Edman: a Classical Biochemical Way to Reveal the N-Terminal Amin

Edman degradation, also known as Edman sequencing, is a biochemical method used to determine the N-terminal amino acid sequence of proteins. This method was first proposed by American biochemist Pehr Edman in 1950, and has been constantly improved and applied in the following decades. The Edman method is usually used to analyze the amino acid sequences of smaller proteins or peptides, and its main steps include:

 

Sample Preparation

First, the target protein or peptide needs to be purified. Typically, the protein is reduced to its free amino acids, which then react with a specialized labeling reagent, such as the Edman reagent (commonly phenyl isothiocyanate), to form a labeled compound with the amino acid-N-terminus.

 

Edman Degradation

The labeled compound is exposed to acidic conditions, leading to a reaction between the N-terminal amino acid and the Edman reagent to form an amino acid ketone derivative. In this reaction, the Edman reagent replaces the amino group at the N-terminus of the target amino acid to form a stable derivative. This process typically goes through 10 to 20 amino acid cycles, with each cycle determining one amino acid.

 

Substitution Reaction

The reacting labeled compound is collected and removed from the reaction vessel through acid-base neutralization.

 

Analysis and Detection

The collected labeled compound is analyzed and detected, usually using techniques such as high-performance liquid chromatography (HPLC) to separate and identify amino acids or amino acid ketone derivatives. Then, the N-terminal amino acid in each cycle can be determined.

 

The main limitation of Edman degradation is that it is suitable for smaller proteins and peptides, usually between 30 and 50 amino acids. For larger proteins, other sequencing methods, such as mass spectrometry, may be required. However, the Edman method remains a useful tool for studying the N-terminal sequences of small proteins and peptides, especially in protein structure and function research.