Reactivation of kinetics of guanidine denatured bovine carbonic anhydrase B.

Denaturation and reactivation of bovine carbonic anhydrase B was studied with particular attention to the anomalous behavior in the transition region (about 2 m guanidine hydrochloride) that had been reported by previous workers. The denaturation curve based on the partition coefficient of gel chromatography was markedly different from the one based on the ultraviolet difference spectroscopy. Intrinsic viscosity and fluorescence intensity were also measured in guanidine solution. Reactivation of esterase activity of the enzyme was over 90% complete with an average half time of 9 ± 1 min when the protein was fully denatured in 5 m guanidine hydrochloride. Similar reactivation from 2 m guanidine solution showed the dependence of the extent of final activity regain on the time of incubation in 2 m guanidine solution. It decreased to a plateau value of 40–50% of the native activity after 24 h in 2 m guanidine. The irreversibly inactivated fraction could be reactivated if it was transferred to 5 m guanidine before reactivation experiment. Renaturation kinetics followed by ultraviolet difference spectroscopy showed a fast phase ($\text{t}\text{1}\text{2}\text{< 30}\text{sec}$) and a slow phase ($\text{t}\text{1}\text{2}\text{= 7 ± 1}\text{min}$).