Phenylthiohydantoin, Edman degradation

Edman degradation (Section 27 13) Method for determining the N terminal amino acid of a peptide or protein It in volves treating the material with phenyl isothiocyanate (CgH5N=C=S) cleaving with acid and then identifying the phenylthiohydantoin (PTH derivative) produced Elastomer (Section 10 11) A synthetic polymer that possesses elasticity... 

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 mol l-1 HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acid identified by comparison to phenylthiohydantoin derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established...

To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 3-25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluo-roacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The deriva-tized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over... 

Ingenious protein sequenators have been devised to carry out the Edman degradation automatically.242 244-246 Each released phenylthiohydantoin is then identified by HPLC or other techniques. Commercial sequenators have often required 5-20 nmol of peptide but new microsequenators can be used with amounts as low as 5-10 picomoles or less.247 248... 

Step 3 Once formed, the thiazolone derivative isomerizes to a more stable phenylthiohydantoin (PTH) derivative, which is isolated and characterized, thereby providing identification of the N-terminal amino acid. The remainder of the peptide (formed in step 2) can be isolated and subjected to a second Edman degradation. 

Gas chromatography is used to analyze volatile derivatives of amino acids. Phenylthiohydantoins (products of Edman degradation) may be analyzed directly by GC but are better resolved if converted to their trimethylsilyl derivatives with N, O-bis(trimethylsilyl) acetamide. Free amino acids are generally converted to their 7V-trifluoroacetyl- -butyl esters or trimethylsilyl derivatives before GC analysis. For best results, all gas chromatography of amino acid derivatives should be done with a glass column and injection port, as contact with metals causes extensive decomposition of the derivatives. 

The Edman degradation method for polypeptide sequence determination. The sequence is determined one amino acid at a time, starting from the amino-terminal end of the polypeptide. First the polypeptide is reacted with phenylisothiocyanate to form a polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino-terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected spectrophoto-metrically. The remaining intact polypeptide, shortened by one amino acid, is then ready for further cycles of this procedure. A more sensitive reagent, dimethylaminoazobenzene isothiocyanate, can be used in place of phenylisothiocyanate. The chemistry is the same. 

The formation of methyl- (MTH) or phenylthiohydantoin (PTH) amino acids is a valuable technique for sequencing of amino acids in peptides and proteins by the Edman degradation procedure [1], HPLC is very useful for the separation of MTH- or PTH-amino acids as adsorption [2,3], reversed-phase [4] and ion-exchange [S] chromatography. 

The Edman degradation removes the N-terminal amino acid, which is identified as a phenylthiohy-dantoin derivative. The first Edman degradation of Val-Phe-Gly-Ala gives the phenylthiohydantoin derived from valine the second gives the phenylthiohydantoin derived from phenylalanine. 

The first cycle of the Edman degradation of Phe-Ala-Ser will give the phenylthiohydantoin of Phe because it is the N-terminal amino acid. 

When insulin is subjected to the Edman degradation, the N-terminal amino acids of the A and B chains (glycine and phenylalanine, respectively) will both be converted to their phenylthiohydantoins. Their structures are ... 

The N-terminal amino acid of a protein can be determined by reacting the protein with dansyl chloride or fluorodinitrobenzene prior to acid hydrolysis. The amino acid sequence of a protein can be determined by Edman degradation which sequentially removes one residue at a time from the N terminus. This uses phenyl isothiocyanate to label the N-terminal amino acid prior to its release from the protein as a cyclic phenylthiohydantoin amino acid. 

The most useful method of N-terminal analysis is called the Edman degradation. This method allows the N-terminal amino acid to be removed and its identity to be determined without hydrolyzing the other peptide bonds. The reaction initially produces a thiazolinone, which is rearranged by aqueous acid to a phenylthiohydantoin for identification by high-performance liquid chromatography ... 

Lequin and Niall [282] described GC analysis of more volatile analogues, pentafluoro-phenylthiohydantoins, which were prepared by modifying the Edman degradation using pentafluorophenylisothiocyanate as a reagent. Except for Arg and His, the derivatives of all amino acids could be chromatographed and separated satisfactorily in a simple column (1.22 m X 2 mm I.D.) packed with either 10% of DC-560 or 2% of OV-25 on Chromo-... 

The most efficient method for sequencing peptides is the Edman degradation. A peptide is treated with phenyl isothiocyanate, followed by acid hydrolysis. The products are the shortened peptide chain and a heterocyclic derivative of the N-terminal amino acid called a phenylthiohydantoin. 

The phenylthiohydantoin derivative is identified by chromatography, by comparing it with phenylthiohydantoin derivatives of the standard amino acids. This gives the identity of the original N-terminal amino acid. The rest of the peptide is cleaved intact, and further Edman degradations are used to identify additional amino acids in the... 

The first two steps in sequencing oxytocin. Each Edman degradation cleaves the N-terminal amino acid and forms its phenylthiohydantoin derivative. The shortened peptide is available... 

The N-terminal residue, i.e., the first amino acid in the sequence of a peptide, can be determined by reaction with phenylisothiocyanate. At neutral pH, this compound reacts with the a-amino group. After mild acid hydrolysis, the reaction product cydizes, releasing the terminal residue as a phenylthiohydantoin (PTH) derivative (the Edman degradation. Fig. 4-1). The derivative can be analyzed to determine its parent amino acid and its quantity. 

The primary structure (i.e., the amino acid sequence) of a protein can be determined by stepwise chemical degradation of the purified protein. By far the most powerful and commonly used technique for doing this is the automated Edman degradation. The amino terminal amino acid residue of the polypeptide is reacted with Edman s reagent (phenylisothiocyanate) to form the phenylthiocar-bamyl derivative, which is removed without hydrolysis of the other peptide bonds by cyclization in anhydrous acid. The amino acid derivative is converted to the more stable phenylthiohydantoin and identified by HPLC. The process can be repeated many times, removing the amino acids from the amino terminus of the polypeptide one residue at a time and identifying them until the entire sequence... 

The sequencing of a peptide (26) uses the well-known Edman degradation (27) of peptides, as shown in Fig. 2.2. The reaction cycle consists of reaction with phenylisothiocyanate followed by treatment with anhydrous TFA to promote cycliza-tion of the intermediate thiourea. Rearrangement induced by treatment with aqueous TFA produces the phenylthiohydantoin (PTH) of the N-terminal amino acid 2.2 and the tmncated peptide 2.3. The sequence is repeated through n cycles until the whole peptide is degraded. The PTHs produced are detected by HPLC-UV and their retention times are compared to those of 20 standard PTHs, one for each natural amino acid. 

The first 34 residues of human parathyroid hormone (PTH) [190] were identified from the Cl mass spectra of their phenylthiohydantoin derivatives by repeated Edman degradations on a Beckmann Sequencer [163]. In a similar way as part of the characterisation of ovine hypothalmic luteinizing hormone-releasing factor (LRF), the trimethylsilyi derivatives of the phenylthiohydantoins from Edman degradation were confirmed by MS [191]. 

Automated Edman degradation of protein and peptide samples were performed using an Applied Biosystems sequencer (Model 470A or 477A) or a Hewlett Packard G1005A sequencer. Each sequencer was fitted with an on-line HPLC analyzer for the identification of phenylthiohydantoin derivatives. 

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