Abstract
Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of similar to 44kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 angstrom and 8.18x10(-7)cm(2)s(-1), respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K-i=4.10nM) followed by ficin and bromelain. CBC possessed 34.7% -helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - H, S, G along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N=.97 +/-.07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative H (-5.78kcalmol(-1)) and positive S (11.01calmol(-1)deg(-1)) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative G (-9.19kcalmol(-1)) value implies a spontaneous CBC-papain interaction.