A comparison of the denaturants urea and guanidine hydrochloride on protein refolding

The effect of the denaturants guanidine hydrochloride (GdnHCl) and urea an enzyme activity has been investigated. Inactivation of hen egg white lysozyme and bovine carbonic anhydrase was monitored by enzyme activity assays, and fluorescence spectroscopy was used to detect conformational changes. The results show that lysozyme and carbonic anhydrase were inactivated at GdnHCl and urea concentrations which were much lower than those required to cause denaturation. Ionic strength effects, examined by addition of NaCl, were found to significantly reduce the activity of lysozyme and carbonic anhydrase to different extents. Denaturant type was also found to affect refolding yields. The refolding of 0.015 mg ml(-1) reduced, denatured lysozyme resulted in a maximum 65% recovery of activity from GdnHCl and 50% from urea. The first order rate constants for refolding were found to be 2.4 X 10(-3) s(-1) and 2.5 X 10(-3) s(-1) for GdnHCl and urea denatured lysozyme respectively. It is believed that the differences in yield from these two denaturants are due to their effect on protein aggregation. Concentrations of GdnHCl greater than 0.01 M present during refolding greatly reduced the yield of active protein.