The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole.

The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5-diazonium-(1H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr-53. This side chain is not accessible to the reagent in F-actin. The modification is compared to similar inhibitions by other reagents.     

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.     

Selective cleavage of N-terminal amino acid in a peptide by polymeric cobalt (III) triene complex

Cellulose was functionalized to incorporate triethylenetetramine group. This was in turn converted into the polymeric analogue of cobalt(III)triene complex. The polymeric complex reacts with peptides resulting in the cleavage of amino end amino acid, thus suggesting the applicability of the polymeric reagent as a solid phase reagent for N-terminal determination.     

Postcolumn fluorometric detection system for liquid chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-L-cysteine reagent

A high-performance liquid chromatographic system for the determination of amino acid and imino acid was developed using a two-step reaction with sodium hypochlorite and o-phthalaldehyde/N-acetyl-L-cysteine (OPTA/AcCys) reagent. This reagent improved the sensitivity in the analysis of proline presumably because the fluorophore is more stable to hypochlorite which has been used for the oxidative cleavage of the imino linkage. The use of OPTA/AcCys facilitated the detection of imino acids at the same concentration level as that of amino acids. The detection limit for all the amino and imino acids was a few picomoles. This detection system, together with cation-exchange chromatographic separation, was applied to the determination of amino and imino acids in biological samples.     

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.     

Free amino acid quantification by LC-MS/MS using derivatization generated isotope-labelled standards

The further development of derivatizing reagents for plasma amino acid quantification by tandem mass spectrometry is described. The succinimide ester of 4-methylpiperazineacetic acid (MPAS), the iTRAQ reagent, was systematically modified to improve tandem mass spectrometer (MS/MS) product ion intensity. 4-Methylpiperazinebutyryl succinimide (MPBS) and dimethylaminobutyryl succinimide (DMABS) afforded one to two orders of magnitude greater MS/MS product ion signal intensity than the MPAS derivative for simple amino acids. CD(3) analogues of the modified derivatizing reagents were evaluated for preparation of amino acid isotope-labelled quantifying standards. Acceptable accuracy and precision was obtained with d(3)-DMABS as the amino acid standards derivatizing reagent. The product ion spectra of the DMABS amino acid derivatives are diagnostic for structural isomers including valine/norvaline, alanine/sarcosine and leucine/isoleucine. Improved analytical sensitivity and specificity afforded by these derivatives may help to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization generated isotope-labelled standards a viable alternative to amino acids analysers.     

Solubilization of Feather Keratin by a Copper-amine Complex

Chicken feather keratin was solubilized by cupri-ethylenediamine treatment and the solubilized products were separated into acidic and basic fractions by ion exchangers. In the solubilized products which had a molecular weight between 10,000 and 60,000, all the original cystine residues disappeared and cysteic acid residues were recovered instead of them but partly. The cupri-ethylenediamine reagent which catalyzed air-oxidation of cystine residues in keratin was removable mostly from the products by dialysis against water. The common copper-amine complexes were ineffective to solubilize feather keratin except for Schweitzer’s reagent. One strongly basic, unusual amino acid was detected in the basic solubilized fraction. This amino acid was eluted after arginine by usual column chromatography.     

Synthesis of ketomethylene amino pseudopeptide analogues via reductive amination of glyoxals derived from alpha-amino acids

The reductive amination of an amino acid derived glyoxal, with the free amino group of a protected amino acid or oligopeptide fragment, has been developed as a simple and efficient method for the preparation of ketomethylene amino pseudo-oligopeptide isosteres Aa psi(COCH2NH)Aa. Trichlorosilane-DMF is the reagent of choice for the reduction.     

A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids

1. An acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine. 2. The method was highly sensitive for the determination of cysteine (in 28.0x10(3)). Homocysteine, glutathione, proline, ornithine and other naturally occurring amino acids tested did not give a similar reaction. 3. The reaction product was stable for at least 3-4hr. at room temperature and the extinction was proportional to the concentration in the range 0.05-0.5mumole of cysteine. 4. The acid ninhydrin reagent also gave yellow products (E(max.) 370-404mmu) with tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and indol-3-ylacetic acid. 5. The method was applied for the determination of cysteine in perchloric acid extracts of rat brain, liver and blood.     

Studies in C-terminal sequencing: New reagents for the synthesis of peptidylthiohydantoins

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Baileyet al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50°C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.     

The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.     

Detection of Protein-Bound Amino Groups in Plant Tissue by Schiff Base Formation and Azo Coupling

This account pertains to the detection of protein-bound amino groups in fresh, freehand sections of bean cotyledons. Two separate reagents were used: (A) p-aminobenz-aldehyde and (B) p-dimethylaminobenzaldehyde. Both of these compounds, by virtue of their free aldehyde groups, condensed with native amino groups to form their respective Schiff bases. The free amino groups of the resultant Schiff base with reagent A were diaz-otized and coupled with an arylamine, whereas the Schiff base produced in the sections treated with reagent B was treated with nitrous acid to yield its nitroso derivative which was finally coupled with H acid to produce a purple-red dye.     

An automatic analyzer for the specific determination of amino sugars

The techniques previously employed for the extraction and determination of amino acids from different matrices are not necessarily optimal for the determination of the amino sugars. An analytical system is described which is a hybrid between the conventional amino acid analyzer and the liquid chromatographic system for the detection of reducing sugars. The major, naturally occurring amino sugars are separated in about 40 min, with sensitivites lying under the nanomole range, without interference from other co-extracted compounds such as amino acids and sugars. The reagent employed is noncorrosive and stable over long periods of time. The amino sugar analyzer can be readily constructed by simple modification of a conventional phenylketonuria or amino acid analyzer.     

Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum

Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.     

采用质谱场解析法从氨基酸废水中发现肽类物质

猪毛酸解提取胱氨酸母液中,含有１７种游离氨基酸。采用酒精析出法,提取了氨基酸,同时采用质谱场解析法证实了还存在小分子肽类物质,它将可在日用化工工业中作为助剂应用。     

Cowpea mosaic virus VPg: sequencing of radiochemically modified protein allows mapping of the gene on B RNA

A partial amino acid sequence of cowpea mosaic virus (CPMV) VPg radiochemically modified by chloramine-T and Bolton-Hunter reagent has been determined. VPg covalently bound to viral RNA chains (VPg-RNA) was iodinated with chloramine-T and Bolton-Hunter reagent to label tyrosine and lysine residues, respectively. [¹²⁵I]VPg-RNA was digested with nuclease P1 and the resulting [¹²⁵I]VPg-pU was purified by SDS-polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Control experiments with chemically synthesized poliovirus VPg showed the feasibility of radiochemical microsequence analysis of protein that had been radiochemically modified by chloramine-T and Bolton-Hunter reagent. Analysis of CPMV [¹²⁵I]VPg-pU revealed the presence of tyrosine residues at position 12 and 14, and of lysine residues at position 3 and 20, respectively. In combination with Edman degradation of unlabeled CPMV VPg, which showed serine and arginine residues to be present at position 1 and 2, respectively, the data obtained allow the precise positioning of VPg within the 200 000 dalton (200 K) polyprotein encoded by CPMV B RNA and the prediction of its entire amino acid sequence. VPg is located at the COOH terminus of its 60 K, membrane-bound,precursor and proximal to the amino terminus of the protease-polymerase domain of the polyprotein. A processing scheme for the 200 K polyprotein is discussed in which Gln-Ser amino acid pairs act as the major signal for proteolytic cleavage.     

Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.     

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.     

Dimethylmaleic anhydride, a specific reagent for protein amino groups

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.     

Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean.

A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.