López-Mas J A, Streitenberger S A, García-Carmona F, Sánchez-Ferrer A
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Spain.
J Chromatogr A. 2001 Mar 9;911(1):47-53. doi: 10.1016/s0021-9673(00)01248-6.
Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography.
以三嗪染料为模型的汽巴克隆蓝F3GA（CB F3GA）与以环糊精为模型的2-羟丙基-β-环糊精（HP-β-CD）之间的相互作用，通过监测伴随结合现象的光谱位移进行了研究。对CB F3GA与HP-β-CD的差示光谱滴定进行基质分析，仅发现两个吸收峰，表明主客体比例为1:1。该HP-β-CD-CB F3GA复合物的解离常数Kd为0.43 mM。利用HP-β-CD与CB F3GA形成包合物的数据，提出了染料亲和色谱中包合物竞争洗脱的概念。当将这一概念应用于从CB F3GA亲和基质上洗脱L-乳酸脱氢酶时，结果表明这是一种有效的洗脱策略。它提供了15倍的纯化因子，回收率为89%，洗脱峰尖锐（80%回收率时为0.8个柱体积），与用NADH特异性洗脱（16倍，78%回收率，1.8个柱体积）的效果相当。此外，新的洗脱策略比用KCl进行的传统非特异性洗脱（4.5倍，1.4个柱体积）具有更好的纯化因子和更尖锐的洗脱峰。因此，包合物竞争洗脱可能是一种在染料亲和色谱中以高回收率和纯化因子洗脱蛋白质的有前景的策略。
