Unfolding of truncated and wild type aspartate aminotransferase studied by size-exclusion chromatography.

The reversible unfolding of globular proteins with increasing concentration of guanidinium chloride (GuCl) can be analysed by size-exclusion chromatography, because the hydrodynamic volume of the proteins increases during unfolding. The dimeric enzyme aspartate aminotransferase (AAT) shows an uncoupled dissociation of the identical subunits followed by the unfolding of the monomers. During the monomer unfolding formation of an intermediate is observed. A monomeric mutant of AAT unfolds with a similar shape of the unfolding transition phase, but is less stable, as shown by a shift of the transition mid-point from 1.7 M GuCl for the wild type to 1.3 M GuCl for the mutant.