C18 Reversed-phase High-performance Liquid Chromatography Research Articles

Forward and reverse (retro) iron(III) or gallium(III) desferrioxamine E and ring-expanded analogues prepared using metal-templated synthesis from endo-hydroxamic acid monomers.

A metal-templated synthesis (MTS) approach was used to preorganize the forward endo-hydroxamic acid monomer 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoic acid (for-PBH) about iron(III) in a 1:3 metal/ligand ratio to furnish the iron(III) siderophore for-[Fe(DFOE)] (ferrioxamine E) following peptide coupling. Substitution of for-PBH with the reverse (retro) hydroxamic acid analogue 3-(6-amino-N-hydroxyhexanamido)propanoic acid (ret-PBH) furnished ret-[Fe(DFOE)] (ret-ferrioxamine E). As isomers, for-[Fe(DFOE)] and ret-[Fe(DFOE)] gave identical mass spectrometry signals ([M + H(+)](+), m/zcalc 654.3, m/zobs 654.3), yet for-[Fe(DFOE)] eluted in a more polar window (tR = 23.44 min) than ret-[Fe(DFOE)] (tR = 28.13 min) on a C18 reverse-phase high-performance liquid chromatography (RP-HPLC) column. for-[Ga(DFOE)] (tR = 22.99 min) and ret-[Ga(DFOE)] (tR = 28.11 min) were prepared using gallium(III) as the metal-ion template and showed the same trend for the retention time. Ring-expanded analogues of for-[Fe(DFOE)] and ret-[Fe(DFOE)] were prepared from endo-hydroxamic acid monomers with one additional methylene unit in the amine-containing region, 4-[(6-aminohexyl)(hydroxy)amino]-4-oxobutanoic acid (for-HBH) or 3-(7-amino-N-hydroxyheptanamido)propanoic acid (ret-HBH), to give the corresponding tris(homoferrioxamine E) macrocycles, for-[Fe(HHDFOE)] or ret-[Fe(HHDFOE)] ([M + H(+)](+), m/zcalc 696.3, m/zobs 696.4). The MTS reaction using a constitutional isomer of for-HBH that transposed the methylene unit to the carboxylic acid containing region, 5-[(5-aminopentyl)(hydroxy)amino]-5-oxopentanoic acid (for-PPH), gave the macrocycle for-[Fe(HPDFOE)] in a yield significantly less than that for for-[Fe(HHDFOE)], with the gallium(III) analogue for-[Ga(HPDFOE)] unable to be detected. The work demonstrates the utility and limits of MTS for the assembly of macrocyclic siderophores from endo-hydroxamic acid monomers. Indirect measures (RP-HPLC order of elution, c log P values, molecular mechanics, and density functional theory calculations) of the relative water solubility of the ligands, the iron(III) macrocycles, and the apomacrocycles were consistent in identifying for-DFOE as the most water-soluble macrocycle from for-DFOE, ret-DFOE, for-HHDFOE, ret-HHDFOE, and for-HPDFOE. From this group, only for-DFOE is known in nature, which could suggest that water solubility is an important trait in its natural selection.

별불가사리 Asterina pectinifera의 유문맹낭 추출물로부터 새로운 2종류의 항균활성 펩타이드의 정제

PAP-1, a novel antimicrobial peptide isolated from pyloric caeca extract of the starfish Asterina pectinifera was purified and characterized. First, the acidified pyloric caeca extract was put through Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient. Among the eluents, RM 60 (retained materials at 60% methanol) showed good antimicrobial activity against Bacillus subtilis and Escherichia coli D31 and was purified in C18 reversed-phase and ion-exchange high-performance liquid chromatography columns. The purification steps yielded two novel peptides showing strong antimicrobial activities. These peptides were named pyloric caeca A. pectinifera peptide 1 and 2 (PAP-1 and PAP-2). For the characterization of the purified peptides, the molecular weights and amino acid sequences were determined by MALDI-TOF MS and Edman degradation. The molecular weights of PAP-1 and PAP-2 were about 2951.54 Da and 2980.15 Da respectively. The amino acid sequences of PAP-1 and PAP-2 were partially determined: AIQNAGES and AIQNAAES, respectively. PAP-2 is an isoform of PAP-1, differing merely by a single residue at position 6 (glycine or alanine). The comparison of the N-terminal amino acid sequences and molecular weights of the peptides with those of other known antimicrobial peptides revealed that PAP-1 and PAP-2 have no homology with any known peptides. These findings suggest that PAP-1 and PAP-2 play a significant role in the innate defense system of starfish pyloric caeca.

