Use of high-speed size-exclusion chromatography for the study of protein folding and stability.
The urea denaturation of sperm whale myoglobin and thermal denaturation of ribonuclease have been 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 have been shown to be 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 two forms are well separated on the column in the transition region. Both the folded and unfolded species are shown to undergo significant swelling in urea. This result suggests 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 ribonuclease is fast relative to the time scale of the chromatographic experiments performed in this study. This is reflected in the fact that only one peak is observed in the elution profiles of ribonuclease in the transition region. Thermally unfolded ribonuclease 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 ribonuclease calculated from the size-exclusion chromatographic experiments (36 +/- 3 kcal/mol) is significantly lower than previously reported values.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D004355:Drug Stability; D006801:Humans; D008970:Molecular Weight; D011487:Protein Conformation; D011506:Proteins; D011817:Rabbits; D012997:Solvents; D013329:Structure-Activity Relationship; D014783:Viscosity
D011506:Proteins; D012997:Solvents