High-performance Liquid Chromatography Purification Research Articles

A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an M13mp10 genome containing the major carcinogen-DNA adduct at a unique site

The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Ventricular atriopeptin. Unmasking of messenger RNA and peptide synthesis by hypertrophy or dexamethasone.

Left ventricular hypertrophy or treatment with dexamethasone caused a 2.5-fold to threefold increase in both immunoreactive atriopeptin (AP) and AP messenger RNA (mRNA), primarily in left ventricular tissue. The combined treatments increased immunoreactive AP and AP mRNA more than either treatment alone. In the animals in which cardiac hypertrophy had been produced by abdominal aortic constriction, there was a decrease in atrial levels of AP and an increase in plasma levels of immunoreactive AP. The increase in left ventricular immunoreactive AP was confirmed by immunohistochemical staining of tissue from hypertrophied and/or dexamethasone-treated rats. The mRNA accumulated in the left ventricle was identical to atrial AP mRNA, as judged by transcriptional start site and by size on Northern blots. Because the mass of ventricular tissue is substantially greater than that of atrial tissue, the induced mRNA levels may represent a total abundance approaching one third of the total AP mRNA in the atria. High performance liquid chromatographic purification of ventricular extracts primarily demonstrated the presence of the high molecular precursor and small amounts of C-terminal peptide AP. Induction of ventricular AP (mRNA and peptide) may represent regression of the tissue to an earlier developmental form. These data provide a unique example of regulation of AP biosynthesis in nonatrial tissue.

Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus

The biosynthesis and posttranslational processing of proenkephalin and the level of preproenkephalin mRNA were investigated in the mossy fiber system of the granule cells of the hippocampus in the presence or absence of a unilateral lesion of the hilus, a procedure that produces an episode of recurrent bilateral hippocampal seizures lasting several hours. Both immunocytochemistry and radioimmunoassay (RIA) have demonstrated that the hilus lesion leads to large bilateral increases in enkephalin immunoreactivity in the mossy fiber system. In the present study, RIA data indicate that following an initial decline in immunoreactivity, enkephalin content within the mossy fibers first begins to increase between 18 and 24 hr after lesioning. Using the technique of in vivo radiolabeling and high-performance liquid chromatographic purification of identified radiolabeled peptides, we observed a 14-fold increase in incorporation of radiolabeled methionine into Met5-enkephalin at 24-30 hr postlesion, as compared with control animals, when Met5-enkephalin was purified from the mossy fiber terminal fields. To examine the posttranslational proteolytic processing of proenkephalin, the biosynthesis of 5 additional Met5-enkephalin-containing peptides was also examined. We determined that the posttranslational processing of proenkephalin did not yield exclusively penta-, hepta-, and octapeptides but larger opioid peptides as well in both control and lesioned animals, and that the ratio of the enkephalin peptides was not altered following the lesion. Measurement of preproenkephalin messenger RNA levels in the granule cells by Northern analysis revealed a marked increase following the lesion. Compared with the control animals, preproenkephalin mRNA was 8.5-fold higher in the contralateral dentate gyrus at 12 hr postlesion and 14- to 15-fold higher by 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoreaction of monofunctional 3-carbethoxypsoralen with DNA: identification and conformational study of the predominant cis-syn furan side monoadduct to thymine.

The main DNA‐3‐CPs furan side adduct has been isolated following acidic hydrolysis of photomodified DNA and subsequent reversed‐phase high performance liquid chromatography purification. This photoadduct has been identified as a thymine‐3‐CPs C4‐cycloadduct Thy<5 4′6 5′, >3‐CPs based on its optical spectroscopic features and of its plasma desorption mass spectrometric characteristics. Moreover, its reversed‐phase high performance liquid chromatography retention time is in agreement with a cis‐syn stereochemistry as compared to the chromatographic properties of the cis‐syn model adduct Thy< 5 4′6 5′ >3‐CPs previously identified. Further support for the cis‐syn stereoconfiguration assignment was provided by fluorescence quenching experiments using iodide and silver ions as external and internal quenchers respectively. These data strongly indicate that the Thy< 5 4′6 5′ >3‐CPs photoadduct is located inside the DNA helix in agreement with its cis‐syn stereochemistry.

