Biophysical properties of human erythrocyte spectrin at alkaline pH: implications for spectrin structure, function, and association.

The effects of pH 6-13 on the conformation and assembly of spectrin were studied by means of analytical ultracentrifugation, circular dichroism (CD), 1H NMR, and UV spectrophotometry. Sedimentation velocity analysis showed that spectrin oligomers dissociate cooperatively into component alpha- and beta-subunits above pH 9.5, and that spectrin tetramers, heterodimers, and monomers adopt more extended and/or expanded shapes above this pH. The dissociation to monomers is mostly completed by pH 10.5 and is used as the basis for purifying the subunits [see Fujita et al. (1998) Biochemistry 37, 272-280]. Along with the dissociation, biphasic unfolding of spectrin was observed above pH 9.5 as detected by CD. The first phase of the transition occurred between pH 9.5 and 11, and the second phase between pH 11 and 13. A similar biphasic dependence was observed for the upfield shift of lysine epsilon-CH2 resonances detected by spin-echo 1H NMR and the spectrophotometric titration of the absorbance at 294 nm. These data indicate that deprotonation of tyrosine and lysine residues is closely correlated with (i) the dissociation of spectrin oligomers into heterodimers, (ii) the dissociation of heterodimers into monomers, and (iii) the unfolding of spectrin. Taken together, our data suggest that hydrophobic and electrostatic interactions involving tyrosine and lysine residues play a critical role in the formation of the alpha-helix of spectrin and assembly of physiologically relevant spectrin oligomers from the two component subunits.