Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.     

A procedure for rapid extraction and high-pressure liquid chromatographic separation of the nucleotides and other small molecules from bacterial cells

A method for the extraction and separation of ribo- and deoxyribonucleosides and nucleotides from Salmonella typhimurium and Escherichia coli is described. Cells are harvested by a new rapid filtration method, and the small molecules are extracted with formic acid. The acid-extracted nucleotides and other uv-absorbing compounds are separated by boronate-affinity chromatography and by reverse-phase and ion-pair high-pressure liquid chromatography. The complete analysis uses the extract from 10 ml of log phase cells and separates the small molecule pool into about 170 components.     

Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.     

High-pressure, reverse-phase partition chromatograhy separation of diethylstilbestrol metabolites and analogs.

Various conjugated and oxidative metabolites of diethylstilbestrol were effectively separated by high-pressure, reverse-phase liquid chromatography on C₈- and C₁₈-hydrocarbon phases with water-methanol gradients.     

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO₄^?, subjected to preliminary purification on a Cu²⁺-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.     

Fluorescent/ultraviolet absorbing ester derivative formation and analysis of eicosanoids by high-pressure liquid chromatography

A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B₂ chromatographs as two species and coelutes with PGF_2α. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF_1α, 6-keto-PGE₁, 13,14-dihydro-6,15-diketo-PGF_1α). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF_1α extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF_1α quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.     

Analysis of endo-beta-N-acetylglucosaminidase activity by high-pressure liquid chromatography on a silica-based chemically bonded octadecyl column

We have devised a new method for assaying the endo-β-N-acetylglucosaminidase activity by using the dansyl asparaginyl oligosaccharide, (Man)₅(GlcNAc)₂-Asn-DNS, as the substrate and analyzing the product, GlcNAc-Asn-DNS, by a reverse-phase high-pressure liquid chromatography using a silica-based chemically bonded octadecyl column (Waters μBondapack C₁₈). The column is eluted with 8% acetonitrile in 25 mm sodium borate buffer, pH 7.5, at 3 ml/min. The effluent is monitored by a Perkin-Elmer LC-75 uv monitor at 213 nm and a Perkin-Elmer LC-1000 fluorescence monitor (excitation, 313 nm; emission, 540 nm). Under these conditions, GlcNAc-Asn-DNS is well separated from (Man)₅(GlcNAc)₂-Asn-DNS and the analysis can be completed in 5 min. The peak height is used to quantify the dansyl derivatives. Under the conditions described above, the lower limit of detection is 0.1 nmol of dansyl glycopeptides.     

Isolation and Partial Characterization of Antagonistic Peptides Produced by Paenibacillus sp. Strain B2 Isolated from the Sorghum Mycorrhizosphere

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B₁, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B₁, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.     

Isolation and Identification of the Coal-Solubilizing Agent Produced by Trametes versicolor

Low-ranked coals were dissolved by using cell extracts derived from liquid cultures of Trametes versicolor. The coal-solubilizing agent (CSA) was separated from the broth components by a multistep isolation procedure including reverse-phase high-pressure liquid chromatography, size exclusion chromatography, ethanol fractionation, and recrystallization. Staircase voltammetry was used to show that two CSA moieties can coordinate to aqueous copper(II) ion. A molecular weight determination (using amperometry) gave an apparent molecular weight of 1.3₄ × 10² g/mol ± 8%. Nuclear magnetic resonance indicated that all protons on CSA are exchangeable in D₂O and that there is only one type of carbon in CSA. The infrared spectrum of recrystallized CSA is identical to that of ammonium oxalate, and X-ray studies confirmed the crystal structure and composition of CSA to be that of ammonium oxalate monohydrate. The equivalent weight of the coal in solution, when the coal was dissolved by ammonium oxalate, is 7,940 g of coal per mol of iron present in the coal.     

Molecular weight separation of proteins and peptides with a new high-pressure liquid chromatography column.

A new high-pressure liquid molecular weight chromatography column was evaluated for its ability to separate proteins and peptides. The column was able to give a linear separation of compounds between 5,000–700,000 M_r. Chromatography of posterior pituitary extracts, tumor-associated fetal antigens, and estrogen receptors demonstrated the ability of the column to separate biological samples.     

High-pressure liquid chromatography of alpha-keto acid 2,4-dinitrophenylhydrazones

Various α-keto acids were separated as their 2,4-dinitrophenylhydrazine derivatives by ion-pair, reverse-phase, high-pressure liquid chromatography. Excellent baseline resolution was obtained for a seven-component homologous series of α-keto acid dinitrophenylhydrazones at increasing carbon-chain length. Branched-chain keto acids were also separated. Resolution of syn and anti isomers of the α-keto acid derivatives was possible. Pyruvate from biological material was located and identity confirmed by an enzymic peak shift technique. Monitoring at 366 nm permits low-level (nanogram) amounts of keto acids to be detected. Ion pair versus ion exchange is discussed with regard to the mechanism of chromatographic separation.     

A radiometric high-pressure liquid chromatography assay for the simultaneous determination of the three main oxidative metabolites of antipyrine in studies in vitro

A quantitative radiometric high-pressure liquid chromatography assay for the estimation of the three main oxidative metabolites of antipyrine in vitro using [3-¹⁴C]antipyrine as substrate is described. Baseline separation of antipyrine, 3-hydroxymethylantipyrine, 4-hydroxyantipyrine, and norphenazone was achieved, after methylation, using a reverse-phase μBondapak C₁₈ column with a mobile phase of 17% acetonitrile in water. The metabolites could be estimated free of interference as confirmed by gas chromatography-mass spectrometry. Unlabeled metabolites were used as recovery standards. Activity could be determined with as little as 100 μg human liver microsomal protein. Maximum velocities for the formation of the three metabolites ranged from 1 to 3.5 nmol product mg^?1 min^?1 with rat liver and from 0.3 to 0.6 nmol product mg^?1 min^?1 with human liver.     

