Elementary plant Biochemistry and Biotechnology (2+1)

Separation and purification of polypeptide chains

 
• Determination of C-terminal and/or N-terminal amino acids reveals the presence of one or more polypeptide chains in a protein.
• If the protein contains more than one polypeptide chain, separation of polypeptide chain is essential.
  
• If the polypeptide chains are connected by disulfide bond, they are cleaved to separate the individual peptide chains.
• The polypeptide is treated with 2-mercaptoethanol (HS-CH2-CH2OH) so that reductive cleavage occurs and the polypeptide chains are separated.
• The resulting free-SH groups are usually alkylated by treatment with iodoacetic acid.
  
• After cleaving the disulfide links using mercaptoethanol, subunits are dissociated using denaturing agents such as urea or guinidinum ion or detergents such as sodium dodecyl sulphate (SDS).
  
• The dissociated subunits are then separated using ion exchange or gel filtration chromatographic method.

Amino acid Sequencing of polypeptides

• The amino acid sequence in polypeptides with 30-40 amino acids can be determined by Edman reaction.
• For polypeptides containing more than 40 amino acids, both enzymatic and chemical methods are employed to break polypeptide chains into smaller peptides.
• The enzyme, trypsin hydrolyses the peptide bond on the carboxyl side of the basic amino acid residues of lysine or arginine.
• The chemical reagent, cyanogens bromide cleaves peptide bond on the carboxyl side of methionine residues.
• The hydrolyzed peptides are separated and the amino acid sequence is determined by Edman reaction.
  
• The hydrolysis of the original polypeptide by two different methods separately gives overlapping regions, from which the sequence is derived