High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2- aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.      

Characterization of antibodies to chicken riboflavin carrier protein: Antigenicity of the tryptic fragments

The antigenecity of tryptic fragments of reduced and carboxymethylated chicken riboflavin carrier protein were studied. The tryptic sites of the native riboflavin carrier protein bound to riboflavin were inaccessible. The molecular weight and the elution profile on high performance liquid chromatography (TSK 545 DEAE) were unaltered at an enzyme to substrate ratio of 1:31. However, carboxymethylated riboflavin carrier protein could be cleaved into 3 or 4 fragments at an enzyme to substrate ratio of 1:250 or 1:125. Chromatographic separation of the tryptic fragments on high pressure liquid chromatography (TSK 545 DEAE) revealed the presence of two fragments with different elution profiles but similar molecular weight 26 ±2 kDa. Only one fragment (associated with peak 2) had the ability to displace chicken riboflavin carrier protein in an homologous chicken riboflavin carrier protein radioimmunoassay. Thus, carboxymethylated ribotlavin carrier protein which does not compete with chicken riboflavin carrier protein in the radioimmunoassay, on mild trypsinization generates a fragment which interacts with chicken riboflavin carrier protein in radioimmunoassay.     

Improved method for rapid purification of protein kinase from streptomycetes

Protein kinase from Streptomyces lincolnensis was purified nearly to homogeneity using a high performance liquid chromatography (HPLC) and a Pharmacia FPLC system. The procedure used employed column chromatography on DE-53, followed by FPLC affinity chromatography with serine- or threonine-Sepharose (prepared as described in this paper) and gel filtration using a Superose 12 or TSK G3000SW column. Starting with 3.5 g of mycelial proteins, ∼ 1 mg of pure enzyme was obtained. The procedure is simple and highly reproducible. The protein kinase thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylaminde gel electrophoresis. The purified protein kinase phosphorylated substrate proteins at the seryl residues.     

Inhibition of maize leaf phosphoenolpyruvate carboxylase by diethyl oxaloacetate

Diethyl oxaloacetate was found to be a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase activity with respect to the substrate phosphoenolpyruvate. The K_i values, based on total diethyl oxaloacetate, decreased with increasing pH, while the K_i values, based on the enol tautomer (average of 4 M), were similar and independent of pH. The results suggest that inhibition is dependent on the enol tautomer. Diethyl oxaloacetate was a weak inhibitor following treatment of the enzyme with dithiothreitol; inhibition could be restored by treatment with diamide, indicating inhibition depends on the reduction state of thiol groups on the enzyme.Abbreviations DTT dithiothreitol - HPLC high performance liquid chromatography - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine     

Metabolism of tritiated gibberellin a(20) in maize

After the application of 2.36 Curies per millimole [2,3-³H]gibberellin A₂₀ (GA₂₀) to 21-day-old maize (Zea mays L., hybrid CM7 × CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of ³H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [³H]GA₁ on the basis of co-chromatography with standard [³H]GA₁ on SiO₂ partition, high resolution isocratic elution reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [³H]GA₈ and C/D ring-rearranged [³H]GA₂₀, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C₁₈ high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA₁ and GA₈, or with retention times where conjugates of GA₂₀ would be expected to elute. Conversion to [³H]GA₁ was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [³H]GA₂₀-like metabolite was the major acidic product, while conversion to [³H]GA₁ was low.     

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55°C. EDTA inhibits the activity, and this can be reactivated with Zn²⁺, Mn²⁺, and partially with Co²⁺. The reducing agents dithiothreitol and β-mercaptoethanol and the divalent cation Cu²⁺ inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K_m of 0.15 mM and a V_max of 151 μmol/min per mg of protein.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

An enzyme-immunoassay of abscisic acid in potato (Solanum commersonii) cultured cells

Estimation of abscisic acid (ABA) content in potato (Solanum commersonii) suspension-cultured cells with an enzyme-immunoassay (EIA) was investigated. In crude extracts of potato cultured cells or even after simple clean-up using C₁₈ cartridge, EIA based on commercial monoclonal antibodies (Idetek, Inc) failed to detect any ABA content. An interference could be removed by partitioning against ethyl acetate after the C₁₈ cartridge so that the EIA yielded an estimate of ABA similar to that determined by high pressure liquid chromatography and gas chromatography-mass spectrometry analysis. These results demonstrate the presence of metabolites in potato cultured cell extract that prevent the binding of ABA to its binding site but not the binding of tracer.Abbreviations ABA abscisic acid - EIA enzyme-immunoassay - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass spectrometry     

Enzymatic conversion of dehydrodivanillin to vanillin by an anaerobic recombinant FE7

