PHOTOHEMOLYSIS OF HUMAN ERYTHROCYTES LABELED IN BAND 3 WITH EOSIN-ISOTHIOCYANATE

Eosin-isothiocyanate (EYNCS) is 50 to 100 times more effective in sensitizing delayed photo-hemolysis of human erythrocytes than is eosin when matched for absorbance in the reaction medium. These dyes are equally efficient in generating singlet oxygen, a potent membrane oxidant. When cells are treated with sensitizer and washed extensively prior to illumination, EYNCS phototoxicity persists, while that of eosin is lost. SDS-gel electrophoresis of membranes from EYNCS-exposed cells shows a large fluorescence signal coincident with band 3 protein that is abolished by pretreatment with H₂DIDS, the inhibitor of anion exchange by band 3. This treatment reduces the photohemolytic potency of EYNCS by over 90%. The marked enhancement of photohemolytic activity upon binding sensitizer to band 3 implicates band 3 itself as a site of photodamage leading to lysis.