Itoh Daisuke, Yoshimoto Noriko, Yamamoto Shuichi
Bio-Process Engineering Laboratory, Graduate School of Medicine, Biomedical Engineering Center (YUBEC), Yamaguchi University, Tokiwadai, Ube 755-8611, Japan.
Curr Protein Pept Sci. 2019;20(1):75-81. doi: 10.2174/1389203718666171024122106.
Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE).
Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces.
The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient.
通过线性梯度洗脱实验（LGE）研究了蛋白质在羟基磷灰石色谱（HAC）中的保留机制。
评估了几种流动相（缓冲液）溶液策略和溶质，以探究羟基磷灰石（HA）颗粒的两个吸附位点的相对贡献，即由于Ca产生的C位点（金属亲和力）和由于PO4产生的P位点（阳离子交换）。当P位点被阻断时，两种碱性蛋白质，溶菌酶（Lys）和核糖核酸酶A（RNase）不被保留，而细胞色素C（Cyt C）和乳铁蛋白（LF）被保留，并且由于P位点的排斥作用被消除，酸性蛋白质的保留也变得更强。从LGE确定的结合位点B值的数量也增加了，这也表明排斥力降低。
HAC中四种碱性蛋白质（RNase、Lys、Cyt C、LF）的选择性（保留）与离子交换色谱中的不同。此外，通过使用NaCl梯度可以调节选择性。
