Analysis and purification of plasmid DNA by reversed-phase high-performance liquid chromatography

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Under our experimental conditions, the retention time depends not on the chain length but rather on the base composition and the secondary structure of the molecule. Because of the torsional strain caused by the supercoiling of the plasmid, more of its bases are accessible for interaction with the hydrophobic stationary phase. This increases the retention time of the supercoiled DNA compared to the relaxed or linear DNA. We have exploited these properties to analyze the quality of plasmid preparations. The method is more sensitive to contaminants than common electrophoretic techniques. Furthermore, we describe a convenient and rapid procedure for purifying plasmid DNA. The highly pure plasmid is biologically more active for most of the enzymatic reactions commonly used in genetic engineering.     

Analysis and purification of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Sizing of DNA fragments is a routine analysis traditionally performed on agarose or polyacrylamide gels. Electrophoretic analysis is labor-intensive with only limited potential for automation. Recovery of DNA fragments from gels is cumbersome. We present data on automated, size-based separation of DNA fragments by ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) - DNA chromatography - on the WAVE DNA Fragment Analysis System with the DNASep cartridge. This system is suitable for accurate and rapid sizing of double-stranded (ds) DNA fragments from 50 to ca. 2000 base pairs (bp). Fluorescently labeled DNA fragments are compatible with the technology. Length-dependent separation of dsDNA fragments is sequence independent and retention times are highly reproducible. The resolving capabilities of DNA chromatography are illustrated by the analysis of multiple DNA size markers. Resolved dsDNA fragments are easily collected and are suitable for downstream applications such as sequencing and cloning. DNA chromatography under denaturing conditions with fluorescently labeled DNA fragments offers a means for the separation and purification of individual strands of dsDNA. Analysis of DNA fragments on the WAVE System is highly automated and requires minimal manual intervention. DNA chromatography offers a reliable and automated alternative to gel electrophoresis for the analysis of DNA fragments.     

Separation of collagen cyanogen bromide-derived peptides by reversed-phase high-performance liquid chromatography.

A new technique for separation of the CNBr cleavage products of collagen is described. It involves the use of a 30 nm pore reversed-phase high-performance liquid chromatography column eluted with a linear gradient of acetonitrile/water containing 0.01 M-heptafluorobutyric acid. The separation of type I, type II and type III CNBr peptides is described. Resolution is particularly good for the low-molecular-weight peptides. The method is fast, quantitative and sensitive, and the complete volatility of the eluent facilitates the recovery of the separated peptides.     

Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis

A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic Acid Purification Cartridge. Salts, failure sequences and synthetic by-products are washed away while the desired, full-length, dimethoxytrityl oligonucleotide remains bound to the cartridge. The trityl group is hydrolyzed from the 5'-end of the oligonucleotide with an acid wash and then the purified oligonucleotide is eluted with 35% methanol. Oligonucleotides are recovered salt-free with purities greater than 95%. NENSORB PREP-purified primers provide superior sequence data compared to similar primers used without purification and equivalent data to primers purified by polyacrylamide gel electrophoresis when used in manual radiometric Sanger sequencing.     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.     

Separation and purification of S49 mouse lymphoma histones by reversed-phase high-performance liquid chromatography

A rapid reversed-phase high-performance liquid chromatography procedure for the fractionation of histones from S49 mouse lymphoma cells is reported. The system utilizes a Vydac C4 macroporous column, heptafluorobutyric acid as solubilizing and ion-pairing agent, and an acetonitrile gradient. All five histone classes and several subclass species are separated, including two H1 species, H2B, two H2A species, H4, and two H3 species. Analytical to multimilligram semipreparative scale fractionations are demonstrated while maintaining resolution of all histone types.     

