Fluorimetric study of the artificial chaperone-assisted renaturation of carbonic anhydrase: A kinetic analysis

It is now well accepted that ionic detergents along with α- or β-cyclodextrins can enhance protein refolding yields. In this report, we evaluated the effect of detergent's tail length on the kinetics of denatured carbonic anhydrase refolding along with determining the rate-limiting step of the whole refolding process. A sensitive fluorimetric technique was also developed to follow up the second-by-second fate of the denatured protein while undergoing refolding. In this technique, inclusion complexes are formed between the correctly refolded CA and the fluorescent active site probe, 5-dimethylaminonaphtalene-1-sulfonamide. By this specific technique, it became evident that the rate of detergent stripping from the CA-detergent mixed micelles that also appeared to be the rate-limiting step depends on the β-CD-detergent association constants which are under the influence of detergent's tail length. Based on these findings, appropriate refolding conditions could be designed to kinetically diminish the rate of off-pathway aggregation.