Chemistry Department, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Chemistry Department, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jul 15;1121:1-8. doi: 10.1016/j.jchromb.2019.05.004. Epub 2019 May 4.
Several approaches were compared for the entrapment of proteins within hydrazide-activated silica for use in affinity microcolumns and high performance affinity chromatography. Human serum albumin (HSA) and concanavalin A (Con A) were used as model proteins for this work. Items considered in this study included the role played by the solution volume, amount of added protein, and use of slurry vs. on-column entrapment on the levels of solute retention and extent of protein immobilization that could be obtained by means of entrapment. The levels of retention and protein immobilization were evaluated by injecting warfarin or 4-methylumbellipheryl α-D-mannopyranoside as solutes with known binding properties for HSA or Con A. Altering both the solution volume and amount of added protein led to an increase of up to 17-fold in the extent of protein immobilization for HSA in slurry-based entrapment; on-column entrapment provided an additional 3.6-fold increase in protein content vs. the optimized slurry method. Similar general trends were seen for Con A. The protein contents obtained by entrapment for HSA or Con A (i.e., up to ~87 and 46 mg/g silica, respectively) were comparable to or higher than levels reported for the covalent immobilization of these proteins onto silica. The retention of warfarin on the entrapped HSA was at least 1.7-fold higher than has been obtained under comparable support and mobile phase conditions when using covalent immobilization. These results indicated that entrapment can be an attractive alternative to covalent immobilization for proteins such as HSA and Con A, with this approach serving as a potential means for obtaining good solute binding and retention in work with affinity microcolumns or related microscale devices.
几种方法被比较用于将蛋白质包埋在酰肼活化的硅胶中，用于亲和微柱和高效亲和层析。人血清白蛋白（HSA）和伴刀豆球蛋白 A（Con A）被用作这项工作的模型蛋白。本研究中考虑的项目包括溶液体积、添加蛋白质的量、以及使用浆体与柱上包埋对通过包埋获得的溶质保留水平和蛋白质固定化程度的影响。保留水平和蛋白质固定化程度通过注射华法林或 4-甲基伞形酮-α-D-甘露吡喃糖苷作为具有已知与 HSA 或 Con A 结合特性的溶质来评估。改变溶液体积和添加蛋白质的量可以使 HSA 在浆体包埋中的蛋白质固定化程度增加高达 17 倍；与优化的浆体方法相比，柱上包埋使蛋白质含量增加了 3.6 倍。Con A 也出现了类似的总体趋势。通过包埋获得的 HSA 或 Con A 的蛋白质含量（即，分别高达约 87 和 46mg/g 硅胶）与共价固定化这些蛋白质到硅胶上的报道水平相当或更高。在类似的支撑体和流动相条件下，华法林在包埋的 HSA 上的保留至少比共价固定化时高 1.7 倍。这些结果表明，与共价固定化相比，蛋白质（如 HSA 和 Con A）的包埋可能是一种有吸引力的替代方法，这种方法可以作为在亲和微柱或相关微尺度设备中获得良好的溶质结合和保留的潜在手段。
