| 31. |
- Kempe, Maria, et al.
(författare)
-
Separation of amino acids, peptides and proteins on molecularly imprinted stationary phases.
- 1995
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 691:1-2, s. 317-323
-
Tidskriftsartikel (refereegranskat)abstract
- Stationary phases, to be used in high-performance liquid chromatography, were tailor-made for the separation of amino acids, peptides and proteins. The stationary phases were prepared by molecular imprinting, applying two different approaches. Low-molecular-mass compounds were imprinted in bulk polymers by copolymerization of functional monomers and cross-linkers in the presence of the compound of interest, the print molecule. These polymers were, after extraction of the print molecule, successfully applied as chiral stationary phases, showing high resolution and load capacity. The development of a surface-imprinting approach for the preparation of stationary phases selective for proteins is also discussed.
|
|
| 32. |
- Kjellström, S, et al.
(författare)
-
On-line coupling of microdialysis sampling with liquid chromatography for the determination of peptide and non-peptide leukotrienes
- 1998
-
Ingår i: Journal of chromatography. A. - 0021-9673. ; 823:1-2, s. 489-496
-
Tidskriftsartikel (refereegranskat)abstract
- An automated on-line sampling method was developed using microdialysis as the simultaneous sampling and sample pre-treatment technique. The extraction fraction values of microdialysis probes sampling different eicosanoids were investigated. The impact of cyclodextrins in the perfusion liquid used for sampling hydrophobic eicosanoids in biological systems was also studied. The total time for one analysis was 7.6 min allowing seven measurements per hour for monitoring kinetic changes in biological systems.
|
|
| 33. |
- Lee, Seungho, et al.
(författare)
-
Development of asymmetrical flow field-flow fractionation-multi angle laser light scattering analysis for molecular mass characterization of cationic potato amylopectin
- 2003
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 1011:1-2, s. 111-123
-
Tidskriftsartikel (refereegranskat)abstract
- The goal of this study is to investigate the applicability of asymmetrical flow field-flow fractionation (AsFlFFF)–multi angle laser light scattering (MALLS), and to develop a method for analysis of cationic potato amylopectin (CPAP) having ultrahigh molecular mass (UHMr). Use of the aqueous carrier having low salt content (3 mM NaN3) resulted in a distortion in AsFlFFF fractograms of CPAP with a general pattern of a sharp rise at the beginning of the elution followed by a long tailing, probably due to combination of attractive and repulsive charge interactions (attractive interaction between CPAP molecules and the channel membrane, and repulsion among cationic CPAP molecules). As the cross flow-rate (Fc) increases, the tailing tends to increase, and the repeatability of the AsFlFFF retention data tends to decrease, which is an indication of the presence of the charge interactions. The tailing gradually decreased, and the repeatability of the AsFlFFF retention data increased, as the salt content of the carrier increased. The distortion of the fractogram finally disappeared at Fc of about 0.2 ml/min and the channel flow-rate (Fout) of about 1 ml/min with the aqueous carrier having the salt content of 40 mM (3 mM NaN3+37 mM NaNO3). The weight-average molecular mass (Mw) and the z-average radius of gyration (z) determined by MALLS were 5.2×107 and 34×101 nm, respectively. With the flow-rate ratio, Fc/Fout kept constant, the degree of the charge interactions (and thus the distortion of fractogram) seems to increase with the cross flow-rate (Fc) and with the sample injection mass. AsFlFFF–MALLS was applied for determination of molecular mass distributions (MrDs) and the sizes of CPAPs prepared by various cooking procedures.
|
|
| 34. |
- Lowe, Christopher R., et al.
(författare)
-
High-performance liquid affinity chromatography of proteins on Cibacron Blue F3G-A bonded silica
- 1981
-
Ingår i: Journal of chromatography. A. - 0021-9673. ; 215, s. 303-316
-
Tidskriftsartikel (refereegranskat)abstract
- The reactive triazine dye, Cibacron Blue F3G-A, has been covalently attached to microparticulate porous silica and used for the resolution of a number of complementary proteins by high-performance liquid affinity chromatography (HPLAC). Cibacron Blue F3G-A was converted to its 6-aminohexyl derivative by reaction with 1,6-diaminohexane and coupled to γ-glycidoxypropyltrimethoxysilane-activated porous silica (5 μm) to generate an adsorbent containing 5.5–6.7 μmol dye/g silica.Cibacron Blue F3G-A silica columns, in conjunction with on-line monitoring of protein concentration and enzyme activity, may be exploited for the high speed fully automated resolution of dehydrogenases, isoenzymes, kinases and other proteins such as pancreatic ribonuclease A from simple or complex mixtures. The examples demonstrate the versatility of HPLAC on silica-immobilised Cibacron Blue F3G-A.
|
|
| 35. |
- Lu, Min, et al.
