Takashi Fujita, Gregory B. Ralston, and Michael B. Morris
Department of Biochemistry, The University of Sydney, Sydney NSW 2006, Australia
Human erythrocyte spectrin is the major protein of the red cell cytoskeleton. The flexible and elastic properties of spectrin are believed to be the origin of the elastic deformability of the erythrocyte. Spectrin consists of a 280 kDa [[alpha]]-chain and a 246 kDa [[beta]]-chain, both of which consist largely of 106-amino-acid repeat motifs. These repeat units are connected in a "tandem-like" fashion by small random coil regions. Spectrin heterodimers, which result from antiparallel side-to-side association of the [[alpha]] and [[beta]] chains, self-associate in a "head-to-head" manner into tetramers, and higher oligomers.
In this study, the self-association of human erythrocyte spectrin has been studied over a wide range of pH (6-12) using 1H NMR, UV spectrophotometry and analytical ultracentrifugation. Also, properties of isolated spectrin subunits were investigated by non-denaturing gel electrophoresis, analytical ultracentrifugation and circular dichroism.
The envelope area of the narrow aliphatic resonances in spin-echo 1H NMR spectra of spectrin increased sharply as the pH was increased from 9.5 to 12. This transition coincided with up field shifts of tyrosine and lysine chemical shifts as well as an increase in UV absorbance at 294 nm. These data indicate that cooperative unfolding of spectrin correlates with the deprotonation of tyrosine and lysine residues of the molecule. Sedimentation velocity experiments showed that between pH 9.5 and 11, tetramers dissociated into dimers and above pH 11 dimers dissociated into individual monomers. This shows that unfolding of the spectrin induced by the deprotonation of tyrosine and lysine residues led to complete dissociation of spectrin dimers and other oligomers. It is likely that protonated tyrosine and lysine residues are involved in the stabilisation of spectrin's domain structures, probably the triple helical bundles of the repeat motifs.
Preparative amounts of spectrin [[alpha]] and [[beta]] subunits have been successfully isolated at high pH (~11) by ion-exchange FPLC followed by size-exclusion FPLC at physiological pH. The method provides similar yields of [[alpha]]- and [[beta]]-subunit, but with superior purity, compared to that obtained with the "conventional method" in which 3M urea is used to dissociate the subunits (Yoshino & Marchesi, 1984). Non-denaturing gel electrophoresis showed that the isolated monomers could form dimers and other oligomers following recombination under physiological conditions. Sedimentation coefficients for the [[alpha]]- and [[beta]]-subunit are ~6.1 S and ~5.6 S, respectively, at pH 7.5, 20[[ring]]C. CD spectra of the purified [[alpha]]-subunit and the reconstituted [[alpha]]+[[beta]] heterodimer were nearly identical to that of native spectrin, while the [[beta]]-subunit has less [[alpha]]-helix content, indicating the relative instability of this subunit.
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