Degradation of the human erythrocyte membrane band 3 studied with monoclonal antibody directed against an epitope on the cytoplasmic fragment of band 3

The mouse hybridoma monoclonal antibody BIII.136 of the IgG2a class is specific for human erythrocyte band-3 protein. It was shown by means of immunoblotting and immunoprecipitation assays that the antibody recognized an epitope located in the cytoplasmic pole of the band-3 molecule within approximately 20 kDa from the N-terminal end. The N-terminal fragments of band-3 protein, migrating in SDS/polyacrylamide gel electrophoresis in the 60-kDa, 40-kDa and 20-kDa regions, were detected with the antibody in untreated red-cell membranes as products of autolysis of band-3 protein. A correlation was found between the amount of these fragments and erythrocyte age, which suggests that partial degradation of band 3 proceeds in vivo during senescence of erythrocytes. The further degradation of band-3 protein in vitro was not observed in intact erythrocytes stored at 4 degrees C, but progressed distinctly after hemolysis of red cells, during washing and storing the membranes.