EPR study of anion radicals of various N-quinonyl amino acids

Summary. Since peptide quinones possess great clinical potential in targeted chemotherapy, several series of novel N-quinonyl amino acids have been synthesized and their first products of reduction were studied by EPR spectroscopy. EPR spectra of the corresponding radical adducts were identified by computer simulation. The dependence between the splitting constants and the chemical structure of the N-quinonyl amino acids anion radicals was examined. Received January 4, 2000; Accepted March 14, 2000     

A modified spray reagent for the detection of amino acids on thin layer chromatography plates

Summary. Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced. Received March 14, 2000 Accepted August 31, 2000     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Mild and effective N-phthaloylation of amino acids

Summary. In the present work various free amino acids, including tryptophan and tyrosine, were effectively N-phthaloylated under reduced pressure and at lower temperature. Moreover, under these conditions, the presence of phthalic acid in phthalic anhydride did not hinder the N-phthaloylation of amino acids. This simple process is economic, environmentally friendly, and suitable for large-scale production.     

Nonprotein amino acids in edible lentil and garden pea seedlings

Summary. Commercial edible seedlings of garden pea (Pisum sativum L.) and lentil (Lens culinaris L.) contain high concentration of nonprotein amino acids and trigonelline. Both seedlings grown in the laboratory or purchased in a supermarket were studied by HPLC. Samples from both origins contained trigonelline, α-aminoadipic acid, homoserine, β-(isoxazolin-5-on-2-yl)-alanine (BIA), and γ-glutamyl-BIA. Garden pea seedlings also contained a uracil-alanine derivative (isowillardiine) in substantial amount. Some of these compounds such as BIA and α-aminoadipic acid have neurotoxic activity. Received December 17, 1999 Accepted February 15, 2000     

Diastereoisomeric 4-substituted acidic amino acids in ferns

Diastereoisomeric 4-substituted acidic amino acids occur in characteristic associations in the green parts of some species of the Filicinae. Subspecies of Phyllitis scolopendrium accumulate 2(S),4(R)-4-methylglutamic acid, 2(S)-4-methyleneglutamic acid and the two diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid, the last two occurring at relative concentrations of 3: 1. All Asplenium species investigated were distinctive in accumulating 2(S),4(R)-4-methylglutamic acid, the two diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid, and the two diastereoisomers of 2(S)-4-hydroxy-2-aminopimelic acid in a characteristic concentration ratio. Some Polystichum species do not accumulate 4-substituted acidic amino acids whereas others accumulate both diastereoisomers of 2(S)-4-hydroxy-4-methylglutamic acid and 'of 2(S)-4-hydroxy-2-aminopimelic acid, and thus resemble Asplenium species. The seasonal variation in the concentration of 4-substituted acidic amino acids in the green parts of Phyllitis, Asplenium and Polystichum species has also been determined.     

Identification of post-translational modified amino acids

Summary Methylated lysines (N -mono-methylated, N -di-methylated and N -tri-methylated) have been identified after derivatization with orthophthaldialdehyde (OPA) by using pre-column and post-column derivatization techniques.Also the N -acetylated lysine and N -formylated lysine have been identified by OPA post-column derivatization techniques but only in free form due to their instability under acidic conditions which are used for protein hydrolysis.Additionally, all the modified amino acids mentioned above have been derivatized with DABITC/PITC, an Edman reagent, and identified as DABTH-derivatives on thin-layer polyamide sheets.     

Lipophilicity of amino acids

Summary The lipophilicity (or hydrophobicity) of amino acids is an important property relevant for protein folding and therefore of great interest in protein engineering. For peptides or peptidomimetics of potential therapeutic interest, lipophilicity is related to absorption and distribution, and thus indirectly relates to their bioactivity. A rationalization of peptide lipophilicity requires basic knowledge of the lipophilicity of the constituting amino acids. In the present contribution we will review methods to measure or calculate the lipophilicities of amino acids, including unusual amino acids, and we will make a comparison between various lipophilicity scales.     

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN⁻). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μM). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol (S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.     

Use of fuzzy clustering technique and matrices to classify amino acids and its impact to Chou's pseudo amino acid composition

In this paper we present a study of classification of the 20 amino acids via a fuzzy clustering technique. In order to calculate distances among the various elements we employ two different distance functions: the Minkowski distance function and the NTV metric. In the clustering procedure we take into account several physical properties of the amino acids. We examine the effect of the number and nature of properties taken into account to the clustering procedure as a function of the degree of similarity and the distance function used. It turns out that one should use the properties that determine in the more important way the behavior of the amino acids and that the use of the appropriate metric can help in defining the separation into groups.     

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.     

Syntheses of fluorinated amino acids: from the classical to the modern concept

Summary A survey of the synthetic pathways leading to the fluorine-containing analogues of amino acids is given. From the great number of syntheses the typical examples are selected and divided into two groups: classical syntheses and the modern ones. The classical ammonolysis of halogeno acids and equivalent reactions are discussed as first, followed by a few examples of oxo amino group transformation. Conversion of the oxo compounds into amino acids richer by one carbon atom is realized by the Strecker and hydantoin syntheses. For the prolongation by two carbons, the Erlenmeyer azlactone method and alkylation of CH-acidic esters are applied. The modern syntheses are represented by direct fluorination by elemental fluorine and other electrophilic fluorinating reagents. Further examples include the applications of the Yarovenko reagent, sulphur tetrafluoride and its derivative DAST. The use of trifluoropyruvates as the fluoro synthons is mentioned briefly. Finally, the examples of the amidocarboxylation method and the syntheses of diverse-fluorinated methionines are shown.     

