Search

Dysregulated oncogenic serine/threonine kinases play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4) and its potential as a new target in MM for clinical applications. PAK4 promoted MM cell growth and survival via activation of MM survival signaling pathways, including the MEK-extracellular signal-regulated kinase pathway. Furthermore, treatment with orally bioavailable PAK4 allosteric modulator (KPT-9274) significantly impacted MM cell growth and survival in a large panel of MM cell lines and primary MM cells alone and in the presence of bone marrow microenvironment. Intriguingly, we have identified FGFR3 as a novel binding partner of PAK4 and observed significant activity of KPT-9274 against t(4;14)-positive MM cells. This set of data supports PAK4 as an oncogene in myeloma and provide the rationale for the clinical evaluation of PAK4 modulator in myeloma.

An exceptional case of suicide with two shots to the head inflicted by a captive-bolt gun is reported here. The pathophysiology observed, and related literature, indicate that the capacity of the victim to fire a second shot depended on the depth of skull penetration by the first shot and the extent of damage to nerve structures. In this case, an ante mortem multi-detector computed tomography was possible. From discussion with the radiologist and a 3D reconstruction, the two-shot suicide scenario could be confirmed., (Copyright © 2011 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3' UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.

This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.

Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was used to detect known point mutations using the method of single-nucleotide primer extension (SNuPE). Three different point mutations in human mitochondrial DNA associated with Leber's hereditary optic neuropathy (LHON) were detected by annealing a primer immediately 5' to the mutation on the template and extending the primer by one fluorescently labeled dideoxy terminator complementary to the mutation. By using two or more differently labeled terminators, both the mutant and wild type could be simultaneously detected. The advantages of using CE-LIF for detecting SNuPE reactions include speed and ease of analysis, absence of radioactivity, and potential for automation.

The potential of a new microanalytical method using magnetic beads (MBs) and commercial capillary electrophoresis (CE) instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens, substrates, etc., has been explored. A small quantity of magnetic beads containing immobilized biomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2-3 mm) of beads was held fixed by a magnet placed in the cartridge of the CE system, without the use of frits. The beads could be replaced after each run, eliminating the need to regenerate the solid support. Two protocols were used for analysis: sequential injection (SI) and SI followed by isotachophoretic (ITP) focusing. Alkaline phosphatase (AP) and HIV-protease were used to demonstrate the SI procedure for enzymatic and inhibition assays. The second protocol, SI/ITP, was employed to quantitate an antigen (mouse mAB) using antibodies (sheep IgG towards mouse AB) immobilized on the beads. The MB-CE method, requiring only femtomole (fmol) quantities of material, can potentially be employed in diagnostic and forensic assays, kinetic studies and searching for inhibitors, ligands, receptors, etc.

A procedure is presented for the separation of underivatized oligosaccharides by capillary electrophoresis (CE) with a phytic acid-borate buffer system. The presence of the phytic acid ion-pairing agent greatly increases resolution between oligosaccharides in the complex mixtures studied, which was demonstrated by the separation of oligosaccharides originating from various immunoglobulin G antibodies and CTLA4Ig, a biologic fusion protein. The conditions also resolve neutral oligosaccharides, usually a major CE limitation. High-performance anion-exchange chromatography with pulsed amperometric detection, a standard technique for oligosaccharide and sugar analysis, is used as a reference method to analyze some of the complex oligosaccharide mixtures.

The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.

This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.

A quantitative competitive polymerase chain reaction (PCR) assay for the detection and quantification of HIV-1 has been developed using capillary electrophoresis. Separation of the PCR products is carried out in coated capillaries filled with replaceable linear polyacrylamide, and detection is performed using laser-induced fluorescence. The quantitative capabilities of this assay are described. We show results from analysis of a noninfectious plasmid encoding the HIV genome and integrated proviral DNA. Potential applications of this assay are discussed.

Methods have been developed for the CE and HPLC analysis of CTLA4Ig, an immunoglobulin fusion protein. Two different LC approaches, size-exclusion (SEC) and "mixed-mode" ion-exchange (ABx), were developed along with a CE method that uses a micellar electrokinetic chromatographic buffer consisting of borate ions, sodium dodecyl sulfate and acetonitrile. These assays measure the presence of several CTLA4Ig-related species, and the observed changes resulting from multiple modes of degradation. In an attempt to identify possible degradation products, collections were taken from the ABx and SEC liquid chromatographic systems and further analyzed by matrix-assisted laser desorption time-of-flight (MALDI TOF) mass spectrometry. Multiple species were detected covering a wide molecular mass range. In addition, the CE method was used to study conformational kinetics between two forms of CTLA4Ig and to estimate the activation energy of the conformer-conformer transition. Pseudo-first-order reaction kinetics were demonstrated for CTLA4Ig samples stressed with papain, H2O2, and sodium dodecyl sulfate/heat.

In this study, the use of capillary isoelectric focusing (cIEF) as a micropreparative tool for protein analysis by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated. A newly designed, automated, collection interface equipped with a fiber-optic UV detector and a sheath flow connection was employed for collection of protein fractions. Multiple fractions were collected during a single cIEF run and further analyzed by MALDI-TOF-MS for mass assignment. The feasibility of the method was tested with a mixture of model proteins with different isoelectric points and molecular masses, and with variants of human hemoglobins differing in pI, but with negligible difference in M(r). Some practical considerations of the collection procedure and subsequent TOF analysis are presented.

