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 × 105 M-1 The affinity of binding (Kd ) 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 .

中文翻译：

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深入了解大豆植物胱抑素的生化特征及其与 Cd+2 和 Ni+2 的相互作用。


植物胱抑素是普遍存在于植物和动物中的半胱氨酸蛋白酶抑制剂。已知它们执行各种重要的生理功能并维持蛋白酶-抗蛋白酶活性的平衡。在本研究中，通过简单的两步策略，使用硫酸铵分级分离和在Sephacryl S-100-HR上进行的凝胶过滤层析，从大豆（Glycine max）中分离和纯化经过初步处理的植物胱抑素，使其达到均质。大豆植物胱抑素(SBPC)的纯化倍数为635，收率为77.6%。在非变性凝胶电泳上观察到单条带，证实纯化的 SBPC 的均质性。通过 SDS-PAGE 测定发现 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。通过等温量热法测定 SBPC-木瓜蛋白酶相互作用的结合化学计量，表明为 1:1 复合物，发现结合常数 (K) 值为 2.78 × 105 M-1。通过 ITC 获得的结合亲和力 (Kd) 值为3.59 × 10-6 M。纯化的 SBPC 在 pH 值 3 至 7 范围内稳定，耐热温度高达 50°C。紫外-可见光和荧光研究表明，SBPC-木瓜蛋白酶复合物形成后构象发生显着变化。此外，还进行了荧光光谱、ANS 结合和酪蛋白分解活性测定，以探索金属离子对 SBPC 的影响，结果表明，与 Cd+2 和 Ni 形成复合物后，随着 SBPC 构象的变化，抑制活性丧失。 +2 。