Single-step procedure for the extraction and purification of leukotrienes B 4, C 4 and D 4

A rapid and simple method for the on-line concentration and high-performance liquid chromatographic (HPLC) purification of the leukotrienes in good yield from biological fluids is described. Readily available antisera are used in conjunction with this system to give a specific and sensitive assay for leukotrienes B 4, C 4 and D 4 with sub-nanogram limits of detection. Tritium-labelled leukotrienes are used as internal standards, both to locate the leukotrienes post-HPLC and to accurately determine recoveries.

A major epitope of synthetic rabbit encephalitogen S24 disclosed through the use of a tritiated peptide probe

The antibody recognition of synthetic rabbit encephalitogen S24 (TTHYGSLPQKG), which had been inactivated by extensive iodination, was fully restored by tritium-iodine exchange during boron hydride reduction. A highly active form of 3H-S24 was obtained by reverse-phase high-performance liquid chromatography (HPLC) purification of the crude product and could be further separated into three continuous subfractions, the latter two of which were fully active from an immunochemical point of view. A major epitope was disclosed through use of the 3H-S24 probe, which required the intact S24 peptide for its expression. Confirmation of two linear epitopes previously encountered, GSLPQK and THYGSL, was also obtained, the latter through the use of the third HPLC subfraction of 3H-S24.

High-efficiency preparative-scale reversed-phase high-performance liquid chromatographic purification of 14C-labelled antibiotics

The 14C-labelled antibiotics [2- 14C]mupirocin, and [thienyl-3- 14C]temocellin cannot be satisfactorily purified on a small scale by conventional methods of chromatography or recrystallisation. Their purification was successfully achieved by high-efficiency preparative-scale reversed-phase high-performance liquid chromatography. The purifications employed 250 mm × 10 mm I.D. or 22 mm I.D. stainless-steel columns packed with Merck LiChrosorb RP-18 (10μm) stationary phase which were eluted with aqueous buffer solutions at flow-rates of 10–25 ml min -1 using conventional analytical instrumentation.

High-performance liquid chromatographic purification of the H4 and MH3 isoenzymes of lactate dehydrogenase from beef heart

High-performance liquid chromatographic purification of the H4 and MH3 isoenzymes of lactate dehydrogenase from beef heart

Preparative high-performance liquid chromatographic purification and structural determination of 1-O-α- d-glucopyranosyl- d-fructose (trehalulose)

The disaccharide 1-O-α- d-glucopyranosyl- d-fructose (trehalulose) was obtained in a 98% pure form, and in good yield by preparative-scale high-performance liquid chromatography (HPLC). Purification was from a complex mixture containing a trisaccharide, sucrose, isomaltulose, glucose and fructose, produced by immobilised microbial cells. Four reversed-phase preparative-scale HPLC columns were used in series with distilled water as the eluent. The structure of trehalulose was shown to be an equilibrium mixture of 1-O-α- d-glucopyranosyl-β- d-fructopyranose (66%) and 1-O-α- d-glucopyranosyl-β- d-fructofuranose (33%) by 13C NMR spectroscopy and other methods. This work demonstrates how gram quantities of pure material can be conveniently obtained using preparative HPLC, even when the components in a mixture are difficult to resolve.

Proenkephalin is processed in a projection-specific manner in the rat central nervous system.

The biosynthesis and posttranslational proteolytic processing of proenkephalin was studied in three projection systems in the rat central nervous system--the caudate-putamen to the globus pallidus, the paraventricular nucleus of the hypothalamus to the median eminence, and the mossy fiber system of the granule cells of the hippocampus. By using the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification coupled with chemical modification, the biosynthesis of six radiolabeled [Met]enkephalin-containing peptides--[Met5]enkephalin, [Met5,Arg6,Gly7,Leu8]enkephalin, [Met5,Arg6,Phe7]enkephalin, metorphamide, peptide E, and BAM 18P--was followed. In each projection system, radiolabeled enkephalins were purified to constant radiochemical specific activity. However, the posttranslational processing of proenkephalin was found to differ between these three systems, as judged by the relative ratio of these peptides. These findings imply that specific, different physiologies and behaviors may be elicited by the enkephalins based upon the specific [Met]enkephalin-containing peptides that are cleaved from proenkephalin and released in synaptic terminal fields.

