Denaturant-induced Unfolding Of Bovine Carbonic Anhydrase B And Re-folding Process And The Role Of Agglomeration

In this paper the unfolding and refolding procedures of bovine carbonic anhydrase B induced by urea and GdnHCl were studied by using intrinsic fluorescence spectroscopy, phase diagram, non-denatured PAGE, size-exclusion chromatography, laser light-scattering and SDS-PAGE.During the unfolding procedure of bovine carbonic anhydrase B induced by urea and GdnHCl, the denaturation solutions kept transparent without forming any precipatate. The intrinsic fluorescence spectroscopy and phase diagram showed that during the unfolding procedure of bovine carbonic anhydrase B induced by urea and GdnHCl, there were one and two intermediates respectively. The results of PAGE, SEC and light scattering showed that during the unfolding procedure induced by urea and GdnHCl, bovine carbonic anhydrase B existed in the denaturation solutions in a form of unimolecular bovine carbonic anhydrase B and bi-molecular bovine carbonic anhydrase B aggregate without any muti-molecular aggregate formed. Meanwhile, the results of SDS-PAGE showed that the bi-molecular bovine carbonic anhydrase B aggregate was formed through the hydrophobic and electrostatic interactions between the unimolecular bovine carbonic anhydrase B.During the refolding procedure of bovine carbonic anhydrase B induced by urea and GdnHCl, when the final urea and GdnHCl concentration in renaturation solution was higher than 1.0 mol/L and 0.7 mol/L respectively, the renaturation solutions kept transparent.; when the final urea or GdnHCl concentration was lower or equal to 1.0 mol/L and 0.7 mol/L respectively, an opaque precipitate was formed in the dilution refolding of urea-induced or GdnHCl-induced bovine carbonic anhydrase B. The intrinsic fluorescence spectroscopy and phase diagram showed that there was both one intermediate during the refolding procedure of bovine carbonic anhydrase B induced by urea and GdnHCl. The results of PAGE, SEC and light scattering showed that during the refolding procedure of bovine carbonic anhydrase B induced by urea, when the final urea concentration in renaturation solution was higher than 2.0 mol/L, bovine carbonic anhydrase B existed in the renaturation solutions in a form of unimolecular bovine carbonic anhydrase B and bi-molecular bovine carbonic anhydrase B aggregate; when the final urea concentration was in the range of 1.0-2.0 mol/L, they existed in a form of unimolecular bovine carbonic anhydrase B, bi-molecular bovine carbonic anhydrase B aggregate and a small amount of multi-molecular bovine carbonic anhydrase B aggregate; and when the final urea concentration was lower or equal to 1.0 mol/L, a transparent supernatant and an opaque precipitate were separately formed in the dilution refolding of urea-induced bovine carbonic anhydrase B, a dynamic dissociation equilibrium existed in the bovine carbonic anhydrase B included in the supernatant and that included in the precipitate and in both the supernatant and precipitate, bovine carbonic anhydrase B existed in a form of unimolecular bovine carbonic anhydrase B, bi-molecular bovine carbonic anhydrase B aggregate and a small amount of multi-molecular bovine carbonic anhydrase B aggregate. During the refolding procedure of bovine carbonic anhydrase B induced by GdnHCl, when the final GdnHCl concentration in renaturation solution was higher than 0.7 mol/L, bovine carbonic anhydrase B existed in the renaturation solutions in a form of unimolecular bovine carbonic anhydrase B and bi-molecular bovine carbonic anhydrase B aggregate; when the final GdnHCl concentration was lower or equal to 0.7 mol/L, a transparent supernatant and an opaque precipitate were separately formed in the dilution refolding of GdnHCl-induced bovine carbonic anhydrase B, a dynamic dissociation equilibrium existed in the bovine carbonic anhydrase B included in the supernatant and that included in the precipitate and in both the supernatant and precipitate, bovine carbonic anhydrase B existed in a form of unimolecular bovine carbonic anhydrase B, bi-molecular bovine carbonic anhydrase B aggregate and a small amount of multi-molecular bovine carbonic anhydrase B aggregate. Meanwhile, the results of SDS-PAGE showed that The bi-molecular and multi-molecular bovine carbonic anhydrase B aggregates were formed through the hydrophobic and electrostatic interactions between the unimolecular bovine carbonic anhydrase B, and only if the concentrations of these components in renaturation solution were higher than a certain value and the urea or GdnHCl concentration in the final renaturation solution was lower than a certain value, an aggregate precipitate could be formed through the non-covalent interactions among these components.The refolding and unfolding procedure of bovine carbonic anhydrase B induced by urea and GdnHCl was irreversible, the refolding procedure of bovine carbonic anhydrase B was much more complex than its unfolding one.