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Biomedical subjects

B L Karger

Publications and source records attributed to B L Karger.

At least 73 records · Page 4Linked to original sources
PubMed1994

Incorporation of hydrophobic selectivity in capillary electrophoresis: analysis of recombinant insulin-like growth factor I variants.

A highly selective electrophoretic system employing differential hydrophobic interaction was evaluated for the quantitative determination of recombinant insulin-like growth factor I (IGF-I) variants. The system consisted of mixed aqueous-organic buffers containing suitable amounts of a zwitterionic detergent. In addition, a neutral hydrophilic coating was attached to the wall of the capillary to minimize analyte adsorption and electroosmotic flow. The zwitterionic detergent acted as a hydrophobic selector, allowing independent optimization of the electrophoretic and hydrophobic selectivities in the separation system. The extent of hydrophobic interaction was conveniently adjusted by varying the type and amount of organic modifier. Complete resolution of a mixture of IGF-I variants with closely related mass-to-charge ratios was achieved. Quantitative analysis of IGF-I process samples agreed well with HPLC results. Finally, the approach was found to be compatible with on-line capillary electrophoresis-mass spectrometry.

Amino Acid Sequence↗
PubMed1994

Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis.

Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.

Base Sequence↗
PubMed1994

Affinity probe capillary electrophoresis: analysis of recombinant human growth hormone with a fluorescent labeled antibody fragment.

A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' fragment of a mouse monoclonal antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled antibody fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence. In these preliminary results, methionyl recombinant human growth hormone (met-rhGH) could be successfully determined down to detection levels of approximately 5 x 10(-12) M. Mono- and dideamidated variants of met-rhGH were detected simultaneously with the nondeamidated form of the antigen as separate peaks.

Electrophoresis↗
PubMed1993

DNA sequencing by capillary electrophoresis: use of a two-laser-two-window intensified diode array detection system.

This paper presents the principles of an instrument designed for DNA sequencing using the standard four-dye-labeled primer approach. The method is based on capillary electrophoresis with laser-induced fluorescence and an intensified diode array detector. An important goal of the instrument design has been a detection system that possesses high sensitivity and high spectral resolution. Based on an analysis of the spectral characteristics of the four standard dye-labeled primers, FAM, JOE, ROX, and TAMRA, the strategy has been to use a two-laser-two-window approach, in which a 488-nm argon ion laser illuminates one window, followed by a 543-nm helium-neon laser illuminating the second window. The two-window approach has no moving parts and permits continuous illumination. Spectral resolution is provided by a grating spectrograph and a cooled intensified diode array. The estimated limit of detection for the standard four dye-labeled primers was found to be in the sample concentration range of 1 x 10(-12) M. To achieve these low levels, complete free-radical polymerization of polyacrylamide has been found to be necessary in order to reduce background noise. In addition, reduction in background noise was accomplished by continual purging of the anodic reservoir in order to prevent electrolysis products from entering the capillary. Separation of DNA sequencing reaction products is demonstrated on a 9% T linear polyacrylamide column.

Base Sequence↗
PubMed1993

Separation of DNA fragments by capillary electrophoresis using replaceable linear polyacrylamide matrices.

The use of low percent (1.5-6% T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23,000 base pairs were readily achieved. Typically, 4 x 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6% T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electro-osmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.

Bacteriophage phi X 174↗
PubMed1993

Characterization and performance of a neutral hydrophilic coating for the capillary electrophoretic separation of biopolymers.

Polyvinylmethylsiloxanediol (50% vinyl) was synthesized and combined with a cross-linker for static coating onto fused-silica columns. After cross-linking and binding to the surface, linear polyacrylamide was grafted to the double bonds of the siloxanediol; subsequently, this linear polymer matrix was cross-linked with formaldehyde. The grafted neutral polymeric layer provided suppression of electroosmotic flow and minimized adsorption. This combination yielded successful open tube and polymer network separations of proteins, peptides and DNA molecules. Very high efficiencies (ca. 1 x 10(6) plates/m) were achieved for open tube protein separations, and hundreds of consecutive runs were performed with minimal change in migration times.

Biopolymers↗
PubMed1993

DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection.

Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32 degrees C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.

