Phenomenon of hot cold hemolysis: chelator induced lysis of sphingomyelinase treated erythrocytes

Abstract

Staphylococcus aureus produces a phospholipase C specific for sphingomyelin (β hemolysin). Erythrocytes with approximately 50% sphingomyelin in their membranes, e.g., from sheep, have been shown to have up to 60% of this phospholipid hydrolyzed by this enzyme at 37 C in isotonic buffered saline without hemolysis. Cooling of sphingomyelinase C treated erythrocytes to 4 C causes complete lysis of the cells, a phenomenon known as hot cold hemolysis. The addition of ethylene diamine tetra acetate (EDTA) to sheep erythrocytes preincubated with sphingomyelinase C was found to induce rapid hemolysis at 37 C. The treated cells became susceptible to chelator induced hemolysis and to hot cold hemolysis simultaneously, and the degree of lysis by both mechanisms increased equally with prolonged preincubation with sphingomyelinase C. Erythrocytes of species not readily susceptible to hot cold hemolysis were equally insusceptible to chelator induced lysis. Chelators of the EDTA series were the most effective, whereas chelators more specific for Ca2+, Zn2+, Fe2+, Cu2+, and Mg2+ were without effect. The rate of chelator induced lysis was dependent on the preincubation period with β hemolysin and on the concentration of chelator added. The optimal concentration of EDTA was found to equal the amount of exogenously added Mg2+, a cation necessary for sphingomyelinase C activity. Hypotonicity increased the rate of chelator induced hemolysis, whereas increasing the osmotic pressure to twice isotonic completely inhibited chelator induced lysis. The data suggest that exogenously added and/or membrane bound divalent cations are important for the stability of sphingomyelin depleted membranes. The phenomenon of hot cold hemolysis may be a consequence of the temperature dependence of divalent ion stabilization.