Resealing of erythrocyte membranes after hypotonic hemolysis

A new quantitative method to study kinetics of resealing of erythrocyte membrane, defined as the recovery of impermeability to dextran, was established. Erythrocyte membrane pre-labelled with 125I were incubated under various conditions. Sealed and unsealed membranes were separated using dextran density gradient centrifugation. Sealed membranes which are impermeable to dextran were recovered from the top of the centrifuge tube, while unsealed membranes were recovered from the bottom of the tube. Degree of resealing was calculated from the counts of 125I recovered from sealed membrane fraction. The resealing of membrane from human erythrocyte after hypotonic hemolysis was dependent on the incubation temperature. Resealing was rapid above 30 degrees C, while it was not observed below 20 degrees C. The process completed within 1 hr at 37 degrees C. Ionic strength of the medium is also an important factor which controls the resealing process. The effect of NaCl in the medium on resealing was biphasic. In the lower range of NaCl (0-50 mM) there was gradual decrease of resealing capacity. Above 50 mM, the capacity was again increased. The mode of resealing of sheep and rabbit erythrocyte membranes was rather different from that observed in human erythrocyte. Under the present experimental condition, these erythrocyte membranes did not reseal well. The immediate resealing, which is observed in human erythrocyte upon hemolysis in the presence of Mg++ or Ca++ ions, was observed neither in sheep erythrocyte membranes nor rabbit ones.