Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins.

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.

Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning.

The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.

Temperature-induced phase partitioning of peptides in water solutions of ethylene oxide and propylene oxide random copolymers.

A thermoseparating random copolymer (Ucon 50-HB-5100) composed of (50%) ethylene oxide and (50%) propylene oxide has been used to form an aqueous two-phase system by heating the polymer-water solution above the cloud point of the copolymer. In the formed two-phase system a water rich top phase is in equilibrium with an aqueous polymer rich bottom phase. The partitioning of amino acids and peptides in this aqueous two-phase system has been studied. Hydrophobic peptides (containing aromatic amino acids) were strongly partitioned to the polymer rich phase, while hydrophilic peptides were enriched in the water rich phase. The effect of temperature on the partitioning was investigated and a decreased partitioning to the polymer rich phase was obtained upon temperature increase. The effect of two salts (NaClO4 and Na2SO4) on the partitioning of a positively charged polypeptide, poly(Lys, Trp), was very strong. With NaClO4 the polypeptide was quantitatively partitioned to the polymer rich phase while with Na2SO4 the polypeptide was partitioned to the water rich phase. Model calculations based on a modified Flory-Huggins theory have been performed to better understand the experimental behavior.

Effects of ions on partitioning of serum albumin and lysozyme in aqueous two-phase systems containing ethylene oxide/propylene oxide co-polymers.

Aqueous two-phase systems composed of ethylene oxide/propylene oxide random co-polymers, EO30/PO70 or Ucon (EO50/PO50), in the top phase and dextran T500 in the bottom phase, have been studied. The cloud point diagram for EO30/PO70 in water solution was determined. EO30/PO70 has a cloud point of 32 degrees C at a concentration of 10% (w/w). The phase diagram for the system EO30/PO70-dextran T500-water was determined. Salt effects have been studied on the partitioning of two model proteins, bovine serum albumin and hen egg white lysozyme, in EO30/PO70-dextran and Ucon-dextran systems. Ions with different hydrophobicity, i.e., with different position in the Hofmeister or lyotropic series, were investigated with reference to their effect on protein partition. The counterion hydrophobicity was shown to have a strong influence on the partitioning of BSA and lysozyme. Most extreme partitioning was obtained with hydrophobic (chaotropic) ions like CIO4- and I-. A comparison of protein partitioning between PEG-dextran and EO30/PO70-dextran has been done. A more extreme protein partitioning was obtained in the EO30/PO70-dextran containing system. Temperature-induced phase separation was studied with EO30/PO70 at 45 degrees C. Both BSA and lysozyme were completely partitioned to the water phase formed above the cloud point of EO30/PO70. Model calculations, based on Flory-Huggins theory of polymer solutions, have been done which could reproduce the salt effect on the protein partitioning in aqueous-two phase system.

Partition of proteins in aqueous polymer two-phase systems and the effect of molecular weight of the polymer.

The partition of substances in aqueous polymer two-phase systems is influenced by the molecular weight of the phase-forming polymers. We investigate how the effect of the molecular weight of the polymers depends on the molecular weight of the partitioned protein. We show that the magnitude of change of the partition is very small for proteins of molecular weights around 10,000, but increases almost linearly up to molecular weights of 250,000.

The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic alpha-helix.

The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.

Compartmentalization of enzymes and distribution of products in aqueous two-phase systems.

Phase separation is a common phenomenon in water solutions of polymers due to "polymer incompatibility." Polymeric aqueous two-phase systems are much used for separations in biochemistry and cell biology. When macromolecules are included in a phase system, it is often possible to obtain a one-sided distribution to one of the phases, i.e., the macromolecule is compartmentalized within one aqueous phase. This chapter describes the thermodynamic forces which govern the partitioning of molecules in aqueous two-phase systems. For a high molecular weight macromolecule, e.g., an enzyme, both enthalpic and entropic effects contribute to a one-sided partitioning. Molecules of low molecular weight will be more evenly distributed between the phases. These mechanisms are significant in biological systems and can be used for enzyme reactors in bioconversions. Enzymatic reactions can take place with enzyme and substrate compartmentalized in one of the phases. A low-molecular weight product which is evenly partitioned between the phases can be continuously removed from the enzyme-substrate compartment. These principles are described in the enzymatic conversion of cellulose in an aqueous two-phase system.

Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide--propylene oxide copolymers.

Endoglucanases (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide-propylene oxide random copolymer (EO(50)PO(50)) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)(4) tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)(2) tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)(4) improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)(2).

Softwood hemicellulose-degrading enzymes from Aspergillus niger: purification and properties of a beta-mannanase.

The enzymes needed for galactomannan hydrolysis, i.e., beta-mannanase, alpha-galactosidase and beta-mannosidase, were produced by the filamentous fungus Aspergillus niger. The beta-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the beta-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger beta-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus beta-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.

Hydrolytic properties of a beta-mannosidase purified from Aspergillus niger.

