title: Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.
abstract: OBJECTIVE
  This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials.
  
  
  METHODS
  Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy.
  
  
  RESULTS
  ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH.
  
  
  CONCLUSIONS
  To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.
authors.affiliation: Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Tooba Naz
authors.initials: TN
authors.lastname: Shamsi
authors.affiliation: Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Romana
authors.initials: R
authors.lastname: Parveen
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Amir
authors.affiliation: Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Mohd Affan
authors.initials: MA
authors.lastname: Baig
authors.affiliation: Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: M Irfan
authors.initials: MI
authors.lastname: Qureshi
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Sher
authors.initials: S
authors.lastname: Ali
authors.affiliation: Department of Biotechnology, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Sadaf
authors.initials: S
authors.lastname: Fatima
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005737:Garlic / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016877:Oxidants / Q000494:pharmacology; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D010940:Plant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D055550:Protein Stability / Q000187:drug effects; D015843:Serpins / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014361:Trypsin Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D003902:Detergents; C053865:Kunitz-type protease inhibitor, plant; D016877:Oxidants; D010455:Peptides; D010940:Plant Proteins; D015843:Serpins; D014361:Trypsin Inhibitors