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 we re studied by means of analytical ultracentrifugation, circular dichro ism (CD), H-1 NMR, and UV spectrophotometry, Sedimentation velocity an alysis showed that spectrin oligomers dissociate cooperatively into co mponent alpha- and beta-subunits above pH 3.5, and that spectrin tetra mers, heterodimers, and monomers adopt more extended and/or expanded s hapes 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 Fu jita er al. (1998) Biochemistry 37, 272-280], Along with the dissociat ion, biphasic unfolding, of spectrin was observed above pH 9.5 as dete cted 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 re sonances detected by spin-echo H-1 NMR and the spectrophotometric titr ation of the absorbance at 294 nm. These data indicate that deprotonat ion of tyrosine and lysine residues is closely correlated with (i) the dissociation of spectrin oligomers into heterodimers, (ii) the dissoc iation of heterodimers into monomers, and (iii) the unfolding of spect rin, Taken together, our data suggest that hydrophobic and electrostat ic interactions involving tyrosine and lysine residues play a critical role in the formation of the rr-helix of spectrin and assembly of phy siologically relevant spectrin oligomers from the two component subuni ts.