Refolding intermediate of guanidine hydrochloride denatured aminoacylase.
The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.
Xie
Qiang
Q
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China.
Zhou
Hai-Meng
HM
D000581:Amidohydrolases / Q000737:chemistry*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D015032:Zinc / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc; D019791:Guanidine