Determination of DOPA,dopamine, DOPAC,epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. By using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxypehnylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). α-Difluoromethyldopa (DFMD) and α-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.     

Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS.     

反相高效液相色谱法测定血浆游离氨基酸

本文报告了采用高效液相色谱法反相梯度洗脱,邻苯二甲醛和β-巯基乙醇柱前衍生化,荧光检测分血浆游离氨基酸。实验采用线性洗脱,在50分钟内可同时测定18种氨基酸,血浆样品的预处理简单,衍生化反应的时间仅需1分30秒,血浆样品的实际进样量少于1μl。本测定方法的精确度高,各个氨基酸保留时间的变异系数平均为0.89%±0.45%(SD),峰面积的变异系数平均为2.06%±1.76%(SD),各个氨基酸的浓度在15—150μmol/L的范围中,线性关系的相关系数平均为0.985±0.0305(SD)。准确性好,各个氨基酸的回收率平均为97.6%±5.1%(SD)。实验还讨论了氨基酸分离时溶液pH值、柱温、离心速度等因素对分析结果的影响。     

Isolation, cloning, and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium

By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.     

Analysis of (3-phenyl,2-thio)hydantoin amino acids by high-performance liquid chromatography: Comparison of three programs with particular reference to the glutamic and aspartic derivatives

(3-Phenyl,2-thio)hydantoin-amino acids are readily separated and quantitated by high-performance liquid chromatography (hplc). Three reversed-phase column procedures were investigated. We found the aspartic and glutamic acid derivatives difficult to separate and quantitate by any of the three until we determined the effects on hplc of several injection solvents and the pH of the elutrient buffers. We found 90% acetic acid or acetonitrile: 90% formic acid (8:2, v:v) satisfactory as injection solvents. Each of the three hplc programs also required pH changes for optimal separation of the aspartic and glutamic acid derivatives from the other (3-phenyl,2-thio)hydantoin-amino acids; none of these had retention times that varied with pH in the ranges investigated. Using the three modified systems, the retention times of 19 commonly found (3-phenyl,2-thio)hydantoin-amino acids were compared. In addition, the N-terminal residues of two model human plasma proteins were chromatographed, fibrinogen and plasminogen. In all of these, the aspartic and glutamic acid derivatives were clearly separable without diminishing resolution of the other amino acids.     

气相色谱-质谱法测定中药沙苑子中的氨基酸

本文利用气相色谱一质谱(GC/MS)联用技术首次对中药沙苑子中的氨基酸进行了定性定量分析。沙苑子经盐酸水解后,对其氨基酸水解液进行羟基脂化和氨基酰化的衍生化反应,利用GC/MS法共测得15种氨基酸,由外标法测定了每种氨基酸的含量。由此得出沙苑子中氨基酸的总质量分数为4.23％。     

Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M(r) of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.     

Immunochemical and biological properties of synthetic peptide fragments from the 124-144 sequence of human leukocyte interferon alpha2]

Three peptides corresponding to the sequences 124-144, 124-138, 129-144 of the human leukocyte interferon alpha 2 (IFN-alpha 2) were synthesized. The synthesis was performed by DCC-HOBT coupling of protected peptide segments in solution. The segments were obtained by the active ester coupling methodology using base-labile 2-[4-(phenylazobenzyl)sulfonyl]ethyl (Pse) group as carboxyterminal protection. After complete deprotection with 1 M methanesulphonic acid in trifluoroacetic acid--thioanisol--m-cresol mixture the peptides were purified by reversed-phase chromatography. The studies of interaction of the peptides with rabbit antiserum against IFN-alpha 2 revealed at least one minor antigenic determinant within the 124-144 region of IFN-alpha 2 amino acid sequence. Rabbit antisera developed against peptides 124-138 and 129-144 showed ability of binding recombinant IFN-alpha 2 and neutralizing its antiviral activity. Free peptides or their conjugates with bovine serum albumine did not display antiviral activity, neither could they inhibit the activity of IFN-alpha 2.     

The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns

Amino acids were reacted with o-phthalaldehyde and 2-mercaptoethanol and were separated using a simple linear gradient from 10 to 65% methanol over 15 min on an octyl silica (C8) column by reversed-phase chromatography. The separation obtained was found to be sensitive to the pH, ionic strength, and tetrahydrofuran concentration of aqueous solvent A [THF: sodium acetate (45 mM), pH 5.7, (4:96)]. These effects were characterized and used to design a rapid (17 min) separation of the amino acids commonly found in acid hydrolysates of proteins. A more involved procedure was used to separate the more complex mixture of amino acids that are found in enzymatic hydrolysates of proteins or in physiological fluids. The simplicity of the methods allows their use on different chromatographic systems with little or no alteration.     

Organics in chimneys and water samples from deep-sea hydrothermal systems: implications for sub-vent biosphere.

Searching for life in extreme terrestrial environments can be a model of that for extraterrestrial life. Submarine hydrothermal system is one of promising sites for the frontier of life on the earth. Here seawater and vent chimnies were collected from deep-sea hydrothermal vents at Suiyo Seamount, Izu-bonin arc, Pacific Ocean as a part of Archaean Park Project. Pure seawater sample of 300 degrees C (purity>97%) could be collected. Dissolved and total hydrolyzable amino acids were determined by ion-exchange HPLC, and their enantiomeric ratio was measured by reversed-phase HPLC for the first time. Glycine and serine were two most abundant amino acids, followed by other proteinous amino acids such as alanine, glutamic acid and aspartic acid. Non-proteinous amino acids were detected as minor constituents. Most of the amino acids detected were of the L-form. Thus amino acids of abiotic origin were quite minor, and most of the amino acids detected were formed biologically. These results, together with analytical results of the vent chimney samples, suggest that there is active microbial activities near the hydrothermal systems.     

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.     

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.     

Isolation, partial characterization and mode of action of acidocin J1229, a bacteriocin produced by Lactobacillus acidophilus JCM 1229

Lactobacillus acidophilus JCM 1229 produces a heat-stable bacteriocin, designated as acidocin J1229, that has a narrow inhibitory spectrum. Production of acidocin J1229 in MRS broth was pH dependent, with maximum activity detected in broth culture maintained at pH 5:0. Acidocin J1229 was purified by ammonium sulphate precipitation and sequential cation exchange and reversed-phase chromatographies. The sequence of the first 24 amino acid residues of the N terminus of acidocin J1229 was determined. The molecular mass of acidocin J1229 as determined by mass spectrometry was 6301 Da. Acidocin J1229 showed a bactericidal effect but not a bacteriolytic effect on sensitive cells. Acidocin J1229 dissipated the membrane potential and the pH gradient in sensitive cells, which affected such proton motive force-dependent processes as amino acid transport. Acidocin J1229 also caused an efflux of glutamate, previously taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic a-helix region that could form hydrophilic pores. These results suggest that acidocin J1229 is a pore-forming peptide that creates cell membrane channels through the 'barrel-stave'mechanism.     

Effect of various amino acids and ammonium salts in a synthetic culture media on the cholera enterotoxin production

The influence of amino acids and ammonium salts on the production of cholera enterotoxin (CT) by 3 Vibrio cholerae strains of different biovars and serogroups was evaluated. As revealed in this study, toxin formation in each of the strains was quantitatively and qualitatively determined by their individual sets of amino acids. The amino acid compositions ensuring the maximum production of CT by the V. cholerae strains under study were formed. The use of ammonium salts as the only source of nitrogen in the composition of a synthetic nutrient medium for the accumulation of CT was shown to be inexpedient.     

Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects

Understanding the hydrophilicity/hydrophobicity of amino acid side chains in peptides/proteins is one the most important aspects of biology. Though many hydrophilicity/hydrophobicity scales have been generated, an "intrinsic" scale has yet to be achieved. "Intrinsic" implies the maximum possible hydrophilicity/hydrophobicity of side chains in the absence of nearest-neighbor or conformational effects that would decrease the full expression of the side-chain hydrophilicity/hydrophobicity when the side chain is in a polypeptide chain. Such a scale is the fundamental starting point for determining the parameters that affect side-chain hydrophobicity and for quantifying such effects in peptides and proteins. A 10-residue peptide sequence, Ac-X-G-A-K-G-A-G-V-G-L-amide, was designed to enable the determination of the intrinsic values, where position X was substituted by all 20 naturally occurring amino acids and norvaline, norleucine, and ornithine. The coefficients were determined by reversed-phase high-performance liquid chromatography using six different mobile phase conditions involving different pH values (2, 5, and 7), ion-pairing reagents, and the presence and absence of different salts. The results show that the intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides (proteins) is independent of pH, buffer conditions, or whether C(8) or C(18) reversed-phase columns were used for 17 side chains (Gly, Ala, Cys, Pro, Val, nVal, Leu, nLeu, Ile, Met, Tyr, Phe, Trp, Ser, Thr, Asn, and Gln) and dependent on pH and buffer conditions, including the type of salt or ion-pairing reagent for potentially charged side chains (Orn, Lys, His, Arg, Asp, and Glu).     

Hybridoma growth,metabolism, and product formation in HEPES-buffered medium: II. Effect of pH

The effects of pH on cell metabolism during the batch growth of hybridomas in flasks were evaluated. Maintaining the pH at 7.2 resulted in a reduction of the maximum antibody and viable cell concentrations by ca. 40% and increased glucose and amino acid metabolic quotients. When the pH was allowed to fall to 6.7 there was further growth and antibody production after glutamine was exhausted using branched-chain amino acids as substrates. Y'[lac/gluc] increased from 1.32 at pH 6.7 to 1.50 at pH 7.2.     

HPLC analysis of free amino acids and amino acids of total proteins in cultured cells: an application to the study of rat Sertoli cell protein metabolism.

A simple, rapid and, sensitive HPLC method, coupled with fluorometric detection, has been worked out and employed to determine the intracellular free amino acid concentrations and the amino acid composition of total proteins in rat Sertoli cell primary culture. Sertoli cells were isolated enzymatically from testes of 20- and 28-day-old rats and cultured at 32 degrees C in Eagle's minimum essential medium. On the second day of culture, cell monolayers were quickly rinsed with ice-cold saline, immediately frozen in liquid nitrogen, accurately harvested, and homogenized in 10% trichloroacetic acid. Tissue free amino acids were determined in the acidic soluble fraction following neutralization, while the precipitate was hydrolyzed for the evaluation of the fractional content of amino acids into total proteins. Amino acid samples were derivatized with o-phthaldialdehyde/3-mercaptopropionic acid and resolved by a linear one-step acetonitrile gradient in 12.5 mM sodium phosphate buffer, pH 7.2, employing a 5-microns particle size reversed-phase column. Fluorescence was monitored with excitation at 330 nm and emission at 450 nm. Under these conditions all major physiological amino acids could be satisfactory separated, identified, and subsequently quantified with the aid of standards. The run time was about 50 min; the linearity was excellent over a large range of concentrations (1-800 pmol) and the lower limit of sensitivity appeared to be 0.5 pmol. This method permits us to demonstrate age-dependent modifications in the intracellular amino acid pool and to adequately evaluate the process of protein synthesis in cultured Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of gliadins from Aegilops squarrosa seeds

The major gliadin components were isolated from the seeds of the diploid species Aegilops squarrosa, a putative source of polyploid wheat D-genome. The isolation procedure included gel-filtration and reversed-phase high-performance liquid chromatography (HPLC). The purified proteins were characterized by electrophoretic mobility in polyacrylamide gel using acid Al-lactate system and a system containing sodium dodecyl sulfate. The amino acid composition of isolated omega-gliadins was determined. Using covalent chromatography on thiopropyl-Sepharose 6B it was found that omega-gliadins of A. squarrosa contain no SH-groups and/or S-S-bonds. The N-terminal amino acid sequences of A. squarrosa gliadins were determined. omega-Gliadins were found to contain three types of N-terminal amino acid sequences, one of which, SRQ, in hexaploid wheat is encoded by 1B chromosome. It was shown that some omega-gliadins of A. squarrosa have blocked N-terminal amino acids. The major component of the gamma-fraction was found to contain an N-terminal sequence of gamma 2 type encoded in polyploid wheat by 1D chromosome. Gliadins with electrophoretic mobility in the beta-zone of the spectrum possess the N-terminal sequence of alpha-type. The results obtained are discussed in terms of the origin of polyploid wheat genomes.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS

Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 μL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6-10.1%. Absolute accuracy as determined by comparison of external reference samples was 82-117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 μmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.