Characterization of the Lateral Interaction between Human Erythrocyte Spectrin Subunits

Abstract

A technique in which the subunits of human erythrocyte spectrin were immobilized on a nitrocellulose membrane was developed to study which domains of the subunit are able to bind to the counterpart subunit. The limited tryptic digestion of the isolated α and β subunits of human erythrocyte spectrin produced eight fragments in the α subunits and nine fragments in the β subunit. Four fragments of the β (80, 60, 44, and 18 kDa) and two of the α (82 and 33 kDa) bound to α and β subunits which were immobilized on nitrocellulose membrane strips, respectively. The binding affinities of all the fragments to the subunits, however, were remarkably lower than that of the mother proteins. The titration of fluorescence anisotropy of N-(1-anilinonaphthyl-4)maleimide which was covalently attached to the subunit by the trypsin-digested fraction of the counterpart subunit also indicate weak binding of the fragments even in solution. These findings suggest that the high-affinity binding of the α subunit to the β subunit to form spectrin αβ dimer occurs only when the binding domains are arrayed along the polypeptide chains at the appropriate positions on the subunits.