Abstract：
Immobilized human serum albumin (HSA) was developed by coupling His-tagged HSA onto Ni2+-coupled magnetizable beads (HSA-beads), allowing the HSA to be easily removed from incubation components. The HSA-beads system provides a rapid and convenient method to study HSA compound binding. In this study, the HSA-beads system was characterized and evaluated as a tool for assessing compound HSA binding properties. The free fraction (fu) values of test compounds measured using HSA-beads were comparable to those determined by equilibrium dialysis (ED), which is commonly used to evaluate albumin binding in vitro. The equilibrium dissociation constant (Kd) values determined for a series of compounds using the HSA-beads method demonstrated good correlation with literature data. This good correlation also suggests that the binding of His-HSA to the beads does not impact the conformations of the two compound binding sites of HSA, as the range of compounds tested encompassed binding to both sites. Furthermore, the Kd values of representative compounds itraconazole and BIRT2584 that were difficult to assess using ED, due to significant cellulose membrane adsorption, were successfully determined. The HSA-beads provide several advantages over ED, such as simple preparation, short assay incubation duration, and the ability to quantify both free and HSA-bound species of the test compound, facilitated by the simple separation of HSA-beads from the solution phase using a magnetic field. These properties render the HSA-beads method suitable for high-throughput studies on compound HSA binding.
摘要：
固定化的人血清白蛋白（HSA）是通过将His标记的HSA偶联到Ni2偶联的可磁化珠（HSA-珠）上而开发的，允许HSA容易地从孵育组分中去除。HSA-珠系统提供了研究HSA化合物结合的快速和方便的方法。在这项研究中,将HSA-珠系统表征并评价为用于评估化合物HSA结合性质的工具。使用HSA-珠测量的测试化合物的游离分数(fu)值与通过平衡透析(ED)测定的那些相当。通常用于体外评估白蛋白结合。使用HSA-珠方法确定的一系列化合物的平衡解离常数（Kd）值显示出与文献数据的良好相关性。这种良好的相关性也表明His-HSA与珠子的结合不会影响HSA的两个化合物结合位点的构象。因为所测试的化合物的范围涵盖与两个位点的结合。此外，难以使用ED评估的代表性化合物伊曲康唑和BIRT2584的Kd值，由于显著的纤维素膜吸附，成功确定。HSA珠提供了超过ED的几个优点，比如简单的准备，短的测定孵育时间，以及定量测试化合物的游离和结合HSA的物种的能力，通过使用磁场从溶液相中简单地分离HSA-珠来促进。这些性质使得HSA-珠方法适用于化合物HSA结合的高通量研究。
