STUDIES OF THE HYDRODYNAMIC VOLUME CHANGES THAT OCCUR DURING REFOLDING OF LYSOZYME USING SIZE-EXCLUSION CHROMATOGRAPHY

A size-exclusion chromatography-based refolding process (SEPROS) has s uccessfully been used to renature lysozyme at high concentrations. Thi s process is based on the different hydrodynamic characteristics of fo lded and unfolded proteins and their interaction with gel filtration m edia. In this paper we have quantified the changes in Stokes radius, h ydrodynamic volume and partition coefficient that occur when lysozyme is refolded from urea in a size-exclusion column. In 8 M urea partiall y folded and unfolded lysozyme were resolved using Superdex 75 HR. The se two species were present at approximately the same concentration. A s the urea concentration was decreased the unfolded species gradually decreased until at 4 M urea only partially folded lysozyme remained, w hich continued to fold on further reduction of the urea concentration. Using these results the initial mechanism for size exclusion chromato graphy protein refolding has been confirmed.