Insight into the biochemical characterization of phytocystatin from Glycine max and its interaction with Cd⁺² and Ni⁺²

Abstract

Phytocystatins are cysteine proteinase inhibitors ubiquitously present in plants and animals. They are known to carry out various significant physiological functions and also maintain the balance of protease-antiprotease activity. In the present disquisition, a phytocystatin after preliminary treatment has been isolated and purified to homogeneity from soybean (Glycine max) by a simple two-step stratagem using ammonium sulfate fractionation and gel filtration chromatography performed on Sephacryl S-100-HR. Soybean phytocystatin (SBPC) was purified with a fold purification of 635 and percent yield of 77.6%. A single band was observed on native gel electrophoresis confirming the homogeneity of the purified SBPC. The molecular weight of SBPC was found to be 19.05 kDa as determined by SDS-PAGE. The SBPC was found to be devoid of carbohydrate moieties and sulfhydryl group content. The binding stoichiometry of SBPC-papain interaction was determined by isothermal calorimetry suggesting 1:1 complex, and the value of binding constant (K) was found to be 2.78 × 10 5 M −1 The affinity of binding (K d ) value obtained through ITC was 3.59 × 10 −6 M. The purified SBPC was found to be stable in the pH range of 3 to 7 and is thermostable up to 50°C. The UV-visible and fluorescence studies showed significant changes in the conformation upon the formation of the SBPC-papain complex. Furthermore, fluorescence spectroscopy, ANS binding, and caseinolytic activity assay were conducted out to explore the effect of metal ions on SBPC which showed that there was a loss in the inhibitory activity along with conformational changes of SBPC upon complex formation with Cd +2 and Ni +2 .

Comparative investigation on interaction between potent antimalarials and human serum albumin using multispectroscopic and computational approaches

“…In this study, the characteristic absorption value of native HSA at 280 nm increased on gradual addition of the compounds (hyperchromic shift) (Figure 2). Any variation in the absorbance of the peak near 280 nm indicates a modification in the proximity of the aromatic amino acid residues when small molecules are present [34]. In the case of the HSA–JMI 346 complex, significant absorbance was observed with hypsochromic shifting (blue shift) of ~3 nm, confirming complex formation.…”