title: Protein unfolding studies of thiol-proteinase inhibitor from goat (Capra hircus) muscle in the presence of urea and GdnHCl as denaturants.
abstract: In recent years, many advances have been made in the understanding of functional and structural characteristics of protein evolution from denaturant-based studies that subject the protein to a change in the microenvironment. This paper reports the chemical denaturation of purified goat muscle cystatin (GMC) a thiol-proteinase inhibitor, using urea and guanidine hydrochloride (GdnHCl). The subtle conformational changes of GMC were monitored by intrinsic fluorescence, extrinsic fluorescence, and CD spectroscopic techniques. Further, the activity of GMC as a function of increasing concentration of denaturants was also studied. It was found that increasing the concentration of GdnHCl significantly enhances the inactivation and unfolding of the inhibitor (GMC). In urea-induced denaturation, the intrinsic and extrinsic fluorescence intensity reveals significant structural changes in the inhibitor. Further, it was found that at low concentrations of urea, up to 0.5-1.0 M: , there was quenching of fluorescence intensity compared with the native form and a red shift of 5 nm was observed up to 5-8 M: . The results presented in this paper suggest that GdnHCl-induced denaturation of GMC follows a simple two-state rule in which native → denatured state transition occurs in a single step. However denaturation with urea proceeds through an intermediate or non-native state.
authors.affiliation: Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Uttar Pradesh, India.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Aatif
authors.forename: Safikur
authors.initials: S
authors.lastname: Rahman
authors.forename: Bilqees
authors.initials: B
authors.lastname: Bano
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006041:Goats; D019791:Guanidine / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D058767:Protein Unfolding / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*
chemical_list: D000817:Anilino Naphthalenesulfonates; D015891:Cystatins; D011480:Protease Inhibitors; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D019791:Guanidine