Separation Of Free Amino Acids Research Articles

The research of free aminoacids of water-soluble protein-polysaccharide complex of oyster mushroom pleurotus ostreatus

Recently, the share of medicinal products of natural origin has increased in the pharmaceutical market of Ukraine. This is due to the fact that the pharmacological effect of natural substances provides a complex of biologically active substances, which has a high therapeutic effect and the minimum number of adverse reactions. Therefore, the study of chemical composition and standardization of natural substances is an important stage in pharmaceutical research.A promising source of active ingredients is the mushroom Pleurotus ostreatus. The chemical composition of this fungus is represented by a large number of biologically active substances, which ensures its many-sided use in folk medicine.Aim. The determination of qualitative and quantitative composition of free amino acids in the composition of water-soluble protein-polysaccharide complex (WPPSC) of oyster mushroom Pleurotus ostreatus was the aim of research.Methods. Physicochemical methods are the most commonly used to study active substances of natural origin. This is due to the fact that they provide high informative, accurate, efficient and reproducible. The method of high performance liquid chromatography (HPLC) was used to analyze the amino acid composition of the WPPSC of oyster mushroom Pleurotus ostreatus. This method is based on the separation of individual components due to different adsorption capacity.Results. Research of the amino acid composition of the WPPSC of oyster mushroom Pleurotus ostreatus showed that the complex contains 10 (7 %) free amino acids, among which 5 are essential. The detected aminoacids are aliphatic, heterocyclic and aromatic compounds. They are presented by glycine, alanine, leucine, isoleucine, threonine, selinum, lysine, arginine, histidine and phenylalanine.Conclusion. The qualitative and quantitative composition of the free amino acids of the WPPSC of oyster mushroom Pleurotus ostreatus was established. The method of determination and separation of free amino acids by the method of high-performance liquid chromatography is proposed. The obtained results are proposed for standardization of the initial substance in the process of production of medicines based on WPPSC of oyster mushroom Pleurotus ostreatus

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 (KGSH) was determined. Examples of chromatographic separation of free amino acids in extracts derived from different parts of the brain are presented.

Development of a gradient micellar liquid chromatographic method eluting from micellar mode to high submicellar mode for the rapid separation of free amino acids

A rapid surfactant-related gradient liquid chromatographic method progressing from a micellar liquid chromatographic mode to a submicellar liquid chromatographic mode has been developed for the quantitative analysis of free amino acids.

Separation of free amino acids and catecholamines in human plasma and rabbit vitreous samples using a new fluorogenic reagent 3‐(4‐bromobenzoyl)‐2‐quinolinecarboxaldehyde with CE‐LIF detection

A sensitive and efficient analysis of amino acids and catecholamines is currently presented with 3-(4-bromobenzoyl)-2-quinolinecarboxaldehyde as a fluorogenic derivatization reagent using CE separation with LIF detection. For good derivatization conditions, the reagent concentrations, pH value, temperature, and reaction time were explored, which were followed by the derivatization reaction in stable yield. The optimal running buffer was composed of mixtures involving 120 mM, pH 9.1 boric acid, 38.5 mM SDS, and 19% ACN v/v. The LOD (S/N=3) was found as low as 0.65 nM. The proposed method was validated by the two-order-magnitude linearity and correlation coefficient ranging from 0.9957 to 0.9998. The accuracy and specificity of this assay were also assured from the spiking of real samples with a standard known concentration. In order to demonstrate its wide range of applications, the method has been applied to the analysis of both human plasma and rabbit vitreous samples.

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4×10 −8 M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.

Preliminary Studies on Peptides in Wine by HPLC

Due to the importance of peptides and the absence of any analytical method for their study in wine using HPLC, an analytical method was developed which includes ultrafiltration for the separation of proteins and low pressure column chromatography using Sephadex LH-20 for separation of free amino acids and phenolic compounds. Peptide derivatives with <i>o</i>-phthalaldehyde were separated by reverse phase HPLC. Principal amino acids from peptides are: glutamic and aspartic acids, serine, glycine, alanine, and lysine.