(författare)
-
Interaction between tryptophan residues and hydrophobically modified dextran - Effect on partitioning of peptides and proteins in aqueous two-phase systems
- 1997
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 766:1-2, s. 99-108
-
Tidskriftsartikel (refereegranskat)abstract
- 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), β-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, NaI 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), β-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-.
|
|
| 36. |
- Medve, József, et al.
(författare)
-
Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography
- 1998
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 808:1-2, s. 153-165
-
Tidskriftsartikel (refereegranskat)abstract
- Trichoderma cellulases appear in several isoforms which makes their purification and analysis difficult. We used fast protein liquid chromatography (FPLC) to purify three major cellulases and to quantitate these enzymes in reconstituted mixtures during cellulose hydrolysis studies (in lack of specific substrates and because of the synergism between the enzymes such analysis is very difficult, if at all possible, with conventional activity measurements). For the analysis methods linear calibration was achieved from 10-15 pmol to 0.5-1 nmol (from 0.5-0.8 to 27-64 μg) for the different enzymes. Due to the high resolution chromatographic media used, our purification methods are simpler and quicker than the usual protocols for cellulase purification. Several isoforms of cellobiohydrolase (CBH) I were purified. The isoforms had different isoelectric points (pI) but their catalytic and adsorption properties were similar. A remarkable feature of CBH I and endoglucanase (EG) II was that their electrophoretically pure preparations gave double peaks during ion-exchange chromatography in certain pH intervals where the two peaks (probably representing two conformations) were transformed into each other by changing pH. This behaviour of cellulases has never been reported before and further explains the difficulties in cellulase purification.
|
|
| 37. |
- Miliotis, Tasso, et al.
(författare)
-
Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- 2000
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 886:1-2, s. 99-110
-
Tidskriftsartikel (refereegranskat)abstract
- An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol. Copyright (C) 2000 Elsevier Science B.V.
|
|
| 38. |
- Mu, HL, et al.
(författare)
-
Response factors of organochlorine compounds in the electrolytic conductivity detector
- 1999
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 849:1, s. 285-292
-
Tidskriftsartikel (refereegranskat)abstract
- In our previous studies we have used electrolytic conductivity detection (ELCD) in the selective analysis of chlorinated fatty acids in marine samples. In order to determine the chlorinated fatty acids quantitatively, we studied the ELCD response factors (RFs) of chlorinated fatty acids and compared them with those of other chlorinated compounds. We also studied the effect of reactor temperature and total gas flow-rate on the RFs. The ELCD RFs of different organochlorine compounds varied significantly at a reactor temperature of 600 degrees C. The variation was reduced at reactor temperatures higher than 850 degrees C. At low reactor temperatures, the RFs of methyl esters of chlorinated fatty acid were much higher than those of the other compounds. Although the gas flow in the reactor was laminar, diffusion was still rapid enough not to cause the varied RFs. Nitrogen-containing chlorinated compounds had lower RFs than compounds without nitrogen, owing to a neutralization of hydrogen chloride by ammonia. (C) 1999 Elsevier Science B.V. All rights reserved.
|
|
| 39. |
- Nilsson, Anna, et al.
(författare)
-
Cutinase-peptide fusions in thermoseparating aqueous two-phase systems - Prediction of partitioning and enhanced tag efficiency by detergent addition
- 2002
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 946:1-2, s. 141-155
-
Tidskriftsartikel (refereegranskat)abstract
- It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide, Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K=1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data.
|
|
| 40. |
- Nilsson, Ulf J.
(författare)
-
Solid-phase extraction for combinatorial libraries
- 2000
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 885:1-2, s. 305-319
-
Tidskriftsartikel (refereegranskat)abstract
- Solid-phase extraction (SPE) has during the last three years emerged as a convenient method for the purification of compound libraries prepared by solution synthesis. The widespread use of SPE in combinatorial chemistry can be explained by straightforward SPE method development facilitated by the availability of numerous commercial SPE resins. High-speed automated SPE is readily accomplished by taking advantage of commercial laboratory robot systems. The present review summarizes and discusses advancements made in the use of different SPE resins and molecule tagging techniques for optimization of ion-exchange, reversed-phase, normal-phase and fluorous-phase SPE in combinatorial chemistry.
|
|