Acrylic Resins↗
PubMed1992

High-performance capillary electrophoresis of SDS-protein complexes using UV-transparent polymer networks.

This paper demonstrates the use of UV-transparent replaceable polymer networks for the separation of SDS-protein complexes on the basis of molecular weight. First, the use of linear (i.e. non-cross-linked) polyacrylamide is shown to provide molecular separation of SDS-protein complexes. A study reveals such columns to yield significantly greater lifetime than cross-linked gels because of the flexibility of the noncovalently attached polymer chains. However, column lifetime was still found to be limited (approximately 20-40 injections), and detection at 214 nm was problematical because of the absorbance of polyacrylamide. UV-transparent polymer networks of dextran and PEG were substituted for polyacrylamide with successful molecular weight sieving of SDS-protein complexes at 214 nm. Due to their low to moderate viscosities, these networks could be routinely replaced leading to the possibility of hundreds of injections with a single column. Migration time reproducibilities of 0.5% RSD or less were found with replacement of the network. Using dextran, calibration plots of peak area vs concentration of standard protein were linear over the range of 0.5 microgram/mL up to at least 0.25 mg/mL. Furthermore, plasma samples could be directly utilized because of the strong solvating power of SDS. Rapid separation of protein mixtures are demonstrated with these UV-transparent polymer networks.

Dextrans↗
PubMed1992

Conformational effects in the reversed-phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-methionyl recombinant human growth hormone (Met-hGH).

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.

Chromatography, Liquid↗
PubMed1992

Capillary electrophoresis.

The past year has seen major advances in capillary electrophoresis in terms of broadening applicability. A variety of successful approaches to peptide/protein and DNA separation and analysis are now available, and techniques for saccharide analysis are developing rapidly. Capillary electrophoresis--mass spectrometry continues to demonstrate its potential as a tool for high-resolution structure analysis.

DNA↗
PubMed1991

Adsorption-desorption isotherm hysteresis of beta-lactoglobulin A with a weakly hydrophobic surface.

Adsorption-desorption isotherms of bovine beta-lactoglobulin A (beta-lact A) on a weakly hydrophobic stationary phase (C1-ether) were measured by frontal analysis. The adsorption isotherms obtained at different pH were found to be dramatically different in shape, column capacity and desorption reversibility. At pH 4.5, an S-shaped adsorption isotherm was observed whereas at pH 6.0 a Langmuir isotherm was found. In addition, the desorption isotherm at pH 6.0 was found to overlap with the adsorption isotherm, and the adsorption-desorption process of beta-lact A under this condition could be characterized by a fully reversible Langmuir model. The desorption isotherm at pH 4.5, however, did not retrace the adsorption isotherm, resulting in hysteresis loops. A higher aggregate (tetramer) of beta-lact A is shown to be in an equilibrium with the beta-lact A protomer (dimer) at pH 4.5 whereas the dimer alone is predominant at pH 6.0. It is further shown that changes in the absorption coefficient between the adsorption and the desorption cycles for the tetramer at pH 4.5 can account for the hysteresis. The results demonstrate that pH can be a sensitive parameter in protein adsorption isotherm behavior and ultimately the behavior of species in preparative-scale chromatography.

Adsorption↗
PubMed1991

Influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis.

The influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis (HPCE) is presented. The major effect of temperature is to shorten the analysis time by decreasing the viscosity, but specific temperature effects on the protein migration behavior were also observed. Myoglobin, under high field (350 V/cm), was essentially temperature stable from 20 to 45 degrees C, but at constant current, a second form of myoglobin could be detected at both 214 and 410 nm. The initial form appeared to correspond to the Fe3+ and the second to the Fe2+ oxidation state of the heme iron. The rate of conversion from Fe3+ to the reduced Fe2+ in myoglobin, under given electrophoretic conditions, followed first-order kinetics with a rate constant at 30 degrees C of 304 s-1. A second protein, alpha-lactalbumin type III, demonstrated a conformational transition that resulted in asymmetric peaks and sigmodial mobility plots versus temperature in the transition region.

Animals↗
PubMed1991

Effect of metal ions on the unfolding kinetics of alpha-lactalbumin on weakly hydrophobic surfaces.

