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Biomedical subjects

F Tjerneld

Publications and source records attributed to F Tjerneld.

At least 55 records · Page 3Linked to original sources
PubMed1996

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.

Bromides↗
PubMed1996

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling.

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from baker's yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzoyl dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na2SO4.

Dextrans↗
PubMed1996

Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms.

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR)-inhibitory substances from bacterial cells. The PCR inhibition of four soft cheeses was examined and three of them were found to be strongly PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inoculated with Listeria monocytogenes in an aqueous two-phase system containing 8% (w/w) PEG 4000 and 8% (w/w) dextran 500, was found to lower the PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sample pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or bottom phase in the aqueous two-phase system. These results show that different soft cheeses contain different types and amounts of PCR-inhibitory substances.

Cheese↗
PubMed1996

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems.

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C12E5 (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, beta-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C12E5 systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.

Buffers↗
PubMed1996

Partitioning of beta-mannanase and alpha-galactosidase from Aspergillus niger in Ucon/Reppal aqueous two-phase systems and using temperature-induced phase separation.

Enzyme partitioning and recovery with a new aqueous two-phase system based on commercially available hydroxypropyl starch Reppal PES 200 and the thermo-separating polymer Ucon 50-HB-5100 was studied. Ucon is an ethylene oxide-propylene oxide random copolymer. A culture supernatant of Aspergillus niger containing extracellular beta-mannanase and alpha-galactosidase was partitioned in two steps. The primary aqueous two-phase system contained Ucon and Reppal as phase forming polymers. The effect on enzyme partitioning of salt composition, salt concentration, pH and polymer concentration was studied with the aim of obtaining optimal partitioning of target enzymes to the phase containing the thermoseparating Ucon polymer. The partitioning of the enzymes could be strongly influenced by addition of the hydrophobic triethyl ammonium ion and the chaotropic perchlorate ion. Also the effect on cationic surfactant, cetyl trimethyl ammonium bromide, on enzyme partitioning was studied. In the second step, temperature induced phase separation was carried out on the isolated Ucon phase. A water phase and a concentrated aqueous Ucon phase were formed. The enzymes were obtained in the water phase almost free of polymer.

Aspergillus niger↗
PubMed1995

Partitioning of proteins in dextran/hydrophobically modified dextran aqueous two-phase systems.

Partitioning of proteins was studied in aqueous two-phase systems composed of the polymers dextran and hydrophobically modified dextran. The modified dextrans were benzoyl dextran with a degree of substitution of 0.17 and valeryl dextran with a degree of substitution of 0.20. Phase diagrams for the systems of dextran/benzoyl dextran and dextran/valeryl dextran were determined at room temperature. The proteins studied were beta-galactosidase, bovine serum albumin, beta-lactoglobulin, lysozyme, myoglobin and cytochrome C. The partition coefficients of a series of salts were determined in dextran/benzoyl dextran two-phase systems. The addition of salts had strong effect on the partitioning of proteins. This effect was related to protein net charge and the position of the ions in the Hofmeister series. Cross partitioning of bovine serum albumin was studied in a dextran/benzoyl dextran aqueous two-phase system.

Animals↗
PubMed1994

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.

Chemical Phenomena↗
PubMed1994

Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method.

A sample treatment method based on an aqueous two-phase system containing polyethylene glycol and dextran was developed for enhancing sensitivity in the detection of Listeria monocytogenes in soft cheese with PCR. The results suggest that the improved detection sensitivity following partitioning of the cheese homogenate in an aqueous two-phase system may be due to partitioning of the PCR inhibitors to the polyethylene glycol phase.

Base Sequence↗
PubMed1992

Interactions in affinity partition studied using fluorescence spectroscopy.