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

BackgroundSilkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Methodology/Principal FindingsSilkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.Conclusions/SignificanceThe study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

Chromatography of bis-(3-sulfopropyl) disulfide and its breakdown products by HPLC coupled with electrochemical detection

A chromatographic method for the detection of bis-(3-sulfopropyl) disulfide (SPS), a common additive in acidic copper plating baths, and its breakdown products is demonstrated. The detection scheme involves a combination of solid-phase extraction for sample pre-treatment, C(18) reversed-phase high-performance liquid chromatography column for separation, and electrochemical sensor for detection of all non-fully oxidized sulfur-containing compounds. We were able to achieve an effective separation and accurately assign chromatographic peaks to all detectable species. Owing to a high sensitivity of the utilized electrochemical detector, detection in low parts per billion range was possible. This can prove crucial for plating bath control, since minute amounts of certain by-products significantly affect the bath performance.

Kinetic performance of reversed-phase C18 high-performance liquid chromatography columns compared by means of the Kinetic Plot Method in pharmaceutically relevant applications

Four fully porous C18 columns (Hypersil Gold, ACE3, Xbridge and Gemini NX), widely employed in the pharmaceutical industry, were compared in terms of efficiency and analysis speed with the Kinetic Plot Method. Weakly basic, medium-sized, N-containing pharmaceutical compounds from GlaxoSmithKline Research and Development were used as test molecules. Isocratic elution was carried out at pH 4.5 and pH 8.0 with acetonitrile as organic modifier. The columns under evaluation included highly pure silica supports (Hypersil Gold, ACE3) and hybrid polymer-silica supports (XBridge, Gemini NX). Both types of columns claim for nearly absent secondary interactions with ionized silanol groups and are therefore applicable in a wide pH range. This is an important feature for method development purposes in pharmaceutical industries. The Kinetic Plot Method was used to compare the support characteristics and assess the kinetic performance of the columns in different experimental conditions. Although the evaluated columns have roughly identical particle diameters (from 3.0 to 3.5 μm) according to their manufacturers, large differences in kinetic performance were observed at pH 4.5 that can be accounted for by different flow resistances, porosities and average particle diameters, experimentally determined from scanning electron microscopy and laser light scattering experiments on loose stationary phase material. The ACE3 column was the best performing support among the evaluated columns, due to its excellent efficiency and average flow resistance. The better performance of the ACE3 column was due to its better packing quality, as could be derived from its impedance plot. Kinetic plots of resolution of a critical pair versus analysis time and column length were established at pH 8.0. These plots can be used as a method development tool to tailor the separation conditions to the required resolution of a given critical pair, combining efficiency and selectivity features of the column.

Characterization of taste-active fractions in red wine combining HPLC fractionation, sensory analysis and ultra performance liquid chromatography coupled with mass spectrometry detection

Five Tempranillo wines exhibiting marked differences in taste and/or astringency were selected for the study. In each wine the non-volatile extract was obtained by freeze-drying and further liquid extraction in order to eliminate remaining volatile compounds. This extract was fractionated by semipreparative C18-reverse phase-high performance liquid chromatography (C18-RP-HPLC) into nine fractions which were freeze-dried, reconstituted with water and sensory assessed for taste attributes and astringency by a specifically trained sensory panel. Results have shown that wine bitterness and astringency cannot be easily related to the bitter and astringent character of the HPLC fractions, what can be due to the existence of perceptual and physicochemical interactions. While the bitter character of the bitterest fractions may be attributed to some flavonols (myricetin, quercetin and their glycosides) the development of a sensitive UPLC–MS method to quantify astringent compounds present in wines has made it possible to demonstrate that proanthocyanidins monomers, dimers, trimers and tetramers, both galloylated or non-galloylated are not relevant compounds for the perceived astringency of the fractions, while cis-aconitic acid, and secondarily vainillic, and syringic acids and ethyl syringate, are the most important molecules driving astringency in two of the fractions (F5 and F6). The identity of the chemicals responsible for the astringency of the third fraction could be assigned to some proanthocyanidins (higher than the tetramer) capable to precipitate with ovalbumin.

