Mechanism of Unfolding of Goat Lung Cystatin During Urea and Guanidine Hydrochloride Induced Denaturation

ArticleinInternational Journal of Peptide Research and Therapeutics 15(1):81-86 · March 2009with6 Reads
Impact Factor: 0.91 · DOI: 10.1007/s10989-008-9166-8

    Abstract

    Cystatins essentially regulate lysosomal cysteine protease besides affecting several physiological processes. In the present
    study, denaturation of a high molecular weight cystatin (Mr 66.4kDa) purified from goat lung (GLC-I) has been studied by
    monitoring its inhibitory activity, intrinsic fluorescence, circular dichroism (CD), and binding of ANS. It was found that
    increasing concentration of GdnHCl significantly enhances the inactivation and unfolding of the purified inhibitor (GLC-I)
    with complete loss of inhibitory activity at 4M GdnHCl. Denaturation of GLC-I in the presence of GdnHCl is accompanied by
    red shift (15nm) of the emission maximum as shown by intrinsic fluorescence. The inhibitory activity of GLC-I was increased
    by 1.5 fold at 2M urea; however, it decreased with further increased of the urea concentration. Intrinsic fluorescence studies
    of GLC-I in the presence of 0–3M urea shows blue shift of 5nm, suggesting stabilization of the inhibitor followed by 5nm
    red shift at higher concentration. ANS binding studies in the presence of urea indicate significant changes in the tertiary
    structure of the inhibitor. Thus, our result shows denaturation profile of GLC-I following simple two state transitions in
    the presence of GdnHCl while it proceeds through an intermediate state in the presence of urea.