Separation of free amino acids by reversed-phase ion-pair chromatography with column switching and isocratic elution

Free amino acids ( FAA) were separated by reversed-phase ion-pair liquid chromatography using a column-switching technique with isocratic elution. The eluted FAA were monitored by fluorescence detection using o-phthalaldehyde reagent. A 5-μm phenyl-bonded silica gel column (5 cm × 4.6 mm I.D.) was used for the hydrophobic and basic amino acids, which were separated within 20 min. A 5-μm octadecyl-bonded silica gel column (25 cm × 4.6 mm I.D.) was used for polar acidic and small amino acids, which were separated within 11 min. The system was applied to the FAA analysis of hydrolysate ob bovine serum albumin.

Utility of copper-containing electrolytes for isotachophoresis of amino acids

Isotachophoretic separation of free amino acids using Cu 2+-diethylene triamine (Den) in high-pH leading electrolytes is investigated. Large changes in relative step heights of free amino acids are seen. Mobility order depends on both Cu 2+ concentration and Cu 2+/Den ratios. Mobility differences seen with CuDen addition can be ascribed to both polyvalent counterion effects and specific interactions forming tertiary complexes. These electrolytes offer a new dimension for mobility variation of complexing amino acids and peptides.

Simple Free Amino Acid Separation by Reversed-Phase Ion-Pair Liquid Chromatography Using Column Switching Technique

Abstract Reversed-phase ion-pair liquid chromatography with copper (II) ions (RP-IPC-Cu) was applied to develop a high speed separation of free amino acids. Dynamic gradient elution in RP-IPC-Cu could not achieve this purpose due to base line shift, therefore a column switching technique was used with a combination of different alkyl-bonded silica gel columns under isocratic elution. The flexibility of eluent components makes it easy to realize a rapid separation of a mixture of targeted amino acids.

Separation of Free Amiimo Acids on Reverse Stationary Phases Using an Alkyl Sulfonate Salt as a Mobile Phase Additive

Abstract Alkylsulfonate (RSO3 −) salts were evaluated as mobile phase additives for the separation of free amino acids on reverse stationary phases using an acidic mobile phase where the amino acids are cations. The enhanced amino acid retention is the result of two major interactions, one being retention of the RSO3 − salt on the stationary phase and the other an ion exchange selectivity between the amino acid analyte cation and the RSO3 − countercation, or other countercations in the mobile phase. Major mobile phase variables are: type and concentration of RSO3 − salt (the studies focused on C8SO3 − salts), presence of organic modifier, type of countercation present, and mobile phase pH and ionic strength. Alkyl modified silica and polystyrenedivinyl-benzene copolymeric reverse stationary phases were compared. A mobile phase gradient, increasing per cent organic modifier was shown to be best, is necessary for separating complex mixtures of polar and nonpolar or basic amino acids. The procedure is applicable to the identification and/or determination of amino acids in mixtures or in peptides after hydrolysis.

Biosynthesis of cyclopentenylglycine from ?-ketopimelate in Idesia polycarpa callus cultures

The nonproteinogenic amino acid, cyclopentenylglycine, is found in certain Flacourtiaceae. This compound may be synthesized by two C1-chain elongations of α-ketoglutarate via α-ketopimelate (C5+2C1) or by condensation of C4 and C3 units (C4+C3), a pathway not involving α-ketopimelate. The following experimental design elucidated the biosynthetic pathway: Idesia polycarpa callus cultures were freshly established from leaf petioles; synthetic α-[1,2-(14)C]ketopimelate was added to the medium and cultures were incubated for 3 weeks. After isolation and separation of free amino acids from the tissues, the radioactivity incorporated into cyclopentenylglycine was determined. The results establish α-ketopimelate as a precursor for cyclopentenylglycine, thus providing evidence for the C5+2C1 biosynthetic path.

Untersuchungen über die Aminosäurezusammensetzung von nichtresorbierten Peptidfraktionen des distalen Dünndarminhaltes von Ratten. 2. Mitt. Verfütterung von Ackerbohnenproteinisolat mit und ohne Dialdehydstärkezusatz und von daraus hergestellten Proteintexturaten

