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Biochemical, Immunological and Kinetic Characterisation of Thiol Protease Inhibitor (Cystatin) from Liver

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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.

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Authors and Affiliations

  1. Department of Biochemistry, SKIMS Medical College, Srinagar, Kashmir, India

    Aaliya Shah

  2. Department of Endocrinology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

    Medha Priyadarshini

  3. Department of Biochemistry, Aligarh Muslim University (AMU), F/O Life Sciences, Aligarh, India

    Mohammad Aatif, Fakhra Amin & Bilqees Bano

  4. PRC, Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia

    Mohd Shahnawaz Khan

Authors
  1. Aaliya Shah
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  2. Medha Priyadarshini
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  3. Mohd Shahnawaz Khan
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  4. Mohammad Aatif
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  5. Fakhra Amin
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  6. Bilqees Bano
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Correspondence to Bilqees Bano.

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Shah, A., Priyadarshini, M., Khan, M.S. et al. Biochemical, Immunological and Kinetic Characterisation of Thiol Protease Inhibitor (Cystatin) from Liver. Appl Biochem Biotechnol 171, 667–675 (2013). https://doi.org/10.1007/s12010-013-0383-z

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  • DOI: https://doi.org/10.1007/s12010-013-0383-z

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