The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis

Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.

A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P(AW) that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P(AW) only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P(AW) prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

Isolation and Characterization of an Extracellular Antimicrobial Protein fromAspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization–tandem mass spectrometry, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization–time-of-flight/mass spectrometry (MALDI–TOF/MS) with subsequent positive identification by reversed-phase HPLC–electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

Separation of Pyridylaminated Oligosaccharides by High Performance Liquid Chromatography on a C30 Reversed-Phase Column

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides and ten high mannose-type PA-oligosaccharides were separated by high performance liquid chromatography (HPLC) on a triacontyl (C30) silica-gel column. The eight fucose-containing PA-oligosaccharides and eight out of ten high mannose-type PA-oligosaccharides were clearly separated by C30 reversed-phase HPLC at a column temperature of 40°C with a fluorescence detector; two high mannose-type PA-oligosaccharides, Man7-GlcNAc2 and Man8-GlcNAc2, were not separated. The two unresolved PA-oligosaccharides were finally separated by C30 reversed-phase HPLC at a column temperature of 10°C. The C30 reversed-phase HPLC is quite useful for the analysis of various types of PA-oligosaccharides, including positional isomers, because of excellent separation compared with that of C18 reversed-phase HPLC. The C30 reversed-phase HPLC system can be applied to HPLC-mass spectrometry for re-confirmation of the structures of the PA-oligosaccharides.

The Effect of Lutein and Zeaxanthin Supplementation on Metabolites of These Carotenoids in the Serum of Persons Aged 60 or Older

To investigate the effect of lutein supplementation at doses of 2.5, 5.0, and 10 mg/d for 6 months on distribution of these carotenoids and their metabolites in the serum of elderly human subjects, with and without age-related macular degeneration. To determine whether supplementation with lutein can interact with the serum levels of other dietary carotenoids, retinol, and alpha-tocopherol. Forty-five subjects received daily supplements of lutein (containing 5% zeaxanthin) for 6 months and were followed up for another 6 months after supplementation. Blood was collected at various intervals and lutein, zeaxanthin, and their metabolites in the sera were quantified by normal-phase high-performance liquid chromatography (HPLC)-UV/visible detection. Other dietary carotenoids, retinol, and alpha-tocopherol were identified and quantified on a C18 reversed phase HPLC column. After 6 months of supplementation with 10 mg of lutein, the increases in the mean serum levels from baseline were: 210 to 1000 nM/L (P < 0.0001) for lutein and 56 to 95 nM/L (P < 0.0001) for zeaxanthin. Similarly, the mean concentrations (nM/L) of carotenoid metabolites increased from 49 to 98 (P < 0.0001) for 3-hydroxy-beta,epsilon-caroten-3'-one (3'-oxolutein); 31 to 80 (P < 0.0001) for 3'-hydroxy-epsilon,epsilon-caroten-3-one; and 19 to 25 (P < 0.0001) for epsilon,epsilon-carotene-3,3'-dione. The serum levels of these carotenoids gradually decline within 6 months after supplementation. The increase in the serum levels of lutein/zeaxanthin correlates with increases in the serum levels of their metabolites that have previously been identified in the ocular tissues. Elderly human subjects with and without AMD can safely take supplements of lutein up to 10 mg/d for 6 months with no apparent toxicity or side effects.

Identification and characterization by high‐performance liquid chromatography/electrospray ionization mass spectrometry of a new variant hemoglobin, Mataro [β134(H12) Val &gt; Ala

This work illustrates the practical use of combined microbore reversed-phase high-performance liquid chromatography (RP-HPLC) with electrospray ionization mass spectrometry (ESI-MS) in protein identification. The approach consisted of the detection of the abnormal beta-globin chain by ESI-MS analysis of mixtures of intact globins, which simultaneously provided their molecular masses. Separation of the polypeptide globin chains was carried out using microbore C4 RP-HPLC on-line with ESI-MS. Direct peptide-mapping ESI-MS without previous chromatographic separation was performed in order to identify tryptic peptides from whole blood. For the sequence confirmation of the abnormal peptide containing the mutation point, C18 RP-HPLC tryptic separation of the globin mixture on-line with collision-induced dissociation (CID) fragmentation was done. The y series ions allowed the identification of the hemoglobin (Hb) variant as [beta134(H12) Val > Ala]. This new Hb was named Hb Mataró, after the city where it was detected.

