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At least 19 recordsLinked to original sources
PubMed1979

Methylthiohydantoin amino acids: chromatographic separation and comparison to phenylthiohydantoin amino acids.

Most phenylthiohydantoin (PTH) amino acids and most methylthiohydantoin (MTH) amino acids may be separated from one another by thin-layer chromatography (TLC) using the same sequential development technique with the same two solvents. Similarly, a single solvent system may be used in high-performance liquid chromatography (HPLC) to separate most PTH-amino acids and most MTH-amino acids. When both TLC and HPLC separations are performed on a sample, all MTH-and PTH-amino acids can be uniquely identified. Since many solid-phase protein sequencing techniques generate both MTH-and PTH-amino acids, these analytical systems simplify identification of the amino acid derivatives. Although the chromatographic properties of MTH-and PTH-amino acids are similar, they are not identical (contrary to a previous report).

Amino Acids↗
PubMed1979

[The separation of the 4-[4-(dimethylamino) phenylazo]phenylthiohydantoin derivatives of leucine and isoleucine on polyamid thin-layer sheets in the picomol range (author's transl)].

The separation of the 4-[4-(dimethylamino)-phenylazo]phenylthiohydantoin derivatives of leucine and isoleucine has so far not been possible. In this paper a method is described which separates these two amino acid derivates in the picomol range on polyamide thin-layer sheets. The system (10% formic acid/ethanol 10:9) can be used either in one or in two dimensions.

Chromatography, Thin Layer↗
PubMed1975

Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography.

A method for the separation of all phenylthiohydantoin (PTH)-amino acids except PTH-arginine and PTH-histidine by high-pressure liquid chromatography on a silica column is described. Elution is performed with a concave solvent gradient from hexane-methanol-propanol (3980:9:11) to methanol-propanol (9:11). A complete run is achieved in 40 min with a pressure drop of 1000 p.s.i. over the 250 mm X 2.1 mm column. Eluted peaks of 2-5 nmole are easily detected by their ultraviolet absorption at 254 nm. This method is superior to existing gas-liquid and thin-layer chromatographic techniques since all PTH-amino acids except PTH-arginine and PTH-histidine may be both separated and quantitated in a single run of 40 min. The use of the technique in conjunction with an automated peptide sequence analyser is illustrated.

Adrenocorticotropic Hormone↗
PubMed1978

Primary structure of the major beta-chain of rat haemoglobins.

The amino acid sequence of the major beta-chain, (II)beta, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the epsilon-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.

Amino Acid Sequence↗
PubMed1975

Rapid identification of amino acid derivatives from the sequenator.

A simple procedure is described for converting thiazolinones, from the protein sequenator, to the more stable phenylthiohydantoins without using the conventional HCI conversion procedure. The thiazolinones are applied to a silica gel plate and converted to phenylthiohydantoins by heating at 140 for 5-10 min, in the presence of heptafluorobutyric acid, prior to chromatography. After chromatography, measurements of the yields of the derivatives on the plate can be made with a variable-wave-length thin-layer chromatography scanner. This is shown to be a useful adjunct for identifications.

Amino Acid Sequence↗
PubMed1977

High-pressure liquid chromatography of amino acids and dipeptides on a tripeptide bonded stationary phase.

A bonded tripeptide (L-valyl-L-phenylalanyl-L-valine) phase has been used as a stationary phase in liquid chromatography. Unique retention orders and retention variations are shown for phenylthiohydantoin (PTH) derivatives of amino acids and isomeric dipeptides using acidic and basic mobile phases. It is shown that the 25 PTH-amino acids studied here have different capacity ratios when 1% citric acid in water (ca. pH 2.5) is used as the mobile phase. The analysis time for these amino acid derivatives could be shortened without a loss in the resolution by adding 5% methanol to the above mobile phase. Of particular importance is the fact that the basic amino acids are eluted first. Isomeric dipeptides have been resolved by using deionized water (pH 5.5), and 1% sodium citrate in water (pH 7.9), as mobile phases. The data show that the nature of the mobile phases, the pH, and the ionic strength are the important factors affecting the selectivity and efficiency of the separations of amino acids and dipeptides.

Amino Acid Sequence↗
PubMed1979

Manual solid phase sequence analysis of polypeptides using 4-N-N,-dimethylaminoazobenzene 4'-isothiocyanate.

A manual solid-phase method for sequence analysis of polypeptides is described. The immobilized polypeptide was subjected to stepwise degradation by Edman-type reagent, using the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate phenyllisothiocyanate double coupling method. The N-terminal amino acids were released (after conversion reaction) as 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin (identified by thin layer chromatography) and phenylthiohydantoin derivatives. The method required 2--10 nmol polypeptide.

Amino Acid Sequence↗
PubMed1978

Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (polybrene), and high pressure liquid chromatography.

