Effect of high concentration of inert cosolutes on the refolding of an enzyme: carbonic anhydrase B in sucrose and ficoll 70.
The kinetics of refolding of carbonic anhydrase II following transfer from a buffer containing 5 m guanidinium chloride to a buffer containing 0.5 m guanidinium chloride were studied by measuring the time-dependent recovery of enzymatic activity. Experiments were carried out in buffer containing concentrations of two "inert" cosolutes, sucrose and Ficoll 70, a sucrose polymer, at concentrations up to 150 g/liter. Data analysis indicates that both cosolutes significantly accelerate the rate of refolding to native or compact near-native conformations, but decrease the fraction of catalytically active enzyme recovered in the limit of long time. According to the simplest model that fits the data, both cosolutes accelerate a competing side reaction yielding inactive compact species. Acceleration of the side reaction by Ficoll is significantly greater than that of sucrose at equal w/v concentrations.
Monterroso
Begoña
B
Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: monterrosob@niddk.nih.gov.
Minton
Allen P
AP
Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
D000818:Animals; D002021:Buffers; D024401:Carbonic Anhydrase I / Q000737:chemistry*; D002384:Catalysis; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D005362:Ficoll / Q000737:chemistry*; D007700:Kinetics; D015233:Models, Statistical; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose / Q000737:chemistry*; D013997:Time Factors
D002021:Buffers; D005362:Ficoll; D013395:Sucrose; D024401:Carbonic Anhydrase I