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B L Karger

Publications and source records attributed to B L Karger.

At least 91 records · Page 5Linked to original sources
PubMed1989

Role of association on protein adsorption isotherms. Beta-lactoglobulin A adsorbed on a weakly hydrophobic surface.

This paper explores the role of association on the adsorption isotherms of beta-lactoglobulin A on a weakly hydrophobic stationary phase at 4 degrees C and mobile phases of 0.85 M and 1 M ammonium sulfate, pH 4.5. The isotherms, obtained by frontal analysis, show an S-shape and the corresponding Scatchard plots indicate positive cooperativity. The slopes and intercepts of the Scatchard plots at low solute concentration are analyzed in terms of two species--a promoter and a higher order stronger adsorbing species. An explicit equation of the isotherm is developed based on this model, and this expression is shown to reproduce the isotherm shape using the appropriate derived parameters. It is further shown from this equation that a Langmuir-shaped adsorption isotherm can be obtained if the higher order associate or aggregate binds weaker to the support than the promoter. These results indicate that protein-protein interactions and the formation of associates can play a significant role on the shape of the isotherm and ultimately on the behavior of the species in preparative scale chromatography.

Adsorption↗
PubMed1989

High-performance capillary electrophoresis in the biological sciences.

High-performance capillary electrophoresis (HPCE), the instrumental approach to electrophoresis, is a method undergoing rapid development at the present time. There is a high expectation that HPCE will become a widely applicable tool within the biochemical community. This review presents principles and instrumentation followed by typical applications of HPCE in the biological area. In the instrumentation area, particular attention is paid to current and future detectors, including laser-induced fluorescence and HPCE-mass spectrometry. In the applications section, both peptide or protein and oligonucleotide high-resolution separations are described for open-tube and polyacrylamide gel capillary columns. The use of isoelectric focusing and electrokinetic chromatography to manipulate separation is also presented. Future directions of methodology are suggested, and it is predicted that in the next few years HPCE will become a complementary tool to liquid chromatography and slab gel electrophoresis.

Biochemical Phenomena↗
PubMed1989

High-performance capillary electrophoresis.

High performance capillary electrophoresis is expected to be the fastest-growing analytical technique since HPLC. The method has already shown its utility in a variety of applications.

Electrophoresis↗
PubMed1989

Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.

Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.

Chromatography, High Pressure Liquid↗
PubMed1988

Rapid separation of DNA restriction fragments using capillary electrophoresis.

Open-tube capillary electrophoresis has been applied to the separation of restriction fragments of DNA with a Tris-borate buffer containing 7 M urea and 0.1% sodium dodecyl sulfate. The importance of sample pretreatment and of the injection of heated samples has been demonstrated. In one separation, a DNA restriction fragment mixture from 72 to 23,130 base pairs (DRIgestTM III) (molecular weight range from 4.6.10(4) to 1.5.10(7] has been electrophoresed in 10 min on a column of 15 cm effective length. Over 600,000 plates have been obtained for individual peaks. Several of the peaks have been identified, by spiking slab gel electrophoretically purified components. Other examples of restriction fragment separations are illustrated in this paper. The results of this study when further validated with full characterization of individual species, open up the possibility of rapid restriction enzyme mapping.

DNA Restriction Enzymes↗
PubMed1988

Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis.

Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)40-60] were baseline resolved and analyzed in less than 8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in less than 20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation.

Electrophoresis↗
PubMed1987

High-performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins.

High-performance capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been developed for the separation and molecular weight (MW) determination of peptides and proteins. In this work, acrylamide was polymerized in fused-silica capillaries of 75 microns I.D. and 10 or 20 cm length. On-line UV detection was employed by placing the capillary in the optical path of the detector. Rapid separations of myoglobin fragments and of a mixture of standard proteins were demonstrated with efficiencies of the order of 40,000 plates for a column, 20 cm in length. Linear plots of log MW vs. mobility for the SDS protein complexes were observed. Variation in the percentage monomer composition (% T) of the polyacrylamide yielded the expected common intercept of log mobility vs. % T, confirming the size separation mechanism in the gel capillaries. The separation of the A and B chains of insulin with a 10-cm column in less than 10 min was also demonstrated.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Protein conformational effects in hydrophobic interaction chromatography. Retention characterization and the role of mobile phase additives and stationary phase hydrophobicity.

