Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-trifluoroethanol solutions.
Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more alpha-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivity indicated that the active sites of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.
Laboratory of Molecular Enzymology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Xiang
X
Wei
Sheng
S
Ding
Yan
Y
Jiang
Xian-Gang
XG
Zeng
Hai-Meng
HM
Zhou
D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D002417:Cattle; D002942:Circular Dichroism / Q000379:methods; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D014270:Trifluoroethanol / Q000737:chemistry*
D014270:Trifluoroethanol; D024402:Carbonic Anhydrase II