Liquid chromatographic studies of the effect of phosphate on the binding properties of silica-immobilized bovine serum albumin.
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 the D- and L-isomers of tryptophan and kynurenine as solutes. The overall binding and chiral selectivity (alphaD,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 alphaD,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 BSA's binding properties and alphaD,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 binding properties of serum albumins.
Tao
W A
WA
Department of Chemistry, Wright State University, Dayton 45435, USA.
Gilpin
R K
RK
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D012822:Silicon Dioxide / Q000737:chemistry*
D010710:Phosphates; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide