Biomedical subjects

Adam Eckhardt

Publications and source records attributed to Adam Eckhardt.

12 recordsLinked to original sources

PubMed2006

Capillary electromigration methods for the study of collagen.

This review paper gives an overview of capillary electromigration methods used in the analysis of collagen. Analyses of the parent chains as well as of the bromcyane and collagenase fragments of collagens are presented. Methods include capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic chromatography as well as combinations of HPLC and capillary electrophoresis, and capillary electrophoresis with mass spectrometry.

Chromatography, High Pressure Liquid↗

PubMed2006

Posttranslational modifications of collagen studied by off-line coupling of HPLC and CE.

A typical example of non-enzymatic change of collagen is glycation (the Maillard reaction, formation of advanced glycation end products) resulting from the reaction of sugars with the epsilon-amino group of lysine. Posttranslational non-enzymatic modifications of collagen by sugars were studied. Collagenous tissues were incubated as a test protein separately with both glucose and ribose. The collagen mixture was digested by bacterial collagenase and separated by reversed-phase HPLC (in a Jupiter Proteo 90 A column). The eluate from this HPLC separation was collected as seven fractions and consecutively analysed by CE in a bare fused silica capillary (57/50 cm x 75 mm id) using 100 mM sodium 1-heptanesulfonate in 100 mM phosphate buffer, pH 2.5 (NaH2PO4 adjusted to pH by phosphoric acid). The chromatographic and electromigration behaviour of individual peptides varied considerably. This off-line HPLC-CE coupling made it possible to discover minor changes in the structure of collagen caused by posttranslational modifications. A new HPLC-CE technique for peptide analysis was developed, and applied to the identification of posttranslational modifications in slowly metabolised test proteins.

Animals↗

PubMed2004

Separation of low-molecular mass peptides by capillary electrophoresis with the use of alkylamines as dynamic coating agents at low pH.

Modification of the silanophilic activity of the inner surface of the capillary wall was studied in a capillary electrophoretic system using alkylamines containing background electrolytes at acid pH. The effect of the following amine additives was investigated: (1) alkyl-alpha,omega-diamines (1,2-diaminoethane, 1,4-diaminobutane, 1,7-diaminoheptane, spermine), (2) polymeric amines (polyethylenimine, polybrene), (3) cationic amine surfactants (cetrimide, hexamethonium bromide). A seven membered test mixture of peptides (Gly-Pro-Ala, Pro-hPro, Gly-Pro-Arg, Gly-Pro-Gln, Lys-Pro-Gly, Asn-Pro-Gly, His-Pro-Gly) possessing one or more amino groups was used for selectivity evaluation. Under optimised concentration of the amine modifiers the selectivity was always improved (except for polybrene), particularly with the fast moving analytes. The best results were obtained with 1,2-diaminoethane and 1,7-diaminoheptane. On the other hand with slowly moving peaks the best separations were obtained with 1,7-diaminoheptane, hexadecyltrimethylammonium bromide and hexamethonium bromide, i.e. with modifiers possessing large aliphatic domains which are likely to be hydrophobically bonded with the separated solutes. The selectivity improvement with fast moving members of the test mixture can be ascribed to the decrease of the electroosmotic flow, while the improved separation with slowly moving peaks appears to reflect the altered interaction with the hydrophobized capillary wall. As expected the endoosmotic flow was in all cases decreased. The practical applicability of using amine based dynamic modifiers of the capillary wall was demonstrated on a natural peptide mixture (bacterial collagenase hydrolysate of collagen types I and III).

Electrophoresis, Capillary↗

PubMed2004

Peptide mapping by capillary electrophoresis with Pluronic F127.

Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide-capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide)(x)(polypropylene oxide)(y)(polyethylene oxide)(z) when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.

Buffers↗

PubMed2004

Plastic substrates based separation channels in electromigration techniques.

Three types of plastic materials (polyester, polyurethane and polymethylmethacrylate) were tested as materials for manufacturing separation columns (polyester and polyurethane capillaries were used) or separation channels (polymethylmethacrylate) in the chip format. A set of 11 fluorescein isothiocyanate amino acid derivatives was used as the test mixture. Using alpha-cyclodextrin additive to the background electrolyte in the case of the chip separation was also tested. The main problem with all plastic separation media was the selectivity of the separation. The best results, practically identical with bare fused silica capillary, were obtained with the polymethylmethacrylate chip, provided that alpha-cyclodextrin in a concentration 40 mmol/l was added to the background electrolyte. An important observation was that in SDS containing background electrolyte all the plastic materials used exhibited a distinct electroosmotic flow, which was ascribe to the sorption of the negatively charged constituents of the background electrolyte to the capillary wall. Regarding the order in which the individual components of the test mixture were brought to the detector only a single change was observed. Histidine migrated in the polystyrene and polymethylmethacrylate separation channels more slowly than in the bare silica or polyurethane based capillaries.

Electrophoresis, Capillary↗

PubMed2004

Chromatographic determination of herbicide residues in various matrices.

The newest results in the use of various extraction techniques and chromatographic methods such as gas-liquid and high-performance liquid chromatography used for the assessment of herbicide residues in various matrices have been compiled and critically evaluated. Practical employments in water and soil research, environmental protection, clinical and food chemistry are presented.

