Capillary electrochromatographic study of the interactions of porphyrin derivatives with amino acids and oligopeptides

Open-tubular capillary electrochromatography (OT-CEC) was used to study the interactions of synthetic (metallo)porphyrin derivatives (immobilized by physical adsorption to the fused-silica capillary wall) with three aromatic amino acids (phenylalanine, tyrosine, tryptophan), three aliphatic amino acids (beta-alanine, proline, valine) and two oligopeptides (diglycine, triglycine). The effective mobilities of amino acids and peptides measured in OT-CEC mode in the acid and alkaline background electrolytes (BGEs) were compared with those obtained by capillary zone electrophoresis (CZE) in the bare fused-silica capillary in the same BGEs. In this way the influence of the peripheral substituents and the character of the central metal atom in porphyrin derivatives on the interactions with amino acids and peptides in the acid and alkaline media was investigated. Three types of noncovalent interactions, axial ligation to the central metal atom, pi-pi stacking and electrostatic repulsion seem to take part in the interactions of analyzed amino acids and peptides with porphyrin derivatives, resulting in a better separation of these analytes by OT-CEC than by CZE.     

Separation of the eleven priority pollutant phenols by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) provides highly efficient separation of the eleven US EPA priority pollutant phenols. All of these phenols are completely resolved in fewer than 15 min in a 100 cm × 75 μm I.D. uncoated fused-silica capillary at 22.5 kV using a pH 9.8 phosphate-borate buffer. Buffer pH is the most critical parameter controlling resolution and separation time. A simple theoretical treatment greatly simplifies the pH optimization procedure. The effects on the separation of buffer concentration, applied voltage, and sample quantity injected were studied. Good calibration data were obtained for phenol concentrations up to 50 mb/l. Limits of detection for all phenols were less than 1 ppm.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Experimental evidence for membrane-mediated protein-protein interaction

Membrane proteins diffuse within the membrane, form oligomers and supramolecular assemblies. Using high-speed atomic force microscopy, we present direct experimental measure of an in-membrane-plane interaction potential between membrane proteins. In purple membranes, ATP-synthase c-rings formed dimers that temporarily dissociated. C-ring dimers revealed subdiffusive motion, while dissociated monomers diffused freely. C-rings center-to-center distance probability distribution allowed the calculation and modeling of an in-membrane-plane energy landscape that presented repulsion at 80 Å, most stable dimer association at 103 Å (−3.5 k_BT strength), and dissociation at 125 Å (−1 k_BT strength). This first experimental data of nonlabeled membrane protein diffusion and the corresponding in-membrane-plane interaction energy landscape characterized membrane protein interaction with an attractive range of several k_BT that reaches to a radius of ∼50 Å within the membrane plane.     

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks

Background

During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system.

Results

A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount.

Conclusions

The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization.

     

Mono-N-acyl-2,6-diaminopimelic acid derivatives: Analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60 mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative—N-fumaryl, Me-ester-DAP—was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.     

Study of the mode of action of endopolygalacturonase from Fusarium moniliforme

One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae. Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan. The dimer was not a substrate for the enzyme. The enzyme was shown to follow Michaelis–Menten behaviour towards the other substrates tested. Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V_max was in the same range as with homogalacturonan. The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from −3 to +2. The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

Toward high-throughput monitoring of metallodrug-protein interaction using capillary electrophoresis in chemically modified capillaries

The performance of capillary electrophoresis (CE) operating with a sulfonated capillary for the separation of protein adducts of anticancer ruthenium(III)-based drugs was evaluated. The coated capillary was shown to yield improved resolution of albumin- and transferrin-bound species of ruthenium compared with that attained with the bare fused-silica capillary. The coating also showed an increased reproducibility of migration times and peak areas and allowed reasonably high efficiency separation of analytes (up to 1300 theoretical plates per meter), which display high affinity toward a fused-silica surface. In addition, due to rather high electroosmotic flow (EOF, > 45 × 10⁻⁵ cm² V⁻¹ s⁻¹) in the coated capillary, it enabled fast counter-EOF monitoring of albumin and transferrin adducts. This benefit, together with requiring only a short flush with the background electrolyte to have migration times reproducible (at < 1.5% relative standard deviation), makes this wall-modified capillary holding promise for CE examination of fast reactions such as those accompanying protein-drug interactions and biotransformations associated with drug delivery via protein binding.     

