Red cell membrane protein changes caused by freezing and the mechanism of cryoprotection by glycerol

Membranes isolated from frozen-thawed erythrocytes and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate have significantly decreased band six, which is the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase. The total membrane protein and sialic acid contents of these membranes are also significantly decreased. The red blood cell membrane protein abnormality is reproduced by suspending membranes in NaCl solutions of increasing molarity. Glycerol prevents the elution of band six in NaCl solutions less than 0.2 M and ameliorates it in solutions of higher ionic strength. When intact cells are suspended in hypertonic salt solution, there is no elution of band six, indicating that exposure of the inner surface of the membrane to toxic concentrations of solutes results in this elution. The data indicate that freezing with its associated hypertonicity induces a specific membrane change which is ameliorated by the addition of glycerol.