Transient association of the first intermediate during the refolding of bovine carbonic anhydrase B.
Many proteins which aggregate during refolding may form transiently populated aggregated states which do not reduce the final recovery of active species. However, the transient association of a folding intermediate will result in reduced refolding rates if the dissociation process occurs slowly. Previous studies on the refolding and aggregation of bovine carbonic anhydrase B (CAB) have shown that the molten globule first intermediate on the CAB folding pathway will form dimers and trimers prior to the formation of large aggregates (Cleland, J. L.; Wang, D. I. C. Biochemistry 1990, 29, 11072-11078; Cleland, J. L.; Wang, D. I. C. In Protein Refolding; Georgiou, G., De-Bernardez-Clark, E., Eds.; ACS Symposium Series 470; American Chemical Society: Washington, DC, 1991; pp 169-179). Refolding of CAB from 5 M guanidine hydrochloride (GuHCl) was achieved at conditions ([CAB]f = 10-33 microM, [GuHCl]f = 1.0 M) which allowed complete recovery of active protein as well as the formation of a transiently populated dimer of the molten globule intermediate on the refolding pathway. A kinetic analysis of CAB refolding provided insight into the mechanism of the association phenomenon. Using the kinetic results, a model of the refolding with transient association was constructed. By adjusting a single variable, the dimer dissociation rate constant, the model prediction fit both the experimentally determined active protein and dimer concentrations. The model developed in this analysis should also be applicable to the refolding of proteins which have been observed to form aggregates during refolding. In particular, the transient association of hydrophobic folding intermediates may also occur during the refolding of other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Biotechnology Process Engineering Center, Department of Chemical Engineering, Cambridge, Massachusetts 02139.
J L
JL
Cleland
D I
DI
Wang
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D011487:Protein Conformation
D006146:Guanidines; D046911:Macromolecular Substances; D002256:Carbonic Anhydrases; D019791:Guanidine