Solubilization and Structural Integrity of the Human Red Cell Membrane

Solubilization of structural protein components of red‐cell ghosts and alterations of their biological activities by treatment with various solvents was attempted. Ghosts were suspended in the solvents and then separated into supernates and residues by high‐speed ultracentrifugation. Treatment with 8 M urea yielded the highest degree of solubilization (60 per cent) and approximately 80 per cent of the protein‐bound sialic acid was found in the supernate. Non‐urea solvents yielded supernates rich in proteins but low in phosphorus and carbohydrates. Urea starch gel electrophoresis demonstrated the heterogeneity of the solubilized material. Serological studies showed high sensitivity of the Rh0(D)‐antigen to treatment with urea, mercaptoethanol and iodoacetamide. The M‐antigen was present in all residues and some M activity was found in the urea supernate. No acetylcholin‐esterase (AChE) activity was detected in the residue and supernate of stroma treated with 8 M urea. To ascertain the critical molarity, red blood cells and ghosts were incubated at increasing urea concentrations (0.5–6.0 M) and complete loss of AChE activity was observed with 4 M urea. Considerable reduction of AChE (70 per cent) was found after mild reduction and alkylation of stroma. When mercaptans and iodoacetamide were tested separately, no significant effect on AChE activity was observed except with high concentrations of cysteine.