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1.
Electromembrane extraction (EME) proved to be a simple and rapid pretreatment method for analysis of amino acids and related compounds in body fluid samples. Body fluids were acidified to the final concentration of 2.5 M acetic acid and served as donor solutions. Amino acids, present as cations in the donor solutions, migrated through a supported liquid membrane (SLM) composed of 1-ethyl-2-nitrobenzene/bis-(2-ethylhexyl)phosphonic acid (85:15 (v/v)) into the lumen of a porous polypropylene hollow fiber (HF) on application of electric field. The HF was filled with 2.5 M acetic acid serving as the acceptor solution. Matrix components in body fluids were efficiently retained on the SLM and did not interfere with subsequent analysis. Capillary electrophoresis with capacitively coupled contactless conductivity detection was used for determination of 17 underivatized amino acids in background electrolyte solution consisting of 2.5 M acetic acid. Parameters of EME, such as composition of SLM, pH and composition of donor and acceptor solution, agitation speed, extraction voltage, and extraction time were studied in detail. At optimized conditions, repeatability of migration times and peak areas of 17 amino acids was better than 0.3% and 13%, respectively, calibration curves were linear in a range of two orders of magnitude (r(2)=0.9968-0.9993) and limits of detection ranged from 0.15 to 10 μM. Endogenous concentrations of 12 amino acids were determined in EME treated human serum, plasma, and whole blood. The method was also suitable for simple and rapid pretreatment and determination of elevated concentrations of selected amino acids, which are markers of severe inborn metabolic disorders. 相似文献
2.
Pyridoxal 5'-phosphate-dependent aminotransferases reversibly catalyzes the transamination reaction in which the alpha-amino group of amino acid 1 is transferred to the 2-oxo acid of amino acid 2 (usually 2-oxoglutarate) to produce the 2-oxo acid of amino acid 1 and amino acid 2 (glutamate). An aminotransferase must thus be able to recognize and bind two kinds of amino acids (amino acids 1 and 2), the side chains of which are different in shape and properties, from among many other small molecules. The dual substrate recognition mechanism has been discovered based on three-dimensional structures of aromatic amino acids, histidinol phosphate, glutamine:phenylpyruvate, acetylornithine, and branched-chain amino acid aminotransferases. There are two representative strategies for dual substrate recognition. An aromatic amino acid aminotransferase prepares charged and neutral pockets for acidic and aromatic side chains, respectively, at the same place by a large-scale rearrangement of the hydrogen-bond network caused by the induced fit. In a branched-chain aminotransferase, the same hydrophobic cavity implanted with hydrophilic sites accommodates both hydrophobic and acidic side chains without side-chain rearrangements of the active-site residues, which is reminiscent of the lock and key mechanism. Dual substrate recognition in other aminotransferases is attained by combining the two representative methods. 相似文献
3.
Following polyacrylamide gel electrophoresis in an acetic/formic acid buffer, pH 2.0, in fabric reinforced gels, amino acids and oligopeptides could successfully be fixed in the gel by freeze-drying. Lyophilization of the fabric reinforced polyacrylamide gel after electrophoresis resulted in a dry film which absorbed ninhydrin solution quickly and uniformly, thus improving the detection limit for amino acids and oligopeptides with molecular weights ranging from 189-1045. Most amino acids were detected with a sensitivity of 0.1-0.25 microgram and for oligopeptides the detection limit was found to be 0.5-5 microgram. 相似文献
4.
Róbert Berkecz Anna R. M. Hyyryläinen Ferenc Fülöp Antal Péter Tamás Janáky Pirjo Vainiotalo Jaana M. H. Pakarinen 《Journal of mass spectrometry : JMS》2010,45(11):1312-1319
Chiral discrimination of seven enantiomeric pairs of β‐3‐homo‐amino acids was studied by using the kinetic method and trimeric metal‐bound complexes, with natural and unnatural α‐amino acids as chiral reference compounds and divalent metal ions (Cu2+ and Ni2+) as the center ions. The β‐3‐homo‐amino acids were selected for this study because, first of all, chiral discrimination of β‐amino acids has not been extensively studied by mass spectrometry. Moreover, these β‐3‐homo‐amino acids studied have different aromatic side chains. Thus, the emphasis was to study the effect of the side chain (electron density of the phenyl ring, as well as the difference between phenyl and benzyl side chains) for the chiral discrimination. The results showed that by the proper choice of a metal ion and a chiral reference compound, all seven enantiomeric pairs of β‐3‐homo‐amino acids could be differentiated. Moreover, it was noted that the β‐3‐homo‐amino acids with benzyl side chains provided higher enantioselectivity than the corresponding phenyl ones. However, increasing or decreasing the electron density of the aromatic ring by different substituents in both the phenyl and benzyl side chains had practically no role for chiral discrimination of β‐3‐homo‐amino acids studied. When copper was used as the central metal, the phenyl side chain containing reference molecules (S)‐2‐amino‐2‐phenylacetic acid (L ‐Phg) and (S)‐2‐amino‐2‐(4‐hydroxyphenyl)‐acetic acid (L ‐4′‐OHPhg) gave rise to an additional copper‐reduced dimeric fragment ion, [CuI(ref)(A)]+. The inclusion of this ion improved noticeably the enantioselectivity values obtained. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
5.
Dynamic protonation equilibria in water of one 4‐methylimidazole molecule as well as for pairs and groups consisting of 4‐methylimidazole, acetic acid and bridging water molecules are studied using Q‐HOP molecular dynamics simulation. We find a qualitatively different protonation behavior of 4‐methylimidazole compared to that of acetic acid. On one hand, deprotonated, neutral 4‐methylimidazole cannot as easily attract a freely diffusing extra proton from solution. Once the proton is bound, however, it remains tightly bound on a time scale of tens of nanoseconds. In a linear chain composed of acetic acid, a separating water molecule and 4‐methylimidazole, an excess proton is equally shared between 4‐methylimidazole and water. When a water molecule is linearly placed between two acetic acid molecules, the excess proton is always found on the central water. On the other hand, an excess proton in a 4‐methylimidazole‐water‐4‐methylimidazole chain is always localized on one of the two 4‐methylimidazoles. These findings are of interest to the discussion of proton transfer along chains of amino acids and water molecules in biomolecules. 相似文献
6.
Chiral separation of unmodified amino acids with non-aqueous capillary electrophoresis based on the ligand-exchange principle 总被引:5,自引:0,他引:5
A ligand exchange mechanism in non-aqueous capillary electrophoresis was employed for the separation of eight unmodified amino acids using chiral complexes of copper(II) with L-proline and L-isoleucine. The electrophoretic medium consisted of 25 mM ammonium acetate and 1 M acetic acid in methanol. We were able to completely separate the enantiomeric pairs of each of the investigated racemic amino acids. We also report the optimization of the separation parameters, such as pH*, composition of the complex, and concentration of the complexing agents. 相似文献
7.
Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts. 相似文献
8.
Determination of haloacetic acids by the combination of non-aqueous capillary electrophoresis and mass spectrometry 总被引:1,自引:0,他引:1
The applicability of capillary electrophoresis (CE) in combination with atmospheric pressure ionization mass spectrometry
(API-MS) is demonstrated for the determination of organic acids and in particular for haloacetic acids. CE-conditions, sheath
flow and MS-parameters were optimized with respect to the separation of the analytes and mass spectrometric sensitivity.
CE/MS turned out to be an attractive alternative for the determination of haloacetic acids to existing methods based on GC-ECD.
Employing CE/MS derivatization is not necessary which saves time and avoids possible sources of errors. In the present work
the sample pre-treatment is performed by liquid-liquid extraction using methyl tert.-butyl ether as the extraction solvent.
The organic phase is brought to dryness in a stream of nitrogen gas and the residue is dissolved in methanol and analyzed
by CE/MS using a mixture of 2-propanol/water 80 : 20 containing triethylamine as the sheath liquid in the interface. Best
results for the separation of all nine possible bromo- and chloroacetic acids together with two internal standards are obtained
with a carrier electrolyte consisting of ammonium acetate/acetic acid in methanol; to resolve the strongly acidic trihaloacetic
acids as well as the less acidic monohaloacetic acids, a careful optimization of the acetic acid content is necessary. The
method was applied to the determination of haloacetic acids in real water samples. With optimized CE and MS conditions detection
limits between 0.3 and 7.6 μg/L in the original water samples were achieved, employing a sample volume of 30 mL.
Received: 4 May 1999 / Revised: 9 June 1999 / Accepted: 12 June 1999 相似文献
9.
Many peptides and proteins, containing Nα-alkylamino acids (including proline) at the second position, are prone to intramolecular aminolysis (IA) with elimination of N-terminal dipeptide sequence as 2,5-diketopiperazines (DKP). We synthesized a series of short peptides, containing N-alkylamino acids at position 2, and studied their stability in the presence of acetic acid and amines. The presence of side chains in the second and the third amino acid residues and alkylation at Nα of the third amino acid residue slowed down IA. Nα-Alkyl residue in the first amino acid residue impeded IA only in peptides, containing three or more residues. Side chains of the first amino acids did not affect significantly the cleavage rates. Acetic acid promoted IA more strongly than aqueous ammonia, while tertiary amines were less effective. Peptides with methionine-S-oxide residues were more labile than the unoxidized analogs, suggesting intramolecular assistance of the S-oxide group in aminolysis. Surprisingly, intermediate compounds of the formula Boc–Met-MeXaa-Sar–NHR underwent rapid cleavage (endopeptolysis) upon attempted acidolytic deprotection. 相似文献
10.
Gao Q Xu W Xu Y Wu D Sun Y Deng F Shen W 《The journal of physical chemistry. B》2008,112(7):2261-2267
In order to disclose the dominant interfacial interaction between amino acids and ordered mesoporous materials, the adsorption behaviors of five amino acids on four mesoporous materials were investigated in aqueous solutions with adjustable amino acid concentration, ion strength, and pH. The selected amino acids were acidic amino acid glutamic acid (Glu), basic amino acid arginine (Arg), and neutral amino acids phenylalanine (Phe), leucine (Leu), and alanine (Ala), and the selected mesoporous materials were SBA-15, Al-SBA-15, CH3(10%)-SBA-15, and CH3(20%)-SBA-15. The adsorption capacities of Glu and Arg were strongly dependent on pH and surface charge of the mesoporous adsorbent. The adsorption of Phe showed pH insensitivity but depended on the surface organic functionalization of mesoporous adsorbent. On the basis of the theoretical analysis about the interaction between amino acid and adsorbent, such a remarkable difference was attributed to the different nature of the interaction between amino acid and adsorbent. Arg could be readily adsorbed on the surface of SBA-15, especially Al-SBA-15, under appropriate pH in which the electrostatic interaction was predominant. The driving force of Phe adsorption on mesoporous adsorbent mainly came from the hydrophobic interaction. Therefore, the adsorption capability of Arg decreased with increasing ion strength of solution, while the adsorption capability of Phe increased with the increasing degree of CH3 functionalization on SBA-15. For neutral amino acid Phe, Ala, and Leu, the adsorption capability increased with the increase of the length of their side chains, which was another evidence of hydrophobic effect. Thus, all the adsorption of amino acids on mesoporous silica materials can be decided by the combined influence of two fundamental interactions: electrostatic attraction and hydrophobic effect. 相似文献
11.
A simple and reliable method, for screening for point mutations in alpha- and beta-human globin chains, is reported here, utilizing capillary zone electrophoresis in isoelectric, acidic buffers. A solution of 50 mM iminodiacetic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme-free, denatured globin (alpha and beta) chains. Due to the low conductivity of such buffers, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. In presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to "micellar electrokinetic chromatography". At this low operative pH, however, charged mutants, involving substitutions of acidic amino acids (Glu and Asp) are not detected, since these residues are extensively protonated. Curiously, however, they are still separated in presence of detergent, due to the large variation in hydrophobicity involved in such mutations. Of the 19 mutants analyzed, all but one were resolved: Hb St Nazaire (beta 103 Phe-->Ile). This is due to the fact that the delta G (in kcal/mol) in the substitution Phe-->Ile is zero, thus no separation can possibly take place between two chains exhibiting the same hydrophobicity parameter. 相似文献
12.
Brian J. Smith 《Journal of computational chemistry》1999,20(4):428-442
Atomic radii have been derived for the common amino acid side chains using a solvent interaction potential (SIP) based on quantum mechanically derived charges. Solvation energies calculated using these parameters are compared with those obtained using other sets of radii and charges, and from alternative methods. The differences from the experimental solvation energies for the nonionizable residues are all less than 10 kJ mol−1. The largest error in the solvation energy occurs for acetic acid (−16.0 kJ mol−1). For the charged side chain systems the difference from experiment are all less than 10 kJ mol−1. SIP parameters for the aminoacetaldehyde derivatives of the common amino acids are presented. These are used in the calculation of the relative binding energies of six benzamidine inhibitors with trypsin. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 428–442, 1999 相似文献
13.
[reaction: see text] N-Methyl amino acid residues in peptides modify several pharmacologically useful parameters, but synthesis of alkylated peptides is hampered by unavailability of N-methylated monomers. The syntheses of four N-methyl amino acids with basic side chains are presented. The side chains of these basic amino acids needed to be specially protected or constructed. This completes the set of 20 common L-amino acid N-methyl derivatives prepared via 5-oxazolidinone intermediates by our group. 相似文献
14.
The applicability of capillary electrophoresis (CE) in combination with atmospheric pressure ionization mass spectrometry (API-MS) is demonstrated for the determination of organic acids and in particular for haloacetic acids. CE-conditions, sheath flow and MS-parameters were optimized with respect to the separation of the analytes and mass spectrometric sensitivity. CE/MS turned out to be an attractive alternative for the determination of haloacetic acids to existing methods based on GC-ECD. Employing CE/MS derivatization is not necessary which saves time and avoids possible sources of errors. In the present work the sample pre-treatment is performed by liquid-liquid extraction using methyl tert.-butyl ether as the extraction solvent. The organic phase is brought to dryness in a stream of nitrogen gas and the residue is dissolved in methanol and analyzed by CE/MS using a mixture of 2-propanol/water 80?:?20 containing triethylamine as the sheath liquid in the interface. Best results for the separation of all nine possible bromo- and chloroacetic acids together with two internal standards are obtained with a carrier electrolyte consisting of ammonium acetate/acetic acid in methanol; to resolve the strongly acidic trihaloacetic acids as well as the less acidic monohaloacetic acids, a careful optimization of the acetic acid content is necessary. The method was applied to the determination of haloacetic acids in real water samples. With optimized CE and MS conditions detection limits between 0.3 and 7.6 μg/L in the original water samples were achieved, employing a sample volume of 30 mL. 相似文献
15.
Two new solvent systems, n-hexane + propionic acid (26:5, v/v) and chloroform + acetone (29:3, v/v), for the rapid resolution and identification of an 18-component mixture of phenylthiohydantoin amino acids are reported. Using these systems certain difficult combinations of phenylthiohydantoin amino acids are resolved. Two more solvent systems, viz chloroform + acetic acid (27:3, v/v) and chloroform + methanol (30:4, v/v), are developed to resolve phenylthiodantoin derivatives of aspartic and glutamic acids. 相似文献
16.
