PURIFICATION OF ERYTHROCYTE SPECTRIN ALPHA-SUBUNIT AND BETA-SUBUNIT AT ALKALINE PH AND STRUCTURAL AND HYDRODYNAMIC PROPERTIES OF THE ISOLATED SUBUNITS

A new method for the isolation of the alpha- and beta-subunits of huma n erythrocyte spectrin was developed, and structural properties and as sociation behavior of the isolated subunits were studied by means of C D, nondenaturing gel electrophoresis, and analytical ultracentrifugati on. The alpha-and beta-subunits were isolated using ion-exchange FPLC (pH 11)followed by size-exclusion FPLC (pH 7.5), having shown that alk aline pH dissociates spectrin polymers to their monomers [see Fujita e t al. (1998) Biochemistry 37, 264-271]. The isolated subunits had alph a-helical content and thermal stability almost equivalent to those of native spectrin and reassembled to form heterodimers and tetramers whi ch were indistinguishable item native spectrin with respect to seconda ry structure content, thermal stability, migration pattern on nondenat uring gels, and sedimentation coefficients. Thus, our data show that t he increase in the structural stability of a heterodimer by associatio n of the two monomers is very small. Sedimentation coefficients for ti le isolated alpha- and beta-subunits were 6.3 and 5.7 S, respectively. The similar frictional ratios (f/f(0)) of the isolated alpha-subunit (2.42) and the beta-subunit (2.45) indicate that the flexibility of bo th these wormlike chains and the range of shapes they can adopt in sol ution are very similar. The f/f(0) value for spectrin dimer (2.41) ind icates that its flexibility is somewhat, but not grossly, reduced comp ared to that of the individual subunits. Consequently, the folded repe at units of the subunits and the flexible connections between them are probably ''in register'' along the length of the dimer.