The Effect of D-Amino Acid-Containing Basic Peptides with Different Hydrophobicity on the Antimicrobial and Cytotoxic Activity

Abstract In order to investigate the structure-activity relationships between the hydrophobicity, differences in the number of positive charged amino acids and D-amino acids as well as their action mechanism with different bio-membranes, we designed and synthesized two sets of 12-mer model peptides originating from an amphiphilic antimicrobial model peptide and a D-amino acid-containing non-amphiphilic peptide. The introduction of D-amino acid in peptides led not only to decreases in the hydrophobicity, helical content, and hemolytic activity, but to an increase in the antimicrobial activity against Gram-positive and -negative bacteria. The relative peptide hydrophobicity was estimated in terms of the peptide elution time, expressed by linear trifluoroacetic acid (TFA)-water to TFA-acetonitrile gradients on C18 reverse-phase high-performance liquid chromatography (RP-HPLC). The retention behavior of model peptides on RP-HPLC is correlated with the activity and selectivity for mammalian cells, Gram-positive and -negative bacteria: The α-helical amphiphilic structure is required for cytolytic ability against mammalian cells, but is not necessary for bacterial activity. Additionally, the peptides have different hydrophobicity regions in which they show their optimum activity against both bacteria and erythrocytes. In the highest hydrophobisity region the peptides display high levels of hemolytic activity. At medium hydrophobicity levels, the peptides are able to display to high levels of activity against Gram-positive bacteria. In the lowest hydrophobisity regions the peptides are able to display high levels of activity against Gram-negative bacteria. This may be useful in designing clinically effective antimicrobial peptides with highly potent activity and high bacterial selectivity.

Analyses of compound libraries obtained by high-throughput parallel synthesis: strategy of quality control by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance techniques

The growing interest in combinatorial chemistry has led to a new source of compounds from which a large number of leads has emerged over recent years. Parallel synthesis, in particular, allows a quick production of a wide number of individual compounds. A rapid analytical control is needed to determine their quality. A strategy using automated, fast reversed-phase C18 high-performance liquid chromatography with diode-array detection (LC–DAD–MS) followed by atmospheric pressure chemical ionisation mass spectrometry (APCI–MS) and NMR has been developed for their characterisation and purity control. Complementary NMR analyses are done on selected compounds to provide a better structural characterisation of the expected compounds and their potential side-products. Validated libraries are then registered in ISIS databases using automated procedures.

Increased plasma malondialdehyde levels in glomerular disease as determined by a fully validated HPLC method

