Spectrin loss during in vitro red cell lysis.
Spectrin was extracted from washed erythrocyte ghosts in 1 mM EDTA buffer (pH 8.0) and purified to homogeneity by gel filtration. Anti-human spectrin was raised in rabbits. Specificity of the antibody was demonstrated by immunodiffusion, immunoelectrophoresis and immunofluorescent techniques. Membrane-free hemolysate prepared by lysing red cells in 5 mM phosphate buffer (pH 8.0) for variable intervals (5--60 min) at 4 degrees C was found to contain spectrin identifiable by immunodiffusion, immunoelectrophoresis, immunofluorescence and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Spectrin was demonstrable in ultracentrifuged membrane-free hemolysate and, in progressively decreasing amounts, in membrane washes. Membrane-free hemolysate contained more spectrin when erythrocytes were lysed for 60 min than for 5 min. The data indicate that a significant amount of spectrin is detached from the membrane following sysis in hypotonic buffer for different time intervals. Spectrin lost in this manner might be part of spectrin attached to the lipid bilayer.
D002458:Cell Fractionation; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D005188:False Negative Reactions; D005455:Fluorescent Antibody Technique; D006461:Hemolysis; D006801:Humans; D007106:Immune Sera; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008565:Membrane Proteins / Q000032:analysis*; D013049:Spectrin / Q000032:analysis*; D013997:Time Factors
D007106:Immune Sera; D008565:Membrane Proteins; D013049:Spectrin