Control of aggregation in protein refolding: the temperature-leap tactic.
The kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4 degrees to 36 degrees, at the relatively high [CAII] of 4 mg/mL. Following dilution to 1 M guanidinium chloride, aggregate formation is very rapid and reduces the formation of active enzyme. The CAII activity yield at 150 min, 20 degrees (approximately 60%), is greater than that at either 4 degrees or 36 degrees. However, if refolding is conducted at 4 degrees, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36 degrees, the yield rises rapidly to 95% at 150 min. This is an example of the "temperature leap" tactic. These results can be understood on the basis of two slow-folding intermediate whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4 degrees, the first intermediate is depleted after 120 min, and the second intermediate rapidly isomerizes to active enzyme on warming. A series of experiments was conducted where the initial (120 min) folding temperature was systematically varied, followed by a "leap" to 36 degrees for 30 additional minutes. With initial incubations from 4 degrees to 12 degrees, the final yield is > 90%, drops rapidly from 12 degrees to 20 degrees, and decreases more gradually to approximately 45% at 36 degrees. The overall results qualitatively fit the simple idea of ordinary temperature-accelerated reactions in competition with hydrophobic aggregation, which is strongly suppressed in the cold. Qualifications are discussed for the temperature-leap approach to find application in refolding other proteins.
Xie
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Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Wetlaufer
D B
DB
D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D003080:Cold Temperature; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D009391:Nephelometry and Turbidimetry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature
D006146:Guanidines; D002256:Carbonic Anhydrases; D019791:Guanidine