Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B. Reaction stoichiometry and refolding model.
Polyethylene glycol (PEG) inhibited aggregation during refolding of bovine carbonic anhydrase B (CAB) through the formation of a nonassociating PEG-intermediate complex. Stoichiometric concentrations of PEG were required for complete recovery of active protein during refolding at aggregating conditions. For example, a PEG (Mr = 3350) to CAB molar ratio ([PEG]/[CAB]) of 2 was sufficient to inhibit aggregation during refolding at 1.0 mg/ml (33.3 microM) protein and 0.5 M guanidine hydrochloride. In addition, the PEG concentration required for enhancement was dependent upon the molecular weight and only molecular weights between 1000 and 8000 were effective in inhibiting aggregation. In the presence of PEG, the rate of refolding was the same as that observed for refolding without the formation of associated species. Refolding in the presence of PEG resulted in the rapid formation of a PEG complex with the molten globule first intermediate, and this PEG-intermediate complex did not aggregate. The CAB refolding kinetics in the presence of PEG were determined and used to develop a model of the PEG enhanced refolding pathway. The mathematical model was validated by independent activity measurements of CAB refolding. This model predicted that PEG enhanced refolding of CAB occurred by a specific interaction of PEG with the molten globule first intermediate to form a nonassociating complex which continued to fold at the same rate as the first intermediate. The predicted pathway and binding properties of PEG indicate that PEG enhanced refolding may be analogous to chaperonin mediated protein folding.
Cleland
J L
JL
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Hedgepeth
C
C
Wang
D I
DI
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; D011092:Polyethylene Glycols; D002256:Carbonic Anhydrases