A beta-mannosidase was purified to homogeneity from the culture filtrate of Aspergillus niger. A specific activity of 500 nkat mg-1 and a 53-fold purification was achieved using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The isolated enzyme has an isoelectric point of 5.0 and appears to be a dimer composed of two 135-kDa subunits. It is a glycoprotein and contains 17% N-linked carbohydrate by weight. Maximal activity was observed at pH 2.4 5.0 and at 70 degrees C. The beta-mannosidase hydrolyzed beta-1,4-linked manno-oligosaccharides of degree of polymerization (DP) 2-6 and also released mannose from polymeric ivory nut mannan and galactomannan. The Km and Vmax values for p-nitrophenyl-beta-D-mannopyranoside were 0.30 mM and 500 nkat mg-1, respectively. Hydrolysis of D-galactose substituted manno-oligosaccharides showed that the beta-mannosidase was able to cleave up to, but not beyond, a side group. An internal peptide sequence of 15 amino acids was highly similar to that of an Aspergillus aculeatus beta-mannosidase belonging to family 2 of glycosyl hydrolases.

High-sensitivity linear dichroism as a tool for equilibrium analysis in biochemistry. Stability constant of DNA--ethidiumbromide complex.

A stoichiometrical application of a sensitive method for linear dichroism (LD) detection is suggested for biochemical purposes. The complex formation between a binding site on a polynucleotide and a ligand may be studied with high precision if the following conditions are fulfilled: (1) The polymer can be given a fixed degree of orientation. (2) The site has a specific orientation with respect to the orientation axis of the polymer (e.g., intercalation). (3) The ligand has an anisotropic optical absorption property. The method was applied to studying the complex between DNA and ethidiumbromide, which was detected by LD with precision of +/- 0.5 X 10(-7) M in a 4 X 10(-4) M DNA solution, i.e., 0.1% occupation of the total site concentration can be detected. The complexation could be explained by a single type of site (n = 0.14 +/- 0.01 sites per nucleotide residue) and a stability constant K1 = (2.5 +/- 1) X 10(5) M-1 at 0.2 M ionic strength. From the specific LD an average angle 60 degrees was concluded between the helix axis and the long axis of the ethidiumbromide molecule. This value formally contradicts the Watson-Crick model or the intercalation model but may be explained by extension and deformation effects on the xhain by the flow.

Linear dichroism studies of binding site structures in solution. Complexes between DNA and basic arylmethane dyes.

The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect to optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K1 ca 10(5) M-1; site density per nucleotide base n1 ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50 degrees to the local DNA helix axis. The complex with fuchsin is weaker (10(4) M-1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C2v symmetry) the angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. The optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydro blue, a perpendicular orientation was observed, possibly due to intercalation.

Interaction between tryptophan residues and hydrophobically modified dextran. Effect on partitioning of peptides and proteins in aqueous two-phase systems.

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan-tryptophan, (tryptophan)3, poly(lysine, tryptophan), beta-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)-benzoyl dextran, PEG-valeryl dextran, dextran-benzoyl dextran and dextran-valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG-dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG-benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, Nal and NaClO4) was determined in PEG-benzoyl dextran and PEG-valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), beta-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I- and ClO4-.

Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems. Effect of fusion localization on endoglucanase I of Trichoderma reesei.

Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-beta-Dglucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.

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 in aqueous two-phase systems consisting of micelle-forming block copolymers from the family of Pluronic block copolymers as one polymer component and dextran T500 as the other component. The Pluronic molecule is a triblock copolymer 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 degrees C for these polymer systems. Since the temperature is an important parameter in Pluronic systems (the block copolymers form micellar-like aggregates at higher temperatures) the partitioning experiments were performed at 5 and 30 degrees C, to explore the effect of temperature-triggered micellization on the partitioning behaviour. The temperatures correspond to the unimeric (single Pluronic chain) and 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 membrane-bound peptide, gramicidin D, and five different proteins were partitioned in the above systems. The proteins were lysozyme, bovine serum albumin, cytochrome c, bacteriorhodopsin and the engineered B domain of staphylococcal protein A, named Z. The Z domain was modified with tryptophan-rich peptide chains in the C-terminal end. It was found that effects of salt dominated over the temperature effect for the water-soluble proteins lysozyme, bovine serum albumin and cytochrome c. A strong temperature effect was observed in the partitioning of the integral membrane protein bacteriorhodopsin, where partitioning towards the more hydrophobic Pluronic phase was higher at 30 degrees C than at 5 degrees C. The membrane-bound peptide gramicidin D partitioned exclusively to the Pluronic phase at both temperatures. The following trends were observed in the partitioning of the Z protein. (i) At the higher temperature, insertion of tryptophan-rich peptides increased the partitioning to the Pluronic phase. (ii) At the lower temperature, lower values of K were observed for ZT2 than for ZT1.

Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers.

