Purification of erythrocyte spectrin alpha- and beta-subunits 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 human erythrocyte spectrin was developed, and structural properties and association behavior of the isolated subunits were studied by means of CD, nondenaturing gel electrophoresis, and analytical ultracentrifugation. The alpha- and beta-subunits were isolated using ion-exchange FPLC (pH 11) followed by size-exclusion FPLC (pH 7.5), having shown that alkaline pH dissociates spectrin polymers to their monomers [see Fujita et al. (1998) Biochemistry 37, 264-271]. The isolated subunits had alpha-helical content and thermal stability almost equivalent to those of native spectrin and reassembled to form heterodimers and tetramers which were indistinguishable from native spectrin with respect to secondary structure content, thermal stability, migration pattern on nondenaturing gels, and sedimentation coefficients. Thus, our data show that the increase in the structural stability of a heterodimer by association of the two monomers is very small. Sedimentation coefficients for the isolated alpha- and beta-subunits were 6.3 and 5.7 S, respectively. The similar frictional ratios (f/f0) of the isolated alpha-subunit (2.42) and the beta-subunit (2.45) indicate that the flexibility of both these wormlike chains and the range of shapes they can adopt in solution are very similar. The f/f0 value for spectrin dimer (2.41) indicates that its flexibility is somewhat, but not grossly, reduced compared to that of the individual subunits. Consequently, the folded repeat units of the subunits and the flexible connections between them are probably "in register" along the length of the dimer.
Fujita
T
T
Department of Biochemistry, University of Sydney, NSW, Australia.
Ralston
G B
GB
Morris
M B
MB
D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012044:Regression Analysis; D013049:Spectrin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014461:Ultracentrifugation
D013049:Spectrin