Spectroscopic Studies on Denaturants and Guluronate Lyase from a Marine Bacterium

Abstract

The denaturation and renaturation of a poly(α-L-guluronate)lyase from a marine bacterium with sodium dodecyl sulfate, guanidine hydrochloride (GHCl), and urea were investigated mainly by reflection of changes in the enzyme activity, far- and near-ultraviolet circular dichroism, and fluorescence. Amino acid analysis of the enzyme was also done to characterize the protein. The enzyme activity was lost with 6 M GHCl and 3 M urea in the assay mixture, but not 3% sodium dodecyl sulfate with the high activity. However, the far-ultraviolet circular dichroic spectra indicated that the secondary structure was hardly affected by treatment with 6 M GHCl, but was influenced by 3 M urea and 3% sodium dodecyl sulfate. By contrast, the near-ultraviolet circular dichroic spectra were attenuated by treatment with 6 M GHCl and 3 M urea, suggesting changes in the environments of tryptophan residues. Removal of the denaturants by dialysis almost restored all of the original protein conformation. The enzyme was more susceptib...