Departamento Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (S2002LRK), Rosario, Argentina.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jul 1;878(21):1831-6. doi: 10.1016/j.jchromb.2010.05.021. Epub 2010 May 24.
The partitioning pattern of bovine trypsinogen (TRPz) and alpha-chymotrypsinogen (ChTRPz) was investigated in a low impact aqueous two-phase system formed by polyethyleneglycol (PEG) and sodium tartrate (NaTart) pH 5.00. ChTRPz exhibited higher partition coefficients than TRPz did in all the assayed systems. The decrease in PEG molecular weight and the increase in tie line length were observed to displace the partitioning equilibrium of both proteins to the top phase, while phase volume ratios in the range 0.5-1.5 showed not to affect protein partitioning behaviour. Systems formed by PEG of molecular weight 600 with composition corresponding to a high tie line length (PEG 12.93%, w/w and NaTart 21.20%, w/w) are able to recover most of both zymogens in the polymer-enriched phase. A crucial role of PEG-protein interaction in the partitioning mechanism was evidenced by isothermal calorimetric titrations. The major content of highly exposed tryptophan rests, present in ChTRPz molecule, could be considered to be determinant of its higher partition coefficient due to a selective charge transfer interaction with PEG molecule. A satisfactory correlation between partition coefficient and protein surface hydrophobicity was observed in systems formed with PEGs of molecular weight above 4000, this finding being relevant in the design of an extraction process employing aqueous two-phase systems.
在 pH 5.00 的聚乙二醇（PEG）和酒石酸钠（NaTart）形成的低影响双水相体系中研究了牛胰蛋白酶原（TRPz）和α-糜蛋白酶原（ChTRPz）的分配模式。在所有测定的体系中，ChTRPz 的分配系数均高于 TRPz。PEG 分子量的降低和连接线长度的增加观察到将两种蛋白质的分配平衡都转移到上相，而在 0.5-1.5 的相体积比范围内不影响蛋白质的分配行为。PEG 分子量为 600 且组成对应于高连接线长度（PEG 12.93%，w/w 和 NaTart 21.20%，w/w）的系统能够在聚合物富集相中回收大部分两种酶原。等温热滴定实验证明 PEG-蛋白质相互作用在分配机制中起关键作用。ChTRPz 分子中存在的大量暴露色氨酸残基可能被认为是其分配系数较高的决定因素，因为它与 PEG 分子之间存在选择性的电荷转移相互作用。在分子量大于 4000 的 PEG 形成的体系中观察到分配系数与蛋白质表面疏水性之间存在令人满意的相关性，这一发现对于采用双水相体系设计提取过程具有重要意义。
