Isolation and purification of phytocystatin from almond: Biochemical, biophysical, and immunological characterization

Abstract

Abstract It is well known that fruit nuts contain wide variety of flavonoids and various proteins, consumption of which has been associated with the reduced risk of chronic diseases. Cystatins, a family of cysteine proteinase inhibitors, ubiquitously present in all cells serve various important and critical physiological functions. In this study a phytocystatin with molecular mass of 63.4 kDa was purified to homogeneity by a three-step process including ammonium sulfate fractionation (50–70%), acetone precipitation, and gel filtration chromatography on Sephacryl S100-HR column. The purified inhibitor migrated as single band under native and SDS-PAGE. The Ki values for purified inhibitor with papain, ficin, and bromelain were found to be 45.45, 83.33, and 90.9 nM, respectively, suggesting higher affinity of the inhibitor for papain as compared to ficin and bromelain. Phytocystatin was stable in broad pH and temperature range. Purified cystatin appeared to be antigenic as observed in western blot analysis. ITC assay data show a binding stoichiometry of 0.870 ± 0.03 sites for cystatin and papain interaction which indicated that cystatin is surrounded by nearly one papain molecule. FTIR, UV, and fluorescence studies showed significant conformational changes on cystatin–papain complex formation. Purified cystatin was found to possess 36.8% α-helical content as observed by CD spectroscopy.