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High‐Pressure Refolding of Disulfide‐Cross‐Linked Lysozyme Aggregates: Thermodynamics and Optimization
Author(s) -
John Richard J. St.,
Carpenter John F.,
Randolph Theodore W.
Publication year - 2002
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0200200
Subject(s) - lysozyme , chemistry , redox , dissolution , guanidine , covalent bond , kinetics , glutathione , protein aggregation , circular dichroism , oxidizing agent , yield (engineering) , chromatography , organic chemistry , biochemistry , enzyme , thermodynamics , physics , quantum mechanics
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.