Chen Chao, Zhao Dawei, Su Zhiguo, Luo Jian, Ma Guanghui, Zhang Songping, Li Xiunan
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China.
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
J Chromatogr A. 2021 Feb 22;1639:461904. doi: 10.1016/j.chroma.2021.461904. Epub 2021 Jan 13.
The adsorption process of bovine serum albumin (BSA), ovalbumin (OVA) and human immunoglobulin G (IgG) on agarose ion-exchange media Q Sepharose FF and two dextran-grafted agarose media including Q Sepharose XL and Capto Q were studied using low field nuclear magnetic resonance (NMR). The T relaxation time was found directly proportional to the pore size and diminished after protein adsorbed, therefore, a theoretical model describing the relationship between protein binding amount and T relaxation signals was established. The model parameters, a, which reflects the contact area between the adsorbed protein and media surface, and the δ, which defined as the ratio of the protein volume to the pore volume after adsorption, were found to describe the pore occupation states of proteins in media with different pore structures very well. For small proteins, such as BSA and OVA, monolayer adsorption occurred on Q Sepharose FF, which has no dextran chains. Therefore, the adsorbed protein only occupied 49.05% of the pore volume for BSA and 25.51% for OVA, and contact area of each protein on the media were also low, suggesting mostly monolayer adsorption occurred. In the contrast, their adsorption to Q Sepharose XL and Capto Q with dextran chains tended to form multilayer adsorption, thus higher contact area was obtained and the pore volumes were almost 100% occupied. For large protein, such as IgG, the adsorption to all these three media was similar and about 30% of the pore volume were occupied, probably due to the similar restriction for IgG to entering the media pore. Results of this study will help to elucidate the relationship between protein adsorption and pore size variation, which present the significance of low field NMR in understanding protein adsorption mechanism.
采用低场核磁共振（NMR）技术研究了牛血清白蛋白（BSA）、卵清蛋白（OVA）和人免疫球蛋白G（IgG）在琼脂糖离子交换介质Q Sepharose FF以及两种葡聚糖接枝琼脂糖介质（Q Sepharose XL和Capto Q）上的吸附过程。发现T2弛豫时间与孔径成正比，且蛋白质吸附后T2弛豫时间减小，因此建立了描述蛋白质结合量与T2弛豫信号之间关系的理论模型。发现模型参数a（反映吸附蛋白质与介质表面的接触面积）和δ（定义为吸附后蛋白质体积与孔体积之比）能够很好地描述不同孔结构介质中蛋白质的孔占据状态。对于小蛋白质，如BSA和OVA，在没有葡聚糖链的Q Sepharose FF上发生单层吸附。因此，吸附的蛋白质仅占据BSA孔体积的49.05%，OVA孔体积的25.51%，并且每种蛋白质在介质上接触面积也较低，表明主要发生单层吸附。相比之下，它们在具有葡聚糖链的Q Sepharose XL和Capto Q上的吸附倾向于形成多层吸附，因此获得了更高的接触面积，并且孔体积几乎被100%占据。对于大蛋白质，如IgG，其在所有这三种介质上的吸附相似，约30%的孔体积被占据，这可能是由于IgG进入介质孔的限制相似。本研究结果将有助于阐明蛋白质吸附与孔径变化之间的关系，这体现了低场NMR在理解蛋白质吸附机制方面的重要性。
