title: Hydroxylamine treatment increases glutathione-protein and protein-protein binding in human erythrocytes.
abstract: Hydroxylamine is a direct-acting hematotoxic agent leading to hemolytic anemia in animals and man. The effect of hydroxylamine on the morphology, sulfhydryl status and membrane skeletal proteins of human erythrocytes were studied. Loss of reduced glutathione (GSH) from the red blood cells was directly proportional to the hydroxylamine concentration used. This loss of GSH was larger than the sum of the increase in the amounts of extracellular glutathione and intracellular oxidized glutathione (GSSG). The extracellular glutathione is mainly present as GSSG, which is in agreement with the fact that only GSSG is exported from the erythrocytes by membrane bound ATPases. Lack of GSSG export was not limited by decreased ATP levels in the erythrocytes and we concluded that the GSH that disappeared did not become available as intracellular GSSG. After reduction of the erythrocyte incubates the lost GSH was almost completely recovered indicating that the lost GSH is present in the cell as protein-glutathione mixed disulfides. Glutathione thus stored within the cell can be quickly recovered by combined thioltransferase and glutathione reductase activity when conditions become more favorable again. SDS-polyacrylamide gel electrophoresis of membrane ghosts from human red cells revealed changes in skeletal proteins with a smearing of bands 1, 2 and 3 to the higher molecular weight end of the gel and the appearance of new monomeric and dimeric hemoglobin bands at about 16 and 30 kD. The observed alterations are probably a consequence of disulfide bridge formation between cellular proteins (mainly hemoglobin) and skeletal proteins as well as between hemoglobin monomers. Exposure of hydroxylamine to erythrocytes caused severe Heinz body formation but the outside morphology of the cells was only marginally altered. The described changes in sulfhydryl status of the red blood cells are likely to play a major role in the premature splenic sequestration of hydroxylamine-damaged erythrocytes.
authors.affiliation: Toxicology Section, University Maastricht, Maastricht, 6200 MD, The Netherlands.
authors.forename: A A
authors.initials: AA
authors.lastname: Spooren
authors.forename: C T
authors.initials: CT
authors.lastname: Evelo
mesh_terms: D000743:Anemia, Hemolytic / Q000097:blood / Q000139:chemically induced; D001798:Blood Proteins / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000737:chemistry; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005978:Glutathione / Q000097:blood*; D006801:Humans; D019811:Hydroxylamine / Q000009:adverse effects / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000097:blood
chemical_list: D001798:Blood Proteins; D013438:Sulfhydryl Compounds; D019811:Hydroxylamine; D005978:Glutathione