Reactive oxygen species and particularly free radical induced lipid peroxidative tissue damage have been implicated in the pathogenesis of various renal diseases. Lipid peroxidation is assessed indirectly by the measurement of secondary products, such as malondialdehyde (MDA), using the widely employed thiobarbituric acid reactive substances (TBARS) method. However, this method lacks sensitivity and specificity. We have therefore developed and validated an HPLC (high-performance liquid chromatography) method for measurement of MDA and applied this to a variety of plasma samples in renal patients. The optimized method involves antioxidant treatment of the plasma sample, followed by a protein precipitation step using trichloroacetic acid, acid hydrolysis and formation of an MDA thiobarbituric acid complex. The MDA-(TBA)2 adduct is separated from other interfering compounds by C18 reverse-phase HPLC techniques, with visible detection at 532 nm. The assay was linear over the ranges 0.25-1.0 microM MDA and the detection limit was 0.06 microM MDA. Within-run precision was <4.5% and between-run precision was <10.0%. MDA plasma concentrations (mean+/-SD) were higher in ESRF diabetic patients (0.32 +/- 0.14 microM, n=20), non-diabetic ESRF patients (0.32 +/- 0.09 microM, n=20), and CRF patients (0.14 +/- 0.06 microM, n=40) compared to healthy controls (0.11 +/- 0.03 microM, n=40), (P < 0.001, P < 0.001 and P = 0.008). Levels were similar in healthy controls with normal renal function and transplanted patients (0.12 +/- 0.03 microM MDA, n=40), (P=NS). No correlation was observed between MDA and creatinine levels (r2 = 0.05, n=80), which suggests that MDA does not correlate with the degree of renal impairment. We matched CRF patients with glomerular and non-glomerular causes of renal failure for creatinine levels and found that MDA levels were higher in patients with glomerulonephritis (0.16 +/- 0.06 microM) than in those with CRF from non-glomerular causes (0.12 +/- 0.04 microM, P = 0.002). We have introduced a reliable and sensitive HPLC technique to enhance the specificity of MDA-(TBA)2 measurement, with a significant improvement in HPLC column life. Using this method, picomole quantities of MDA can be detected in plasma. We have shown that MDA levels are significantly raised in patients with CRF due to glomerulonephritis, regardless of serum creatinine, which suggests that there is oxidative injury independent of any possible MDA retention due to renal impairment.

Rapid HPLC screening method for contaminants found in implicated L-tryptophan associated with eosinophilia myalgia syndrome and adulterated rapeseed oil associated with toxic oil syndrome.

In 1981 a massive food-borne epidemic, termed the toxic oil syndrome (TOS), occurred in Spain. Eight years later a closely related disease, the eosinophilia myalgia syndrome (EMS), was reported in the USA with many additional cases being reported worldwide. Although EMS was linked to the ingestion of contaminated L-tryptophan and TOS to aniline denatured rapeseed oil, the etiological agent(s) responsible for both diseases remains unknown. However, contaminants in both the oil and the dietary supplement are believed to have triggered these diseases, and there has been much speculation that a common contaminant may have caused both epidemics. In this report, methods for the facile preparation and HPLC analysis of EMS-implicated L-tryptophan and adulterated rapeseed oil samples associated with TOS are described which allow a direct comparison between the contaminants of both foodstuffs. A combination of solvent and solid phase extraction methods are demonstrated along with the application of C18 reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with on-line UV and MS detection. These methods have allowed us to determine for the first time, based upon this work, that there are no detectable common contaminants that possess a UV response, between EMS implicated L-tryptophan and TOS implicated rapeseed oil samples.

Isolation, characterization, and heterologous expression of the novel lantibiotic epicidin 280 and analysis of its biosynthetic gene cluster.

Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C18 reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 +/- 1.5 and 3,136 +/- 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332-4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.

A mass spectrometric study of the heterogeneity of the monomer subunit of Lumbricus terrestris hemoglobin

The subunits of the hemoglobin of Lumbricus terrestris consist of heme-binding globin chains, designated a, b, c, and d, and linker chains. The sequence of chain d, which is also referred to as a monomer subunit, has been reported by Shishikura et al. (Biol. Chem.1987, 262, 3123–3131). This subunit has been found to be heterogeneous and in this study three fractions were separated by C18 reverse-phase high-performance liquid chromatography. The major chain d1 with molecular weight 15,993. 5 ± 2. 1 u possesses S7 instead of G7, the partial sequence RDIIDD (33–38) instead of KGILRE, E instead of Q in positions 23 and 58, and T84 instead of A84. The C-terminus is K140 instead of D141I142. Chain d3 with molecular weight 15,963. 2 ± 1. 6 u showed high sequence homology with chain d1, differing apparently only in residue 84 where A84 replaces T84. Analysis of the third chromatographie fraction revealed two additional chains with molecular weights of 15,996. 1 ± 1. 4 and 15,937. 6 ± 1. 2 u. These are designated chains d2 and d4, respectively, but their sequence assignments are not yet certain.

Characterization of a mutant recombinant S100 protein using electrospray ionization mass spectrometry.

Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC). Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting. The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected. One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified. The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10. The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation. The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10. Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon. Another stop codon (TGA) located 27 base pairs downstream halts translation. The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass. Readthrough occurs in strains of E. coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.