Purification and characterization of buffalo brain cystatin

Abstract

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions.

Aims: To purify and characterize Thiol protease inhibitor from buffalo brain and to compare its properties with respect to tissue and organ difference from other mammalian cystatins.

Main methods: Inhibitor has been isolated and purified using alkaline treatment; ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with a % yield of 64.13 and fold purification of 384.72.The inhibitor was studied by U.V and fluorescence spectroscopy. Papain inhibitory activity was measured using casein as substrate.

Key finding: The molecular weight of the buffalo brain cystatin (BC), determined by gel filtration and SDS PAGE came out to be 43.6 KDa and 44.20 KDa respectively. BC was found to be stable in broad pH and temperature range. The inhibitor was devoid of any sulphydryl group and carbohydrate content. These properties led to conclusion that BC is variant of type-I cystatin. The stokes radius and diffusion coefficient of the inhibitor were found to be 27 A° and 8.1 x 10⁻⁷ cm²/sec respectively, the f/f₀ ratio was 1.12 signifying that purified cystatin is nearly globular in shape. Kinetic data revealed binding stoichiometry of BC with papain as 1:1. The Ki value with papain ficin and bromelain were found to be 1, 1.85 and 2.25 nM respectively suggesting that cystatin has higher affinity with papain as compared to ficin and bromelain. The fluorescence and UV spectra of BC- papain complex showed significant conformational changes indicative of perturbation in the micro environment of aromatic amino acid residues on the formation of complex.

Significance: This work proliferates our knowledge about cystatins of the mammalian brain on the basis of their physiochemical properties.