J. Biol. Chem., Vol. 265, Issue 11, 5967-5970, 04, 1990
The detection of kinetic intermediate(s) during refolding of rhodanese
S Tandon and PM Horowitz
University of Texas Health Science Center, Department of Biochemistry, San Antonio 78284.
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.
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