The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state.
Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.
Xie
Qiang
Q
Department of Biological Sciences and Biotechnology, Faculty of Science, Tsinghua University, 100084, Beijing, PR China,
Guo
Tai
T
Lu
Jie
J
Zhou
Hai-Meng
HM
D000581:Amidohydrolases / Q000737:chemistry*; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000737:chemistry* / Q000494:pharmacology; D007477:Ions; D007668:Kidney / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012492:Salts / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013552:Swine; D015032:Zinc / Q000737:chemistry
D007477:Ions; D012492:Salts; D011189:Potassium Chloride; D001120:Arginine; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc; D019791:Guanidine