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4.2. AMINO ACID COMPOSITION
The amino acid composition of proteins and peptides is normally deduced
from their genetic codes. However, unknown proteins or peptides (or more
complex materials such as biological tissue, cells etc.) are normally subjected
to conventional amino acid analysis. This is an analytical process involving
the following steps:
1.
Total acid hydrolysis (6 M HCl, 110
°
C, 20 hours). This will hydrolyse
almost all peptide linkages
2.
HPLC separation of free amino acids (ion exchange or reverse phase)
3.
Trp: Tryptophan is destroyed in acid hydrolysis, and a separate alkaline
hydrolysis is needed in this case.
4.
Asp and Gln: The carboxamide (-(C=O)-NH
2
)) of the side chains in
asparagine and glutamine is unstable in acid and hydrolyses to form
aspartic acid (Asp) and glutamic acid (Glu), respectively. Hence, what is
quantified by HPLC as Asp is in reality the sum of Asp and Asn. The same
goes for glutamic acid. (‘Gln’ = Gln + Glu)
Example: HPLC separation of a hydrolysed protein: