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