Department of Biotechnology, University of Malakand, Lower Dir, Khyber Pakhtunkhwa, Pakistan.
Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
Biotechnol Prog. 2019 Jul;35(4):e2828. doi: 10.1002/btpr.2828. Epub 2019 May 6.
Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.
疏水相互作用层析是一种重要且有效的纯化策略，通常用于多种生物分子的纯化。为了有效地分离这些生物分子，需要对蛋白质相互作用行为有基本的了解。利用胶体扩展的德热那乌、朗道、维韦和奥弗贝克计算，研究了模型蛋白质与市售疏水色谱材料（即 Toyopearl Phenyl 650C 和 Toyopearl Butyl 650C）的相互作用行为。通过接触角和zeta 电位测量，获得了所选模型蛋白质的物理化学性质。通过在破碎的珠子上采用静止液滴法和在完整的珠子上采用毛细渗透法（CPM），实现了色谱材料的接触角。进一步利用表面性质计算蛋白质与色谱支撑物的相互作用。从两种方法确定的接触角值计算出的蛋白质与色谱材料的二次能量最小值与真实色谱的保留体积相关。从静止液滴法获得的输入值计算出的每种蛋白质与色谱材料的二次能量最小值均高于 CPM。例如，免疫球蛋白 G 的二次能量最小值为 0.17 kT，而通过静止液滴法和 CPM 分别获得的二次能量最小值为 0.11 kT。对于所有蛋白质，Toyopearl Butyl 650C 和 Toyopearl Phenyl 650C 的能量最小值的平均相对值分别为 1.51 kT 和 1.29 kT，作为两种方法估计二次能量最小值的转换因子。