We have studied the conformational behavior of alpha-lactalbumin (alpha-LACT) in hydrophobic interaction chromatography (HIC). Retention characterization in terms of Z (slope of plot of ln k' vs. ln phi B, where k' is the capacity factor and phi B is the volume fraction of mobile phase B) has been explored, and the relationship of Z to other slopes, such as S (slope of the plot of ln k' vs. phi B) has been derived. The reasons for the sensitivity of Z to conformational change are discussed. The enhanced broadening of alpha-LACT in a temperature transition region of conformational change has been studied by spectral analysis using on-line photodiode array detection. The influence of Ca2+ and Mg2+ addition to the mobile phase is further explored. Since alpha-LACT is a calcium binding protein, addition of this metal leads to stabilization, i.e. higher column temperatures are required for conformational change. On the other hand, addition of Mg2+ appears to destabilize the protein. We have explored the use of a more hydrophobic support, C2-(ethyl) ether phase, for the elution of alpha-LACT. In this case, two widely separated peaks are observed. By spectral analysis the first peak is shown to be native and the later eluted, broad second peak to be an unfolded mixture of species. As previously observed in reversed-phase liquid chromatography, the second peak grows at the expense of the first, as the column temperature is raised. The second peak also grows as the contact time of the protein with the surface increases. This behavior can be ascribed to the conformational change of alpha-LACT in the column, the late eluted species under the second peak binding significantly more strongly to the phase than the native peak. Reinjection of the late eluted fraction reveals that reformation of the native species takes place in solution within 30 min. As before, addition of Ca2+ reduces the extent of unfolding under any specific condition. These results add further to our understanding and ability to control conformational changes in high-performance liquid chromatography.

Animals↗
PubMed1986

High-performance immobilized-metal affinity chromatography of proteins on iminodiacetic acid silica-based bonded phases.

High-performance separations of proteins, based on immobilized-metal affinity chromatography (HPIMAC), are described. The stationary phase consisted of iminodiacetic acid (IDA) chelate groups, bonded to small particle, wide pore silica gel by means of a polyether hydrophilic leash. After loading the column with metal, retention of proteins was achieved by protein-metal complexation at high concentrations of sodium sulfate. Elution was accomplished by addition of competitive complexing ligands, such as ammonia at constant pH, and/or by a decreasing pH gradient of a specially designed buffer system to maintain buffer capacity constant throughout the gradient. Selective separations, based on differences in the number of histidine residues present on the surface of the proteins, are described. The application of HPIMAC in the separation and purification of structurally similar proteins is presented. The potential application of IDA columns in three chromatographic modes (HPIMA, hydrophobic interaction, and cation exchange) is also described.

Ammonia↗
PubMed1986

Thermal behavior of proteins in high-performance hydrophobic-interaction chromatography. On-line spectroscopic and chromatographic characterization.

The thermal behavior of a series of standard proteins in hydrophobic interaction chromatography on a previously developed weakly hydrophobic ether-bonded phase column has been studied. Depending on the temperature and protein, conformational changes can occur in the chromatographic system. Methods for recognizing these conformational effects are presented, including retention and peak width variations with temperature, and Z values (the slope of the plot of log k' vs. log %B solvent. The Z value is shown to be a general index characterizing protein retention as a function of salt concentration. In addition, on-line UV spectroscopic analysis, (absorbance ratios and second derivative spectroscopy) with a photodiode array detector, is shown to corroborate chromatographic trends. Lysozyme maintains a stable conformation over the temperature range 10-40 degrees C, whereas beta-lactoglobulin A has a conformational transition between 25 degrees C and 40 degrees C. Calcium-depleted alpha-lactalbumin, a rather labile species, maintains a stable conformation up to ca. 20 degrees C, and then undergoes structural changes. Finally, cytochrome c appears to be relatively stable up to ca. 65 degrees C. Since conformational changes for this protein occur at ca. 35 degrees C on more hydrophobic phases, the extent of hydrophobicity of the stationary phase is important for maintenance of the native state. Based on this work, hydrophobic-interaction chromatography at sub-ambient temperatures appears promising.

