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.     

Sensitization of amino acid derivatives obtained from Edman degradation with radioactively-labeled iodohistamine

The minimum amount of proteins and peptides required for sequencing is constantly decreasing as more sensitive microsequencing methods are developed. The sensitization of and Edman degradation product is one such method. We took the 2-anilino 5-thiazolinone amino acid intermediates obtained from Edman degradation by conventional sequencing procedures, and quantitatively reacted them with a primary amine. The amine used was radioactive [125I]iodohistamine, which affords highly sensitive detection. The labeled amino acid derivatives were separated by thin layer chromatography. Ten femtomoles of a labeled derivative can be detected by autoradiography.     

On the synthesis of a 32P-labelled Edman reagent for the sensitive identification of amino-acid derivatives

A phosphorus-32 containing derivative of phenylisothiocyanate was prepared to increase the sensitivity of amino-acid sequence determination. The respective compound 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide showed about the same reactivity, stability, and polarity as the Edman reagent itself. A repetitive yield of 94% was obtained in the stepwise degradation of insulin B chain using a solid phase sequencer. The synthesis of this radioactive reagent was achieved within 5 h but with a specific activity of 1 Ci/mol. Eight amino acids were reacted with the 32P-labelled reagent and identified by autoradiography after two dimensional thin-layer chromatography.     

A novel,simple, and sensitive colorimetric method to determine aromatic amino acid aminotransferase activity using the Salkowski reagent

This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of l-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented.     

Sequence determination of eglin c using combined microtechniques of amino acid analysis,peptide isolation,and automatic Edman degradation

The complete amino acid sequence of a proteinase inhibitor, eglin c (M_r 8100), has been determined with less than 150 μg of the protein using the following microtechniques: (a) amino acid analysis with a low-nanogram amount of protein hydrolysate using dimethylaminoazobenzene sulfonyl chloride, (b) peptide isolation at the picomole level using the dimethylaminoazobenzene isothyiocyanate (DABITC) precolumn derivatization method, and (c) automatic Edman degradation. One amino acid residue has been corrected for the previously reported sequence. The Contribution of each technique to the microsequencing is discussed. In addition, a new high-performance liquid chromatography system that gives a complete baseline separation of all phenylthiohydantoin-amino acids is described.     

Instability of orthophthalaldehyde reagent for amino acid analysis

Determination of amino acids by reversed-phase chromatography of the adduct with orthophthalaldehyde and a thiol is rapid and sensitive. The major recognized adverse feature of this method is the instability of the reaction product, which requires precise control of reaction timing and chromatographic parameters for reliable quantitative application. We report another source of major variability: reagent instability. Deterioration of reagent was noted as low peak heights and peak broadening and was predictable if the premixed reagent was left at room temperature. Restoration of sharp chromatograms was accomplished by addition of mercaptoethanol or sodium metabisulfite. Reagent which was chromatographically inert contained minimal free thiol by direct assay. Free thiol disappearance was markedly slowed by addition of a chelating agent. Excess mercaptoethanol was deleterious. We conclude that reagent deterioration represents oxidation of the thiol, may be reversed by rereduction with minimal thiol or bisulfite, and may be minimized by inclusion of a metal chelator in the reagent.     

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.     

A two-step, one-pot synthesis of diverse N-pyruvoyl amino acid derivatives using the Ugi reaction

A 100-member combinatorial library of -ketoamides, which was designed to search potent cysteine protease inhibitors, was generated by a parallel solution-phase synthesis. A two-step one-pot synthesis, in which the Ugi reaction followed by pyridinium dichromate (PDC) oxidation was employed in the same reaction vessel, easily afforded the -ketoamides in a short time.     

A sensitive method for quantitation of gamma-hydroxybutyric acid and gamma-butyrolactone in brain by electron capture gas chromatography.

A new and sensitive method for the quantitation of γ-hydroxybutyric acid (GHB) and its lactone precursor γ-butyrolactone (GBL), has been developed and successfully utilized to determine the endogenous content of these compounds in a single rat brain. The method involves conversion of endogenous GHB into GBL and extracting the GBL with CHCl₃. The concentrated CHCl₃ extract is treated with BF₃ methanol reagent to produce methyl γ-hydroxybutyrate. Introduction of electron capturing groups was accomplished by further reacting the methyl γ-hydroxybutyrate with heptafluorobutyric anhydride in the presence of pyridine. Prior to quantitation by electron capture gas chromatography, the sample was cleaned up by thin layer chromatography (tlc) using a preabsorbent plate which removed many extraneous peaks as well as CHCl₃ used as the solvent. The effciency of the procedure was evaluated by carrying [1-¹⁴C]GBL through the derivatization. This indicated that about 15% of the starting labeled GBL was converted to the final electron capturing product. δ-Valerolactone was used as an internal standard.     

Reductive N,N-dimethylation of amino acid and peptide derivatives using methanol as the carbonyl source

Formaldehyde is readily formed from methanol in the presence of palladium-on-charcoal and air. The use of this methanolic formaldehyde solution for the reductive methylation of amino groups in amino acid and peptide derivatives by catalytic hydrogenation has been studied and found to be superior to the use of aqueous formaldehyde because no contaminating paraformaldehyde is present. Some data on N-isopropylation are given.     

