Peptide Edman sequential degradation

Substitution of alkaline cyanates by isocyanates allows the preparation of 3-substituted hydantoias, both from amino acids (64) and amino nitriles (65). The related reaction between a-amino acids and phenyl isothiocyanate to yield 5-substituted 3-phenyl-2-thiohydantoiQS has been used for the analytical characterization of amino acids, and is the basis of the Edman method for the sequential degradation of peptides with concomitant identification of the /V-terminal amino acid. 

Phenyl isothiocyanate has also been utilized to analyze small quantities of peptides or short-chain peptides (88) by carrying out sequential degradation with detection by dansylation. Using dansyl-Edman degradation, Leadbeater and Bruce-Ward (60) identified 60 tryptic peptides of/8-casein that had previously been separated by high-voltage paper electrophoresis. 

Shortening of peptide Edman degradation charide chain Sequential removal of Removal of 2 glycotripeptides >80 ... 

Edman degradation. Sequential degradation of peptides beginning at the iV-tenninal residue based on the reaction of phenylisocyanate with the a-amino group of the terminal amino acid of the peptide chain. 

At the time of the conclusion of this study the elucidation of the structure of a nonapeptide was regarded as a major achievement. It could be realized only because of the advances made in the methodology of peptide research during the preceding years, for instance purification by Craig distribution or sequential degradation by the Edman method. The approach that yielded the even more complex structure of insuhn [13], namely partial acid hydrolysis followed by... 

A more useful procedure, in that it allows sequential determination of the A-terminal amino acids in a peptide, is the Edman degradation. This process removes the A-terminal amino acid, but leaves the rest of the chain intact, so allowing further reactions to be applied. The reagent used here is phenyl isothiocyanate. 

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. 

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. 

Dithiocarboxylates are particularly useful reagents in the thioacylation of the terminal amino group in peptides. Combination with a subsequent cleavage by anhydrous TEA results in a modified Edman degradation and a protocol has been worked out with conditions that are compatible with the ones required for an automatic sequential analysis (Scheme 4). ... 

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). 

Describe the sequential Edman degradation method and the automated determination of the amino acid sequences of peptides. 

An example of a reaction on a solid-phase matrix, the Merrifield resin, is shown in Fig. 7.14. The peptide fragments are covalently bonded, either onto a poiymer membrane or onto micrometer sized beads. The solid support is then immersed into a liquid phase and Edman degradation is carried out by sequentially adding the required reagents and removing the products for analysis. 

---