Amino Acids

, Volume 38, Issue 4, pp 1001–1010 | Cite as

Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization

Original Article


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 Ǻ, 7.87 × 10−7 cm2 s−1 and 3.83 s, respectively. It was stable in pH range 3–10 and up to 70°C (critical temperature, Ea = 21 kJ mol−1). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain (Ki = 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% α 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.


Goat pancreas cystatin Kinetics of inhibition Amino acid sequence Mammalian cystatins Characterization 


  1. Abrahamson M, Barrett AJ, Salvessen G et al (1986) Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem 261:11282–11289PubMedGoogle Scholar
  2. Abrahamson M, Alvarez-Fernandez C, Nathanson M (2003) Cystatins. Biochem Soc Symp 70:179–199PubMedGoogle Scholar
  3. Allen B, Blum M, Cunningham A, Tu GC et al (1990) Aligand induced, temperature dependent conformational change in penicillopepcin. J Biol Chem 265:5060–5065PubMedGoogle Scholar
  4. Andrews P (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J 91:222–233PubMedGoogle Scholar
  5. Ansorge S, Kirschke H, Friedrich K (1977) Conversion of proinsulin into insulin by cathepsins B and L from rat liver lysosomes. Acta Biol Med Ger 36:1723–1727PubMedGoogle Scholar
  6. Balbin M, Hall A, Grubb A, Mason RW et al (1994) Structural and functional characterization of two allelic variants of human cystatins D sharing a characteristic inhibition spectrum against mammalian cysteine proetinases. J Biol Chem 223:245–253Google Scholar
  7. Barrett AJ, Rawlings ND, O’Brien EA (2001) The MEROPS database as a protease information system. J Struct Biol 134:95–102. doi:10.1006/jsbi.2000.4332 CrossRefPubMedGoogle Scholar
  8. Björk I, Ylinenjärvi K (1990) Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin. Biochem 29:1770–1776. doi:10.1021/bi00459a016 CrossRefGoogle Scholar
  9. Bjork I, Pol E, Raub-Segall E, Abrahamson M et al (1994) Differential changes in association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes. Biochem J 299:219–225PubMedGoogle Scholar
  10. Björk I, Alriksson E, Ylinenjärvi K (1989) Kinetics of binding of chicken cystatin to papain. Biochem 28:1568–1573. doi:10.1021/bi00430a022 CrossRefGoogle Scholar
  11. Bode W, Engh R, Musil D et al (1988) The 2.0 Ǻ X-ray crystal structure of chicken egg white cystatins and its possible mode of interaction with cystaeine proteinases. EMBO J 7:2593–2599PubMedGoogle Scholar
  12. Burstein EA, Vedenkina NS, Ivkova MN (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol 18:263–279. doi:10.1111/j.1751-1097.1973.tb06422.x CrossRefPubMedGoogle Scholar
  13. Chen YH, Yang JT, Martinez H (1972) Determination of the secondary structure of proteins by circular dichroism and optical rotatory dispersion. Biochem 11:4120–4131. doi:10.1021/bi00772a015 CrossRefGoogle Scholar
  14. Cimerman N, Ksorok MD, Korant BD et al (1996) Characterization of cystatin C from bovine parotid glands: cysteine proteinase inhibition and antiviral properties. Biol Chem Hoppe Seyler 377:19–23PubMedGoogle Scholar
  15. Cohen DH, Feiner H, Jenson O, Frangione B (1983) Amyloid fibril in hereditary cerebral hemorrhages with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, gamma trace. J Exp Med 158:653. doi:10.1084/jem.158.2.623 CrossRefGoogle Scholar
  16. Dubois M, Gilles MA, Hamilton JK et al (1956) Pfizer Flocon 4800 Procedurephenol/sulphuric acid method. Anal Chem 28:350–354. doi:10.1021/ac60111a017 CrossRefGoogle Scholar
  17. Edman P, Begg G (1967) A protein sequenator. Eur J Biochem 1:80–91. doi:10.1111/j.1432-1033.1967.tb00047.x CrossRefPubMedGoogle Scholar
  18. Ellman R (1969) Tissue sulphydryl groups. Biochem Methods 19:446–451Google Scholar
  19. Gally JA, Edelman GM (1962) The effect of temperature on the fluorescence of some aromatic amino acids and proteins. Biochim Biophys Acta 60:499–509. doi:10.1016/0006-3002(62)90869-7 CrossRefPubMedGoogle Scholar
  20. Green GDJ, Kembhavi AA, Davies ME et al (1984) Cystatin-like proteinase inhibitors from human liver. Biochem J 218:939–946PubMedGoogle Scholar
  21. Halangk W, Lerch MM, Brandt-Nedela B et al (2000) Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J Clin Invest 106:773–781. doi:10.1172/JCI9411 CrossRefPubMedGoogle Scholar
  22. Jarvinen M, Rinnie A (1982) Human spleen cysteine proteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants. Biochim Biophys Acta 708:210–217PubMedGoogle Scholar
  23. Khan S Md, Bano B (2009) Mechanism of unfolding of goat lung cystatin during urea and guanidine induced denaturation. Int J Pept Res Therap. doi:10.1007/s10989-008-9166-8