Synthesis of side chain-protected amino acid phenylthiohydantoins and their use in quantitative solid-phase Edman degradation

Solid-phase Edman degradation of synthetic peptidyl-resins has been used advantageously to detect errors of deletion which might occur during Merrifield peptide synthesis. To facilitate complete quantitation of the resulting phenylthiohydantoin(PTH)-amino acids, the PTH derivatives of the following side chain-protected amino acid residues have been synthesized: Arg(Tos), Asp(OBzl), Cys(3,4-(CH3)2-Bzl), Glu(OBzl), Lys(2-ClZ), Ser(Bzl), Thr(Bzl), Tyr(2-BrZ), and Tyr(2,6-Cl2Bzl). For each derivative, a melting point, elemental analysis, and extinction coefficient were obtained. With these new compounds as HPLC standards, an unequivocal assignment and quantification of each side chain protected amino acid was possible. A quantitative analysis was performed for six model peptides with the general formula Ala-X-Leu-Y-Ala-Gly-NHCH2-resin (where X and Y represented different side chain-protected amino acyl residues). We have found solid-phase Edman degradation to be a useful aid for the characterization of peptides when they are used unpurified as synthetic antigens.     

Determination of amino acid pairs sensitive to variants in human copper-transporting ATPase 2

In this study, we use our probabilistic approach to analyze the amino acid pairs in human copper-transporting ATPase 2 (ATP7B) in order to determine which amino acid pairs are more sensitive to 125 variants from missense mutant human ATP7B. The results show 97.6% of 125 variants occur at randomly unpredictable amino acid pairs, which account for 80.9% of amino acid pairs in ATP7B, and the chance of occurring of variant is about 9 times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, the randomly unpredictable amino acid pairs are more sensitive to variants in human ATP7B.     

The transport of amino acids,amino acid derivatives and ions across ion-exchange membranes

The passage of inorganic salts, glucose, amino acids and peptides across polystyrene-backed double membranes (negative-positive fixed-charge junctions) was studied in a two-compartment cell and compared to a known cellular system, the Ehrlich-Lettre ascites carcinoma. It was concluded that passage proceeds by a 1 : 1 exchange of diffusing ions in the membrane. The more rapidly transported systems reflected an increased probability of exchange in all cases, as evidenced both by a saturation effect and by the degree to which the space charge was perturbed at the membrane. The amino group (as NH₃⁺) of the amino acid involved in the exchange process was vital for transport. The presence of a second amino group, either ionized or as -NH₂, accelerated the exchange. The presence of electron-attracting groups on the side chain or of a methyl group on the alpha carbon also facilitated passage. Cation dependence was seen. Passage was hindered by a second carboxyl group, an alcoholic group, or a lengthened side chain. Use of double membranes permits experimental electrodic transport modelling and may facilitate design of a drug delivery system.     

Docking of oxalyl aryl amino benzoic acid derivatives into PTP1B

Protein Tyrosine Phosphatases (PTPs) that function as negative regulators of the insulin signaling cascade have been identified as novel targets for the therapeutic enhancement of insulin action in insulin resistant disease states. Reducing Protein Tyrosine Phosphatase1B (PTP1B) abundance not only enhances insulin sensitivity and improves glucose metabolism but also protects against obesity induced by high fat feeding. PTP1B inhibitors such as Formylchromone derivatives, 1, 2-Naphthoquinone derivatives and Oxalyl aryl amino benzoic derivatives may eventually find an important clinical role as insulin sensitizers in the management of Type-II Diabetes and metabolic syndrome. We have carried out docking of modified oxalyl aryl amino benzoic acid derivatives into three dimensional structure of PTP1B using BioMed CAChe 6.1. These compounds exhibit good selectivity for PTP1B over most of phosphatases in selectivity panel such as SHP-2, LAR, CD45 and TCPTP found in literature. This series of compounds identified the amino acid residues such as Gly220 and Arg221 are important for achieving specificity via H-bonding interactions. Lipophilic side chain of methionine in modified oxalyl aryl amino benzoic acid derivative [1b (a2, b2, c1, d)] lies in closer vicinity of hydrophobic region of protein consisted of Meth258 and Phe52 in comparison to active ligand. Docking Score in [1b (a2, b2, c1, d)] is -131.740Kcal/mol much better than active ligand score -98.584Kcal/mol. This information can be exploited to design PTP1B specific inhibitors.     

The preparation of amino bile acid derivatives

Bile acid derivatives have been prepared by reaction of their mixed anhydrides with diaminoethane. The new compounds have side chains modified by amide bond formation which extends the side chain by two carbon atoms and terminates in a primary amino group. Important properties of the new compounds are their solubility in acid, and their ability to act as nucleophiles in the carbodiimide reaction or mixed anhydride reaction. The derivatives have been used to prepare high molar ratio immunogens with bovine serum albumin and to prepare I labelled ligands for radioimmunoassay     

Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptides with fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.