Sárközy Dániel, Guttman András
Horváth Csaba Memorial Laboratory of Bioseparation Sciences, Research Center for Molecular Medicine, Faculty of Medicine, Doctoral School of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, 8200 Veszprem, Hungary.
Gels. 2024 Dec 7;10(12):805. doi: 10.3390/gels10120805.
Hydrogels like agarose have long been used as sieving media for the electrophoresis-based analysis of biopolymers. During gelation, the individual agarose strands tend to form hydrogen-bond mediated double-helical structures, allowing thermal reversibility and adjustable pore sizes for molecular sieving applications. The addition of tetrahydroxyborate to the agarose matrix results in transitional chemical cross-linking, offering an additional pore size adjusting option. Separation of SDS-proteins during gel electrophoresis is an activated process defined by the interplay between viscosity, gelation/cross-link formation/distortion, and sample conformation. In this paper, the subunits of a therapeutic monoclonal antibody were separated by capillary SDS agarose gel electrophoresis at different temperatures. The viscosity of the separation matrix was also measured at all temperatures. In both instances, Arrhenius plots were used to obtain the activation energy values. It was counterintuitively found that larger SDS-protein complexes required lower activation energies while their low-molecular-weight counterparts needed higher activation energy for their electromigration through the sieving matrix. As a first approximation, we considered this phenomenon the result of the electric force-driven distortion of the millisecond range lifetime reticulations by the larger and consequently more heavily charged electromigrating molecules. In the meantime, the sieving properties of the gel were still maintained, i.e., they allowed for the size-based separation of the sample components, proving the existence of the reticulations. Information about the activation energy sheds light on the possible deformation of the sieving matrix and the solute molecules. In addition, the activation energy requirement study helped in optimizing the separation temperature, e.g., with our sample mixture, the highest resolution was obtained for the high-molecular-weight fragments, i.e., between the non-glycosylated heavy chain and heavy-chain subunits at 25 °C (lower E requirement), while 55 °C was optimal for the lower-molecular-weight light chain and non-glycosylated heavy chain pair (lower E requirement). Future research directions and possible applications are also proposed.
长期以来，像琼脂糖这样的水凝胶一直被用作筛分介质，用于基于电泳的生物聚合物分析。在凝胶化过程中，单个琼脂糖链倾向于形成氢键介导的双螺旋结构，从而实现热可逆性，并为分子筛分应用提供可调节的孔径。向琼脂糖基质中添加四羟基硼酸盐会导致过渡性化学交联，提供了另一种孔径调节选项。凝胶电泳过程中SDS-蛋白质的分离是一个活化过程，由粘度、凝胶化/交联形成/变形和样品构象之间的相互作用决定。在本文中，一种治疗性单克隆抗体的亚基在不同温度下通过毛细管SDS琼脂糖凝胶电泳进行了分离。在所有温度下还测量了分离基质的粘度。在这两种情况下，均使用阿累尼乌斯图来获得活化能值。与直觉相反的是，发现较大的SDS-蛋白质复合物在通过筛分基质进行电迁移时需要较低的活化能，而其低分子量对应物则需要较高的活化能。作为初步近似，我们认为这种现象是由于较大且因此带电量更高的电迁移分子对毫秒级寿命网状结构产生电力驱动变形的结果。与此同时，凝胶的筛分特性仍然得以保持，即它们允许基于大小对样品成分进行分离，证明了网状结构的存在。关于活化能的信息揭示了筛分基质和溶质分子可能的变形情况。此外，活化能需求研究有助于优化分离温度，例如，对于我们的样品混合物，在25°C（较低的活化能需求）下，高分子量片段（即非糖基化重链和重链亚基之间）获得了最高分辨率，而对于低分子量轻链和非糖基化重链对，55°C是最佳温度（较低的活化能需求）。还提出了未来的研究方向和可能的应用。