Enzymatic degradation of dehydrodivanillin (DDV) was studied using high performance liquid chromatography (HPLC) with an anaerobic DDV-degrading recombinant FE7 under both aerobic and anaerobic conditions. When 200 mg of FE7 cells were mixed with 40 μg DDV in 1 ml phosphate buffer (0.01 M, pH 7.0) and 10 mM mercaptoethanol and incubated at 37°C for 24 h under an O₂-free CO₂ atmosphere, about 20 μg of DDV was decomposed. Only 12 μg DDV could be degraded when the same reaction was done under aerobic conditions, suggesting that the reaction occurs more easily under anaerobic than aerobic conditions. Enzymatic degradation of DDV was performed using a cell-free extract as a crude enzyme solution under aerobic conditions in a similar way. A reaction product detected and analysed by thin layer, high performance liquid and gas chromatographies and mass spectrometry was found to be vanillin from enzymatic reaction mixture. This enzymatic activity was not detected in either the culture supernatant or the heat-inactivated control. These results suggest that there may be an intracellular enzyme system which is involved in the conversion of DDV to vanillin. This is the first report to study the enzymatic degradation of DDV by anaerobes.     

Identification of endogenous gibberellins in navel orange shoots

Eight gibberellins (GAs) were identified from vegetative shoots of navel orange trees (Citrus sinensis L. Osbeck cv Washington) after sequential purification by reverse-phase C₁₈ high performance liquid chromatography, Nucleosil 5N(CH₃)₂ high performance liquid chromatography, and capillary gas chromatography-mass spectrometry. GA₁, GA₁₇, GA₁₉, GA₂₀, GA₂₉, and iso-GA₃ were identified based on the full scan mass spectra and Kovats retention indices. GA₈ was tentatively identified based on the comparison of the full scan mass spectra with the published spectra. GA₄₄ was tentatively identified from the characteristic masses at the correct Kovats retention index.     

Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Abstract Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.     

An Activator Stimulating the Hydrolysis of Phospholipids by Phospholipase B from Torulaspora delbrueckii

An activator stimulating the enzymatic hydrolysis of phospholipids was purified to a homogeneous state from autolyzed Torulaspora delbrueckii cell washings. Autolyzed cell washings were extracted with chloroform and ethanol, and the activator was purified about 130-fold by sequential column chromatographies on DEAE-Sephacel, Sephacryl S-300, and TSK gel G 3000 SW (high performance liquid chromatography, HPLC). The molecular weight of the activator was about 175,000 as estimated by gel filtration on HPLC. However, the purified activator gave two protein bands corresponding to molecular weights from 102,000 to 129,000 and from 71,000 to 88,000, respectively, on SDS- polyacrylamide gel electrophoresis, when stained with silver stain reagent and periodic acid-Shiff (PAS) reagent. The activator was sensitive to heat treatment at 70°C for lOmin. The purified activator had no enzymatic activity, but stimulated the hydrolysis of phospholipids by water-soluble and membrane-bound phospholipases B if the substrates were pre-incubated with the activator. No stimulation of hydrolysis by the enzyme was observed when the activator was pre-incubated with the enzyme. The hydrolytic rate of phosphatidylcholine by the enzyme at acidic pH (pH 2.6) depended upon the amount of activator added. On the other hand, the hydrolytic rate at alkaline pH (pH 7.6) was stimulated greatly by more than 0.04 nmol of the activator.     

Simultaneous determination of plasma phenytoin and its primary hydroxylated metabolites in carbon tetrachloride-intoxicated rats by high-performance liquid chromatography

We have developed a simple and sensitive method for the simultaneous determination of phenytoin (PHT), 5(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in rat plasma by high-performance liquid chromatography. The three substances were separated on a reversed-phase column (5 μm TSK gel ODS-80TM, 250 mm × 4.6 mm I.D.) using acetonitrile-0.008 M NaH₂PO₄ (pH 6) (35:65, v/v) as a mobile phase at a flow-rate of 0.8 ml/min. Absorbance was monitored at 215 nm. The quantification limit was 50 ng/ml for each of PHT, m-HPPH and p-HPPH. The mean recoveries for DPH, m-HPPH and p-HPPH from plasma were 95.6±3.6, 94.5±4.2 and 98.6±2.9%, respectively.     

Rapid,sensitive and specific electron capture—gas chromatographic method for the quantitation of 6-chloro-2-(1-piperazinyl)pyrazine in biological fluids

A highly specific and sensitive gas chromatographic method for the determination of 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), a central serotonin-like agent, in biological fluids is described. MK-212 and a related internal standard are extracted into benzene from an alkaline solution, back-extracted into acid and then re-extracted into benzene at an alkaline pH. The amines are converted to the trifluoroacetyl derivatives (characterized by gas—liquid chromatography—mass spectrometry), chromatographed and detected with a ⁶³Ni electron capture detector. The sensitivity of the method is such that 10 ng of drug can be measured per aliquot of biological fluid. The precision and accuracy of the method are well within acceptable limits. Specificity of analysis was established by gas—liquid chromatography—mass spectrometry techniques.     