Chromatography↗
PubMed1986

Conformational effects in the high-performance liquid chromatography of proteins. Further studies of the reversed-phase chromatographic behavior of ribonuclease A.

This paper is a continuation of an examination of the conformational effects of ribonuclease A (RNase A) in reversed-phase liquid chromatography. RNase A is a particularly good example of reversible conformational refolding during chromatographic elution. Absorbance ratio measurements with a photodiode-array detector are consistent with a two-state model in which an early eluted broad band is associated with the folded or native state and a late eluted sharp band is associated with an unfolded state. By varying the mobile phase flow-rate while maintaining the mean capacity factor (k') of the protein in gradient elution constant, the apparent rate constant of refolding in the mobile phase was measured and found to be comparable with that reported in the literature. It has been further shown that, as the gradient steepness parameter b or mean k' value is altered, the apparent rate constant also changes, as a result of variation in mobile phase composition. The apparent rate constant as a function of temperature was also found to reach a maximum at 20 degrees C. Based on the results of this model system, it is possible to predict optimum conditions for elution of species with sharp elution peaks when reversible refolding takes place in the column.

Chemical Phenomena↗
PubMed1985

High-performance hydrophobic interaction chromatography: purification of rat liver carbamoylphosphate synthetase I and ornithine transcarbamoylase.

The applicability of high-performance hydrophobic interaction chromatography using newly developed silica-based ether-bonded phases is demonstrated in the purification of the rat liver enzymes carbamoylphosphate synthetase I and ornithine transcarbamoylase from crude mitochondrial extracts. As a result of the mild adsorption/elution conditions in this high-performance chromatographic mode, the enzymes are recovered in 20 min with 3- to 15-fold increases in specific activity. Since the enzymes are labile and may aggregate in solution, in one case up to Mr 330,000, this rapid purification demonstrates the potential of hydrophobic interaction chromatography in complex biological systems.

Animals↗
PubMed1985

High-performance hydrophobic-interaction chromatography on ether-bonded phases. Chromatographic characteristics and gradient optimization.

This paper is a continuation of the evaluation of hydrophilic ether columns for the separation of proteins by hydrophobic-interaction chromatography. In this approach, linear salt gradients of decreasing concentration of ammonium sulfate yield sharp chromatographic peaks with high mass recovery and maintenance of biological activity. Mild adsorption conditions are indicated by the minimal changes in chromatographic peak area of native proteins as a function of contact time by the biopolymer with the stationary phase. Further evidence of minimal kinetic processes under the chromatographic conditions are seen in the constancy of isocratic retention with mobile phase flow-rate and sample load up to 2 mg. Based on the well-behaved chromatographic characteristics, we have explored gradient optimization in terms of the Snyder model for gradient elution. It is shown that changes in retention, peak capacity and peak height follow predicted gradient time dependencies. Moreover, the influence of particle diameter and column length are found to be in agreement with expected behavior, based on the model. As a consequence of the agreement, prediction of conditions for optimum separation for a particular problem are possible. Other studies examine the influence of specific ion effects, e.g., Mg2+ binding to protein, which can override retention based on predicted surface tension behavior. Some characteristics of the ether column for hydrophobic-interaction chromatography are shown, e.g., column stability at pH 8 and sample capacity.

Chemical Phenomena↗
PubMed1985

Conformational effects in the reversed-phase liquid chromatography of ribonuclease A.

