Aqueous two-phase systems containing self-associating block copolymers - Partitioning of hydrophilic and hydrophobic biomolecules

A series of proteins and one membrane-bound peptide have been partitioned i n aqueous two-phase systems consisting of micelle-forming block copolymers from the family of Pluronic block copolymers as one polymer component and d extran T500 as the other component. The Pluronic molecule is a triblock cop olymer of the type PEO-PPO-PEO, where PEO and PPO are poly(ethylene oxide) and poly(propylene oxide), respectively. Two different Pluronic copolymers were used, P105 and F68, and the phase diagrams were determined at 30 degre es C for these polymer systems. Since the temperature is an important param eter in Pluronic systems (the block copolymers form micellar-like aggregate s at higher temperatures) the partitioning experiments were performed at 5 and 30 degrees C, to explore the effect of temperature-triggered micellizat ion on the partitioning behaviour. The temperatures correspond to the unime ric (single Pluronic chain) Emd the micellar states of the P105 polymer at the concentrations used. The degree of micellization in the F68 system was lower than that in the P105 system, as revealed by the phase behaviour. A m embrane-bound peptide, gramicidin D, and five different proteins were parti tioned in the above systems. The proteins were lysozyme, bovine serum album in, cytochrome c, bacteriorhodopsin and the engineered B domain of staphylo coccal protein A, named Z. The Z domain was mortified with tryptophan-rich peptide chains in the C-terminal end. It was found that effects of salt dom inated over the temperature effect for the water-soluble proteins lysozyme, bovine serum albumin and cytochrome c. A strong temperature effect was obs erved in the partitioning of the integral membrane protein bacteriorhodopsi n, where partitioning towards the more hydrophobic Pluronic phase was highe r at 30 degrees C than at 5 degrees C. The membrane-bound peptide gramicidi n D partitioned exclusively to the Pluronic phase at both temperatures. The following trends were observed in the partitioning of the Z protein. (i) A t the higher temperature, insertion of tryptophan-rich peptides increased t he partitioning to the Pluronic phase. (ii) At the lower temperature, lower values of K were observed for ZT2 than for ZT1. (C) 1999 Elsevier Science B.V. All rights reserved.