色谱

色谱 ›› 2018, Vol. 36 ›› Issue (5): 480-486.DOI: 10.3724/SP.J.1123.2017.12014

• 研究论文 • 上一篇    下一篇

载液组成对于卵白蛋白在非对称流场流分离通道中膜吸附和聚集行为的影响

梁启慧1,2, 杨奕2, 邵兵2, 高也1, 宋宇1, 韩南银1   

  1. 1. 北京大学药学院, 北京 100191;
    2. 北京疾病预防控制中心, 北京 100013
  • 收稿日期:2017-12-13 出版日期:2018-05-08 发布日期:2014-03-21
  • 通讯作者: 韩南银.E-mail:nanyin.han@pku.edu.cn
  • 基金资助:

    北京市自然科学基金（7162088）.

Impact of carrier flow composition on membrane adsorption and aggregation of ovalbumin in asymmetrical flow field-flow fractionation

LIANG Qihui1,2, YANG Yi2, SHAO Bing2, GAO Ye1, SONG Yu1, HAN Nanyin1   

  1. 1. School of Pharmaceutical Sciences, Peking University, Beijing 100191 China;
    2. Beijing Center for Disease Prevention and Control, Beijing 100013 China
  • Received:2017-12-13 Online:2018-05-08 Published:2014-03-21
  • Supported by:

    Beijing Natural Science Foundation (No. 7162088).

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摘要：

非对称流场流分离技术对于蛋白质等生物大分子的分析具有温和、分离范围广的特点。然而，在场流分离通道中，受载液组成的影响而产生的蛋白质与通道膜的相互作用和蛋白质在通道内的聚集行为，会影响分析物的回收率和尺寸形态，这些现象一定程度上限制了场流分离仪器的进一步应用。该文研究了载液组成对于卵白蛋白在非对称流场流分离中膜吸附和聚集行为的影响。考察了不同pH （6.2、7.4、8.2）、阳离子种类（Na+、K+、Mg2+）及多种离子强度（0~0.1 mol/L）等条件对卵白蛋白洗脱过程的影响。结果表明a）载液的离子强度越大，卵白蛋白的吸附和聚集行为越严重；b） pH和蛋白质的等电点pI的相对大小决定了蛋白质的表面电荷，从而影响蛋白质的吸附聚集行为；c）二价阳离子Mg2+更易引发通道中蛋白质的吸附和聚集。这些结果有助于今后使用非对称流场流分离技术分析蛋白质样品时，改善载液组成以获得更高的回收率，降低蛋白质聚集作用，对AF4更广泛地应用于蛋白质生化分析中有较好的参考价值。

关键词: 蛋白质, 蛋白质聚集, 非对称场流分离, 离子强度, 载液

Abstract:

Asymmetrical flow field-flow fractionation (AF4) is a kind of moderate separation technology for the analysis of macromolecules, including proteins with a wide range of sizes. In the separation channel, the membrane adsorption and aggregation of proteins affected by the carrier fluid (CF) composition lead to changes in analyte recovery and size distribution, thereby restricting the application of AF4 to biomolecules. Different pH levels (6.2, 7.4, 8.2), several types of cations (Na+, K+, Mg2+) and various ion strengths (0-0.1 mol/L)were studied to demonstrate the influence of carrier fluid composition on the membrane adsorption and aggregation of proteins. The results revealed the following:a) higher ion strength of CF resulted in a greater degree of membrane adsorption and aggregation; b) the zeta potential, determined by the pI of the protein and the pH of the CF, influenced the adsorption and aggregation; c) divalent cations (Mg2+) caused serious adsorption and aggregation. The experimental results can help us achieve better recovery and mitigate aggregate formation by using the optimal CF components in future AF4 studies. Moreover, the findings indicate that AF4 would find extensive application in protein biochemistry assays.

Key words: asymmetrical flow field-flow fractionation (AF4), carrier fluid, ion strength, protein, protein aggregation

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