Phenylisothiocyanate, Edman degradation

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 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... 

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. 

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...

Sequential analysis can be accomplished by using the Edman technique. Treatment of an intact polypeptide with phenylisothiocyanate derivatizes the N- amino acid leaving the rest of the peptide intact for further Edman degradation. Large chains must be fragmented into shorter peptides, more easy to work with chemically. Cleavage of peptide bonds at specific amino acid residues is accomplished using enzymes such as trypsin (Lys, Arg), chymotrypsin (aromatics), and carboxypeptidase (C-terminus amino acids). 

Each fragment is sequenced through repeated cycles of a procedure called the Edman degradation. In this method phenylisothiocyanate (PITC), often referred to as Edman s reagent, reacts with the N-terminal residue of each fragment. 

If the phenylisothiocyanate method is used, the cyclization and release of the N-terminal derivative occur under mild conditions that leave the rest of the chain intact. It is therefore possible to repeat the procedure on this shortened peptide to determine the second residue in the sequence, and so on. Like most chemical reactions, Edman degradation is not 100% efficient, meaning that at each step there is a chance of additional peptide hydrolysis or incomplete reaction thus the method is not reliable for peptides with more than 60 residues. However, this can be overcome by generating a series of peptides by specific protease pretreatment (see next section) and then separating the peptide fragments prior to using Edman analysis. 

As everybody knows, a sequencing machine digests the amino acid chain starting at the N-terminus and it identifies the amino acid derivatives via a connected HPLC. A requirement is a free N-terminus. The Pierce Catalog, for example, describes the chemistry of the Edman degradation of peptides with phenylisothiocyanate. Baumann (1990) compares the effectiveness of different sequencing techniques. 

Edman degradation (Section 24.5A) A method for determining the A -terminal amino acid in a peptide. The peptide is treated with phenylisothiocyanate (CgHs — N = C = S), which reacts with the A terminal residue to form a derivative that is then cleaved from the peptide with acid and identified. Automated sequencers use the Edman degradation method. 

Methods have now been developed that allow just such a step-by-step sequencing of peptides. One effective way is called the Edman degradation, after Pehr Edman (1916-1977), and uses the molecule phenylisothiocyanate (Fig. 23.34). Isocyanates have appeared before (p. 918), and an isothiocyanate is just the sulfur analogue of an isocyanate. 

FIGURE 23.36 The Edman degradation procedure for sequencing polyamino acids uses phenylisothiocyanate.The amino terminus is identified by the structure of the phenylthiohydantoin formed. Notice that this method does not destroy the peptide chain as the Sanger procedure does. 

The classical method is the Edman degradation reaction. It involves stepwise degradation of peptides with phenylisothiocyanate (cf. 1.2.4.2.3) or suitable derivatives, e. g. dimethylaminoa-zobenzene isothiocyanate (DABITC). The resultant phenylthiohydantoin is either identified directly or the amino acid is recovered. The stepwise reactions are performed in solution or on peptide bound to a carrier, i.e. to a sohd phase. Both approaches have been automated ( sequencer ). Carriers used include resins containing amino groups (e. g. amino polystyrene) or glass beads treated with amino alkylsiloxane ... 

PrrC (Section 19.6) Phenylisothiocyanate, used in the Edman degradation of proteins. 

The guanidinated inhibitor retained only one free amino group, which was on the terminal leucine. The absence of other reactive groups made it possible to subject this derivative to the Edman degradation. This in turn made it possible to determine the role of the amino acids near the amino terminus in the inhibiting activity. Four steps of Edman degradation were carried out as described in EXPERIMENTAL PROCEDURE. Another portion of the material was used as a control of nonspecific loss of activity due to exposure to the Edman reagents this sample was subjected to identical treatment except for omission of the phenylisothiocyanate. 

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