In vivo effects of human dialyzable leukocyte lysate: III. Modulation of the spleen cell proliferative response to antigen by components of leukocyte dialysates and an initial characterization of an ampliative nucleoside

The augmentative effects of isolated components of human dialyzable leukocyte lysates upon the proliferative response to antigen were investigated. Sequential Sephadex G-25 and Bio-Gel P-4 chromatography separated five distinct fractions which, 24 hr after injection into Keyhole limpet hemacyanin (KLH)-sensitive mice, either augmented or suppressed the in vitro spleen cell proliferative response to KLH. An amplifier molecule was isolated from one of the augmentative fractions by high-pressure, reverse-phase liquid chromatography. Preliminary structural analysis of the amplifier component indicated a nucleoside structure, similar to—but possibly distinct from—thymidine.     

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.     

Isogenic variants of Escherichia coli with altered morphology have peptidoglycan with identical muropeptide composition.

The peptidoglycan compositions of three isogenic morphological mutants of Escherichia coli were determined by high-pressure liquid chromatography analysis. The muropeptide compositions of the peptidoglycan of these mutants were the same, indicating that the shape of E. coli is not (solely) determined by the chemical composition of the peptidoglycan. Furthermore, it appeared that the muropeptide composition of the peptidoglycan was not affected by growth temperature.     

Acetate-Activating Enzymes of Bradyrhizobium japonicum Bacteroids

Acetyl coenzyme A (acetyl-CoA) synthetase and acetate kinase were localized within the soluble portion of Bradyrhizobium japonicum bacteroids, and no appreciable activity was found elsewhere in the nodule. The presence of each acetate-activating enzyme was confirmed by separation of the two enzyme activities on a hydroxylapatite column, by substrate dependence of each enzyme in both the forward and reverse directions, by substrate specificity, by inhibition patterns, and also by identification of the reaction products by C₁₈ reverse-phase high-pressure liquid chromatography. Phosphotransacetylase activity, found in the soluble portion of the bacteroid, was dependent on the presence of potassium and was inhibited by added sodium. The greatest acetyl-CoA hydrolase activity was found in the root nodule cytosol, although appreciable activity also was found within the bacteroids. The combined specific activities of acetyl-CoA synthetase and acetate kinase-phosphotransacetylase were approximate to that of the pyruvate dehydrogenase complex, thus providing B. japonicum with sufficient capacity to generate acetyl-CoA.     

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

The metabolism of 13-cis-[11-³H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.     

High-pressure liquid chromatographic determination of 13-cis-retinoic acid and all-trans-retinoic acid in human plasma

A simple and selective method is described for the determination of 13-cis-retinoic acid and all-trans-retinoic acid in plasma. The assay involves lyophilization of the sample, extraction of the residue with methanol, and separation of the retinoids by reverse-phase high-pressure liquid chromatography. The procedure can detect as little as 0.05 μg of either compound. It is capable of being readily modified to determine the amount of retinoic acid in other tissues as well as tissue concentrations of other retinoids. Using this assay, the absorption and disappearance from human plasma of an oral dose of 13-cis-retinoic acid have been followed.     

Catabolic Fate of Streptomyces viridosporus T7A-Produced, Acid-Precipitable Polymeric Lignin upon Incubation with Ligninolytic Streptomyces Species and Phanerochaete chrysosporium

Degradation of ground and hot-water-extracted corn stover (Zea mays) lignocellulose by Streptomyces viridosporus T7A generates a water-soluble lignin degradation intermediate termed acid-precipitable polymeric lignin (APPL). The further catabolism of T7A-APPL by S. viridosporus T7A, S. badius 252, and S. setonii 75Vi2 was followed for 3 weeks in aerated shake flask cultures at 37°C in a yeast extract-glucose medium containing 0.05% (wt/vol) T7A-APPL. APPL catabolism by Phanerochaete chrysosporium was followed in stationary cultures in a low-nitrogen medium containing 1% (wt/vol) glucose and 0.05% (wt/vol) T7A-APPL. Metabolism of the APPL was followed by turbidometric assay (600 nm) and by direct measurement of APPL recoverable from the medium. Accumulation and disappearance of soluble low-molecular-weight products of APPL catabolism were followed by gas-liquid chromatography and by high-pressure liquid chromatography, utilizing a diode array detector. Identified and quantified compounds present in culture media included p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, protocatechuic acid, vanillic acid, and vanillin. The further catabolism of these APPL-derived aromatic compounds varied with the culture examined, and only S. setonii and P. chrysosporium completely degraded all of them. Some new intermediates of APPL metabolism also appeared in culture media, but the patterns were culture specific. Additional evidence from high-pressure liquid chromatography analyses indicated that one strain, S. badius, converted a water-soluble fraction evident by high-pressure liquid chromatography (7 to 10 min retention time range) into new products appearing at shorter retention times. Mineralization of a [¹⁴C-lignin]APPL was also followed. The percent ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-APPL, ¹⁴C-labeled water-soluble products, and cell mass-associated radioactivity, were determined for each microorganism after 1 and 3 weeks of incubation in bubbler tube cultures at 37°C. P. chrysosporium evolved the most ¹⁴CO₂ (10%), and S. viridosporus gave the greatest decrease in recoverable ¹⁴C-APPL (23%). The results show that S. badius was not able to significantly degrade the APPL, while the other microorganisms demonstrated various APPL-degrading abilities. The significance of these findings relative to the fate of APPLs in nature was discussed.     

Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography.

An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, non-destructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by fluorometry down to a concentration of 1.5 ng/ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBA₂ and NMRI mice.