N-terminal Analysis Edman Degradation

The Ai-terminus of a polypeptide can be determined by reaction with dansyl chloride, with Edman s reagent or with an aminopeptidase. 

1-dimethyl aminophthalene-5-sulfonyl chloride (dansyl chloride) reacts with primary amine groups in the polypeptide chain and forms a dansyl polypeptide. Upon acidic hydrolysis, all peptide bonds in the chain are cleaved and the Ai-terminal residue of the peptide is liberated in the form of a dansyl-amino acid (Fig. 7.1). This amino acid derivative is highly fluorescent and can thus be detected with very high sensitivity. 

In contrast to other processes for A-terminal analysis, Edman degradation only leads to cleavage of the A-terminal residue, whilst leaving the remaining polypeptide intact. The reaction can be repeated in a cyclical fashion and each time the A-terminus is cleaved, extracted into the organic phase and identified. With this process, relatively short peptides with 40 to 60 residues can be sequenced (section 7.7). 

Sequential analysis of A-termini can also be achieved by enzymatic reactions. An exopeptidase cleaves a terminal polypeptide residue. An aminopeptidase, for example, cleaves A-terminal residues (Fig. 7.3a). Aminopeptidases, however, have only a limited use for the determination of an amino acid sequence. Due to their high specificity, only selected amino acids are cleaved and these at different rates. Some residues are not cleaved by certain aminopeptidases, in which case the sequencing experiment stops. Some amino acid residues may be more resistant to the enzyme than others. The different cleavage rates make unambiguous sequence determination difficult. 

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