MECHANISM OF FREE RADICAL-INDUCED HEMOLYSIS OF HUMAN ERYTHROCYTES - HEMOLYSIS BY WATER-SOLUBLE RADICAL INITIATOR

Hemolysis of human erythrocytes induced by free radicals initiated fro m water-soluble, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) ha s been investigated. The formation of the radical detected as DMPO (5, 5-dimethyl-1-pyrroline N-oxide) adduct depended on temperature and AAP H concentration in a similar manner as hemolysis. The curve for the fo rmation of DMPO-radical adduct, however, did not correspond directly t o the hemolysis curve. The product of thiobarbituric acid-reactive mat erials, which reflect the extent of lipid peroxidation, could not be r elated directly to the hemolysis curve, too. During the hemolysis, the fluidity of the erythrocyte membrane did not change in appearance. To study whether band 3 proteins participate in the hemolysis or not, eo sin-5-maleimide (EMI)-labeled ghosts were incubated in the presence of AAPH. High molecular weight band 3 was formed, and the induced circul ar dichrosim spectrum of the bound EMI was changed, indicating a confo rmational change of band 3. It was observed that ascorbic acid suppres sed the hemolysis and the oxidation of band 3 dose dependently to prod uce an induction period. This result shows that specifically blocking band 3 oxidation inhibits the hemolysis, despite lipid peroxidation. F urther, it was observed that the EMI-labeled erythrocytes revealed dis tinct clusters by incubation with AAPH. This means a redistribution of band 3 proteins to form hemolytic holes in the membrane. However, the time course of the conformational change of band 3 during the redistr ibution was not also correspondent to the hemolysis curve, These resul ts indicate that either Lipid peroxidation or redistribution of oxidiz ed band 3 is not attributed only by itself to the hemolysis. Thus, the hemolysis was interpreted by a simple competitive reaction model betw een lipid peroxidation and redistribution of oxidized band 3. This mod el explained well the hemolysis curves.