Identification of Pea Gibberellins by Studying [14C]GA12-Aldehyde Metabolism

Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with [(14)C]GA(12)-7-aldehyde ([(14)C]GA(12)ald) would serve as appropriate [(14)C]markers for the recovery of naturally-occurring gibberellins (GAs). The [(14)C]GA(12)ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using [4,5-(14)C]mevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high performance liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as [(14)C]markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co-eluting with each [(14)C]marker. These [(14)C] and UV-absorbing peaks were shown to contain GA(53), GA(44), GA(20), GA(19), and GA(17) by GC-MS. The finding of GA(53) is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using [(14)C]GA(12)ald metabolites as [(14)C]markers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species.

High-performance liquid chromatographic purification of a ribonucleoprotein complex and its protein component

This paper describes the rapid purification by high-performance liquid chromatographic techniques of milligram quantities of the 7 S ribonucleoprotein complex (RNP 7 S) and its protein component from tench ( Tinca tinca) oocyte extracts. High-performance gel permeation chromatography (with a TSK 3000SW column) was found to be unacceptable because of multiple contaminants which coelute with RNP 7 S. In contrast, semipreparative high-performance DEAE ion-exchange chromatography was found to give an excellent separation of the 7 S complex which could be directly adapted to a preparative scale providing rapid purification (less than 1 h) of milligram quantities of the complex. Agarose electrophoresis followed by specific staining of protein and nucleic acid was found to be a convenient and rapid means of evaluating the purification. Finally, reverse-phase high-performance liquid chromatography was found suitable for the purification of the protein component of the 7 S complex.

Formation of didesmethylethimizol (4-5-dicarbamoyl-1-ethylimidazole) in Man

The chemical structure of an ethimizol metabolite found earlier in human urine and saliva was established as 4,5-dicarbamoyl-1-ethylimidazole (didesmethylethimizol). The treatment of biological samples comprised a solid-phase extraction, high-performance thin-layer chromatographic separation and high-performance liquid chromatographic purification techniques. The identification of the metabolite was based on the comparison of a mass spectrum of the substance isolated from the biological fluid with the mass spectra of synthetised reference compounds.

HPLC purification of the natural ATPase inhibitor from the yeast Candida utilis

A rapid method of preparation of the natural ATPase inhibitor (IF 1) from the mitochondria of the yeast Candida utilis has been developed. It involved high performance liquid chromatography (HPLC) as the final step of purification. An active form of Candida utilis IF 1 was obtained, free of contaminant. Its properties are compared with those of IF 1 from other sources.

Enkephalin Biosynthesis and Processing during Lactation

The biosynthesis of three Met-enkephalin-containing peptides was studied in the hypothalamo-hypophysial system of normal and lactating female rats by the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification of radiolabeled peptides. Met-enkephalin biosynthesis was stimulated in the lactating animals; however, the products derived from the post-translational processing of the enkephalin precursor appeared to remain constant between the two states, as judged by the ratio of radioactivity present in Met5-enkephalin, Met5Arg6Gly7Leu8-enkephalin and Met5Arg6Phe7-enkephalin. Additionally, 8-fold more radioactive enkephalin was harvested from the median eminence than the posterior pituitary. Measurement of preproenkephalin mRNA in the two states also revealed an increase during lactation. All of these data suggest a physiologically important role for the enkephalins in the regulation of anterior pituitary function.

Use of a C 4 column for reversed-phase high-performance liquid chromatographic purification of synthetic oligonucleotides

The small-pore C 18 column materials commonly used for reversed-phase high-performance liquid chromatographic purification of synthetic oligodeoxyribonucleotides do not yield DNA sufficiently pure for some applications. Separation and purification on large-pore C 4 columns was investigated. Model studies presented demonstrate excellent resolution of synthetic DNAs of similar sequence. Also presented are the results of purification of larger compounds.

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 m M Tris, 2 m M EGTA, 0.5 m M dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 m M potassium dihydrogen phosphate, 2 m M EGTA, 0.5 m M dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate—urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708.

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.

High-performance liquid chromatographic purification of deoxynucleoside 5′-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2′-deoxynucleoside 5′-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP,dGTP,dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N 6-etheno-dATP (εdATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pur dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. εdAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.

Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking.

The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.