane. The first step (1) represents a rapid reversible combining of digitonin with specific binding-sites on the membrane. The second step (2) is prelytic, related to the time required for bound digitonin molecules to alter the membrane structure so much that hemolysis may take place; this step has a transition temperature at 26 degrees C, probably related to the "melting" of specific membrane structures at that temperature. The third step (3) is hemolytic, and comprises the changes within the digitonin-altered membrane during hemolysis; it is stongly influenced by temperature. Lowering of temperature slows down the rate of hemolysis and increases the quantity of digitonin required to obtain a fixed extent of hemolysis. It appears that two molecules of digitonin combine with a single binding-site on the outer face of the membrane in a digitonin-membrane complex.