    1.1.1.363 | guanidine-HCl |
between 0.9 and 1.2 M denaturant the enzyme undergoes a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurrs at denaturant concentrations above 1.4 M. In the denaturant concentration range of 1.7-1.9 M the fluorescence change occurrs in two distinct steps. The first step involves a large, very rapid drop in fluorescence whose rate is strongly dependent on the denaturant concentration. This is followed by a small, relatively slow rise in the emission intensity, the rate of which is independent of denaturant concentration. Enzymatic activity is lost with a denaturant-concentration-dependent rate, which is approx. 3times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regains up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates are able to reactivate only minimally and in fact are found to aggregate and precipitate out of solution |
721694 |