  24. Kunitz M (1947) Crystalline soya bean trypsin inhibitor, general properties. J Physiol 30:291–310Google Scholar
  25. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132. doi:10.1016/0022-2836(82)90515-0 CrossRefPubMedGoogle Scholar
  26. Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0 CrossRefPubMedGoogle Scholar
  27. Laurent TC, Killander J (1964) A theory of gel filtration and its experimental verification. J Chromatogr A 14:317–330. doi:10.1016/S0021-9673(00)86636-4 CrossRefGoogle Scholar
  28. Layne E (1957) Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol 3:447–455. doi:10.1016/S0076-6879(57)03413-8 CrossRefGoogle Scholar
  29. Li F, An M, Baynes TL (2000) Rainbow trout (Oncorhynchus mykiss) cystatins C: expression in Escheichia coli and properties of the recombinant protease inhibitor. Comp Biochem Physiol B Biochem Mol Biol 125:493–502. doi:10.1016/S0305-0491(00)00156-5 CrossRefPubMedGoogle Scholar
  30. Lindahl P, Abrahamson M, Bjoerk I (1992) Interaction of recombinant human cystatin C with cysteine proteinases papain and actinidin. Biochem J 28:49–55Google Scholar
  31. Lowry OH, Rosenbrough NJ, Farr AL et al (1951) Protein determination with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  32. Nesternko MV, Tilley M, Upton SJ (1994) A simple modification of Blum’ silver stain method allows for 30 minutes detection of protein in polyacrylamide gels. J Biochem Biophys Methods 28:239–242. doi:10.1016/0165-022X(94)90020-5 CrossRefGoogle Scholar
  33. Ni J, Abrahamson M, Zhang M et al (1997) Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins. J Biol Chem 272:10853–10858. doi:10.1074/jbc.272.30.18823 CrossRefPubMedGoogle Scholar
  34. Ni J, Fernandez MA, Danielsson L et al (1998) Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. J Biol Chem 273:24797–24804. doi:10.1074/jbc.273.38.24797 CrossRefPubMedGoogle Scholar
  35. Nicklin MJH, Barrett AJ (1984) Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg white cystatins. Biochem J 223:245–253PubMedGoogle Scholar
  36. Ohkubo I, Kurachi K, Takasawa T et al (1984) Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen. Biochemistry 23:5691–5697. doi:10.1021/bi00319a005 CrossRefPubMedGoogle Scholar
  37. Olsson S, Ek B, Bjork I (1999) The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C. Biochim Biophys Acta 1432:73–81PubMedGoogle Scholar
  38. Otto K, Riesenkonig H (1975) Improved purification of cathepsin B1 and cathepsin B2. Biochim Biophys Acta 379:462–475PubMedGoogle Scholar
  39. Pace CN (1975) The stability of globular proteins. CRC Crit Rev Biochem 3:1–43. doi:10.3109/10409237509102551 CrossRefPubMedGoogle Scholar
  40. Rashid F, Sharma S, Bano B (2006) Detailed biochemical characterization of human placental cystatins (HPC). Placenta 2:822–831. doi:10.1016/j.placenta.2005.09.005 CrossRefGoogle Scholar
  41. Schurmann G, Haspel J, Grumet M et al (2001) Cell adhesion molecule L1 in folded (horseshoe) and extended conformations. Mol Cell Biol 12:1765–1773Google Scholar
  42. Shahid PB, Zehra S, Bano B (2005) Purification and characterization of kininogens from sheep plasma. Protein J 24:95–102. doi:10.1007/s10930-004-1516-6 CrossRefGoogle Scholar
  43. Sotiropoulou G, Anisowicz A, Sagar R (1997) Identification, cloning and characterization of cystatins M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem 272:903–910. doi:10.1074/jbc.272.2.903 CrossRefPubMedGoogle Scholar
  44. Stubbs MT, Laber B, Bode W et al (1990) The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J 9(6):1939–1947PubMedGoogle Scholar
  45. Sumbul S, Bano B (2006) Purification and characterization of high molecular mass and low molecular mass cystatins from goat brain. Neurochem Res 31:1327–1336. doi:10.1007/s11064-006-9175-y CrossRefPubMedGoogle Scholar
  46. Turk B, Turk V, Turk D (1997) Structural and functional aspects of papain like cysteine proteinases and their protein inhibitors. Biol Chem 378:141–150PubMedGoogle Scholar
  47. Ylonen A, Rinne A, Herttuainen J et al (1999) Atlantic salmon (Salmo salar L.) skin contains a novel kininogen ans another cysteine proteinase inhibitor. Eur J Biochem 266:1066–1072. doi:10.1046/j.1432-1327.1999.00950.x CrossRefPubMedGoogle Scholar
  48. Zehra S, Shahid PB, Bano B (2005) Isolation, characterization and kinetics of goat cystatins. Comp Biochem Physiol B Biochem Mol Biol 142:361–368Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Department of Biochemistry, Faculty of Life SciencesAligarh Muslim UniversityAligarhIndia

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