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.     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Selective isolation of free and blocked amino-terminal peptides from enzymatic digestion of proteins

A general method for the selective isolation of free and blocked amino-terminal peptides from proteins is described. The rationale behind the methodology is based on the reasoning that if a protein, which has all its free amino groups blocked by citraconylation, is digested with a protease, all peptides, except those derived from the amino terminus, will have a free amino group. Reaction of such a digest with 1-fluoro-2,4-dinitrobenzene (Dnp-F) followed by removal of citraconyl groups by acid treatment and removal of dinitrophenyl (Dnp) groups from histidine and tyrosine side chains by thiolysis will result in dinitrophenylation of all alpha-amino groups of peptides generated from internal cleavages, leaving only peptides derived from the amino terminus without a Dnp group. The strong adsorption of Dnp groups to polystyrene is used to selectively elute the underivatized amino-terminal peptides from such a column. It is also demonstrated how selective isolation of amino-terminal peptides can be used to determine whether a protein has a free or blocked amino terminus.     

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.     

An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography.

The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.     

Carbonyldiimidazole (CDI) mediated synthesis of Nα-protected amino acid azides: application to the one-pot preparation of ureidopeptides

Synthesis of Nα-protected amino acyl azides starting from corresponding acids via the carbonyldiimidazole (CDI) activation is described. The protocol is extended for a one-pot preparation of ureido peptides that circumvents the isolation of acyl azide and isocyanate intermediates. The reaction was accomplished without using any additives and base. The protocol is simple, clean, high yielding and free from racemization.     

Gas chromatographic determination of free amino acids and amino acids attached to transfer RNA in biological samples

A method was developed to analyze quantitatively free amino acids and amino acids attached to transfer RNA (tRNA) in tissue samples by gas chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization. Total cellular aminoacyl-tRNA was extracted from rabbit reticulocytes and liver by a modified phenol extraction method under conditions which were designed to prevent deacylation of the attached amino acids. After deacylation and separation from tRNA by pressure ultrafiltration, eighteen amino acids were determined by gas chromatography as their N-heptafluorobutyryl isobutyl derivatives.     

Simple and rapid procedure suitable for quantitative isolation of low and high molecular weight extracellular nucleic acids

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100-10000 b.p. DNA fragments and 50-10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost-effective.     

Nutrient medium for the isolation and cultivation of pneumococcus

A culture medium for the isolation and cultivation of pneumococci, produced in a solid or liquid form and based on raw material unsuitable for use as foodstuff (human placenta), has been developed. The amino acid composition of the medium has been studied. The medium has been found to contain 19 amino acids, to be free from ballast serum proteins and blood, and to ensure the good growth of pneumococci isolated from pathological material, the formation of the normal capsule, as well as active biological properties. The medium has proved to create elective and selective conditions enhancing the effectiveness of investigations and simplifying the isolation of pneumococci in the microbiological examination of patients.     

Simple and Rapid Procedure Suitable for Quantitative Isolation of Low and High Molecular Weight Extracellular Nucleic Acids

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100–10000 b.p. DNA fragments and 50–10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost‐effective.     

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.     

Gradient-thickness thin-layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples

A thin-layer chromatographic method for quantitative isolation of free fatty acids is described. This method appears to be more satisfactory than existing methods in offering the combination of advantages of specificity, simplicity, rapidity, reproducibility, accuracy, high sensitivity, and applicability as a preparative technique. The method involves chromatography on a thin-layer plate on which the layer of Silica Gel G decreases linearly in thickness from 1000 micro at the base to 125 micro at the upper end. This gradient-thickness design allows the separation and densitometric quantitation of very small traces of free fatty acids from relatively large and complex lipid samples in a single chromatographic step. The method has been shown to be applicable directly to the crude total lipid extracts of several mammalian tissues. It appears to generate little if any artifactual free fatty acids from the breakdown of complex lipids, in contrast to the undesirable behavior of silicic acid columns in this respect. Gradient-thickness thin-layer chromatography promises to be useful for the quantitative isolation of trace amounts not only of other types of lipids but also of classes of compounds other than lipids.     

Utilization of 15N-labelled yeast hydrolysate in Lactococcus lactis IL1403 culture indicates co-consumption of peptide-bound and free amino acids with simultaneous efflux of free amino acids

Lactococcus lactis subsp. lactis IL1403 was grown in medium containing unlabelled free amino acids and ¹⁵N-labelled yeast hydrolysate to gain insight into the role of peptides as a source of amino acids under conditions where free amino acids are abundant. A mathematical model was composed to estimate the fluxes of free and peptide-derived amino acids into and out of the intracellular amino acid pool. We observed co-consumption of peptides and free amino acids and a considerable efflux of most free amino acids during growth. We did not observe significant differences between the peptide consumption patterns of essential and non-essential amino acids, which suggests that the incorporation of a particular amino acid is more dependent on its availability in a readily assimilated form than the organism’s auxotrophy for it. For most amino acids the contribution of peptide-bound forms to the formation of biomass was initially between 30 and 60 % with the remainder originating from free amino acids. During the later stages of fermentation we observed a decrease in the utilization of peptide-bound amino acids, thus indicating that the more readily assimilated peptides are gradually exhausted from the medium during growth.     

