Features of partitioning pattern of two pancreatic enzymatic precursors: Trypsinogen and chymotrypsinogen in polyethyleneglycol–sodium citrate aqueous biphasic systems

The partitioning behaviour of bovine trypsinogen and alpha-chymotrypsinogen, enzymatic precursors with similar physicochemical properties, was investigated in different polyethyleneglycol/sodium citrate aqueous two-phase systems. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length and temperature was also examined. The increase of pH and the decrease of polyethyleneglycol molecular weight displaced the partitioning equilibrium of both proteins to the top phase. An enthalpy–entropy compensation pattern was observed, indicating the participation of water molecules in the partitioning mechanism. TRPz phase equilibrium showed to be more displaced to the citrate-rich phase than ChTRPz for most of the assayed systems. From a practical view, the aqueous two-phase system formed by polyethylenglycol of molecular weight 1450 and sodium citrate pH 8.20 showed the best capability for separating both proteins. When a mixture formed by equal quantities of both zymogens was partitioned in this system, significant recoveries (about 60%) were obtained. Purity values were improved significantly (84–89%) by either developing a second extractive step or increasing the top–bottom volume ratio.