Degradation, Edman

The cycle of reactions in the Edman procedure can be summarised as follows  

Methods in Peptide and Protein Sequence Analysis, Elsevier, Amsterdam, 1981. 

which incorporates substantial modifications in the design of the commercial Beckman instrument coupled with the computer assisted identification of phenylthiohydantoin derivatives by reversed-phase HPLC. These methods have enabled HunkapUler and Hood to determine 24 residues of the mouse fibroblast interferon amino-acid sequence starting with only 0-6 fig of material. This remarkable achievement is now being succeeded by developments in the same laboratory. A machine of even greater sensitivity, based on coupling and cleavage in the gas phase, has been constructed. The sensitivity of the automatic amino-acid sequencer is apparently beginning to surpass most of the analytical techniques commonly used to detect proteins. 

Peptide mixtures have been successfully sequenced by a combination of Edman degradation and field desorption mass spectrometry. The idea is really akin to the classical subtractive Edman method. iV-terminal residues are removed by manual Edman d radation and the new masses of the peptides determined on the 

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FIGURE 27 12 Identifica tion of the N terminal ammo acid of a peptide by Edman degradation... 

Step 3 Once formed the thiazolone derivative isomerizes to a more stable phenylthiohydantom (PTH) derivative which IS isolated and characterized thereby providing identification of the N terminal ammo acid The remainder of the peptide (formed m step 2) can be isolated and subjected to a second Edman degradation... 

Only the N terminal amide bond is broken m the Edman degradation the rest of the peptide chain remains intact It can be isolated and subjected to a second Edman procedure to determine its new N terminus We can proceed along a peptide chain by beginning with the N terminus and determining each ammo acid m order The sequence is given directly by the structure of the PTH derivative formed m each successive degradation... 

Modem methods of peptide sequencing follow a strategy similar to that used to sequence insulin but are automated and can be carried out on a small scale A key feature is repetitive N terminal identification using the Edman degradation... 

Somatostatin is a tetradecapeptide of the hypothalamus that inhibits the release of pituitary growth hormone Its ammo acid sequence has been determined by a combination of Edman degradations and enzymic hydrolysis expenments On the basis of the following data deduce the pnmary structure of somatostatin... 

Edman degradation (Section 27 13) Method for determining the N terminal amino acid of a peptide or protein It in volves treating the material with phenyl isothiocyanate (CgH5N=C=S) cleaving with acid and then identifying the phenylthiohydantoin (PTH derivative) produced Elastomer (Section 10 11) A synthetic polymer that possesses elasticity... 

A major advance was devised by Pehr Edrnan (University of Lund, Sweden) that has become the standard method for N-terminal residue analysis. The Edman degradation is based on the chemistry shown in Figure 27.12. A peptide reacts with phenyl isothiocyanate to give a phenylthiocarbamoyl (PTC) derivative, as shown in the first step. This PTC derivative is then treated with an acid in an anhydrous medium (Edrnan used nitrornethane saturated with hydrogen chloride) to cleave the amide bond between the N-terminal anino acid and the remainder of the peptide. No other peptide bonds are cleaved in this step as amide bond hydrolysis requires water. When the PTC derivative is treated with acid in an anhydrous medium, the sulfur atom of the C=S unit acts as... 

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 /xg. 

Figure 26.4 MECHANISM Mechanism of the Edman degradation for N-terminal analysis of peptides.

Draw the structure of the PTH derivative that would be formed on Edman degradation of angiotensin II (Problem 26.12). 

ATZ Derivative (Section 26.6) An anilinothiazolinone, formed from an amino acid during Edman degradation of a peptide. 

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. 

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