Spectrin degradation in intact red blood cells by phenylhydrazine

Abstract

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 α- and β-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 monobromobirnane 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 α- and β-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.