Shi Qinghong, Zhou Ying, Sun Yan
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China.
Biotechnol Prog. 2005 Mar-Apr;21(2):516-23. doi: 10.1021/bp049735o.
The ion-exchange equilibrium and the dependence of the parameters in the steric mass-action (SMA) model on salt concentration and buffer pH around the isoelectric point of protein were studied. Bovine serum albumin (BSA, isoelectric point = 5.4) was used as a model protein and DEAE Sepharose FF as an ion exchanger. Finite batch adsorption experiments and isocratic elution chromatography were performed for the determination of the model parameters (i.e., characteristic charge, equilibrium constant, and steric factor). The results showed that pH had significant effects on the parameters. With an increase of pH from 4.5 to 6.5, the characteristic charge increased from 0.9 to 3.0 and leveled off as a plateau at pH above 5.5. The charge groups in the contact region of protein surface were considered to play a crucial role on the characteristic charge. The decrease of pH and increase of salt concentration lowered the absolute value of the zeta potential of the protein surface and led to a decrease of the equilibrium constant. The steric factor remained unchanged at about 31 at pH 5.5 and 6.0 and increased to 44.5 at pH 5.0 and 96.8 at pH 4.5, mainly as a result of the lower adsorption capacity of BSA at pH <5.5. Furthermore, the increase of the molecular volume of BSA at pH 4.5 would be an additional reason for the increase of the steric factor. Taking into account the effect of the pH and salt concentration on these parameters, the SMA model described the ion exchange equilibrium of protein more accurately.
研究了离子交换平衡以及空间质量作用（SMA）模型中参数对蛋白质等电点附近盐浓度和缓冲液pH值的依赖性。以牛血清白蛋白（BSA，等电点 = 5.4）作为模型蛋白，DEAE琼脂糖FF作为离子交换剂。进行了有限批次吸附实验和等度洗脱色谱分析以确定模型参数（即特征电荷、平衡常数和空间因子）。结果表明，pH值对这些参数有显著影响。随着pH值从4.5增加到6.5，特征电荷从0.9增加到3.0，并在pH高于5.5时趋于平稳。蛋白质表面接触区域的电荷基团被认为对特征电荷起着关键作用。pH值的降低和盐浓度的增加降低了蛋白质表面zeta电位的绝对值，并导致平衡常数减小。空间因子在pH 5.5和6.0时保持在约31不变，在pH 5.0时增加到44.5，在pH 4.5时增加到96.8，这主要是由于BSA在pH <5.5时吸附能力较低。此外，BSA在pH 4.5时分子体积的增加将是空间因子增加的另一个原因。考虑到pH值和盐浓度对这些参数的影响，SMA模型能更准确地描述蛋白质的离子交换平衡。