Rats were fed diets containing 20% protein in the form of casein, protein isolate from sunflower seeds, protein isolate from field beans, model protein texturate made from field-bean protein isolate or model protein texturate made from field-bean protein isolate treated with dialdehyde starch and a nitrogenfree diet. The content in the distal small intestine of the rats was collected 3 hours after feeding, and the amino-acid compositions of the TCA-precipitable and TCA-soluble peptides (after separation of free amino acids) of the content were studied. The comparison of the amino-acid compositions of the two peptide fractions with the respective dietary protein showed no agreement. On the contrary, the comparison of the amino-acid compositions of the two peptide fractions determined after protein-free diet with those found after protein ingestion were in good agreement, independently of the kind of dietary protein. The comparison of the two peptide fractions with one another revealed an increased degree of proteolytic decomposition of the TCA-soluble peptides. As compared to the other protein-containing diets and to the protein-free diet, diets containing field-bean protein caused an increase of TCA-precipitable peptides and an increase of glycine in both fractions. This was also observed in model protein texturates containing only 30% field-bean protein. Treatment with dialdehyde starch produces certain alterations of the basic amino acids and their accumulation in the fractions of undigested peptide.

Chromatographic Separation of Free Amino Acids in Table Sirups

Abstract Qualitative chromatographic methods for the separation of free amino acids in table sirups are presented to aid in the development of chemical indices of composition which may be useful in establishing the identity of sirups and detecting their adulteration. Free amino acids in 2 table sirups were isolated on ion exchange columns and eluted with dilute ammonia. The concentrated amino acid mixture in the eluate was spotted directly on silica gel G plates for TLC analysis, or the amino acids were converted to their N-trifluoroacetyl n-butyl esters for GLC analysis. As many as 16 amino acids were qualitatively separated and identified and a potential for quantitative analysis was demonstrated.

Amino acids in pupa of tsetse fly ( Glossina palpalis)

1. 1. High-voltage paper electrophoretic and paper chromatographic separation of free amino acids in the pupa of Glossina palpalis gave a minimum of seventeen ninhydrin-positive spots. 2. 2. Most of these ninhydrin-reacting substances are neutral amino acids, and α-alanine has the highest concentration.

Proline metabolism in Leishmania tarentolae

Leptomonads of Leishmania tarentolae were able to grow in Trager's defined medium C deficient in glucose or l-proline, but not without both. In addition, inositol and glycine could be deleted without any adverse effect as long as either glucose or l-proline was present. Cells could grow in methionine-free media only if l-proline was present. Respiration studies indicated that l-proline, glutamate, and aspartate were the only amino acids in medium C capable of stimulating O 2 uptake by L. tarentolae. Methionine and serine appeared to inhibit respiration. d-proline and hydroxyproline could not substitute for the l form. Thin layer chromatography separation of free amino acids from cells incubated in 14C l-proline contained radioactive proline, glutamate, and alanine. Separations of tricarboxylic acid cycle acids contained a number of radioactive intermediates including α-ketoglutarate and malate as well as pyruvate. Although Δ 1-pyrroline-5-carboxylic acid could not be found in chromatograms, an increased positive O-aminobenzaldehyde reaction was found in cell extracts incubated in l-proline. The above results suggest the presence of a proline-glutamate interconversion oxidation pathway similar to the proline oxidase metabolic system found in a variety of organisms. Alanihe- 14C was found in cells incubated with 14C l-proline. Alanine is thought to be derived from pyruvate because of: (a) The presence of 14C Krebs cycle acids, (including malate) and pyruvate; (b) demonstration of an NADP dependent malic enzyme, and (c) formation of only 14C alanine by cells incubated in 14C sodium pyruvate.

Studies on the protein metabolism of Culex pipiens L.—IV. Separation of free amino acids and peptides in adult mosquitoes by column chromatography

Separation of free amino acids and peptides in methanol extracts of adult mosquitoes ( Culex pipiens and C. fatigans) was accomplished with Dowex-50 column chromatography in a gradient elution system. In addition to the amino acids previously reported, tryptophan and phenylalanine were detected and leucine and isoleucine could be separated distinctly. The relative concentrations of the various amino acids are in good agreement with those previously estimated by paper chromatography. Furthermore, the detailed analyses revealed the presence of at least thirty different peptides and amino acid derivatives, which amount to about 14–19 per cent of the total ninhydrin-positive components. Some preliminary data on the amino acid compositions of these compounds are given. The present results do not suggest any unusual features, at least not at the level of free amino acids and peptides, which could be responsible for the initiation of ovarian development in autogenous mosquitoes.