The ultrastructural organization of the contractile peripheral protein layer of the human erythroycte membrane

Erythroycte ghosts fixed in glutaraldehyde were dehydrated in (a) alcohol or acetone, (b) propylene glycol followed by Epon and embedded in an epoxy resin. A water-soluble urea/glutaraldehyde mixture was also used. The aim was to study the structure of the peripheral protein layer, which contains spectrin and actin, in the absence of OsO4 induced denaturation changes. Ghost membranes prepared in this way had an asymmetrical quadrilaminar structure. A layer of amorphous peripheral protein +/- 18 nm in width covered the entire inner face of the membrane in the form of a coarse meshwork in both Wash I (haemoglobin-containing) and haemoglobin-free ghosts. Cations (Mg2+ or Ca2+, or Mg2+ plus ATP) had no apparent effect on its fine structure. In contrast, the corresponding layer in OsO4-fixed membranes was represented by scanty, fuzzy material attached to the unit membrane only at irregular intervals. The results demonstrate the superior ability of glutaraldehyde to preserve the peripheral protein layer in thin sections, and afford further support for the view that much of this protein normally exists in an unpolymerized state.