Methods Edman degradation

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...

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. 

One example of practical application of the above solution involves determination of allergens in sesame seeds, for which 2-D electrophoresis and Edman degradation methods have been employed (Beyer et al., 2002) (Section 3.4 contains more information on this issue). 

M. Tornqvist, J. Mowrer, S. Jensen and L. Ehren-berg, Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation method, Anal. Biochem., 154, 255-266 (1996). 

A primary focus of future research should be determination of the primary (10) structure of acrosin. Protein sequencing by the classical Edman degradation method and recombinant DNA methods will be needed to elucidate completely the primary structure of acrosin. Knowledge of the primary structure is of fundamental inq>ortance for additional studies on the functional properties of the enzyme. 

Two step screening was done for the 430,000 library beads. In the first step, the beads stained by lpM NBD-DCA were selected. The fluorescent beads were picked up by a capillary using an inverted fluorescent microscope for observation. The 399 peptide beads obtained were then washed with ethanol. After washing, the beads still fluorescing were excluded since they had bound irreversibly to the NBD-DCA. Thus, only 18 beads were tested in the second step. In the second step, the beads were subjected to a competitive reaction to select only the peptides binding to the DCA moiety of the NBD-DCA. They were suspended in a 5mM DCA solution containing 5% ethanol for 3h and only those five beads for which the fluorescence decreased were selected. Finally, the sequences of the five beads obtained were determined by a standard Edman degradation method. 

The Edman degradation method becomes more difficult as the number of amino acids increases. In most proteins, the chain is more than 100 residues long. For sequencing, it is usually necessary to break a long polypeptide chain into fragments, ranging from 20 to 50 residues for reasons that will be explained later. 

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

Edman degradation Method for determining the N-terminal amino acid of a peptide or protein. It involves treating the material with... 

The most widely used procedure for identifying the N-terminal amino acid in a peptide is the Edman degradation method (developed by Pehr Edman of the University of Lund,... 

Sweden). Used repetitively, the Edman degradation method can be used to sequence peptides up to about 60 residues in length. The process works so well that machines called amino acid sequencers have been developed to carry out the Edman degradation process in automated cycles. 

Edman degradation (Section 24.5A) A method for determining the A -terminal amino acid in a peptide. The peptide is treated with phenylisothiocyanate (CgHs — N = C = S), which reacts with the A terminal residue to form a derivative that is then cleaved from the peptide with acid and identified. Automated sequencers use the Edman degradation method. 

Undoubtedly, solid-phase methods have found their most important applications in the field of protein chemistry, especially for peptide synthesis. Another very important and successful application in this area is the sequencing of peptides and proteins by the solid-phase Edman degradation method. It can be said that the method is even better established than solid-phase peptide synthesis and that the uncertainties observed in solid-phase peptide synthesis do not exist in the solid-phase Edman degradation. Reviews of work in this area are available (Laursen, 1975 Laursen and Horn, 1977 Mross and Doolittle, 1977). 

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