On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.
On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.
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DOI：
10.1007/s00216-016-9677-7
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发表时间：
2016-08
影响因子：
4.3
通讯作者：
Hage DS
中科院分区：
文献类型：
--
作者：
Anguizola J;Bi C;Koke M;Jackson A;Hage DS
建立了一种基于高效亲和层析的蛋白质包埋固定α-酸性糖蛋白(AGP)的柱上方法。使用含有肼活化的多孔硅胶的微柱，以温和氧化的糖原为封端剂，对可溶性AGP进行了物理包埋。对三种柱上包封法进行了评价，并与以前的浆料包封法进行了比较。最终选择的方法用于制备1 cm×2.1mmI.D.亲和微柱，其含有多达21(±4)μg琼脂糖蛋白，可用于超过15 0个样品的应用过程。在这些亲和微柱上进行前沿分析和区带洗脱研究，以检查各种药物与包裹的AGP的结合。对这些药物也进行了现场选择性竞争研究。结果表明，这些药物-蛋白质体系的研究结果与以前的观察结果和文献报道的结合常数很好地吻合。这项研究中开发的包埋方法将对未来个性化药物领域的工作以及高通量筛选药物与AGP或其他蛋白质的相互作用有用。
An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (± 4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins.