Separation of D-amino acid-containing peptide phenylseptin using 3,3′-phenyl-1,1′-binaphthyl-18-crown-6-ether columns

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3′-phenyl-1,1′-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (−), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (−) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH₃⁺ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.     

Simple and sensitive liquid chromatography-tandem mass spectrometry method for determination of the S(+)- and R(-)-enantiomers of baclofen in human plasma and cerebrospinal fluid

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine the enantiomers of the muscle relaxant baclofen in human plasma and cerebrospinal fluid (CSF) has been developed. A commercially available ultrafiltration membrane is used to prepare the sample. A chiral CROWNPAK CR(+) stationary phase column is then used to perform complete resolution of the S(+)- and R(-)-enantiomers of baclofen. This method was used to analyze human plasma and CSF spiked with baclofen, and the calibration curves for both biologic samples were linear over a concentration range of 0.15-150 ng enantiomer/ml. The lower limit of quantification was 0.15 ng enantiomer/ml in both fluids. Finally, the method was tested with an artificial CSF as an alternative to authentic human CSF. The results showed that no matrix effects and no interfering peaks were observed using this artificial CSF.     

The separation of D/L amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate

A method for determining the optical purity of amino acids using HPLC and precolumn derivatization is described. (+)-1-(1-Naphthyl)ethyl isocyanate reacts with racemic amino acids, in high yield, to form naphthylethyl carbamoyl derivatives. The resulting diastereoisomeric pairs were separated on reversed-phase C18 columns and detected fluorometrically. Excitation maxima for naphthylethyl carbamoyl aspartic acid were 235 and 297 nm. The emission maximum was at 333 nm. Using a filter fluorometer with a zinc or cadmium lamp, less than 1 pmol of a D amino acid can be measured in the presence of 1000-fold excess of the L isomer. The column can also be monitored at lower sensitivity, using an ultraviolet detector operating at or near the absorption maximum of 222 nm. Chromatographic data are presented on the resolution of 17 amino acid pairs.     

Amino acids analysis using a monolithic silica column after derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)

A fast HPLC method using a monolithic silica column was developed for the measurement of amino acids. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a monolithic silica column (MonoClad C18-HS, 250 mm × 3 mm I.D.). The separation of 19 NBD-amino acids was achieved within 18 min, which was only one-fifth of the time taken by the methods using a conventional particle-packed column, with the gradient elution of a mobile phase at the flow rate of 1.4 mL/min. The sensitivity was good with a limit of detection for the individual amino acids ranging from 2.94 to 53.4 fmol. The calibration curves for all the amino acids were found to be linear in the range of 200 fmol to 20 pmol with correlation coefficients of 0.997 or better. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Bioanalytical method for the simultaneous determination of d- and l-serine in human plasma by LC/MS/MS

d-Serine is an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors. Plasma concentrations of d-serine and the ratio of d-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the d- and l-isomers of serine in human plasma. Since d- and l-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. d- and l-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. d-Serine (m/z 106.1 > 60.0), l-serine (m/z 106.1 > 60.1) and dl-serine-d₃ (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of d- and l-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01–10 μg/mL (d-serine) and 0.1–100 μg/mL (l-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of d- and l-serine in human plasma were 0.124–0.199 and 7.97–13.1 μg/mL, respectively.     

Monolithic column-based reversed-phase liquid chromatography separation for amino acid assay in microdialysates and cerebral spinal fluid

The development of a HPLC method using a monolithic C18 column is described using fluorescence detection for the assay of 21 amino acids and related substances with derivatisation using ortho-phthaldialdehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA). The method employs a tertiary gradient and has a run time of 24 min. Linearity (r2) for each amino acid was found to be greater than 0.99 up to a 10 microM concentration; reproducibility across all analyses (relative standard deviation (R.S.D.)) was between 0.97 and 6.7% and limit of detection (LOD) between 30 and 300 fmol on column. This method has been applied to the analysis of amino acids in both spinal microdialysis and cerebral spinal fluid samples.     

Quantitative analysis of 17 amino acids in the connective tissue of patients with pelvic organ prolapse using capillary electrophoresis-tandem mass spectrometry

The simultaneous determination of 17 amino acids in connective tissue using capillary electrophoresis is described in this study. Separation was carried out on a fused silica capillary column (80 cm x 50 mm i.d.) with 1M formic acid as the running electrolyte. The detection was conducted on a mass spectrometer by selective reaction monitoring (SRM) mode via an electrospray ionization source. Tissue samples were prepared by reduction and acid hydrolysis to extract amino acids; over 84.3% recovery was seen for all compounds. The method allowed for sensitive, reproducible, and reliable quantification, and all 17 amino acids were separated using this method. Good linearity over the investigated concentration ranges was observed, with values of R higher than 0.993 for all the analytes. Precision and accuracy examined at three concentration levels ranged from 0.2% to 19.5% and 84.1% to 120.0%, respectively. Matrix effects were also tested and ranged from -9.1% to 15.4%. The validated method was applied to the quantitation of 17 amino acids in pelvic connective tissue of pelvic organ prolapsed patients. Methionine, glutamine, and histidine were significantly higher in the experimental patients compared to the controls. This suggests that changes in the amino acid concentrations within the connective tissue could be a factor in the genesis of pelvic organ prolapse. Therefore, this method is potentially applicable for amino acid analysis in tissue, providing a more complete understanding of pelvic organ prolapse.     

