Use of high-speed size-exclusion chromatography for the study of protein folding and stability

Abstract

The urea denaturation of sperm whale myoglobin and the thermal denaturation of [bovine pancreatic] RNase were studied by following the associated volume changes by size-exclusion chromatography on a Toya Soda TSK 3000SW gel permeation column. The permeation properties of the gel were invariant in the following solvent systems: 0.2 M NaCl; 8.0 M urea-0.2 M NaCl; and 6.0 M guanidinium chloride (GdmCl). A precise measurement of the volume changes associated with solvent-induced protein denaturation is thus practicable. The column was calibrated in the above solvent systems by using 12 well-characterized proteins as standards. In the case of the denaturation of myoglobin by urea, the rate of equilibration of folded and unfolded species is slow on the time scale of the chromatographic experiment, and the 2 forms are well separated on the column in the transition region. Both the folded and unfolded species undergo significant swelling in urea, suggesting that the view of denaturation based solely on the preferential solvation of the unfolded protein is incorrect. The rate of interconversion between folded and unfolded RNase is fast relative to the time scale of the chromatographic experiments. This is reflected in the fact that only 1 peak is observed in the elution profiles of RNase in the transition region. Thermally unfolded RNase has a smaller volume than the unfolded state in urea or GdmCl, suggesting that it has residual structure. The van''t Hoff .DELTA.H for the thermal unfolding of RNase calculated from the size-exclusion chromatographic experiments (36 .+-. 3 kcal/mol) is significantly lower than previously reported values. There are probably contributions to the enthalpy of unfolding which have negligible volume changes.