Effect of Chromatographic Conditions on Enantioseparation of Bovine Serum Albumin Chiral Stationary Phase in HPLC and Thermodynamic Studies

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA‐CSP) by high‐performance liquid chromatography (HPLC) in reversed‐phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N‐(2, 4‐dinitrophenyl)‐serine (DNP‐ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R_s) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N‐(2, 4‐dinitrophenyl)‐proline (DNP‐pro) decrease along with increasing 1‐propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods. Chirality 25:487–492, 2013. © 2013 Wiley Periodicals, Inc.     

Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analysis of the resulting pyrimidine derivatives

Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom⁷Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom⁷Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-¹⁴C]guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH₄H₂PO₄ (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH₄H₂PO₄ (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, about 90% of the material was formylated. When stored at 24°C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom⁷Gua are in equilibrium. The rom⁷Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species.     

Three TRH-Like Molecules Are Released from Rat Hypothalamus In Vitro

TRH-like immunoreactivity distinct from TRH is present in various tissues and fluids. In order to determine whether TRH-like molecules are secreted by the hypothalamus, we analyzed tissues and media from hypothalamic slices incubated in Krebs Ringer bicarbonate. Media from basal or high KCl conditions contained 3 TRH-like molecules evidenced by reverse phase high performance liquid chromatography followed by TRH radioimmunoassay. Peak I corresponded to authentic TRH (73% of total immunoreactivity) and peaks II and III had a higher retention time. These additional TRH-like forms were neither detected in hypothalamic tissue nor in tissue or medium from olfactory bulb. Gel filtration analysis of hypothalamic media revealed only one TRH-like peak eluting as TRH, suggesting that the molecular weights of peaks II and III are similar to that of TRH. Peak II retention time was similar to that of pglu-phe-proNH₂. We analysed if they could be produced by post secretory metabolism of TRH. Incubation of hypothalamic slices with [³H-Pro]-TRH did not produce radioactive species comigrating with peaks II or III. However, it induced rapid degradation to [³H-Pro]-his-prodiketopiperazine ([³H]-HPDKP). Inhibitor profile suggested that pyroglutamyl aminopeptidase II, but not pyroglutamyl aminopeptidase I, is responsible for [³H]-HPDKP production. These data are consistent with the hypothesis that pyroglutamyl aminopeptidase II is the main aminopeptidase degrading TRH in hypothalamic extracellular fluid. Furthermore, we suggest that the hypothalamus releases additional TRH-like molecules, one of them possibly pglu-phe-proNH₂, which may participate in control of adenohypophyseal secretions.     

Catalytic activity of maize leaf phosphoenolpyruvate carboxylase in relation to oligomerization

The relationship between the state of oligomerization and activity of purified maize leaf phosphoenolpyruvate carboxylase using size exclusion high performance liquid chromatography was examined. Maximum activities of 35 to 38 micromoles per minute per milligram protein were found when 100% of the enzyme was in its tetrameric form. The effects of the sulfhydryl group modifiers CuCl₂ and p-chloromercuribenzoate on enzyme inhibition and the state of aggregation of the protein complex were examined. Aggregation of the enzyme is temperature and pH sensitive with low temperature and high pH favoring depolymerization. Stability of the tetrameric form is largely dependent upon histidyl residues, and to some extent this explains the biphasic response of enzyme activity to changes in MgCl₂ concentrations. Modification of the tetramer's histidyl residues by the inhibitor diethylpyrocarbonate (0.125 millimolar) results in its dissociation to the dimeric form and loss of activity. Subsequent treatment with 0.4 molar hydroxylamine results in reassociation to the tetramer and restoration of enzymic activity.     

Purification and Characterization of an Arylamine N-Acetyltransferase from the Bacteria Aeromonas hydrophilia

N-acetyltransferase from Aeromonas hydrophilia was purified by ultrafiltration, DEAE-Sephacel, gel filtration chromatography on Sephadex G-100, and DEAE-5pw on high performance liquid chromatography, as judged by sodium dodecyl sulfate-polyacrylamine gel electrophoresis (SDS-PAGE) on a 12.% (wt/vol) slab gel. The enzyme had a molecular mass 44.9 kDa. The purified enzyme was thermostable at 37°C for 1 h with a half-life 28 min at 37°C, and displayed optimum activity at 37°C and pH 7.0. The K _m and V _max values for 2-aminofluorene were determined to be 0.896 mM and 2.456 nmol/min/mg protein, respectively. Among a series of divalent cations and salts, Zn²⁺, Ca²⁺, and Fe²⁺ were demonstrated to be the most potent inhibitors. Received: 10 November 1997 / Accepted: 17 February 1998     

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD⁺-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD⁺-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C₂–C₈) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K _m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point