Tannic acid as a marker for membrane sidedness of human red cell vesicles

Membrane sidedness of human erythrocytes was investigated in inside-out vesicles (IOV's), ghosts and intact cells by means of transmission electron microscopy (e.m.) after tannic acid fixation. No gross difference in appearance of either membrane surface was observed when IOV's were subjected to conventional e.m. preparation. This included in addition to tannic acid, a double fixation with glutaraldehyde and osmium, followed by "en bloc" and thin section staining with uranyl acetate and lead citrate. By contrast, if IOV's were treated with a high EDTA concentration (2-5 mM) before tannic fixation, granular, electron-dense deposits were found on one of the surfaces. The presence of such a material was unaffected by neuraminidase treatment prior to the EDTA step. On the hand, red cells show no electron-dense deposits when exposed to EDTA (5 mM) unless they presented a light cytoplasm and an altered membrane appearance. Such a material was only observed on the inner membrane surface. Furthermore, a similar distribution of such deposits following EDTA treatment was also found in white ghosts before being induced to vesiculate. These results indicate that tannic acid can be employed as a marker for the cytoplasmic surface of the human erythrocyte membrane when used in combination with EDTA.