Department of Chemical and Process Engineering, Rzeszów University of Technology, 35-959 Rzeszów, Poland.
Institute of Macromolecular Chemistry, Czech Academy of Sciences, 162 00 Prague, Czech Republic.
Molecules. 2024 Oct 13;29(20):4858. doi: 10.3390/molecules29204858.
Protein adsorption behavior was examined on poly(-isopropylacrylamide-co-sodium methacrylate)-based hydrogels at different temperatures: 5, 20, and 37 °C, and pH: 4.5, 7, and 9.2. The hydrogels, whose covalent skeleton contains pendant anionic units due to the presence of the sodium methacrylate co-monomer, exhibited both thermo- and pH-sensitivity with different extents, which depended on the content of ionizable moieties and the cross-linker density. The hydrogel composition, temperature, and pH influenced the zeta potential of the hydrogels and their swelling properties. The proteins selected for the study, i.e., bovine serum albumin (BSA), ovalbumin (OVA), lysozyme (LYZ), and a monoclonal antibody (mAb2), differed in their aminoacidic composition and conformation, thus in isoelectric point, molecular weight, electrostatic charge, and hydrophobicity. Therefore, the response of their adsorption behavior to changes in the solution properties and the hydrogel composition was different. LYZ exhibited the strongest adsorption of all proteins with a maximum at pH 7 (189.5 mg ggel-1); adsorption of BSA and OVA reached maximum at pH 4.5 (24.4 and 23.5 mg ggel-1), whereas mAb2 was strongly adsorbed at 9.2 (21.7 mg ggel-1). This indicated the possibility of using the hydrogels for pH-mediated separation of proteins differing in charge under mild conditions in a water-rich environment of both the liquid solution and the adsorbed phase. The adsorption affinity of all proteins increased with temperature, which was attributed to the synergistic effects of attractive electrostatic and hydrophobic interactions. That effect was particularly marked for mAb2, for which the temperature change from 5 to 37 °C caused a twentyfold increase in adsorption. In all cases, the proteins could be released from the hydrogel surface by a reduction in temperature, an increase in pH, or a combination of both. This allows for the elimination of the use of salt solution as a desorbing agent, whose presence renders the recycling of buffering solutions difficult.
在不同温度（5、20 和 37°C）和 pH 值（4.5、7 和 9.2）下，研究了聚（异丙基丙烯酰胺-co-甲基丙烯酸钠）基水凝胶上的蛋白质吸附行为。由于存在甲基丙烯酸钠共聚单体，水凝胶的共价骨架含有悬挂的阴离子单元，表现出不同程度的温度和 pH 敏感性，这取决于可离子化部分的含量和交联剂密度。水凝胶组成、温度和 pH 值影响水凝胶的 ζ 电位和溶胀性能。选择用于研究的蛋白质，即牛血清白蛋白（BSA）、卵清蛋白（OVA）、溶菌酶（LYZ）和单克隆抗体（mAb2），在氨基酸组成和构象上有所不同，因此在等电点、分子量、静电荷和疏水性上也有所不同。因此，它们的吸附行为对溶液性质和水凝胶组成变化的响应也不同。LYZ 是所有蛋白质中吸附能力最强的，在 pH 值为 7 时达到最大值（189.5 mg ggel-1）；BSA 和 OVA 的吸附在 pH 值为 4.5 时达到最大值（24.4 和 23.5 mg ggel-1），而 mAb2 在 pH 值为 9.2 时被强烈吸附（21.7 mg ggel-1）。这表明有可能在富含水的环境中，在温和的条件下，使用水凝胶通过 pH 介导分离电荷不同的蛋白质。所有蛋白质的吸附亲和力随温度升高而增加，这归因于静电和疏水相互作用的协同效应。这种效应在 mAb2 中尤为明显，温度从 5°C 升高到 37°C 导致吸附量增加了二十倍。在所有情况下，通过降低温度、提高 pH 值或两者结合都可以将蛋白质从水凝胶表面释放出来。这使得无需使用盐溶液作为洗脱剂，而盐溶液的存在会使缓冲溶液的回收变得困难。
