Sodium borohydride as a reducing agent for preparing ninhydrin reagent for amino acid analysis

Sodium borohydride, in place of stannous chloride or titanous chloride, is effective in the preparation of the ninhydrin reagent for automated amino acid analysis. The reagent, coupled with dimethyl sulfoxide as a solvent, a very stable ninhydrin solution which did not form precipitates in the flow lines of the analyzer.     

Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-phthaldialdehyde/2-mercaptoethanol reagent.

Previous reports (Drescher, D.G., and Lee, K.S. (1978) Anal. Biochem. 84, 559-569; Lee, K.S., and Drescher, D.G. (1978) Int. J. Biochem. 9, 457-467) have shown that high performance liquid chromatographic analysis of amino acids with the o-phthaldialdehyde/2-mercaptoethanol reagent (OPA/2-ME) is one of the most sensitive procedures currently available for micro amino acid analysis. In the present paper, methods are presented for the modification of cysteine and cystine in proteins for micro amino acid analysis using OPA/2-ME. Cysteine and cystine, which both show low fluorescence with OPA/2-ME, are converted to cysteic acid with performic acid directly, or to S-3-sulfopropylcysteine with 1,3-propane sultone after reduction of cystine with tri-n-butylphosphine. Cysteic acid and S-3-sulfopropylcysteine form highly fluorescent adducts with OPA/2-ME. The formation of S-3-sulfopropylcysteine in proteins and the subsequent hydrolysis of the proteins with methanesulfonic acid are particularly useful for complete amino acid analysis at the picomole level using a single sample.     

Possible site-specific reagent for the general amino acid transport system of Saccharomyces cerevisiae.

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids. The amino acid analogue N-delta-chloroacetyl-L-ornithine (NCAO) has been tested as potential site specific reagent for this system. L-Tryptophan, which is transported exclusively by the general transport system, was used as a substrate. In the presence of glucose as an energy source, NCAO inhibited tryptophan transport competitively (Ki = 80 micrometer) during short time intervals (1-2 min), but adding 100 micrometer NCAO to a yeast cell suspension resulted in a time-dependent activation of tryptophan transport during the first 15 min of treatment. Following the activation a time-dependent decay of tryptophan transport activity occurred. Approximately 80% inactivation of the system was observed after 90 min. When a yeast cell suspension was treated with NCAO in the absence of an energy source, an 80% inactivation of tryptophan transport occurred in 90 min. The inactivation was noncompetitive (Ki congruent to 60 micrometer) and could not be reversed by the removal of the NCAO. Addition of a five-fold excess of L-lysine during NCAO treatment or prevented inactivation of tryptophan transport. Under parallel conditions of incubation, other closely related transport systems were not inhibited by NCAO.     

Kinetic analysis of the fluorescence reaction of histamine with orthophthalaldehyde

Histamine reacts with orthophthalaldehyde (OPA) in an alkaline medium to form an unstable fluorescent adduct (F_base). Acidification of the solution gives a stable adduct (F_acid). In order to elucidate the mechanism of this fluorescence reaction, a kinetic study of this reaction was carried out. Although F_base was believed to be the precursor of F_acid, it was shown not to be the precursor of F_acid owing to the effects of the reaction time in an alkaline medium and OPA concentration on the yields of F_base and F_acid. The kinetic analysis of the formation and degradation of F_base revealed the pathway of the fluorescence reaction. On the basis of the results obtained in this study, the mechanism of the fluorescence reaction is proposed.     

Use of Marfey's reagent and analogs for chiral amino acid analysis: assessment and applications to natural products and biological systems

The present paper describes an updated knowledge and status on Marfey's reagent (MR), 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDNP-L-Ala-NH(2)). The reagent is used for pre-column derivatization of amino acids followed by HPLC separation of the diastereomers so formed. Emphasis is put on the design and application of structural variants which are synthesized by introducing different (other than L-Ala-NH(2)) L- and D-amino acid amides and amino acids in the 1,5-difluoro-2,4-dinitro benzene (DFDNB) moiety, as the chiral auxiliary. Advantages, disadvantages, the required precautions and suitability of the approach for the separation of multi component mixtures of DL-amino acids are assessed. Use of two dimensional (2D) techniques, in particular online HPLC in combination with various mass spectrometry techniques is discussed as well as methods designated 'advanced Marfey's method' and 'C(3) Marfey's method'. Application of MR and its variants for the determination of the stereochemistry of protein and non-protein amino acids in bioactive natural products isolated from living organisms (bacteria including blue-green algae, filamentous fungi, plants, marine sponges, invertebrates and vertebrates), in physiological samples including human beings, and in biologically relevant synthetic peptides are presented. In an outlook future applications are envisaged.     

