Conventional Amino Acid Analysis Research Articles

A micro procedure for the estimation of the protein content of mucus glycoproteins

A sensitive automated procedure for the estimation of the protein content of glycoproteins has been developed using 0.5-mg samples (10–250 μg of protein). The method employs a modification of the conventional amino acid analysis using the unresolved neutral and acidic amino acid peak to estimate the total protein content. Hexosamines, sialic acid, and amide amino acids do not interfere.

A rapid chromatographic method for the determination of omega-N-methylarginines in protein and muscle tissues.

A rapid and sensitive chromatographic method is described for the complete separation and determination of NG-methylarginine, NG,NG-dimethlarginine, NG,N'G-dimethylarginine, and related compounds in protein and muscle tissue hydrolysates. This method is designed to be used both with conventional amino acid analyzers using a single column (10 X 0.9 cm) of Durrum type DC-6A resin at 51 degrees C, one buffer system (0.21 M sodium citrate pH 5.40), and one buffer flow rate (70 ml/h) and with the accelerated and fully automatic Beckman Spinco model 121M amino acid analyzer using a 10 X 0.28 cm microcolumn packed with the Beckman type AA-10 resin and operated with the same buffer at 7.9 ml/h. This procedure was successfully applied to the determination of omega-N-methylarginines in various animal tissues, and the presence of four as yet unidentified ninhydrin-positive compounds is reported.

Improved chromatographic separations on anion-exchange resins: II. Separation of uronic acids in acetate medium and detection with a noncorrosive reagent

An ion-exchange chromatographic system is described which is capable of separating complex uronic acid mixtures in about 2.5 hr. The system has the following advantages: (i) The resin is commercially available; (ii) no column temperature or buffer changes are needed during a run; (iii) the detection reagent is highly sensitive and also noncorrosive; and (iv) organic contaminants such as amino acids and reducing monosaccharides do not interfere with the separations. Several hundred samples can be run without column regeneration or equilibration. Furthermore, the system can be easily built by modification of existing conventional amino acid or sugar analyzers.

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.

Hair as an index of protein malnutrition.

Hair samples from seven sick Ghanaian children were analyzed for amino acids. Cystine was determined by a procedure of Friedman using tributylphosphine and 2-vinylpyridine to change residues of cystine (and cysteine, if present) to S-beta-(2-pyridylethyl)-L-cysteine (2-PEC). This acid-stable derivative is released by normal acid hydrolysis and is eluted as a well-resolved peak before lysine in conventional ion-exchange amino acid analysis. The average cystine content of six children suffering from kwashiorkor or marasmic kwashiorkor was found to be about 20% less than that of the one remaining child, whose protein nutrition was judged adequate. In view of conflicting evidence of the relation of hair cystine content and nutrition, we believe further definitive studies of this subject are urgent. No other substantial difference in amino acid composition was noted.

Tryptic peptide analysis of normal and mutant forms of hypoxanthine phosphoribosyltransferase from HeLa cells.

Hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) can be purified 5-to 10,000-fold from extracts of HeLa (human) cells by a three-step procedure consisting of high-speed centrifugation, adsorption to Sepharose-conjugated HPRT antibody, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Purified enzyme labeled in vivo with radioactive lysine, arginine, or methionine was digested with trypsin and the tryptic peptides were separated by column chromatography on Bio-Rad cation exchanger Aminex A-5. Less than 50 ng (2 pmol) of HPRT is required to produce a tryptic peptide pattern. A methionine-labeled peptide was identified as the COOH-terminus because it was not labeled with either lysine or arginine. We have compared the tryptic peptide patterns of normal HeLaHPRT and a crossreacting HPRT protein lacking enzyme activity from HeLa mutant H23 [Milman et al. (1976) Proc. Natl. Acad. Sci. USA 73, 4589--4593]. The mutant protein has a new lysine-labeled peptide, but the chromatography patterns of arginine- or methionine-labeled peptides appear identical to those of the normal protein. The appearance in the H23 mutant HPRT protein of a new tryptic peptide provides strong evidence for a mutation in the HPRT structural gene. The tryptic peptide patterns were used to determine the total number of residues of labeled amino acid in the protein, and the values are reasonably consistent with those determined by conventional amino acid analysis pf erythrocyte HPRT.

