Mechanism of hypotonic hemolysis of human erythrocytes.
A mechanism of hemolytic hole formation during rapid hemolysis in a hypotonic medium has been investigated using eosin-5-maleimide (EMI) as a probe. The EMI-labeled erythrocytes revealed a distinct cluster and/or ring of intense fluorescence staining in a hypotonic 5 mM Hepes buffer (pH 7.4), but not in an isotonic buffer containing 150 mM KCl. This EMI cluster indicates an association of band 3 proteins, which correspond to a hemolytic hole. The hole was confirmed by an atomic force microscopy image. The erythrocytes showed a single large hole in the membrane. By the use of EMI-labeled ghosts, it was observed that the lateral clustering of band 3 was accompanied by a biphasic change of fluorescence intensity of EMI. This biphasic change is interpreted as the hemolytic hole formation by band 3, followed by a disappearance of the hole accompanied by band 3 diffusion or distribution within membrane. The latter event corresponds to a spontaneous membrane seal. When a cytoplasmic domain of band 3 was digested with trypsin, or when SH groups in the cytoplasm-facing components of the membrane were also labeled by EMI, no fluorescence change was observed. These results suggest that the association and/or dissociation of band 3 proteins in a hypotonic medium are strongly influenced by cytoplasmic domains. The apparent biphasic change of the fluorescence intensity in the hypotonic medium was well explained by assuming three events: swelling, clustering of band 3, and sealing accompanied by band 3 redistribution.
Sato
Y
Y
Pharmaceutical Institute, Tohoku University, Sendai, Japan.
Yamakose
H
H
Suzuki
Y
Y
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects*; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D007038:Hypotonic Solutions; D066298:In Vitro Techniques; D007700:Kinetics; D011189:Potassium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D001457:Anion Exchange Protein 1, Erythrocyte; D007038:Hypotonic Solutions; D011189:Potassium Chloride; C033531:eosin maleimide; D004801:Eosine Yellowish-(YS)