title: High-pressure refolding of disulfide-cross-linked lysozyme aggregates: thermodynamics and optimization.
abstract: Previous exploratory work revealed that high pressure (200 MPa), 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 study we varied the redox conditions and levels of guanidine hydrochloride (GdnHCl) in the refolding buffer. Maximum refolding yields of 80% were seen at 1 M GdnHCl; higher concentrations did not increase refolding yields further. A maximum in 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 8 h, respectively. Estimates of the free energy of unfolding of lysozyme in GdnHCl solutions at 200 MPa suggested that the native state of lysozyme is strongly favored (ca.18.6 kJ/mol) under the conditions used for dissolution and refolding.
authors.affiliation: Department of Chemical Engineering, University of Colorado, Boulder, Colorado 80309, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: St  John
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D019803:Glutathione Disulfide / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011312:Pressure; D011487:Protein Conformation; D017510:Protein Folding; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D009113:Muramidase; D019791:Guanidine; D019803:Glutathione Disulfide