Hemolytic properties under hydrostatic pressure of neuraminidase or protease-treated human erythrocytes

We investigated the hemolytic properties under high pressure (200 MPa) of human erythrocytes in which sialic acids and glycopeptides had been removed from membrane surface by using neuraminidase and proteolytic enzymes such as trypsin and chymotrypsin, respectively. The degree of hemolysis increased in proportion to the amounts of sialic acids or glycopeptides released from intact erythrocytes. Studies of the time course of hemolysis showed that upon enzymatic digestion erythrocyte membranes became more fragile against high pressure. Such fragility decreased in the presence of chlorpromazine and trifluo-perazine but was unaffected by chlorpromazine methiodide or indomethacin. Furthermore, the effect of cross-linking of membrane proteins by diamide on the fragility was examined. The degree of hemolysis at 200 MPa increased upon removal of sialic acids from red cells in which spectrin is mainly cross-linked, but did not upon enzymatic digestion of red cells in which glycophorins, in addition to cross-linking of themselves, are included in the large-molecular-weight aggregates formed by cross-linking of the membrane skeleton with transmembrane proteins. In the latter case, however, upon reduction of the cross-linking by dithiothreitol the effect of enzymatic digestion appeared again. On the other hand, such an enzymatic digestion effect on osmotic hemolysis was not observed either in intact erythrocytes or in diamide-treated red cells. These results suggest that the interaction of the cytoplasmic domains of glycophorins with cytoskeletal proteins may be weakened by enzymatic digestion of the exofacial domains of glycophorins.