Department of Pharmaceutical Sciences, Health Sciences Center, Federal University of Pernambuco, 50740-520 Recife, PE, Brazil.
Department of Fundamental Chemistry, Center for Exact and Nature Sciences, Federal University of Pernambuco, 50740-560 Recife, PE, Brazil.
Eur J Pharm Sci. 2019 Oct 1;138:105018. doi: 10.1016/j.ejps.2019.105018. Epub 2019 Jul 29.
The search for plasma proteins precipitation methods has been increasing due to the plasma protein therapeutic needs in world-wide. Thus, this work evaluates the tannic acid (TA) ability to precipitate proteins from human plasma. In this study, TA-plasma protein complexes were studied at different pH conditions, tannin/plasma ratio and reaction mixing time. The complexes formed from combinations of TA and plasma proteins were analyzed by gel electrophoresis, protein quantification, particle size, charge, mass spectrometry, microscopic image, and circular dichroism. It was possible to verify the precipitate formation in all tested pH values, with high precipitation at pH 5. The native PAGE analysis showed three mainly bands corresponding independent of the pHs used. It was possible to observe a gradual growing of precipitate protein in the first precipitation process (P1) when increased the TA/plasma ratio. 15 min of incubation was enough to precipitate 72.3% of proteins. Spectroscopic analyzes showed albumin signals and the electron microscopy analysis of IgG-TA confirmed the compact form of a precipitate. According to CD, formation of the IgG-TA complexes does not cause a major structural change of the protein. From the results obtained, it was possible to establish some parameters for plasma proteins precipitation using TA.
由于全球对血浆蛋白治疗的需求不断增加,寻找血浆蛋白沉淀方法的研究也在不断增加。因此,本工作评估了鞣酸(TA)沉淀人血浆中蛋白质的能力。在这项研究中,在不同的 pH 值条件下、鞣酸/血浆比和反应混合时间下研究了 TA-血浆蛋白复合物。通过凝胶电泳、蛋白质定量、粒径、电荷、质谱、显微镜图像和圆二色性分析了 TA 和血浆蛋白结合形成的复合物。可以在所有测试的 pH 值下验证沉淀的形成,在 pH 5 时沉淀效果最高。在不依赖于使用的 pH 值的情况下,天然 PAGE 分析显示了三个主要条带。当增加 TA/血浆比时,可以观察到在第一次沉淀过程(P1)中沉淀蛋白逐渐增加。孵育 15 分钟即可沉淀 72.3%的蛋白质。光谱分析显示了白蛋白信号,并且 IgG-TA 的电子显微镜分析证实了沉淀的紧凑形式。根据 CD,IgG-TA 复合物的形成不会导致蛋白质的主要结构变化。根据实验结果,可以使用 TA 确定一些沉淀血浆蛋白的参数。