Chapter 27: Amino Acids, Peptides and Proteins

Sequencing Peptides

• In order to determine which amino acids are present in a protein, the protein has to be broken apart.
• This requires that the amide bonds are hydrolysed to the component amino acids (review including mechanism ?)

• Chemical hydrolysis using 6M HCl / heat will break all the amide bonds (i.e. it is not selective).
• The amino acids are then separated using chromatography (this tells us which amino acids are present)
• Reaction with ninhydrin allows the molar ratios of the amino acids to be determined. (i.e. how much is present)
• Enzymatic hydrolysis using proteases is more selective and can be used to break the protein into fragments rather than individual amino acids.

As examples:
• Trypsin cleaves amides where the C=O is part of lysine or arginine.
• Chymotrypsin cleaves amides where the C=O unit has an aromatic side chain : phenylalanine, tyrosine, tryptophan.
• Carboxypeptidases cleaves only at the C-terminus (this allows the C-terminus to be identified)
• End group analysis means that a "molecular flag" is attached to the N-terminus so allowing it to be recognised.
• Once this information has been collected, the pieces needed to be analysed and the sequence carefully deduced.