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Purification, Characterization and Kinetics of Thiol Protease Inhibitor from Goat (Capra hircus) Lung

Mohd Shahnawaz Khan and Bilqees Bano*

Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India; E-mail: banobilqees@gmail.com

* To whom correspondence should be addressed.

Received July 4, 2008; Revision received December 15, 2008
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°C. GLC-I was found to possess 49% α-helical structure by CD spectroscopy. The inhibitor–papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies.
KEY WORDS: goat lung cystatin, ion-exchange chromatography, kinetics, circular dichroism, ELISA, fluorescence spectroscopy

DOI: 10.1134/S0006297909070116