The effect of metal ion [Ca2+ and Zn(NH3)4(2+)] on the unfolding kinetics of bovine-alpha-lactalbumin (alpha-LACT) on a weakly hydrophobic chromatographic surface (ethyl polyether phase bonded on porous silica, C2 ether) has been studied using surface intrinsic fluorescence spectroscopy and liquid chromatography. Chromatographic results on the C2 ether phase revealed two peaks for alpha-LACT, the first being the folded and the second an unfolded conformation, as determined by fluorescence spectroscopy. The retention time for the second peak was found to depend on the specific metal additive in the mobile phase. Fluorescence studies showed a slow change in emission maximum from ca. 330 nm to 350 nm and a 5-fold increase in emission intensity for the adsorbed protein in the unfolded state. By following the fluorescence emission intensity at a given wavelength during the unfolding process, biphasic kinetics were observed with the kinetic constants depending on the specific metal-ion additive. In addition, solution refolding rates of the desorbed, unfolded species were measured and found to be consistent with literature refolding rate constants.

Animals↗
PubMed1990

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields.

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.

Acrylic Resins↗
PubMed1990

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis.

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.

Base Sequence↗
PubMed1990

Intrinsic fluorescence studies of the kinetic mechanism of unfolding of alpha-lactalbumin on weakly hydrophobic chromatographic surfaces.

The kinetic mechanism of unfolding of calcium depleted bovine alpha-lactalbumin adsorbed on two weakly hydrophobic chromatographic surfaces, methyl- and ethyl-polyether phases bonded to porous silica, with a solution phase of 3 M ammonium sulfate at pH 6.3, has been determined using intrinsic fluorescence and liquid chromatography (LC). The adsorbent has been packed into quartz flow cells which are used for both fluorescence measurements and as a microcolumn for LC. The LC measurements revealed two peaks for alpha-lactalbumin on both phases, the first being folded and the second unfolded. The rate of unfolding was measured to be 1.75.10(-3) min-1 on the Cl-ether and 7.42.10(-3) min-1 on the C2-ether phase. Fluorescence studies revealed a slow change in emission maximum from ca. 330 nm to 350 nm and a 4-fold increase in intensity for the protein adsorbed on the two supports. Variation of fluorescence intensity at a given wavelength revealed biphasic kinetics in which the rate law on the surface was deduced as F in equilibrium X----U, where F is the folded form, U an unfolded form and X an intermediate. The normalized emission spectra of the three species were calculated and it was found that there was approximately a 20-nm-red shift in the position of the maximum from F to U. The emission maximum for X was close to U on both columns; however, the normalized intensity for X was between F and U. Activation enthalpies and entropies were determined from the temperature dependence of the microscopic rate constants. The formation of the intermediate on the C1-ether phase was entropy driven whereas on the C2-ether phase it was enthalpy driven. Finally, the solution refolding rates of U desorbed from the two supports were found to be identical. The differences observed in the surface kinetics of unfolding on the two supports are related to the hydrophobic differences of the adsorbents.

Adsorption↗
PubMed1990

Reversed-phase chromatographic behavior of proteins in different unfolded states.

A series of standard small globular proteins in different unfolded states was studied by gradient reversed-phase liquid chromatography. The retention parameters Z [slope of log capacity factor (k') vs. log molar concentration of organic modifier, 1-propanol, in the mobile phase] and log I (the value of log k' at 1 M 1-propanol) were derived from gradient retention data. Each protein in four different conformational states, i.e., folded, chromatographic surface-unfolded, urea-unfolded and disulfide-bridge reduced-unfolded, showed a variation of 10-fold in Z and up to 10(12)-fold in I values. For the different states of all the proteins studied, the order of Z and I values was as follows: folded much less than surface-unfolded less than urea-unfolded less than reduced-unfolded. The differences in the values of the coefficients suggest, in agreement with literature reports, that proteins with their disulfide bridges cleaved have the largest degree of unfolding. In addition, the Z and I values and solution refolding kinetics all suggest that chromatographic surface-unfolded proteins have a lower degree of unfolding than their urea-unfolded forms. It was also found that an additional chemical cross-link in lysozyme caused a significant decrease in the first-order rate constant of the surface-induced unfolding process.

Animals↗