A Simple Gasometric Method for Determination of Free Amino Acid Contents in Biological Fluids

A simple method for the determination of total amino acid contents in biological fluids which contain various amino acids, peptides and proteins is described. This method is based on the determination in a Gilson differential respirometer of CO₂ gas evolved by the reaction of chloramin-T with α-amino-carboxylic acids in 5% trichloroacetic acid. The use of the trichloroacetic acid greatly suppressed interference by amines and ammonia, thus making this method appreciable to the determination of free amino acids in biological fluids without prior separation. Samples containing 1~20 μmoles of free amino acids can be assayed with accuracy of ±3%. This method was applied to the study of proteolytic digestion of casein.     

Analysis of free amino acids in mammalian brain extracts

An optimized method for analysis of free amino acids using a modified lithium-citrate buffer system with a Hitachi L-8800 amino acid analyzer is described. It demonstrates clear advantages over the sodium-citrate buffer system commonly used for the analysis of protein hydrolysates. A sample pretreatment technique for amino acid analysis of brain extracts is also discussed. The focus has been placed on the possibility of quantitative determination of the reduced form of glutathione (GSH) with simultaneous analysis of all other amino acids in brain extracts. The method was validated and calibration coefficient (K _GSH) was determined. Examples of chromatographic separation of free amino acids in extracts derived from different parts of the brain are presented.     

氨基酸生产和海洋生物的氨基酸资源开发

氨基酸在医药、食品、饲料等领域有着极为重要和广泛的用途,世界上氨基酸总需求量以５～１０％递增,市场竞争十分激烈。生物资源提取、化学合成、生物合成和综合法是生产氨基酸的４种技术,目前的发展趋势为生物合成和综合法,特别是将现代生物工程技术应用于氨基酸生产。另外,氨基酸生产领域另一个新的倾向是海洋生物氨基酸资源的开发和应用,尤其是海洋生物所产生的特殊氨基酸、肽及其衍生物的开发,同时,综合利用海产品加工后的废弃物来生产氨基酸也受到重视。     

Accumulation and remobilization of amino acids during senescence of detached and attached leaves: in planta analysis of tryptophan levels by recombinant luminescent bacteria

The process of leaf senescence is biochemically characterized by the transition from nutrient assimilation to nutrient remobilization. The nutrient drain by developing vegetative and reproductive structures has been implicated in senescence induction. The steady-state levels of amino acids in senescing leaves are dependent on the rate of their release during protein degradation and on the rate of efflux into growing structures. To determine the possible regulatory role of amino acid content in leaf senescence, an in planta non-destructive, semi-quantitative method for the analysis of endogenous levels of free amino acids has been developed. The method is based on in vivo bioluminescence of amino acid-requiring strains of recombinant Escherichia coli carrying the lux gene. The luminescence signal was found to be proportional to the levels of added exogenous tryptophan and to the free amino acid levels in the plant tissues analysed. During the senescence of tobacco flowers and of detached leaves of oats and Arabidopsis, a progressive increase in the levels of free amino acids was monitored. By contrast to the detached leaves, the attached oat leaves displayed a decrease in the levels of free amino acids during senescence. In Arabidopsis, both the attached and detached leaves exhibited a similar pattern of gradual increase in amino acid content during senescence. The differences between the sink-source balance of the two species and the possible relationships between amino acid content and leaf senescence are discussed.     

The effect of the antibiotic AL-87 on the amino acid yield from Staphylococcus aureus 209P cells]

Antibiotic AL-87 has been studied for its effect on the composition of intracellular free amino acids and of amino acids in culture fluid of Staphylococcus aureus 209 P. It is established that the content of amino acids in the culture fluid of S. aureus 209 P is doubled due to antibiotics, while the content of intracellular free amino acids considerably decreases. Spectrum of free amino acids of S. aureus 209 P is presented by 17 basic amino acids. When there is a sub-bacteriostatic concentration of the antibiotic in the medium all free amino acids tend to leave the cells, the content of aspartic acid, serine, threonine and leucine in the medium being increased. Data obtained when studying the effect of antibiotic AL-87 on the composition of free amino acids of Staphylococcus agree well with the previously obtained results from the study of the fatty acid composition of cells. In the light of these data it may be supposed that an increase of the membrane permeability and as a result of it an outlet of amino acids into the medium is one of constituents of the mechanism of antibiotic AL-87 action on Staphylococcus cells.     

不同肝脏疾病患者血浆游离氨基酸水平改变的模式

采用DNS -Cl荧光标记氨基酸 ,聚酰胺薄膜层析 ,分析了慢性血吸虫病肝肿大、晚期血吸虫病肝肿大 ,慢活肝 ,慢迁肝以及肝硬化等五种不同肝损伤患者血浆游离氨基酸水平 ,结果发现它们血浆游离氨基酸改变 ,均有一定的特异的模式