Capillary electrophoresis of chitooligosaccharides in acidic solution: Simple determination using a quaternary-ammonium-modified column and indirect photometric detection with Crystal Violet

Five chitosan oligosaccharides were separated in acidic aqueous solution by capillary electrophoresis (CE) with indirect photometric detection using a positively coated capillary. Electrophoretic mobility of the chitooligosaccharides (COSs) depended on the number of monomer units in acidic aqueous solution, similar to other polyelectrolyte oligomers. The separation was developed in nitric acid aqueous solution at pH 3.0 with 1 mM Crystal Violet, using a capillary positively coated with N-trimethoxypropyl-N,N,N-trimethylammonium chloride. The limit of the detection for chitooligosaccharides with two to six saccharide chains was less than 5 μM. CE determination of an enzymatically hydrolyzed COS agreed with results from HPLC.     

Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Acid Tolerance Response Induced by Organic Acids at 20°C: Optimization and Modeling

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20°C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t_s), the log-linear decline (k_max), and the magnitude of a critical component (C). The magnitude of k_max gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.     

A rapid assay for the binding of actinomycin to DNA.

The theory of countercurrent distribution (CCD) was reviewed and extended. The separation function for the fundamental distribution of CCD was presented in the form n = t²(αk₁+β)²/αk₁(β?1)² where n is the number of transfers, t the abscissa of the standard normal distribution, α = v_m/v₈ the phase ratio, β = k₁/k₂≥ 1 the separation factor, and k₁ the partition coefficient of the more radidly moving component; n was found to be minimal on the condition αk₁ = β. The separation function for the single withdrawal of CCD was obtained in the form N = u + 1 = t²{(αk₁ + 1)^1/2 + [β(αk₁ + β)]^1/2}²/(β ? 1)²+ 1, where N is the number of partition units. From this equation it appears that N is minimal when αk₁ = 0. Compared with the former separation functions presented in the literature, these separation functions have the advantage of giving directly the relationships among the phase ratio, the absolute partition coefficient, the separation factor, the resolution degree, and the number of transfers or partition units required. In addition, the dependencies of the elution volumes and the widths of the elution curves on α, β, and the partition coefficients were considered mathematically by means of differential calculus. The elution volumes were found to have minima at certain αk₁ values. The standard deviations, on the contrary, did not have minima in respect to αk₁. The theory presented can be used for selecting proper operating conditions while separating chemical compounds.     

Direct and fast capillary zone electrophoretic method for the determination of Gleevec and its main metabolite in human urine

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 microm I.D.x60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of -25 kV, a capillary temperature of 25 degrees C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4-30.0 mg l(-1) concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l(-1) for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.     

Influence of an initial addition of lactic acid on growth, acid production and their coupling for batch cultures of Lactobacillus helveticus

Growth and production coupling for L. helveticus growing at constant pH?on a medium largely supplemented with yeast extract and peptons has been examined as a function of initial lactic acid addition: these conditions allowed to avoid side effects of cultivation pH?and nitrogen source deficiency on coupling. Growth of L. helveticus strain milano was reduced by half for a free lactic acid concentration of 0.34?g.l^?1; both growth and production were fully inhibited for a critical end-product concentration of 83?g.l^?1, despite the fact that lactose was still available. The extent of autolysis 8?h after growth ceased was practically independent of initial lactic acid concentration: the same conclusion held for the part played by a growth-associated mechanism in total acid production. During the slowing down phase of growth, production was non-growth-associated: production rate remained close to its maximal value, while growth rate declined.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

Simultaneous determination of l- and d-lactic acid in plasma by capillary electrophoresis

A novel method for simultaneous determination of d- and l-lactic acids in plasma was presented by capillary electrophoresis with photodiode array detection at 195nm. The separation was performed in an uncoated fused-silica capillary. The parameters influencing the resolution and the migration time of lactic acids were optimized. When 150mM phosphate-Tris buffer (pH 7.0) consisting of 220mM 2-hydroxypropyl-beta-cyclodextrin and 0.2mM tetradecyltrimethylammonium bromide was utilized as the running buffer, highly effective chiral separation of d- and l-lactic acids was achieved at about 42min at an effective voltage of -25kV. The resolution of lactic acid enantiomers was >/=1.25. The limits of detection of d- and l-lactic acids in standard solution without any pretreatment were 80 and 50muM (S/N=3), respectively. Sample pretreatment was preceded by protein-removal procedure with acetonitrile. With a pre-concentration procedure by 10 times, the limits of detection of d- and l-lactic acids were 20 and 15muM (S/N=10), respectively. The satisfactory analytical performance of the proposed method was validated.     

