The role of pi-acidic and pi-basic chiral stationary phases in the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids

The application of 3,5-dimethylphenyl-carbamoylated-beta-cyclodextrin (Cyclobond I 2000 DMP) and 2,6-dinitro-4-trifluoromethylphenyl-ether-beta-cyclodextrin-based (Cyclobond DNP) chiral stationary phases for the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids is reported. The investigated amino acids were saturated or unsaturated alicyclic beta-3-homo-amino acids and bicyclic beta-amino acids. Prior to chromatographic analyses, all amino acids were transformed to N-3,5-dinitrobenzoyl- or N-3,5-dimethylbenzoyl form to ensure a pi-acidic or pi-basic function and to enhance the pi-acidic-pi-basic interactions between analytes and chiral selectors. Chromatographic results are given as retention, separation and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The sequence of elution of the enantiomers was determined in some cases.     

Classes and narrowing selectivity of olfactory receptor neurons of Xenopus laevis tadpoles

In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.     

Solution structure of a novel calcium binding protein, MTH1880, from Methanobacterium thermoautotrophicum

MTH1880 is a hypothetical protein from Methanobacterium thermoautotrophicum, a target organism of structural genomics. The solution structure determined by NMR spectroscopy demonstrates a typical alpha + beta-fold found in many proteins with different functions. The molecular surface of the protein reveals a small, highly acidic pocket comprising loop B (Asp36, Asp37, Asp38), the end of beta2 (Glu39), and loop D (Ser57, Ser58, Ser61), indicating that the protein would have a possible cation binding site. The NMR resonances of several amino acids within the acidic binding pocket in MTH1880, shifted upon addition of calcium ion. This calcium binding motif and overall topology of MTH1880 differ from those of other calcium binding proteins. MTH1880 did not show a calcium-induced conformational change typical of calcium sensor proteins. Therefore, we propose that the MTH1880 protein contains a novel motif for calcium-specific binding, and may function as a calcium buffering protein.     

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues

Summary. In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates. Received September 22, 1999 Accepted July 5, 2000     

Electronic Properties of the Amino Acid Side Chains Contribute to the Structural Preferences in Protein Folding

Abstract

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: α-helix, β-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (eσ), and a composite of these two scales (ε). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for α-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

Quantitative gas chromatographic analysis of amino acids on a short glass capillary column

A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run.Hydrolyzed peptides have been analyzed. The analyses were performed with a precision varying between 1 and 6% (mean relative standard deviation) depending on the number of amino acid residues in the peptide. The amount taken for analysis was 20–300 μg. The results agree with the known sequences of the peptides and with the analyses by ion-exchange chromatography except for cysteine. This amino acid can be analyzed after modification as S-methylated cysteine.     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Exogenous calcium affects nitrogen metabolism in root-zone hypoxia-stressed muskmelon roots and enhances short-term hypoxia tolerance

We investigated the effects of short-term root-zone hypoxic stress and exogenous calcium application or deficiency in an anoxic nutrient solution on nitrogen metabolism in the roots of the muskmelon cultivar Xiyu No. 1. Seedlings grown in the nutrient solution under hypoxic stress for 6 d displayed significantly reduced plant growth and soluble protein concentrations. However, NO₃⁻ uptake rate and activities of nitrate reductase and glutamate synthase were significantly increased. We also found higher amounts of nitrate, ammonium, amino acids, heat-stable proteins, polyamines, H₂O₂, as well as higher polyamine oxidase activity in the roots. In comparison to the reactions seen under hypoxic stress, exogenous calcium application led to a marked increase in plant weights, photosynthesis parameters, NO₃⁻ uptake rate and contents of nitrate, ammonium, amino acids (e.g., glutamic acid, proline, glycine, cystine, γ-aminobutyric acid), soluble and heat-stable proteins, free spermine, and insoluble bound polyamines. Meanwhile, exogenous calcium application resulted in significantly increased activities for nitrate reductase, glutamine synthetase, and glutamate synthase but decreased activities for diamine and polyamine oxidase, as well as lower H₂O₂ content in roots during exposure to hypoxia. However, calcium deficiency in the nutrient solution decreased plant weight, photosynthesis parameters, NO₃⁻ reduction, amino acids (e.g., alanine, aspartic acid, glutamic acid, γ-aminobutyric acid), protein, all polyamines except for free putrescine, and the activities of glutamate synthase and glutamine synthetase. Additionally, there was an increase in the NO₃⁻ uptake rate, polyamine oxidase activity and H₂O₂ contents under hypoxia-Ca. Simultaneously, exogenous calcium had little effect on nitrate absorption and transformation, photosynthetic parameters, and plant growth under normoxic conditions. These results suggest that calcium confers short-term hypoxia tolerance in muskmelon, most likely by promoting nitrate uptake and accelerating its transformation into amino acids, heat-stable proteins or polyamines, as well as by decreasing polyamine degradation in muskmelon seedlings.     

New approach for amino acid profiling in human plasma by selective fluorescence derivatization

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.     

猪血粉分离氨基酸的研究

本文报道了从猪血粉中提取分离七种氨基酸(L—Phe、L—Tyr、L—His·HCl、L—lys·HAC、L—Arg·HCl、L—leu、L—Val)的小试工艺。通过合成一系列专用树脂(AAS—1、AAS—2、D083)对现行工艺脱酸、脱色及层析分离等过程作了较大改进,使猪血粉分离氨基酸的生产工艺全部树脂化,并使氨基酸收率大幅度提高。经周期性实验,每批投料130g猪血粉,七种氨基酸总回收率平均达34.9%。(占血粉量)。经自检产品质量基本符合注射用结晶氨基酸原料标准。     

Analytical Procedures for the Sequential Extraction of 14C-Labeled Constituents from Leaves, Bark and Wood of Cottonwood Plants

A procedure for the extraction, separation, and measurement of photosynthetically fixed ¹⁴C in up to 8 chemical fractions (CHCl), amino acids, organic acids, sugars, protein, starch, hemicellulose, and residue) from small samples (1 to 100 mg) of cottonwood (Populus deltoides Bartr.) leaf material is described. The different chemical fractions are extracted in a sequence of chemical, ion exchange, and enzymatic steps. The ¹⁴C-activity in these major fractions is then determined with liquid scintillation spectrometry. These major fractions (e.g., sugars, amino acids, organic acids) can be further separated into their individual chemical components by standard thin-layer or gas- chromatographic methods for quantitative analysis if specific activities are desired. The major advantage of the procedure is that many chemical fractions can be sequentially separated with good reproducibility from a small amount of plant material without transfer of the material from the original homogenizer or centrifuge tube.     

Isolation and quantitation of isotopically labeled amino acids from biological samples

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phtaaldialdehyde (OPA), which is known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Becuase the OPA probe cannot be removed after isolation of the derivative, we used 9-fluorenylmethylchloroformate (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken byb gas-phase acid hydrolysis (103% recovery after 5 h at 150°C: S.D = 3.5%, n = 14). Run time (injection to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 μl could be injcted, representing approximately 200 μl of deproteinized plasma. The methods were linear up to injection of 0.5 μmol of all amino acids (OPA: r²=0.995−0.999; FMOC: r²=0.992−0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50–500 nmol), was less than 3% above 100 mmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.     

Postcolumn fluorometric detection system for liquid chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-L-cysteine reagent

A high-performance liquid chromatographic system for the determination of amino acid and imino acid was developed using a two-step reaction with sodium hypochlorite and o-phthalaldehyde/N-acetyl-L-cysteine (OPTA/AcCys) reagent. This reagent improved the sensitivity in the analysis of proline presumably because the fluorophore is more stable to hypochlorite which has been used for the oxidative cleavage of the imino linkage. The use of OPTA/AcCys facilitated the detection of imino acids at the same concentration level as that of amino acids. The detection limit for all the amino and imino acids was a few picomoles. This detection system, together with cation-exchange chromatographic separation, was applied to the determination of amino and imino acids in biological samples.     

Isolation of individual amino acids from various microbiological sources using reversed-phase high-performance liquid chromatography

A new method for the preparative isolation of individual amino acids on a milligram scale based on reversed-phase high-performance liquid chromatography (RP-HPLC) after pre-column derivatization with carbobenzoxychloride (ZCl) has been developed. The chromatographic procedure was tested by the investigation of jack bean urease hydrolysate. The method has been applied to the preparative separation of Z-amino acids (from 10 up to 16) obtained from protein hydrolysates of various sources (green microalgae, blue-green algae, halophilic and methylotrophic microorganisms) and was proved to be reliable by the separation of deuterated amino acids (enrichment 97–99%) from Methylobacillus flagellatum (due to the bioconversion of CD₃OD and D₂O). Independent of the biological source of the protein, the amino acids were isolated with high recovery (from 68% up to 89%) and chromatographic purity (from 96% up to 99%). The method was also applied for the isolation of phenylalanine and leucine excreted by amino-acid overproducing microorganisms.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.     

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.     

Electronic properties of the amino acid side chains contribute to the structural preferences in protein folding.

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: alpha-helix, beta-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (esigma), and a composite of these two scales (epsilon). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for alpha-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

Reverse-phase liquid chromatographic analysis of amino acids after reaction with o-phthalaldehyde

Precolumn formation of o-phthalaldehyde (OPA) derivatives of amino acids, followed by reverse-phase separation and fluorescent detection, provides rapid, sensitive amino acid analysis. Eighteen OPA-amino acid derivatives are resolved on a Micropack MCH 5 column and can be measured at picomole levels. Ease of derivative preparation and separation makes liquid chromatographic analysis of OPA-amino acids a convenient and improved technique for measuring or confirming the presence of low levels of amino acids in aqueous solutions. Use of the method was demonstrated by measuring low concentrations of amino acids released from zooplankters stressed by contaminants.