Zhen Weng Xiuyuan Ni Dan Yang Jiao Wang Weikang Chen 《Journal of photochemistry and photobiology. A, Chemistry》2009,201(2-3):151-156
Polymerizations of vinyl acetate are photocatalyzed by TiO2 nanoparticles in presence of carboxylic acids including propionic acid, n-butyric acid and pivalic acid. Nuclear magnetic resonance (NMR) analysis using 13C-labeled n-butyric acid as the probing molecule demonstrates that the polymerization of vinyl acetate is initiated by alkyl radicals generated from photocatalytic decarboxylation of the carboxylic acid. A universal mechanism is established with extending the photo-Kolbe reaction from acetic acid to the carboxylic acids with longer chains. Kinetics studies find that n-butyric acid has higher initiation rate than acetic acid, indicating more efficient decarboxylation for butyric acid than acetic acid in their aqueous solutions. It is proved that carboxylates participate in the decarboxylation. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra are obtained with aqueous solutions of the carboxylic acids in contact with a layer of the TiO2 nanoparticles, and the observations are discussed with respect to the interaction between the TiO2 and carboxylic acids. 相似文献
17.
Summary Direct, simple and reliable means for characterization of the common amino acids, using mixtures of structural analogues of amino acids and combinations of low-voltage electrophoresis (1.04 mol dm–3 formic acid) and chromatography (tert-butanol/methanol/pyridine/formic acid/water, 33:43:9.6:0.4:20, v/v, methanol/pyridine/formic acid/water, 70:9.6:0.4:20, v/v, or tertpentanol/methylethylketone/pyridine/water, 37.5:37.5:5:20, v/v) on amorphous cellulose thin-layer are given. The efficiency of the procedures is evaluated in various examples.Abbreviations used: TLC = thin-layer chromatography, TLE = thin-layer electrophoresis, TLP = thin-layer plate; other abbreviations: see Table I and end of text.for structural analogues of amino acids and glucosamine, see Table I. 相似文献
18.
Takatsune Yoshida Takao Aoyagi Etsuo Kokufuta Teruo Okano 《Journal of polymer science. Part A, Polymer chemistry》2003,41(6):779-787
The synthesis and characterization of thermoresponsive hydrogels on the basis of N‐isopropylacrylamide (IPAAm) copolymers crosslinked with biodegradable poly(amino acids) are described. This hydrogel was prepared with two kinds of reactive IPAAm‐based copolymers containing poly(amino acids) as the side‐chain groups and activated ester groups. We introduced the graft chains by decarboxylation polymerization of amino acid N‐carboxyanhydrides initiated from lateral amino groups in the PIPAAm copolymer. The hydrogels easily crosslinked with degradable poly(amino acid) chains by only mixing the copolymer aqueous solutions. The gelling method in this study would provide some of the following innovative features: (1) no necessary removal of unreacted monomers and so forth, (2) simpler loading of drugs into the hydrogels (only mixing when gelling), and (3) easier insertion into the body. On the basis of the swelling ratio measurement of the hydrogel, large volume changes dependent on temperature changes were observed. Moreover, the enzymatic temperature‐dependent degradation was confirmed. The results suggested that these hydrogels could be used for an injectable or implantable matrix of temperature‐modulated drug release. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 779–787, 2003 相似文献
19.
The hydrogen-bonding abilities of a few amino acid side chains have been studied through aggregation of methylamine, methanol,
and acetic acid (as model molecules) with formo- and thioformo- hydroxamic acids using ab initio calculations. Forty six aggregates
representing all possible H-bond interactions between these amino acid side chain groups and two most stable keto and enol
tautomeric forms of both hydroxamic acids have been optimized. Although participation of conventional H-bond donors and acceptors
leads to significant stabilization energies, yet C–H···O, C–H···N, S–H···O, and S–H···N etc. unconventional H-bonds also contribute
to stabilize interactions in many aggregates. Strength of H-bonds of the molecules with formo- and thioformo- hydroxamic acid
studied follows the order acetic acid > methylamine > methanol. A comparative study of atomic charges and orbital interactions
employing NBO analysis has been carried out to explore the role of bond polarizations, charge transfer, and electron delocalizations
as contributors to stabilization energy. 相似文献
20.
Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach 总被引:1,自引:0,他引:1
Pollegioni L Sacchi S Caldinelli L Boselli A Pilone MS Piubelli L Molla G 《Current protein & peptide science》2007,8(6):600-618
D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications. 相似文献