Degradation reactions Edman

Underlined sequences indicate amino acid sequences used for the generation of degenerate primers. Bracketed question marks represent blank cycles from the Edman degradation reaction. Additional sequence was obtained after blank cycles in all cases except the Glu-C-1 and Glu-C-2 peptides. 

The sequence of an amino acid in a protein is determined sequentially from the N-terminus. The N-terminus amino acid is identified by the Edman degradation reaction developed at Rockefeller University and later automated in Melbourne, Australia, by Edman and his collaborators (1950, 1967). 

Fig. 4-1 Edman degradation reaction for the identification of an N-terminai amino acid residue in a peptide. Sequentiai appiication of the reaction ieads to determination of the sequence of the whole peptide.

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. 

Collect fractions and identify labeled peptide(s) by scintillation counting of 1/10 of each fraction. Monitor OD220 to assess the concentration of peptides in each fraction. As a rule of thumb, 1 nmol of a 10-mer peptide yields an OD220 of about 0.2 Thus, a peak of 0.002 OD contains 10 pmol of peptide, which should be sufficient for microsequencing However, it is advisable to work with OD peaks of about O.l-.Ol for a better sensitivity of the Edman-degradation reaction and detection of the phenylthioazolinone (PTH) denvatives of amino acids Lyophilize the labeled fraction(s) in glass tubes... 

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 ... 

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. 

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. 

Diels-Alder reaction, 493 El reaction, 391-392 ElcB reaction, 393 E2 reaction, 386 Edman degradation, 1032 electrophilic addition reaction, 147-148. 188-189 electrophilic aromatic substitution, 548-549 enamine formation, 713 enol formation, 843-844 ester hydrolysis, 809-811 ester reduction, 812 FAD reactions. 1134-1135 fat catabolism, 1133-1136 fat hydrolysis, 1130-1132 Fischer esterification reaction, 796 Friedel-Crafts acylation reaction, 557-558... 

Peptide sequencers automatically carry out all the reactions of the Edman degradation procedure under controlled conditions, and a typical scheme is described below. The released N-terminal derivatives are then analysed by reverse-phase HPLC. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

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. 

The stepwise chemical degradation of peptide chains with Edman s method represents one of the truly significant additions to the inventory of methods in modem biochemistry. Without this procedure the rapid development within recent years of our knowledge about stmcture and function of proteins would have been impossible (see Attempts based on different chemical reactions have not yet yielded practical methods 56-62)... 

Recently, Laursen realized the concept of a novel Peptide-Sequencer based on the chemistry of the Edman degradation, adapted to solid phase chemistry. The peptide under investigation is covalently linked to a resin packed into a column. The reactions are carried out by pumping the reagents and solvents through the column as required. This method appears to be particularly suitable for shorter peptides and may be regarded as an excellent supplement to the Sequencer based on Edman s design. 

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... 

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