We have combined the use of a nonprotein carrier (Polybrene), high pressure liquid chromatography, and modifications in Edman chemistry with the improvements of a commercial spinning cup sequenator suggested by Wittmann-Liebold [Wittmann-Liebold, B. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 1415] to analyze amino acid phenylthiohydantoins obtained from automated Edman degradation of microquantities of polypeptide directly without the use of radiolabel. This approach has allowed us to determine the sequence of the N-terminal 47 residues of sperm whale myoglobin starting with 200 pmol of protein, 77 residues of an antibody light chain with 5 nmole of protein, and 54 residues of an antibody heavy chain with 8 nmol of protein. In addition, we completely sequenced a hydrophobic 14-residue peptide at the 1.5-nmol level. Our technique of direct analysis for microsamples is capable of providing routine, extende N-terminal sequence analysis for nanomole and subnanomole levels of polypeptides and protines, and it also is applicable to analysis of more classical sample quantities.

Amino Acid Sequence↗
PubMed1977

Amino acid sequence of the precursor region of MOPC-315 mouse immunoglobulin heavy chain.

Partially purified mRNA coding for the MOPC-315 heavy immunoglobulin alpha chain was translated in a reticulocyte lysate containing 20 labeled amino acids. Radiolabeled MOPC-315 heavy chain precursor protein, purified by preparative gel electrophoresis and immunoprecipitation, was sequenced by Edman degradation. The labeled phenylthiohydantoin amino acid obtained in each cycle was identified and quantitated by high-pressure liquid chromatography. The precursor sequence of 18 amino acids, Met-Lys-Val-Leu-Ser-Leu-Leu-Tyr-Leu-Leu-Thr-Ala-Ile-Pro-His-Ile-Met-Ser, preceded the sequence corresponding to the NH2 terminus of the mature secreted heavy chain.

Amino Acid Sequence↗
PubMed1979

Characterization of alkylamine-sensitive site in alpha 2-macroglobulin.

Methylamine reacts with the plasma protease inhibitor, alpha 2-macroglobulin, to form an irreversible, covalent modification. Quantitation of the reaction indicates 3.9 +/- (SD) 0.4 reactive sites per native tetrameric protein (Mr = 725,000) or one site per subunit. The reaction is selective and specific in that only 1 or 2 labeled peptides are observed on radioautography of peptide maps derived from [14C]methylamine-treated alpha 2-macroglobulin. A single chymotryptic peptide was isolated in 56% overall yield from the labeled protein. The peptide sequence by Edman degradation was found to be Gly-Cys-Gly-Glu-X-Asn-Met-(Val, Leu), in which X was the only radiolabeled phenylthiohydantoin derivative. Amino acid analysis and mass spectral analysis of the derivative suggests that X is gamma-glutamylmethylamide. Because glutamic acid and glutamine residues do not normally react with alkylamines, this work presents presumptive evidence for an alternative activated center in selected proteins.

Binding Sites↗
PubMed1978

Amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase.

The amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase was determined in order to compare it with those in phosphorylases from other sources having different regulatory properties. The potato enzyme was reduced by NaBH4 in the presence of urea, carboxymethylated, and digested with chymotrypsin and trypsin. Pyridoxyl peptides were isolated by the differential procedure using paper electrophoresis or DEAE-cellulose column chromatography. In Edman degradation of these peptides, pyridoxyllysine was identified as the phenylthiohydantoin derivative of pyridoxyllysine using a combination of thin-layer chromatography and the Pauli reaction. The sequence around pyridoxyllysine, comprising 57 amino acid residues, was determined except for a region with 6 amino acid residues. The pyridoxal 5'-phosphate binding site in potato phosphorylase showed a high homology with those of the rabbit muscle and yeast enzymes. This finding suggests that the cofactor should be directly related to the essential process of phosphorylase action.

Amino Acid Sequence↗
PubMed1978

The primary structure of bovine pancreatic phospholipase A2.

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.

Amino Acid Sequence↗
PubMed1979

Precursor sequence of MOPC-315 mouse immunoglobulin heavy and light chains.

Partially purified mRNA coding for the MOPC-315 heavy (alpha) or light (lambda 2) immunoglobulin chain was translated in a nuclease-treated reticulocyte lysate containing 20 labeled amino acids. Radiolabeled precursor heavy and light chains, purified by immunoprecipitation and preparative gel electrophoresis, were subjected to Edman degradation. The labeled phenylthiohydantoin derivatives obtained in each degradative cycle were identified and quantitated by high pressure liquid chromatography. Both heavy and light chain precursor segments were hydrophobic in nature; however, they were not homolgous in sequence. To establish whether COOH-terminal proteolytic processing of the heavy chain might also be occurring during secretion, the cyanogen bromide peptides of the heavy chain precursor were compared to those of the mature secreted heavy chain. The results indicated that the COOH termini of the two chains were identical.

Amino Acid Sequence↗