PHOTOHEMOLYSIS OF HUMAN ERYTHROCYTES LABELED IN BAND 3 WITH EOSIN-ISOTHIOCYANATE |
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Authors: | John P. Pooler Albert W. Girotti |
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Affiliation: | Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA and department of Biochemistry, The Medical College of Wisconsin, Milwaukee, WI 53226, USA |
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Abstract: | 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. |
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