Purification, characterization and kinetics of thiol protease inhibitor from goat (Capra hircus) lung.
In the present study, two molecular forms of goat lung cystatin (GLC), I and II, were purified to homogeneity by a two-step procedure including ammonium sulfate precipitation (40-60%) and ion exchange chromatography. The inhibitor forms migrated as single bands under native and SDS-PAGE with and without reducing agent giving molecular mass of 66.4 and 76.4 kDa, respectively. GLC-I possesses 0.07% and GLC-II 2.3% carbohydrate content and no -SH groups. GLC-I showed greater affinity for papain than for ficin and bromelain. Immunological studies showed that the inhibitor was pure and there was cross reactivity between anti-GLC-I serum and goat brain cystatin. Both inhibitor forms were stable in the pH range of 3-10 and up to 75 degrees C. GLC-I was found to possess 49% alpha-helical structure by CD spectroscopy. The inhibitor-papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies.
Khan
Mohd Shahnawaz
MS
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India.
Bano
Bilqees
B
D000818:Animals; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006041:Goats / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D055550:Protein Stability
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors