Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis.
Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.
Cleland
J L
JL
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Wang
D I
DI
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004548:Elasticity; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation
D007527:Isoenzymes; D046911:Macromolecular Substances; D002256:Carbonic Anhydrases