title: Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders.
abstract: In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.
authors.affiliation: Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: Anas
authors.initials: A
authors.lastname: Shamsi
authors.affiliation: Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: Azaj
authors.initials: A
authors.lastname: Ahmed
authors.affiliation: Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India. Electronic address: bbbano17@gmail.com.
authors.forename: Bilqees
authors.initials: B
authors.lastname: Bano
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001963:Bromelains / Q000037:antagonists & inhibitors / Q000737:chemistry; D002020:Buffaloes; D015891:Cystatins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D005361:Ficain / Q000037:antagonists & inhibitors / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry* / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D055550:Protein Stability; D016415:Sequence Alignment; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D015891:Cystatins; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D001963:Bromelains; D010206:Papain; D005361:Ficain
keywords: Cystatin; N-terminal analysis; Papain