Purification and biochemical characterization of phytocystatin from Brassica alba

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two-step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S-100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180-fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10^-7 cm²s^-1 respectively. The isolated phytocystatin was found to be stable in the pH range of 6-8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non-competitive type of inhibition and inhibited papain more efficiently (K_i = 3 × 10^-7 M) than ficin (K_i = 6.6 × 10^-7 M) and bromelain (Ki = 7.7 × 10^-7 M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content.