Reagents phenylthiohydantoine

FIGURE 5.19 N-Tertninal analysis using Edman s reagent, phenylisothiocyanate. Phenylisothiocyanate combines with the N-terminus of a peptide under mildly alkaline conditions to form a phenylthiocarbamoyl substitution. Upon treatment with TFA (trifluo-roacetic acid), this cyclizes to release the N-terminal amino acid residue as a thiazolinone derivative, but the other peptide bonds are not hydrolyzed. Organic extraction and treatment with aqueous acid yield the N-terminal amino acid as a phenylthiohydantoin (PTH) derivative. 

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. 

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 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... 

Edman s reagent is also used to determine the amino acid sequence of a polypeptide chain from the N-terminal by subjecting the polypeptide to repeated cycles of Edman degradation. After every cycle, the newly liberated phenylthiohydantoin (PTH) amino acid was identified. The sequence of peptides containing 30-40 amino acids can be determined using a sequencer by adopting the Edman s degradation method. 

Contained ala + gly + cys + glu + arg + ile + N HT Carboxypeptidase A liberated isoleucine Treatment with phenylisothiocyanate (PITC, the Edman reagent) yielded the phenylthiohydantoin derivative of glycine (PTH-glycine)... 

Thin-layer chromatography (TLC) provides convenient routine analytical support of synthesis and other amino-acid interests and has been used in the Mosher procedure just described. It is most generally used for free amino acids and peptides, with spray reagents based on ninhydrin, or on the above derivatives ( post-TLC derivatisation ). Dansyl and phenylthiohydantoin (PTH) derivatives have been used for many years for identifying amino acids in mixtures by TLC ( pre-TLC... 

There are several ways to identify the N-terminal amino acid of a peptide or protein. One of the most widely used methods is to treat the protein with phenyl isothiocyanate (PITC), more commonly known as Edman s reagent. This reagent reacts with the N-terminal amino group, and the resulting thiazolinone derivative is cleaved from the protein under mildly acidic conditions. The thiazolinone derivative is extracted into an organic solvent and in the presence of acid, rearranges to a more stable phenylthiohydantoin (PTH). 

The amount of Edman reagent must exactly match the amount of N-termini in the first reaction. If there is too little Edman reagent, some of the N-termini will not react. If there is too much, some of the second amino acid will react. In either case, there will be a small amount of contaminating phenylthiohydantoin (PTH) derivatives. This error grows with the number of... 

Separation of phenylthiohydantoin (PTH) amino acid derivatives, which are produced in the sequential analysis of proteins by the Edman degradation, is required. Ashraf-Khorassani etal. showed [21] how SFC on a cyanopropyl-modified silica column allowed the separation of more than twenty PTH-amino acids if an ion-pairing reagent (tetramethylammonium hydroxide) was present at low concentration in the methanol-modified CO2 mobile phase. 

Chemical reagents that induce degradation of a peptide allow various constituent amino acid residues to be identified. Ninhydrin is a common reagent used to indicate the presence of amino acids. Disulfide linkages can be cleaved with peroxyformic acid or with mercaptoethanol. Sanger s reagent forms a compound that allows the N-terminal amino acid to be identified. Dansyl chloride also reacts with the N-terminal amino acid to form a readily identifiable compound. Phenylisothiocyanate reacts with the N-terminal amino acid to form a phenylthiohydantoin derivative, which is readily identified. 

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