Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride.
The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8-1.0 mol/L and 0.3-0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8-1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3-0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.
Tang
Hong-Min
HM
College of Life Science, Yunnan University, Kunming, China.
Yu
Hong
H
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry*; D002942:Circular Dichroism; D033364:Decapoda / Q000201:enzymology; D019791:Guanidine / Q000494:pharmacology*; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000817:Anilino Naphthalenesulfonates; D001122:Arginine Kinase; D019791:Guanidine