Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase in the presence of guanidine-HCL at low temperature

Denaturation of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase induced by incubation with guanidine-HCl (GdnHCl) at 4°C was examined. Enzyme activity was amplified by 2.5 times in 0.2 M GdnHCl; such an increase in activity was also detected in NaCl and KCl at concentrations less than 1 M, but not in urea. With increasing amount of GdnHCl, the enzyme lost half and all of its activity in 1.0 M and 1.6 M GdnHCl, respectively. Notable changes in fluorescence spectra of Trp residue and FAD cofactor besides circular dichroism in far UV region were detected and insufficiently correlated to the inactivation. Based on changes in fluorescence intensities and molecular ellipticity, a two-state transition equilibrium was suggested; transition midpoints were roughly at 1.7 M GdnHCl. In GdnHCl at concentrations above 2 M, the enzyme released FAD and formed inactive aggregate. Effects by GdnHCl at concentrations less than 1.4 M were mostly cancelled by its removal. Most results were similar to those from studies on the enzyme being one of components of pyruvate dehydrogenase complex.