Capillary electrophoresis incorporating hydrophobic selectivity is shown to be a powerful technique for separating closely related peptide species. In this work, hydrophobic interaction was induced through the addition of suitable amounts of a zwitterionic detergent (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and further modified with organic solvents. A neutral, hydrophilic-coated capillary was used to minimize electroosmotic flow. Two test solutes, Met15- and Leu15-gastrin, were employed to probe hydrophobic selectivity with various electrophoretic conditions. The nature and concentration of the detergent and the organic modifier were varied to adjust the selectivity. Operation near the critical micelle concentration of the zwitterionic detergent in the presence of acetonitrile or various alcohols produced the highest hydrophobic selectivity among the conditions studied. The zwitterionic detergent approach was also briefly compared to the use of non-ionic detergents for hydrophobic selectivity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

This paper explores the influence of mobile-phase and temperature effects on the gradient elution reversed phase chromatographic behavior of proteins. Using LiChrospher SI 500, bonded with n-butyl groups and a gradient in 1-propanol, 10 mM H3PO4, rapid separation and high mass recovery were obtained for a series of globular proteins. This protein separation and recovery are compared to those obtained when acetonitrile and acetonitrile plus 10 mM H3PO4 are used as eluting gradient solvents. In general, acetonitrile yielded lower recovery than 1-propanol, particularly for the more hydrophobic proteins, e.g., ovalbumin. For all three gradient solvents, little difference was observed in retention or recovery when the n-alkyl chain of the bonded phase varied. On the other hand, relative to the n-alkyl phases, a significantly lower retention of all proteins was found on more hydrophilic phases, e.g., cyano and nonendcapped n-butyl, when acetonitrile was the organic modifier, while in the case of 1-propanol, no retention difference was observed. Thus, column comparisons depend on the protein/mobile-phase combinations examined. The role of column temperature was also studied, and it was found that for certain proteins dramatic changes in peak shape occurred as a function of temperature. The influences of ionic strength and salt type were also studied. Protein mass recovery was shown to decrease with an increase in salt concentration; moreover, perchlorate was shown to have a larger effect in this regard than phosphate. In addition, salt concentration and type were found to influence peak shape greatly in certain cases. The results indicate the strong influences of mobile phase and temperature on chromatographic behavior, and some of the options available when this behavior is not satisfactory. Several protein separations are presented illustrating the power of the reversed phase approach.

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.

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.

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.

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.

High-performance ion-pair partition chromatography is shown to be a versatile, efficient method for separating sulfonamides. For a group of fourteen sulfa drugs varying widely in pKA and hydrophobicity, the effect of mobile phase composition, counterion composition, pH, and ionic strength on their ion-pair partition chromatographic separation using tetrabutylammonium as the counterion and n-butanol-n-heptane as the mobile phase is shown. Wide variation in k' and a is possible by changing these parameters. Silica columns coated with buffered aqueous solutions of tetrabutylammonium sulfate resulted in efficiencies of 4000-6000 theoretical plates per 25 cm. These columns are stable for long periods of time, and can be stripped and re-used in the adsorption mode with little or no loss in efficiency. Several chromatograms are presented in order to illustrate the performance of ion-pair partition chromatography.

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.

Whenever proteins are found in environments different from those provided by physiological conditions, structural alterations can occur which can dramatically affect their adsorption and chromatographic behavior. The resultant behavior is often kinetically controlled and thus dependent on such factors as contact time of the protein with the adsorbent surface. Examples are given of the appearance of multiple peaks from seemingly pure species, as a result of these structural changes. In one case (papain in reversed-phase LC), multiple peaks are shown to arise from different conformational states. In a second case (beta-lactoglobulin A in hydrophobic interaction chromatography), a series of three peaks is a result of self-association or aggregation. Finally, recent work on an examination of structural changes of proteins on chromatographic supports, by means of intrinsic fluorescence and HPLC, is presented. The value of these studies for the elucidation of the retention mechanism in HPLC and the assessment of purity of proteins is demonstrated.

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.

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.

An automated high-performance liquid chromatographic column-switching system is presented for the analysis of antiepileptic drugs in serum. Initial results show that a reversed-phase extraction column works best overall when fitted with screens versus frits, and when packed with porous 30-micron particles as opposed to a pellicular packing of similar size or with smaller porous particles. The continuous analysis of primidone for over 2000 serum samples is achieved at a rate of twelve samples per hour with a single analytical column. An analogous boxcar high-performance liquid chromatographic system is also assembled and used to analyze two of four injected antiepileptic drugs at a rate of 40 samples per hour. For 1000 of these analyses, the coefficient of variation is 1% without an internal standard.

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.

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.

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.

A methodology involving equilibrium dialysis and gas chromatography has been developed to measure concurrently the concentration of the dialyzable (i.e., free) fractions of thyroxine and 3,5,3'-triiodothyronine in serum. The electron capture response of the N,0-diheptafluorobutyryl methyl ester derivatives of these hormones allows their detection and quantitation in the picogram range (detection limit approx. 0.2pg). Purification of the dialysate is achieved using a cation-exchange column which is pretreated with diiodothyronine. Adsorption problems on the glassware are eliminated by silanization. The methodology also resolves 3,3',5'-triiodothyronine (reverse T3).

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.

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.