Previous exploratory work revealed that high pressure (200MPa0,in combination with oxido-shuffling agents such as glutathione,effectively refolds covalently cross-linked aggregates of lysozyme into catalytically active native molecules,at concentrations up to 2 mg/mL(1).To understand further and optimize this process,in the current wtudy we varied the redox conditions and levels of guanidine hydorchloride (GdnHCl) in the refolding buffer.Maximum refolding yields of 80% were seen at 1M GdnHCl;higher concentrations did not increase refolding yields further.A maximumin refolding yield was observed at redox conditions with a 1:1 ratio of oxidized to reduced glutathione (GSSG:GSH).Yields decreased dramatically at more oxidizing conditions ([GSSG]>[GSH]).Kinetics of dissolution and refolding of covalently cross-linked aggregates of lysozyme depended strongly on redox conditions.At GSSG:GSH ratios of 4:1,1:1,and 1:16,lysozyme dissolved and refolded with time constants of 62,20,and 8h,respectively.Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200MPa suggested that the native state of lysozyme is stronlgy favored (ca.18.6 kJ/mol)under the conditions used for dissolutiona nd refolding.
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