Proteins The Edman degradation

The Edman degradation uses phenyl isothiocyanate to remove and identify the N-terminal amino acid of peptides or proteins. The Edman degradation can be repeated and automated so that the sequence of up to 20 to 30 N-terminal amino acids can be determined. 

DETERMINATION OF THE AMINO ACID SEQUENCE OF PROTEINS 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. 

The ability to visualize spots on a 2D gel, while useful as a fingerprint, is not the same as protein identification. Protein sequencing by the Edman degradation technique is the classical means of determining... 

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... 

Because many proteins are modified at the N terminus, blocking application of the Edman degradation, it would be useful to have a similar method for sequencing from the C terminus. It has been difficult to devise a suitable strategy, but there has been some success.252-254... 

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 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. 

By the primary structure of a peptide or protein, we mean its amino acid sequence. Complete hydrolysis gives the amino acid content. The N-terminal amino acid can be identified by the Sanger method, using 2,4-dinitrofluorobenzene. The Edman degradation uses phenyl isothiocyanate to clip off one amino acid at a time from the N-terminus. Other reagents selectively cleave peptide chains at certain amino acid links. A... 

Lamkin et al. [276] studied in detail the GC analysis of silylated methylthiohydantoins of all protein amino acids. They effected the silylation with BSA-acetonitrile (1 3) at 100°C for 10 min. They separated the products in a simple column packed with 2% of OV-17 on Gas-Chrom Q at 145—230°C, and Fig. 5.20 illustrates the results. The authors used a flame photometric detector, sensitive to sulphur-containing compounds, in order to ensure sensitive and selective detection. Minor incidental peaks that were often noticed during the analysis of the samples obtained by the Edman degradation of proteins with the use of an FID did not appear and the peak of the solvent was not detected. The baseline stability was good and the response was linear over a range of two orders of magnitude of concentration. Asn and Phe were the only unresolved pair Arg, as in previous instances, did not form a volatile derivative. 

The formation of PTH-amino adds by the Edman degradation [4] of peptides and proteins or by successive modifications of the method constitutes the most commonly used technique for the study of the structure of biologically active polypeptides today. Identification of PTH-amino acids in mixtures may be successfully achieved by TLC. Quantitative determination is based on UV absorption (detection limit 0.1 jLg at 270 nm). An alternative is offered by the chlorine/toUdine test, which is very useful because the minimal amount required for detection is about 0.5 (xg. 

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