Spectrin degradation in intact red blood cells by phenylhydrazine.
The effects of phenylhydrazine on intact red cells and on red cell ghost membrane proteins were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In intact red cells 1 mM phenylhydrazine induced a marked decrease in intensity of the alpha- and beta-bands of spectrin without the formation of high molecular weight materials. Phenylhydrazine was also responsible for cross-linking of hemoglobin, which is apparent by the appearance of two new broad bands on the gel. Membrane glycoproteins were unaffected. Electrophoretic patterns of cytoskeletal proteins from phenylhydrazine-treated red cells obtained on two-dimensional SDS-polyacrylamide gels and stained with Coomassie blue or fluorescently labeled with monobromobimane indicated the presence of a new band between bands 4.2 and 5 at 60-65 kilodaltons (K). An immunoelectrophoretic blotting procedure utilizing polyclonal IgG antibodies for alpha- and beta-spectrin of the red cell cytoskeletal proteins revealed that the band observed at 60-65 K in the two-dimensional SDS-PAGE studies reacted with the antibodies. The presence or absence of glucose in the incubation medium and modification of oxyhemoglobin to met- or carboxyhemoglobin in the red cells did not protect the phenylhydrazine-mediated degradation of the major cytoskeletal proteins. Metal chelators and antioxidants had no effect on membrane protein changes. Ghost red cell proteins did not undergo changes at 1 mM phenylhydrazine in the presence or absence of hemoglobin, although at 5 mM phenylhydrazine the appearance of a faint high molecular weight band was observed. These results indicate that spectrin degradation without significant polymerization can be induced by phenylhydrazine.
D000975:Antioxidants / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000032:analysis; D010659:Phenylhydrazines / Q000494:pharmacology*; D013049:Spectrin / Q000097:blood*
D000975:Antioxidants; D002614:Chelating Agents; D006454:Hemoglobins; D008565:Membrane Proteins; D010659:Phenylhydrazines; C030299:phenylhydrazine; D013049:Spectrin