Fluorimetric study of the artificial chaperone-assisted renaturation of carbonic anhydrase: a kinetic analysis.
It is now well accepted that ionic detergents along with alpha- or beta-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 beta-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.
Khodarahmi
Reza
R
Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, The University of Tehran, Tehran, Iran.
Yazdanparast
Razieh
R
D000818:Animals; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism; D003619:Dansyl Compounds / Q000494:pharmacology; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D019791:Guanidine / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008823:Micelles; D018832:Molecular Chaperones / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D003619:Dansyl Compounds; D003902:Detergents; D005456:Fluorescent Dyes; D007477:Ions; D008823:Micelles; D018832:Molecular Chaperones; C001026:5-dimethylaminonaphthalene-1-sulfonamide; D002256:Carbonic Anhydrases; D019791:Guanidine