Development of enhanced capacity affinity microcolumns by using a hybrid of protein cross-linking/modification and immobilization.
Development of enhanced capacity affinity microcolumns by using a hybrid of protein cross-linking/modification and immobilization.
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DOI：
10.1016/j.chroma.2015.04.051
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发表时间：
2015-06-26
期刊：
影响因子：
--
通讯作者：
Hage DS
中科院分区：
文献类型：
--
作者：
Zheng X;Podariu M;Bi C;Hage DS
通过结合蛋白质交联/修饰和共价固定，研究了一种杂交方法来提高蛋白质亲和柱的结合能力和活性。考虑了该方法在药物-蛋白质相互作用研究中的各种应用以及与亲和微柱的使用。本研究以人血清白蛋白（HSA）为模型蛋白。双马来烯亚胺（BMH，一种同双官能团的马来烯亚胺）被用来修饰和/或通过存在于该蛋白上的单个游离巯基来交联HSA。当比较用BMH制备的亲和支架与仅用共价固定制备的参考支架时，蛋白质含量增加了75-113%。我们进一步发现，几种已知与HSA结合的药物（如华法林、维拉帕米和卡马西平）在用BMH处理的HSA微柱上的滞留量显著增加（即，蛋白基滞留量增加70-100%）。这些经bmh处理的HSA微柱用于手性分离和超快速亲和萃取，以测量药物/蛋白质混合物中的游离药物组分，后者方法给出的关联平衡常数与文献值吻合良好。此外，我们还发现，用bmh处理的HSA微柱可以检测HSA与乙酸的可逆结合，乙酸可以与该蛋白上的游离巯基不可逆结合。同样的杂交固定方法可以扩展到其他蛋白质或可能需要具有增强结合能力和活性的基于蛋白质的亲和柱的替代应用。
A hybrid method was examined for increasing the binding capacity and activity of protein-based affinity columns by using a combination of protein cross-linking/modification and covalent immobilization. Various applications of this approach in the study of drug-protein interactions and in use with affinity microcolumns were considered. Human serum albumin (HSA) was utilized as a model protein for this work. Bismaleimidohexane (BMH, a homobifunctional maleimide) was used to modify and/or cross-link HSA through the single free sulfhydryl group that is present on this protein. Up to a 75-113% increase in protein content was obtained when comparing affinity supports that were prepared with BMH versus reference supports that were made by using only covalent immobilization. Several drugs that are known to bind HSA (e.g., warfarin, verapamil and carbamazepine) were further found to have a significant increase in retention on HSA microcolumns that were treated with BMH (i.e., a 70-100% increase in protein-based retention). These BMH-treated HSA microcolumns were used in chiral separations and in ultrafast affinity extraction to measure free drug fractions in drug/protein mixtures, with the latter method giving association equilibrium constants that had good agreement with literature values. In addition, it was found that the reversible binding of HSA with ethacrynic acid, an agent that can combine irreversibly with the free sulfhydryl group on this protein, could be examined by using the BMH-treated HSA microcolumns. The same hybrid immobilization method could be extended to other proteins or alternative applications that may require protein-based affinity columns with enhanced binding capacities and activities.
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影响因子：
2.9
作者：
Jackson AJ;Xuan H;Hage DS
通讯作者：
Hage DS
DOI：
10.1016/j.chroma.2010.02.026
发表时间：
2010-04-23
期刊：
Journal of chromatography. A
影响因子：
--
作者：
Mallik R;Yoo MJ;Briscoe CJ;Hage DS
通讯作者：
Hage DS
DOI：
10.1007/978-1-4939-2447-9_21
发表时间：
2015-01-01
期刊：
AFFINITY CHROMATOGRAPHY: METHODS AND PROTOCOLS, 3RD EDITION
影响因子：
--
作者：
Matsuda, Ryan;Anguizola, Jeanethe;Hage, David S.
通讯作者：
Hage, David S.
影响因子：
3.1
作者：
FEHSKE, KJ;MULLER, WE
通讯作者：
MULLER, WE
影响因子：
2.9
作者：
OHLSON, S;LUNDBLAD, A;ZOPF, D
通讯作者：
ZOPF, D