This paper examines the reversed-phase liquid chromatographic behavior of ribonuclease A (RNase) using an n-butyl chemically bonded phase and a gradient of 10 mM H3PO4 and l-propanol. At a column temperature of 25 degrees C, a broad band followed by an overlapped late-eluting sharp peak is observed. As the temperature is raised, the sharp peak grows at the expense of the broad band until at 37 degrees C, only a single narrow-eluting band is found. Using an absorbance ratio of A288/A254, it is demonstrated that the broad band represents a folded or native state of RNase and the late-eluting band a denatured state. Based on postcolumn absorbance ratio changes in the denatured state as a function of time and the known behavior of the protein, reversible refolding or renaturation is proposed to take place in solution. RNase is denatured upon adsorbing to the bonded phase, and upon migration down the column, reversible refolding takes place in the mobile phase. The relaxation time for native state formation is assumed to be comparable to the time spent by RNase in the mobile phase. As temperature is raised, both the native and denatured states exist at equilibrium in solution, thus slowing the refolding process, until at 37 degrees C only the denatured peak appears. Changes in peak shape with flow rate provide further evidence for this model. The use of HCl or H2SO4 instead of H3PO4 yields similar results except that the temperature at which only the denatured peak is observed follows the order of salt stabilization of the native state.

1-Propanol↗
PubMed1984

Kinetics of unfolding of proteins on hydrophobic surfaces in reversed-phase liquid chromatography.

As a continuation of previous studies, we present in this paper measurements on the kinetics of denaturation of papain, soybean trypsin inhibitor and lysozyme on n-butyl-bonded silica gel surfaces used in reversed-phase liquid chromatography (RPLC). In all cases, native and denatured peaks widely separated from one another are observed. The rate constants for denaturation or unfolding are determined by the measurement of the peak area of the native protein as a function of the incubation time that the species spends on the bonded-phase surface. The results reveal that a slow denaturation step occurs with a half-life of ca. 15 min. In addition, studies of denaturation as a function of the amount of 1-propanol in the initial mobile phase suggest an additional unfolding step when the protein comes in contact with the bonded-phase surface. The extent of this latter step decreases as the concentration of 1-propanol increases, further suggesting that 1-propanol sorption on the bonded stationary phase may play a role in this behavior. Other studies are conducted with alpha-chymotrypsinogen, in which injection is made after the start of the gradient. The extent of denaturation is observed to be a function of the organic modifier employed. The results of this paper provide insight into the denaturation process in RPLC and suggest approaches to minimize this behavior.

Chemical Phenomena↗
PubMed1984

Wide-pore silica-based ether-bonded phases for separation of proteins by high-performance hydrophobic-interaction and size-exclusion chromatography.

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure identical to Si-(CH2)3-O-(CH2-CH2-O)n-R, where n = 1, 2, 3 and R = methyl, ethyl or n-butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g., from 3 M ammonium sulfate (pH 6.0, 25 degrees C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g., 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[eta]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.

Chromatography, Gel↗
PubMed1984

Sequence analysis of derivatized peptides by high-performance liquid chromatography-mass spectrometry.

N-Acetyl-N,O,S-permethylated derivatives of oligopeptides were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using a moving belt interface. A heated-gas nebulizer was employed for sample deposition, thus permitting the effective use of a water-methanol gradient covering the range from 5% to 95% water at mobile phase flow-rates of 0.5 ml/min. We demonstrate in this paper that it is possible to sequence the octapeptide derived from the C-chain of glucagon by HPLC-MS analysis of a permethylated enzymatic hydrolysate of this peptide using overlap information from the mass spectral patterns. Moreover, it is shown that peptides not readily amenable to analysis by gas chromatography-MS can be analyzed using this approach. Preliminary results suggest that N-acetyl-methyl ester derivatives of oligopeptides may in specific cases also be a useful alternative for HPLC-MS analysis of complex oligopeptide mixtures.

Amino Acid Sequence↗