Polypeptides Edman degradation

Edman degradation A method of amino acid sequencing in proteins in which successive V-terminal amino acids are removed from the polypeptide chain and identified. 

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...

FIGURE 3-25 Steps in sequencing a polypeptide, (a) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger s method for identifying the amino-terminal residue is shown here, (b) The Edman degradation procedure reveals... 

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... 

The length of polypeptide that can be accurately sequenced by the Edman degradation depends on the... 

Determination of the amino-terminal residue of a polypeptide by Edman degradation. 

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. 

Edman degradation. A systematic method of sequencing proteins, proceeding by stepwise removal of single amino acids from the amino terminal of a polypeptide chain. 

Conventionally, the first attribute known about an enzyme used to be its function, usually in a crude extract. This property was screened for in microbial cultures or in tissue samples. The crude extract was then purified to homogeneity and the protein subjected to biochemical studies to learn of its pH and T profiles, its pi and subunit composition, catalytically important residues, and other properties. Proteolytic digestion of the protein with subsequent Edman degradation led to the primary sequence, but no information on the secondary structures such as a-heli-ces and [5-sheets or the folding in three dimensions of the polypeptide chain. The primary sequence could have been used to deduct the gene sequence but, with the degeneration of the code, several possibilities for certain amino acids occur, which makes prediction of the gene sequence a risk. 

Write the equations for the Edman degradation of a given tri- or polypeptide. 

Polypeptides of up to approximately 25 residues can be sequenced by the technique of mass spectrometry (MS), which involves an ionization technique called fast atom bombardment (FAB) in concert with a tandem mass spectrometer (two mass spectrometers coupled in series). The sequence of the polypeptide can be obtained from the molecular masses of the various fragments produced in the ionization stage in only a few minutes compared to the hour required for just one cycle of Edman degradation. In addition, mass spectrometry can be used to sequence several polypeptides in a mixture, alleviating the need to completely... 

The feature that makes the Edman degradation so useful is that the new polypeptide, with one fewer amino acid, can be isolated and submitted to the process again, allow-... 

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. 

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. 

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). 

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