Rapid analysis of amino acids using pre-column derivatization

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.     

Synthesis,characterization and antimicrobial activity of β-aminovinylphosphonium salts derived from aromatic amino acids

This study presents the synthesis, characterization and antimicrobial activity evaluation of β-aminovinyl-phosphonium salts derived from aromatic amino acids. The compounds were prepared in the reaction of (formylmethyl)triphenylphosphonium chloride with various aromatic amino acids and their structures were determined by elemental analysis, IR and NMR spectroscopy. All synthesized compounds were tested for their antibacterial and antifungal activity.     

Biochemical characterization of the vital activity ofYersinia pseudotuberculosis in a culture medium as a function of the temperature of cultivation

A biochemical evaluation of the accumulation of amino acids and organic acids in the culture medium during the cultivation of Yersinia pseudotuberculosis str. 2781 as a function of the temperature has been made by methods of quantitative chromatographic analysis. A considerable decrease in the biosynthesis of the products of vital activity under investigation at 37°C as compared with the cultivation of the same microorganism at 6°C has been found. This permits the assumption that the temperature of cultivation of Y. pseudotuberculosis is a factor regulating the biosynthesis of the amino acids and metabolites of the Krebs cycle necessary for its vital activity.     

Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis

Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.     

Methods of TLC Resolution of Enantiomeric Amino Acids And Their Derivatives

Abstract

It has been detected that racemization of amino acids in metabolically stable proteins of living mammals takes place (1–4) and as a consequence the protein-structure function relationship may be altered (5). Thus in vitro and in vivo analysis of racemic amino acids is very important. Besides enantiomeric purity becomes very significant when only one of the enantiomers has a potential biological activity for example L-DOPA (3,4-dihydroxypheny 1 alanine) is an important drug for treating Parkingson's disease. The measurement of specific rotation is a common and well accepted method for evaluating hydrophile-hydrophile interactions between the second carboxyl group of amino acid and the alkaline substructures of adenine (probably hydrogen-bond formation); hydrophobe-hydrophobe interactions between the apolar sidechain of the amino acid and the corresponding adenine substructures.     

氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化

为改善二氧化硅载体材料本身的生物相容性和疏水性,维持包埋生物分子的活性,本文对水解前驱体3-氨基丙基三甲氧基硅烷进行氨基酸分子改性。具体过程包括N-Fmoc-L-缬氨酸和氯化亚砜反应生成N-Fmoc-L-缬氨酰氯,再和3-氨基丙基三甲氧基硅烷反应生成N-（3-三甲氧基硅基）丙基-N′-Fmoc-L-缬氨酰胺后。然后去除Fmoc,得到N-（3-三甲氧基硅基）丙基-L-缬氨酰胺作为氨基酸修饰的硅源前驱体。通过IR、MS、1H-NMR等分析测试手段对合成得到的各个化合物的结构进行了表征。利用正硅酸甲酯（TMOS）和N-（3-三甲氧基硅基）丙基-L-缬氨酰胺为复合硅源,经过溶胶-凝胶过程来包埋了胰蛋白酶,研究得到最适的固定化条件为,N-（3-三甲氧基硅基）丙基-L-缬氨酰胺的含量为15mol%。在该条件下,固定化胰蛋白酶活力的绝对值是199U,游离酶的酶活力的绝对值是103U, 四甲氧基硅烷直接包埋的固定化酶活力的活性是38 U。在该条件下,杂化硅源得到的固定化酶的活性是以四甲氧基硅烷水解前驱体的固定化酶活性的5倍,杂化硅源固定化胰蛋白酶的最相比游离酶,酶的最高活力提高的几乎2倍。这些结果表明氨基酸分子对水解前驱体修饰以后,水解产生的固定化载体具有良好的生物相容性。通过改性载体制备的固定化酶,对甲醇变性剂的稳定性,对酸碱的抵抗性及热稳定性也有明显地提高。     

Gas chromatography of amino acids: Use of a fused-silica capillary column in the determination of plasma amino acids

A capillary chromatographic procedure using a fused silica column is described which can be used to quantitatively determine amino acids in plasma following the pre-chromatographic “clean-up” described in a recent paper [1]. In substituting this procedure for that involving a packed column, advantage has been taken of the greater resolving power to separate amino acids from background component peaks. In order to extend this advantage and provide a sound basis for quantitative analysis, the technique of cold on-column injection was employed. As a result, good precision of standard analysis was obtained with relative standard deviation (RSD) values for all amino acids of less than 4%. Application of the entire procedure to plasma samples yields RSD values of better than 10% for all amino acids with recoveries ranging from 72% to 104%. Simultaneous determination of plasma amino acid levels by gas chromatography (GC) using capillary columns and by classical ion exchange (CIE) showed reasonable agreement. Statistical evaluation showed no significant difference between twelve amino acids. Values for the remaining two, namely, phenylalanine and histidine are significantly different (p < 0.005). Comparison of the values obtained from GC capillary and packed columns reveals no significant difference between fourteen amino acids. Significant differences exist between results for phenylalanine and tyrosine (p < 0.001). It is concluded that there is good agreement between data obtained by GC capillary and CIE techniques and that differences between results for phenylalanine and histidine are method related.     