Fluorescence titration has been used to determine the binding constant and number of binding sites for the textile triazine dye Procion Yellow HE-3G to lactate dehydrogenase from rabbit muscle (E.C. 1.1.1.27). Triazine dye was either free in solution or attached to one of the polymer carriers, polyethylene glycol or dextran. Titrations were performed in solutions of buffer, dextran, and polyethylene glycol. Aqueous two-phase systems composed of polyethylene glycol and dextran were prepared and the binding constant and number of binding sites for ligand polyethylene glycol-Procion Yellow to lactate dehydrogenase were determined in both upper and lower phases of these systems. Affinity partition of lactate dehydrogenase in a PEG-dextran system was also performed using PEG-Procion Yellow as ligand, and partition coefficients of lactate dehydrogenase showed good agreement with theoretical partition coefficients calculated from the binding constant and number of binding sites obtained from fluorescence titration. The advantage of using fluorescence titration to determine affinity of a polymer ligand for a protein is that measurement of binding strength can be made in the actual environment encountered by protein-ligand complex during the purification process.

Algorithms↗
PubMed1992

Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning.

Synthesis of conjugates of the ethylene oxide/propylene oxide copolymer UCON 50-HB-5100 and the triazine dyes Cibacron Blue F3G-A and Procion Yellow HE-3G is described. The UCON-dye conjugate of Procion Yellow HE-3G is used as a ligand for affinity partitioning of glucose-6-phosphate dehydrogenase from bakers' yeast. The enzyme is first partitioned in a two-phase system composed of UCON, UCON-ligand and dextran, and the two phases isolated in separate containers. A small amount of salt is then added to the upper phase, which contains the UCON-ligand-enzyme complex, and the temperature increased above the cloud point of the UCON polymer to give a new two-phase system. The new two-phase system consists of an upper salt/water phase containing free enzyme and a lower UCON/water phase containing free UCON-ligand. Temperature-induced phase partitioning is thus seen to be of much assistance in dissociating enzyme-ligand complex, recovering enzyme and recycling UCON-ligand.

Chemical Phenomena↗
PubMed1991

Enzyme purification using temperature-induced phase formation.

A new type of aqueous two-phase system composed of an ethylene oxide and propylene oxide random co-polymer, UCON 50-HB-5100, as the upper phase polymer and either dextran or hydroxypropyl starch as the lower phase polymer has been characterized and used to purify 3-phosphoglycerate kinase (EC 2.7.2.3) and hexokinase (EC 2.7.1.1) from bakers' yeast. The UCON 50-HB-5100 polymer has a cloud point of 55 degrees C at which temperature it phase separates from water. This cloud point can be lowered to 40 degrees C by the addition of 0.2 M sodium sulfate salt. The low cloud point of this UCON polymer makes it possible to obtain the target enzymes in a water and buffer solution, and to recover and recycle the UCON 50-HB-5100 polymer. The phase diagrams for the systems UCON 50-HB-5100/Dextran T500 and UCON 50-HB-5100/hydroxypropyl starch have been determined. Yeast homogenate was first partitioned in a system composed of a top phase containing UCON 50-HB-5100 and a bottom phase containing either dextran or hydroxypropyl starch. The top phase containing the enzyme free of cell debris was removed and the temperature increased above the cloud point of the UCON until a new two phase system composed of water as the top phase and a concentrated liquid UCON 50-HB-5100 bottom phase was formed. The water phase containing the enzyme was removed and the bottom phase containing the UCON 50-HB-5100 could be recycled to perform a second extraction.

Hexokinase↗
PubMed1991

Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.

Cell Membrane↗
PubMed1990

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.

Cell Fractionation↗
PubMed1990

Separation processes in biotechnology. Aqueous two-phase separations.

Aqueous two-phase systems are useful for separation of a wide range of water-compatible substances (from peptides to cells). The selectivity of the separation normally increases with the size of the partitioned molecules or particles. The partition and separation capacity can be influenced in a number of ways, including electric charge, hydrophobicity, or specific ligand binding. Because of the simpleness in operation and high capacity, aqueous two-phase systems are well suited for large-scale purification of biomaterials such as enzymes and other specific proteins.

Animals↗
PubMed1990

Aqueous two-phase systems for biotechnical use.

The different kinds of aqueous two-phase systems for accepted or potential use in biotechnology are summarized. Some properties of interest for the extractive use are discussed.

Biotechnology↗
PubMed1987

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.

Dextrans↗