Purification of erythrocyte spectrin alpha- and beta-subunits at alkaline pH and structural and hydrodynamic properties of the isolated subunits

Biochemistry
T FujitaM B Morris

Abstract

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 flexi...Continue Reading

References

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Citations

Jun 29, 2012·Biochemistry·K S VishwanathaB A Eipper
Oct 26, 2001·Protein Expression and Purification·D G Kakhniashvili, S R Goodman

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