The role of choline phospholipids in hypertonic cryohemolysis

Hemolysis resulting from a warm-to-cold temperature shift in a hypertonic environment (hypertonic cryohemolysis) is studied with the use of phospholipases as membrane probes of the phospholipids of the outer leaflet of the bilayer. Bee venom phospholipase A2 which attacks only phosphatidylcholine (PC) in the intact erythrocyte results in inhibition of cryohemolysis produced by both hypertonic sodium chloride and sucrose. In both cases, about 25% of the loss of PC occurs before any such inhibition, suggesting the possibility of functionally separate domains of PC in the outer leaflet of the bilayer. Sphingomyelinase also attacks only sphingomyelin in the intact erythrocyte and results in inhibition of cryohemolysis due to hypertonic sodium chloride but not of that due to sucrose. In each case, inhibition of the enzymatic hydrolysis by EDTA abolished the effect on cryohemolysis. It is postulated that cryohemolysis is inhibited when phospholipid interaction with membrane (cytoskeletal) proteins are abolished, but present knowledge of membrane structure does not permit a detailed mechanism to be proposed.