Multiple liquid-liquid partition: II. Theory of Martin-Synge distribution (MSD)

The theory of Martin-Synge distribution (MSD) was refined, with special attention being focused upon the derivation of the separation functions. The separation function for the fundamental distribution of MSD was obtained in the form v = t²(αk₁ + 1)(αk₁ + β)[(αk₁ + 1)^1/2 + (αk₁ + β)^1/2]²/αk₁(β ? 1)², where ν is the number of aliquots v_m driven through the apparatus, t the abscissa of the standard normal distribution, α = v_m/v₈ the phase ratio, β = k₁/k₂≥ 1 the separation factor, and k₁ the partition coefficient of the more rapidly moving component; ν was shown to have minima at given αk₁ values. The separation function of the single withdrawal of MSD was presented in the form N = u + 1 = t²(2αk₁ + β + 1)²/(β ? 1)²+ 1, where N is the number of partition units; N is minimal when αk₁ = 0. The elution volumes and standard deviations of the two compounds to be separated were mathematically analyzed in a manner similar to that previously presented when dealing with the theory of counter-current distribution (CCD). As in CCD, the elution volumes in MSD were found to have minima at given αk₁ values. However, the standard deviations of the elution curves also have minima in respect to αk₁ in MSD, which is a different situation as compared to CCD. The selection of optimal operating conditions was found to be more critical in MSD than in CCD.     

Laccase-catalyzed oxidative polymerization of aniline dimer (N-phenyl-1,4-phenylenediamine) in aqueous micellar solution of sodium dodecylbenzenesulfonate

We have studied oxidative polymerization of aniline dimer (N-phenyl-1,4-phenylenediamine) catalyzed by high-redox potential laccase isolated from the fungi Trametes hirsuta (Wulfen) Pilát CF-28. Enzymatic aniline dimer polymerization was performed in aqueous micellar solution of sodium dodecylbenzenesulfonate, the atmospheric oxygen serving as an oxidizer. The resultant dispersion was stable for at least 6 months. The products synthesized were characterized using Fourier transform infrared and UV–vis spectroscopies. MALDI TOF analysis has shown that aniline dimers polymerize to mainly form aniline oligomers with the m/z ratio up to 2180, which corresponds to a polymerization degree of 24 (in terms of aniline subunits). Enzymatically formed aniline oligomers consist for the most part of para-directed units in the form of emeraldine salt. The end product structure depends on the reaction medium pH. Transmission electron microscopy has revealed granular nanoparticles of the reaction product.     

Microemulsion and micellar electrokinetic chromatography of steroids

A mixture of ten steroids was separated by microemulsion and micellar (SDS and glycodeoxyholate) electrokinetic chromatography systems. Separations were done on a 50 cm (to the detector) × 50 μm I.D. fused-silica capillary. Complete separation of all the test compounds in the micellar mode was obtained with glycodeoxycholate (50 mM) in 25 mM borate buffer, pH 6.5, as the micelle-forming agent. The best results, however, were obtained using microemulsion electrokinetic chromatography in which higher aliphatic alcohols were used as the microemulsion-forming modifiers. The system consisted of n-hexanol (0.81%), SDS (3.31%) and n-butanol (6.61%) in 20 mM phosphate buffer, pH 10.0 (89.28%, w/w). In the microemulsion mode, linear calibration for steroid standards was obtained in the concentration range 3 × 10⁻⁴ − 3 × 10⁻⁵ mol l⁻¹ with a detection limit of 1 pmol. The method was validated and applied to an 11β-hydroxysteroid dehydrogenase assay in tissues.     

Simultaneous determination of key bioactive components in Hedyotis diffusa by capillary electrophoresis

A capillary zone electrophoresis (CZE) method based on systematic one-variable-at-a time approach was developed for the analysis of four important bioactive components (geniposidic acid, ursolic acid, quercetin and p-coumaric acid) in the extract of Hedyotis diffusa (HD). Separations were carried out in a fused-silica capillary tube with peak detection at 214 nm. Good separation was achieved using a 20 mM borate buffer containing 5% acetonitrile as organic modifier and pH adjusted to 10.0. Operating voltage was 15 kV and temperature was maintained at 25 degrees C while hydrodynamic injection was 5s. A good linearity, with correlation coefficients in the ranges of 0.997-0.999 was obtained in the calibration curves of each standard. Relative standard deviation (R.S.D.) of migration time was between 0.32 and 0.70% and deviation of corrected peak area was between 8.84 and 11.99%. These results indicate that this method could be used for rapid and simultaneous analysis of the bioactive components in HD and other herbal products.