Altered intestinal transport of amino acids in cirrhotic rats: the effect of insulin-like growth factor-I

The intestine is an important target organ for insulin-like growth factor-I (IGF-I), an anabolic hormone synthesized in the liver upon growth hormone (GH) stimulation. Levels of IGF-I are reduced in cirrhosis, and altered GH/IGF-I axis may contribute to malnutrition in cirrhotic patients. Our aim was to study Na(+)-dependent jejunal transport of amino acids (L-leucine, L-proline, L-glutamic acid, and L-cysteine) in cirrhotic rats and to analyze the effect of IGF-I on this function. IGF-I or saline was administered for 2 wk to rats with CCl(4)-induced cirrhosis and saline was administered to healthy control rats. Transport of amino acids was assessed in brush-border membrane vesicles (BBMV) using (14)C- or (35)S-labeled amino acids, and the kinetic constants V(max) and K(t) were determined. Na(+)-independent uptake of L-leucine, L-proline, L-glutamic acid, and L-cysteine by BBMV was similar in all groups. Na(+)-dependent uptake of all four amino acids was significantly diminished in cirrhotic rats compared with both controls and IGF-I-treated cirrhotic rats. The latter two groups exhibited similar V(max) and K(t), whereas untreated cirrhotic rats had reduced V(max) and increased K(t) compared with normal controls and IGF-I-treated cirrhotic animals. In conclusion, the transport of all four tested amino acids by BBMV is impaired in cirrhotic rats, and low doses of IGF-I can correct this defect.     

Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition

The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.     

Enantiomer separation of amino acids in immunoaffinity micro LC-MS

Chiral immunoaffinity microbore columns were directly interfaced with MS detection, and the effect of column length and temperature on the enantiomer separation of a number of underivatized aromatic and aliphatic amino acids was investigated utilizing an antibody chiral stationary phase that had been prepared by immobilizing a monoclonal anti-D-amino acid antibody onto silica. The stronger affinity of the antibody towards aromatic and bulky amino acids allowed separation of such analytes in a 0.75 x 150 mm column, while an increase in column length enabled separation of more weakly bound compounds. The strength of interaction between chiral selector and analytes could be modulated conveniently by lowering the temperature. For the first time, simultaneous enantiomer separation of mixtures of amino acids was achieved on antibody-based chiral stationary phases using extracted ion chromatograms.     

Are proton symports in yeast directly linked to H(+)-ATPase acidification?

Transport of amino acids in Saccharomyces cerevisiae is an H(+)-driven secondary active transport. Inhibitors of the plasma membrane H(+)-ATPase, particularly heavy water, diethylstilbestrol and suloctidil, were shown to affect the H(+)-extruding ATPase activity as well as the ATP-hydrolyzing activity, to a similar degree as they inhibited the transport of amino acids. The inhibitors had virtually no effect on the membrane electric potential or on the delta pH which constitute the thermodynamically relevant source of energy for these transports. Transport of acidic amino acids was affected much more than that of the neutral and especially of the basic ones. The effects were greater with higher amino acid concentrations. All this is taken as evidence that the amino acid carriers respond kinetically to the presence of protons directly at the membrane site where they are extruded by the H(+)-ATPase, rather than to the overall protonmotive force.     

Chiral aminophosphonate eluent for enantiomeric analysis of amino acids

An aqueous solution of the (+)-monoethyl ester of N-(l′-hydroxymethyl-)propyl-α-aminobenzylphosphonic acid has been proposed as a suitable chiral eluent for enantiomeric analysis of amino acids by ligand-exchange chromatography. Asymmetric synthesis of the chiral selector using (−)-(R)-2-aminobutan-1-ol as a starting reactant is described. The dependence of the parameters of separation of valine enantiomers on concentration of the complexing ion, pH, and temperature has been investigated. It is shown that the order in which enantiomers are eluted from a column depends on the concentration of the complexing ion and pH. © 1996 Wiley-Liss, Inc.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Effects of pH strategy on endo- and exo-metabolome profiles and sodium potassium hydrogen ports of beta-lactamase-producing Bacillus licheniformis

The effects of pH strategy on endo- and exo-metabolome profiling of beta-lactamase-producing Bacillus licheniformis were investigated at controlled-pH (pH(C) = 6.5, 6.75, 7.0, 7.25, 7.5) and uncontrolled-pH (pH(UC) = 7.5) values using a glucose-based defined medium. The cell concentration profiles were not affected by the pH considerably within the investigated range. The highest enzyme activities were obtained as A = 54 U cm(-)(3) at pH(C) = 6.75 among the controlled-pH operations and as A = 57 U cm(-3) at the uncontrolled-pH pH(UC) = 7.5. At all conditions, oxygen transfer resistances were more effective, whereas the limitation increased in the beta-lactamase production phase. Total intracellular amino acid concentrations ranged between 0.142 and 6.766 kg m(-3) (0.0058-0.277 g g(cell)(-1)), and their concentrations in terms of kg m(-3) were, at most, 580-fold higher than the extracellular concentrations. Methionine/cysteine concentrations were generally higher than the other intracellular amino acids, whereas asparagine concentration was the highest in the fermentation broth. From Na(+), K(+), and H(+) ion profiles, Na(+)-K(+) antiport and Na(+)-H(+) symport were found to be present within the system, and a correlation was found between organic acid transport and Na(+)-H(+) symport. Intracellular organic acid concentrations in terms of kg m(-3) were, at most, 20-fold higher than that of the extracellular, and with the increase in pH, extracellular acetic acid concentration increased and lactic acid concentration decreased. Average permeability coefficient values of organic acids were found to be in the range from 4.10 x 10(-7) to 4.32 x 10(-6) cm s(-1) for the growth phase (0 < t < 6 h) and decreased at least 3-fold in the beta-lactamase production phase (8 < t < 15 h), indicating the considerable structural change of the lipid membrane during the fermentation.     

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.