Generation of peptides suitable for sequence analysis by proteolytic cleavage in reversed-phase high-performance liquid chromatography solvents

A methodological study of practical importance to protein sequencing has been carried out. Peptide mapping and sequence analysis of the cleavage products of reduced and carboxymethylated ribonuclease have been applied to the study of the activity and specificity of trypsin, chymotrypsin, elastase, lysyl endopeptidase (Achromobacter protease I), endoproteinase Arg-C (from mouse submaxillary gland), Staphylococcus aureus V8 protease, pepsin, and thermolysin in the presence of 20% methanol, ethanol, 2-propanol, and acetonitrile at 22 and 37 degrees C. The peptide bond specificities were retained, and the activities were generally unaffected or moderately reduced at 22 degrees C and pH 8. At 37 degrees C the activity of chymotrypsin, endoproteinase Arg-C, V8 protease at pH 4, and pepsin was substantially reduced and decreased in the order methanol, ethanol, 2-propanol, and acetonitrile. The activity of thermolysin at 55 degrees C was reduced very little in the presence of 20% organic solvent and 50 mM Ca2+. In low calcium and 20% 2-propanol at 22 degrees C the activity of thermolysin was restricted to the complete and specific cleavage of peptide bonds N-terminally of Phe, Ile, and Leu. The experiments suggest that secondary proteolytic digestions can be carried out directly in reversed-phase-HPLC fractions, and that organic cosolvents can be applied to control the degree of proteolysis. Moreover, the denaturing potential of these solvents might be useful in the degradation of proteins resistant to proteolysis, for example, in studies aimed at identification of disulfide bridges.     

Combined paper and reversed-phase high-performance liquid chromatography method for the study of pregnenolone and progesterone metabolites

A method for the separation and quantitation of steroid metabolites, obtained from enzymatic conversions of tritiated pregnenolone or progesterone, is described. As the first step, paper chromatography is used and the different zones obtained, as monitored by radiochromatogram scanning, are then eluted separately from the paper. The final resolution is achieved by reversed-phase high-performance liquid chromatography in 35% acetonitrile with UV detection at 215 nm. The method has been successfully applied to the study of metabolite patterns obtained from steroid conversion produced by testicular biopsy material under conditions of in vitro incubations.     

Identification of substance P metabolites using a combination of reversed-phase high-performance liquid chromatography and capillary electrophoresis

Gradient elution reversed-phase high-performance liquid chromatographic and capillary electrophoretic separations were optimised to separate substance P (SP) and twelve of its fragments. The methods were applied to a study of the in vivo metabolism of substance P in the rat after intrastriatal injection of the peptide (10 nmol). SP and significant amounts of its N-terminal fragments, SP(1-7) and SP(1-4), were detected but no major C-terminal fragments could be identified. At the concentration studied, the metabolism of SP was shown to follow zero order elimination kinetics with a rate of decay of 0.2 nmol/min. As we have shown that SP(1–4) and SP(1–7) can be produced in vivo in the striatum in relatively large amounts, it is conceivable that these fragments contribute to the overall pharmacological pattern of activity of the parent peptide.     

Optimum separation condition of peptides in reversed-phase liquid chromatography

An efficient optimization method was suggested to separate biologically active peptides by RP-HPLC. In this work, the binary mobile phase of water and acetonitrile was used with the buffer of trifluoroacetic acid (TFA). The elution profiles were calculated by the plate theory based on the linear and quadratic equations of retention factor, lnk=A+BF, lnk=A+BF+CF(2), and F was the vol.% of acetonitrile. We modified the plate theory to calculate elution profile in both isocratic and gradient mode. From the final calculated results, the first mobile phase composition was water in 0.1% TFA/acetonitrile in 0.1% TFA, 81/19vol.%, then after 7-8 min, the second composition of mobile phase was linearly changed to 79/21vol.%, and finally after 8 min, it was kept at the isocratic mode. In the experimental conditions, the agreement between the experimental data and the calculated values was relatively good.     

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L⁻¹. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z^eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0.

• •For lactoferrin (pI=8–9), the electrophoretic mobility (μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption.
• •For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z^eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l⁻¹, respectively. Accordingly, one to six phosphates were bound per molecule, respectively.
• •For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z^eff was only discrete values +5 for I in the range 0.001–0.020 mol l⁻¹ and about +3 in the range 0.050–0.500 mol l⁻¹, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength.

Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.     

Aggregation and secondary structure of synthetic amyloid beta A4 peptides of Alzheimer's disease

The deposition of amyloid beta A4 in the brain is a major pathological hallmark of Alzheimer's disease. Amyloid beta A4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates. Using synthetic peptides corresponding to the natural beta A4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of beta A4. A comparison of amino-terminally truncated beta A4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid beta A4. Infrared spectroscopy of beta A4 peptides in the solid state shows that their secondary structure consists of a beta-turn flanked by two strands of antiparallel beta-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative beta-turn positions. Limited proteolysis of these analogs allowed a localization of the central beta-turn at residues 26 to 29 of the entire sequence. Purified beta A4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a beta-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a beta-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the beta A4 sequence. This model is sustained by the properties of beta A4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between beta A4 peptides.     

