Article

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

International Journal of Peptide Research and Therapeutics (Impact Factor: 1.28). 01/2009; 15(1):81-86. 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.

0 Bookmarks
 · 
72 Views
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.
    Applied biochemistry and biotechnology 07/2013; · 1.94 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A thiol proteinase inhibitor from Capra hircus (goat) pancreas (PTPI) isolated by ammonium sulphate precipitation (20-80%) and gel filtration chromatography on Sephacryl S-100HR, with 20.4% yield and 500-fold purification, gave molecular mass of 44 kDa determined by its electrophoretic and gel filtration behavior, respectively. The stokes radius, diffusion and sedimentation coefficients of PTPI were 27.3 A, 7.87 x 10(-7) cm(2) s(-1) and 3.83 s, respectively. It was stable in pH range 3-10 and up to 70 degrees C (critical temperature, E (a) = 21 kJ mol(-1)). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain (K ( i ) = 5.88 nM). The partial amino acid sequence analysis showed that it shared good homology with bovine parotid and skin cystatin C. PTPI possessed 17.18% alpha helical content assessed by CD spectroscopy. The hydropathy plot of first 24 residues suggested that most amino acids of this stretch might be in the hydrophobic core of the protein.
    Amino Acids 08/2009; 38(4):1001-10. · 3.91 Impact Factor