Air Pollutants↗

PubMed2003

Comparison of the electrophoretic separation of proteins in capillaries with different inner diameter.

Two fused-silica capillaries of considerably different inner diameter (75 and 10 microm) were used for the separation of a set of five standard proteins. The separations were run in acid pH (50 mM phosphate buffer, pH 2.5). Generally better separations (with minor tailing only) were obtained using a standard capillary [27 cm (20 cm effective length)x 75 microm I.D.] in comparison with a narrow bore capillary [27 cm (20 cm effective length)x 10 microm I.D.]. The conditions of the electrophoretic separation were the same for both types of capillaries (25 degrees C; 10 kV; positive polarity at the inlet). The sequence of the proteins was cytochrome c, albumin, transferrin followed by a partly resolved peak of catalase and chymotrypsinogen A. In the narrow bore capillary severe tailing was observed--tailing factor ranged from 2.11 to 5.54 or 1.67 to 2.53 depending on the concentration of the analytes injected (2 or 0.2 mg/ml of each test compound injected). The relative [delta(deltaG(0))] values of the interaction with the capillary wall in the small bore capillary (with cytochrome c taken as initial standard) ranged from -0.74 to -1.04 kJ/mol. The problem of assaying the speed of the endoosmotic flow (EOF) in both capillary types was thoroughly investigated using thiourea and dithiothreitol as EOF markers. It was revealed that if thiourea is used as the EOF marker, the obtained value was dependent on the concentration of the marker injected. Optimum conditions for the EOF determination in acid buffer were specified. The higher speed of the EOF in the narrow bore capillary (10 microm) as compared to the 75 microm I.D. capillary is discussed.

Electrophoresis, Capillary↗

PubMed2003

Comparison of standard capillary and chip separations of sodium dodecylsulfate-protein complexes.

Conditions for converting a set of five standard proteins to electrochemically active sodium dodecylsulfate (SDS) complexes were worked out with the aim of using such complexes for conductivity detection with a a chip electrophoresis system. The results obtained were compared with standard capillary electrophoresis (37 cm (effective length 30 cm) x 75 microm I.D. capillary, 10 kV, negative polarity at the inlet). The chip separations were run at 500 V per chip (100 V/cm) as compared to the standard capillary arrangement, which was run at 266.6 V/cm. For the capillary set-up the protein complexes were prepared in aqueous solution (Milli-Q water) made 10 mM with respect to SDS. If the SDS concentration was increased to 50 mM, the separation in the capillary was incomplete. On the other hand with the chip system both approaches yielded acceptable results. The chip separations were slightly (but not distinctly) shorter and offered better separations than the standard set-up. The concentration of the surfactant used for the preparation the complexes results in alternations of the elution sequence, which is preserved if the chip separation is used instead of the capillary set-up. Apparently the full capacity of protein-SDS binding is not exploited for the preparation of the adducts.

Electrophoresis, Capillary↗

PubMed2003

Binding of environmental pollutants to the corn protein zein studied by high-performance liquid chromatography.

The interaction of 16 ring-substituted phenols and anilines with the corn protein zein was studied by reversed-phase high-performance liquid chromatography by preparing silica- and alumina-based stationary phases coated with various concentrations of zein. The relationship between the strength of interaction and the physicochemical parameters of solutes was elucidated by principal component analysis followed by the nonlinear mapping technique. The binding of each phenol and aniline derivative to zein has been demonstrated. It was established that the electrostatical parameters of solutes exert the highest influence on the interaction and the involvement of hydrophobic binding forces is of secondary importance. The binding characteristics of phenol and aniline derivatives were different.

Chromatography, High Pressure Liquid↗

PubMed2003

Insoluble eggshell matrix proteins--their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatography.

Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2-4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer.

Animals↗

PubMed2002

The effect of sodium dodecyl sulfate and Pluronic F127 on the electrophoretic separation of protein and polypeptide test mixtures at acid pH.

Using a test mixture consisting of standard proteins (cytochrome c, chymotrypsinogen A, hen egg albumin, bovine serum albumin, aldolase, catalase and ferritin) and synthetic polypeptides (polylysine, polyaspartic, polyglutamic acid and polyproline) it was revealed that using sodium dodecyl sulfate (SDS) as background electrolyte modifier at acid pH (2.5) allows selective separation of highly positively charged polypeptides (polylysine) provided that their relative molecular mass is sufficiently low (3300 Da). The altered elution sequence of standard proteins as compared to a separation done without SDS may help their identification. Addition of Pluronic F127 offers clear-cut separations of standard proteins up to a relative molecular mass of 5 x 10(4) Da and allows to reveal protein/polypeptide microheterogeneity where applicable. None of the systems tested is suitable for the separation of acidic polypeptides and polyproline.

Buffers↗

PubMedDate not supplied

Matrices for capillary gel electrophoresis--a brief overview of uncommon gels.

This article gives an overview of uncommon replaceable matrices (gels) for capillary gel electrophoresis. This electrophoretic technique is useful mainly for the separation and analysis of biopolymers-nucleic acids and their fragments, and proteins/peptides. Commonly used gels are not reviewed. Those mentioned and discussed here are gels containing saccharides, newly developed acrylamide-based gels and thermoadjustable viscosity polymers, namely triblock copolymers and grafted polyacrylamide.

Acrylamides↗