The use of o-phthalaldehyde for fluorescence detection in conventional amino acid analyzers : Sub-nanomole sensitivity in the analysis of phenylthio-hydantoin-amino acids

A conventional amino acid analyzer has been modified for fluorescence detection using o-phthalaldehyde. The original ninhydrin system is retained, and the amino acid analyzer can be used with either of the detection systems. The fluorescence detection is used for identification of PTH-amino acids after back hydrolysis, when nanomole amounts of proteins are sequenced. Impurities in the water and the chemicals used for buffer preparations are limiting factors for the sensitivity of the fluorescence detection system. A 100-fold increase in sensitivity above the original ninhydrin system is obtained.

GAS CHROMATOGRAPHIC ANALYSIS OF SILK AND WOOL TREATED WITH ARYL GLYCIDYL ETHERS

Silk and wool fibers were treated with phenyl glycidyl ether (PGE) or p-tolyl glycidyl ether (TGE) in the presence of aqueous neutral salt according to Shiozaki and Tanaka, and the gas-liquid chromatography (GC) of the hydrolysates of the treated silk and wool fibers were studied for the identification and quantification of the artifact amino acids produced. The GC analysis was carried out by the linear temperature-programmed method on OV-17 and on Dexsil 300 GC columns, after the amino acids in the hydrolysate being converted to n-butyl esters of N-trifluoroacetyl (BTFA) derivatives. The peaks of O-(2-hydroxy-3-phenoxypropyl)tyrosine (PGE-TYR) and O-(2-hydroxy-3-p-tolyloxypropyl)tyrosine (TGE-TYR), which were not detected with a conventional automatic amino acid analyzer, were observed in the gas chromatograms of the silk and wool fibers treated with, PGE and TGE, respectively, and the Kovats retention indices of these peaks were determined. The Δ I values of PGE-TYR and TGE-TYR were about equal to each other and much higher than those of O-2-hydroxypropyltyrosine and O-2-hydroxybutyltyrosine, previously reported. The retention indices of TGE-TYR were the highest among those of amino acids ever reported. The GC analysis of the PGE-and TGE-treated wool fibers also indicated that 2, 3-dihydroxypropyl phenyl ether and 2, 3-dihydroxypropyl p-tolyl ether were present in their acid hydrolysates, respectively. This finding clearly demonstrates that, in addition to the tyrosine residues, the acidic amino acid residues in wool react with aryl glycidyl ethers.

High-performance liquid chromatographic analysis of amino acids and peptide-hormone hydrolysates in the picomole range

A high-pressure liquid chromatographic system for the separation of amino acids and peptide-hormone hydrolysates is described. A modified high-pressure liquid chromatograph (Hewlett-Packard, model 1010B) equipped with an automatic gradient-generating system, and the applications of luorescamine and ion-exchange resins of particle size 8 ± 2 μm permit the detection of amino acids in the picomole range with good resolution. Compared with conventional amino acid analyzers, the times for separation are reduced by a factor of 3–4.

Collagen reduction by sodium borohydride: Effects of reconstitution, maturation and lathyrism

This chapter emphasizes on the methods for detecting and quantifying the mature hydroxypyridinium cross-links, but also describes the procedures for quantifying them together with the borohydride-reducible cross-linking residues in the same sample of collagen. To measure cross-linking amino acids in a collagen hydrolyzate using a conventional amino acid analyzer with ninhydrin detection, the bulk amino acids must be largely removed to avoid plugging the reaction coil. Tissue is thoroughly reacted with sodium borohydride to convert all intermediate cross-linking residues to their reduced, acid-stable forms. More vigorous reduction conditions may be used than when preparing profiles of tritium-labeled cross-links using NaB3H4 and direct amino acid analysis. This chapter presents the direct quantitation of hydroxypyridinium cross-links in tissue hydrolyzates by HPLC and fluorescence detection. The following method based on reverse-phase high-performance liquid chromatography can resolve and quantify the HP and LP forms of hydroxypyridinium residue down to 1 pmol directly in hydrolyzates of whole connective tissue, without any preliminary clean-up or enrichment step.