Phenylisothiocyanate, Edman

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. 

Edman degradation (Section 26.6) A method for N-terminal sequencing of peptide chains by treatment with Af-phenylisothiocyanate. 

PITC (Section 26.6) Phenylisothiocyanate used in the Edman degradation. 

Amino acid sequencing may be carried out in a number of ways. The most widely used is the Edman degradation procedure in which phenylisothiocyanate is used to react with the amino acid residue at the amine end of the protein chain. This derivatized residue is removed from the remainder of the protein and converted to a phenylhydantoin derivative which is identified by using, for example, HPLC. 

Figure 4-6. The Edman reaction. Phenylisothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenyithiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.

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... 

Sequencing is a stepwise process of identifying the specific amino acids at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman s reagent, is... 

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

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. 

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. 

Figure 8.11) via the intermediacy of an activated dithiocarbamate D. Phenylisothiocyanate enables the Edman degradation of peptides (Figure 8.14). 

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... 

Phenylisothiocyanate, C6Hs-N=C=S Used in the Edman degradation of peptides to identify N-terminal amino acids (Section 26.6). 

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. 

Figure 5-2. Chemistry of the Edman degradation. In the Edman degradation, peptides undergo reaction with phenylisothiocyanate which generates a phenylthiocarbamylpeptide adduct. This adduct is cleaved to release the...

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)... 

---