Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II.

The denaturation and renaturation of carbonic anhydrase II (CAII) has been studied in several laboratories. Both thermodynamic and kinetic evidence support the existence of at least two intermediates between denatured and native protein. Previous studies have shown that on rapid dilution of a CAII solution from 5 M to 1 M guanidinium chloride, aggregation strongly competes with renaturation at higher protein concentrations, suggesting an upper limit for [CAII] of approximately 0.1%. Our experiments show 60% renaturation at 0.4% [CAII] and that aggregate formation is partially reversible. This yield can be substantially increased by several surfactant additives, including simple alkanols as well as micelle-forming surfactants. Effective surfactants (promoters) act by suppressing initial aggregate formation, not by dissolving aggregates. Promoters act on either the first folding intermediate (I1) or oligomers thereof. Eight of the 18 surfactants examined showed promoter activity, and no correlation was evident between promoter activity and chemical structure or surface tension lowering. These results indicate discrimination (molecular recognition) by I1 and/or its oligomers.