1. Derivatization of terminal amino acid-The chemical reaction is performed to labeled terminal amino group with compounds such as sanger reagent 1-fluoro-2,4-dinitrobenzene (DFNB) and dansyl chloride. In most of the case these reagents also label free amino group present on basic amino acids such as lysine and arginine. In a reaction mechanism given in Figure 38.3, dinitrofluorobenzene reacts with the free amine group to form dinitrophenyl-amino acid complex.   

2. hydrolyse the protein-Acid hydrolysis of dinitrophenyl-amino acid complex leads to the breaking of peptide bond to release dinitrophenyl-amino acid complex in solution.

3. Separation and analysis of derivatized amino acids-A HPLC or TLC separation of complex and comparing with the standard amino acids.

 

Figure 33.5: Derivatization of N-terminal amino acid with sanger reagent