Edman degradation

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Diagram of the Edman degradation process

The Edman degradation is a method of protein sequencing that allows for the sequential identification of amino acids in a peptide. This technique was developed by Pehr Edman in the 1950s and has been a fundamental tool in biochemistry for determining the primary structure of proteins.

Principle

The Edman degradation involves the selective cleavage of the N-terminal amino acid of a peptide, which is then identified as a phenylthiohydantoin (PTH) derivative. This process can be repeated multiple times to sequence the entire peptide.

Step 1: Coupling

In the first step, the peptide is treated with phenyl isothiocyanate (PITC) under mildly alkaline conditions. This reagent reacts with the free amino group of the N-terminal amino acid to form a phenylthiocarbamoyl derivative.

Step 2: Cleavage

The peptide is then treated with anhydrous acid, typically trifluoroacetic acid (TFA), which cleaves the N-terminal amino acid as a cyclic phenylthiohydantoin (PTH) derivative, leaving the rest of the peptide intact.

Step 3: Identification

The PTH-amino acid is identified using chromatography or mass spectrometry. This identification step is crucial for determining the sequence of the peptide.

Advantages and Limitations

The Edman degradation is advantageous for its ability to sequence peptides with high accuracy. However, it has limitations, such as the inability to sequence peptides longer than about 50 residues due to incomplete cleavage and side reactions. Additionally, it requires a free N-terminal amino group, which may not be available in all peptides.

Applications

Edman degradation has been widely used in the field of proteomics for the analysis of protein structure and function. It has been instrumental in the sequencing of many important proteins and in the study of enzyme mechanisms.

Related pages

• Protein sequencing
• Amino acid
• Peptide
• Phenyl isothiocyanate
• Chromatography