Changes in plasma and urinary taurine and amino acids in runners immediately and 24 h after a marathon

Summary. Changes in urinary and plasma taurine and amino acids have been evaluated in trained runners competing in the Rotterdam Marathon, 1998, both immediately after completing the event and 24 h after recovery. There were significant changes in the urinary amino acids excretion, the majority showing a significant decrease both immediately at the completion of the Marathon and after 24 h recovery. In contrast urinary taurine excretion increased immediately post Marathon, although not significantly as the range of results was wide. Such changes in urinary taurine correlated with percentage changes in plasma creatine kinase both immediately post race, (r = 0.972, P < 0.001), and 24 h later (r = 0.872, P < 0.001), possibly indicating that the source of the taurine was muscle. Significant correlations between the individual values for urinary and plasma amino acids in all of the athletes were calculated for taurine (r = 0.528), glycine (r = 0.853), threonine (r = 0.749), alanine (r = 0.747), serine (r = 0.620), glutamine (0.614), arginine (r = 0.507), histidine (r = 0.470) and valine (r = 0.486). Changes in the mean plasma concentrations of amino acids were comparable to our previously published data (Ward et al., 1999) the majority showing significant decreases immediately and 24 h post Marathon, such an adaptation being due primarily to their utilisation for gluconeogenesis. However, in contrast, the mean taurine concentrations were significantly elevated both post race, P < 0.01 and after 24 h, P < 0.05. The physiological response by the muscle to exhaustive exercise, particularly with regard to changes in plasma and urinary taurine concentrations remain to be elucidated, but is probably related to muscle function impairment. The increase in taurine urinary excretion could be used as an indicator of muscle damage occurring during exhaustive exercise. Whether taurine supplementation would minimise such changes is an interesting scientific question and merits investigation. Received January 6, 2000 / Accepted February 1, 2000     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Use of amino acids by Plasmodium relictum oocysts in vitro.

A comparison was made of the in vitro growth of the gut of Culex tarsalis in Grace's insect culture medium, supplemented with fetal bovine serum in the presence of dividing cells of Antheraea eucalypti, with a similar preparation of a gut infected with oocysts of the avian parasite, Plasmodium relictum. In the latter case, after 16 hr, significant decreases occurred in the concentration of arginine, asparagine, and glutamine combined, glutamic acid, glycine, histidine, lysine, proline, and serine. Lower and less marked decreased concentrations of alanine, β-alanine, cystine, isoleucine, leucine, methionine, ornithine, phenylalanine, threonine, tryptophane, tyrosine, and valine also took place. This indicated utilization of certain amino acids by the developing oocysts of P. relictum in the presence of metabolizing insect cells.     

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.     

Synthesis,electrochemical and spectral properties of some ω-N-quinonyl amino acids

Summary. Four series of ω-N-quinonyl amino acids were synthesized by Michael-like additions. The quinones include 2-phenylthio-1,4-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and 2,3-dichloro-1,4-naphthoquinone. These modified amino acids can be used for post chain assembly modifications of biologically active peptides, which target the quinonic drug to a cancer damaged area. The electron-transfer capabilities of the modified amino acids were probed by cyclic voltammetry measurements. The results described in this paper show that the novel N-quinonyl amino acids are effective in producing semiquinone radicals similarly to the unconjugated quinones themselves. A direct relation was found between the first reduction potentials of the quinones and their reactivity towards the ω-amino acids. The successful generation of stable semiquinone radicals by the novel quinone derivatives is a prerequisite for the manifestation of site-directed antitumor activity of corresponding quinone-peptide conjugates. Received January 3, 2001 Accepted March 28, 2001     

Quantitative determination of free intracellular amino acids in single human polymorphonuclear leucocytes: Recent developments in sample preparation and high-performance liquid chromatography

The described procedure allows quantitative, highly precise and reproducible analysis of free amino acid concentrations in single polymorphonuclear leucocytes (PMLs). This method is superior to previously described procedures with regard to sample size, PML separation, sample preparation and stability, as well as the chosen fluorescence high-performance liquid chromatography procedure, and can satisfy the high demands for ultra-sensitive and comprehensive amino acid analysis, especially for the continuous surveillance of severe diseases and organ dysfunction.     

When quinones meet amino acids: chemical, physical and biological consequences

Summary. Quinones and amino acids are usually compartmentally separated in living systems, however there are several junctions in which they meet, react and influence. It occurs mainly in wounded, cut or crushed plant material during harvest, ensiling or disintegrating cells. Diffusing polyphenols are oxidized by polyphenol oxidases (PPOs) to quinonic compounds, which associate reversibly or irreversibly with amino acids and proteins. The reaction takes place with the free nucleophilic functional groups such as sulfhydryl, amine, amide, indole and imidazole substituents. It results in imine formation, in 1,4-Michael addition via nitrogen or sulphur and in Strecker degradation forming aldehydes. The formation and activity of quinone–amino acids conjugates influences the colour, taste, and aroma of foods. Physical and physiological phenomena such as browning of foods, discoloration of plants during processing, alteration of solubility and digestibility, formation of humic substances, germicidal activity, cytotoxicity and more occur when quinones from disintegrating cells meet amino acids. The mechanisms of toxicity and the pathways by which PCBs may be activated and act as a cancer initiator include oxidation to the corresponding quinones and reaction with amino acids or peptides. Sclerotization of insect cuticle is a biochemical process involving also the reaction between quinones and amino acid derivatives.