Protein area occupancy at the center of the red blood cell membrane (1)
The area occupancy of protein and lipid at the center of the human RBC plasma membrane, a much-studied membrane with direct relevance to other cell types were determined. RBC plasma membranes were shaved by using a combination of high-pH washes with 10 mM NaOH and proteolytic digestion with proteinase K to remove loops and domains extending beyond the lipid characteristic groups of the membrane bilayer surface. The 10 mM NaOH wash was the most effective and efficient wash medium. The procedure of using a 10 mM NaOH wash before shaving with 3.0 mg/ml proteinase K in 10 mM NaOH for 24 h also resulted in the elimination of contamination from hemoglobin. The time progression of membrane shaving was extended to 48 h of digestion with proteinase K to establish completion of the shaving. A buoyant density plateau was reached by 24 h. The immunoblot analysis for band 3, glycophorins A and B, and spectrin revealed that by 24 h of shaving with proteinase K, a significant amount of extramembranous material had been successfully removed from the cytoplasmic surface, the extracellular surface, and the cytoskeleton of the RBC open ghost membrane samples.
Based on the dry weight measurements reported in Table 1, the area occupancy of the RBC plasma membrane was determined to be 20 +/- 3% protein and 79 +/- 5% lipid.
Based on the buoyant density measurements, the buoyant density measurements yielded an area occupancy of 23 +/- 0.003% protein and 77 +/- 0.003% lipid.
The measured value of at least ~23% of the RBC plasma membrane area occupied by protein transmembrane regions is in close agreement with previous estimates.