Figure 1.

Reduction of Hsp33 is not sufficient to cause substrate release from Hsp33. Light scattering measurements of luciferase (140 nM) were performed either in the absence (a) or presence of 210 nM active Hsp33 dimers (b, c) at 43°C. At the time point indicated by the arrow, 5 mM DTT was added to reactions a and b, and the incubation was continued at 43°C. Inset: DTT induces the fast reduction of Hsp33's thiol groups. Dimeric oxidized Hsp33 in complex with thermally unfolded luciferase (left panel) or oxidized Hsp33 alone (right panel) was incubated with 5 mM DTT at 25°C. At the time points indicated, aliquots were withdrawn and free thiol groups were covalently modified with the 500 Da molecule AMS as described. Similar results were obtained at 43°C, albeit with significantly faster reduction rates.