Protein involvement in structural transition of erythrocyte ghosts. Use of thermal gel analysis to detect protein aggregation.

In this study, it is shown that systematic temperature-induced protein aggregation occurs on the erythrocyte membrane by intermolecular disulfide bond formation. Specific protein bands disappear from acrylamide gel profiles over rather narrow temperature regions. The aggregation appears to be the result of irreversible structural transitions of the membrane, which can be seen in a sensitive scanning calorimeter. When this method of thermal gel analysis is used, the results suggest that spectrin is a participant in the A transition, that bands 2.1, 4.1, and 4.2 and the cytoplasma portion of 3 are involved in the B transition, and that the transmembrane portion of band 3 may undergo changes in the C transition, previously shown to occur in the anion transport domain of the membrane. The aggregation of specific proteins in the narrow temperature region of these transitions persists as the transitions are moved around on the temperature axis by varying solution conditions. The assignment of particular proteins to specific transitions is reinforced by selective extraction of membrane proteins. Large variations in both the calorimetry and the aggregation pattern occur as salt concentration is increased from 77 mosm to 310 mosm, which is manifested in the splitting of the B transition into two separate transitions, B1 and B2. It is speculated that this occurs as the result of a structural change which may involve components of the cytoskeletal network.