PTH derivatives of amino acids

It was claimed [573] that for complicated samples, such as the separation of 20 phenylthiohydantoin (PTH) derivatives of amino acids, the optimization of many parameters simultaneously is required to achieve sufficient selectivity. However, in ref. [573] the pH was optimized separately, before starting the complete three-parameter optimization with two continuous parameters and one discrete one. 

Figure 6.8 Experimental variation of the retention of 23 phenylthiohydantoin (PTH) derivatives of amino acids with mobile phase composition in RPLC. Mobile phase mixtures of acetonitrile and 0.05M aqueous sodium nitrate buffer (pH — 5.81). All mobile phases contain 3% THF. Stationary phase ODS silica. Solutes D = aspartic acid C-OH = cysteic acid E = glutamic acid N = asparagine S = serine T = threonine G = glycine H = histidine Q = glutamine R = arginine A = alanine METS = methionine sulphone ABA = a-aminobutyric acid Y = tyrosine P = proline V = valine M = methionine NV = norvaline I = isoleucine F = phenylalanine L = leucine W = tryptophan K = lysine. Figure taken from ref. [610]. Reprinted with permission.

Phenylisothiocyanate (PITC). The use of PITC (Edman Reagent) to form thiohydantoin (PTH) derivatives of amino acids for protein sequencing is well known. The development of reversed phase systems for the separation of these PTH derivatives has resulted in the introduction of both isocratic and gradient elution systems (Fig. 11.2.11). A microbore HPLC unit for PTH-amino acid analysis is now available along with the recently introduced gas phase sequenator... 

PTH derivatives of amino acids Aqueous mixture of 0.5 M sodium azide, 0.01 M iodine, 0.5 M potassium iodide 0.5 [5]... 

Separation and identification of derivatives of amino acids such as DNP-, PTH-, dansyl-, and DABITC-, is very important, particularly in the primary structure determination of peptides and proteins. Adequate description of the preparation of PTH- (76-79), dansyl (80-82), and DNP-amino acids (83-86) is available in the literature, and the methods of identification of N-terminal amino acids by TLC and other techniques have been reviewed by various workers (87-91). The present section describes briefly the preparation of such derivatives and TLC resolution data reported in recent years. 

Amino acids and their derivatives] resolution of amino acids and dieir derivatives on impregnated layers, 406-408 resolution of enantiomeric mixtures of amino acids and their derivatives, 411-419 separation of amino acid derivatives, 398-406 DABITC derivatives of amino acids, 403-406 dansyl amino acids, 402 dinitrophenyl-amino acids, 406 PTH amino acids, 399-402 separation of amino acids, 389-398 chromatographic techniques, 393-395 preparation of test materials, 389-392 TLC system for amino acids, 395-398 Aminoglycosides, 457-461 Amino-modified precoated silica layers, 114,117-119... 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

We have selected many unnatural a-amino acids to study their sequencing profiles. Table III summarizes the RT of 74 PTH derivatives of both natural and unnatural amino acids. Through Table III it is easy to select 4(M-5 amino acids with ART greater than 0.10 min as building blocks to construct peptide libraries. This greatly increases the diversity of the peptide libraries that can be generated. 

Somatostatin is a tetradecapeptide and so is composed of 14 amino acids. The fact that Edman degradation gave the PTH derivative of alanine identifies this as the N-terminal amino acid. A major piece of information is the amino acid sequence of a hexapeptide obtained by partial hydrolysis ... 

Today s predominance of the gas phase sequencer for sequence analysis is partly due to the advancement of PTH-amino acid analysis on HPLC, in which the analysis takes only 10 min to complete using reversed-phase column.70-72 Thus, a gas phase sequencer with an on-line PTH-analyzer can perform a single Edman degradation cycle within one hour. With the use of a microbore column and a data station which handles various data manipulations, a few pmol of PTH-amino acid can be quantitatively analyzed. Although the minimum detection level of amino acid derivatives has improved significantly in the last 10 years (103-104-fold enhancement), further improvement is expected with the use of a fluorescent compound, i.e., fluorescein isothiocyanate (FITC), and the laser detection technique. 

Conversion of half-cystine residues in proteins and peptides to the S-methyl derivatives is advantageous in subsequent studies of amino acid sequence. Under the usual conditions of acid hydrolysis ( 2.1), S-methylcysteine is recovered in a 90% yield (Heinrikson 1971). The phenylthiohydantoin of S-methylcysteine is readily identified by routine thin layer chromatography procedures (Rochat et al. 1970). With the increasing use of the sequenator, PTH-S-methylcysteine offers a marked advantage over derivatives such as PTH-cysteic acid, or PTFl-carboxymethylcysteine, which have to be identified by special techniques (Edman 1960, 1970). S-methylcysteinyl residues provide a new point of cleavage for cyanogen bromide (5). 

Determining the structure of a peptide or protein is carried out in several steps. The identity and amount of each amino acid present in a peptide is determined by amino acid analysis. The peptide is hydrolyzed to its constituent a-amino acids, which are then separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forma an easily identifiable phenylthvobydantoin (PTH) derivative of the N-terminal amino acid. A series of sequential Edman degradations allows the sequencing of a peptide chain up to 50 residues in length. 

The DNP-amino acids, after separation into individual spots on the chromatographic plate, can be eluted from the scraped off area by adding 4 ml of water to the material in a small tube. The tube is heated at 50° in a water bath for 15 minutes and centrifuged to clear the solution. The color is read against known standards at 360 nm. Direct estimation of DNP-, PTH-, and DANS-amino acids separated on the thin-layer plate can be performed by fluorescence and fluorescence quenching techniques (P8). It is also possible to convert unmodified amino acids, separated on a silica gel G chromatographic plate, into DNP-amino acids by in situ conversion as was described in Section 4.7.18. The DNP-derivatives can then be developed in the second dimension and the spots analyzed quantitatively. 

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