Liquid Chromatographic Studies of the Effect of Phosphate onthe Binding Properties of Silica-Immobilized Bovine Serum Albumin

摘要

High-performance liquid chromatography has been used toexamine how phosphate ions affect the binding properties ofbovine serum a~bumrn ~SN~ ~rnm ~ze4 ts porotis s~1ica. In doing this, the time dependence of the protein to reach conformational uilibrium is measured as a function of the concentration of phosphatein the eluent using the D- and L-isomers of tryptophanand kynurenine as solutes. The overall binding and chiralselectivity (aD,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 the L-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 ctD,L at 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 OSA’s binding properties and CLD,L similar 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 bindingproperties of serum albumins.