Nitrotyrosine internal standard for amino acid analysis.

3-Nitrotyrosine is shown to be suitable as an internal standard for amino acid analysis. It is stable to acid hydrolysis, gives a color yield of 1.01 with ninhydrin which obeys Beer's Law, elutes in a position which is resolved from other amino acids on both Spinco and Durrum amino acid analyzers, and is commercially available. A method is also described for the preparation of a stable, pure, standardized stock solution of 3-nitrotyrosine.     

Sensitization of Edman amino acid derivatives using the fluorescent reagent, 4-aminofluorescein

The ability to analyze amino acid derivatives at the femtomole level is one of the most interesting challenges in the field of protein microsequencing. 2-Anilino-5-thiazolinone amino acids, obtained by Edman degradation, were quantitatively derivatized with fluorescent primary amines. The most fluorescent reagent tested was 4-aminofluorescein. The amino acid derivatives sensitized with this reagent were separated using reversed-phase high-performance liquid chromatography and identified at the 100 attomole level. Incorporation of this method into the operation of a conventional automated sequencer is also described.     

Application of hexafluoroacetone as protecting and activating reagent in amino acid and peptide chemistry

Summary Using hexafluoroacetone as protecting and activating reagent, multifunctional amino acids like aspartic acid can be functionalized regioselectively. This strategy offers i.a. a two-step synthesis for aspartame and preparatively simple access to multifunctional natural and unnatural amino acids, like 4-oxo-L-amino acids, 5-diazo-4-oxo-L-amino acids, 4-substituted L-proline derivatives and various heterocyclic L-amino acids. On application of this strategy to amino diacetic acid N-substituted glycines become readily available.     

Analysis of gamma-carboxyglutamic acid via amino acid analysis.

Modifications of the analysis of protein-bound residues of γ-carboxyglutamic acid (Gla) via alkaline hydrolysis are presented. The method described allows easier sample manipulation than that heretofore required and insures quantitative recovery of hydrolyzed amino acids. A possible explanation of the shoulder which sometimes appears near Gla on some amino acid analyzers is presented.     

Bayesian analysis of amino acid substitution models

Models of amino acid substitution present challenges beyond those often faced with the analysis of DNA sequences. The alignments of amino acid sequences are often small, whereas the number of parameters to be estimated is potentially large when compared with the number of free parameters for nucleotide substitution models. Most approaches to the analysis of amino acid alignments have focused on the use of fixed amino acid models in which all of the potentially free parameters are fixed to values estimated from a large number of sequences. Often, these fixed amino acid models are specific to a gene or taxonomic group (e.g. the Mtmam model, which has parameters that are specific to mammalian mitochondrial gene sequences). Although the fixed amino acid models succeed in reducing the number of free parameters to be estimated--indeed, they reduce the number of free parameters from approximately 200 to 0--it is possible that none of the currently available fixed amino acid models is appropriate for a specific alignment. Here, we present four approaches to the analysis of amino acid sequences. First, we explore the use of a general time reversible model of amino acid substitution using a Dirichlet prior probability distribution on the 190 exchangeability parameters. Second, we then explore the behaviour of prior probability distributions that are'centred' on the rates specified by the fixed amino acid model. Third, we consider a mixture of fixed amino acid models. Finally, we consider constraints on the exchangeability parameters as partitions,similar to how nucleotide substitution models are specified, and place a Dirichlet process prior model on all the possible partitioning schemes.     

Free amino acid analysis of five microalgae

The HPLC separation of fluorescent o-phtaldialdehyde (OPA) derivatives has been applied to the assay of free amino acids from five microalgae commonly used in aquaculture: Tetraselmis suecica, Skeletonema costatum, Chaetoceros calcitrans, Thalassiosira sp. and Isochrysis galbana, as part an assessment of their potential use in cosmetic products. Thirteen free amino acids were analyzed using High Performance Liquid Chromatography. There were considerable differences between species. However, four amino acids were responsible for more than 60% total concentration in all species: ASP, GLU, ARG and TYR; the next most important (accounting for less than 30%) were: ALA, VAL, PHE and LYS. This revised version was published online in June 2006 with corrections to the Cover Date.