In this study we present a new aqueous two-phase system where both polymers are thermoseparating. In this system it is possible to recycle both polymers by temperature induced phase separation, which is an improvement of the aqueous two-phase system previously reported where one of the polymers was thermoseparating and the other polymer was dextran or a starch derivative. The polymers used in this work are EO50PO50, a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random copolymer of EO and PO with aliphatic C14H29-groups coupled to each end of the polymer (HM-EOPO). In water solution both polymers will phase separate above a critical temperature (cloud point for EO50PO50 50 degrees C, HM-EOPO, 14 degrees C) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM-EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous two-phase system is formed analogous to poly(ethylene glycol) (PEG)/dextran system. The partitioning of three proteins, bovine serum albumin, lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM-EOPO system and how the partitioning is affected by salt additions. Protein partitioning is affected by salts in similar way as in traditional PEG/dextran system. Recombinant apolipoprotein A-1 has been purified from a cell free E. coli fermentation solution. Protein concentrations of 20 and 63 mg/ml were used, and the target protein could be concentrated in the HM-EOPO phase with purification factors of 6.6 and 7.3 giving the yields 66 and 45%, respectively. Recycling of both copolymers by thermoseparation was investigated. In protein free systems 73 and 97.5% of the EO50PO50 and HM-EOPO polymer could be recycled respectively. Both polymers were recycled after aqueous two-phase extraction of apolipoprotein A-1 from a cell free E. coli fermentation solution. Apolipoprotein A-1 was extracted to the HM-EOPO phase with contaminating proteins in the EO50PO50 phase. The yield (78%) and purification factor (5.5) of apolipoprotein A-1 was constant during three polymer recyclings. This new phase system based on two thermoseparating polymers is of great interest in large scale extractions where polymer recycling is of increasing importance.

Effects of fused tryptophan rich peptides to a recombinant protein. A domain on the partitioning in polyethylene glycol-dextran and Ucon-dextran aqueous two-phase systems.

Genetic engineering has been used to construct fusion proteins with tryptophan containing peptides. The peptides and the fusion proteins have been partitioned in aqueous two-phase systems of poly(ethylene glycol) (PEG)-dextran and Ucon-dextran. The studied model protein was ZZT0, where Z is an engineered domain of domain B of staphylococcal protein A. The specially designed hydrophobic peptides, Ala-Trp-Trp-Pro (T1) and (Ala-Trp-Trp-Pro)2 (T2), have been inserted into ZZT0, to give the peptide-protein fusions ZZT1 and ZZT2. In the experimental studies it was found that T1 and T2 preferred the PEG phase and even more the Ucon phase over the dextran phase. For T2 the partitioning was more one sided than for T1. For the fusion proteins, ZZT1 and ZZT2, the partitioning was enhanced into the PEG or Ucon rich phase as compared to ZZT0. The effects were lower than expected from independent contributions to the partition coefficient from the protein and the peptides. A heterogeneous lattice model was used to calculate theoretical peptide and protein partition coefficients. The calculations could reproduce the qualitative features of the experimental data. The model results suggest that a part of these experimentally observed effects is due to a depletion zone, i.e. a zone of reduced polymer concentration around the protein. The experimental results indicate a further reduction of the partition coefficient, beyond that predicted by the lattice calculations. A possible folding of the inserted peptide is discussed as a plausible mechanism for this further reduction in the partition coefficient.

Application of temperature-induced phase partitioning at ambient temperature for enzyme purification.

Aqueous two-phase partition and temperature-induced phase separation using a non-ionic, random copolymer composed of 20% ethylene oxide, 80% propylene oxide (EO20 PO80) has been used for purification of glucose-6-phosphate dehydrogenase, hexokinase and 3-phosphoglycerate kinase from bakers' yeast. This EO20PO80 copolymer has a cloud point of 18 degrees C, at which temperature it phase separates from water. Enzymes were first partitioned at 4 degrees C in an initial EO20PO80-dextran T500 aqueous two-phase system. This system had an upper copolymer-rich phase and a lower dextran-rich phase. After phase separation had occurred the upper EO20PO80-rich phase was removed and placed at 24 degrees C. This resulted in formation of a new two-phase system with an upper water phase and a lower phase containing 98% copolymer and 2% water. Enzymes were recovered exclusively in upper water phase leaving a polymer-rich lower phase free of contamination. The phase diagram for the system EO20PO80 and dextran T500 at 4 degrees C has been determined.

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy.

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.

Partition of macromolecules and cell particles in aqueous two-phase systems based on hydroxypropyl starch and poly(ethylene glycol).

The partition behavior of proteins, nucleic acids, cell membranes, cell organelles and whole cells has been studied in liquid-liquid two-phase systems composed of water, poly-(ethylene glycol), and an hydroxypropyl starch. The properties of the systems are in many respects comparable with the traditional poly(ethylene glycol)-dextran systems, but the cost is reduced to around one-fifth.