Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering

Role of disulfide bridges in phytase's unfolding-refolding was probed using dynamic light scattering. Phytase was unfolded by guanidinium chloride and then refolded by removing the denaturant by dialysis. Thiol reagents prevented refolding; thus, disulfide bridge formation is an integral step in phytase folding. Catalytic demise of phytase after unfolding and refolding in presence of Tris(2-carboxyethyl)phosphine (TCEP) indicates that disulfide bridges are necessary for refolding. The hydrodynamic radius (rh) of active and unfolded phytase is 4 and 14 nm, respectively. Removal of denaturant through dialysis refolds phytase; its rh shifts back to 4 nm. When TCEP remains in the refolding media, the rh remains high. The unfolded phytase when diluted in assay medium refolds as a function of time at 25 and 37 °C, but not at higher temperature. Monitoring rh under denaturing and renaturing condition gives an accurate measure of the folding status of phytase.