The effect of EDTA, cations, and various buffers on the morphology of erythrocyte membranes: an electron microscopic study

Abstract

Membranes of human erythrocytes were prepared by stepwise osmotic hemolysis in Ca2+ free solutions. Examinations with the electron microscope after negative staining showed some short, conelike 24°C, on the surface of about 20% of the ghosts, while 80% were round, intact spheres. After Ca2+ treatment, all membranes were round and intact. After exposure to ethylenediaminetetraacetic acid (EDTA) (1.0 mM, pH 7.4), the entire ghost surface was covered with long, thin extrusions called stromalytic forms (about 460 p/cell). Their sizes, shapes, and fine structure are described. Exposure to ionic calcium (1.4 x 10-4M) abolished the EDTA induced stromalytic forms. A second exposure to EDTA reversed this Ca2+ effect. ATP, like EDTA, produced stromalytic forms. EDTA induced stromalytic forms were also abolished by Zn2+, , La3+ and Nd3+ at concentrations of 1-5 x 10-4M. Mg2+ at 10-2M was ineffective. Ghosts were prepared by graded lysis in various buffers. Those prepared in phosphate were the most stable and provided consistent EDTA effects and Ca2+ reversal. Ghosts in Tris HCl showed breakdown unless salt was added. Moderately satisfactory ghosts were also obtained in Hepes NaOH buffer and salt.