The enzyme rhodanese can be reactivated after denaturation in guanidinium chloride.
For the first time, the enzyme rhodanese has been refolded after denaturation in guanidinium chloride (GdmHCl). Renaturation was by either (a) direct dilution into the assay, (b) intermediate dilution into buffer, or (c) dialysis followed by concentration and centrifugation. Method (c) preferentially retained active enzyme whose specific activity was 1140 IU/mg, which fell to 898 IU/mg after 6 days. The specific activity of native enzyme is 710 IU/mg. Progress curves were linear for the dialyzed enzyme, and kinetic analysis showed it had the same Km for thiosulfate as the native enzyme, but apparently displayed a higher turnover number. Progress curves for denatured enzyme directly diluted into assay mix showed as many as three phases: a lag during which no product formed; a first order reactivation; and an apparently linear steady state. An induction period was determined by extrapolating the steady-state line to the time axis. The percent reactivation fell to 7% (t1/2 = 10 min) as the time increased between GdmHCl dilution and the start of the assay, independent of the presence of thiosulfate. The induction period, which decreased to zero as the incubation time increased, was retained in the presence of thiosulfate. There were no observable differences between native and renatured protein by electrophoresis or fluorescence spectroscopy. Previous reports of some refolding of urea-denatured rhodanese (Stellwagen, E. (1979) J. Mol. Biol. 135, 217-229) were confirmed, extended, and compared with results using GdmHCl. A working hypothesis is that rhodanese refolding involves intermediates that partition into active and inactive products. These intermediates may result from nucleation of the two rhodanese domains, which exposes hydrophobic surfaces that become the interdomain interface in the correctly folded protein.
Horowitz
P M
PM
Simon
D
D
D000818:Animals; D002417:Cattle; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011489:Protein Denaturation; D013466:Sulfurtransferases / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
D006146:Guanidines; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D019791:Guanidine