A new micellar aqueous two-phase partitioning system (ATPS) for the separation of proteins

Partitioning of six typical globular proteins with molecular weights ranging from 12.6 to 250 kDa was investigated using an aqueous two-phase system formed by heating a solution containing the individual proteins and n-dodecyldimethylphosphine oxide (APO12) above the cloud point of the nonionic surfactant (approximately 40 °C). The partition coefficient, Kp, was much greater at 55 than 45 °C and depended on both APO12 and protein concentrations. The value of Kp for bovine β-lactoglobulin (β-L) varied from 2 to 60, and was larger for 1.0 mg/mL solutions than for ovalbumin (2× greater), bovine serum albumin (3× greater) and lysozyme (12× greater). Catalase and cytochrome c were apparently denatured in the presence of 20 mg/mL of APO12 and were not investigated. Large values of Kp for β-L resulted when the pH of APO12 mixtures containing phospholipids and either a cationic or anionic surfactant in molar ratios of 10:0.5:1.0 was partitioned above or below the isoelectric point of the protein, respectively. The affinity of the proteins for the APO12 micelle was responsible for partitioning of the proteins into the upper phase. Finally, DSC studies with β-L showed that the denaturing action of n-decyldimethylphosphine oxide (APO10) below 61 °C and APO12 at 22 °C was reversed by dilution or dialysis, respectively.