Liquid chromatographic studies of the effect of phosphate on the binding properties of silica-immobilized bovine serum albumin
Liquid chromatographic studies of the effect of phosphate on the binding properties of silica-immobilized bovine serum albumin
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DOI：
10.1093/chromsci/39.5.205
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发表时间：
2001-05-01
影响因子：
1.3
通讯作者：
Gilpin, RK
中科院分区：
文献类型：
--
作者：
Tao, WA;Gilpin, RK
用高效液相色谱法研究了磷酸根离子对固定在多孔二氧化硅上的牛血清白蛋白(BSA)结合性能的影响。在此过程中，使用色氨酸和犬尿氨酸的色氨酸和L-异构体作为溶质，测量蛋白质达到构象平衡的时间依赖关系，作为洗脱液中磷酸盐浓度的函数。蛋白质对这些溶质的总结合和手性选择性(αD，L)似乎与两种效应有关：一种是位置选择性的，只影响l-异构体的保留；另一种是非选择性的，影响两种对映体的保留。浓度依赖数据的一个有趣特征是αD，Lat中磷浓度(即10到50 mM磷酸盐)的最大值，表明协同和拮抗结合效应。在这个浓度范围内的磷酸盐洗脱液提供了选择性优势，而浓度较高的洗脱液则缩短了蛋白质或柱子达到平衡所需的时间。最后一组研究还使用了四种不同的缓冲体系(即，硼酸盐、碳酸盐、醋酸盐和砷酸盐淋洗液)。虽然硼酸洗脱液影响牛血清白蛋白的结合性能，αD，L与磷酸盐洗脱液相似，但其他缓冲液的分离效果较差。这项研究的观察结果有助于优化对固定化BSA进行的分离，以及解决与阴离子如何影响血清白蛋白的天然结合特性相关的生物学和机制问题。
High-performance liquid chromatography has been used to examine how phosphate ions affect the binding properties of bovine serum albumin (BSA) immobilized to porous silica. In doing this, the time dependence of the protein to reach conformational equilibrium is measured as a function of the concentration of phosphate in the eluent using theD- andL-isomers of tryptophan and kynurenine as solutes. The overall binding and chiral selectivity (αD,L) of the protein toward these solutes appear to be related to two types of effects: one being those that are site-selective and only influence the retention of theL-isomers and the other being those that are nonselective and influence the retention of both enantiomers. An interesting feature of the concentration-dependent data is a maximum in αD,Lat intermediate phosphate concentrations (i.e., 10 to 50mM phosphate) indicative of both cooperative and antagonistic binding effects. Phosphate eluents within this concentration range provide selectivity advantages, and those at higher concentrations decrease the time required for the protein or column to reach equilibrium. A final set of studies has also been carried out using four alternate buffer systems (i.e., borate, carbonate, acetate, and arsenate eluents). Although the borate eluents affect the BSA's binding properties and αD,Lsimilar to the phosphate eluents, the other buffers result in poor separations. Observations from this study are useful in helping to optimize separations carried out on immobilized BSA as well as addressing biological and mechanistic questions related to how anions influence the native binding properties of serum albumins.