Application of reversed-phase high-performance liquid chromatography and capillary zone electrophoresis to the peptide mapping of pepsin isoenzymes

A combination of reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) was used for the characterization of peptide maps of swine pepsin after its digestion with α-chymotrypsin. Peptide maps obtained by both methods were compared and five selected chromatographic peaks were identified on an electrophoreogram. The different order of peaks found in RP-HPLC compared to CZE confirmed the complementarity of these two methods. More peptide fragments were resolved by RP-HPLC, which was also found to be less sensitive to salt content in peptide mixtures, than by CZE, but only CZE was able to separate and identify phosphorylated and dephosphorylated peptide fragments of swine pepsin digest. CZE peptides faster separation than RP-HPLC, however, the salts have to be removed by ultrafiltration or by RP-HPLC pre-separation prior to CZE analysis. Combined use of RP-HPLC and CZE for peptide mapping makes it possible to distinguish between the phosphorylated and dephosphorylated forms of swine pepsin. This is important from a diagnostic point of view, because pepsin phosphorylation may be associated with gastric cancer.     

Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

A rapid and sensitive reversed-phase HPLC procedure is reported which allows the simultaneous separation and quantitation of LTC₄, 11t-LTC₄, LTD₄, LTB₄, 12epi,6t,8c-LTB₄, 6t-LTB₄ + 12epi,6t-LTB₄, two trihydroxy-eicosatetraenoic acids tentatively identified as 20-OH-LTB₄ and 20-OH,12epi,6t,8c-LTB₄ and several not yet identified 15-series leukotrienes produced by the cytosol of porcine polymorphonuclear leukocytes.     

Partial purification of a specific inhibitor of the insulin-like growth factors by reversed-phase high-performance liquid chromatography

We report here preliminary data using reversed-phase high-performance liquid chromatography for the purification of a specific inhibitor (a molecular weight 16,000–18,000 protein) of the insulin-like growth factor (IGF) or somatomedin family. Crude inhibitor prepared from Cohn fraction IV-1 of human serum was first partially purified using an IGF/CH-Sepharose 4B affinity column. Following elution of the bound inhibitor and resuspension in 0.1% aqueous trifluroacetic acid (mobile phase A), it was injected (100 μl; 2.0 mg protein) onto a Brownlee Aquapore RP-300 column. Application of a linear gradient from 0% to 100% mobile phase B (45% isopropanol−0.1% trifluoroacetic acid) resulted in elution of two peaks of inhibitor activity between 31% and 34% isopropanol associated with a major homogeneous protein peak and a minor heterogeneous protein peak. No inhibitor was recovered when an acetonitrile gradient was used instead of isopropanol, indicating that the inhibitor is very hydrophobic. These data suggest that high-performance liquid chromatography offers a simple procedure for the potential purification of IGF inhibitor(s) from normal human serum.     

Determination of rat liver triglycerides by gas–liquid chromatography and reversed-phase high-performance liquid chromatography

Rats fed with a fat-free or an olive oil-rich diet were employed to compare the response of two chromatographic techniques in the determination of rat liver triglyceride (TG) molecular species composition. Gas–liquid chromatography (GLC) on polarizable liquid phase and reversed-phase high-performance liquid chromatography (RP-HPLC) have been commonly employed for TG analysis, obtaining a similar number of chromatographic peaks when used for animal tissue TG determination. In the present study similar results were achieved with regard to most relevant chromatographic peaks, however, important differences were found in the content of minor TGs. Indeed, RP-HPLC permitted separation of long chain polyunsaturated fatty acids, which were not detected by GLC, while the latter technique reported a higher number of myristoyl-containing TG species. RP-HPLC analysis reported a greater number of TGs, with more similarity to a random composition, made up from the liver fatty acid composition. Therefore, it was concluded that utilization of both techniques would be helpful for liver TG analysis as the use of only one of them does not provide a complete profile of liver TGs. Nevertheless RP-HPLC seems to be more useful for this purpose since revealed a more extensive profile.