Sensitive analysis of amino acids in injection liquor by reversed-phase HPLC

A convenient derivatization method of amino acids with l-fluoro-2,4-dimtrobenzene as reaction reagent and a separation system were described. The derivative amino acids were separated on a specific chemically bonded phase column with a simple linear gradient elution consisting of aqueous buffer and methanol. The eluate was detected by common ultraviolet absorption detector at 360 nm. The detection limits of amino acids were as low as 10 picomole. This method has been successfully applied to assay amino acid injection liquor used in hospital. It has good repro-ducibility and precision. The procedures avoid the requirements of particular derivative equipment and analyzer employed in conventional amino acid analysis.     

Determination of enantiomeric purity in biogenic11C-labelled amino acids by aminoacylation of tRNA

In syntheses of naturally occurring amino acids labelled with short-lived radionuclides, such as¹¹C, the determination of the enantiomeric purity presents problems. The aminoacyl coupling of L-amino acids to tRNA followed by a separation with gel filtration of the tRNA-amino acid complex and free amino acid, was here shown to be an adequate method for determining the enantiomeric purity of [methyl-¹¹C]-methionine even in the pmol range. This method has also been used for the determination of the specific radioactivity of the¹¹C-labelled methionine. The method is probably valid for other naturally occurring amino acids and it might also be of interest for enantiomeric separations of L- and D-amino acids of high specific radioactivity.     

Applications of gas-liquid chromatography in protein chemistry. Determination of C-terminal sequences on nanomolar amounts of proteins.

A method for the determination of C-terminal amino acids and C-terminal amino acid sequences in nanomolar amounts of proteins is described, based on carboxypeptidase A digestion of the protein, followed by removal of the partially digested protein and quantitative gas-liquid chromatographic determination of the amino acids released after known time intervals. Sequences deduced from the kinetics of release of specific amino acids are compared with the known C-terminal sequences of well-characterized proteins.     

Automated Analysis of O-Phthalal-Dehyde Derivatives of Amino Acids in Physiological Fluids by Reverse Phase High Performance Liquid Chromatography

Abstract

An automated method is described for the determination of free amino acids in biological fluids using precolumn deriva-tization with o-phthalaldehyde and reverse phase high performance liquid chromatography. Chromatographic separation of amino acids is accomplished in 24 minutes (cycle time 44 minutes). As little as 1.5 pmol of most commonly occurring amino acids can be accurately quantified. Accuracy and reproducibility are optimized by automating the derivatization-injection sequence and by correcting for variations in the fluorescence response of each amino acid in each run. A total of 31 analyses can be completed in 24 hours on a single column (7 standards and 24 unknowns). The method can be used in the general determination of free amino acids in biological fluids, or can be further accelerated and used for the quantitation of specific amino acids simply by altering the elution conditions.     

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 alpha-amino acids, beta-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither beta-, gamma-, nor delta-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong basis for the application of MALDI TOF/TOF MS/MS in qualitative and quantitative analysis of amino and organic acids, including application in clinical medicine.     

Chiral separation and determination of the optical purity of thiamphenicol-related chiral compound by capillary zone electrophoresis with cyclodextrins as chiral selectors

Summary One classical method for quantitation of amino acids in proteins is hydrolysis of the proteins and determination of the free amino acids. Although the drastic experimental conditions necessary for complete hydrolysis always cause degradation of some of the amino acids, if mild hydrolysis conditions are used, a mixture of amino acids and oligopeptides is obtained. If these conditions are adequately tuned, the oligopeptides are almost exclusively dipeptides. For this reason we have initiated a study to find a derivatizing agent suitable for the analysis of amino acids and dipeptides by an absolute method of quantitation already tested for amino acids. FMOC-Cl was found to be a suitable derivatizing agent for this purpose.     

Amino acid analysis of physiological fluids by high-performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography

The suitability of pre-column derivatization with phenylisothiocyanate followed by high-performance liquid chromatography was investigated as a means of analyzing free amino acids in plasma and other physiological fluids. A comparison was made between this method and a conventional ion-exchange method. The correlation coefficient for all the amino acids tested was greater than 0.9, except for proline and tryptophan. Various forms of sample preparation were tried for plasma and amniotic fluid; it was finally decided that protein precipitation with acetonitrile was most suitable. Ultrafiltration was used for cerebrospinal fluid preparation while urine was treated the same as a standard mixture. The retention times relative to the internal standard (nor-leucine) are given for over 90 compounds. Some of these were chromatographed underivatized because they are known to be present in some physiological fluids and absorb at 254 nm because of their aromaticity. The imprecision for this method compared favourably with the standard ion-exchange method although each had specific amino acids for which the imprecision was poor. The technique is suitable for the same routine clinical analysis purposes as high-resolution ion-exchange chromatography. It also offers the advantages of speed of analysis, sensitivity and equipment versatility over the conventional ion-exchange methods.     

