Immobilization of α1-acid glycoprotein for chromatographic studies of drug-protein binding
Immobilization of α1-acid glycoprotein for chromatographic studies of drug-protein binding
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DOI：
10.1016/j.ab.2005.08.025
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发表时间：
2005-11-15
影响因子：
2.9
通讯作者：
Hage, DS
中科院分区：
文献类型：
--
作者：
Xuan, H;Hage, DS
开发并优化了一种制备用于药物-蛋白质结合研究的固定化 α(1)-酸性糖蛋白 (AGP) 的新方法。在这种方法中，在温和条件下使用高碘酸盐氧化 AGP 中的碳水化合物链，以附着到酰肼活化的载体上。该氧化选择的最终条件包括 5.0 mg/mL AGP 在 4 摄氏度和 pH 7.0 下与 5-20 mM 高碘酸反应 10 分钟。这些条件有助于最大限度地固定 AGP，而不显着影响其活性。通过使用该方法将 AGP 连接到二氧化硅以用于高性能亲和色谱和自竞争区带洗脱研究来评估该方法。在使用 R-和 S-普萘洛尔时，固定化 AGP 上的两种对映体仅观察到一种类型的结合位点，这与之前使用可溶性 AGP 的研究一致。在 pH 7.4 和 37 摄氏度下，测量固定化 AGP 与 R-和 S-普萘洛尔的缔合平衡常数分别为 2.7 x 10(6) 和 4.2 x 10(6) M-1，在 5 至 37 摄氏度之间获得线性范特霍夫图。使用其他药物进行的工作也表明固定化 AGP 和可溶性 AGP 的行为之间具有良好的一致性。这项工作中描述的相同固定方法可用于将 AGP 连接到其他材料，例如用于表面等离子体共振或替代生物传感器的材料。 (c) 2005 Elsevier Inc. 保留所有权利。
A new method for preparing immobilized alpha(1)-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M-1, respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors. (c) 2005 Elsevier Inc. All rights reserved.