Methods to determine primary structures: The primary structure determination of protein has multiple stages. A protein needs to go through following stages for elucidation of its sequence as well as bonding pattern. These stages are schematically given in Figure 33.3. Over-all, the complex protein first needs to break into the subunits, and sequential release of amino acids from N-terminus of each fragment following edman-degradation method. At the end, the sequence of each fragment can be put together to deduce the complete amino acid sequence of protein. The details of each stage is as follows-

Figure 33.3: Over-View of the different stages in sequencing of a protein.

Stage 1. Breaking Disulphide Bonds: In protein two cysteine amino acids are linked by a disulphide linkage. The disulphide linkage interfere with the complete sequencing procedure as it doesn’t allow the release of cleaved amino acid from the peptide chain. There are two approaches two disrupt the disulphide linkage in a protein sequence (Figure 33.4). In first approach, protein is oxidized with a performic acid to produce two cysteic acid residues. In another approach, protein is reduced by dithiothreitol (DTT) or β-mercaptoethanol (β-me) to form two cysteine followed by treatment with iodoacetate to form carboxymethyl-cysteine. Formation of carboxymethyl-cysteine stops the re-formation of disulphide bond.