Department of Biochemistry, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
J Mol Recognit. 2019 Oct;32(10):e2787. doi: 10.1002/jmr.2787. Epub 2019 Jun 10.
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 M The affinity of binding (K ) value obtained through ITC was 3.59 × 10 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 and Ni .
植物胱抑素是广泛存在于植物和动物中的半胱氨酸蛋白酶抑制剂。它们具有多种重要的生理功能，并且还维持着蛋白酶-抗蛋白酶活性的平衡。在本研究中，通过使用硫酸铵分级和凝胶过滤色谱（在 Sephacryl S-100-HR 上进行）的简单两步策略，从大豆（Glycine max）中初步处理后分离和纯化了一种植物胱抑素。大豆植物胱抑素（SBPC）经过 635 倍的纯化和 77.6%的产率得到纯化。在天然凝胶电泳中观察到单一条带，证实了纯化的 SBPC 的均一性。通过 SDS-PAGE 确定 SBPC 的分子量为 19.05 kDa。发现 SBPC 不含碳水化合物部分和巯基含量。通过等温量热法确定了 SBPC-木瓜蛋白酶相互作用的结合化学计量比，表明为 1:1 复合物，并且结合常数（K）的值为 2.78 × 10 M。通过 ITC 获得的结合亲和力（K ）值为 3.59 × 10 M。发现纯化的 SBPC 在 pH 值为 3 至 7 的范围内稳定，并且在高达 50°C 的温度下热稳定。紫外可见和荧光研究表明，在形成 SBPC-木瓜蛋白酶复合物时，构象发生了显著变化。此外，进行了荧光光谱、ANS 结合和酪蛋白水解活性测定，以研究金属离子对 SBPC 的影响，结果表明，在与 Cd 和 Ni 形成复合物时，SBPC 的抑制活性丧失，同时发生构象变化。
