Heat-induced dissociation of human erythrocyte spectrin dimer into monomers

Human erythrocyte spectrin heated above 49 degrees C could be separated into two fractions by DEAE-Toyopearl column chromatography at room temperature. The first fraction eluting with the salt gradient was predominantly the alpha subunit, indicating a heat-induced dissociation of the spectrin alpha beta dimer into monomers. The second fraction, obtained with 0.5 M NaOH after salt elution, consisted of high-molecular-weight proteins in addition to alpha and beta subunits, which were visualized by gel electrophoresis with sodium dodecyl sulfate. The isolated beta subunit when heated above 48 degrees C could also be separated into two fractions by column chromatography. About 30% of the protein eluted with the salt solution and the rest of the proteins were in the alkali eluate in which high molecular weight protein bands also appeared, indicating a heat-induced aggregation of the beta subunits. Almost all the isolated alpha subunit, however, eluted out with the salt solution, even though the subunit was heated at 52 degrees C. Studies of the binding of subunits to inside-out vesicles indicate that the isolated beta subunit was denatured irreversibly by heating; on the other hand, the alpha subunit kept its binding ability after heating above 50 degrees C. These findings are attributed to the heat-induced dissociation of the spectrin molecules into alpha and beta subunits at 49-50 degrees C, and eventual aggregation of the denatured beta subunits.