Mechanism of Free Radical-Induced Hemolysis of Human Erythrocytes. II. : Hemolysis by Lipid-Soluble Radical Initiator

Hemolysis of human erythrocytes induced by free radicals initiated from lipid-soluble 2, 2'-azobis(2, 4-dimethylvaleronitrile) (ADVN) was examined under various conditions. From the ESR spectra of the spin-labeled erythrocytes, it was found that the fluidity of the membrane did not change during the radical-induced hemolysis. The curves of the time courses of the extent of oxidation and the conformational change of band 3 proteins were hyperbolic, though the hemolysis curves were sigmoidal. In spite of the necessity of lipid peroxidation, the peroxidation did not seem to relate directly to the hemolysis. It was observed that the hemolytic holes were formed by a lateral clustering of band 3, an anion exchange protein in erythrocytes. The competitive reaction model between lipid peroxidation and the redistribution of oxidized band 3 proteins, which was previously presented for the hemolysis initiated from water-soluble, 2, 2'-azobis(amidinopropane)dihydrochloride (AAPH), could explain well the curves for the hemolysis by ADVN. Further, the rates of lipid peroxidation at various concentrations of ADVN and AAPH were calculated on the basis of the hemolysis curves, and they were compared with the experimental values estimated from the curves for the lipid peroxidation. The curves which showed a dependence of the calculated rate constant on the concentration of radical initiators were similar to those of the experimental values. These results indicate that the competitive reaction model is appropriately represents the hemolysis induced by free radicals which also originated from lipid-soluble initiators.