The detection of kinetic intermediate(s) during refolding of rhodanese.
Recent studies showed that the enzyme rhodanese could be reversibly unfolded in guanidinium chloride (GdmCl) if aggregation and oxidation were minimized. Further, these equilibrium studies suggested the presence of intermediate(s) during refolding (Tandon, S., and Horowitz, P. (1989) J. Biol. Chem. 264, 9859-9866). The present work shows that native and refolded enzymes are very similar in structural and functional characteristics. Kinetics of denaturation/renaturation were used to detect the folding intermediate(s). The shift in fluorescence wavelength maximum was used to monitor the structural changes during the process. First order plots of the structural changes during unfolding and refolding show nonlinear curves. The refolding occurs in at least two phases. The first phase is very fast (t1/2 much less than 30 s) and accounts for the partial regain in the structure but not in the activity. The second phase is slow (t1/2 = 2.9 h) during which the enzyme fully regains its structure along with the activity. The fractional renaturation of rhodanese due to the fast phase, monitored in various concentrations of GdmCl, describes a transition centered at 3.5 M GdmCl which is very similar to the higher of the two transitions observed in the reversible refolding. All of these findings support the presence of detectable intermediate(s) during folding of rhodanese.
Tandon
S
S
University of Texas Health Science Center, Department of Biochemistry, San Antonio 78284.
Horowitz
P M
PM
D000818:Animals; D002417:Cattle; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013466:Sulfurtransferases / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*; D013997:Time Factors; D014364:Tryptophan
D006146:Guanidines; D014364:Tryptophan; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D019791:Guanidine