Abstract
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.
Highlights
Rhodanese is an example of the growing refolded after denaturation in guanidinium chloride number of enzymes that have been recognized to be folded (GdmHCl).Renaturation wasby either (a)direct dilu- into structural domains
Thisenzyme is interesting since the protein is flexible and conformational changes appeatro be correlated ularly important because of reports that they denature rhodanese by different pathways [15].The present results show, for the first time, that rhodanese can be refolded after denaturation in GdmHCl to give a product with a high specific activity, and several parameters of the refolded and thenative enzyme have been compared
The results presented here indicate that, under the appropriate conditions, rhodanese is capable of being refolded to a high specific activity product after denaturation in urea or guanidine
Summary
The ability to regain active enzyme from the denatured state would greatly increase the usefulness of rhodanese as a model for study of the functional role of protein dynamics and domain interactions. This hasbeen held not tobe possible for the form of the enzyme normally isolated because under most circumstances it appears to be structurally labile [4, 10, 11] and to conformationally relax to inactive species [4, 10]. These intermediates may result from nucleation of the two rhodanese domains, which exposes hydrophobic surfaces that become the interdomain interface in the correctlyfolded protein
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