Specificity of Carboxypeptidases from Actinomucor elegans and Their Debittering Effect on Soybean Protein Hydrolysates

The specificities of carboxypeptidases from Actinomucor elegans were investigated by determining enzymatic activities at pH 7.0 and pH 4.0 with 16 Z-dipeptides and three Z-tripeptides as substrates. The debittering effect was evaluated and the free amino acid compositions of the soybean protein hydrolysates were analyzed before and after treatment with A. elegans extract at pH 7.0 and pH 4.0, with carboxypeptidases from Aspergillus oryzae as control. The results of the enzyme activity determinations indicated that carboxypeptidases from A. elegans prefer hydrophobic substrates, such as Z-Phe-Leu, Z-Phe-Tyr-Leu, and Z-Phe-Tyr. The sensory evaluation and free amino acid composition analysis showed that these carboxypeptidases are efficient tools for decreasing the bitterness of peptides because they liberated the fewest free amino acids, which consisted of 73% hydrophobic amino acids, under acidic conditions. Carboxypeptidases from A. elegans display promising prospects for future applications in the protein hydrolysate industry.     

Amino acid δ13C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry: paleodietary implications from intra‐individual comparisons

We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (δ¹³C) of individual amino acids in hair proteins and bone collagen using the LC‐IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound‐specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context by the δ¹³C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used as a proxy for bone collagen at the amino acid level, this validates compound‐specific isotope studies using hair as a model for palaeodietary reconstruction. Our results suggest that a small offset observed in the bulk δ¹³C values of the hair and bone samples may be attributed to two factors: (i) amino acid compositional differences between hair and bone proteins, and (ii) differential turnover rates of the tissues and the amino acid pools contributing to their synthesis. This application proposes that hair may be a useful complementary or alternative source of compound‐specific paleodietary information. Copyright © 2010 John Wiley & Sons, Ltd.     

中国梨木虱分泌物中氨基酸的分离与分析

利用离子交换色谱法分离了中国梨木虱分泌物中的氨基酸;利用毛细管气相色谱法对梨木虱分泌物中氨基酸的三氯乙酰了酯衍生物进行了分析。用标准样对照定性,内标法定量。分泌物中共检出13种氨基酸。     

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization.

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicyclic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde-3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.     

Analysis of photo-oxidized amino acids in tryptic peptides of calf lens gamma-II crystallin.

Insoluble and crosslinked proteins and increased pigmentation in the eye lens are features of aging and cataracts. Determining the amino acids which are involved in insolubilization, crosslinking and visible light scattering will shed light on the mechanisms by which cataracts form. Calf lens gamma-II crystallin was irradiated at 295 nm, digested and separated into tryptic peptides. Additional tryptic peptides were found in the digest of irradiated gamma-II which were not present in the dark control digest. These peptides were identified by amino acid sequencing and shown to correspond to expected tryptic fragments of the protein, indicating more facile digestion in the UV-irradiated protein than in dark controls. Amino acid analysis of the irradiated protein and peptides showed losses of histidine, methionine and cysteine residues as compared to control samples. Tryptophan, which is not detected by amino acid analysis, was also found to be reactive since losses in its fluorescence intensity were observed after irradiation. Some of the photochemically active amino acids had lower than expected responses in amino acid sequencing experiments. This suggested specific sites of photochemical activity in the various peptides. The evidence for peptide crosslinks is also discussed.     

Advantages of compound‐specific stable isotope measurements over bulk measurements in studies on plant uptake of intact amino acids

Increasing interest in the ability of plants to take up amino acids has given rise to questions on the accuracy of the commonly used bulk method to measure and calculate amino acid uptake. This method uses bulk measurements of ¹³C and ¹⁵N enrichment in plant tissues after application of dual‐labelled amino acids but some authors have recommended the use of compound‐specific stable isotope (CSI) analysis of the plants' amino acids instead. However, there has never been a direct evaluation of both methods. We conducted a field study applying dual‐labelled (¹³C, ¹⁵N) amino acids (glycine, valine, tyrosine and lysine) to soil of a Plantago lanceolata monoculture. Root and shoot samples were collected 24 h after label application and the isotope composition of the plant tissues was investigated using bulk and CSI measurements. Enrichment of ¹³C in the case of CSI measurements was limited to the applied amino acids, showing that no additional ¹³C had been incorporated into the plants' amino acid pool via the uptake of tracer‐derived C‐fragments. Compared with this rather conservative indicator of amino acid uptake, the ¹³C enrichment of bulk measurements was 8, 5, 1.6 and 6 times higher for fine roots, storage roots, shoot and the whole plant, respectively. These findings show that the additional uptake of tracer‐derived C‐fragments will result in a considerable overestimation of amino acid uptake in the case of bulk measurements. We therefore highly recommend the use of CSI measurements for future amino acid uptake studies due to their higher accuracy. Copyright © 2009 John Wiley & Sons, Ltd.