Phenylthiohydantoin Amino Acids Research Articles

A device coupled to a modified sequenator for the automated conversion of anilinothiazolinones into PTH amino acids

A device for the automated conversion of anilinothiazolinones of amino acids to their phenylthiohydantoin derivatives has been incorporated into a modifled Beckman sequenator. The construction of the device and its cyclic operating principle are described, and a program for its operation is given. The automated conversion procedure has been used in the sequenator for 4 years, and its significant advantages over manual conversion with respect to the improved yields and purity of phenylthiohydantoin amino acids are discussed.

Advances in the analysis of amino acid phenylthiohydantoins by high performance liquid chromatography

All the organic soluble amino acid phenylthiohydantoins are eluted (but not totally resolved) in under 40 min on either a Zorbax ODS or Permaphase ETH column. The superior resolution of the Zorbax ODS column and the complimentary characteristic of the Permaphase ETH column allow the isocratic analysis of 15 phenylthiohydantoins in less than 20 min when the columns are operated simultaneously. The water soluble PTH-His and -Arg are analyzed separately in under 10 min with the Zorbax ODS column. Five picomoles of a derivative may be detected with a sample to noise ratio of 6, HPLC is the most sensitive, practical method for PTH analysis.

High-pressure liquid chromatographic analysis of amino acid phenylthiohydantoins: Comparison with other techniques

High-pressure liquid chromatography, utilizing reverse phase μ Bondapak C18 columns and elution with increasing acetonitrile concentrations, has been used to resolve amino acid phenylthiohydantoins obtained from the automated Edman degradation of proteins. Assignment of identity to residues which are difficult to distinguish or identify conclusively by other conventional techniques is easily achieved by high-pressure liquid chromatographic techniques. The use of high-pressure liquid chromatography, in parallel with gas-liquid and polyamide thin-layer chromatography, allows unequivocal assignments of identity to amino acid phenylthiohydantoins obtained in protein sequencing. Single protein sequence determinations can be extended by 20 to 100% by the use of high-pressure liquid chromatography with rapid, accurate, and quantitative identifications of amino acid phenylthiohydantoins.

High sensitivity amino acid sequence determination. Application to proteins eluted from polyacrylamide gels.

An automatic solid-phase procedure is described for determining the amino-terminal amino acid sequence of very small quantities of proteins. The sample is covalently attached to an inert support so that mechanical and physical losses during sequencing are eliminated. High sensitivity is achieved by using an initial coupling with high specific activity phenyl [35S]isothiocyanate followed by a longer reaction with the unlabeled reagent. The radioactive phenylthiohydantoins are identified by autoradiography after two-dimensional thin-layer chromatography. Unlabeled phenylthiohydantoin-amino acids are added to each fraction to assist in the identification and to act as carriers, hence reducing absorptive and extractive losses of the small quantities of sample. The method may be used on proteins eluted from polyacrylamide gels containing sodium dodecyl sulfate without removal of the detergent. Sequences of up to 20 residues have been obtained on quantities of protein ranging from 2.5 to 70 pmol. Results from proteins of hitherto unknown sequence are included.

Investigation of the covalent bond structure of peptide-matrix systems by Edman degradation of support-fixed peptides.

Two model peptides Ala-Ser-Asp-Tyr-Leu and Ala-Ser-Glu-Tyr-Leu have been synthesized and coupled with an without protecting groups to the amino groups of polystyrene resins. The yield in the binding operation was 93-97% by using water-soluble carbodiimides as condensing reagents. Edman degradation of the products and quantitative estimation of the amino acid phenylthio-hydantoins, as well as amino acid analysis of the partially and completely degraded resins, showed about 7-10% of the fixed peptides not to be involved in the degradation procedure. That part of the material which was not involved in the Edman degradation during the first step also remained inactive in the following degradation operations. The analytical data presented in the paper explain not only the course of the Edman degradation of peptides linked via the side-chain carboxylic groups to the resin, but they also describe the chemical reactivity of the various carboxylic groups in the binding operation. 22% of the peptides are bound to the resin by side-chain carboxylic groups in the case of the aspartate-containing peptide, 26-27% in the case of the glutamate-containing peptide. 75-77% and 72-75% respectively of the peptides are fixed to the resin via the C-terminal carboxylic group. No more than 3% of the fixed material may be bound bifunctionally to the support.

A simple and inexpensive high pressure liquid chromatographie system as an adjunct to gas chromatography in identification of amino acid phenylthiohydantoins

A simple and inexpensive system has been developed in our laboratory for the identification and quantitation of amino acid phenylthiohydantoins by high pressure liquid chromatography. Isocratic methodology has been employed, so that expensive complex gradient makers can be avoided. The method is rapid, and retention times of 10 min or less are obtained in all but one case. This paper presents the rationale and details of the method, and demonstrates a comparison between gas-liquid chromatograms and high pressure liquid chromatograms of selected residues from actual automated sequence analyses of both sperm whale myoglobin and egg white lysozyme. The method is intended to complement rather than replace gas-liquid chromatography, as it cannot completely resolve all 20 phenylthiohydantoins, but rather identifies easily those residues which are difficult or impossible by gas chromatography.

The complete amino acid sequences of both subunits of the sweet protein monellin.

The amino acid sequences of both chains of the sweet protein Monellin have been determined. Since chain separation could not be accomplished easily, cyanogen bromide cleavage at the only methionine residue (in the B-chain) was performed and the three products obtained after cyanogen bromide cleavage were separated. For the identification of amino acid phenylthiohydantoins, high performance liquid chromatography was employed. Thus 37 out of a total of 44 residues of the A chain and 40 out of a total of 42 residues of the large CNBr fragment of the B chain could be determined after Edman degradation of the polypeptides on an automated sequenator.

The sequence determination of a protein in a micro scale: the sequence analysis of ribosomal protein L34 of Escherichia coli.

The complete amino acid sequence of ribosomal protein L34 has been established by improved micro techniques with 3 mg of the lyophilized protein. The protein was digested with trypsin, thermolysin and chymotrypsin and the resulting peptides were isolated from fingerprints performed on cellulose thin-layer plates. The amino acid sequences of the peptides were determined by the combined micro dansyl-Edman technique using 5 - 10 nmol per sample. Aspartic acid and glutamic acid were distinguished from their amides by use of the color reaction of ninhydrin with the respective amino acid phenylthiohydantoins.

Separation of phenylthiohydantoin-amino acids by highpressure 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 rain with a pressure drop of 1000 p.s.i. over the 250 mm × 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.

Crotalus adamanteus phospholipase A 2-α: Subunit structure, NH 2-terminal sequence, and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A 2-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A 2-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH 2-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH 2-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.

A rapid and quantitative determination of amino acid phenylthiohydantoins by direct densitometry on thin-layer chromatogram

Thin-layer chromatograms on silica gel plate of amino acid phenylthiohydantoins were simultaneously used for identification and quantitation. With a scanning, two-wavelength densitometer, linear calibration curves have been obtained for 15 different derivatives, which can be employed for a rapid and simple quantitation of sample spots. A satisfactory application of the method to the determination of amino-terminal residues of bovine α-chymotrypsin is described.

Open-flask alkaline hydrolysis of amino acid phenylthiohydantoin derivatives

It is suggested that the open-flask system for the acid hydrolysis of proteins described earlier can also be used to advantage for the alkaline hydrolysis of amino acid phenylthiohydantoin derivatives obtained in protein sequential analysis according to Edman. Results obtained under nitrogen and argon atmospheres are reported for the hydrolysis of 0.5 μmole of each phenylthiohydantoin derivative, which is the amount of protein that is usually employed in an automatic sequenator.

Improved thin-layer chromatography technique for the identification of phenylthiohydantoin amino acids

Improved thin-layer chromatography technique for the identification of phenylthiohydantoin amino acids

Subnanomole detection of phenylthiohydantoins of amino acids after thin-layer chromatography

Subnanomole detection of phenylthiohydantoins of amino acids after thin-layer chromatography

Micropolyamide thin-layer chromatography of phenylthiohydantoin amino acids (PTH) at subnanomolar level. A rapid microtechnique for simultaneous multisample identification after automated Edman degradations

Based on our previous method (6) and improvements made by Summers et al. (8), we developed a tlc-technique for two-dimensional separation of 24 most common PTH 1 1 The following nonstandard abbreviations were used: PTH, phenylthiohydantoin; CM-Cys, S-carboxymethylcysteine; CySO 3H, cysteic acid; Metsf, methionine sulfone; N-Phe, N-phenylthiourea; N,N-DPhe, N,N-diphenylthiourea. -amino acids obtained after manual or automated Edman degradations. Using a fluorescent indicator (8) and double-faced micropolyamide sheets (5 × 5 cm) our method allows the complete identification of all derivatives in about 30 min including His/Arg/CySO 3H 1 and Leu/Ile PTHs not separable by Summers' technique. Offering the same lower limit of detection at 254 nm (0.05–0.2 n m), in contrast to the dansyl-Edman technique, the micropolyamide tlc in addition to direct Edman degradation allows correct placing of Gln and Asn within sequences. We also set up a method for identification in 20–30 min of 10 individual PTH samples on the same 5 × 5-cm sheet by consecutively applying two solvent systems in identical direction. Compared with gc the advantages of this convenient and inexpensive micromethod consist in completeness of separations, increase of sensitivity, and in simultaneous processing of many samples.

Detection of sub-nanomole amounts of phenylthiohydantoins of amino acids

Detection of sub-nanomole amounts of phenylthiohydantoins of amino acids

Analysis of amino acid phenylthiohydantoins by high speed liquid chromatography

Summary A high speed liquid chromatography (HSLC) method has been developed which permits the analysis of all the PTH amino acids in the subnanomolar range on a single column in about 30 min. Although resolution of some derivatives is not entirely satisfactory at this stage of development the HSLC method appears to be well-suited for the analysis of multiple samples produced by the automated protein sequencer particularly when used in conjunction with the gas chromatographic procedure.

Acetylation of phenylthiohydantoins of amino acids

The acetylation of all the common amino acid phenylthiohydantoins has been studied and the chromatographic behaviour of the derivatives compared with that of the phenylthiohydantoins. All of the phenylthiohydantoins (except that of proline) examined could be converted into acetylated products but a variety of reaction conditions was necessary. In some cases, these acetylated derivatives were better suited than the parent phenylthiohydantoins to gas chromatographic analysis.

Thin-layer chromatography of sub-nanomole amounts of phenylthiohydantoin (PTH) amino acids on polyamide sheets

Thin-layer chromatography of PTH amino acids on polyamide sheets with a fluorescent indicator gave a 50–100-fold increase in sensitivity when compared to thin-layer polyamide plates developed by spray techniques. Resolution of the individual residues in a standard mixture remains comparable to that described previously. Using this technique, the two-dimensional chromatography of each residue from automated sequence analysis is quite feasible even when small amounts of material are available.

Miniature thin-layer chromatography of phenylthiohydantoin amino acids application of automatic Edman degradation

Miniature thin-layer chromatography of phenylthiohydantoin amino acids application of automatic Edman degradation