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 degrees C). The partition coefficient, Kp, was much greater at 55 than 45 degrees C and depended on both APO12 and protein concentrations. The value of Kp for bovine beta-lactoglobulin (beta-L) varied from 2 to 60, and was larger for 1.0mg/mL solutions than for ovalbumin (2x greater), bovine serum albumin (3x greater) and lysozyme (12x greater). Catalase and cytochrome c were apparently denatured in the presence of 20mg/mL of APO12 and were not investigated. Large values of Kp for beta-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 beta-L showed that the denaturing action of n-decyldimethylphosphine oxide (APO10) below 61 degrees C and APO12 at 22 degrees C was reversed by dilution or dialysis, respectively.
Kresheck
Gordon C
GC
Department of Chemistry, University of Colorado at Colorado Springs, Colorado Springs, CO 80933-7150, USA. gkreshec@uccs.edu
Wang
Zheng
Z
D002152:Calorimetry, Differential Scanning; D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods*; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry / Q000302:isolation & purification; D008823:Micelles; D009113:Muramidase / Q000737:chemistry / Q000302:isolation & purification; D010047:Ovalbumin / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
D003574:Cytochrome c Group; D007782:Lactoglobulins; D008823:Micelles; D011506:Proteins; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; D002374:Catalase; D009113:Muramidase