| Arginine Kinase (ATP: L-arginine phosphotransferase EC 2.7.3.3), is a member of highly conserved family of phosphotransferases. It is widely used as a model protein in the investigation of proteins'folding and unfolding. Osmolytes are a series of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. However, the protective effects of osmolytes on arginine kinase folding and unfolding haven't been reported. In this study, we focused on analyzing the mechanism of their protective effects on AK and we provided insight into the role of chemical chaperones that act against guanidine denaturation.We extracted AK from locust (Migratoria manilensis) and purified it by Sephacryl S-300 and DEAE-Sephadex A50 chromatography. In this study, we investigated the mechanism of AK unfolding and the protective effects of the four osmolytes-proline, sucrose, DMSO and glycerol by measuring AK's activity, observing the fluorescence emission spectra and ANS-binding fluorescence spectra. The results showed that all the osmolytes applied in this study effectively prevented AK unfolding and inactivation in a concentration-dependent manner by reducing the inactivation rate constant ki, increasing the transition free energy changesΔΔGi, increasing the value for the midpoint of denaturation Cm and stabilizing the native conformation of AK. Moreover, during the AK refolding, osmolytes remarkably inhibited aggregation and induced AK to folding into the correct conformation.All the results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.
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